Abstract 2794: Mutation of IDH1/2 is strongly associated with MGMT methylation in adult astrocytic and oligodendroglial tumors
Authors: Mulholland Shani A. , Malley Deborah S. , Hamoudi Rifat , Kocialkowski Sylvia , Pearson Danita , Collins V.Peter , Ichimura Koichi 
Published: June 2012

Background: Astrocytic, oligodendroglial and oligoastrocytic tumors are major subtypes of human gliomas, which are the most common malignant primary brain tumor. IDH1/IDH2 mutations are found in more than 50% of WHO grade II and III tumors in each of these three subtypes and is considered to be an early change. Presence of MGMT methylation has been shown to predict a good response to temozolomide and improved survival. Various protocols for MGMT methylation testing are utilized in the clinic, however there is a lack of consensus on how MGMT CpG island (CGI) should be analysed. The aim of this study was to 1) define the optimal target region for MGMT methylation-testing by bisulfite modification and pyrosequencing 2) profile methylation at each CpG in MGMT CGI in a large cohort of gliomas and 3) correlate MGMT methylation with other genetic abnormalities including IDH1/IDH2 mutations.

Method: The methylation status of each CpG in the MGMT CGI was determined and compared with MGMT mRNA expression in 22 glioblastoma xenografts, 13 glioma cell lines and 6 normal brain tissues. A luciferase assay was used to investigate selected CpGs for their role in transcription. An optimized pyrosequencing assay was then used to investigate 406 astrocytic and oligodendroglial tumors of all major types. A normal mixture model was applied to the data points and the optimal cut-off value to judge MGMT methylation in clinical samples was determined.

Results: We identified a 120 bp region containing 16 CpG sites to be most critical in transcriptional control. We determined an optimal cut-off of value of 22% to judge MGMT methylation in clinical samples. Using this criterion, methylation in this region was found in 58% of 406 gliomas. Pilocytic astrocytomas had a low incidence of MGMT methylation (6.8%). All the other tumor types had methylation incidence of 50-87%. When compared with the pattern of genetic alterations, we found that all tumors with IDH1/IDH2 mutations had MGMT methylation. In astrocytoma grade II and all oligodendrogliomas, MGMT methylation and IDH mutations showed 100% concordance. Conversely, the majority of glioblastomas with MGMT methylation did not have IDH1/IDH2 mutations, suggesting MGMT methylation can occur independently of IDH mutation in glioblastoma.

Conclusion: We propose a robust pyrosequencing assay to accurately assess MGMT methylation: 16 CpGs sites using a cut-off value of 22%. We showed that all IDH mutations were associated with MGMT methylation, while some tumors with MGMT methylation did not harbor IDH mutations. We therefore propose that MGMT methylation may be the earliest change in the development of astrocytomas and oligodendrogliomas, preceding IDH1/IDH2 mutations. This research highlights the importance of integrating epigenetic and genetic alteration data to study the pathogenesis of a disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2794. doi:10.1158/1538-7445.AM2011-2794