Surveillance of Frameshift Neoantigen-Specific T Cells During Tumor Development in Patients With Lynch Syndrome and Mismatch Repair Deficient Colorectal Cancer

Introduction: Lynch syndrome (LS) patients have up to 80% lifetime risk of developing cancers with high microsatellite instability (MSI-H). T cell surveillance throughout mismatch repair deficient (MMRd) colon cancer development is evidenced by i) increased mutation load in MMRd non-neoplastic tissue in LS carriers and ii) a significant correlation between increased T cell infiltration in normal epithelium of LS patients and delayed onset of colorectal cancer. Our group and others previously demonstrated that unrepaired insertion/deletion (indel) mutations in microsatellites caused by loss of MMR protein expression generate frameshift (fs)-neoantigen peptides that are highly conserved among MSI-H colon, endometrial, and gastric cancers. Methods: We aimed to i) measure fs-neoantigen expression in normal, premalignant, and tumor tissue from patients with LS-associated and sporadic MMRd colon cancer and ii) assess the frequency of fs-specific T cells circulating in peripheral blood and infiltrating MMRd colon lesions. Peripheral blood was collected at the time of colonoscopy to isolate peripheral blood mononuclear cells (PBMCs). Bulk RNA and whole-exome sequencing were performed on normal, adenoma, and carcinoma. Leveraging an in vitro neoantigen-specific T cell expansion and stimulation assay developed by our team for patient PBMCs and tumor infiltrating lymphocytes (TILs), immunogenicity of 9 shared fs-peptides in patient peripheral blood was assessed. Results: We detected fs-neoantigen expression in LS patients throughout MMRd carcinogenesis (normal, adenoma and MMRd carcinoma). In vitro neoantigen-specific T cell expansion and stimulation assays of LS patients with and without adenomas on colonoscopy revealed detectable fs-specific T cells in circulation. We also demonstrated that fs-specific T cells are present in the primary tumor, draining lymph nodes, and metastases of LS patients with colorectal cancer by TCR sequencing (Figure 1). Conclusion: We believe this effort will inform target selection for fs-neoantigen-based vaccination to prevent LS-associated tumor development. However, breakthrough tumor growth in the presence of these fs-specific T cell clones suggests suboptimal T cell surveillance in some patients. We plan to leverage single-cell RNA sequencing, spatial transcriptomics, and multiplexed immunohistochemistry to characterize T cell dysfunction and the immunosuppressive tumor microenvironment during MMRd tumor development in an effort to explain how MMRd lesions escape T cell surveillance.Figure 1.: Assessment of fs-neoantigen expression at different stages of tumor development for MMRd CRC using bulk RNA sequencing (Illumina Stranded Total RNA Library) from normal, precancerous (adenoma), and colon tumor tissue from Lynch syndrome patients. Analysis targeted immunogenic indel mutations in genes frequently mutated in microsatellite unstable (MSI-H) tumors shown previously by our group (PMID: 33259803).