Development of a Quantitative Methylation-Specific Droplet Digital PCR Assay for Detecting Dickkopf-related Protein 3

Objective: The detection and monitoring of DNA methylation status in circulating tumor cell DNA (ctDNA) provides critical insights into cancer diagnosis and progression. The methylation status of the Dickkopf-related protein 3 (DKK3) promoter region is correlated with the metastasis and recurrence of multiple cancers. Thus, detecting the methylation status via non-invasive methods is essential for the diagnosis and prognosis of cancers. Using a droplet digital polymerase chain reaction approach, we have developed a highly sensitive and quantitative measurement of methylated and unmethylated DKK3 derived from circulating cell-free DNA (ccfDNA). Results: : We confirmed the specificity of droplet digital methylation specific polymerase chain reaction (ddMSP). We selected the optimal bisulfite conversion method using commercially available kits. We validated the ddMSP analysis system by analyzing the methylation status of genomic DNA extracted from cultured mesothelioma cells and mesothelial cells. Our system quantified approximately 30 copies of cell-free DNA per 4 mL, which is sufficient for detecting ctDNA. Finally, we quantified methylated and unmethylated DKK3 copies in ccfDNA from 21 patients with malignant mesothelioma.