Immunological and Molecular Insights into Acinar-Ductal Metaplasia and Atypical Flat Lesions as Precursor Lesions of Pancreatic Ductal Adenocarcinoma

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) is known to develop through a stepwise progression from precursor lesions, such as pancreatic intraepithelial neoplasias (PanIN) or intraductal papillary mucinous neoplasms. An alternative carcinogenic pathway has been proposed via transformation of acinar cells, with acinar-ductal metaplasia (ADM) and atypical flat lesions (AFL). Defining the characteristics of PDAC precursors is crucial to better understand PDAC carcinogenesis. Methods 15 KC ( Ptf1a Cre/+ , Kras LSLG12D/+ ) and 15 KPC-like mice ( Ptf1a Cre/+ , Kras LSLG12D/+ , Trp53 LoxP/LoxP , referred as fKPC hereafter) were sacrificed at different time points. A meticulous morphological evaluation was performed to define different pancreatic lesion types. Multiplex immunofluorescence staining was applied to define the characteristics of the immune and stromal microenvironment of the lesions (referred as TME hereafter) using variable markers. In order to investigate the association between the genetic alterations and the components of the microenvironment, all lesion types were subjected to next-generation sequencing (NGS) using a 20 genes-panel Results Multiplex immunofluorescence staining revealed that AFL had the most intense immune cell infiltration compared to PanIN and ADM. AFL were infiltrated by higher number of CD4 + helper T cells, FOXP3 + regulatory T cells and CD19 + B cells compared to all analyzed lesions. They displayed and more CD8 + cytotoxic T cells than PDAC, while peripheral and central PDAC tissues were infiltrated by macrophages in higher frequency. In addition, AFL had more prominent αSMA-expressing myofibroblastic cancer-associated fibroblasts-rich stroma than other lesions. PDAC had higher CXCL12 expression and more common CD109 + cells than other lesions. In NGS analysis, none of the lesions in fKPC mice revealed additional coding mutations, while the preneoplastic lesions in 7 KC mice showed variable coding alterations in 16 different genes. The most frequently affected genes were Arid1a, Rnf43 , and Pik3ca ; in contrast, coding Gnas, Braf , and Idh1 mutations were not found. PDAC precursors in KC mice showed more dense infiltration of adaptive immune cells than in fKPC mice, supporting the immunosuppressive role of Trp53 alterations. Conclusions Our study highlights the unique immunological and stromal features of AFL. Moreover, reinforcing their potential as precursor lesions, ADM and AFL exhibit variable alterations in the genes that have a critical role in PDAC carcinogenesis.