NCM 2023 Houston: Nanopore sequencing reveal structural heterogeneity in canine osteosarcoma

Osteosarcoma (OS) is an aggressive bone malignancy in 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 outcomes. Whole genome sequencing (WGS) revealed that a hallmark of OS is a highly rearranged genome with complex copy number aberrations. In many respects, canine and human OS are similar. A significant difference is that initial surgery in canine OS allowed us to explore tumor evolution unperturbed by treatment. Here we performed bulk WGS of multiple tumor and normal sites in two canines to analyze the genetic and epigenetic evolution of osteosarcoma. In both canines, structural variation analysis using Severus revealed chromothripsis-like rearrangements in chr11 leading to the deletion of Cdkn2a, a known cell cycle regulator and tumor suppressor. We further detected multi-break rearrangements in chr5, chr8, and chr10. The multi-site analysis identified subclonal structural alterations in TP53 and ATRX genes. Direct phasing of heterozygous germline variants revealed copy-neutral loss-of-heterozygosity events and long identical-by-descent blocks. Haplotype-specific copy-number analysis showed different aneuploidy patterns in both canines, with primarily tetraploid karyotype and partial chromosome arm losses. Our analysis allowed us to identify clonal and subclonal genetic and epigenetic factors involved in tumor progression and evolution in canine OS, which can potentially translate to human disease.