ArticleBMC cancer2021
Development of a liquid biopsy based purely quantitative digital droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer patients.
Article in BMC cancer, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed, 31 citations in OpenAlex.
- Biological basis and clinical translation prospects of circulating cell-free DNA in precision management of breast cancer (Review).Oncology letters · 2026Review
- Diagnostic Accuracy of Blood-Based cfDNA/ctDNA Tests for Colorectal Cancer-A Systematic Review and Meta-Analysis.Cancers · 2026Review
- A scoping review of DNA methylation biomarkers for non-invasive detection of colorectal cancer at the CpG site level.Clinical epigenetics · 2026Article
- A liquid biopsy-based multi-methylation marker panel for minimally invasive gastric cancer screening.Clinical epigenetics · 2026Article
- Epi-liquidomics: redefining cancer diagnostics through epigenetic profiling.Frontiers in epigenetics and epigenomics · 2026Review
- Genome-wide methylation analysis of circulating cell-free DNA identifies an episignature in lung cancer: validation of EMP2 as a potential biomarker.Clinical epigenetics · 2025Article
- DNA Methylation in Ovarian and Endometrial Cancers: Predictive and Mechanistic Roles in PARP Inhibitor and ICI Response.Cancer science · 2025Review
- A Novel Droplet Digital PCR Assay for BRCA1 and RAD51C Methylation: Advancing Homologous Recombination Deficiency Detection in Ovarian Cancer.The Journal of molecular diagnostics : JMD · 2025Article
- Comparative Analysis of Two Digital PCR Platforms for Detecting DNA Methylation in Patient Samples.Cell biochemistry and function · 2025Article
- Recent advances on gene-related DNA methylation in cancer diagnosis, prognosis, and treatment: a clinical perspective.Clinical epigenetics · 2025Review
- Research Progress of DNA Methylation Markers for Endometrial Carcinoma Diagnosis.Journal of Cancer · 2025Review
- Review
- Methylation‑sensitive restriction enzyme‑droplet digital PCR assay for the one‑step highly sensitive analysis of DNA methylation hotspots.International journal of molecular medicine · 2024Article
- Hypermethylation Loci of ZNF671, IRF8, and OTX1 as Potential Urine-Based Predictive Biomarkers for Bladder Cancer.Diagnostics (Basel, Switzerland) · 2024Article
- Plasma Cell-Free Tumor Methylome as a Biomarker in Solid Tumors: Biology and Applications.Current oncology (Toronto, Ont.) · 2024Review
- The utility of liquid biopsy-based methylation biomarkers for colorectal cancer detection.Frontiers in oncology · 2024Article
- Current and emerging applications of liquid biopsy in pan-cancer.Translational oncology · 2023Article
- Combined SERS Microfluidic Chip with Gold Nanocone Array for Effective Early Lung Cancer Prognosis in Mice Model.International journal of nanomedicine · 2023Article
- Emerging roles of cystathionine β-synthase in various forms of cancer.Redox biology · 2022Review
- DNA Methylation Biomarkers for Prediction of Response to Platinum-Based Chemotherapy: Where Do We Stand?Cancers · 2022Review
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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