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Background   MutL Homolog 1  ( MLH1 ) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). The strong correlation between methylation status and cancer development and progression has led to a growing interest in the use of methylation markers in circulating tumor DNA (ctDNA) for early cancer detection and longitudinal monitoring. As cancer-specific DNA methylation changes in body fluids are limited, it is particularly challenging to develop clinically applicable liquid biopsy methodologies with high sensitivity and specificity. The purpose of this study was to develop a fit-for-purpose methylation sensitive restriction enzyme (MSRE) based digital droplet PCR (ddPCR) assay to examine  MLH1  promoter methylation in ctDNA in advanced CRC.    Methods  Primers and probes were designed to amplify CpG sites of the  MLH1  promoter. Methylated and unmethylated control genomic DNA were sheared to mimic ctDNA and subjected to MSRE  HpaII  digestion. Plasma samples from 20 healthy donors and 28 CRC patients were analyzed with the optimized MSRE procedure using ddPCR.    Results  Using methylated and unmethylated controls, we optimized the conditions for  HpaII  enzyme digestion to ensure complete digestion and avoid false positives. Based on the results from the ddPCR assay using 1 ng circulating cell-free DNA (cfDNA) input from healthy donors or CRC samples, ROC curves were generated with an area under the curve (AUC) value of 0.965 (95% CI: 0.94, 0.99). The statistically optimal assay sensitivity and specificity was achieved when 8 positive droplets were used as acceptance criteria (78% sensitivity and 100% specificity, 95% CI: 0.45, 0.95). A tiered-based cutoff (20, 50, 80% percentile based) was applied to distinguish CRC samples with different methylation level.    Conclusions  Our study demonstrated that the liquid biopsy assay for  MLH1  promoter methylation detection using purely quantitative ddPCR is a simple and highly sensitive procedure that provides reliable methylation detection in ctDNA. The MSRE ddPCR approach can also be applied to other genes of interest where methylation patterns could reveal clinically relevant information for future clinical biomarker and/or companion diagnostic development.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12885-021-08497-x.

bmc cancer

Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients