Abstract
BackgroundMutL 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.MethodsPrimers 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.ResultsUsing 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.ConclusionsOur 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.
Highlights
MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC)
We developed a methylation sensitive restriction enzyme (MSRE) digested assay to absolutely quantify the MLH1 promoter methylation in circulating tumor DNA (ctDNA) using highly sensitive digital droplet PCR (ddPCR) in advanced stage CRC samples
In ddPCR assay, using sheared synthetic DNA, MLH1 methylation was able to be detected using as low as 0.096 ng of DNA (Fig. 1b)
Summary
MutL Homolog 1 (MLH1) promotor methylation is associated with microsatellite instability high colorectal cancer (CRC). Methylation changes are present in a variety of cancers, and occur early in carcinogenesis, typically repressing the expression of tumor suppressor genes. Acquired promoter hypermethylation often occurs with global hypermethylation of gene promoters known as CpG island methylator phenotype [1]. These epigenetic changes are highly pervasive across a tumor type and can be a very consistent feature of cancer in contrast to mutations, which typically occur at a wide range of sites. 15% of CRC cases are MSI-High (MSI-H); 3% of which include hereditary polyposis colorectal cancer (HNPCC) or Lynch syndrome and sporadic MSI-H CRC, typically caused by somatic methylation of the MLH1 gene promoter, making up the remaining 12% of cases [2, 3]. The assessment of MLH1 promoter methylation status will add essential clinical value across the cancer types
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