SpliceMutr Enables Pan-Cancer Analysis of Splicing-Derived Neoantigen Burden in Tumors

Aberrant alternative splicing can generate neoantigens, which can themselves stimulate immune responses and surveillance. Previous methods for quantifying splicing-derived neoantigens are limited by independent references and potential batch effects. Here, we introduce SpliceMutr, a bioinformatics approach and pipeline for identifying splicing-derived neoantigens from tumor and normal data. SpliceMutr facilitates the identification of tumor-specific antigenic splice variants, predicts MHC-binding affinity, and estimates splicing antigenicity scores per gene. By applying this tool to transcriptomic data from The Cancer Genome Atlas, we generate splicing-derived neoantigens and neoantigenicity scores per sample and across all cancer types and find numerous correlations between splicing antigenicity and well-established biomarkers of antitumor immunity. Notably, carriers of mutations within splicing machinery genes have higher splicing antigenicity, which provides support for our approach. Further analysis of splicing antigenicity in cohorts of patients with melanoma treated with mono- or combined immune checkpoint inhibition suggests that the abundance of splicing antigens is reduced post-treatment from baseline in patients who progress. We also observe increased splicing antigenicity in responders to immunotherapy, which may relate to an increased capacity to mount an immune response to splicing-derived antigens. We find the splicing antigenicity to be higher in tumor samples when compared with normal, that mutations in the splicing machinery result in increased splicing antigenicity in some cancers, and higher splicing antigenicity is associated with positive response to immune checkpoint inhibitor therapies. Furthermore, this new computational pipeline provides novel analytical capabilities for splicing antigenicity and is openly available for further immuno-oncology analysis. Significance: SpliceMutr shows that splicing antigenicity changes in response to ICI therapies and that native modulation of the splicing machinery through mutations increases the contribution of splicing to the neoantigen load of some The Cancer Genome Atlas cancer subtypes. Future studies of the relationship between splicing antigenicity and immune checkpoint inhibitor response pan-cancer are essential to establish the interplay between antigen heterogeneity and immunotherapy regimen on patient response.