BACKGROUND: Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. RESULTS: A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). CONCLUSIONS: Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.

Center of Oncocytogenomics Institute of Medical Biochemistry and Laboratory Diagnostics General University Hospital and 1st Faculty of Medicine of Charles University Prague U Nemocnice 499 2 128 00 Prague Czech Republic

Department of HLA Institute of Hematology and Blood Transfusion U Nemocnice 2094 1 128 00 Prague Czech Republic

Department of Neurosurgery and Neurooncology 1st Faculty of Medicine of Charles University and Military University Hospital Prague U Vojenske Nemocnice 1200 169 02 Prague Czech Republic

Department of Pathology 1st Faculty of Medicine of Charles University and Military University Hospital Prague U Vojenske Nemocnice 1200 169 02 Prague Czech Republic

The Clinical Trials and Research Department General University Hospital and 1st Faculty of Medicine of Charles University Prague U Nemocnice 499 2 128 00 Prague Czech Republic

The Fingerland Department of Pathology Charles University Faculty of Medicine in Hradec Kralove and University Hospital Hradec Kralove Sokolska 581 500 05 Hradec Kralove Czech Republic

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