Abstract While continued therapeutic improvement and innovation have greatly improved survival rates of pediatric cancer, survivors face a broad spectrum of adverse health outcomes such as accelerated physiological aging and subsequent neoplasms (SN) due to a multitude of treatment exposures. Here we investigate the effects of therapy-related genotoxicity on clonal hematopoiesis (CH, an aging biomarker) and SN development in long-term survivors of childhood cancer. Elevated mutation rates associated with therapy-related CH were previously identified in 8. 6% of childhood cancer survivors by bulk sample deep sequencing. By performing single-cell multi-omics profiling of DNA amplicons and cell-surface proteins on cryopreserved blood samples from 108 CH-positive survivors, we found that CH mutations were enriched in distinct immune cell populations. For example, STAT3 Y640F and TP53 R273S mutations were predominantly present in CD8+ T cells while DNMT3A F752Lfs and KRAS L19F were enriched in CD3+ double-negative T cells and CD14 monocyte cells, respectively. To investigate whether mutation-positive cells have altered transcription programming, we performed single-cell full-length transcriptome sequencing on three samples with STAT3 Y640F mutation. Pre-sorted CD8+ cells (4, 396 to 5, 416 cells per sample) were profiled, and clustering by marker gene expression revealed 11-14 subpopulations per sample which included naive, central memory, effector memory, and terminally differentiated effector memory CD8 T cells. STAT3-mutant cells were present in 3 out of 13 (23%), 1 out of 14 (7%), and 2 out of 11 (18%) clusters in these samples. Four out of the five (80%) mutant-positive clusters exhibited high expression of cytotoxic markers, including GZMB, GZMH, FGFBP2 and NKG7, while the fifth showed high expression of stress response markers from the FOS/JUN pathway (JUN, JUNB, and FOS). To investigate how previous cancer therapy affected SN development, we employed whole-genome, exome and RNA sequencing on tumor samples of 200 breast, meningioma, and thyroid SNs, which developed a median of 26. 7 years after childhood cancer diagnosis. Meningioma and thyroid SNs were enriched for rearrangement driver alterations compared to their de novo counterparts from The Cancer Genome Atlas and other cohorts, including NF2-disrupting alterations and kinase fusions potentially induced by radiation. Radiation was also associated with increased insertion-deletion signature ID5 across SNs. Prior treatment with nitrogen mustards was associated with an elevated age-related SBS5-like signature in breast and meningioma SNs. In meningioma, platinum treatment was associated with NF2 splice-site variants. Together, these results demonstrate the long-term impact of childhood cancer treatment on clonal hematopoiesis and the genomes of SNs developed in adulthood, which may guide efforts to treat and prevent long-term adverse events. Citation Format: Jinghui Zhang. Therapy-related mutagenesis in long-term survivors of childhood cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (8Suppl): Abstract nr SY31-01.
Jinghui Zhang (Fri,) studied this question.