Published online before print June 13, 2008

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Journal of Molecular Diagnostics 2008, Vol. 10, No. 4
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.070169

Validation of Real-Time Methylation-Specific PCR to Determine O⁶-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma

Ilse Vlassenbroeck^*, Stéphane Califice^*, Annie-Claire Diserens, Eugenia Migliavacca, Josef Straub^*, Ivano Di Stefano^*, Fabrice Moreau^*, Marie-France Hamou, Isabelle Renard^*, Mauro Delorenzi, Bruno Flamion^¶, James DiGuiseppi^*, Katja Bierau^* and Monika E. Hegi

From OncoMethylome Sciences SA, * Liège, Belgium; the Laboratory of Tumor Biology and Genetics, Centre Romand de Neurochirurgie, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland; the Swiss Institute of Bioinformatics, Geneva, Switzerland; the National Center of Competence in Research Molecular Oncology at the Swiss Institute of Experimental Cancer Research, Epalinges, Switzerland; and the Molecular Physiology Research Unit, ^¶ Laboratory of Physiology and Pharmacology, Facultés Universitaires Notre-Dame de la Paix, University of Namur, Namur, Belgium

Epigenetic silencing of the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the β-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen’s kappa = 0.80; 95% confidence interval, 0.82–0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

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