SummaryAccurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Ovarian Cancer is the deadliest gynecologic malignancy. Despite recent advances in cancer diagnostics, it is a cancer that is often detected late. After treatment with debulking surgery and chemotherapy, 80% of patients have tumor relapse. Early detection efforts have been largely unsuccessful and the only effective prevention strategy in high risk patients is surgical removal of the fallopian tube and ovaries. Careful pathologic studies of prophylactic risk reducing bilateral salpingo-oophorectomy (RRSO) has demonstrated that the origins of HGSC is likely within secretory cells of the fimbriae in the fallopian tube. This is based on numerous lines of correlative evidence including the identification of abnormal regions in the fallopian tube called serous tubal intraepithelial cancer (STIC) in 10-15% of patients undergoing RRSO, and the presence of STIC in half of patients undergoing debulking surgeries for HGSC. Finally, p53 mutations have been found to be identical in STIC and in the concurrent invasive disease indicating that HGSC and the STIC likely arise from the same clone. TCGA analysis has demonstrated that HCSC is a disease characterized by genomic and chromosomal instability harboring copy number alteration, including amplifications, deletions and more complex chromosomal rearrangements which impact multiple genes and pathways. Aberrantly segregating chromosomes that lag behind the spindle apparatus fail to become part of the primary nucleus (PN) and are instead enclosed by a self-assembled nuclear membrane, creating a structure called the micronucleus (MN). Spontaneous breakdown of the MN causes cytoplasmic DNA sensing and activation of the cGAS-STING pathway an important innate cellular defense mechanism to foreign cytoplasmic DNA. To understand the early genomic and signaling events in the development of HGSC, we are interrogating FFPE tissue from our archives from MSKCC patients who have undergone RRSO, hormone ablation surgeries or surgical debulking and were found to have benign fallopian tube, STIC lesions or invasive disease with STIC. We have performed a pilot experiment of 20 patient samples of different genetic backgrounds. Intriguingly, cGAS signaling was observed only rarely in the STICs indicating low rates of CIN in STICs, however we note that even in early invasive disease, we observe cGAS positivity. Moreover, we observe robust CD8+ T cell localization to early invasive disease. Taken together, these data are consistent with a model where CIN and subsequent cGAS/STING pathway activation drives the transition from pre-invasive to invasive disease with early immune recognition that is lost in invasive disease. We propose a model of ovarian cancer development where fallopian tube secretory transition from a p53 WT SCOUT to p53 mutant state STIL acquire a proliferative capacity forming a STIC and then develop CIN transitioning to invasive disease. Citation Format: Duaa Hassan Al-Rawi, Herman Chui, Sorhab Shah, Samuel Bakhoum. Interrogating chromosomal instability in the earliest steps of ovarian cancer development [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 806.

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