Quantitative, qualitative and spatial analysis of lymphocyte infiltration in periampullary and pancreatic...

Immunotherapeutic modalities are currently revolutionizing cancer treatment. In pancreatic cancer, however, early clinical trials have been disappointing. The optimization of immunotherapeutic strategies requires better understanding of the inflammatory tumor microenvironment. Therefore, the aim of our study was to perform a detailed in situ description of lymphocyte infiltration patterns in resected pancreatic and other periampullary cancers. Multiplexed immunofluorescence imaging was applied to tissue microarrays with tumors from a cohort of 175 patients with resected periampullary adenocarcinoma. A panel of immune cell markers including CD4, CD8α, FoxP3, CD20, CD45RO and pan-cytokeratin was applied to allow for simultaneous spatial analysis of multiple lymphocyte populations. The majority of lymphocyte populations were significantly more abundant in intestinal (I-type) compared to pancreatobiliary (PB-type) tumors. Hierarchical cluster analysis revealed several immune cell signatures of potential clinical relevance. Notably, in the stromal compartment of PB-type tumors, high infiltration of B cells, CD8α⁺ CD45RO⁺ and single-positive CD4⁺ T cells, but low levels of FoxP3⁺ CD45RO^high and single-positive CD8α⁺ T cells were associated with improved overall survival (OS). The study also defined prognostic relevant topographical patterns of lymphocytic infiltration, in particular proximity of CD8α⁺ cells to cancer cells. Moreover, the presence of lymphocytes with potential T-helper capacities (CD4⁺ ) in the nearest vicinity to CD8α⁺ cells was associated with a prolonged OS. Our data demonstrate that the composition and clinical impact of immune infiltrates in periampullary adenocarcinoma differ by morphological type as well as localization. Furthermore, spatial in situ analysis identified potential immunological mechanisms of prognostic significance.