Abstract B022: Tumor-enriched splicing events exhibit histology-specific prevalence in pediatric CNS tumors and encode tumor-specific antigens with immunotherapeutic potential

Abstract Alternative splicing is frequently dysregulated in pediatric central nervous system (CNS) tumors, including high-grade gliomas (HGGs) and diffuse midline gliomas (DMGs) which have poor survival outcomes and urgently need new therapeutic strategies. Aberrant splicing events are increasingly being explored as sources of tumor-specific antigens (TSAs) for immunotherapy, yet the tumor-specific splicing landscape in pediatric CNS cancers remains critically underexplored. In this study, we quantified alternative splice events in primary CNS tumors from the Pediatric Brain Tumor Atlas (PBTA) and compared expression to that in healthy brain tissue to identify tumor-enriched splice events (TESEs). Events were detected from RNA-seq data of PBTA CNS tumors (n=1,375) and healthy brain samples from GTEx (<40 years, n=2,642), the Evo-Devo atlas (n=228), and pediatric brain tissue (n=7) using replicate Multivariate Analysis of Transcript Splicing (rMATS-turbo). We limited analysis to exon inclusion (SE), retained introns (RI), and alternative 3’ and 5’ splice site (A3SS, A5SS) events involving annotated splice sites in GENCODE v39. TESEs were defined by an absolute difference in percent spliced-in (|ΔPSI|) > 0.3 between tumors and all healthy brain samples, while oncofetal TESEs were defined as those meeting the same |ΔPSI| threshold compared to postnatal brain samples only. TESEs were annotated with Pfam protein domains and UniProt topological domains to identify those encoding functional and extracellular (EC) domains, respectively. We identified 3,315 recurrent TESEs (>10% of tumors), most of which (77.1%) involved increased exon skipping (n=1,451) or inclusion (n=1,105) in tumors. Of these, 1,483 (44.7%) were classified as oncofetal, and comprised the majority of TESEs in medulloblastoma (MB, 65.9%), other CNS embryonal tumors (65.3%), and oligodendrogliomas (52.2%). Clustering of tumors by TESE PSI values grouped samples broadly into tumor histologies (i.e., MB, ependymoma, HGG) or distinct molecular subtypes (e.g. within Atypical Teratoid Rhabdoid Tumor), suggesting conserved patterns of splicing aberrations. 113 recurrent TESEs encoded protein extracellular domains in 71 unique genes, including 28 genes in HGG and DMG tumors. Overall, we identified hundreds of pan-pediatric CNS cancer, histology-, and subtype-specific splicing aberrations affecting protein domains, and a subset with predicted cell surface localization, nominating candidate TSAs. Future work will seek to validate the protein expression of TESE-derived EC peptides as well as predict presentation of intracellular TESE-derived peptides by MHC molecules. Citation Format: Ryan J Corbett, Patricia J Sullivan, Ammar S Naqvi, Alex Sickler, Bicna Song, Chao Di, Bo Zhang, Chuwei Zhong, Brian Rood, Jo Lynne Rokita. Tumor-enriched splicing events exhibit histology-specific prevalence in pediatric CNS tumors and encode tumor-specific antigens with immunotherapeutic potential [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr B022.