Methylguanine Methyltransferase Methylation Research Articles

Cognitive functioning in a cohort of high-grade glioma patients.

High grade gliomas are associated with cognitive problems. The aim of the study was to investigate cognitive functioning in a cohort of patients with high grade glioma, according to isocitrate dehydrogenase (IDH) and methyl guanine methyl transferase (MGMT) status and other clinical characteristics. The patients with the high-grade glioma treated in Slovenia in given period of time were included in study. Postoperatively they completed neuropsychological assessment consisting of Slovenian Verbal Learning Test, Slovenian Controlled Oral Word Association Test, Trail Making Test Part A and B and self-evaluation questionnaire. We analysed results (z-scores and dichotomized results) also according to IDH mutation and MGMT methylation. We examined differences between groups using T-test, Mann-Whitney U, χ2 and Kendall's Tau tests. Out of 275 patients in the cohort, we included 90. Forty-six percent of patients were unable to participate due to poor performance status and other conditions related to tumour. Patients with the IDH mutation were younger, with better performance status, larger proportions of grade III tumours and MGMT methylation. In this group cognitive functioning is significantly better in the domains of immediate recall, short delayed recall and delayed recall, and in the fields of executive functioning and recognition. There were no differences in cognitive functioning in regard to MGMT status. Grade III tumours were associated with more frequent MGMT methylation. Self-assessment proved week tool, associated only with immediate recall. We found no differences in cognitive functioning according to MGMT status, but cognition was better when IDH mutation was present. In a cohort study of patients with high-grade glioma, almost half were unable to participate in a study, which points to an overrepresentation of patients with better cognitive functioning in the research.

EPID-24. TIMING OF STANDARD-OF-CARE CHEMO-RADIATION THERAPY FOLLOWING SURGICAL RESECTION OR STEREOTACTIC BIOPSY

Abstract OBJECT The literature examining the optimal timing for initiating post-surgical therapy after glioblastoma surgery is brimming with inconsistencies, with reports of both deleterious effects and benefits in the delay of post-surgical therapy at various time intervals. Moreover, there is currently limited data pertinent to the post-Stupp regimen era. Here, we use the National Cancer Database (NCDB) to explore this matter, with a focus on patients who underwent the standard-of-care (SOC) Stupp regimen. METHODS The study cohort included newly glioblastoma patients from the NCDB, with exclusion of patients age < 40 [as proxy for isocitrate dehydrogenase (IDH) mutated astrocytoma] and stratified for Methyl-guanine-methyl transferase (MGMT) promotor methylation. Timing of initiating SOC was grouped by 0-2, 2-4, 4-6, 6-8, and > 8 weeks from surgery. Biopsy and craniotomy patients were analyzed separately. The Kaplan-Meier method and Cox proportional hazards regression were applied for survival analysis. RESULTS We identified 6511 glioblastoma patients who underwent SOC therapy with available MGMT methylation status. SOC was initiated in 228 (3.5%), 2478 (38.1%), 2954 (45.4%), 646 (9.9%), and 205 (3.2%) patients at 0-2, 2-4, 4-6, 6-8, and >8 weeks after surgery. For craniotomy patients, initiation of SOC at 0-2 weeks was associated with an increased risk of death relative to SOC initiation at 4-6 weeks (HR: 1.19, p = 0.036). These results remain robust after controlling for the extent of resection, tumor size, and tumor location (HR: 1.19, p = 0.044). Such association was not observed in patients who underwent stereotactic biopsy (HR: 1.27, p = 0.148). Patients who received SOC at 6-8 weeks post-craniotomy (HR: 0.96, p = 0.420) or biopsy (HR: 0.93, p = 0.628) did not appear to worsen survival outcomes. CONCLUSION Optimal timing for initiation of SOC differ for craniotomy and needle biopsy patients. Initiating SOC within 2 weeks after craniotomy could lead to worse survival outcomes for GBM patients.

64-Year-Old Woman With Aphasia and Troponin Elevation

64-Year-Old Woman With Aphasia and Troponin Elevation

Radiological model based on the standard magnetic resonance sequences for detecting methylguanine methyltransferase methylation in glioma using texture analysis.

This study aims to build a radiological model based on standard MR sequences for detecting methylguanine methyltransferase (MGMT) methylation in gliomas using texture analysis. A retrospective cross‐sectional study was undertaken in a cohort of 53 glioma patients who underwent standard preoperative magnetic resonance (MR) imaging. Conventional visual radiographic features and clinical factors were compared between MGMT promoter methylated and unmethylated groups. Texture analysis extracted the top five most powerful texture features of MR images in each sequence quantitatively for detecting the MGMT promoter methylation status. The radiomic signature (Radscore) was generated by a linear combination of the five features and estimates in each sequence. The combined model based on each Radscore was established using multivariate logistic regression analysis. A receiver operating characteristic (ROC) curve, nomogram, calibration, and decision curve analysis (DCA) were used to evaluate the performance of the model. No significant differences were observed in any of the visual radiographic features or clinical factors between different MGMT methylated statuses. The top five most powerful features were selected from a total of 396 texture features of T1, contrast‐enhanced T1, T2, and T2 FLAIR. Each sequence’s Radscore can distinguish MGMT methylated status. A combined model based on Radscores showed differentiation between methylated MGMT and unmethylated MGMT both in the glioblastoma (GBM) dataset as well as the dataset for all other gliomas. The area under the ROC curve values for the combined model was 0.818, with 90.5% sensitivity and 72.7% specificity, in the GBM dataset, and 0.833, with 70.2% sensitivity and 90.6% specificity, in the overall gliomas dataset. Nomogram, calibration, and DCA also validated the performance of the combined model. The combined model based on texture features could be considered as a noninvasive imaging marker for detecting MGMT methylation status in glioma.

Prognostic and predictive impact of MGMT promoter methylation in grade 3 gliomas

BackgroundGrade 3 gliomas are aggressive primary brain tumors. Promoter methylation of methyl guanine methyl transferase (MGMT) has been associated with a favorable prognosis in patients with glioblastoma, but the impact of MGMT promoter methylation in patients with grade 3 gliomas is less clear. The purpose of the present study was to evaluate the utilization of MGMT testing in patients with Grade 3 glioma, as well its prognostic and predictive value. MethodsThe National Cancer Database (NCDB) was queried (2004–2016) for patients with newly diagnosed grade 3 glioma without 1p19q codeletion. Statistics included Kaplan-Meier overall survival (OS) analysis, along with Cox proportional hazards modeling. ResultsOf 20,488 total patients, 1,209 (5.0%) had MGMT testing. Of these patients, 561 (46.4%) were MGMT methylated (mMGMT), and 648 (53.6%) were MGMT unmethylated (uMGMT). mMGMT patients experienced greater median overall survival (OS) than both uMGMT patients as well as patients with no MGMT status reported (p < 0.05 for both). mMGMT was associated with improved OS for patients receiving adjuvant chemoradiation or adjuvant radiation, but not for patients receiving adjuvant chemotherapy or no adjuvant treatment. ConclusionsThis is the largest study to date describing the utilization of and outcomes for mMGMT patients with grade 3 glioma. The present results demonstrate that mMGMT is a prognostic factor and possibly a predictive biomarker, and is currently under-utilized within the US. MGMT methylation status could be used to risk-stratify and select patients for treatment intensification. Importance of studyThe present study is the largest of its kind to examine the prognostic and predictive impact of MGMT methylation (mMGMT) amongst patients with Grade 3 Glioma. The results suggest that mMGMT is prognostic, as amongst all patients, mMGMT was associated with improved overall survival. These results also suggest that mMGMT is predictive, as patients treated with adjuvant chemoradiation or adjuvant radiation therapy did have improved overall survival with mMGMT, though there was no difference in overall survival observes amongst patients receiving adjuvant chemotherapy or those patients receiving no adjuvant treatment. The study also found that only 5% of patients nationwide with Grade 3 Glioma are tested for MGMT.

Management of Gliomas: Individualized Treatment Options

Optimizing treatment for patients with low-grade gliomas should focus on the role of radiation and chemotherapy, as well as the prognostic impact of molecular diagnostics (1p/19q and IDH status). For anaplastic oligodendroglioma, focus should be placed on molecular markers (particularly 1p/19q status) and combination treatment with chemotherapy (temozolomide or PCV [procarbazine, lomustine, and vincristine]) and radiation. For patients with malignant glioblastomas, the role of methylguanine methyl-transferase (MGMT) methylation status has become increasingly important to treatment decisions. MGMT methylation status should be considered in elderly patients and/or those with low performance status (methylated patients benefit from temozolomide) and a hypofractionated radiation schedule should be used.

Validation of a PCR-Based MGMT Promoter Methylation Assay and Comparison With Reference Lab Results

Abstract Introduction Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor. Treatment of GBM and other high-grade gliomas consists of surgical resection followed by radiation and temozolomide (TMZ) chemotherapy. TMZ alkylates purine DNA bases, causing DNA damage and eventually cell death. Alkylation at O6-gauanine can be reversed by the DNA repair enzyme methylguanine methyltransferase (MGMT), inhibiting the desired cytotoxic effect. MGMT is epigenetically silenced by methylation of a CpG island in its promoter. Many high-grade gliomas are hypomethylated at the MGMT promoter. MGMT promoter methylation has emerged as a biomarker for response to alkylating chemotherapy. Multiple clinical trials demonstrate that the patients most likely to benefit from TMZ therapy have a hypermethylated MGMT promoter. MGMT promoter status is now incorporated into adjuvant therapy guidelines. Our institution receives approximately 70 to 80 high-grade glioma resections per year. The goal of this study is to validate a PCR-based MGMT promoter methylation assay and to compare results to those previously obtained at a well-known reference lab. Methods We extracted DNA from high-grade gliomas specimens (FFPE) or used commercially available methylated and unmethylated DNA controls. DNA was treated with bisulfite to convert unmethylated cytosine into uracil. Promoter methylation was detected with real-time quantitative PCR using a primer/probe set specific for methylated CpG sites within the MGMT promoter (meMGMT, published in Ida et al 2017) or to a housekeeping gene (COL2A1). Results Using control methylated DNA, we demonstrated that our assay consistently detects meMGMT and COL2A1 at DNA inputs greater than 5 ng. Both within- and between-run imprecision was minimal with little variation in crossing thresholds (Cts) for both COL2A1 (%CV at 50 ng input: 7.9% [within-run], 0.3% [between-run]) and MGMT (%CV at 50 ng input: 0.6% [within-run], 0.4% [between-run]). Delta Ct values were consistent across different levels of DNA input. Analysis of 15 cases from our archives (8 positive, 7 negative) demonstrates that our results (delta Ct values) correlate (R2 = 0.9) with the reference lab methylation score (&lt;2 considered unmethylated). Separation between positive and negative cases was good with minimal overlap; positive cases had delta Cts of 0.2 to 7.6, while negative cases had delta Cts of 7.1 to 13.6 or had no meMGMT amplification. ROC curve analysis showed an AUC of 0.96, and using the cutoff of delta Ct &lt;7.6, our assay had 93% (14/15) agreement with the reference lab, a sensitivity of 100%, and a specificity of 71%. The single negative case that was misclassified had a reference lab methylation score of 1.86, near their cutoff of &lt;2. Additional patient samples are pending. Conclusions Our PCR-based MGMT methylation assay has excellent analytical sensitivity and low imprecision. Preliminary analysis of patient samples shows that our results are concordant with historic reference lab results.

¹⁸F-fluorodeoxyglucose and ¹¹C-methionine positron emission tomography in relation to methyl-guanine methyltransferase promoter methylation in high-grade gliomas.

Methylation status of the methyl-guanine methyltransferase (MGMT) promoter is associated with a favorable response to a DNA alkylating agent in high-grade gliomas. We analyzed PET scans of patients with high-grade gliomas to determine whether the MGMT methylation status affects the tumor metabolic characteristics. Twenty-three patients with high-grade glioma, who were initially examined with 11C-methionine (MET) and 18F-fluorodeoxyglucose (FDG) PET, were retrospectively enrolled. MET and FDG PET images were coregistered to each other and quantitative uptake of MET or FDG was assessed using tumor-to-normal uptake ratio of the cortex (TNR). TNRs for MET and FDG PET were compared between the two groups classified by MGMT promoter methylation status. Maximum TNR(FDG) of the MGMT methylated group was significantly higher than that of the MGMT unmethylated group (1.80±0.90 vs. 1.29±0.19; P=0.02). The MGMT methylated group also showed a trend for increased mean TNRFDG compared with the unmethylated group (0.85±0.21 vs. 0.72±0.11; P=0.10). There was no significant difference in TNR(MET) between the groups. In subgroup analyses with WHO grade 3 and 4, a trend for higher maximum TNR(FDG) was found in the MGMT methylated group compared with the unmethylated group. The MGMT methylated group showed higher glucose metabolism compared with the unmethylated group, whereas MET uptake did not show a significant difference. This suggests that MGMT methylation in high-grade gliomas could affect the tumor glucose metabolism. Thus, MGMT methylation status can cause a discrepancy in the prognostic prediction of high-grade gliomas by FDG PET, especially in patients scheduled for DNA alkylating chemotherapeutics.

High-grade glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

High-grade glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Prediction of methylguanine methyltransferase promoter methylation in glioblastoma using dynamic contrast-enhanced magnetic resonance and diffusion tensor imaging.

The methylation status of the methylguanine methyltransferase (MGMT) promoter has been associated with treatment response in glioblastoma. The authors aimed to assess whether MGMT methylation status can be predicted by dynamic contrast-enhanced (DCE) MRI and diffusion tensor imaging (DTI). This retrospective study included 43 patients with pathologically diagnosed glioblastoma who had undergone preoperative DCE-MRI and DTI and whose MGMT methylation status was available. The imaging features were qualitatively assessed using conventional MR images. Regions of interest analyses for DCE-MRI permeability parameters (transfer constant [Ktrans], rate transfer coefficient [Kep], and volume fraction of extravascular extracellular space [Ve]) and DTI parameters (apparent diffusion coefficient [ADC] and fractional anisotropy [FA]) were performed on the enhancing solid portion of the glioblastoma. Chi-square or Mann-Whitney tests were used to evaluate relationships between MGMT methylation and imaging parameters. The authors performed receiver operating characteristic curve analysis to find the optimal cutoff value for the presence of MGMT methylation. MGMT methylation was not significantly associated with any imaging features on conventional MR images. Ktrans values were significantly higher in the MGMT methylated group (median 0.091 vs 0.053 min(-1), p = 0.018). However, Kep, Ve, ADC, and FA were not significantly different between the 2 groups. The optimal cutoff value for the presence of MGMT methylation was Ktrans > 0.086 min(-1) with an area under the curve of 0.756, a sensitivity of 56.3%, and a specificity of 85.2%. Ktrans may serve as a potential imaging biomarker to predict MGMT methylation status preoperatively in glioblastoma; however, further investigation with a larger cohort is necessary.

Methylation of O6-Methyl Guanine Methyltransferase Gene Promoter in Meningiomas - Comparison between Tumor Grades I, II, and III

Meningiomas are the second most common primary intracranial tumors after gliomas. Epigenetic biomarkers such as DNA methylation, which is found in many tumors and is thus important in tumorigenesis can help diagnose meningiomas and predict response to adjuvant chemotherapy. We investigated aberrant O6- methyl guanine methyltransferase (MGMT) methylation in meningiomas. Sixty-one patients were classified according to the WHO grading, and MGMT promoter methylation status was examined via the methylation-Specific PCR(MSP) method. MGMT promoter methylation was found in 22.2% of grade I, 35% of grade I with atypical features, 36% of grade II, and 42.9% of grade III tumors. There was an increase, albeit not statistically significant, in MGMT methylation with a rise in the tumor grade. Higher methylation levels were also observed in the male gender.

Continuing the search for MR imaging biomarkers for MGMT promoter methylation status: conventional and perfusion MRI revisited

Continuing the search for MR imaging biomarkers for MGMT promoter methylation status: conventional and perfusion MRI revisited

Serum DNA can define tumor-specific genetic and epigenetic markers in gliomas of various grades

We evaluated whether cell-free circulating DNA can be used as a noninvasive approach for detection of genetic/epigenetic alterations in brain tumors during the course of the disease. Paired tumor-serum samples from 70 patients with either high-grade astrocytomas (n = 41) or oligodendrogliomas of various grades were analyzed. The median interval between surgery and serum sampling was 1 month (range 0.5-168 months). DNA was extracted from whole blood, serum, and paraffin-embedded tumor sections. Loss of heterozygosity (LOH) in chromosomes 1p, 19q, and 10q was assessed by polymerase chain reaction (PCR)-based microsatellite analysis. The methylation status of O(6)-methyl guanine methyltransferase (MGMT) and phosphatase and tensin homolog promoters was studied by methylation-specific PCR. LOH and/or methylation that could identify DNA as tumor-specific was found in 80.5% of astrocytic tumors and in all oligodendrogliomas. The rate of serum detection of these biomarkers was 51% and 55%, respectively, with specificity around 100%. The rate of serum detection did not differ between low- and high-grade oligodendrogliomas. Statistically significant tumor-serum concordance was found for MGMT methylation in both astrocytic tumors (83%; P < .001) and oligodendroglial tumors (72%; P < .003) and for LOH of 10q (79%; P < .002) and 1p (62%; P < .03) in oligodendrogliomas. We conclude that serum DNA in glial tumors is informative for both LOH and aberrant gene promoter methylation analysis during the course of the disease. The sensitivity is moderate and specificity is high for both low- and high-grade tumors. Future studies should identify a panel of biomarkers that bear the highest potential for clinical application.

Real-time polymerase chain reaction technique determines absolute copy number of plasma methylated MGMT gene promoter copies in newly diagnosed malignant glioma patients

22073 Background: New methods for disease surveillance, quantification, and prognosis in malignant glioma are needed. Recent work has demonstrated that malignant gliomas release their DNA into the blood which bears the genetic and epigenetic aberrations in the intracranial lesion. We determined the actual plasma copy number of the methylated chemoresistance gene methylguanine methyltransferase (MGMT) in patients enrolled in clinical trials for malignant glioma as a prelude to biomarker development. Methods: This prospective NABTT CNS Consortium study was designed to determine if tumor specific plasma DNA could serve as an accurate biomarker. Plasma was collected from newly diagnosed high grade glioma patients prior to therapy and with each follow-up MRI. DNA was isolated from plasma of the first 19 patients enrolled and bisulfite treated. Quantitative PCR was performed with methylation-sensitive primers selective for the methylated MGMT promoter. A S. cerevisiae actin gene spike-in quantitative PCR technique was used to control for variations in DNA loss during the process. Resuts: Baseline plasma from 19 patients demonstrated an overall mean of 2.74 × 105 copies/mL (SEM: 7.67 × 104) of the MGMT promoter. The range of tumor specific DNA in these samples was 1.14 × 104 to 1.22 × 106 copies/mL. One of 19 patients demonstrated no detectable MGMT methylation. Conclusions: Tumor DNA in the blood of high grade glioma patients offers a unique platform to develop a biomarker to satisfy the needs of evolving treatments. This provides the possibility to assess tumor burden in a manner distinct from MRI scans which rely heavily on blood-brain barrier status. This study will eventually yield important information on initial tumor specific DNA concentrations in plasma and changes over the course of the patients' illness. No significant financial relationships to disclose.

Methylguanine Methyltransferase Testing in Glioblastoma: When and How?

Controversy remains on the optimal treatment schedule of the alkylating agent temozolomide (TMZ) in malignant glioma and the value of methylguanine methyltransferase (MGMT) determination. This repair enzyme is key in reverting lethal DNA damage induced by alkylating agent chemotherapy, and in the absence of this enzyme, cells are more susceptible to TMZ. In this issue of the Journal of Clinical Oncology, Chinot et al report on a (too) small phase II study investigating a week-on and week-off dosedense TMZ administration regimen as neoadjuvant chemotherapy before definitive radiation in 29 eligible patients with unresectable glioblastoma. The aim of this alternative dose-dense administration schedule is not only to increase the dose-intensity by more than two-fold, but to overcome drug resistance by depleting the tumor cells of MGMT. In peripheral blood mononuclear cells, it has been shown that repeated and continuous exposure to TMZ will exhaust the cells of MGMT, but whether this also applies to tumor cells remains to be demonstrated. The results of this study were disappointing. Median survival was only 6 months (95% CI, 1 to 11), with only 28% of patients alive at 1 year. Even when considering that this is a poor prognosis group of patients, these results appear inferior to standard therapy, including concomitant chemoradiotherapy, where a median survival of 9 months was reported for patients with unresectable tumors. Chinot et al attempted to determine MGMT expression in 25 patients by immunohistochemistry, choosing an arbitrary cutoff based on the median percentage of cells staining positively. They found that 6 of 11 patients with low MGMT expression, but only one of the 14 patients with high MGMT expression responded. The study emphasizes again the importance of MGMT to predict a response to the alkylating agent therapy. The predictive value of MGMT for benefit from alkylating agent chemotherapy, whether this be a nitrosourea or temozolomide, has now been repeatedly shown. Methylation of the MGMT gene promoter has been found to be a predictive marker for benefit from TMZ treatment in patients treated with temozolomide chemotherapy. Analysis of a representative subgroup of patients in a randomized trial showed that the addition of TMZ only benefited patients with a methylated (and thus silenced) MGMT gene promoter, while overall survival and progression-free survival was comparable for TMZ-treated patients with a functional MGMT gene to patients having been treated with initial radiotherapy alone. Based on this data, one may want to conclude that patients without MGMT promoter methylation should not be treated with alkylating agent chemotherapy. In these patients, TMZ has marginal activity at best, and alternative strategies may offer better chances. However, outside clinical trials, alternative strategies are currently not available. Nevertheless, the treatment, although generally well tolerated, carries its risks for toxicity. Also, in countries without reimbursement for chemotherapy, some patients may choose to forego the expense for temozolomide in the absence of MGMT promoter methylation. Furthermore, this analysis was performed retrospectively, and although very provocative and suggestive, requires prospective validation. In an ongoing Intergroup trial by the Radiation Therapy Oncology Group and European Organisation for Research and Treatment of Cancer (RTOG 0525/EORTC 26052-22053), patients with newly diagnosed glioblastoma are stratified by MGMT methylation status before random assignment to a TMZ administration schedule (standard daily dose for 5 days or a dose-dense treatment for 3 of 4 weeks). To be eligible for this trial, patients must have undergone at least partial tumor resection, and tumor material must be available for centralized MGMT promoter methylation determination. Thus, not only efficient trial management is necessary, but also the goodwill and collaboration of the neurosurgeons and pathologists at many sites. Repeatedly, investigators advocate performing MGMT determination using the simpler immunohistochemistry staining, rather than a methylation-specific polymerase chain reaction assay (MSP) of the function of the MGMT gene. Although some associations may be found by immunohistochemical MGMT expression, this method has many shortcomings. Heterogeneous MGMT expression within tumors, and high levels of MGMT protein present in infiltrating lymphocytes, microglia, and blood vessels limit the reproducibility of the method. More important, MGMT is an inducible enzyme that may be upregulated after chemotherapy or radiotherapy, thus the expression of the protein at diagnosis might not reflect the functional protein during therapy. The MSP assay requires small quantities of DNA that can be extracted from paraffin-embedded tissue samples or, better yet, from cryopreserved tissue samples. In glioma, the presence of a methylated MGMT allele can be attributed solely to the neoplastic cells, irrespective of contamination of nontumor tissue. In our opinion, determination JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 25 NUMBER 12 APRIL 2