Abstract
BackgroundPromoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter benefit from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methylation is of importance for diagnostic decisions. We experienced that different methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specific PCR assays and pyrosequencing.ResultsTo better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfite sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specific pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350–1354, 2000. https://doi.org/10.1056/NEJM200011093431901) and Felsberg et al. (Clin Cancer Res 15(21):6683–6693, 2009. https://doi.org/10.1158/1078-0432.CCR-08-2801) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfite sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status.ConclusionOur integrated analysis allows to evaluate and redefine co-methylation domains within the MGMT promoter and to rationalize the practical impact on assays used in daily routine diagnostics.
Highlights
Promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma
Following the cutoff values for the therascreen MGMT Pyro Kit as suggested by Reifenberger and colleagues, we regard an average methylation percentage larger than 8% as methylated for diagnosis [23]. For both “methylated” Methylation-specific PCR (MSP), any visible PCR product will signify a methylation of the MGMT promoter
Comparing MSP results using primer pairs published by Esteller et al (2000) and primer pairs published by Felsberg et al (2009), we observed concordance, i.e., presence or absence of PCR products in both MSPs, for 58 tumors, while discordant results, i.e., absence of PCR product in one of the MSPs, were obtained for 22 tumors (Additional file 1: Table S1) [18, 19]
Summary
Promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Methylation-induced downregulation or silencing of MGMT inhibits its DNA repair mechanism of alkyl group removal, thereby making tumor cells sensitive to cytotoxic alkylating agents like temozolomide which improves patients’ overall survival [5, 6] Another genetic alteration in astrocytic tumors that confers a significant survival benefit is a mutation of isocitrate dehydrogenase (IDH). Point mutations in the IDH1 or IDH2 gene are correlated with a better overall survival and while they occur frequently in lower-grade astrocytomas (WHO grade II and III) and secondary glioblastomas, they are rare (< 10%) in primary glioblastomas [7] This genetic influence on prognosis needs to be considered when therapies in astrocytic tumors are compared or a predictive parameter like the MGMT promoter methylation is determined
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