Abstract 4192: CervicalMethDx: A precision DNA methylation test to identify advanced disease risk in cervical cancer screening algorithms

Abstract Cervical cancer is one of the most common cancers in women. Despite progress in prevention through Human Papilloma Virus [HPV] vaccination and success in early detection of cervical cancer through cytologic screening and HPV detection, there remains an unequal burden in low-resource settings in developed and developing countries. Applying novel methylation-based detection methods, we validated a panel of three human methylated genes (ZNF516, FKBP6 and INTS1) on discarded cervical liquid cytology samples from clinical laboratories in the United States during the development of the CervicalMethDx test. We hypothesized that the CervicalMethDx test can identify HPV positive women most likely to be diagnosed with Cervical Intraepithelial Neoplasia (CIN) grades 2 and 3 by anatomic pathologists, before they are referred to colposcopy-driven biopsies. We assessed DNA methylation by Quantitative Real Time Methylation Specific PCR (QMSP) analysis of sodium bisulfite-modified genomic DNA. Primers and probes were previously designed to specifically amplify the promoters of the 3 genes of interest and the promoter of a reference gene, β-actin, to assess DNA input. We performed blinded retrospective studies on well-characterized, discarded, HPV-positive clinical samples in PreservCyt sample transport media (Thin Prep, Hologic), comparing DNA methylation levels in samples from CIN2 and CIN3 cases (346), and 120 controls without evidence of Intraepithelial Lesions or Malignancy (NILM) as an endpoint. Our results showed that the CervicalMethDx test can correctly classify 96% of CIN2 (n=197) samples with 93% Sensitivity, 100% Specificity, and an AUC of 0.96 as well as 95% of CIN3 (n=149) samples with 91% Sensitivity, 100% Specificity, and an AUC of 0.96. Moreover, the assay correctly classified 94% of CIN2-CIN3 samples combined (n=346) with 92% Sensitivity, 100% Specificity, an AUC of 0.96, and a 100% positive predictive value (PPV), when compared to samples with NILM (n=120). The CervicalMethDx test high PPV supports the addition of this low-cost molecular test to cervical cancer screening algorithms worldwide. Our results suggest that the CervicalMethDx test is a new and valuable tool to stratify HPV positive women prior to colposcopy-driven biopsies in developed countries and ablative treatment in developing countries, most of which are unnecessary. These results warrant further evaluation of the CervicalMethDX test in prospective, population-based studies, assessing the use of precision DNA methylation algorithms to triage women before colposcopy-driven biopsies or ablative treatments worldwide. Citation Format: Laura Palmieri, Fernando T. Zamuner, Dieila Giomo De Lima, Keerthana Gosala, Eli Winkler, Yash Prashar, Ana Purcell-Wiltz, Amanda García-Negrón, Ashley Ramos-Lopez, David Sidransky, Mariana Brait, Rafael Guerrero-Preston. CervicalMethDx: A precision DNA methylation test to identify advanced disease risk in cervical cancer screening algorithms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4192.