An evaluation of cell-free tumor DNA from 20 mL of peritoneal fluid for homologous recombination deficiency (HRD) testing in newly diagnosed ovarian cancer (OC).

5577 Background: HRD testing at diagnosis is crucial for patients with advanced high grade OC (HGOC). HRD status is defined by a BRCA1 or BRCA2 pathogenic variant, or high genomic instability score (GIS). These genomic tests are performed on tumor samples but result in non-contributive tests in 10-19% of cases. We have previously shown that ascites yields high quality tumor derived cell-free DNA (cfDNA) suitable for HRD testing. Here we present results from an updated prospective series of HRD testing (BRCAm+/-GIS) performed on 20ml of peritoneal fluid obtained at diagnostic laparoscopy for suspected advanced EOC. Methods: We collected 20ml of free peritoneal fluid, or in absence of free fluid, 20ml of peritoneal washings from patients undergoing laparoscopy for suspicion of EOC into Streck tubes. cfDNA was extracted after centrifugation and quantified. Next generation sequencing (NGS) was performed for TP53, BRCA1/2 and other genes, and GIS assessed by shallow whole genome sequencing (sWGS). Where available genomic profile from cfDNA was compared to FFPE tumor results. Results: A total of 48 patients with suspected advanced EOC were included. Most patients had stage III/IV (N=47) EOC. 45/48 pts had >50ml free peritoneal fluid at diagnostic laparoscopy, the other 3 benefited from a peritoneal washing to retrieve cfDNA. cfDNA was detectable in 96% (46/48) of peritoneal samples, including in 2 of 3 washings. cfDNA concentration extracted from just 20ml of peritoneal fluid or washings was high at a median of 3450 ng/ml. Importantly all 46 contributive cfDNA samples contained tumor DNA as confirmed by the detection of a pathogenic variant with high allelic frequencies (median VAF=0.52). The most common variant was TP53 detected in 83% (38/46) of samples. On final histology, most patients had high grade serous or endometrioid OC (HGOC) (39/48), there were 6 low grade and 3 others. Among HGOC patients 95% had a TP53 pathogenic variant, 23% (9/39) and 15% (6/39) had a BRCA1 or BRCA2 pathogenic variant, respectively. Other relevant variants included CCNE1amplification (N=3), 2 ATMmutations and 1 RAD51 mutation. Importantly, concordance between variant calling on peritoneal cfDNA and DNA from FFPE was high. However, NGS performed on cfDNA was able to salvage 4 non contributive FFPE-based NGS tests. So far 14 cfDNA samples have been subjected to sWGS and all yielded an informative GIS, sWGS on the rest of the cohort is ongoing and will be presented. Conclusions: At diagnosis 95% of patients with advanced HGOC present with some peritoneal free fluid. Only 20ml of fluid or peritoneal washing yields large quantities of high quality tumor derived cfDNA that is suitable for genomic profiling and can salvage non-contributive tumor based testing. This approach could provide a new diagnostic pathway for HRD testing in advanced OC, while sparing precious tumor samples.