Abstract Background Ependymomas (EPNs) account for approximately 9% of all primary central nervous system tumors in children, with peak incidence in those under five years old. These tumors are highly heterogeneous and classified into distinct subtypes, such as myxopapillary (MPE), posterior fossa A (PFA), classic spinal, supratentorial YAP1-fused and ZFTA-fused EPN. The clinical behavior varies widely, with PFA EPNs representing the most prevalent and aggressive subtype in young children. While ongoing efforts continue to explore the tumor immune microenvironment, the diversity and functional architecture of B cells in pediatric EPN remain unknown. Defining B cell phenotypes across EPN subtypes may uncover new insights into oncogenic pathways and novel opportunities for targeted immunotherapies. Methods We analyzed non-targeted bulk RNA-sequencing data from over 2, 000 pediatric brain tumors using bcRflow, a Nextflow pipeline for reconstructing B cell receptor (BCR) repertoires. This cohort included 179 EPN samples spanning all major subtypes. For each tumor, we examined B cell isotype distribution, clonal expansion, class switch recombination (CSR), somatic hypermutation (SHM) rates, and overall repertoire diversity using diversity metrics such as Chao1, ACE, and the inverse Simpson index. Results Across all samples, EPN demonstrated relatively lower BCR repertoire diversity quantified by diversity indices including Chao1, ACE, and inverse Simpson index. EPNs were further characterized by a dominance of hyperexpanded B cell clonotypes. The majority of the BCR repertoire space was occupied by clones within the 1–10 and 11–100 frequency bins, indicating the expansion of select clonal populations rather than a broadly polyclonal response. Within EPN subtypes, there was notable heterogeneity in isotype usage, IGHV gene usage, and CSR dynamics. The frequency of IgM-expressing B cells is comparatively low across all subtypes. None of the subtypes display IgM as the most abundant isotype, indicating that most B cells within these tumors have undergone CSR away from the naïve IgM phenotype. Conclusions Pediatric EPNs exhibit markedly restricted BCR diversity compared to other pediatric brain tumors, suggesting a limited and potentially oligoclonal B cell repertoire. This indicates a focused, potentially tumor antigen-driven response. Most infiltrating B cells within the samples had already undergone class-switching, with IGHA1 and IGHG1 isotypes predominating over IgM. This shift away from naïve B cells points to immune engagement shaped by unique tumor antigens. Importantly, the immune architecture varied substantially across subtypes like PFA, MPE, spinal, YAP1-fused and ZFTA-fused EPNs. This highlights that EPNs are not immunologically uniform and may reflect distinct immunological pressures and potential therapeutic vulnerabilities. Taken together, these findings reveal a previously underappreciated layer of immune complexity in EPNs and opens new avenues for subtype-informed diagnostics and immunotherapeutic development. Citation Format: Amelia Stepniak, Stephen C. Frederico, Sydney Jackson, Itay Raphael, Dhivyaa Rajasundaram, Gary Kohanbash. Immune heterogeneity across pediatric ependymoma subtypes revealed by B cell repertoire profiling 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 (18Suppl₂): Abstract nr A042.
Stepniak et al. (Thu,) studied this question.