Abstract Acquired treatment resistance in homologous recombination (HR) deficient high-grade serous ovarian cancer (HGSOC) commonly involves restoration of HR, often through secondary, somatic reversion mutations in HR genes. Our recent analysis of HR deficient, end-stage HGSOC found that most resistance mechanisms, including reversion mutations, are subclonal. The aim of this study is to investigate how the subclonal, reversion-negative cells persist during treatment. We are investigating single cell sequencing approaches that enable genotyping of individual cells, such that the phenotype of cells without reversions can be studied. In a pilot experiment we have used two HGSOC cell lines with known reversion mutations. To genotype individual cells and thus overcome the limitations of standard single-cell RNA sequencing (scRNA-seq), we combined short-read whole-transcriptome scRNA-seq with long-read, targeted single-cell cDNA sequencing on an Oxford Nanopore MinION using a 44-gene hybridization capture panel. A custom bioinformatics pipeline, incorporating Flexiplex for multiallelic codon resolution, annotated cell barcodes with their HR driver, reversion and TP53 mutations. Genotypes were then transferred to the short-read dataset following outlier and doublet filtering, and transcriptomic differences between cells with and without reversions were identified through differential gene expression analysis. We have repeated this protocol on a patient ascites sample. Prior short-read DNA sequencing from the cell lines and matched tumor samples defined the driver BRCA1 mutation, reversion events, pathogenic TP53 mutations, and whether there was loss of heterozygosity (LOH) at the BRCA1 loci. We successfully genotyped BRCA1 driver and reversion alleles in both cell lines in the long-read data. Rare cells heterozygous wildtype for the BRCA1 driver mutation were detected, suggesting either back-reversion events or subclones that escaped LOH - cells that were undetectable by bulk short-read methods. Cell genotypes were mapped to the short-read whole transcriptome data, and transcriptional programs that may support cell survival were identified in the non-reverted cells. Our approach demonstrates the utility of unbiased long-read sequencing for detecting BRCA1/2 reversions at single-cell resolution, including potential back-reversion subclones that are challenging to identify through bulk sequencing approaches. The differential gene expression results have identified potential resistance mechanisms. Ongoing work includes incorporation of fusion detection, structural-variant calling, and splice isoform analysis in our bioinformatics pipeline to further elucidate resistance pathways. We anticipate this work will result in an increased understanding of the resistance mechanisms that arise in HGSOC patients with HR deficiency, including identification of subclonal resistance mechanisms that cannot be detected through bulk sequencing approaches. Citation Format: Lauren Tjoeka, Stuart Bencraig, David Yoannidis, Madelynne Willis, Joy Hendley, AOCS Study Group, Timothy Semple, Alicia Oshlack, Elizabeth L. Christie. Characterizing resistance at single cell resolution in HR deficient HGSOC abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl): Abstract nr PR019.
Tjoeka et al. (Fri,) studied this question.