Integrated Single-Cell Whole-Genome Sequencing and Spatial Transcriptomics Reveal Intratumoral Heterogeneity in Ovarian Cancer.

The mortality rate of ovarian cancer remains disproportionately high compared with its incidence. This is partly due to a high level of intratumoral heterogeneity, driven by genomic instability, that promotes disease recurrence and treatment failure. In this study, we describe degrees of heterogeneity revealed by single-cell whole-genome sequencing and spatial transcriptomics (ST) of five late-stage, treatment-naïve primary epithelial ovarian carcinomas, including high-grade serous and clear-cell subtypes. All samples exhibited widespread copy-number (CN) aberrations, with the greatest intraspecimen diversification in regions of CN gain. Diversification was also associated with whole-genome doubling in all samples. In two samples, we identify persistent, clonal pseudodiploid cells evolutionarily consistent with a premalignant phenotype. In multiclonal samples, we interpret clonal evolution in the context of single-cell CN, loss of heterozygosity analysis, and somatic mutations and correlate these with tissue histology and gene expression programs. In one high-grade serous carcinoma, we identify functionally consequential CN alterations that contribute to molecular diversity, cell proliferation, and inflammation in a minor clone that persisted without major expansion alongside a more complex major clone. In another clear-cell carcinoma, we describe a complex evolutionary history, including a spontaneous functional reversion of a CTNNB1 driver mutation in a secondary clone, which correlated with a switch in oncogenic expression programs. These examples highlight various consequences of genomic instability on clonal heterogeneity and plasticity in ovarian cancer. SIGNIFICANCE: We utilize single-cell DNA sequencing and ST to illustrate the wide extent of intratumoral heterogeneity within late-stage ovarian tumors. We describe several consequences of chromosomal instability, including divergent biology in multiclonal tumors, persistence of a premalignant cell population, and functional reversion of an oncogenic driver mutation.