Multiplex immunophenotyping reveals unique characteristics of pancreatic neoplasms

Abstract

Introduction: The tumor microenvironment of pancreatic adenocarcinoma (PDA) is characterized by a dense stroma and infiltration with primarily immunosuppressive cells. While much is known about the immune cellular changes occurring during tumorigenesis in murine models of PDA, data in humans is lacking. We sought to identify difference in the immune microenvironment of neoplastic and pre-neoplastic lesions using multiplex immunohistochemistry. Method: Operative pancreatic tissue was collected from 289 patients undergoing pancreatectomy at the University of Michigan. Slides were prepared from formalin-fixed, paraffin embedded tissue which were subjected to serial rounds of primary and secondary antibody staining followed by tyramide signal amplification. Result: Analysis was performed on tissue from patients with normal pancreata (n=42), mucinous cystic neoplasms (MCN;n=22), intraductal papillary mucinous neoplasms (IPMN;n=80), pancreatic intraepithelial neoplasia (PanIN;n=37) and PDA (n=108). IPMNs and normal pancreata had significantly more epithelial cells when compared to MCN and PDA. In IPMNs, these cells were more frequently positive for the checkpoint inhibitor PD-L1 (mean = 16.4%; p<0.0001). Mucinous lesions (MCN and IPMN) had significantly more antigen presenting cells than other lesion types (mean = 4.6% and 3.2%, respectively; p=0.0048). While there were no differences in relative infiltration of cytotoxic T lymphocytes, PDA had a far greater overall and relative number of regulatory T lymphocytes (Treg) (mean = 17%; p<0.0001). The accumulation of Tregs paralleled progression of neoplasia with increasing infiltration in normal pancreata, PanIN and PDA (1%, 3%, 17%, respectively; p<0.0001). Conclusion: A better understanding of the immune microenvironment of PDA is needed for the development of novel immunotherapeutic approaches. These data demonstrate an accumulation of Tregs as tumors progress from preinvasive to invasive cancer, suggesting a potential avenue for intervention.