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

Abstract

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.

Emerging roles of cystathionine β-synthase in various forms of cancer

“…Refs. [ [417] , [418] , [419] , [420] , [421] , [422] , [423] , [424] , [425] ]. Thus, a similar detection of CBS is not beyond the realms of possibilities.…”

Dynamic liquid biopsy components as predictive and prognostic biomarkers in colorectal cancer

“…In addition to this, the detection rate of targeted methylated genes was also found to increase significantly with increasing stage indicating the significance of these cfDNA methylation markers as screening and diagnostic tools in CRC [ 135 ]. On the other hand, several markers such as MutL homolog 1(MLH1) promoter methylation markers [ 136 ] and Septin 9 (SEPT 9) [ 137 ] have also been identified as suitable markers with high sensitivities (78% and 87%) and specificities (100% to 90.6%) to differentiate CRC patients from heathy controls. In addition to this, two separate studies have documented that Secretin Receptor (SCTR) hypermethylation and Transmembrane Protein 240 (TMEM240) promoter hypermethylation is detected only in cfDNA samples of CRC patients but not in healthy controls [ 138 ].…”

Four methylation-driven genes detected by linear discriminant analysis model from early-stage colorectal cancer and their methylation levels in cell-free DNA

“…As a result, although some genes have increased methylation levels in tumor tissues, few of them are released into the blood from tissue cells before stage IV. However, this reason cannot explain the fact that some methylated sites in cfDNA of CRC patients are specific and significantly different from those of healthy normal people ( 68 , 69 ).…”