Abstract AP05: ULTRA–DEEP SEQUENCING DETECTS OVARIAN CANCER CELLS IN PERITONEAL FLUID AND REVEALS SOMATIC TP53 MUTATIONS IN NON–CANCEROUS TISSUES
Authors: Krimmel JD , Schmitt MW , Kennedy SR , Harrell MI , Agnew KJ , Loeb LA , Swisher EM , Risques RA 
Published: June 2017

High–grade serous ovarian carcinoma (HGSOC), the most common and most aggressive type of ovarian cancer, is characterized by near–universal prevalence of TP53 mutations, even at the early stages of the disease. Thus, the detection of tumor–specific TP53 mutations in clinical samples could potentially enable early cancer detection, monitoring, and screening. A main limitation, however, is the high error rate of conventional DNA sequencing technologies (estimated at 1 in 1000 nucleotides), which precludes the identification of low frequency tumor mutant molecules from technical error. Duplex Sequencing is a next–generation sequencing technology that uniquely tags each strand of a DNA molecule and quantifies mutations only when present in both strands of DNA. This reduces the error rate of sequencing to 1 in 10 million nucleotides, an unprecedented sensitivity that could enable the detection of extremely low frequency tumor mutant molecules by ultra–deep sequencing. The goal of this study was to provide proof–of–principle of the ability of Duplex Sequencing to detect extremely rare TP53 mutated cancer cells disseminated into the peritoneal cavity of women with HGSOC. We analyzed 17 peritoneal fluid samples from women with HGSOC and known TP53 tumor mutation (cases) and 20 peritoneal fluid samples from women without cancer (controls). The tumor–specific TP53 mutation was detected in matched peritoneal fluid from 94% of women with HGSOC (16/17), including 2 patients with occult tubal intraepithelial neoplasia, 7 patients with early stage HGSOC, and 8 with negative peritoneal fluid cytopathology. Tumor–specific alleles were detected as low as 1 mutant molecule per 24,737 normal genomes. Additionally, we detected extremely low–frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cases from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age–associated, low frequency TP53 mutations were also found in 100% of matched peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic cancer). Our results demonstrate the ability of Duplex Sequencing to detect very rare cancer cells and provide evidence of widespread, low frequency, age–associated somatic TP53 mutation in non–cancerous tissue. These mutations likely represent a premalignant mutational background that accumulates in cancer and aging.

Citation Format: Krimmel JD, Schmitt MW, Kennedy SR, Harrell MI, Agnew KJ, Loeb LA, Swisher EM, Risques RA. ULTRA–DEEP SEQUENCING DETECTS OVARIAN CANCER CELLS IN PERITONEAL FLUID AND REVEALS SOMATIC TP53 MUTATIONS IN NON–CANCEROUS TISSUES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP05.