Discovery and validation of frameshift-derived neopeptides in Lynch syndrome: paving the way for novel cancer prevention strategies.

Background: Lynch syndrome (LS), caused by germline pathogenic variants in the mismatch repair genes, leads to high rates of frameshift-derived neopeptide (FSDN) expression due to microsatellite instability (MSI). While colorectal cancer (CRC) prevention is effective, most LS-related tumors lack such strategies. Cancer vaccines targeting FSDNs offer a promising approach for immune interception in LS. This study aimed to identify and validate LS-related FSDNs to develop vaccines for cancer prevention.

Methods: We identified LS-related coding MS mutations and predicted FSDN with high coverage on common Human Leukocyte Antigen (HLA)-I and II alleles. We validated FSDN-associated mutations in colorectal adenomas (CrAD), endometrial cancers (EC), and CRC samples from patients with LS, non-LS tumors, and cell lines. Immunogenicity was assessed through interferon (IFN)-γ enzyme-linked immunospot and flow cytometry analysis of tissue-infiltrating lymphocytes (TILs) from LS carriers.

Results: We prioritized 53 HLA-I and 45 HLA-II FSDNs in MSI tumors using in silico predictions. Validation revealed 86.7% of FSDN-associated mutations present in LS-CRC samples, with a median of 7.67 (6.5-9) mutations in CrADs and 6.02 (2-10) in CRCs. Sequencing of CrAD and EC samples showed 95% and 77.5% of predicted FSDN-associated mutations, respectively. MSI cancer cell lines transcribed 69.8% of FSDNs. Immunogenicity assays showed that 71% of potential FSDNs elicited IFN-γ responses, with a median of 7.37 (1-10.75) HLA-I and 6 (2-5.75) HLA-II FSDNs per patient. After prioritizing 24 FSDN, in a cohort of 19 LS-derived samples (4 CrAD and 15 normal mucosa), 52% (10/19) demonstrated T-cell reactivity to an HLA-I neoantigen pool. CD8+CD137+ activation markers increased significantly (p=0.037) over time and peptide-specific cells were detected by pentamer staining.

Conclusions: Our predicted FSDN set has optimal coverage among LS carriers and can induce IFN-γ inflammatory responses in LS-derived TILs, offering an opportunity for vaccine development.