title: MethyLight droplet digital PCR for detection and absolute quantification of infrequently methylated alleles.
abstract: Aberrant DNA methylation is a common epigenetic alteration found in colorectal adenomas and cancers and plays a role in cancer initiation and progression. Aberrantly methylated DNA loci can also be found infrequently present in normal colon tissue, where they seem to have potential to be used as colorectal cancer (CRC) risk biomarkers. However, detection and precise quantification of the infrequent methylation events seen in normal colon is likely beyond the capability of commonly used PCR technologies. To determine the potential for methylated DNA loci as CRC risk biomarkers, we developed MethyLight droplet digital PCR (ddPCR) assays and compared their performance to the widely used conventional MethyLight PCR. Our analyses demonstrated the capacity of MethyLight ddPCR to detect a single methylated NTRK3 allele from among more than 3125 unmethylated alleles, 25-fold more sensitive than conventional MethyLight PCR. The MethyLight ddPCR assay detected as little as 19 and 38 haploid genome equivalents of methylated EVL and methylated NTRK3, respectively, which far exceeded conventional MethyLight PCR (379 haploid genome equivalents for both genes). When assessing methylated EVL levels in CRC tissue samples, MethyLight ddPCR reduced coefficients of variation (CV) to 6-65% of CVs seen with conventional MethyLight PCR. Importantly, we showed the ability of MethyLight ddPCR to detect infrequently methylated EVL alleles in normal colon mucosa samples that could not be detected by conventional MethyLight PCR. This study suggests that the sensitivity and precision of methylation detection by MethyLight ddPCR enhances the potential of methylated alleles for use as CRC risk biomarkers.
authors.affiliation: a Clinical Research Division; Fred Hutchinson Cancer Research Center ; Seattle , WA USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Yu
authors.forename: Kelly T
authors.initials: KT
authors.lastname: Carter
authors.forename: Karen W
authors.initials: KW
authors.lastname: Makar
authors.forename: Kathy
authors.initials: K
authors.lastname: Vickers
authors.forename: Cornelia M
authors.initials: CM
authors.lastname: Ulrich
authors.forename: Robert E
authors.initials: RE
authors.lastname: Schoen
authors.forename: Dean
authors.initials: D
authors.lastname: Brenner
authors.forename: Sanford D
authors.initials: SD
authors.lastname: Markowitz
authors.forename: William M
authors.initials: WM
authors.lastname: Grady
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics*; D019175:DNA Methylation; D018450:Disease Progression; D006238:Haploidy; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020812:Receptor, trkC / Q000235:genetics; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; C484449:EVL protein, human; D020812:Receptor, trkC
keywords: DNA methylation; MethyLight; colorectal cancer; droplet digital PCR; field cancerization effect; risk biomarkers