MGMT promoter methylation in patients with glioblastoma: is methylation-sensitive high-resolution melting superior to methylation-sensitive polymerase chain reaction assay?

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In Brief

The authors assessed MGMT promoter methylation results obtained by methylation-sensitive PCR (MS-PCR) and high-resolution melting (MS-HRM) methods to determine whether MS-HRM overcomes the limitations of MS-PCR. They found that MS-HRM was superior to MS-PCR for predicting survival outcome in 75 GBM patients with and without MGMT promoter methylation. Based on the results of multivariate Cox analysis, MS-HRM revealed independent prognostic factors. The authors suggest that MS-HRM is optimal for assessing the MGMT promoter methylation status and that it represents an alternative to MS-PCR.

ABBREVIATIONS AUC = area under the curve; GBM = glioblastoma; KPS = Karnofsky Performance Status; MGMT = O6-methylguanine-DNA methyltransferase; MS-HRM = methylation-sensitive high-resolution melting; MS-PCR = methylation-sensitive polymerase chain reaction; OS = overall survival; PFS = progression-free survival; ROC = receiver operating characteristic; TMZ = temozolomide.

Article Information

Correspondence Shinji Yamashita: University of Miyazaki, Japan. shinjy@med.miyazaki-u.ac.jp.

INCLUDE WHEN CITING Published online May 4, 2018; DOI: 10.3171/2017.11.JNS171710.

Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.

© AANS, except where prohibited by US copyright law.

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Figures

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    Glioma cell cultures. Region of interest in the MGMT promoter and MS-HRM analysis of glioma cell lines. A: MGMT promoter methylation was analyzed by MS-HRM and MS-PCR. For MS-HRM, we designed 3 primer sets to evaluate CpG sites 72–89. B: Dot-plot of the methylation level of CpG sites analyzed by MS-HRM using primers 1, 2, and 3 and by cloning-based bisulfite sequencing. With primers 1 and 2 there was a strong correlation with the results of cloning-based bisulfite sequencing (primer 1: r = 0.959; primer 2: r = 0.960; both p < 0.01, primer 3: r = 0.649, p = 0.163). C: Cloning-based bisulfite sequencing results of the MGMT promoter region in U87. Black and white indicate methylated and unmethylated CpG dinucleotides, respectively. The methylation level of each CpG site was calculated from the methylation status of 25 clones. The CpG sites analyzed by MS-HRM primers 1 (red), 2 (blue), and 3 (green) are identified at the bottom of the illustration. D: Western blots showing a strong LN18 band, a faint T98G band, and no band for the 4 other cell lines. The expression patterns were inversely correlated with the methylation levels evaluated by MS-HRM using primers 1 and 2. Figure is available in color online only.

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    MS-HRM analysis of surgical samples. ROC analysis was applied to evaluate the sensitivity and specificity for predicting OS ≥ 18 months. MS-HRM was with primers 1, 2, and 3 to assess the MGMT promoter methylation status. The AUC was largest with primer 1.

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    Surgical samples. Comparison of results obtained with MS-HRM and MSP. A: Kaplan-Meier estimates of PFS and OS of 75 GBM patients based on the MGMT promoter methylation status determined by MS-HRM and MSP. Based on the log-rank test for PFS the MS-HRM and MS-PCR values were significantly different (MS-HRM: p = 0.00023; MS-PCR: p = 0.0035), as were the values for OS (MS-HRM: p = 0.00019; MS-PCR: p = 0.00028). B: ROC curves were calculated on the basis of MGMT promoter methylation determined by MS-HRM and MS-PCR (MSP) for PFS ≥ 10 months and OS ≥ 18 months. For both PFS and OS, the AUC was larger with MS-HRM than with MS-PCR. C: Scatterplot showing the methylation status of 75 patients classified as MS-PCR negative/MS-HRM negative (n = 34), MS-PCR positive/MS-HRM negative (n = 10), MS-PCR negative/MS-HRM positive (n = 3), and MS-PCR positive/MS-HMS positive (n = 28). Among MS-HRM-negative samples, the methylation level was significantly higher in MS-PCR-positive samples than in MS-PCR-negative samples (p < 0.01). D: Standard samples prepared to evaluate the methylation level by MS-HRM were analyzed by MS-PCR assay. Samples with 5% methylation featured a faint positive band, suggesting that the appropriate cutoff value for MS-PCR is 5%. M = methylated; U = unmethylated. Figure is available in color online only.

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