Serum cell free DNA based duplex methyLight assay for liquid biopsy based detection of epithelial ovarian cancer

Background: Ovarian cancer, being the third most common gynaecological cancer in India, exhibits poor diagnosis and prognosis due to its non-specific early symptoms and late-stage disease presentation. Aberrant CpG island hypermethylation represents an initial and key hallmark of carcinogenesis, enabling the silencing of tumour suppressor genes and serving as a promising detectable biomarker in different body fluids for cancer diagnosis. Aberrant methylation of tumour suppressor genes RUNX3 and ZNF154 is frequently observed in ovarian cancer, offering promise as minimally invasive biomarkers in circulating cell-free DNA. Methods: RUNX3 and ZNF154 are identified as potential hypermethylated tumour suppressor candidates in epithelial ovarian cancer. Targeted NGS/BSAS was performed to confirm that these hypermethylated CpG-rich regions were aberrantly methylated in EOC. With this preliminary data, we developed and validated a duplex MethyLight assay (quantitative fluorescence-based real-time PCR) to simultaneously detect hypermethylation of both genes in n-100 (T-70, N-30) tissue and 70 matched serum samples (T-50, N-20). Our present study developed and validated a duplex MethyLight assay targeting these genes (with COL2A1 as endogenous control) in both tissue and serum cfDNA cohorts to assess their diagnostic performance.​ ROC curves were built employing calculated Percentage of Methylated Reference values from MethyLight assays to evaluate the diagnostic performance of the assay. In the tissue cohort, the sensitivity and specificity of ZNF154 are reported as 89% and 90%, respectively, with RUNX3 showing 87% and 83%, respectively. In serum cfDNA samples, RUNX3 demonstrated a sensitivity and specificity of 84% and 90%, respectively, and ZNF154 demonstrated a sensitivity and specificity of 90% and 92%, respectively. The duplex panel, which is based on either gene hypermethylation, outperformed singleplex markers, achieving 92% sensitivity and 94% specificity with an AUC of 0.95 for early-stage EOC in the serum cohort.​ The duplex panel demonstrated significant clinicopathological correlation with p<0.05. Conclusion: This duplex MethyLight-based pilot study demonstrates robust diagnostic performance for EOC diagnosis in cfDNA, supporting its potential as minimally invasive screening tool. Furthermore, multi-centre validation is necessary for its clinical translation.