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

Objectives: 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. TP53 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 TP53 somatic mutations in blood and Pap test DNA are associated with HGSOC in women with and without germline BRCA1 or BRCA2 (BRCA) mutations. Methods: Peripheral blood and Pap samples were collected from 30 women with HGSOC (10 with a germline BRCA mutation) and 40 women without cancer (20 with a germline BRCA 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 TP53 coding region (mean depth ~3300x) using duplex sequencing for error correction. Mutations were characterized based on pathogenicity and presence in the UMD TP53 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. Results: Total 436 coding TP53 mutations were identified in DNA from Pap tests, and 262 mutations were identified in the DNA from blood. Pap tests demonstrated an overall higher TP53 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 BRCA mutations (2.4x10-6 vs 1.4x10-6, p=0.01). Women with BRCA 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 TP53 mutation frequency was associated with HGSOC in patients with germline BRCA mutations (OR: 4.2, 95% CI: 1.2-14.3). There were no significant differences between HGSOC and non-cancer cases when examining TP53 mutations in blood samples. Among 22 cases with known TP53 tumor mutations, the tumor mutation was identified in six Pap samples (27%) and zero blood samples. Conclusions: 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 TP53 mutations at a higher rate in women with germline BRCA mutations and HGSOC compared to women without cancer. Somatic TP53 mutation frequency using age-related thresholds should be further explored as a biomarker for ovarian cancer risk in high-risk women.