Glioblastoma Recurrence and the Role of O6-Methylguanine-DNA Methyltransferase Promoter Methylation.
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ABSTRACT:
SUBMITTER: Storey K
PROVIDER: S-EPMC6732255 | biostudies-other | 2019 Feb
REPOSITORIES: biostudies-other
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Project description:BackgroundPromoter methylation of the DNA repair gene, O-6-methylguanine-DNA methyltransferase (MGMT), is associated with improved treatment outcome for newly diagnosed glioblastoma (GBM) treated with standard chemoradiation. To determine the prognostic significance of MGMT protein expression as assessed by immunohistochemistry (IHC) and its relationship with methylation, we analyzed MGMT expression and promoter methylation with survival in a retrospective patient cohort.MethodsWe identified 418 patients with newly diagnosed GBM at University of California Los Angeles Kaiser Permanente Los Angeles, nearly all of whom received chemoradiation, and determined MGMT expression by IHC, and MGMT promoter methylation by methylation-specific PCR (MSP) and bisulfite sequencing (BiSEQ) of 24 neighboring CpG sites.ResultsWith use of the median percentage of cells staining by IHC as the threshold, patients with <30% staining had progression-free survival (PFS) of 10.9 months and overall survival (OS) of 20.5 months, compared with PFS of 7.8 months (P < .0001) and OS of 16.7 months (P < .0001) among patients with ≥30% staining. Inter- and intrareader correlation of IHC staining was high. Promoter methylation status by MSP was correlated with IHC staining. However, low IHC staining was frequently observed in the absence of promoter methylation. Increased methylation density determined by BiSEQ correlated with both decreased IHC staining and increased survival, providing a practical semiquantitative alternative to MSP. On the basis of multivariate analysis validated by bootstrap analysis, patients with tandem promoter methylation and low expression demonstrated improved OS and PFS, compared with the other combinations.ConclusionsOptimal assessment of MGMT status as a prognostic biomarker for patients with newly diagnosed GBM treated with chemoradiation requires determination of both promoter methylation and IHC protein expression.
| S-EPMC3578486 | biostudies-literature
Project description:BACKGROUND:Treatment of glioblastoma (GB), the most common malignant primary brain tumor in adults, can include alkylating chemo-therapeutic agents. Two molecular biomarkers of treatment response are MGMT (O6-methylguanine-DNA methyltransferase) promoter methylation and IDH (isocitrate dehydrogenase) mutations, which prevent repair of tumor cell DNA damage caused by alkylating chemotherapy. The status of MGMT promoter methylation and IDH mutation are associated with longer survival and a better response to chemotherapy. OBJECTIVE:Assess the prognostic value of MGMT methylation status and IDH mutation in adult Saudi glioblastoma patients. DESIGN:Retrospective, comparative survival analysis. SETTING:Tertiary care center. PATIENTS AND METHODS:The status of the MGMT promoter methylation and IDH mutation was assessed in adult patients diagnosed with GB between 2006 and 2019. A PCR-based assay was used to analyze for methylation of the MGMT promoter. A qualitative assay combining PCR clamping and amplification refractory mutation system technology was used to search for any of the 12 most common mutations in IDH1 and IDH2. Differences in survival were compared between those with and without MGMT promoter methylation and IDH mutation and between other subgroups. MAIN OUTCOME MEASURES:Survival of GB patients relative to MGMT promoter methylation and IDH mutation status. SAMPLE SIZE:146 patients (80 males and 66 females). RESULTS:Of 43 (29.5%) cases tested for MGMT promoter methylation, 14 (32.5%) were positive. Of 65 (44.5%) cases screened for IDH mutation, 6 cases (9.2%) tested positive. The 36-month survival rate was 47% for the MGMT methylated cohort compared to 27% for their unmethylated counterparts. The 18-month survival rate for the IDH-mutant was 75% compared to 48% for their IDH-wildtype counterparts. CONCLUSION:The findings confirm the positive impact of both MGMT promoter methylation and IDH mutation on the overall survival of Saudi GB patients. LIMITATIONS:Single institute study with relatively few tested cases. CONFLICT OF INTEREST:None.
| S-EPMC6894451 | biostudies-literature
Project description:Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.
| S-EPMC2438202 | biostudies-literature
Project description:BackgroundGlioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine-DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide.ObjectivesTo determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who are treated with temozolomide.Search methodsWe searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies, we additionally searched NHS Economic Evaluation Database (EED) up to December 2014.Selection criteriaEligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiotherapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies and cost analysis.Data collection and analysisTwo review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios (RHR), using an imputed value of the correlation between results based on the same individuals.Main resultsWe included 32 independent cohorts involving 3474 people that compared two or more methods. We found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds (RHR 1.31, 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR 1.36, 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR 1.14, 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarised 190 articles describing a single method for measuring MGMT promoter methylation status.Authors' conclusionsPSQ and MSP appear more prognostic for overall survival than IHC. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.
| S-EPMC8078495 | biostudies-literature
Project description:BackgroundEpigenetic silencing of the MGMT gene by promoter methylation is associated with loss of MGMT expression, diminished DNA-repair activity and longer overall survival in patients with glioblastoma who, in addition to radiotherapy, received alkylating chemotherapy with carmustine or temozolomide. We describe and validate a rapid methylation sensitive quantitative PCR assay (MS-qLNAPCR) using Locked Nucleic Acid (LNA) modified primers and an imprinted gene as a reference.MethodsAn analysis was made of a database of 159 GBM patients followed between April 2004 and October 2008. After bisulfite treatment, methylated and unmethylated CpGs were recognized by LNA primers and molecular beacon probes. The SNURF promoter of an imprinted gene mapped on 15q12, was used as a reference. This approach was used because imprinted genes have a balanced copy number of methylated and unmethylated alleles, and this feature allows an easy and a precise normalization.ResultsConcordance between already described nested MS-PCR and MS-qLNAPCR was found in 158 of 159 samples (99.4%). The MS-qLNAPCR assay showed a PCR efficiency of 102% and a sensitivity of 0.01% for LNA modified primers, while unmodified primers revealed lower efficiency (69%) and lower sensitivity (0.1%). MGMT promoter was found to be methylated using MS-qLNAPCR in 70 patients (44.02%), and completely unmethylated in 89 samples (55.97%). Median overall survival was of 24 months, being 20 months and 36 months, in patients with MGMT unmethylated and methylated, respectively. Considering MGMT methylation data provided by MS-qLNAPCR as a binary variable, overall survival was different between patients with GBM samples harboring MGMT promoter unmethylated and other patients with any percentage of MGMT methylation (p = 0.003). This difference was retained using other cut off values for MGMT methylation rate (i.e. 10% and 20% of methylated allele), while the difference was lost when 50% of MGMT methylated allele was used as cut-off.ConclusionsWe report and clinically validate an accurate, robust, and cost effective MS-qLNAPCR protocol for the detection and quantification of methylated MGMT alleles in GBM samples. Using MS-qLNAPCR we demonstrate that even low levels of MGMT promoter methylation have to be taken into account to predict response to temozolomide-chemotherapy.
| S-EPMC2843669 | biostudies-literature
Project description:O6-methylguanine-DNA methyltransferase (MGMT) is a ubiquitous protein responsible for repair of O6-alkylguanine, a mutagenic, carcinogenic and toxic lesion. To characterize the elements responsible for the regulation of the MGMT gene, a 2.6 kb Sstl fragment isolated from a genomic clone, was shown to contain 5' flanking sequences of the gene. The promoter activity of this fragment as well as various subfragments were tested in NIH 3T3 mouse fibroblasts by transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to these fragments. Maximal promoter activity was observed in a 1.2 kb 3' terminal fragment, which contains the first untranslated exon. The transcription initiation site was identified in this fragment by primer extension and S1 mapping. Sequence analysis of this fragment showed the absence of TATA and CAAT boxes but an abundance of extremely GC-rich sequences, including ten GC hexanucleotide motifs 5'CCGCCC. Reduced CAT expression with the minimal promoter sequence suggests the presence of multiple regulatory elements.
| S-EPMC329112 | biostudies-other
Project description:ObjectivesTo non-invasively predict the coexistence of isocitrate dehydrogenase (IDH) mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in adult-type diffuse gliomas using apparent diffusion coefficient (ADC) histogram and direct ADC measurements and compare the diagnostic performances of the two methods.Materials and methodsA total of 118 patients with adult-type diffuse glioma who underwent preoperative brain magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) were included in this retrospective study. The patient group included 40 patients with coexisting IDH mutation and MGMT promoter methylation (IDHmut/MGMTmet) and 78 patients with other molecular status, including 32 patients with IDH wildtype and MGMT promoter methylation (IDHwt/MGMTmet), one patient with IDH mutation and unmethylated MGMT promoter (IDHmut/MGMTunmet), and 45 patients with IDH wildtype and unmethylated MGMT promoter (IDHwt/MGMTunmet). ADC histogram parameters of gliomas were extracted by delineating the region of interest (ROI) in solid components of tumors. The minimum and mean ADC of direct ADC measurements were calculated by placing three rounded or elliptic ROIs in solid components of gliomas. Receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) were used to evaluate the diagnostic performances of the two methods.ResultsThe 10th percentile, median, mean, root mean squared, 90th percentile, skewness, kurtosis, and minimum of ADC histogram analysis and minimum and mean ADC of direct measurements were significantly different between IDHmut/MGMTmet and the other glioma group (P < 0.001 to P = 0.003). In terms of single factors, 10th percentile of ADC histogram analysis had the best diagnostic efficiency (AUC = 0.860), followed by mean ADC obtained by direct measurements (AUC = 0.844). The logistic regression model combining ADC histogram parameters and direct measurements had the best diagnostic efficiency (AUC = 0.938), followed by the logistic regression model combining the ADC histogram parameters with statistically significant difference (AUC = 0.916) and the logistic regression model combining minimum ADC and mean ADC (AUC = 0.851).ConclusionBoth ADC histogram analysis and direct measurements have potential value in predicting the coexistence of IDHmut and MGMTmet in adult-type diffuse glioma. The diagnostic performance of ADC histogram analysis was better than that of direct ADC measurements. The combination of the two methods showed the best diagnostic performance.
| S-EPMC9875074 | biostudies-literature
Project description:This study aimed to investigate the influence of miR-221-3p and O6-methylguanine-DNA methyltransferase (MGMT) interaction in human hepatocellular carcinoma (HCC), thereby revealing a novel molecular mechanism of hepatic carcinogenesis involving miR-221-3p and MGMT.Fluorescence qPCR and immunoblot assays were performed to determine the expression of RNA and protein in HCC tissues and cell lines. We also employed the firefly and Renilla luciferase assay to verify the target relationship between miR-221-3p and MGMT mRNA. Assessments including the MTT assay, wound-healing assay, transwell assay, colony foci formation experiment, and flow cytometric experiment were carried out to determine the viability, migration, invasion, proliferation, cell cycle progression, and apoptosis of SMMC-7721 and BEL-7404 cell lines with the modulated expression of miR-221-3p and MGMT. Compared to healthy tissues and cell line HL7702, miR-221-3p was significantly upregulated but MGMT was significantly downregulated in carcinomas and cancerous cell lines. Forced miR-221-3p overexpression was found to enhance the proliferation, migration, invasion, and clonogenicity of cell lines, but it suppressed cell apoptosis. Findings also revealed that forced miR-221-3p overexpression had little effect on cell cycle progression. After MGMT was confirmed to be atarget gene of miR-221-3p, it was found that the forced upregulation of miR-221-3p downregulated MGMT mRNA and protein levels significantly. MiR-221-3p was identified as an HCC promoting factor, and it specifically inhibited the expression of the MGMT. Besides, the upregulation of miR-221-3p had apositive influence on HCC pathogenesis by inhibiting MGMT expression.
| S-EPMC7644250 | biostudies-literature
Project description: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.
| S-EPMC3397507 | biostudies-literature
Project description:Trained immunity is the heightened state of innate immune memory that enhances immune response resulting in nonspecific protection. Epigenetic changes and metabolic reprogramming are critical steps that regulate trained immunity. In this study, we reported the involvement of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme of lesion induced by alkylating agents, in regulation the trained immunity induced by β-glucan (BG). Pharmacological inhibition or silencing of MGMT expression altered LPS stimulated pro-inflammatory cytokine productions in BG-trained bone marrow derived macrophages (BMMs). Targeted deletion of Mgmt in BMMs resulted in reduction of the trained responses both in vitro and in vivo models. The transcriptomic analysis revealed that the dampening trained immunity in MGMT KO BMMs is partially mediated by ATM/FXR/AMPK axis affecting the MAPK/mTOR/HIF1α pathways and the reduction in glycolysis function. Taken together, a failure to resolve a DNA damage may have consequences for innate immune memory.
2024-01-23 | GSE231419 | GEO