Tumour Infiltrating Lymphocytes and Immune-Related Genes as Predictors of Outcome in Pancreatic Adenocarcinoma

Background: Pancreatic Carcinoma (PC) is a lethal disease with a poor prognosis. Pancreatic Carcinoma is characterized by a desmoplastic, highly heterogeneous and immune-suppressive microenvironment that hinders antitumour immunity. The aim of this study is to investigate the correlation between PC patient prognosis and the presence of tumour infiltrating lymphocytes and expression of 521 immune system genes. Materials and Method: Fresh PC specimens were obtained from patients (n=12) undergoing surgical resection at the Department of Medical, Surgical & Health Sciences, Cattinara Teaching Hospital, Trieste University, between 2005 and 2015. Prognosis of primary PC patients was determined using clinical data and Kaplan-Meier curves. Overall survival (OS) was measured from the time of surgery to the time of death or the last follow up visit. A more in-depth analysis of the 12 patients revealed two groups with different disease free survival (DFS) and/or OS: six patients with an OS between 25 and 66 months were classified as “good cases”, while six with OS between 2 and 9 months or DFS between 1 and 2 months were classified as “worse cases”. Intratumoural CD3+, CD8+, and CD20+ lymphocytes were examined by immunohistochemistry. The results were correlated with gene expression profile using the digital multiplexed NanoString nCounter analysis system (NanoString Technologies, Seattle, WA, USA). Results: Our data showed that the CD3 level was statistically higher in the good prognosis group compared to the worse prognosis group (p=0.0267). Three primary PC patients with a good prognosis and three with a worse prognosis were then chosen for mRNA analysis by PanCancer Immune Profile Panel multiplex gene expression analysis. Twenty immune system genes were significantly differentially expressed in patients with a good prognosis relative to patients with a worse prognosis: TLR2 and TLR7 (Toll-like receptor superfamily); CD4, CD37, FOXP3, PTPRC (B cell and T cell signalling); IRF5, IRF8, STAT1, TFE3 (transcription factors); ANP32B, CCND3 (cell cycle); BTK (B cell development); TNF, TNFRF1A (TNF superfamily); HCK (leukocyte function); C1QA (complement system); BAX, PNMA1 (apoptosis); IKBKE (NFB pathway). Differential expression was more than twice log 2 for TLR7, TNF, C1QA, FOXP3, and CD37. Conclusion: The key findings from this study, that longer-surviving PC patients had higher levels of intratumoural TILs and overexpressed five immune markers (TLR7, TNF, C1QA, FOXP3, CD37), could have two main uses. Together with an assessment of TIL levels, such an immune system gene panel constitutes a potential prognostic tool to permit a risk-based stratification of pancreatic tumour patients into personalized treatment protocols towards improving the current abysmal clinical outcome of these patients.