Quantitative Analysis of MGMT Promoter Methylation in Glioblastoma Multiforme
Bin Yang, Rosemarie Read, Raymond Tubbs. Cleveland Clinic, Cleveland, OH
Background: O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that specifically removes alkyl groups from the O6 position of guanine in DNA. Repair of O6-alkylguanine adducts by tumour cells has been implicated in drug resistance since it reduces the cytotoxicity of alkylating chemotherapeutic agents. Recent clinical trial reveals that glioblastoma patients with MGMT methylation had much better response to temozolomide and median survival nearly doubled than those without MGMT methylation. In order to provide this molecular biomarker in guidance of chemosensitivity, we have developed and validated MGMT methylation assay using pyrosequencing on small biopsy specimens of glioblastoma multiforme.
Design: Promoter methylation of MGMT was quantitatively analyzed in 43 cases of glioblastoma multiforme and 10 cases of non-neoplastic epilepsy brain tissue. MGMT methylation profile is analyzed quantitatively using Pyro Q96 on paraffin-embedded biopsy tissues. The analytical sensitivity and minimal requirement of DNA volume and biopsy size is evaluated. Two cell lines, one harboring of MGMT methylation and another free of MGMT methylation were included as positive and negative controls. The sensitivity between pyrosequencing and methylation-specific PCR was also compared.
Results: Using a cutoff at 10%, methylation of MGMT was identified in 33% (11/33) cases of glioblastoma and 0% of the non-neoplastic epilepsy brain tissue. The range of percentage of methylation of any CpG island in MGMT promoter is 33-95% with a mean of 65%. By a series dilution of a methylated cancer cell line with an unmethylated normal cell line, pyrosequencing can detect 5% of tumor cells harboring MGMT methylation. The minimal amount of genomic DNA required to be able to successfully detect MGMT methylation by pyrosequencing is at 100 ng (approximately 3,000 cells). In comparison with MSP, pyrosequencing is comparably sensitive with less false-positive cases and also provide quantitative methylation value of each CpG island.
Conclusions: We have studied and validated the quantitative MGMT methylation assay on small biopsy tissue from patients with glioblastoma. We demonstrate that pyrosequencing detection of MGMT methylation has an analytical sensitivity suitable for clinical utility. MGMT methylation assay can provide a useful molecular biomarker for prediction of chemosensitivity in patients with glioblastoma multiforme.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 250, Tuesday Afternoon