Abstract 169: Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas
Authors: Huang Zhihao , Mandelkow Tim , Müller Jan H. , Marx Andreas H. , Krech Till , Sauter Guido , Ebner Julia , Lennartz Maximilian , Simon Ronald , Kluth Martina , Hube-Magg Claudia , Minner Sarah , Bady Elena , Blessin Niclas C. 
Published: March 2024

Abstract
Background: Although there is raising evidence that the density and the level of immune checkpoint expression of 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 cell subpopulations and their spatial interplay is lacking.
Material and methods: To comprehensively assess the density, level of immune checkpoint expression, and spatial interplay of 48 immune checkpoint expressing intraepithelial leukocyte subpopulations in 46 carcinoma entities, 4740 tumor samples in a tissue microarray format were stained with 21 antibodies using our BLEACH&STAIN mfIHC approach. A deep learning-based framework comprising 18 different convolutional neuronal networks (U-Net and DeepLabv3+) were used for image analysis.
Results: 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 example, 88 (±90) cells/mm2 were found in tubular breast cancer, 661 (±729) cells/mm2 in colorectal cancer, and 1280 (±1334) to 2325 (±2131) cells/mm2 in squamous cell cancers from various origins. The abundance of T-cell subpopulations, M2 macrophages, and dendritic cells was highly concordant (range r=0.49 to 0.92) across all tumor entities, while the density of M1 macrophages was unrelated (r=0.1, p&lt;0.001). Unsupervised hierarchical clustering across all tumor entities revealed a cluster A of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells, thus a highly inflamed tumor microenvironment that was characterized by the strongest interaction between intraepithelial CD8+ T-cell and CD4+ T-helper cells, M2 macrophages, as well as dendritic cells (DC, p&lt;0.002 each) along with the highest expression level of TIM3, PD-1, CLTA-4 and PD-L1. In contrast, a cluster C of 2245 patients with the lowest intraepithelial immune cell infiltration was characterized by a significantly increased M1 proportion (p&lt;0.001) and an enriched proportion of regulatory T-cells (p&lt;0.001). Across all analyzed tumor entities, the intraepithelial highly inflamed cluster A was significantly linked to low pT (p&lt;0.001).
Conclusion: The data from this study provide a comprehensive characterization of intraepithelial immune cells across 46 different human carcinomas and identify an inflamed phenotype defined 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, PD-L1, and CTLA-4 expression that is linked to a favorable tumor phenotype.
Citation Format: Zhihao Huang, Tim Mandelkow, Jan H. Müller, Andreas H. Marx, Till Krech, Guido Sauter, Julia Ebner, Maximilian Lennartz, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Elena Bady, Niclas C. Blessin. Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 169.