Validating a clinically based MS-MLPA threshold through comparison with Sanger sequencing in glioblastoma patients

Abstract 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 cut-off 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 MGMT₂15, MGMT₁90, and MGMT₁24 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 MGMT₂15; p 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.