Redefining phenotypic heterogeneity of pancreatic ductal adenocarcinoma: A bottom-up approach

Background Pancreatic ductal adenocarcinoma (PDAC) tumor inter-patient heterogeneity has been well described with two major prognostic subtypes (classical and basal-like). An important intra-patient heterogeneity has been reported but has not yet been extensively studied due to the lack of standardized, reproducible and easily accessible high throughput methods. Material and Methods We built an immunohistochemical (IHC) tool capable of differentiating RNA-defined classical and basal-like tumors by selecting relevant antibodies using a multi-step process. The successive stages of i) an in-silico selection from a review literature and a bulk transcriptome analysis of 309 PDACs, ii) a tumor-specific selection from 30 patient-derived xenografts followed by iii) the validation on tissue microarrays in 50 PDAC were conducted. We used our final IHC panel on two independent cohorts of resected PDAC (n=95, whole-slide, n=148, tissue microarrays) for external validation. After digitization and registration of pathology slides, we performed a tile-based-analysis in tumor and pre-neoplastic epithelial areas and a k-means clustering to identify relevant marker combinations. Results Sequential marker selection led to the following panel: GATA6, CLDN18, TFF1, MUC16, S100A2, KRT17, PanBasal. Four different phenotypes were identified: 1 classical, 1 intermediate (KRT17+) and 2 basal-like (MUC16+ vs S100A2+) with specific biological properties. The presence of a minor basal contingent drastically reduced overall survival, even in classical predominant PDACs (HR=2.36, p=0.01). Analysis of preneoplastic lesions suggested that pancreatic carcinogenesis may follow a progressive evolution from classical toward a basal through an early intermediate phenotype. Conclusion Our IHC panel redefined and easily assessed the high degree of intra- and inter-tumoral heterogeneity of PDAC. ### Competing Interest Statement CN received consulting fees from Amgen, AstraZeneca, Baxter, Bristol-Myers Squibb, Fresenius Kabi, Incyte Biosciences, Merck, MSD, Mundipharma, Mylan, Novartis, Nutricia, OSE Immunotherapeutics, Pierre Fabre, Roche, Sanofi, Servier, Viatris, grants from OSE Immunotherapeutics, AstraZeneca, Bristol-Myers Squibb, Fresenius Kabi, Nutricia and support for attending meetings and/or travel from Merck, MSD, Mylan/Viatris, OSE Immunotherapeutics, Pierre Fabre. MH received consulting fees from Amgen and Pierre Fabre. ND and JI are co-founders of Predicting Med. VR, LDM, AC, FD, JR, YB, TB, MR, RN and JC declare no conflicts of interests.