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

Abstract

OBJECTIVE:

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a prognostic factor in adults with glioblastoma (GBM); it also yields information that is useful for clinical decision-making in elderly GBM patients. While pyrosequencing is the gold standard for the evaluation of the methylation status of MGMT, methylation-sensitive polymerase chain reaction (MS-PCR) assay continues to be used widely. Although MS-PCR results exhibited a good correlation with the prognosis of patients with GBM treated under the Stupp protocol, interpretation of the bands is based on subjective judgment, and the assay cannot be used to analyze heterogeneously methylated samples. We assessed whether methylation-sensitive high-resolution melting (MS-HRM) is an alternative to MS-PCR.

METHODS:

The authors prepared 3 primer sets that covered CpG 72–89 for MS-HRM analysis to determine the methylation levels of 6 human glioma cell lines. The results were validated by bisulfite sequencing of cloned alleles. The authors also subjected surgical samples from 75 GBM patients treated with temozolomide (TMZ) to MS-HRM to assess the MGMT methylation status and compared the findings with MS-PCR results using receiver operating characteristic (ROC), univariate, and multivariate analyses.

RESULTS:

There was a strong correlation between the methylation levels of the 6 glioma cell lines evaluated by MSHRM and by bisulfite sequencing; with primers 1 and 2, the correlation was significant (r = 0.959 and r = 0.960, respectively, p < 0.01). Based on log-rank analysis, MS-HRM was significantly better than MS-PCR for predicting progressionfree survival (PFS) and overall survival (OS) based on the methylation status of the MGMT promoter (PFS predicted by MS-HRM and MS-PCR = 0.00023 and 0.0035, respectively; OS = 0.00019 and 0.00028, respectively). ROC analysis showed that the area under the curve was larger with MS-HRM than with MS-PCR (PFS: 0.723 vs 0.635; OS: 0.716 vs 0.695). Based on multivariate Cox regression analysis, MS-HRM was significantly better than MS-PCR for predicting the treatment outcome (MS-HRM vs MS-PCR: PFS, p = 0.001 vs 0.207; OS, p = 0.013 vs 0.135).

CONCLUSIONS:

The authors’ findings show that MS-HRM is superior to MS-PCR for the detection of MGMT promoter methylation. They suggest MS-HRM as an alternative to MS-PCR for assessing the prognosis of patients with GBM.

• Authors: Shinji Yamashita, Kiyotaka Yokogami, Fumitaka Matsumoto, Kiyotaka Saito, Asako Mizuguchi, Hajime Ohta, Hideo Takeshima
• Journal: Journal of neurosurgery (J Neurosurg) Vol. 130 Issue 3 Pg. 780-788 (05 04 2018) ISSN: 1933-0693 [Electronic] United States
• PMID: 29726772 (Publication Type: Journal Article)

Chemical References

• Antineoplastic Agents, Alkylating
• DNA Primers
• Tumor Suppressor Proteins
• DNA Modification Methylases
• MGMT protein, human
• DNA Repair Enzymes
• Temozolomide

Topics

• Adult
• Aged
• Aged, 80 and over
• Alleles
• Antineoplastic Agents, Alkylating (therapeutic use)
• Brain Neoplasms (genetics, surgery, therapy)
• Cell Line, Tumor
• DNA Methylation (genetics)
• DNA Modification Methylases (genetics)
• DNA Primers
• DNA Repair Enzymes (genetics)
• Female
• Glioblastoma (genetics, surgery, therapy)
• Humans
• Male
• Middle Aged
• Mutation (genetics)
• Nucleic Acid Denaturation
• Polymerase Chain Reaction (methods)
• Predictive Value of Tests
• Progression-Free Survival
• ROC Curve
• Temozolomide (therapeutic use)
• Treatment Outcome
• Tumor Suppressor Proteins (genetics)