Prevalence, immune checkpoint expression and spatial interplay of immune cells is linked to favourable tumor phenotype in 4915 human carcinomas

Although there is raising evidence that immune cell subpopulations that are in direct contact to the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patient's outcome, a comprehensive assessment of intraepithelial immune cells and their spatial interplay is lacking. To assess intraepithelial leukocyte densities, immune checkpoint expression, and spatial interactions in 43 carcinoma entities, 4915 tumor samples in a tissue microarray format were analyzed using a deep learning framework and BLEACH&STAIN multiplex fluorescence immunohistochemistry (mfIHC). This approach enabled single-cell resolution quantification of 21 biomarkers through seven sequential staining and imaging rounds. Immune and tumor cells were classified into 54 subpopulations. The mean intraepithelial immune cell density of CD8+cytotoxic T-cells, CD4+T-helper cells, FOXP3+Tregs, CD20+B-cells, M1/M2-macrophages and CD11c+dendritic cells varied markedly between tumor entities and individual tumors. For instance, 88(±90) cells/mm2 were found in tubular breast cancer, 661(±729) cells/mm2 in colorectal cancer, and up to 2325(±2131) cells/mm2 in squamous cell cancers from various origins. Unsupervised cluster analysis revealed a "cluster a" of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells that was characterized by a unique interaction profile along with the highest immune checkpoint expression. Across all analyzed tumor entities, the intraepithelial highly inflamed cluster a was significantly linked to low pT (p<0.001). The data from this study provide a comprehensive characterization of intraepithelial immune cells across 43 different human carcinomas and identify an inflamed pan-cancer phenotype characterized by strong interactions of intraepithelial CD8+cytotoxic T-cells, CD4+T-cells, dendritic cells, and M2 macrophages along with highest levels of TIM3, PD-1, and CTLA-4 expression that is linked to favorable tumor phenotype.