MGMT CpG island is invariably methylated in adult astrocytic and oligodendroglial tumors with IDH1 or IDH2 mutations

Abstract

We have previously identified a region containing 16 CpGs within the MGMT CpG islands which is critical for the transcriptional control of MGMT (Malley, Acta Neuropathol 2011). To investigate the patterns and incidence of MGMT methylation in astrocytic and oligodendroglial tumors, we quantitatively assessed methylation at these 16 CpGs using bisulfite modification followed by pyrosequencing of 362 gliomas not treated with temozolomide, and correlated the findings with previously identified patterns of genetic abnormalities, patients' age and survival. The MGMT gene was considered to be methylated when the mean methylation of the 16 CpGs was 10% or higher. This cut-off value distinguished diffuse astrocytomas with high and low MGMT expression. Within each tumor type, the patterns of methylation were highly variable and also highly heterogeneous across the 16 CpGs. A high incidence of MGMT methylation was observed in all subtypes of gliomas included in this study. Among a subset of 97 tumors where conventional methylation-specific PCR (MSP) was also applied, methylation was detected by both methods in 54 tumors, while the pyrosequencing results identified a further 17 tumors. No additional cases were found using MSP alone, indicating that pyrosequencing is a robust method for methylation analysis. All tumors with IDH1/IDH2 mutations except two had MGMT methylation, while there were many tumors with MGMT methylation, particularly primary glioblastomas, which had no mutations of IDH1/2. We suggest that MGMT methylation may be one of the earliest events in the development of astrocytic and oligodendroglial tumors.Gliomas are the most common group of primary brain tumors consisting of clinically and biologically distinct subtypes. The three main subgroups of glioma are astrocytic, oligodendroglial and mixed gliomas, which are further malignancy graded into up to four grades according to the WHO classification. 1 Glioblastomas (WHO grade IV) are the most malignant and common astrocytic brain tumor in adults, the overall median survival being only just over 14 months despite modern combined therapies. 2 Most glioblastomas are primary glioblastoma (pGB), while secondary glioblastoma (sGB) generally develops by progression from astrocytomas WHO grade II or III. Patients with astrocytic or oligodendroglial tumors WHO grade II and III survive longer, however they always recur and are ultimately fatal.Among the many genetic and epigenetic abnormalities commonly found in adult gliomas, three have been reported to predict response to treatment and/or prognosis: IDH1/ IDH2 mutations, concurrent deletion of the entire 1p and 19q chromosomal arms (total 1p/19q loss) and MGMT methylation. 3,4 With the exception of pGB, mutations of either IDH1 or IDH2 occur in more than 50% of adult astrocytic, oligodendroglial and oligoastrocytic tumors. [5][6][7][8] It has been shown that the presence of IDH1/2 mutations is associated with longer patient survival. 5-7,9,10 IDH1/2 mutations have been proposed as the earliest genetic changes in a ...

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients

“…In contrast to the results obtained for the whole series, in these G-CIMP-tumors CpG 25 However, due to these G-CIMP-tumors included various grades and subtypes of glioma, the analysis of the prognostic value of CpG 25, STP27 model and CpG 165 methylation particularly in GBMs with a G-CIMP-profile (n = 125) revealed no association with OS for methylation of either CpG or the MGMT-STP27 model (P [ 0.05). This lack of prognostic value of CpG 25 methylation determined by HM-450K could be confirmed in 166 primary GBMs with wildtype IDH (G-CIMP-) reported by Mulholland et al [27] Fig. 3a).…”

“…In addition, a recent report by van den Bent and collaborators reveals one of these CpGs (cg12981137, CpG 25) as the strongest differentially methylated CpG associated with survival in the RT-PCV arm of the EORTC-26951 study [23]. It is worth noting that the CpG 25 is included in the reverse primer of the MSP and qMSP assays, and is close to the CpG region evaluated by pyrosequencing [11,[25][26][27][28], the techniques most widely used to detect methylation of the MGMT promoter.…”

Upregulating mutations in the TERT promoter commonly occur in adult malignant gliomas and are strongly associated with total 1p19q loss

“…In addition, 12 established glioma cell lines (U87MG, U118MG, U178MG, U251MG, U373MG, TP265MG, TP365MG, TP483MG, TP336MG, T98G, H4 and A172) were also included for analysis. The mutational status of IDH, TP53, CDKN2A, EGFR, PTEN as well as MGMT CpG island methylation (18) and 1p19q copy number in the Validation cohort and the glioma cell lines have previously been published [14,25,26,32]. The study was approved by the respective local institutional review boards.…”

“…The methylation status of MGMT was analyzed by bisulfite modification of tumor genomic DNA followed by pyrosequencing essentially as described with some modification [26]. Methylation was determined when the average levels of methylation in the 16 CpG sites examined were above 10 %.…”

“…To minimize confounding factors such as IDH1 mutations, which is known to be a favorable prognostic factor in pGBM [14], cases who met the following criteria were subjected to the analysis: (1) age: 18-70 years old, (2) absence of clinically apparent preceding low grade gliomas to exclude sGBMs, (3) no IDH1/2 mutations, (4) with follow-up period of 3 months or more, and (5) KPS value of 70 or more. As all the Validation cohort patients were initially treated prior to the temozolomide era [26], none of these cohort cases were included in the survival analysis.…”