Prognostic Impact and Spatial Interplay of Immune Cells in Urothelial Cancer

Background and objective: Quantity and the spatial relationship of specific immune cell types can provide prognostic information in bladder cancer. The objective of the study was to characterize the spatial interplay and prognostic role of different immune cell subpopulations in bladder cancer. Methods: A total of 2463 urothelial bladder carcinomas were immunostained with 21 antibodies using BLEACH&STAIN multiplex fluorescence immunohistochemistry in a tissue microarray format and analyzed using a framework of neuronal networks for an image analysis. Spatial immune parameters were compared with histopathological parameters and overall survival data. Key findings and limitations: The identification of > 300 different immune cell subpopulations and the characterization of their spatial relationship resulted in numerous spatial interaction patterns. Thirty-nine immune parameters showed prognostic significance in univariate analyses, of which 16 were independent from pT, pN, and histological grade in muscle-invasive bladder cancer. Among all these parameters, the strongest association with prolonged overall survival was identified for intraepithelial CD8(+) cytotoxic T cells (time-dependent area under receiver operating characteristic curve [AUC]: 0.70), while stromal CD8(+) T cells were less relevant (AUC: 0.65). A favorable prognosis of inflamed cancers with high levels of "exhaustion markers" suggests that TIM3, PD-L1, PD-1, and CTLA-4 on immune cells do not hinder antitumoral immune response in tumors rich of tumor infiltrating immune cells. Conclusions and clinical implications: The density of intraepithelial CD8(+) T cells was the strongest prognostic feature in muscle-invasive bladder cancer. Given that tumor cell killing by CD8(+) cytotoxic T lymphocytes through direct cell-to-cell-contacts represents the "terminal end route" of antitumor immunity, the quantity of "tumor cell adjacent CD8(+) T cells" may constitute a surrogate for the efficiency of cancer recognition by the immune system that can be measured straightaway in routine pathology as the CD8 labeling index. Patient summary: Quantification of intraepithelial CD8(+) T cells, the strongest prognosticfeature identified in muscle-invasive bladder cancer, can easily be assessed by brightfield immunohistochemistry and is therefore "ready to use" for routine pathology.