Cellular phone use and brain tumor: a meta-analysis

Methylation of the promoter region of the O ⁶ -methylguanine-DNA methyltransferase (MGMT) gene is known to be predictive of response to temozolomide treatment in patients with glioblastoma. Contrastingly, little is known about variation in the methylation status of the MGMT promoter after treatment or across different regions of the same tumor. About 22 samples from 10 patients who had undergone multiple resections of a glioblastoma were examined with promoter sequencing. Of these, 20 were also analyzed using Methylation Specific PCR (MSP). The methylation status of the MGMT promoter was altered in the specimens obtained pre and post treatment in 2 of 9 samples as assessed by MSP and 7 out of 10 patients as assessed by promoter sequencing. In four patients, the MGMT promoter was unmethylated at primary surgery, but displayed some methylation (32, 44, 12, and 4%) on post-treatment sampling. Alteration in MSP status from unmethylated to methylated was also observed in 2 of these 4 patients. In another patient, methylation increased from 40% on initial sampling to 68% on the second sample. The remaining two patients initially demonstrated some degree of methylation (72% and 12%); subsequent sampling showed no methylation of the MGMT promoter. To ensure variable methylation status was not due to intra-tumoral variability, three to four specimens were sampled from different regions of large glioblastomas (n = 7). Promoter sequencing revealed minimal variation in methylation in all but two sites examined. Immunohistochemistry also demonstrated minimal change in MGMT expression across the tumors. This suggests that variation in MGMT promoter methylation can occur within the same tumor after treatment, necessitating caution in clinical decision-making based on this analysis.     

MGMT promoter methylation testing to predict overall survival in people with glioblastoma treated with temozolomide: a comprehensive meta-analysis based on a Cochrane Systematic Review

BackgroundThe DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) causes resistance of tumor cells to alkylating agents. It is a predictive biomarker in high-grade gliomas treated with temozolomide, however, there is no consensus on which test method, methylation sites, and cutoff values to use.MethodsWe performed a Cochrane Review to examine studies using different techniques to measure MGMT and predict survival in glioblastoma patients treated with temozolomide. Eligible longitudinal studies included (i) adults with glioblastoma treated with temozolomide with or without radiotherapy, or surgery; (ii) where MGMT status was determined in tumor tissue, and assessed by 1 or more technique; and (iii) where overall survival was an outcome parameter, with sufficient information to estimate hazard ratios (HRs). Two or more methods were compared in 32 independent cohorts with 3474 patients.ResultsMethylation-specific PCR (MSP) and pyrosequencing (PSQ) techniques were more prognostic than immunohistochemistry for MGMT protein, and PSQ is a slightly better predictor than MSP.ConclusionsWe cannot draw strong conclusions about use of frozen tissue vs formalin-fixed paraffin-embedded in MSP and PSQ. Also, our meta-analysis does not provide strong evidence about the best CpG sites or threshold. MSP has been studied mainly for CpG sites 76-80 and 84-87 and PSQ at CpG sites ranging from 72 to 95. A cutoff threshold of 9% for CpG sites 74-78 performed better than higher thresholds of 28% or 29% in 2 of the 3 good-quality studies. About 190 studies were identified presenting HRs from survival analysis in patients in which MGMT methylation was measured by 1 technique only.     

Prognostic value of O6-methylguanine-DNA methyltransferase status in glioblastoma patients, assessed by five different methods

This multicenter study assesses the value of O⁶-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.     

MGMT promoter methylation level in newly diagnosed low-grade glioma is a predictor of hypermutation at recurrence

BackgroundEmerging data suggest that a subset of patients with diffuse isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG) who receive adjuvant temozolomide (TMZ) recur with hypermutation in association with malignant progression to higher-grade tumors. It is currently unclear why some TMZ-treated LGG patients recur with hypermutation while others do not. MGMT encodes O6-methylguanine-DNA methyltransferase, a DNA repair protein that removes cytotoxic and potentially mutagenic lesions induced by TMZ. Here, we hypothesize that epigenetic silencing of MGMT by promoter methylation facilitates TMZ-induced mutagenesis in LGG patients and contributes to development of hypermutation at recurrence.MethodsWe utilize a quantitative deep sequencing assay to characterize MGMT promoter methylation in 109 surgical tissue specimens from initial tumors and post-treatment recurrences of 37 TMZ-treated LGG patients. We utilize methylation arrays to validate our sequencing assay, RNA sequencing to assess the relationship between methylation and gene expression, and exome sequencing to determine hypermutation status.ResultsMethylation level at the MGMT promoter is significantly higher in initial tumors of patients that develop hypermutation at recurrence relative to initial tumors of patients that do not (45.7% vs 34.8%, P = 0.027). Methylation level in initial tumors can predict hypermutation at recurrence in univariate models and multivariate models that incorporate patient age and molecular subtype.ConclusionsThese findings reveal a mechanistic basis for observed differences in patient susceptibility to TMZ-driven hypermutation. Furthermore, they establish MGMT promoter methylation level as a potential biomarker to inform clinical management of LGG patients, including monitoring and treatment decisions, by predicting risk of hypermutation at recurrence.     

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

BackgroundO⁶-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide.Patients and methodsWe evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing.ResultsThresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008).ConclusionsMethyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.     

<Emphasis Type="Italic">MGMT</Emphasis> promoter methylation in malignant gliomas

The O⁶-methylguanine-DNA methyltransferase (MGMT) gene is located at chromosome 10q26 and codes for a DNA repair enzyme that—if active—can counteract the effects of alkylating chemotherapy. Malignant gliomas often have the MGMT gene inactivated due to aberrant methylation of its promoter region. The assessment of the MGMT promoter methylation status has become of clinical relevance as a molecular marker associated with response to alkylating chemotherapy and prolonged survival of glioblastoma patients. MGMT promoter methylation testing is also on the merge of being used as a marker for patient selection within clinical trials, e.g., the current CENTRIC trial that is specifically focusing on patients with MGMT promoter-methylated glioblastomas. In anaplastic gliomas, MGMT promoter methylation is a favorable prognostic marker independent of the type of therapy, i.e., radio- or chemotherapy. This occurrence might be associated with the high incidence of other prognostically favorable molecular markers in these tumors, such as IDH1 mutation, 1p/19q deletion or yet to be identified novel aberrations. A variety of different methods are being used to assess MGMT promoter methylation in clinical samples, which may give rise to inter-laboratory variations in test results. Immunohistochemical determination of MGMT protein expression has not proven reliable for diagnostic purposes. This brief review article aims to summarize the main aspects of MGMT promoter methylation testing in contemporary neuro-oncology, in particular its value as a clinically useful molecular marker, putting it into the context of other molecular markers of clinical use in gliomas of adult patients.     

Assessment of MGMT promoter methylation status in pleomorphic xanthoastrocytoma

Although pleomorphic xanthoastrocytoma (PXA) is currently designed as a grade II glioma according to the WHO classification, a significant percentage of the tumors undergo malignant progression. In this study, the MGMT methylation status was examined in 11 PXA patients to determine if a biologic rationale exists to support the use of temozolomide (TMZ) for treatment of aggressive PXA. There were 9 cases of PXA grade II and 2 cases of PXA with anaplastic features. In the MGMT methylation study, only 2 (18.1%) of the 11 tumor samples tested by MS-qLNAPCR were positive for MGMT promoter methylation. In contrast, other cases, including PXAs with anaplastic features, were unmethylated. In addition, a tumor recurrence was found to be unmethylated. Thus, MGMT promoter methylation is not frequent in PXA and our results raise doubts about the benefits of treating indistinctly aggressive PXA with TMZ.     

MGMT gene promoter methylation correlates with tolerance of temozolomide treatment in melanoma but not with clinical outcome

Background:

Despite limited clinical efficacy, treatment with dacarbazine or temozolomide (TMZ) remains the standard therapy for metastatic melanoma. In glioblastoma, promoter methylation of the counteracting DNA repair enzyme O⁶-methylguanine-DNA-methyltransferase (MGMT) correlates with survival of patients exposed to TMZ in combination with radiotherapy. For melanoma, data are limited and controversial.

Methods:

Biopsy samples from 122 patients with metastatic melanoma being treated with TMZ in two multicenter studies of the Dermatologic Cooperative Oncology Group were investigated for MGMT promoter methylation. We used the COBRA (combined bisulphite restriction analysis) technique to determine aberrant methylation of CpG islands in small amounts of genomic DNA isolated from paraffin-embedded tissue sections. To detect aberrant methylation, bisulphite-treated DNA was amplified by PCR, enzyme restricted, and visualised by gel electrophoresis.

Results:

Correlation with clinical data from 117 evaluable patients in a best-response evaluation indicated no statistically significant association between MGMT promoter methylation status and response. A methylated MGMT promoter was observed in 34.8% of responders and 23.4% of non-responders (P=0.29). In addition, no survival advantage for patients with a methylated MGMT promoter was detectable (P=0.79). Interestingly, we found a significant correlation between MGMT methylation and tolerance of therapy. Patients with a methylated MGMT promoter had more severe adverse events, requiring more TMZ dose reductions or discontinuations (P=0.007; OR 2.7 (95% CI: 1.32–5.7)). Analysis of MGMT promoter methylation comparing primaries and different metastases over the clinical course revealed no statistical difference (P=0.49).

Conclusions:

In advanced melanoma MGMT promoter, methylation correlates with tolerance of therapy, but not with clinical outcome.     

<Emphasis Type="Italic">MGMT</Emphasis> promoter methylation in non-neoplastic brain

O⁶–methylguanine–DNA–methyltransferase (MGMT) is mainly regulated by cytosine–guanine island promoter methylation that is believed to occur only in neoplastic tissue. The present study was undertaken to investigate whether methylation occurs also in non-neoplastic brains by collecting 45 non-neoplastic brains from autopsies and 56 lobectomy specimens from epileptic surgeries. The promoter methylation status of MGMT was studied by methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), while protein expression was studied by immunohistochemical stain (IHC). The methylation rates, as determined by MSP and PSQ, were 3.0 % (3/101) and 2.9 % (2/69), respectively. Of note, no case had positive result concomitantly from both MSP and PSQ (3 were MSP+/PSQ? and 2 were MSP?/PSQ+), and all the positive samples were further confirmed by cloning and Sanger sequencing. All the methylated cases, except for those having indeterminate IHC results from autopsy specimens, revealed no loss of MGMT protein expression and similar staining pattern to that of the unmethylated cases. In conclusion, the current study demonstrated that MGMT promoter methylation could occur in a low percentage of non-neoplastic brains but did not affect the status of protein expression, which could be regarded as a normal variation in non-neoplastic brains.     

Extent of MGMT promoter methylation correlates with outcome in glioblastomas given temozolomide and radiotherapy

Background:

Epigenetic silencing of O⁶-methylguanine-DNA-methyltransferase (MGMT) by promoter methylation is associated with improved survival in glioblastomas treated with alkylating agents. In this study, we investigated MGMT promoter methylation in glioblastomas treated with temozolomide and radiotherapy in a single UK treatment centre.

Methods:

Quantitative methylation data at individual CpG sites were obtained by pyrosequencing for 109 glioblastomas.

Results:

Median overall survival (OS) was 12.4 months with 2-year survival of 17.9%. Pyrosequencing data were reproducible with archival samples yielding data for all glioblastomas. Variation in methylation patterns of discrete CpG sites and intratumoral methylation heterogeneity were observed. A total of 58 out of 109 glioblastomas showed average methylation >non-neoplastic brain in at least one clinical sample; 86% had homogeneous methylation status in multiple samples. Methylation was an independent prognostic factor associated with prolonged progression-free survival (PFS) and OS. Cases with methylation more than 35% had the longest survival (median PFS 19.2; OS 26.2 months, 2-year survival of 59.7%). Significant differences in PFS were seen between those with intermediate or high methylation and unmethylated cases, whereas cases with low, intermediate or high methylation all showed significantly different OS.

Conclusions:

These data indicate that MGMT methylation is prognostically significant in glioblastomas given chemoradiotherapy in the routine clinic; furthermore, the extent of methylation may be used to provide additional prognostic stratification.     

Methylation of MGMT in malignant pleural mesothelioma occurs in a subset of patients and is associated with the T allele of the rs16906252 MGMT promoter SNP

Silencing of the DNA repair gene O⁶-methylguanine DNA methyltransferase (MGMT) by promoter methylation is an early event in several human cancers. MGMT removes alkyl adducts from the O⁶ position of guanine thereby preventing G > A mutations in the genome. For this reason, MGMT promoter methylation predicts a favorable outcome for glioblastoma patients treated with alkylating agents. In this study, we investigated whether MGMT becomes silenced by promoter methylation in malignant pleural mesothelioma (MPM), an aggressive cancer of the pleura associated with a poor prognosis. Ninety-five samples from patients diagnosed with MPM were studied. These samples were genotyped for the MGMT rs16906252 promoter SNP using high-resolution melting, and methylation status was analyzed using SMART-MSP and confirmed by Sanger sequencing. The SMART-MSP assay was designed to provide information on the allelic methylation status in samples heterozygous for rs16906252. MGMT immunohistochemistry was performed on samples showing no methylation, monoallelic methylation, and biallelic methylation. Thirteen of the 95 MPM samples (13.7%) were methylation positive and a strong association with the T allele of the rs16906252 SNP (P < 0.001) was observed. Detection of the protein was found to be dependent not only on the allelic methylation status but also on the methylation level, and complete silencing was observed in only one sample, showing biallelic methylation and a methylation level close to 100%. In conclusion, methylation of the MGMT promoter occurs in a subset of MPM patients and is associated with the T allele of the MGMT rs16906252 SNP. However, complete silencing of MGMT in MPM is a rare event.     

<Emphasis Type="Italic">MGMT</Emphasis> promoter hypermethylation is a frequent,early, and consistent event in astrocytoma progression,and not correlated with <Emphasis Type="Italic">TP53</Emphasis> mutation

Hypermethylation of the MGMT gene promoter and mutation of the TP53 tumor-suppressor gene are frequently present in diffuse astrocytomas. However, there is only anecdotal information about MGMT methylation status and TP53 mutations during progression of low-grade diffuse astrocytoma (AII) to anaplastic astrocytoma (AIII) and secondary glioblastoma (sGB). In this study biopsy specimens from 51 patients with astrocytic tumors with radiologically proved progression from low to high-grade malignancy were investigated for the presence and consistency of MGMT promoter hypermethylation and TP53 mutations. For 27 patients biopsy samples both of primary tumors and their recurrences were available. For the other 24 patients histology of either the low-grade lesion or the high-grade recurrence was available. It was found that MGMT promoter hypermethylation and TP53 mutations are both frequent and early events in the progression of astrocytomas and that their status is consistent over time. No correlation was found between MGMT methylation status and the presence of TP53 mutations. In addition, no correlation was found between MGMT promoter hypermethylation and the type of TP53 mutations. These results argue against the putative TP53 G:C>A:T transition mutations suggested to occur preferentially in MGMT hypermethylated tumors.     

Correlation of Clinical Features and Methylation Status of MGMT Gene Promoter in Glioblastomas

In an effort to extend the potential relationship between the methylation status of MGMT promoter and response to CENU therapy, we examined the methylation status of MGMT promoter in 44 patients with glioblastomas. Tumor specimens were obtained during surgery before adjuvant treatment, frozen and stored at -80 degrees C until for DNA extraction process. DNA methylation patterns in the CpG island of the MGMT gene were determined in every tumor by methylation specific PCR (MSP). These results were then related to overall survival and response to alkylating agents using statistical analysis. Methylation of the MGMT promoter was detected in 68% of tumors, and 96.7% of methylated tumors exhibited also an unmethylated status. There was no relationship between the methylation status of the MGMT promoter and overall survival and response to alkylating agents. Our observations do not lead us to consider promoter methylation of MGMT gene as a prognostic factor of responsiveness to alkylating agents in glioblastomas.     

The MGMT promoter SNP rs16906252 is a risk factor for MGMT methylation in glioblastoma and is predictive of response to temozolomide

BackgroundPromoter methylation of O⁶-methylguanine-DNA methyltransferase (MGMT) is an important predictive biomarker in glioblastoma. The T variant of the MGMT promoter-enhancer single nucleotide polymorphism (SNP; rs16906252) has been associated with the presence of MGMT promoter methylation in other cancers. We examined the association of the T allele of rs16906252 with glioblastoma development, tumor MGMT methylation, MGMT protein expression, and survival outcomes.MethodsTwo independent temozolomide-treated glioblastoma cohorts—one Australian (Australian Genomics and Clinical Outcomes of Glioma, n = 163) and the other American (University of California Los Angeles/Kaiser Permanente Los Angeles, n = 159)—were studied. Allelic bisulphite sequencing was used to determine if methylation was specific to the T allele. Additionally, we compared the incidence of the T allele between glioblastoma cases and matched controls to assess whether it was a risk factor for developing MGMT methylated glioblastoma.ResultsCarriage of the T allele of the rs16906252 SNP was associated with both MGMT methylation and low MGMT protein expression and predicted significantly longer survival in temozolomide-treated patients with both MGMT methylated and nonmethylated glioblastoma. Methylation was linked to the T allele, inferring that the T variant plays a key role in the acquisition of MGMT methylation. Carriage of the T allele was associated with a significantly elevated risk of developing glioblastoma (adjusted odds ratio, 1.96; P = .013), increasing further when glioblastoma was classified by the presence of MGMT methylation (adjusted odds ratio, 2.86; P = .001).ConclusionsThe T allele of the rs16906252 SNP is a key determinant in the acquisition of MGMT methylation in glioblastoma. Temozolomide-treated patients with the rs16906252 T genotype have better survival, irrespective of tumor methylation status.     

MGMT promoter methylation status in clival chordoma

Chordomas are rare, slow-growing neoplasms, characterized by locally aggressive growth patterns and high local recurrence rates. To the best of our knowledge, the MGMT promoter methylation status has not been studied in a population of patients with chordomas to determine if a biologic rationale exists to support the use of temozolomide. We here show for the first time that methylation of MGMT promoter is present in a significant portion or recurring clival chordomas; on the contrary in clival chordomas without recurrence MGMT promoter was always unmethylated (p = 0.0317). Although these observations need to be confirmed in a larger study population, our results (1) indicate that methylation of MGMT promoter is present in a significant portion of recurring chordomas, and (2) prompt further investigation into the potential role of temozolomide as an adjuvant treatment of these tumors.     

Combined analysis of O6-methylguanine-DNA methyltransferase protein expression and promoter methylation provides optimized prognostication of glioblastoma outcome

Background

Promoter 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.

Methods

We 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.

Results

With 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.

Conclusions

Optimal 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.