Identification of pre-existing ubiquitous neoantigen-reactive tumor-infiltrating T-cells in a patient with metastatic pancreatic neuroendocrine tumor

Abstract

Background Mutation-derived neoantigens, typically identified in primary tumors, are emerging therapeutic targets for personalized cancer vaccines and adoptive T-cell therapies. However, clinical efficacy of neoantigen-directed therapies in patients with metastatic disease remains limited, partly due to inter-site genetic heterogeneity. We investigated whether ubiquitous neoantigens–derived from mutations shared across all tumor sites–could provide more effective, durable targets, particularly in patients undergoing resection of metastatic lesions. Methods Whole-exome and RNA sequencing were performed on 14 tumor samples (primary and 13 synchronous nodal metastases) from a treatment-naïve patient with pancreatic neuroendocrine tumor (PNET). Ubiquitous mutations were identified bioinformatically, and their immunogenicity assessed using in-vitro stimulation of autologous peripheral blood mononuclear cells followed by IFN-γ ELISpot assay. Neoantigen-specific T-cell clonotypes were further identified by HLA-tetramer staining and single-cell RNA/TCR sequencing. Neoantigen-reactive clonotypes identified in peripheral blood were tracked across multiple metastatic sites using bulk TCRβ repertoire sequencing. Results Among 1,195 non-synonymous mutations detected, eight were shared across all 14 tumor sites. Of these, one encoded a neoantigen that elicited a reproducible IFN-γ ELISpot response in peripheral blood, confirming its immunogenicity. Further, we identified the corresponding neoantigen-reactive TCR clonotypes in blood. Comparison with bulk TCRβ repertoires from eight metastatic sites showed that these clonotypes were present in every site analyzed, with evidence of local clonal expansion. Conclusion This study provides direct evidence that a single ubiquitous mutation-derived neoantigen can generate systemic T-cell responses and clonotype expansion across multiple metastatic sites in a TMB-low, TIL-low tumor. Our findings support incorporating mutation-sharing status across metastases as a key criterion for neoantigen selection in cancer vaccines and adoptive T-cell therapies. This approach could inform the design of neoantigen-directed immunotherapies in metastatic PNET and potentially other metastatic solid tumors. What is already known on this topic Neoantigen-directed therapies, such as personalized cancer vaccines or adoptive T-cell transfer, can induce anti-tumor responses but have shown limited success in metastatic disease. One major barrier is genetic heterogeneity between tumor sites, suggesting that targeting ubiquitous mutations–those shared across all tumor sites–may improve the efficacy of such therapies. What this study adds In one patient with metastatic pancreatic neuroendocrine tumor involving 13 lymph nodes, we identified eight ubiquitous mutations, one of which generated a detectable neoantigen-specific T-cell response in blood. The corresponding T-cell clonotypes were found across all metastatic sites analyzed and showed evidence of clonal expansion, providing direct evidence of systemic and local recognition of a shared neoantigen in a TMB-low/TIL-low cancer. How this study might affect research, practice or policy These findings support incorporating mutation sharing across metastases as a key criterion in neoantigen selection for cancer vaccines and adoptive T-cell therapies. This strategy could enhance the relevance and durability of neoantigen- directed approaches in patients with metastatic disease.