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. 2009 Sep;27(9):858-63.
doi: 10.1038/nbt.1559. Epub 2009 Aug 16.

Sensitive digital quantification of DNA methylation in clinical samples

Meng Li  1 , Wei-Dong Chen, Nickolas Papadopoulos, Steven N Goodman, Niels Christian Bjerregaard, Søren Laurberg, Bernard Levin, Hartmut Juhl, Nadir Arber, Helen Moinova, Kris Durkee, Kerstin Schmidt, Yiping He, Frank Diehl, Victor E Velculescu, Shibin Zhou, Luis A Diaz Jr, Kenneth W Kinzler, Sanford D Markowitz, Bert Vogelstein
Affiliations

Sensitive digital quantification of DNA methylation in clinical samples

Meng Li et al. Nat Biotechnol. 2009 Sep.

Abstract

Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by flow cytometry or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in approximately 5,000 unmethylated molecules in DNA from plasma or fecal samples. Using methylated vimentin as a biomarker in plasma samples, methyl-BEAMing detected 59% of cancer cases. In early-stage colorectal cancers, this sensitivity was four times more than that obtained by assaying serum-carcinoembryonic antigen (CEA). With stool samples, methyl-BEAMing detected 41% of cancers and 45% of advanced adenomas. In addition to diagnostic and prognostic applications, this digital quantification of rare methylation events should be applicable to preclinical assessment of new epigenetic biomarkers and quantitative analyses in epigenetic research.

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Conflict of interest statement

COMPETING INTERESTS STATEMENT

The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturebiotechnology/.

Figures

Figure 1
Figure 1
Schematic and examples of digital quantification of rare DNA methylation in clinical samples. (a) Schematic. (b) Representative results of Methyl-BEAMing obtained with flow cytometry. The number of beads representing methylated, unmethylated, mixture, and virgin beads are indicated in the top right corner of each box.
Figure 1
Figure 1
Schematic and examples of digital quantification of rare DNA methylation in clinical samples. (a) Schematic. (b) Representative results of Methyl-BEAMing obtained with flow cytometry. The number of beads representing methylated, unmethylated, mixture, and virgin beads are indicated in the top right corner of each box.
Figure 2
Figure 2
Vimentin Methyl-BEAMing of plasma from colorectal cancer patients. (a) The number of methylated vimentin fragments in 2 ml plasma from normal, age and sex-matched control patients. (b) The number of methylated vimentin fragments in 2 ml plasma from colorectal cancer patients of various stages. (c) Receiver operating characteristic (ROC) curves based on the number of methylated vimentin fragments for the indicated classes of samples. (d) ROC curves based on CEA level for the indicated classes of samples. The dotted lines in (a) (b) (c) represent 1 methylated fragment per 2 ml plasma DNA. The doted line in (d) represents 5 ng/ml CEA level.
Figure 3
Figure 3
Vimentin Methyl-BEAMing of fecal DNA from colorectal tumor patients. (a) The fraction of methylated vimentin fragments in 4 g feces from normal, age and sex-matched control patients. (b) The fraction of methylated vimentin fragments in 4 g feces from patients with colorectal adenomas (c) The fraction of methylated vimentin fragments in 4 g feces from patients with colorectal carcinomas (Duke’s A or B red; Duke’s C or D, blue). The dotted line represents 2% methylated fragments. (d) Receiver operating characteristic (ROC) curves based on the fraction of methylated vimentin fragments for the indicated classes of samples.
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