title: Detection of MGMT promoter methylation in glioblastoma using pyrosequencing.
abstract: Recent clinical trials on patients with glioblastoma revealed that O6-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.
authors.affiliation: Robert J. Tomsick Pathology & Laboratory Medicine Institute, Cleveland Clinic Cleveland, OH, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Xie
authors.affiliation: Robert J. Tomsick Pathology & Laboratory Medicine Institute, Cleveland Clinic Cleveland, OH, USA.
authors.forename: Raymond
authors.initials: R
authors.lastname: Tubbs
authors.affiliation: Robert J. Tomsick Pathology & Laboratory Medicine Institute, Cleveland Clinic Cleveland, OH, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
mesh_terms: D044963:Biopsy, Fine-Needle; D001932:Brain Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D005909:Glioblastoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D006801:Humans; D016612:Paraffin Embedding; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes
keywords: MGMT; epigenetics; glioblastoma; methylation; pyrosequencing