TP53 mutations in Pap test DNA are a potential biomarker of ovarian cancer risk in high-risk women (226)

Abstract

<b>Objectives:</b> The use of Pap tests as a molecular diagnostic for high-grade serous ovarian carcinoma (HGSOC) is a promising concept, but studies to detect tumor DNA report limited sensitivity. <i>TP53</i> somatic mutations from clonal expansions in non-neoplastic tissue can be detected in Pap tests and blood and could potentially be utilized for cancer risk assessment. We aimed to determine whether <i>TP53</i> somatic mutations in blood and Pap test DNA are associated with HGSOC in women with and without germline <i>BRCA1</i> or <i>BRCA2 (BRCA)</i> mutations. <b>Methods:</b> Peripheral blood and Pap samples were collected from 30 women with HGSOC (10 with a germline <i>BRCA</i> mutation) and 40 women without cancer (20 with a germline <i>BRCA</i> mutation) who underwent gynecologic surgery at the University of Washington. Genomic DNA from buffy coats and Pap cell pellets was used for ultra-deep sequencing of the <i>TP53</i> coding region (mean depth ~3300x) using duplex sequencing for error correction. Mutations were characterized based on pathogenicity and presence in the UMD <i>TP53</i> Cancer Database. Mutation frequencies (total coding mutations divided by nucleotides sequenced) were compared between groups using t-tests. Fitted linear regression models were used to explore age-related trends, and logistic regression models were used to predict cancer with adjustments for age. <b>Results:</b> Total 436 coding <i>TP53</i> mutations were identified in DNA from Pap tests, and 262 mutations were identified in the DNA from blood. Pap tests demonstrated an overall higher <i>TP53</i> mutation frequency in patients with HGSOC than in those without cancer (2.0x10^-6 vs 1.4x10^-6, p=0.03), but this effect was driven by and limited to patients with <i>BRCA</i> mutations (2.4x10^-6 vs 1.4x10^-6, p=0.01). Women with <i>BRCA</i> mutations and HGSOC also demonstrated a greater frequency of mutations affecting the DNA-binding domain, predicted to be pathogenic, and frequently found in cancer (referred to as ‘cancerlike') (all p<0.05). Cancer-like mutations increased with age in Pap test samples from women without cancer (p<0.01, R²=0.23) but not in patients with HGSOC. Age-adjusted logistic regression confirmed that cancer-like <i>TP53</i> mutation frequency was associated with HGSOC in patients with germline <i>BRCA</i> mutations (OR: 4.2, 95% CI: 1.2-14.3). There were no significant differences between HGSOC and non-cancer cases when examining <i>TP53</i> mutations in blood samples. Among 22 cases with known <i>TP53</i> tumor mutations, the tumor mutation was identified in six Pap samples (27%) and zero blood samples. <b>Conclusions:</b> The identification of HGSOC-specific mutations in Pap tests, even with highly accurate DNA sequencing methods, has limited sensitivity for cancer detection. However, Pap tests can detect abundant low-frequency cancer-like <i>TP53</i> mutations at a higher rate in women with germline <i>BRCA</i> mutations and HGSOC compared to women without cancer. Somatic <i>TP53</i> mutation frequency using age-related thresholds should be further explored as a biomarker for ovarian cancer risk in high-risk women.