Background Patients with Lynch syndrome (LS), characterized by germline inactivation of one allele of the mismatch repair (MMR) genes, have an increased risk (up to 80%) of developing cancers with high microsatellite instability (MSI-H).¹ MSI-H tumors account for 15–30% of all colon, endometrial, and gastric cancers.² Due to unrepaired frameshift (fs) mutations, these cancers possess a high tumor mutational burden which has enabled microsatellite instability to emerge as a biomarker of immune checkpoint blockade (ICB) response. However, there is still a significant population of MSI-H patients unresponsive to ICB treatment.^3,4 Therefore, novel strategies to prevent LS-associated tumor development and predict ICB resistance are urgently needed. T cell surveillance in LS is evidenced by (i) elevated fs-loads in MMR-deficient (MMRd) nonneoplastic tissue⁵ and (ii) a correlation between increased T cell infiltration into normal mucosa of LS patients and delayed onset of colorectal cancer.⁶ We previously characterized unique immunogenic fs-peptides shared in MSI-H cancers⁷ and identified T cell receptors (TCRs) specific to MSI-H-associated fs-peptides. Utilizing TCR sequencing, we demonstrated that fs-specific T cells were present in the primary tumor, draining lymph nodes, and metastases of an LS patient. However, tumor growth in the presence of these fs-specific T cell clones suggests suboptimal T cell surveillance.

Methods Our hypothesis is that MMRd precancerous and malignant lesions expressing fs-neoantigens escape immune surveillance due to suppression of T cell infiltration and activity. We investigated whether this is the result of immunosuppressive activity from innate immune cell populations and/or T cell dysfunction. Using normal, precancerous, and tumor tissue collected from our cohort of LS and sporadic MSI-H cancer patients we are leveraging whole-exome sequencing, spatial transcriptomics, and multiplexed immunohistochemistry.

Results This has allowed us to begin to define the spatiotemporal landscape of fs-neoantigen expression, innate immune cell localization, and T cell dysfunction in the course of MSI-H tumor development to explain how MMRd lesions escape T cell surveillance. Additionally, our analysis of MSI-H tumors from The Cancer Genome Atlas (TCGA) with high fs-neoantigen loads but low cytotoxic T lymphocyte (CTL) signatures (assessing granzyme and perforin expression) have revealed lower immune-stimulatory macrophage and higher activated mast cell-related gene signatures. We have also observed high frequencies of exhausted (PD-1+TIM-3+) tumor infiltrating lymphocytes in advanced MSI-H tumors assessed by flow cytometry.

Conclusions This work supports the development of fs-neoantigen-based vaccination/immunomodulation strategies to prevent LS-associated tumor development and identifies potential ICB resistance biomarkers for advanced MSI-H tumors.

References

1. Roudko V, Cimen Bozkus C, Greenbaum B, Lucas A, Samstein R, Bhardwaj N, Lynch Syndrome and MSI-H Cancers: From Mechanisms to ‘Off-The-Shelf’ Cancer Vaccines. Frontiers in Immunology. 2021;12:3946.

2. Bonneville R, Krook MA, Kautto EA, Miya J, Wing MR, Chen HZ, et al., Landscape of Microsatellite Instability Across 39 Cancer Types. JCO Precis Oncol. 2017.

3. Bellone S, Roque DM, Siegel ER, Buza N, Hui P, Bonazzoli E, et al., A phase 2 evaluation of pembrolizumab for recurrent Lynch-like versus sporadic endometrial cancers with microsatellite instability. Cancer. 2021.

4. Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, et al. PD-1 Blockade in Tumors with Mismatch-Repair Deficiency. N Engl J Med. 2015;372:2509–20.

5. Lee BCH, Robinson PS, Coorens THH, Yan HHN, Olafsson S, Lee-Six H, et al., Mutational landscape of normal epithelial cells in Lynch Syndrome patients. Nat Commun. 2022;13:2710.

6. Bohaumilitzky L, Kluck K, Hüneburg R, Gallon R, Nattermann J, Kirchner M, et al., The Different Immune Profiles of Normal Colonic Mucosa in Cancer-Free Lynch Syndrome Carriers and Lynch Syndrome Colorectal Cancer Patients. Gastroenterology. 2022;162:907–19.

7. Roudko V, Bozkus CC, Orfanelli T, McClain CB, Carr C, O’Donnell T, et al., Shared Immunogenic Poly-Epitope Frameshift Mutations in Microsatellite Unstable Tumors. Cell. 2020;183:1634–49.

Ethics Approval The study was approved by the Icahn School of Medicine at Mount Sinai Institutional Review Board, approval numbers: STUDY-21–01317 and STUDY-19–00936-CR001. Informed consent was given by all participants in this research study.