Clinical Chemistry
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Clinical Chemistry 55: 1559-1563, 2009. First published June 18, 2009; 10.1373/clinchem.2008.122937
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(Clinical Chemistry. 2009;55:1559-1563.)
© 2009 American Association for Clinical Chemistry, Inc.

Brief Communications

Comparative Analysis of PCR-Based Biomarker Assay Methods for Colorectal Polyp Detection from Fecal DNA

Christoph Ausch1,2,1, Young-Ho Kim1,3,1, Karen D. Tsuchiya4,5, Slavomir Dzieciatkowski1, Mary K. Washington6, Christos Paraskeva7, Jerry Radich1 and William M. Grady1,a

1 Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA;2 Institute for Surgical Oncology, Cluster for Translational Oncology, Ludwig Boltzmann Research Institute, Danube Hospital, Vienna, Austria;3 Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea;4 Division of Clinical Research, Fred Hutchinson Cancer Research Center, Department of Laboratories, Seattle Childrens Hospital;5 Department of Laboratory Medicine, University of Washington, Seattle, WA;6 Department of Pathology, Vanderbilt University Medical School, Nashville, TN, USA;7 University of Bristol, Bristol, UK;

aaddress correspondence to this author at: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., D4-100, Seattle, WA 98109. Fax 206-667-2917; e-mail wgrady{at}fhcrc.org.


Abstract

Background: Aberrantly methylated genes are promising biomarkers for the detection of colon adenomas and colorectal cancers (CRCs). The optimal assay type and specific methylated genes for these assays remain to be determined.

Methods: We used genomewide microarray-based assays to identify methylated genes as candidate biomarkers for colon neoplasms. The frequency of aberrant methylation of these genes in primary tumors was assessed with methylation-specific PCR (MSP). The limits of detection and specificities for different types of PCR-based assays were then assessed with the most promising genes identified in this screen. Finally, we assessed the best-performing MSP assay as an early-detection marker using fecal DNA samples.

Results: ITGA4 [integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)] was identified as a novel gene frequently methylated in CRC. Methylated ITGA4 is present in 75% of colon adenomas (n = 36) and 92% of colon adenocarcinomas (n = 75). Comparison of end point MSP, end point MSP with clamped primers, and quantitative fluorescent MSP (qMSP) approaches revealed that both types of end point MSP assays could routinely detect as little as 70 pg DNA, whereas the qMSP assay could routinely detect as little as 7 pg. A fecal DNA qMSP assay for methylated ITGA4 can detect 69% of individuals with colon adenomas (n = 13) with a diagnostic specificity of 79% (n = 28).

Conclusions: Methylated ITGA4 is a promising marker gene for the early detection of colonic neoplasms. qMSP has the lowest limit of detection of the MSP assay types tested, and a qMSP assay that detects methylated ITGA4 has potential as an early-detection assay for colon neoplasms.






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