Background

Gliomas are the commonest group of primary brain tumours. They include astrocytic (including glioblastoma), oligodendroglial and mixed gliomas. IDH1/IDH2 mutations are found in more than 50% of astrocytic and oligodendroglial tumours of grade II and III and considered to be an early change. Presence of MGMT methylation has been shown to predict a good response to temozolomide and improved survival. A problem for clinical MGMT methylation testing is the lack of consensus on what area of the 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 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. The optimised pyrosequencing assay was then used to investigate 406 astrocytic and oligodendroglial tumours of all major types.

Results

We identified a 120 bp region containing 16 CpG sites to be most critical in transcriptional control. Methylation in this region was present in 58% of 406 gliomas. We found that MGMT methylation was 100% concordant with IDH1/IDH2 mutations in astrocytoma grade II and all oligodendrogliomas. All glioblastomas with IDH1/IDH2 mutations also had MGMT methylation, while the majority of glioblastomas with MGMT methylation did not have IDH1/IDH2 mutations.

Conclusion

We propose a robust pyrosequencing assay to accurately assess MGMT methylation. Our data suggests that MGMT methylation is the earliest change in the development of astrocytomas and oligodendrogliomas, preceding IDH1/IDH2 mutations.