Abstract B010: Concurrent analysis of multiple methylated CpG sites using multiplex MethyLight assay for epithelial ovarian cancer detection

Abstract Objective: Epithelial ovarian cancer (EOC), a heterogeneous and aggressive gynaecologic malignancy, remains a leading cause of cancer-related death due to the lack of reliable, sensitive, and specific early diagnostic biomarkers. Methods: This study employed a cost-effective, CpG site-specific Amplification Refractory Mutation System PCR (ARMS-PCR) to assess the methylation frequency of six diagnostically potent CpG sites (cg18878992, cg00480298, cg029572270, cg10636246, cg10061138, and cg12910797) in 45 EOC samples and 25 normal controls. Methylation patterns (methylated, unmethylated, partially methylated) were confirmed through image analysis and Combined Bisulfite Restriction Analysis (COBRA). Three top methylated CpG sites were chosen to form two biomarker panels, which were subsequently tested for diagnostic efficiency. Furthermore, a TaqMan-based quantitative PCR (MethyLight) assay was developed, integrating the three most hypermethylated CpG sites (cg02957270, cg00480298, and cg10061138) in a triplex format. Results: ARMS-PCR revealed the diagnostic potential of two hypermethylated CpG sites (cg02957270, cg10061138) and one hypomethylated site (cg10061138). The sensitivity of the singleplex and multiplex panels reached 80%, 73.3%, and 82.3%, respectively. The MethyLight assay demonstrated an enhanced sensitivity of 86% and specificity of 92% for the multiplex panel (cg02957270 and cg00480298) along with control col2A1. ROC curve analysis revealed AUC values of 0.96, 0.91, and 0.84 for the singleplex assays and 0.97 for the multiplex panel. While ARMS-PCR showed limited correlation with clinicopathological characteristics, significant associations were found in the MethyLight assay (p < 0.05). Conclusions: This discovery opens a new frontier in DNA methylation research, suggesting the potential to harness clinically relevant, independent CpG sites, in hypermethylated state for the development of novel biomarkers. Citation Format: Deepa Bisht, Manisha Sachan. Concurrent analysis of multiple methylated CpG sites using multiplex MethyLight assay for epithelial ovarian cancer detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B010.