Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas

Abstract

-Heine-Universit€ at D€ usseldorf, D€ usseldorf, GermanyHypermethylation of the DNA repair gene O 6 -methyl-guanine DNA methyltransferase (MGMT) has been linked to prolonged survival in glioblastoma patients treated with alkylating agents. It was aimed to analyze prospectively whether the MGMT status of malignant gliomas could be determined from small-sized stereotactic biopsies (maximum volume: 1 mm 3 ). Special attention was directed towards the intratumoral distribution of the MGMT promoter methylation, the MGMT protein expression and potential correlations between both. Twenty-five adult patients were included (20 patients with primary World Health Organisation (WHO) Grade III or IV malignant gliomas, 5 patients with secondary malignant gliomas). About 2-4 biopsy specimens per tumor were collected from different sites within the tumor. Promoter methylation of the MGMT gene was assessed by methylation-specific PCR (MSP) and sodium bisulfite sequencing in each of the collected specimens (overall number of specimens: 69). Both methods were validated for application in small-sized tissue samples (1 mm 3 ). The MGMT protein expression was analyzed by immunohistochemistry. The overall MGMT promoter methylation rate was 30% in the de novo group and 80% in the tumor progression group. The success rates of MSP and sequencing were 100% and 80%, respectively. Sequence analysis and MSP exhibited 100% concordant findings. No differences in MGMT promoter methylation were detected between the different samples of each individual tumor in 24 of 25 patients. One false negative result was obtained due to the contamination of the biopsy specimen by necrotic tissue. Tissue samples taken from different sites of each individual tumor (13 tumors investigated) exhibited equal or highly similar MGMT protein expression. No correlation between MGMT protein expression and MGMT promoter methylation was observed. The MGMT promoter methylation status of malignant gliomas can be reliably determined from small-sized stereotactic biopsies. The methylation profile, as defined by MSP and sodium bisulfite sequencing, constitutes a homogeneous marker throughout malignant gliomas. The lack of correlation between MGMT status and MGMT protein expression needs further evaluation. ' 2007 Wiley-Liss, Inc.Key words: MGMT; promoter methylation; stereotactic biopsy; MGMT protein; immunohistochemistry; malignant glioma; glioblastoma; chemotherapy MGMT is a ubiquitous DNA-repair enzyme in normal human tissues. It removes and accepts alkyl groups from the O 6 position of methylguanine without affecting DNA integrity. MGMT exerts its reparative function in a stochiometric fashion, without requiring any additional cofactor and by finally inactivating itself. Overexpression of the MGMT gene in brain tumors confers resistance to alkylating chemotherapeutic agents such as temozolomide by removing chemotherapy-induced cytotoxic DNA lesions. 1 Epigenetic silencing of the MGMT gene by promoter methylation has been shown to be an independent predictor of response to...

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

“…Unlike the MSP assay used in the majority of clinical studies, pyrosequencing allows highly reproducible quantitative evaluation of methylation at discrete CpG sites thereby providing more information on promoter methylation status and facilitating analysis of specific methylation patterns. Analysis is fast and cost effective and, unlike conventional bisulphite sequencing (Grasbon-Frodl et al, 2007;Mikeska et al, 2007;Parkinson et al, 2008), is practical in a diagnostic environment. In our assay, the PCR primers are methylation independent and as little as 5% methylated DNA in a mixture of methylated and unmethylated DNA may be detected.…”

MGMT promoter methylation in malignant gliomas: ready for personalized medicine?

“…18 Third, many authors have failed to identify a correlation between MGMT promoter methylation assessed by MSP and protein levels in glioma tissue assessed by immunochemistry. 18,[24][25][26] One explanation for this lack of correlation is the considerable and highly variable contamination of glioma tissue sections with non-neoplastic cells expressing MGMT that are not always easy to distinguish from tumor cells (Figure 3). …”

“…Stereotactic biopsies should be controlled for tumor content, and usually only yield sufficient DNA when obtained frozen. 25 Tissues should be fixed in buffered formalin. Overfixation decreases the quality of the DNA owing to formation of cross-links, and can impede successful testing.…”

“…MSP and sodium bisulfate sequencing showed identical results, and only one tumor showed inconsistent results between biopsies. 25 Little is known regarding therapy-induced changes in MGMT promoter methylation or MGMT expression levels in the tumor. Neither dexamethsone nor irradiation induced MGMT gene transcription in glioma cells in vitro.…”

A Phase II Study of Temozolomide in Patients with Advanced Aerodigestive Tract and Colorectal Cancers and Methylation of theO6-Methylguanine-DNA Methyltransferase Promoter

“…Studies evaluating MGMT promoter methylation within glioblastoma lesions as well as in paired primary and recurrent glioblastoma samples suggested that MGMT promoter methylation could be a homogeneous marker throughout malignant gliomas (48) and that most recurrent lesions retained the methylation profile from the primary lesions (49). However, considering the increasing awareness of tumor heterogeneity (50) and the positive predictive value (77%) for serum assay observed in our study, the serum methylation assay may provide a possible alternative for patients with no available tumor samples.…”