Sensitive digital quantification of DNA methylation in clinical samples

Abstract

Abnormally methylated genes are increasingly being used as cancer biomarkers 1, 2 . For clinical applications, it is important to precisely determine the number of methylated molecules in the analyzed sample. We here describe a digital approach that can enumerate one methylated molecule out of ~5000 unmethylated molecules. Individual DNA fragments can be amplified and analyzed either by flow cytometry or next generation sequencing instruments. Using methylated vimentin as a biomarker, we tested 191 plasma samples and detected cancer cases with 59% sensitivity (95% CI, 48%-70%) and 93% specificity (95% CI, 86%-97%). Using the same assay, we analyzed 80 stool samples and demonstrated 45% sensitivity for detecting colorectal adenomas (23%-68%), 41% COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturebiotechnology/. NIH Public Access Author ManuscriptNat Biotechnol. Author manuscript; available in PMC 2010 March 30. Published in final edited form as:Nat Biotechnol. 2009 September ; 27(9): 858-863. doi:10.1038/nbt.1559. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sensitivity for detecting cancer (21%-64%), and 95% specificity (82%-99%). This digital quantification of rare methylation events should be applicable to diagnostic evaluations of clinical samples, to preclinical assessments of new epigenetic biomarkers, and to quantitative analyses of epigenetic biology.In humans, DNA methylation is largely restricted to cytosines within 5′-CpG dinucleotides. This covalent modification of DNA functions as an important mediator of gene regulation and, together with covalent modifications of histone proteins, forms the cornerstone for the burgeoning field of epigenetics. Though cancers are globally hypomethylated 3 , specific regions of genes have been shown to be hypermethylated in association with transcription silencing 4 . In addition to its implications for gene regulation, DNA methylation is providing a new generation of cancer biomarkers 5 . Though mutant sequences provide exquisitely specific biomarkers of this class 6, 7 , their utility is compromised by their heterogeneity: the same gene can be mutationally inactivated through many different mechanisms or mutated at many different positions. In contrast, DNA hypermethylation in cancers often affects identical residues in the regulatory regions of particular genes, providing major advantages in biomarker test design. Accordingly, many studies have employed DNA methylation of specific genes for diagnostics development 2,4,5,8 . Such diagnostic tests can in principle be used for early detection of cancers, for assessing prognosis, and for determining the effects of therapy or detecting residual disease.The majority of diagnostic tests based on DNA methylation have employed bisulfite to convert cytosine residues to uracils. This conversion alters the sequence of DNA 9 , providing an opportunity to assess DNA methylat...

Advances in epigenetic biomarker research in colorectal cancer

“…Since then, many studies have been devoted to vimentin methylation. Follow-up studies have obtained similar results and thus have reinforced the utility of vimentin as a standalone biomarker [47,54,55,59,61] . This has led to the commercialization of a single-gene stool kit for CRC detection based on vimentin methylation.…”

Epigenetic Alteration of DNA in Mucosal Wash Fluid Predicts Invasiveness of Colorectal Tumors

“…For example, promoter hypermethylation is associated with the silencing of various cancer-related genes (14,15), and aberrant methylation of the CpG islands of genes in stool and serum/plasma can be used as a molecular marker for detection of colorectal tumors (16)(17)(18)(19)(20). On the contrary, because DNA methylation is an early event (21)(22)(23), it does not enable one to distinguish between premalignant lesions and invasive tumors.…”

Aberrant DNA methylation of tumor‐related genes in oral rinse

“…In addition, the detection methods for DNA methylation, such as MSP, Q-MSP, MethyLight, and Methyl-BEAMing, are based on PCR and, thus, are rapid and sensitive methods for detecting cancer in clinical samples. 35,36 Moreover, aberrant DNA methylation can be detected before tumors are clinically evident 37 ; for example, aberrant methylation of tumor suppressor genes was detected in DNA from sputum in 100% of patients with squamous cell carcinoma of the lung up to 3 years before clinical diagnosis. 38 Thus, the DNA methylation status of oral rinse samples may be a useful marker not only for tumor detection but also for predicting the future risk of OSCC.…”