Multiplex Immunohistochemistry and High-Throughput Image Analysis for Evaluation of Spatial Tumor Immune Cell Markers in Human Breast Cancer: Preliminary Results from the Nashville Breast Health Study

Abstract Purpose Tumor-infiltrating lymphocytes (TILs) have emerged as predictive biomarkers for cancer prognosis. The clinicopathological significance of spatial TIL subpopulations is not well studied due to lack of high-throughput scalable methodology for large population studies.Methods We established a fluorescent multiplex immunohistochemistry (mIHC) method coupled with computer-assisted high-throughput quantitative analysis. We then evaluated associations of six TILs markers (CD3, CD8, CD20, CD56, FOXP3, and PD-L1) with breast cancer prognosis among 188 tumor samples from the Nashville Breast Health Study. Results Our 5-plex fluorescent mIHC workflow was reliable, highly sensitive, non-interfering, and balanced labeling for three biomarkers per tissue section, which is applicable for high-throughput imaging quantification of spatial TILs in regular laboratory settings. In this study we found that the increased intratumoral CD56+ cells were associated with worse overall survival (OS) (HR, 4.89; 95% CI: 1.26-18.95, highest vs lowest tertiles; Ptrend = 0.022), suggesting the subset of immunosuppressive NK cell phenotypes within tumor bed. Increased stromal PD-L1+ cells (HR, 0.01; 95% CI, 0.00-0.89; Ptrend = 0.042) and a high stromal CD8+/FOXP3+ ratio (HR, 0.00; 95% CI, 0.00-0.12; Ptrend = 0.036) were associated with more favorable OS in stage III-IV breast cancer patients. Conclusion We established a reliable 5-plex fluorescent mIHC and showed that CD56+, PD-L1, and CD8+/FOXP3+ ratio may be important biomarkers for breast cancer prognosis. Further studies with a larger sample size are warrant to further elucidate the association between TILs and breast cancer outcomes.