2 research outputs found
Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics
Background: Promoter 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 beneft from treatment with alkylating agents, such as temozolomide, and
show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methyla‑
tion is of importance for diagnostic decisions. We experienced that diferent 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-specifc PCR assays and pyrosequencing.
Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep
bisulfte 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-specifc 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 bisulfte 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.
Conclusion: Our integrated analysis allows to evaluate and redefne co-methylation domains within the MGMT pro‑
moter and to rationalize the practical impact on assays used in daily routine diagnostics
Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics
Background: Promoter 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. Results: To 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. Conclusion: Our 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.Funding: Open Access funding enabled and organized by Projekt DEAL. This work was supported by the German Epigenome Programme (DEEP) of the Federal Ministry of Education and Research in Germany (BMBF) (01KU1216F). MS is supported by the BMBF project de.NBI-epi (031L0101D) and the EU H2020 project SYSCID (733100