Clinical Chemistry
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Clinical Chemistry 54: 414-423, 2008. First published December 18, 2007; 10.1373/clinchem.2007.095992
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(Clinical Chemistry. 2008;54:414-423.)
© 2008 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening

Catherine Lofton-Day1,a,1, Fabian Model2,1, Theo DeVos1, Reimo Tetzner2, Juergen Distler2, Matthias Schuster2, Xiaoling Song1, Ralf Lesche2, Volker Liebenberg2, Matthias Ebert3, Bela Molnar4, Robert Grützmann5, Christian Pilarsky5 and Andrew Sledziewski1

1 Epigenomics, Inc., Seattle, WA; 2 Epigenomics AG, Berlin, Germany; 3 Technical University, Munich, Germany; 4 Semmelweis University, Budapest, Hungary; 5 Technical University of Dresden, Dresden, Germany.

aAddress correspondence to this author at: Epigenomics, Inc., 1000 Seneca St., Suite 300, Seattle, WA 98101. e-mail clofton{at}us.epigenomics.com.

Background: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma.

Methods: We first used restriction enzyme–based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range.

Results: Restriction enzyme–based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%–73%] of plasma samples from CRC patients and not detected in 69% (62%–76%) of the controls. The corresponding results for NGFR were 51% (42%–60%) and 84% (77%–89%); for SEPT9, the values were 69% (60%–77%) and 86% (80%–91%).

Conclusions: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.




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