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Background: Epigenetic inactivation of O6-methylguanine-methyltransferase (MGMT) gene by methylation of its promoter is predictive of Temozolomid (TMZ) response in glioblastoma (GBM). MGMT is located on chromosome 10q26 and the loss of chromosome 10q is observed in 70% of GBMs. In this study, we assessed the hypothesis that the dual inactivation of MGMT, by hypermethylation of MGMT promoter and by loss the long arm of chromosome 10 (10q), may confer greater sensitivity to TMZ., Methods: A total of 149 tumor samples from patients diagnosed with GBM based on the WHO 2016 classification were included in this retrospective study between November 2016 and December 2018. Methylation status of MGMT promoter was evaluated by pyrosequencing and status of chromosome 10q was assessed by array comparative genomic hybridization., Results: Glioblastoma patients with chromosome 10q loss associated with hypermethylation of MGMT promoter had significantly longer overall survival (OS) (P = .0024) and progression-free survival (PFS) (P = .031). Indeed, median OS of patients with dual inactivation of MGMT was 21.5 months compared to 12 months and 8.1 months for groups with single MGMT inactivation by hypermethylation and by 10q loss, respectively. The group with no MGMT inactivation had 9.5 months OS. Moreover, all long-term survivors with persistent response to TMZ treatment (OS ≥ 30 months) displayed dual inactivation of MGMT., Conclusions: Our data suggest that the molecular subgroup characterized by the dual inactivation of MGMT receives greater benefit from TMZ treatment. The results of our study may be of immediate clinical interest since chromosome 10q status and methylation of MGMT promoter are commonly determined in routine practice., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Pyrosequencing is recognized as a strong technique to analyze the MGMT status of glioblastoma patients. The most commonly used assay, quantifies the methylation levels of CpGs 74 to 78. A more recent CE-marked In Vitro Diagnostic Medical Device (CE-IVD) assay, Therascreen, analyzes CpGs 76-79., Methods: We performed a comparison of these two assays to evaluate the potential impact of this shift in analyzed CpGs. Therascreen analysis was centrally performed for 102 glioblastoma patients, who were part of a prospective multicenter trial., Results: A strong correlation was observed for the mean values of the 4 or 5 analyzed CpGs, with lower values recorded using the Therascreen assay, especially for values greater than 20%. When considering a classification in 3 categories (> 12%: methylated; ⩽ 8%: unmethylated; 9-12%: grey zone), 93% of patients were identically classified between the two assays. Using a binary classification, 95% and 97% of patients were identically classified with cut-offs of 8% and 12%, respectively. A strong prognostic significance was observed for both assays: median overall survival were 15.9 months and 34.9 months for respectively unmethylated and methylated patients with either test., Conclusion: The results demonstrate that these assays may be used interchangeably.

Background: The goal of this prospective multicentric trial was to validate a technique that allowed for MGMT promoter methylation analysis in routine clinical practice., Methods: The MGMT status of 139 glioblastoma patients, whom had received standard first line treatment, was determined using pyrosequencing (PSQ) and a semi-quantitative Methylation-specific PCR (sqMS-PCR) method, using both frozen and formalin-fixed paraffin-embedded FFPE samples. Eight participating centers locally performed the analysis, including external quality controls., Results: There was a strong correlation between results from FFPE and frozen samples. With cut-offs of 12% and 13%, 98% and 91% of samples were identically classified with PSQ and sqMS-PCR respectively. In 12% of cases frozen samples were excluded because they had a low percentage of tumor cells. In 5-6% of cases the analysis was not feasible on FFPE samples. The optimized risk cut-offs were higher in both techniques when using FFPE samples, in comparison to frozen samples. For sqMS-PCR, we validated a cut-off between 13-15% to dichotomize patients. For PSQ, patients with a low level of methylation (<= 8%) had a median progression-free survival under 9 months, as compared with more than 15.5 months for those with a level above 12%. For intermediate values (9-12%), more discordant results between FFPE and frozen samples were observed and there was not a clear benefit of temozolomide treatment, which indicated a "grey zone"., Conclusions: MGMT status can reliably be investigated in local laboratories. PSQ is the ideal choice as proven by strong interlaboratory reproducibility, along with threshold agreements across independent studies., Competing Interests: There is no conflicts of interest for the authors and acknowledged contributors.

Malignant gliomas are the most common primary brain tumors. Grade III and IV gliomas harboring wild-type IDH1/2 are the most aggressive. In addition to surgery and radiotherapy, concomitant and adjuvant chemotherapy with temozolomide (TMZ) significantly improves overall survival (OS). The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) promoter is predictive of TMZ response and a prognostic marker of cancer outcome. However, the promoter regions the methylation of which correlates best with survival in aggressive glioma and whether the promoter methylation status predictive value could be refined or improved by other MGMT-associated molecular markers are not precisely known. In a cohort of 87 malignant gliomas treated with radiotherapy and TMZ-based chemotherapy, we retrospectively determined the MGMT promoter methylation status, genotyped single nucleotide polymorphisms (SNPs) in the promoter region and quantified MGMT mRNA expression level. Each of these variables was correlated with each other and with the patients' OS. We found that methylation of the CpG sites within MGMT exon 1 best correlated with OS and MGMT expression levels, and confirmed MGMT methylation as a stronger independent prognostic factor compared to MGMT transcription levels. Our main finding is that the presence of only the A allele at the rs34180180 SNP in the tumor was significantly associated with shorter OS, independently of the MGMT methylation status. In conclusion, in the clinic, rs34180180 SNP genotyping could improve the prognostic value of the MGMT promoter methylation assay in patients with aggressive glioma treated with TMZ., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Consistently reported prognostic factors for glioblastoma (GBM) are age, extent of surgery, performance status, IDH1 mutational status, and MGMT promoter methylation status. We aimed to integrate biological and clinical prognostic factors into a nomogram intended to predict the survival time of an individual GBM patient treated with a standard regimen. In a previous study we showed that the methylation status of the DGKI promoter identified patients with MGMT-methylated tumors that responded poorly to the standard regimen. We further evaluated the potential prognostic value of DGKI methylation status., Methods: 399 patients with newly diagnosed GBM and treated with a standard regimen were retrospectively included in this study. Survival modelling was performed on two patient populations: intention-to-treat population of all included patients (population 1) and MGMT-methylated patients (population 2). Cox proportional hazard models were fitted to identify the main prognostic factors. A nomogram was developed for population 1. The prognostic value of DGKI promoter methylation status was evaluated on population 1 and population 2., Results: The nomogram-based stratification of the cohort identified two risk groups (high/low) with significantly different median survival. We validated the prognostic value of DGKI methylation status for MGMT-methylated patients. We also demonstrated that the DGKI methylation status identified 22% of poorly responding patients in the low-risk group defined by the nomogram., Conclusions: Our results improve the conventional MGMT stratification of GBM patients receiving standard treatment. These results could help the interpretation of published or ongoing clinical trial outcomes and refine patient recruitment in the future.

Glioblastoma (GBM) is the most malignant type of primary brain tumor with a very poor prognosis. The actual standard protocol of treatment for GBM patients consists of radiotherapy and concomitant temozolomide (TMZ). However, the therapeutic efficacy of this treatment is limited due to tumor recurrence and TMZ resistance. Recently isolated, glioma stem-like cells (GSCs) are thought to represent the population of tumorigenic cells responsible for GBM resistance and recurrence following surgery and chemotherapy. In addition, MGMT (O6-methylguanine-methyltransferase) methylation is considered as one of the principal mechanisms contributing to TMZ sensitivity of GBM. In this study we have isolated GSCs from 10 adult GBM patients and investigated the relationship between MGMT methylation status and Temozolomide (TMZ) sensitivity of these lines grown either in stem-like or differentiation promoting conditions. Sensitivity to TMZ was significantly associated with MGMT methylation status in cells committed to differentiation but not in stem-like cells. In addition, patients harboring highly methylated MGMT promoters had a longer overall survival. These results reveal the importance of the differentiation process when considering the predictive value of MGMT status in GSCs for clinical response to TMZ.

This multicenter study assesses the value of O(6)-methylguanine-DNA methyltransferase (MGMT) status for predicting overall survival in glioblastoma patients. Five methods are used, to identify the approach with the best prognostic value. Eighty-one tumors were obtained from patients with glioblastomas treated by surgery and radiotherapy with concomitant temozolomide (TMZ) followed by adjuvant TMZ. MGMT promoter methylation was assessed by qualitative methyl-specific polymerase chain reaction (MSP), semiquantitative methyl-specific polymerase chain reaction (SQ-MSP), and pyrosequencing, while MGMT expression was measured at the RNA level by quantitative real-time PCR (Q-RT-PCR) and at the protein level by immunohistochemistry (IHC). MGMT promoter methylation as evaluated by MSP, SQ-MSP, and pyrosequencing was significantly correlated with overall survival. The best predictive value was obtained by pyrosequencing of one specific CpG position. Overall survival was 14 and 25 months for patients with percentages of methylation below and above the median, respectively. In contrast, MGMT status determined by Q-RT-PCR and IHC showed little or no correlation with overall survival, respectively. These results confirm the prognostic value of MGMT promoter methylation in glioblastoma patients initially treated with TMZ. SQ-MSP allowed better discrimination than classical MSP, and pyrosequencing represented a good option.