Prognostic significance of CD8+ T cell Spatial Biomarkers in ER+ and ER− breast cancer: A retrospective cohort study

Andrew E. Walker, Ricky W. Johnstone, Xiaohua Gao, Qichen Wang, Gabriela De la Cruz, Didong Li, Charles M. Perou, Joel Saltz, James Steve Marron, Katherine A. Hoadley, Melissa A. Troester

Abstract: Background Tumor infiltrating lymphocytes (TILs) are prognostic in triple-negative breast cancer, but not estrogen receptor (ER) positive cancers which comprise 70%–80% of breast cancers. This is due to the relatively low immune infiltration in ER-positive tumors. However, few studies have explored the prognostic impact of lower abundance TILs evaluated using spatial methods. The objective of this study was to explore whether the distribution of lymphocytes with respect to tumor cells predicts prognosis. Methods and findings In this retrospective cohort study, we used multiplex immunofluorescent (IF)-stained images of tissue microarray cores (stained for cytokeratin [Ck], CD8, and FoxP3) obtained from 1,467 study participants to compute distance-based visual morphometry for epithelial and immune cells, including two new metrics, proximity and consistency. Proximity and consistency are defined as functions of the mean and variance of nearest neighbor distances between Ck+ tumor cells and CD8+ T cells. Prognostic significance of proximity and consistency were compared to lymphocyte counts using log-rank tests of differences in Kaplan–Meier survival curves and Cox proportional hazards models. Better recurrence-free survival (RFS) was observed for both ER+ and ER− breast cancers with high proximity and consistency of CD8+ T cells. Among ER− breast cancers, proximity had the highest RFS hazard ratio (HR 1.84, 95% CI [1.18, 2.87]; p = 0.0069) compared to count and consistency. Among ER-positive participants, RFS hazard ratios for proximity and consistency were 2.04 (95% CI [1.39, 2.98]; p = 0.0003) and 1.82 (95% CI [1.23, 2.69]; p = 0.0026), respectively. These associations were stronger than those observed for lymphocyte count (HR 1.35, 95% CI [0.92, 1.98]; p = 0.1289). Independent prognostic value was demonstrated by controlling clinical and demographic variables such as age, tumor grade, stage, ER status, progesterone receptor status, and human epidermal growth factor receptor 2 status. These IF-derived spatial metrics were also associated with established TILs metrics and RNA expression-based measures of tumor adaptive immune response. Though these results are promising, our exploration of the tumor immune microenvironment was limited by the small number of immune markers available for our data. Conclusions Spatial characteristics described by proximity and consistency are frequently associated with recurrence irrespective of ER status. The prognostic significance of proximity in ER+ breast cancer implies that spatial parameters may identify individuals who would benefit from immune therapy; up to 75% of breast cancers experience T cell proximity suggestive of immune susceptibility.

External IDs:doi:10.1371/journal.pmed.1004647