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Sensitive digital quantification of DNA methylation in clinical samples

Nature Biotechnology volume 27, pages 858–863 (2009)Cite this article

Abstract

Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers1,2. 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 cytometry3 or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in ∼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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Figure 1: Digital quantification of rare DNA methylation in clinical samples by methyl-BEAMing.
Figure 2: Methyl-BEAMing to detect methylation of the vimentin gene using plasma from colorectal cancer patients.
Figure 3: Methyl-BEAMing to detect methylation of the vimentin gene using fecal DNA from colorectal tumor patients.

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Acknowledgements

We thank B. Berger for helpful discussions; D. Edelstein for the help with plasma collection; and M. Whalen and L. Kasturi for expert technical assistance. This work was supported by the Virginia and D.K. Ludwig Fund for Cancer Research; the Miracle Foundation; the Edelstein Fund; the US National Colorectal Cancer Research Alliance; The US National Institutes of Health grants CA43460,CA62924 and CA120237; The Danish Cancer Society; The Danish Research Council; and The Institute of Experimental Clinical Research, Aarhus University.

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Authors and Affiliations

  1. The Ludwig Center for Cancer Genetics and Therapeutics, Howard Hughes Medical Institute and Sidney Kimmel Cancer Center at the Johns Hopkins Medical Institutions, Baltimore, Maryland, USA

    Meng Li, Nickolas Papadopoulos, Kerstin Schmidt, Yiping He, Frank Diehl, Victor E Velculescu, Shibin Zhou, Luis A Diaz Jr, Kenneth W Kinzler & Bert Vogelstein

  2. Department of Medicine and Ireland Cancer Center, Case Western Reserve University and Case Medical Center of University Hospitals of Cleveland and Howard Hughes Medical Institute, Cleveland, Ohio, USA

    Wei-dong Chen, Helen Moinova & Sanford D Markowitz

  3. Department of Biostatistics, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA

    Steven N Goodman

  4. Department of Surgery P, Aarhus University Hospital, Aarhus, Denmark

    Niels Christian Bjerregaard & Søren Laurberg

  5. U.T.M.D. Anderson Cancer Center, Houston, Texas, USA

    Bernard Levin

  6. Indivumed, Center for Cancer Research at Israelitic Hospital, Hamburg, Germany

    Hartmut Juhl

  7. Tel Aviv University, Tel Aviv Medical Center and Sackler School of Medicine and Integrated Cancer Prevention Center, Tel Aviv, Israel

    Nadir Arber

  8. Exact Sciences Corp., Marlborough, Massachusetts, USA

    Kris Durkee

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  1. Meng Li
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Contributions

M.L., S.D.M., S.N.G., K.W.K. and B.V. designed the project. M.L. developed the methyl-BEAMing assay and performed the experiments on plasma and fecal DNA. N.P., M.L. and Y.H. performed the Solexa sequencing experiments. H.M. performed the MSP assay. K.D. purified fecal DNA. S.N.G. provided statistical analysis. H.J., L.A.D., N.C.B., S.L., N.A. and K.S. collected clinical samples. W.-d.C., S.Z, V.E.V., F.D. and B.L. made intellectual contributions to the project. B.V., M.L. and S.D.M. wrote the manuscript.

Corresponding author

Correspondence to Sanford D Markowitz.

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Competing interests

Technology for digital quantification of methylated DNA is the subject of a patent application from Johns Hopkins and Case Western Universities that include S.D.M., M.L., W.D.C., B.V. and K.W.K. as inventors. Under agreements between the Johns Hopkins University, Genzyme, Exact Sciences, Beckman, Inostics, and Invitrogen, K.W.K., B.V., M.L., and F.D. are entitled to a share of the royalties received by the University on sales of products related to BEAMing. Under agreements between Case Western University and Exact Sciences, S.D.M. and W.D.C. are entitled to a share of the royalties received on sales of products related to methylated vimentin DNA. Johns Hopkins University, K.W.K. and B.V. own stock in Genzyme and K.W.K., B.V., N.P., F.D. and L.D. own stock in Inostics, both of which are subject to certain restrictions under Johns Hopkins University policy. The terms of these arrangements are being managed by the universities in accordance with their conflict of interest policies.

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Li, M., Chen, Wd., Papadopoulos, N. et al. Sensitive digital quantification of DNA methylation in clinical samples. Nat Biotechnol 27, 858–863 (2009). https://doi.org/10.1038/nbt.1559

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