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Background: Keratin 17 (K17) is a negative prognostic biomarker, overexpressed in the biologically most aggressive forms of pancreatic ductal adenocarcinoma (PDAC). In other anatomic sites and disease processes, K17 expression also correlates with immune cell infiltrates and could block T-cell infiltration. Thus, we hypothesized that K17 expression correlates with the inflammatory microenvironment in PDAC. In this study, we aimed to determine the relationship between the stromal immune cell infiltrates and K17 expression, using multiplexed immunohistochemistry (mIHC) and our suite of deep learning tools to quantitatively evaluate the expression of four biomarkers of T-cells and macrophages in PDAC. Methods: mIHC was performed on representative sections of 201 primary PDACs from Stony Brook University Hospital, Thomas Jefferson University Hospital, Cedars Sinai Medical Center, and from a national cohort (KYT, Pancreatic Cancer Action Network, and Perthera). Antibodies for CD4 (helper T-cells), CD8 (cytotoxic T-cells), CD16 (pan-macrophage), CD163 (M2 macrophages), pancytokeratin, and K17 were provided by Roche Diagnostics Corporation through a sponsored research agreement. mIHC was performed on a Discovery Ultra Autostainer (Roche), using horseradish peroxidase (HRP) and alkaline phosphatase (AP)-based protocols with multiple chromogens (Red: CD4, Purple: CD8, Yellow: CD16, Green: CD163, Teal: pancytokeratin, and Brown: K17) to enable multispectral imaging of diverse immune cell populations within the cancer microenvironment. A deep learning analysis workflow was used to detect and classify stromal inflammatory cells, in whole slide images (WSIs), generated using an Olympus VS120 digital microscope (Olympus, Tokyo, Japan). Pixel-wise predictions from a color auto-encoder (ColorAE) union UNET anchor UNET model were combined to create multi-class masks that were further analyzed to perform detection and classification. Results: The analysis of the inflammatory microenvironment focused on defining immune cell infiltrates located within 25 microns of the closest K17-positive versus K17-negative tumor cell in each representative section. Across the sum of K17-positive and negative zones/section, CD4 cell counts ranged from 0-10,617 (mean 2,709), CD8 cell counts ranged from 63-28,596 (mean 6,745), CD16 cell counts ranged from 4-7,797 (mean 3,024), and CD163 cell counts ranged from 35-34,696 (mean 14,968). CD4 T-helper cells, CD8 cytotoxic T cells, and CD16 macrophages were more numerous (respectively, p=0.0012; p=<0.0001; p=<0.0001) in K17-negative tumor zones compared to K17-positive zones. By contrast, the number of CD163 (M2) tumor-promoting macrophages was greater in K17 positive zones (p=0.0019). Conclusion: K17 expression by tumor cells impacts the chronic inflammatory microenvironment, shielding tumor cells from immune cell mediated cytotoxic responses, while recruiting tumor-promoting M2 macrophages, indicating that K17 impacts the immune response as a fundamental hallmark of aggression in PDAC. Citation Format: Lyanne Oblein, Michael Horowitz, Mahmudul Hasan, Sruthi Babu, Mariana Torrente-Goncalves, Lucia Roa, Jaymie Oentoro, Jason Harper, Xin Yao Zheng, Wei Jiang, Andrew Hendifar, Natalie Moshayedi, Brent Larson, Veronica Placencio-Hickok, Edik Blais, Emmanuel Petricoin, Joel Saltz, Natalia D. Marchenko, Luisa F. Escobar-Hoyos, Kenneth Shroyer. Keratin 17 excludes CD8-positive T cells and recruits CD163-positive macrophages in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C071.

Background: Clinically and histologically identical pancreatic ductal adenocarcinomas (PDACs) differ in response to treatment, disease progression, and survival. Clinical trials are testing the use of either keratin 17 (K17) or GATA6 as surrogate prognostic and predictive biomarkers of PDAC. Still, there is a need to contrast their expression and prognostic value in the same cohort of cases. K17, a biomarker of the most aggressive subtype of PDAC, promotes tumor growth and chemoresistance. GATA6 is a transcription factor necessary for normal pancreatic development, and a surrogate marker of the classical subtype of PDAC, associated with longer-term survival. Our goal was to test the hypothesis that there is a reciprocal relationship between K17 and GATA6 expression and that their detection informs prognostic stratification. Methods: Stony Brook University Hospital PDAC patients were selected, and representative sections were processed for dual-staining by indirect immunoperoxidase methods using antibodies to K17 (KDx Diagnostics) and GATA6 (R&D Systems AF1700). Manual scoring of the proportion of cells with strong (2+) nuclear and/or cytoplasmic staining was determined for K17 versus nuclear staining for GATA6 (PathSQ). Automated image analysis of GATA6 expression was further scored using QuPath v0.3.2 on annotated PDAC sections. Cut-off values for the best COX model for each marker were established to stratify for survival. Results: K17 expression was observed in cancer epithelial cells and in few benign proliferative ducts of chronic pancreatitis but no staining was detected in stromal or acinar cells. By contrast, GATA6 was detected in the nuclei of most cancer epithelial cells and was also consistently detected in benign ductal cells, acinar cells, and stromal cells. Defining high K17 cases as those with staining in ≥10% of tumor cells, high K17 correlated with shorter overall survival (p=0.03), supporting its use as a negative prognostic biomarker. Due to the high level of expression of GATA6 in PDAC cells (range 60-100% of tumor cells across all cases), however, no threshold was found to provide prognostic value. By dual-color IHC, K17 positive cells almost always had strong GATA6 nuclear staining but GATA6 was also detected in all K17-negative tumor cells. Computational analysis demonstrated 98% concordance with manual scoring for high GATA6 and 90% concordance with manual scoring for low GATA6. Conclusions: GATA6 was expressed in most PDAC tumor cells, as well as in benign stromal and epithelial cells. However, K17 expression was focal, relatively tumor cell-specific, and was not detected in most benign cellular components. Although high K17 status identified cases with relatively short survival, a survival threshold for GATA6 could not be detected. Contradiction in our observations of GATA6 compared to previous reports could result from differences in case selection or immunohistochemical protocol. Thus, further work is needed to explore the relationship between K17 and GATA6 as complementary biomarkers of PDAC patient survival. Citation Format: Michael Horowitz, Lyanne Oblein, Jaymie Oentoro, Saumya Kasliwal, Natalia D. Marchenko, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Comparison of keratin 17 and GATA6 as prognostic markers in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B049.

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