PROJECT SUMMARY Checkpoint blockade immunotherapy has induced dramatic responses in treatment refractory cancers by targeting neoantigens encoded by cancer-specific mutations. Neoantigen load predicts immunotherapeutic response, validating antigen identification as a rational strategy of biomarker discovery. Pancreatic adenocarcinoma (PDAC) however has shown limited efficacy to checkpoint blockade immunotherapy due to presumed neoantigen paucity. However, systematic antigen discovery in PDAC is lacking. 3% of PDAC patients survive > 5 years (long term survivors). As T cell immunity may explain this extreme outcome, they represent the ideal cohort for deep antigen discovery. Using genomic, molecular, and cellular immunoprofiling, computational evolutionary modeling, and neoantigen discovery in these rare long term survivors (n=82), we have discovered that neoantigen quality, but not quantity, is prognostic of survival, and that long term survivors evidence lasting neoantigen-specific T cell immunity. The scientific objectives of this proposal are to address questions essential to translate these findings - 1) is there stage and treatment- specific neoantigen heterogeneity, 2) can neoantigens be identified in the peripheral blood, and 3) can neoantigen quality predict response to immunotherapy. The translational objective is to develop novel tissue and blood-based biomarkers for rational patient and target selection for immunotherapy. The proposal utilizes several highly unique tissue collection strategies ? a) laparoscopic multi-site biopsies to identify stage-specific heterogeneity, b) serial pre- and post-chemotherapy assessment for treatment-specific modulation, and c) evaluation of neoantigen quality as a predictive biomarker on a large, immunotherapeutic PDAC trial. This initiative will also for the first time develop the ability to identify neoantigens in circulating exosomes for blood-based biomarker assessment. The research methodology employs next generation sequencing, transcriptional profiling, computational biophysical modeling, clonotypic T cell profiling, neoantigen discovery, and functional assessments to evaluate the prognostic, predictive, and therapeutic potential of neoantigens. The team comprises of world-class expert junior and senior investigators in a broad range of highly relevant disciplines from Memorial Sloan Kettering Cancer Center, Meyer Cancer Center at the Weill Cornell Medical Center, Tisch Cancer Institute at the Icahn School of Medicine at Mount Sinai, and the Institute for Advanced Study. This initiative is directly relevant to the primary objective to develop novel biomarkers and T cell antigenic targets for the successful application of immunotherapy in PDAC.