Abstract 782: Mapping therapy-related clonal hematopoiesis across immune cell populations in long-term survivors of pediatric cancer by single-cell multi-omics profiling

Abstract Recently we and others reported high prevalence of clonal hematopoiesis (CH) in long-term survivors of pediatric cancers by performing ultra-deep panel sequencing of peripheral blood samples, a phenomena significantly associated with exposures to radiotherapy and chemotherapeutic agents. Motivated by a preliminary finding that a therapy-related STAT3 CH mutations were highly enriched in T-cells of 3 Hodgkin’s Lymphoma (HL) survivors, we initiated a systematic investigation to map the CH mutations across different immune cells populations. Blood samples of 108 survivors of 13 pediatric cancer types (4 hematological, 8 solid and 1 central nervous system (CNS) tumor types), known to harbor CH mutations discovered by prior bulk sample analysis, were selected for multi-omics single-cell profiling with Mission Bio Tapestri assay. For mutation screening, we designed a panel of 29 amplicons across 12 genes, covering 52 CH mutations detected in bulk samples. Amongst the 23 completed samples, we identified 99 mutations in CH-genes (DNMT3A, STAT3, TP53, KRAS, TET2, and ASXL1), and 67.7% of those were found in all cell types and the rest were present in at least two cell types. We discovered that a subset of CH mutations was enriched (FDR < 0.05) in certain cell types across several samples. For example, STAT3 Y640F mutations were consistently enriched in CD8 T-cells in 6 out of 7 HL samples with a mean fold change of 18.5. DNMT3A mutations were enriched in cells from both myeloid (monocytes) and lymphoid (CD4/8 T-cells and NK-cells) lineages and affected samples from five cancer types. Specifically, we found that DNMT3A mutations were enriched in monocytes in 2 HL and 2 acute lymphoblastic leukemia (ALL) cases with an average fold change of 2.3; and in CD8 T-cells in 5 samples from Wilms tumor, soft tissue sarcoma, ALL, HL, and CNS with an average fold change of 4.9. KRAS L19F was found to be enriched in monocytes and NK-cells in an ALL sample. By contrast, we observed a uniform distribution of some TP53 mutations across all cell types, suggesting that they confer comparable selective advantage across all immune cell types, consistent with the biological function of TP53 in cell cycle. To our knowledge, this is the first large-scale multi-omics single-cell profiling of CH mutations in long-term survivors of pediatric cancer. The differential clonal expansion in immune cell populations demonstrates the potential impact of CH mutations on immune cell dysfunction. This suggests that CH mutations may contribute to long-term adverse health outcomes related to the immune system in long-term survivors beyond the direct cause of secondary neoplasms, which will warrant future investigations using the valuable data resource established through this study. Citation Format: Sivaraman Natarajan, Nadezhda V. Terekhanova, Kohei Hagiwara, Bensheng Ju, Li Dong, Zhaoming Wang, Jinghui Zhang, John Easton. Mapping therapy-related clonal hematopoiesis across immune cell populations in long-term survivors of pediatric cancer by single-cell multi-omics profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 782.