Published online before print January 29, 2009

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Copyright © 2009 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2009.080109

Accepted for publication December 12, 2008.

Technical Advances

High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis

Marija Balic^*@, Martin Pichler^*, Jasmin Strutz^*, Ellen Heitzer^*, Christoph Ausch, Hellmut Samonigg^*, Richard J. Cote, and Nadia Dandachi^*

From the Division of Oncology,* Department of Internal Medicine, Medical University of Graz, Graz, Austria; the Department of Surgery, Ludwig Boltzmann Research Institute of Surgical Oncology, Danube Hospital, Vienna, Austria; and the Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California

^@ To whom correspondence should be addressed. E-mail: marija.balic{at}meduni-graz.at.

	Abstract

High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O⁶-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh or formalin-fixed paraffin-embedded human cancer cell lines. Then we used these assays for the analysis of MGMT and APC promoter methylation in a subset of archival formalin-fixed paraffin-embedded colorectal tumor specimens. All samples with promoter methylation of MGMT or APC and randomly selected samples without promoter methylation were analyzed twice. All results generated by HRM were validated with MGMT and APC MethyLight assays. APC and MGMT promoter methylation data were consistent and reproducible throughout the dilutions and all three replicates in the methylated DNA dilution matrix and between two experiments in clinical samples. There was high concordance between HRM and MethyLight results. HRM for APC promoter methylation revealed consistent results between fresh and formalin-fixed paraffin-embedded human cancer cell line DNA. The methylation status in archival tumor specimens from patients with colorectal cancer can therefore be determined with high quality by HRM. The ability to analyze archival tissues greatly facilitates further research and its clinical implementation.

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