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. 2015 Jan 1;8(1):636-42.
eCollection 2015.

Detection of MGMT promoter methylation in glioblastoma using pyrosequencing

Hao Xie  1 , Raymond Tubbs  1 , Bin Yang  1
Affiliations

Detection of MGMT promoter methylation in glioblastoma using pyrosequencing

Hao Xie et al. Int J Clin Exp Pathol. .

Abstract

Recent clinical trials on patients with glioblastoma revealed that O(6)-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient's response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.

Keywords: MGMT; epigenetics; glioblastoma; methylation; pyrosequencing.

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Figures

Figure 1
Figure 1
Pyrograms for MGMT methylation pyrosequencing test from the brain tissues of an epilepsy patient (Panel A) and a glioma patient (Panel B).
Figure 2
Figure 2
Mean MGMT methylation of tissue specimens from epilepsy patients (E1-E10) and glioma patients (G1-G43). The dash line represents a 10% cutoff.
Figure 3
Figure 3
The relationship between biopsy size and mean DNA yields.
See this image and copyright information in PMC

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