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Supplementary Figure 3: Association of chemokine expression with a T cell-inflamed phenotype was validated in a second independent PDAC patient cohort

Supplementary Table 1: Top 99 genes associating with CD8+ TIL counts in primary resected cases

Purpose:The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC.Experimental Design:Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing.Results:Among all known human chemokines, a coregulated set of four (CCL4, CCL5, CXCL9, and CXCL10) was strongly associated with CD8+ T-cell infiltration (P < 0.001). Expression of this “4-chemokine signature” positively correlated with transcriptional metrics of T-cell activation (ZAP70, ITK, and IL2RB), cytolytic activity (GZMA and PRF1), and immunosuppression (PDL1, PD1, CTLA4, TIM3, TIGIT, LAG3, FASLG, and IDO1). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell–inflamed phenotype across primary PDAC and PDAC liver metastases.Conclusions:A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.

Purpose: The molecular drivers of antitumor immunity in pancreatic ductal adenocarcinoma (PDAC) are poorly understood, posing a major obstacle for the identification of patients potentially amenable for immune-checkpoint blockade or other novel strategies. Here, we explore the association of chemokine expression with effector T-cell infiltration in PDAC. Experimental Design: Discovery cohorts comprised 113 primary resected PDAC and 107 PDAC liver metastases. Validation cohorts comprised 182 PDAC from The Cancer Genome Atlas and 92 PDACs from the Australian International Cancer Genome Consortium. We explored associations between immune cell counts by immunohistochemistry, chemokine expression, and transcriptional hallmarks of antitumor immunity by RNA sequencing (RNA-seq), and mutational burden by whole-genome sequencing. Results: Among all known human chemokines, a coregulated set of four (CCL4, CCL5, CXCL9, and CXCL10) was strongly associated with CD8+ T-cell infiltration (P < 0.001). Expression of this “4-chemokine signature” positively correlated with transcriptional metrics of T-cell activation (ZAP70, ITK, and IL2RB), cytolytic activity (GZMA and PRF1), and immunosuppression (PDL1, PD1, CTLA4, TIM3, TIGIT, LAG3, FASLG, and IDO1). Furthermore, the 4-chemokine signature marked tumors with increased T-cell activation scores (MHC I presentation, T-cell/APC costimulation) and elevated expression of innate immune sensing pathways involved in T-cell priming (STING and NLRP3 inflammasome pathways, BATF3-driven dendritic cells). Importantly, expression of this 4-chemokine signature was consistently indicative of a T-cell–inflamed phenotype across primary PDAC and PDAC liver metastases. Conclusions: A conserved 4-chemokine signature marks resectable and metastatic PDAC tumors with an active antitumor phenotype. This could have implications for the appropriate selection of PDAC patients in immunotherapy trials.

Pancreatic cancer continues to have the highest mortality rate of all solid cancers, with a 5-year overall survival of approximately 8%. Despite recent progress in other malignancies, immunotherapy remains ineffective against pancreatic cancer. Previous work from our group and others has shown that tumors with mismatch repair (MMR) and homologous repair (HR) deficiency have increased immune activity, likely attributable to the inherently increased mutational and neoantigen load in these patients. However, there remains a subset of pancreatic cancer patients with increased CD8+ T cell infiltration, despite lacking these specific mutational signatures. Furthermore, an increasing body of evidence has shown that mutational burden and neoantigen load only partially explain the T cell-inflamed phenotype seen in tumors, implicating the presence of additional mechanisms that drive T cell infiltration in cancer. Several recent studies have highlighted the importance of proper CD8+ T cell priming by antigen presenting cells (APCs), particularly Batf3+ dendritic cells (DCs), and subsequent tumor infiltration via chemokines for immunotherapy to be effective. In this study, we investigated the involvement of chemokines in cytotoxic T lymphocyte infiltration in pancreatic cancer. Using a bioinformatics-driven approach, we analyzed 78 treatment-naïve, primary pancreas cancer resections for associations between CD8+ tumour infiltrating lymphocytes (TILs) and chemokine expression by RNAseq. A panel of chemokines, including CCL4, CCL5, CXCL9, CXCL10, and CXCL11, was highly associated with CD8+ TILs (p < 0.001). Segregating 173 tumour-enriched patient RNAseq samples based on expression of CXCL9 and CXCL10, we found those with higher expression of these chemokines had increased immune activation signatures. Importantly, this included increased MHC I presentation, presence of Batf3+ DCs, and T cell/APC co-stimulation (p < 0.001), while we observed no differences in conventional predictors of CD8+ T cell infiltration such as SNV counts or neoantigens between groups. These results were consistent across ICGC and TCGA data sets. Moreover, these results were also recapitulated in 72 tumor-enriched liver metastases, suggesting an underlying immunobiology that may occur in both primary and metastatic sites. The cellular sources of these chemokines, as determined by immunohistochemical analysis, and the role of tumor-sensing innate immune pathways leading to CD8+ T cell priming, by pathway and differential gene expression analysis, are currently being investigated. Taken together, these results demonstrate a potential role for these chemokines in recruiting CD8+ T cells in pancreatic cancer. Understanding mediators driving cytotoxic T cell infiltration will help identify mechanisms leading to proper CD8+ T cell priming and homing into tumors, to stratify patients amenable for known and novel immunotherapies. Citation Format: Joan Miguel Romero, Barbara Grünwald, Ashton Connor, Gun Ho Jang, Prashant Bavi, Aaditeya Jhaveri, Mehdi Masoomian, Sandra Fischer, Amy Zhang, Robert E. Denroche, Tracy McGaha, Faiyaz Notta, Pamela Ohashi, Grainne O'Kane, Julie Wilson, Jennifer Knox, Steven Gallinger. Mediators of CD8+ cytotoxic T lymphocyte infiltration in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1500.