Defining optimal cutoff value of MGMT promoter methylation by ROC analysis for clinical setting in glioblastoma patients

Abstract

Resistance to temozolomide (TMZ) chemotherapy poses a significant challenge in the treatment of glioblastoma (GBM). Hypermethylation in O-methylguanine-DNA methyltransferase (MGMT) promoter is thought to play a critical role in this resistance. Pyrosequencing (PSQ) has been shown to be accurate and robust for MGMT promoter methylation testing. The unresolved issue is the determination of a cut-off value for dichotomization of quantitative MGMT PSQ results into "MGMT methylated" and "MGMT unmethylated" patient subgroups as a basis for further treatment decisions. In this study, receiver operating characteristic (ROC) curve analysis was used to identify an optimal cutoff of MGMT promoter methylation by testing mean percentage of methylation of 4 CpG islands (76-79) within MGMT exon 1. The area under the ROC (AUC) as well as the best cutoff to classify the methylation were calculated. Positive likelihood ratio (LR+) was chosen as a diagnostic parameter for defining an optimal cut-off. Meanwhile, we also analyzed whether mean percentage of methylation at the investigated CpG islands could be regarded as a marker for evaluating prognostication. ROC analysis showed that the optimal threshold was 12.5% (sensitivity: 60.87%; specificity: 76%) in response to the largest LR+ 2.54. 12.5% was established to distinguish MGMT promoter methylation, which was confirmed using validation set. According to the cutoff value, the MGMT promoter methylation was found in 58.3% of GBM. Mean methylation level of the investigated CpG sites strong correlated with overall survival (OS), which means GBM patients with a high level of methylation survived longer than those with low level of methylation(log-rank test, P = 0.017). In conclusion, ROC curve analysis enables the best cutoff for discriminating MGMT promoter methylation status. LR+ can be used as a key factor that evaluates cutoff. The promoter methylation level of MGMT by PSQ in GBM patients had prognostic value.

Combinations of four or more CpGs methylation present equivalent predictive value for MGMT expression and temozolomide therapeutic prognosis in gliomas

“…The PSQ testing has been regarded as the gold standard for MGMT promoter methylation testing and wildly used in reported studies and clinical practice . However, which CpGs should be included in the MGMT methylation PSQ testing and analysis is still a controversial issue.…”

“…In glioblastoma tissues, individual CpG 27, 32, 73, 75, 79 and 80 were reported to be significantly correlated to the MGMT mRNA expression, and combinations of CpGs 32‐33 and CpGs 72‐83 were concordant with MGMT mRNA expression . But, there was no consensus on which individual CpG or CpG combinations should be used in MGMT methylation PSQ testing, and the combinations of CpGs 72‐83, 72‐80, 72‐77, 74‐78, 74‐89, 76‐79, and 80‐83 were used in distinct studies . Here, we systematically compared the predictive value of all combinations within the CpGs 72‐82 on the MGMT mRNA expression through analyzing paired samples with both the MGMT methylation PSQ testing and mRNA expression data.…”

“…The template used in the MGMT PSQ testing was prepared as previously described . Briefly, bisulfite‐treated DNA was preamplified with the primers (a) F‐primer 5′‐GTT TYG GAT ATG TTG GGA TAG TT‐3′; (b) biotinylated R‐primer 5′‐biotin‐ACR ACC CAA ACA CTC ACC AA‐3′.…”

“…However, along with the high heterogeneity of the CpGs methylation gradually identified, MSP‐based methods were unable to reflect such heterogeneity . Currently, pyrosequencing (PSQ) has been developed to be a stable technique, offering a valid, reliable and quick evaluation of both fresh frozen and formalin fixed paraffin embedded (FFPE) specimens …”

“…Various number of CpGs from four to eighteen has been used as CpG combinations in reported studies (Figure ) . Among which, the commonly adopted combinations are CpGs 76‐79 and 74‐78, which are currently used in Qiagen commercial kits.…”