Quantitative DNA melting analysis with hybridization probes (qDMA-HP) as a novel approach to assess MGMT promoter methylation in malignant glioma.

purposeQuantifying MGMT methylation in malignant glioma is vital for clinical decision-making. Methylated MGMT indicates a better prognosis and enhances the effectiveness of temozolomide therapy. However, there is ongoing debate regarding the best assay methods, targeted CpG sites, and optimal methylation cutoffs for predicting patient survival. This pilot study evaluates the novel qDMA-HP approach for quantifying MGMT methylation.

methodsAsymmetric multiplex PCR with methylation-independent primers and TaqMan probes, interrogating short CpG loci, followed by post-amplification melting of probe/amplicon hybrids, was used for quantitation of methylated and unmethylated MGMT loci. Hybridization probes were also used to minimize bias toward amplification of unmethylated sequences. The methylation cutoffs were determined programmatically.

resultsWe compared the prognostic efficiency of six loci of MGMT methylation. Cutoff Finder software determined the optimal methylation cutoff for CpGs 74-77 to be 9.5%: methylation of this locus increased the median PFS from 9 to 15 months (HR = 0.37, p = 0.0029) and the median OS from 18 to 50 months (HR = 0.34, p = 0.0062). The results of ROC analysis were as follows: AUC = 0.683, LR + = 3.02 (for PFS); AUC = 0.678, LR + = 1.87 (for OS). Multivariate analysis showed that methylation of CpGs 74-77 was an independent favorable prognostic marker for malignant glioma (PFS: HR = 0.25, p = 0.0031; OS: HR = 0.28, p = 0.0156), which was confirmed in the validation set for PFS.

conclusionqDMA-HP is comparable to pyrosequencing in prognostic effectiveness but is simpler and more cost-effective, suggesting its potential for broad clinical use.