Altered Expression of MGMT in High-Grade Gliomas Results from the Combined Effect of Epigenetic and Genetic Aberrations

Abstract

MGMT downregulation in high-grade gliomas (HGG) has been mostly attributed to aberrant promoter methylation and is associated with increased sensitivity to alkylating agent-based chemotherapy. However, HGG harboring 10q deletions also benefit from treatment with alkylating agents. Because the MGMT gene is mapped at 10q26, we hypothesized that both epigenetic and genetic alterations might affect its expression and predict response to chemotherapy. To test this hypothesis, promoter methylation and mRNA levels of MGMT were determined by quantitative methylation-specific PCR (qMSP) or methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) and quantitative RT-PCR, respectively, in a retrospective series of 61 HGG. MGMT/chromosome 10 copy number variations were determined by FISH or MS-MLPA analysis. Molecular findings were correlated with clinical parameters to assess their predictive value. Overall, MGMT methylation ratios assessed by qMSP and MS-MLPA were inversely correlated with mRNA expression levels (best coefficient value obtained with MS-MLPA). By FISH analysis in 68.3% of the cases there was loss of 10q26.1 and in 15% of the cases polysomy was demonstrated; the latter displayed the highest levels of transcript. When genetic and epigenetic data were combined, cases with MGMT promoter methylation and MGMT loss depicted the lowest transcript levels, although an impact in response to alkylating agent chemotherapy was not apparent. Cooperation between epigenetic (promoter methylation) and genetic (monosomy, locus deletion) changes affecting MGMT in HGG is required for effective MGMT silencing. Hence, evaluation of copy number alterations might add relevant prognostic and predictive information concerning response to alkylating agent-based chemotherapy.

A novel DNA methylation panel accurately detects colorectal cancer independently of molecular pathway

“…DNA was extracted from FFPE sections that contained at least 70% neoplastic cells, using phenol-chloroform conventional method as described previously Ramalho-Carvalho et al [25]. DNA was quantified using NanoDrop ND-1000 ® (NanoDrop Technologies, DE, USA) spectrophotometer and modified with sodium bisulfite, using the EZ DNA Methylation-Gold ™ Kit (Zymo Research, Orange, CA, USA) according to manufacturer's instructions.…”

Prognosis of glioblastoma with faint MGMT methylation-specific PCR product

“…The above changes could cause incomplete downregulation of MGMT protein expression. In addition to methylation of CpG islands in the promoter region, which is the most significant mechanism of MGMT regulation that suppresses MGMT transcription [26,27], other mechanisms, such as MGMT genetic changes [28,29], histone modifications [30,31], p53 derangement [32,33] and post-transcriptional regulation [34,35] could also lead to MGMT silencing. Absence of MGMT protein expression (IHC-) in 15.8 % of our unmethylated (MSP-) GBM cases (Table 3) would be the result of other MGMT silencing mechanisms.…”

Impact ofO6-methylguanine-DNA methyltransferase expression on the drug resistance of clear cell renal cell carcinoma

“…Recent studies have shown that the expression of MGMT could be regulated by several signal pathways. MGMT expression is highly regulated via promoter methylation (29,32). Overall 97 CpG islands have been identified in the MGMT promoter.…”