Abstract 3888: Exploring intra-tumor heterogeneity in malignant peripheral nerve sheath tumors through single-cell multi-omics

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are a rare subtype of sarcomas arising from peripheral nerves, occurring either sporadically or in association with neurofibromatosis type 1 (NF1). Although comprising only 5-10% of soft tissue sarcomas, MPNSTs are highly aggressive and have limited treatment options, with 5-year survival rates of 34%-60%. Intra-Tumor Heterogeneity (ITH) is a key driver of tumor evolution and treatment resistance. However, prior genomic analyses of MPNSTs are often limited to a single sample per patient and have not explored ITH in detail. To fill this gap, we developed a multi-omics and single-cell integration pipeline to analyze bulk Whole-Genome Sequencing (WGS), single-nuclei DNA (snDNA) and RNA (snRNA) sequencing, and multi-regional shallow-coverage WGS derived from multiple Laser Capture Microdissection (LCM) spots within tissue sections. We applied this integrative multi-omics approach to analyze 63 samples collected from 17 patients. Somatic mutations were called in WGS using a two-out-of-three consensus approach (MuSE2, Strelka2, Mutect2), and Copy Number Aberrations (CNA) were analyzed using the Battenberg algorithm. Phylogenetic trees were reconstructed using Dirichlet Process-based methods and PyClone. We identified low-to-moderate mutational burden (median = 1.23 mutations/Mb, range: 0.52–2.34 mutations/Mb) but significant chromosomal aberrations and varied levels of genome instability. Whole genome doubling was highly prevalent (47/63 samples; 14/17 patients) and many samples showed widespread loss of heterozygosity (median = 26%, range: 4%-93%). Among the 17 patients, 5 exhibited a linear evolution pattern, while the majority demonstrated branching evolution. Cross-referencing CNA profiles across bulk WGS, snDNA (ASCAT.sc), LCM, and snRNA (inferCNV) data validated WGS-derived CNAs and demonstrated subclonal CNA co-occurrence within individual cells or subclones. Genotyping WGS-derived mutation clusters in snDNA revealed that co-occurrence of mutation pairs within single cells was exclusively observed when mutations belonged to the same cluster or lineage, further validating our bulk WGS-derived phylogenetic trees. Our findings reveal that MPNSTs are primarily driven by chromosomal instability, with ITH further elucidated through cross-referenced and refined phylogenetic tree reconstruction at single-cell resolution. Future work would focus on 3D phylogenetic tree reconstruction using spatial-genomics information derived from LCM and integrating single-cell transcriptomics. Additionally, incorporating spatial transcriptomics could provide deeper insights into the tumor microenvironment and its role in driving ITH. These efforts will further advance the understanding of MPNST biology and evolution, with potential implications for targeted therapeutic strategies. Citation Format: Yidan Pan, Yixiao Cheng, Chunxu Gao, Haixi Yan, Zhihui Zhang, Leah Weber, Tom Lesluyes, Cristina Cotobal Martin, Isidro Cortes-Ciriano, Nischalan Pillay, David T. Miller, Adrienne M. Flanagan, Maxime Tarabichi, Peter Van Loo. Exploring intra-tumor heterogeneity in malignant peripheral nerve sheath tumors through single-cell multi-omics [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 3888.