Abstract 801: Surprisingly conserved copy numbers from cell to cell within structurally complex tumors challenge the unstable genome hypothesis
Authors: Rajan Sanjana , Zaccaria Simone , Cannon Matthew , Cam Maren , Gross Amy , Raphael Benjamin , Roberts Ryan D. 
Published: June 2022

Abstract
Osteosarcoma is an aggressive disease that exhibits almost universal p53 inactivation and a high level of genomic complexity. The chaotic genome of most osteosarcomas has led many to assume that this complexity must accumulate under conditions of significant genomic/chromosomal instability. However, such conclusions have been drawn primarily from single-timepoint bulk-tumor analyses. More recent studies have questioned this assumption of chromosomal instability, hypothesizing that genomic complexity could arise from an early catastrophic event followed by faithful propagation of a highly complex, malignancy-promoting genome.
To determine whether osteosarcoma tumors show evidence of instability, we performed single-cell whole-genome sequencing of &gt;14,000 osteosarcoma cells obtained from 15 distinct lesions resected from 8 individual patients. Using the CHISEL algorithm, we inferred allele- and haplotype-specific copy numbers from this sequencing data. To evaluate how the SCNA profiles changed during the evolutionary pressures associated with metastatic dissemination and therapy, we compared the single-cell SCNA profiles of paired samples collected from both primary sites and metastases and from pre-treatment and relapsed specimens. We likewise compared profiles of patient tumors propagated orthotopically in mouse tibias to those grown within the lung to evaluate for tissue-dependent emergence of rare subclones.
Despite extensive structural variations that give rise to highly complex patterns of SCNA, the cells within each tumor showed remarkably little heterogeneity. We found similar evidence of chromosomal stability when we reconstructed phylogenetic trees to identify the evolutionary relationships of cells collected from metastatic lesions or at relapse. Despite being evolutionarily distant, nearly all variants could be assigned to the trunk of the phylogenetic tree, with only modest changes occurring in the branches. This result suggests that nearly all SCNAs were acquired in an early event, followed by selection of advantageous patterns with subsequent preservation of that particular SCNA pattern. Analysis of bulk whole-genome sequencing from serially collected patient samples supports the preservation of SCNA profiles over time.
This work demonstrates the power of combining single-cell DNA sequencing with an allele- and haplotype-specific CNV inference algorithm. Our approach clarifies longstanding questions about the genetics of osteosarcoma initiation and progression, calling into question previous assumptions of genomic instability inferred from single-timepoint bulk sequencing data. These results suggest that an isolated, early catastrophic event, rather than sustained genomic instability, gives rise to the complex genome that characterizes osteosarcoma tumors.
Citation Format: Sanjana Rajan, Simone Zaccaria, Matthew Cannon, Maren Cam, Amy Gross, Benjamin Raphael, Ryan D. Roberts. Surprisingly conserved copy numbers from cell to cell within structurally complex tumors challenge the unstable genome hypothesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 801.