Abstract 5665: Native nanopore sequencing of multiple tumor sites reveals genetic and epigenetic intra-tumor heterogeneity in canine osteosarcoma

Abstract

Abstract Osteosarcoma (OS) is an aggressive bone malignancy affecting children and young adults. Implementation and optimization of neoadjuvant chemotherapy led to significant improvements in outcome, but patients with recurrence or metastases at diagnosis continue to have poor prognosis. Whole genome sequencing (WGS) revealed that a hallmark of OS is a highly rearranged genome with complex copy number aberrations rather than point mutations. A better understanding of the key genomic and epigenetic factors driving clonal evolution may provide insights into the tumor progression and the therapy response. Canine OS recapitulates the majority of the hallmark genetic and molecular factors of human OS and is recognized as an informative model. A significant difference is that initial surgery in canine OS allows us to explore tumor evolution unperturbed by treatment, providing invaluable resources to study the genetic and molecular landscape of tumor evolution. Here, we present the first study to date in which we performed bulk WGS of multiple tumor sites and distal normal tissue in two dogs using nanopore long-read sequencing to analyze the genetic and epigenetic evolution of OS. In both subjects, somatic structural variation detection using Severus revealed chromothripsis-like rearrangements in chr11, leading to the deletion of Cdkn2a, a known cell cycle regulator and tumor suppressor. Interestingly, in one of the subjects, the majority of the SVs (n = 131) shared across all the samples were localized on chr5, coinciding with perturbed copy number profiles and bi-allelic frequency of the SNVs, with all mutations identified using complementary bioinformatics analysis of the same native nanopore reads. The multi-site sample analysis identified subclonal structural alterations in TP53 and ATRX genes. With phased nanopore reads, we observed subject-specific copy-neutral loss-of-heterozygosity events and long identical-by-descent blocks. Haplotype-specific copy-number analysis of tumors showed different aneuploidy patterns in both subjects, highlighted clonal compositions of samples from distinct sites, and identified regions of copy-number heterogeneity. Furthermore, phased nanopore reads also revealed haplotype-specific 5mC and 5hmC methylation differences across tumor samples, where samples from different sites of the same tumor demonstrate distinct methylation patterns that overlap DLG2, RUNX2, KDR, and other genes known as mutational hotspots in canine/human OS. Overall, we showcase how a single sequencing assay allows for a joint analysis of genetic and epigenetic tumor alterations, thus providing a richer understanding of the drivers of somatic evolution and their association with the molecular landscape in the canine OS. These findings can be translated into human disease and shed light on tumor progression and therapy response. Citation Format: Ayse Keskus, Sergey Aganezov, Tanveer Ahmad, Xiaoguang Dai, Robert Walker, John Beaulaurier, Scott Hickey, Sissel Juul, Amy Kathleen LeBlanc, Paul S. Meltzer, Mikhail Kolmogorov. Native nanopore sequencing of multiple tumor sites reveals genetic and epigenetic intra-tumor heterogeneity in canine osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5665.