Abstract 6424: Harnessing circulating neoantigen-specific T cells for immune surveillance and cancer prevention in Lynch syndrome

Abstract Background: Lynch syndrome (LS) is a hereditary cancer predisposition caused by germline mutations in mismatch repair (MMR) genes, with secondary somatic alterations leading to MMR deficiency (dMMR) and increased mutation rates. These mutations often result in frameshift peptides (FSPs) that act as immunogenic neoantigens (neoAgs), triggering immune responses and expanding specific T cell clonotypes. NeoAg-specific T cells in LS carriers have significant potential for tumor surveillance. This study hypothesizes that these T cells are detectable in the blood, allowing for precise identification of T cell receptors (TCRs) and the exploration of their anti-tumor properties through transcriptomic and clonal analysis. Methods: Peripheral blood mononuclear cells (PBMCs) from 20 LS carriers, including 10 cancer survivors and 10 cancer-free individuals (previvors), were analyzed for neoAg-specific T cell responses. Interferon-gamma (IFN-γ) production was measured using ELISPOT assays. Patient-derived tumor organoids (PDTOs) were co-cultured with peptide-stimulated PBMCs in a microfluidic system to assess T cell cytotoxicity. MHC-I dextramer staining identified circulating neoAg-specific T cells. Single-cell RNA and TCR sequencing mapped tumor-targeting capabilities and TCR clonality. Results: NeoAg-specific T cells from LS carriers showed strong IFN-γ responses, varying by HLA type, with ex vivo priming enhancing activity across all HLAs. PBMCs from both baseline and follow-up samples exhibited significant tumor-killing potency against PDTOs, though LS survivors displayed slightly reduced cytotoxicity over time. Survivors also exhibited increased pro-inflammatory cytokines but decreased Granzyme B, indicating reduced cytotoxicity and potential immunosuppressive shifts. NeoAg-specific T cells, detected in over 1% of PBMCs, were analyzed via single-cell RNA and TCR sequencing. Profiling of these T cells revealed functional states, including proliferative effector cells with tissue-residency markers and progression toward terminal exhaustion, with higher clonality driven by hyperexpanded TCR clones compared to pan-T cells. Public neoAg-specific TCR clonotypes shared across LS carriers were identified by analyzing archival TCR sequencing data from 277 PBMC samples and 14 colorectal mucosal biopsies from an independent project. A total of 309 neoAg-specific clonotypes were shared among previvors and 537 among survivors, including seven hyperexpanded dMMR CRC-related clones. Of these, four were identified in adenocarcinoma lesions, while 29 were detected in tubular adenoma lesions. Conclusion: This study underscores the presence and functionality of circulating neoAg-specific T cells in LS carriers, highlighting their potential as biomarkers for immune monitoring, cancer surveillance, and tumor targeting. Citation Format: Fahriye Duzagac, Nan Deng, Ana Bolivar, Mahira Zaheer, Araceli Garcia Gonzalez, Abel Martel Martel, Jacklyn V Thompson, Laura Reyes Uribe, Alexandre Reuben, Krishna M Sinha, Eduardo Vilar. Harnessing circulating neoantigen-specific T cells for immune surveillance and cancer prevention in Lynch syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6424.