Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

Abstract

Objectives: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma.

Background: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response.

Methods: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry.

Results: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis.

Discussion and conclusion: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

Figure 1

Comparison of the MGMT promoter methylation status of GBM cases analyzed using MSP and PyroS. Five CpG sites were analyzed using PyroS, and 8 CpG sites were analyzed using MSP. For PyroS, the MGMT methylation status was scored according to the average percentage of specifically methylated CpG sites as unmethylated (U) when <10% CpG sites were methylated, intermediate (I) when 10 to 26% CpG sites were methylated, or methylated (M) when ≥27% of CpG sites were methylated. For MSP, the MGMT methylation status was scored as methylated (M) or unmethylated (U) according to the presence or absence of the specific PCR amplification.

Figure 2

Correlation between relative gene expression level of MGMT and promoter methylation status determined using MSP (A) and PyroS (B). MGMT expression levels in the GBM samples were determined relative to non-neoplastic tissues. The horizontal bars show the median values for the relative expression of MGMT in each group. The Mann-Whitney test was used to evaluate the differences in the relative expression level between the methylated and unmethylated groups.

Figure 3

Heatmap of the MGMT analyses showing methylation status as assessed by MSP and PyroS, quantitative real-time PCR (qRT-PCR) for MGMT relative gene expression and immunohistochemistry (IHC) for MGMT protein expression. MSP and PyroS: green, methylated; yellow, intermediate; red, unmethylated; and white, not determined. qRT-PCR: green, low expression; red, high expression. IHC: green, negative staining; red, positive staining.

Figure 4

Kaplan-Meier curves showing the overall survival of GBM patients submitted to adjuvant therapy (radiotherapy and/or chemotherapy) and grouped according to MGMT promoter methylation status as determined by MSP (A) and PyroS (B). The difference in overall survival times between the methylated and unmethylated groups was statistically significant for both methods (log-rank test: p = 0.025 for MSP and p = 0.004 for PyroS).