Cell-free DNA from ascites identifies clinically relevant variants and tumour evolution in patients with advanced ovarian cancer

Background The emergence of targeted therapies and predictive biomarkers is transforming the ovarian cancer treatment paradigm. However, the demand for high quality, tumour-enriched samples for biomarker profiling can be limited by access to adequate tissue samples. The use of cell-free DNA (cfDNA) in ascites presents a potential solution to this clinical challenge. Methods A unique set of sequential ascites-derived cfDNA samples (26 samples from 15 human participants) were collected from people with ovarian cancer (age range 36-82 years). cfDNA was sequenced using targeted next-generation sequencing, along with matched DNA from ascites-derived tumour cells (n=5) and archived FFPE-tissue from surgery (n=5). Results Similar tumour purity, variant detection and reference alignment were achieved with cfDNA when compared to FFPE and ascites derived tumour cell DNA, as well as improved coverage. No artefactual single-base mutation signatures were identified in cfDNA. Combined analysis of large-scale genomic alterations, loss of heterozygosity and tumour mutation burden identified 6 cases of high genomic instability (including 4 with pathogenic variants in BRCA1 and BRCA2). Copy number profiles and subclone prevalence changed between sequential ascites samples, particularly in a case study where deletions and chromothripsis in Chr17p13.1 and Chr8q resulted in changes in clinically relevant TP53 and MYC variants over time. Conclusions Ascites cfDNA successfully identified clinically actionable information, concordant to tissue biopsies, enabling opportunistic molecular profiling. These findings advocate for analysis of ascites cfDNA in lieu of accessing tumour tissue via biopsy. ### Competing Interest Statement KW declares potential financial conflict of interest due to stock ownership in the following companies that are developing cell-free DNA based clinical assays: Guardant Heath; Exact Sciences; EpiGenomics AG. No other authors have conflicts of interest to declare. ### Funding Statement This research was supported by a number of anonymous philanthropic donations, and in part by the Intramural Research Program of the NIH and the Adam J Berry Memorial Fund through the Australian Academy of Science. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Human Research Ethics Committee of Prince of Wales Hospital gave ethical approval of this work (HC19-001) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All deidentified data produced in the present study may be made available upon reasonable request to the authors.