Detection of MGMT promoter methylation in malignant gliomas.

e23131 Background: The O6-methylguanine methyltransferase (MGMT) is a DNA repair enzyme that is responsible for de-alkylation of naturally occurring O6-methylguanines in DNA and aids in the prevention of errors during DNA replication and transcription. Epigenetic silencing of the MGMT compromises DNA repair making tumor cells more sensitive to alkylating drugs and resulting in better response and longer survival in patients with glioma. We have validated the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA)-based method to determine the MGMT methylation status in patients with glioma. Methods: Twenty-two formalin fixed paraffin embedded (FFPE) brain tissue samples from glioma cases, 7 FFPE brain tissue samples from non-tumor cases and 23 FFPE non-tumor samples from other organ sites were used in the study. MS-MLPA probemix ME012-X1 (MRC-Holland, Amsterdam, The Netherlands) was used to detect the methylation status in the MGMT promoter. The ME012-X1 contains 6 MS-MLPA probes that detect the methylation status of the MGMT gene promoter based on sequence recognition by the methylation-sensitive restriction enzyme HhaI. Greater than 40% methylation in one or MS-MLPA probes in the sample indicated hypermethylation. Results: MGMT methylation status in all 52 tissue specimens was determined. Hypermethylation was not detected in the 7 FFPE normal brain tissue samples and 23 FFPE non-tumor samples from other organ sites, as expected. Of the 22 glioma cases 14 were hypermethylated in one or more probe locations. Hypermethylation was not detected in the rest of the 8 glioma cases. The comparison of our results of the 22 glioma cases to reference laboratory results done by MethyLight assay, revealed discrepancies in two samples: one sample identified as hypermethylated MGMT by MS-MLPA was negative by MethyLight assay, and the other result was vice versa. This discrepancy was due to the differences in locations of the primer/probes between two assays. The MS-MLPA assay showed 100 % reproducibility and was capable of detecting hypermethylation in FFPE specimen with at least 50% tumor cellularity. Conclusions: The MS-MLPA assay represents a reliable and sensitive detection method to analyze promoter hypermethylation of MGMT.