Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma.
Epigenetic silencing of the DNA repair protein O(6)-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 beta-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.
OncoMethylome Sciences SA, Tour 5 GIGA (B34), +3, Avenue de l'Hôpital 11, 4000 Liège, Belgium.
Ilse
I
Vlassenbroeck
Stéphane
S
Califice
Annie-Claire
AC
Diserens
Eugenia
E
Migliavacca
Josef
J
Straub
Ivano
I
Di Stefano
Fabrice
F
Moreau
Marie-France
MF
Hamou
Isabelle
I
Renard
Mauro
M
Delorenzi
Bruno
B
Flamion
James
J
DiGuiseppi
Katja
K
Bierau
Monika E
ME
Hegi
D019175:DNA Methylation; D005910:Glioma / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D019853:O(6)-Methylguanine-DNA Methyltransferase