MGMT and Whole-Genome DNA Methylation Impacts on Diagnosis, Prognosis and Therapy of Glioblastoma Multiforme

Abstract

Epigenetic changes in DNA methylation contribute to the development of many diseases, including cancer. In glioblastoma multiforme, the most prevalent primary brain cancer and an incurable tumor with a median survival time of 15 months, a single epigenetic modification, the methylation of the O⁶-Methylguanine-DNA Methyltransferase (MGMT) gene, is a valid biomarker for predicting response to therapy with alkylating agents and also, independently, prognosis. More recently, the progress from single gene to whole-genome analysis of DNA methylation has allowed a better subclassification of glioblastomas. Here, we review the clinically relevant information that can be obtained by studying MGMT gene and whole-genome DNA methylation changes in glioblastomas, also highlighting benefits, including those of liquid biopsy, and pitfalls of the different detection methods. Finally, we discuss how changes in DNA methylation, especially in glioblastomas bearing mutations in the Isocitrate Dehydrogenase (IDH) 1 and 2 genes, can be exploited as targets for tailoring therapy.

Keywords: IDH1/2 mutations; MGMT gene methylation; glioblastoma multiforme; liquid biopsy; whole-genome methylation profiling.

Figure 1

Antagonistic action between temozolomide (TMZ) and O⁶-Methylguanine-DNA Methyltransferase (MGMT) enzymatic activity. (A) TMZ drug (blue circle) modifies guanine (G) in O⁶-methylguanine (O⁶-MeG), creating mismatches in DNA sequence and, in turn, DNA breaks. Unmethylated MGMT promoter is associated with MGMT gene expression and MGMT protein synthesis (green rectangle). MGMT removes methyl-group on guanosine, re-establishing correct sequence. This event is associated with TMZ resistance, tumor growth and poor prognosis. (B) When MGMT promoter is methylated, the absence of MGMT transcription and synthesis results in TMZ tumor sensitivity, with accumulation of DNA mismatches and, in turn, cell death.

Figure 2

Graphic representation of MGMT gene regulatory region and output of the technologies used to analyze MGMT methylation after sodium bisulfite conversion. (A) MGMT gene promoter (blue) and exon 1 (red) are represented. Focus on bisulfite-converted sequence analyzed by different techniques: in yellow rectangles, primers used to perform pyrosequencing; in blue rectangles, primers used in methylation-specific PCR (MSP); in green rectangles, primers used in real-time PCR. (B) Graphical representation of MSP output: a glioblastoma sample (S1), an internal methylated (CT) and a non-methylated (CT-) controls, and a control without DNA (B) are depicted, each including a lane signed with «+» (methylated DNA specific primers) and a lane signed with «−» (un-methylated DNA specific primers). (C) Graph indicating MGMT methylation status predicted by methylome analysis. (D) Capillary electrophoresis indicating output of pyrosequencing analysis of MGMT promoter methylation in 2 glioblastoma samples.

Figure 3

Methylome workflow and data analysis: schematic representation of the analytic steps to perform the EPIC array. DNA is converted with sodium bisulfite, pre-amplificated and fragmented. DNA is, then, hybridized on a specific array with specific probes recognizing the single CG both if methylated or if not methylated. The technology analyzes 850,000 CpG sites tracing a sort of tumor barcode. Output raw data are analyzed using different bioinformatic pipelines to extrapolate information useful for the characterization of the tumor, such as the methylation status of the MGMT gene, the copy number variations (CNVs), the co-deletion 1p-19q, the presence of functional mutations in the Isocitrate Dehydrogenase (IDH) 1 and 2 genes. Using a specific comparison algorithm, the analyzed methylomic profile is compared with others present in the database for a more precise classification of the tumor.

Figure 4

Nanopore sequencing strategy. As the first step, the genomic DNA is fragmented and then subjected to end repair and dA tailing. To allow DNA passage through flow cell pores, DNA is ligated to a protein adapter. Each flow cell may contain from 512 to 10,700 channels that may be potentially suitable for sequencing from 10–20 Gigabases to up to 100 Gigabases. Each channel is linked to a specific electrode, and a constant voltage is applied across the membrane. When double-stranded DNA molecules cross the pores, the voltage changes according to the specific single nucleotide passing the membrane. The difference in electric potential between the two sides of the membrane is converted into the specific nucleotide that may be identified in real-time during nanopore runs.