MGMT Promoter Methylation Testing Research Articles

Molecular Testing in Gliomas: What is Necessary in Routine Clinical Practice?

A number of molecular characteristics are essential for accurate diagnosis and prognostication in glioma. The 2021 WHO classification of brain tumors and recent Food and Drug Administration (FDA) pathology agnostic drug approvals highlight the importance of molecular testing in the management of glioma. For diffuse gliomas, it is important to identify IDH mutations, given the favorable clinical behavior and potential for using FDA approved IDH inhibitors in the near future. MGMT promoter methylation testing is the most established molecular marker for response to temozolomide in IDH wild-type glioblastoma and in turn impacts overall survival. Moreover, identification of certain mutations and molecular markers, such as BRAF V600E, hypermutation or elevated tumor-mutational burden and NTRK fusions allow for the use of FDA approved agents that are tumor-agnostic. Finally, molecular testing opens options for clinical trials that are essential for diseases with limited treatment options like gliomas.

‘The Reports of My Death Are Greatly Exaggerated’—Evaluating the Effect of Necrosis on MGMT Promoter Methylation Testing in High-Grade Glioma

(1) Background: MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation remains an important predictive biomarker in high-grade gliomas (HGGs). The influence of necrosis on the fidelity of MGMT promoter (MGMTp) hypermethylation testing is currently unknown. Therefore, our study aims to evaluate the effect of varying degrees of necrosis on MGMTp status, as determined by pyrosequencing, in a series of primary and recurrent HGGs; (2) Methods: Within each case, the most viable blocks (assigned as ‘true’ MGMTp status) and the most necrotic block were determined by histopathology review. MGMTp status was determined by pyrosequencing. Comparisons of MGMTp status were made between the most viable and most necrotic blocks. (3) Results: 163 samples from 64 patients with HGGs were analyzed. MGMTp status was maintained in 84.6% of primary and 78.3% of recurrent HGGs between the most viable and necrotic blocks. A threshold of ≥60% tumor cellularity was established at which MGMTp status was unaltered, irrespective of the degree of necrosis. (4) Conclusions: MGMTp methylation status, as determined by pyrosequencing, does not appear to be influenced by necrosis in the majority of cases at a cellularity of at least 60%. Further investigation into the role of intratumoral heterogeneity on MGMTp status will increase our understanding of this predictive marker.

QLTI-14. CAN VARIANT ALLELIC FREQUENCIES OF DRIVER MUTATIONS PREDICT FALSE NEGATIVE MGMT PYROSEQUENCING RESULTS IN ADULT-TYPE DIFFUSE GLIOMAS?

Abstract MGMT promoter methylation predicts favorable temozolomide (TMZ) response in gliomas. Accurate test results depend on adequate tumor cellularity in analyzed samples, which is usually estimated (with sub-optimal accuracy) via light microscopy. We evaluated driver mutation variant allelic frequency (VAF) as a quantitative metric for tumor cellularity in MGMT promoter methylation testing. In a cohort of 691 adult-type diffuse gliomas, MGMT promoter methylation was tested by either pyrosequencing (N = 445) or DNA methylation array (N = 246). VAF of TERT and IDH driver mutations was quantified by next generation sequencing (NGS). We compared frequency of methylated (positive) and unmethylated (negative) results (Fisher’s exact test) and promoter methylation levels (unpaired t-test) according to VAF. We correlated MGMT promoter methylation, VAF, visually estimated tumor cellularity, and cellularity inferred from VAF via linear regression. We assessed survival (Log-Rank test) for 201 patients with IDH-wildtype GBM treated with TMZ. In samples with VAF <0.325 (identified by CutoffFinder), pyrosequencing yielded fewer positive results (37% versus 56%, p < 0.0001) and lower methylation levels (13% versus 19%, p < 0.0001) than samples with VAF≥0.325. Methylation array showed less frequent positive results at low VAF for IDH-mutant astrocytoma, but not for IDH-wildtype GBM. Microscopic examination tended to overestimate tumor cellularity, especially when VAF was low (R2 = 0.558). GBMs with unmethylated MGMT by pyrosequencing and TERT VAF <0.145 showed better responses to TMZ versus cases with VAF≥0.145 (median survival = 16.8 versus 13.3 months). Twelve pyrosequencing samples were re-tested by DNA methylation array and droplet digital PCR (ddPCR). RESULTS: changed for 2/6 samples with TERT VAF≤0.1, and 0/6 samples with VAF >0.1. Driver mutation VAF is useful for quality assurance in MGMT promoter methylation testing. Tumor samples with low VAF are at greater risk of false-negative results by pyrosequencing, which can be corrected by DNA methylation array and ddPCR.

Variant allelic frequencies of driver mutations can identify gliomas with potentially false-negative MGMT promoter methylation results

MGMT promoter methylation testing is required for prognosis and predicting temozolomide response in gliomas. Accurate results depend on sufficient tumor cellularity, but histologic estimates of cellularity are subjective. We sought to determine whether driver mutation variant allelic frequency (VAF) could serve as a more objective metric for cellularity and identify possible false-negative MGMT samples. Among 691 adult-type diffuse gliomas, MGMT promoter methylation was assessed by pyrosequencing (N = 445) or DNA methylation array (N = 246); VAFs of TERT and IDH driver mutations were assessed by next generation sequencing. MGMT results were analyzed in relation to VAF. By pyrosequencing, 56% of all gliomas with driver mutation VAF ≥ 0.325 had MGMT promoter methylation, versus only 37% with VAF < 0.325 (p < 0.0001). The mean MGMT promoter pyrosequencing score was 19.3% for samples with VAF VAF ≥ 0.325, versus 12.7% for samples with VAF < 0.325 (p < 0.0001). Optimal VAF cutoffs differed among glioma subtypes (IDH wildtype glioblastoma: 0.12–0.18, IDH mutant astrocytoma: ~0.33, IDH mutant and 1p/19q co-deleted oligodendroglioma: 0.3–0.4). Methylation array was more sensitive for MGMT promoter methylation at lower VAFs than pyrosequencing. Microscopic examination tended to overestimate tumor cellularity when VAF was low. Re-testing low-VAF cases with methylation array and droplet digital PCR (ddPCR) confirmed that a subset of them had originally been false-negative. We conclude that driver mutation VAF is a useful quality assurance metric when evaluating MGMT promoter methylation tests, as it can help identify possible false-negative cases.

Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

The O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status and clinical outcomes of Ewing sarcoma patients treated with irinotecan and temozolomide.

There remains an unmet need to identify molecular biomarkers in Ewing sarcoma (ES). We sought to assess the influence of the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation on response and progression-free survival (PFS) following initiation of irinotecan and temozolomide (IT), PFS following initiation of vincristine, doxorubicin, and cyclophosphamide alternating with ifosfamide and etoposide (VDC-IE), and overall survival (OS). Data of advanced ES patients, treated with IT were retrospectively collected. Patients were required to have progression after prior VDC-IE. MGMT promoter methylation was assessed on non-decalcified Formalin-fixed paraffin embedded (FFPE) tissue using methylation sensitive restriction enzyme-quantitative PCR (MSRE-qPCR). Survival was estimated by the Kaplan-Meier method. A total of 20 ES patients underwent MGMT promoter methylation testing, and were eligible for analysis. Five patients (25%) had methylated MGMT, whereas the remaining (15; 75%) had unmethylated promoter. Five (25%) had objective response to IT, with no observed difference by promoter methylation (p = 0.76). Median PFS from initiation of IT for methylated vs. unmethylated MGMT patients was 4.9 and 1.2 months, respectively, p = 0.69. Median PFS from date of initiation of VDC-IE was significantly superior in the methylated group; 27.8 vs. 8.6 months, p = 0.034. Median OS was superior but not statistically significant in the methylated group. MGMT-promoter methylation did not correlate with clinical activity or outcomes following the IT regimen for advanced ES. However, methylated MGMT predicted significantly superior PFS following initiation of the standard VDC-IE protocol.

Prognostic value of O 6-methylguanine-DNA methyltransferase methylation in isocitrate dehydrogenase mutant gliomas.

BackgroundPatients with isocitrate dehydrogenase (IDH) mutant gliomas have been associated with longer survival time than those that are IDH wild-type. Previous studies have shown the prognostic value of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation for glioblastoma multiforme (GBM), which are predominantly IDH wild-type. Little is known of the prognostic value of MGMT methylation status for IDH mutant gliomas.MethodsWe retrospectively identified IDH mutant gliomas patients between 2011 and 2020 that were tested for MGMT promoter methylation. We generated Kaplan–Meier estimator curves and performed Cox proportional hazard models for overall survival (OS) and progression-free survival (PFS) to compare the outcomes of MGMT promoter methylated versus MGMT unmethylated patients.ResultsOf 419 IDH mutant gliomas with MGMT promoter methylation testing, we identified 54 GBMs, 223 astrocytomas, and 142 oligodendrogliomas. 62.3% patients had MGMT methylated tumors while 37.7% were MGMT unmethylated. On Kaplan–Meier analysis, median OS for all MGMT methylated patients was 17.7 years and 14.6 years for unmethylated patients. Median PFS for all MGMT methylated patients was 7.0 years and for unmethylated patients 5.2 years. After univariate subgroup analysis, MGMT methylation is only prognostic for OS and PFS in GBM, and for OS in anaplastic oligodendroglioma and anaplastic oligodendroglioma for OS. In multivariate analysis, MGMT unmethylated GBM patients carry a higher risk of death (HR 7.72, 95% CI 2.10–28.33) and recurrence (HR 3.85, 95% CI 1.35–10.96).Conclusions MGMT promoter methylation is associated with better OS and PFS for IDH mutant GBM. MGMT promoter methylation testing for other IDH mutant glioma subtypes may not provide additional information on prognostication.

The MGMT (O6-methylguanine–DNA methyltransferase) promotes methylation and clinical outcomes of salvage temozolomide and irinotecan chemotherapy in progressive Ewing sarcoma: A preliminary report.

e23507 Background: There remains an unmet need to identify prognostic and predictive molecular biomarkers in advance Ewing sarcoma (ES). We sought to assess the influence of MGMT promoter methylation status on response rate, time to progression (TTP), and overall survival (OS) following salvage irinotecan and temozolomide (IT) chemotherapy. Methods: Data of advanced ES patients, treated with IT chemotherapy from Jan, 2014 to Jan, 2020 were retrospectively collected. Patients were required to have previously received and progressed after vincristine, doxorubicin, and cyclophosphamide alternating with ifosfamide and etoposide (VDC-IE). MGMT promoter methylation status was assessed by Methylation Sensitive Restriction Enzyme quantitative-PCR (MSRE-qPCR) on non-decalcified Formalin-fixed paraffin embedded (FFPE) tissue using internally developed primers and the OneStep qMethyl Kit (ZYMO RESEARCH CORP). Responses were assessed by response evaluation criteria in solid tumors (RECIST v. 1.1). TTP and OS were assessed by the Kaplan-Meier method. Survival comparisons were performed by the Log-rank test. Results: Herein, we present data of the preliminary analysis of the first 18 patients who underwent MGMT promoter methylation testing. Patients had a median age of 18 years (range: 5-34 years), and were predominantly male ( n=11; 61%). The primary tumor was located in the pelvis in 9 patients (50%), femur in 3 (17%), tibia in 2 (11%), kidney in 2 (11%), chest wall in one (6%), and scapula in one patient (6%). IT was given in a second ( n=15) or third-line setting ( n=3). At the time of initiation of IT, 13 patients (72%) had distant metastasis, and 5 (28%) had unresectable local progression in the pelvis ( n=4) or chest wall ( n=1). The mean percentage of MGMT promoter methylation was 29% (range: 3-98%). Five patients (28%) had methylated MGMT promoter, whereas the remaining had partially methylated ( n=6; 33%) or unmethylated ( n=7;39%) promoter. Five patients (28%) had objective response, with no observed difference according to MGMT promoter methylation ( p=0.58 for comparison between methylated and unmethylated/ partially methylated). Median TTP was 4.9 and 2.2 months for patients with methylated and partially methylated/ unmethylated MGMT respectively; p=0.76. The corresponding median OS was 69.4 and 14.3 months in favor of the methylated group; p=0.3. Conclusions: This preliminary data suggests a possible prognostic role for MGMT promoter methylation, with a markedly extended median OS for the methylated group. Nevertheless, the median OS difference did not reach statistical significance in this preliminary analysis. We plan to report data of the final analysis after finalizing MGMT testing for the rest of our study patients.

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.

Socioeconomic Disparities Associated With MGMT Promoter Methylation Testing for Patients With Glioblastoma

This cohort study evaluates the national practice patterns of MGMT gene testing and identified potential factors associated with access to testing.

Quality assurance in neuropathology: Experiences from the round robin trials on IDH mutation and MGMT promoter methylation testing launched by the Quality Assurance Initiative Pathology (QuIP) in 2018 and 2019.

We here report on the first neuropathological round robin trials initiated by the Quality Assurance Initiative Pathology (QuIP) in Germany in the years 2018 and 2019. Testing services as external laboratory controls were offered for IDH1-R132H immunohistochemistry in 2018 followed by a molecular trial for IDH1 and IDH2 mutations in 2019 including the rare mutational variants. Also in 2019, a trial on MGMT promoter methylation testing was offered. On a national scale, trial offers were well received with around 40 participating institutions. The international announcement of the molecular IDH1/IDH2 mutational trial achieved only moderate European outspread. Success rates in all three trials were excellent (IDH1-R132H immunohistochemistry 2018: 94%, 18 out of 20 possible points required; IDH1/IDH2 mutational status 2019: 100%, 19 out of 20 possible points required; MGMT promoter methylation 2019: 94%, 19 out of 20 possible points required) indicating that quality standards are high in the broad majority of the institutions. Trial participation also involved filling in a questionnaire asking for background information on local testing procedures. We here present a first assessment of the information collected providing unique insights in the landscape of molecular testing in neuropathology. Derived from this information we identify future challenges and provide an outlook on the development of quality assurance in the field of neuropathology.

Combinations of four or more CpGs methylation present equivalent predictive value for MGMT expression and temozolomide therapeutic prognosis in gliomas

The pyrosequencing (PSQ) has been regarded as the gold standard for MGMT promoter methylation testing in gliomas. However, various CpG combinations are currently used in clinical practice. We aimed to clarify how and how many CpGs combined is robust enough to predict MGMT mRNA expression and therapeutic prognosis of patients. Total 223 patients with WHO III/IV gliomas were enrolled from Chinese Glioma Genome Atlas, including two independent cohorts, the eight-site cohort (with CpGs 75-82 tested) and the seven-site cohort (with CpGs 72-78 tested). Spearman's correlation and ROC curves were employed to investigate the value of different CpG combinations on predicting MGMT mRNA expression. The ROC curves and Kaplan-Meier steps were performed to compare the TMZ therapeutic prognostic values of different CpG combinations. The methylation level of all individual CpG and CpG combinations for the eleven CpGs (CpGs 72-82), significantly correlated to MGMT mRNA expression (Spearman, all P<0.0001), could effectively predict the mRNA expression (AUC, 0.86-0.91 in the eight-site cohort, 0.83-0.90 in the seven-site cohort). Moreover, the correlation coefficients and the predictive values presented equivalent when four or more CpGs combinedly used (AUC, 0.88-0.90 in the eight-site cohort, 0.87-0.88 in the seven-site cohort). Finally, similar results were also observed when using selected CpG combinations to predict therapeutic prognosis of patients. Four-CpG combinations of pyrosequencing are sufficient for evaluating the methylation status of MGMT and predicting therapeutic prognosis in gliomas.

Defining optimal cutoff value of MGMT promoter methylation by ROC analysis for clinical setting in glioblastoma patients.

Resistance to temozolomide (TMZ) chemotherapy poses a significant challenge in the treatment of glioblastoma (GBM). Hypermethylation in O6-methylguanine-DNA methyltransferase (MGMT) promoter is thought to play a critical role in this resistance. Pyrosequencing (PSQ) has been shown to be accurate and robust for MGMT promoter methylation testing. The unresolved issue is the determination of a cut-off value for dichotomization of quantitative MGMT PSQ results into "MGMT methylated" and "MGMT unmethylated" patient subgroups as a basis for further treatment decisions. In this study, receiver operating characteristic (ROC) curve analysis was used to identify an optimal cutoff of MGMT promoter methylation by testing mean percentage of methylation of 4 CpG islands (76-79) within MGMT exon 1. The area under the ROC (AUC) as well as the best cutoff to classify the methylation were calculated. Positive likelihood ratio (LR+) was chosen as a diagnostic parameter for defining an optimal cut-off. Meanwhile, we also analyzed whether mean percentage of methylation at the investigated CpG islands could be regarded as a marker for evaluating prognostication. ROC analysis showed that the optimal threshold was 12.5% (sensitivity: 60.87%; specificity: 76%) in response to the largest LR+ 2.54. 12.5% was established to distinguish MGMT promoter methylation, which was confirmed using validation set. According to the cutoff value, the MGMT promoter methylation was found in 58.3% of GBM. Mean methylation level of the investigated CpG sites strong correlated with overall survival (OS), which means GBM patients with a high level of methylation survived longer than those with low level of methylation(log-rank test, P = 0.017). In conclusion, ROC curve analysis enables the best cutoff for discriminating MGMT promoter methylation status. LR+ can be used as a key factor that evaluates cutoff. The promoter methylation level of MGMT by PSQ in GBM patients had prognostic value.

Different diagnostic values of imaging parameters to predict pseudoprogression in glioblastoma subgroups stratified by MGMT promoter methylation

The aim of this study was to determine whether diffusion and perfusion imaging parameters demonstrate different diagnostic values for predicting pseudoprogression between glioblastoma subgroups stratified by O6-mythylguanine-DNA methyltransferase (MGMT) promoter methylation status. We enrolled seventy-five glioblastoma patients that had presented with enlarged contrast-enhanced lesions on magnetic resonance imaging (MRI) one month after completing concurrent chemoradiotherapy and undergoing MGMT promoter methylation testing. The imaging parameters included 10 or 90% histogram cutoffs of apparent diffusion coefficient (ADC10), normalized cerebral blood volume (nCBV90), and initial area under the time signal-intensity curve (IAUC90). The results of the areas under the receiver operating characteristic curve (AUCs) with cross-validation were compared between MGMT methylation and unmethylation groups. MR imaging parameters demonstrated a trend toward higher accuracy in the MGMT promoter methylation group than in the unmethylation group (cross-validated AUCs = 0.70-0.95 and 0.56-0.87, respectively). The combination of MGMT methylation status with imaging parameters improved the AUCs from 0.70 to 0.75-0.90 for both readers in comparison with MGMT methylation status alone. The probability of pseudoprogression was highest (95.7%) when nCBV90 was below 4.02 in the MGMT promoter methylation group. MR imaging parameters could be stronger predictors of pseudoprogression in glioblastoma patients with the methylated MGMT promoter than in patients with the unmethylated MGMT promoter. • The glioblastoma subgroup was stratified according to MGMT promoter methylation status. • Diagnostic values of diffusion and perfusion parameters for predicting pseudoprogression were compared. • Imaging parameters showed higher diagnostic accuracy in the MGMT promoter methylation group. • Imaging parameters were independent to MGMT promoter methylation status for predicting pseudoprogression. • Imaging biomarkers might demonstrate different diagnostic values according to MGMT promoter methylation.

Abstract 11: Advancing methylation profiling in neuropathology: Diagnosis and clinical management

Abstract Introduction: Although molecular profiling is increasingly being applied to improve subgroup classification and to provide novel prognostic and predictive biomarkers, clinical neuropathology practice is largely based on morphology and immunohistochemistry. Current molecular methods play only a small role in determining the diagnosis itself. Methods: For molecular subclassification of tumors at NYU neuropathology and to improve diagnostic accuracy, we introduced genome-wide methylation profiling through Illumina Infinium HumanMethylation 450k array that can detect methylation marks from the DNA extracted from formalin-fixed paraffin embedded tissues. To this effort, an in-house pipeline was established in-house pipeline, which includes morphologic review, sample preparation, molecular profiling and bioinformatics analysis. We compared the methylation profiles to a reference cohort of 2150 cases from 77 tumor entities previously profiled and analyzed at German Cancer Research Center using a random forest algorithm and customized bioinformatics packages, which were shared between our institutions. Selected copy number variants (CNV) and mutations were confirmed by Fluorescence in situ Hybridization (FISH) or sequencing, and mutation specific immunohistochemistry, respectively. Results: We profiled 60 difficult in-house or consult adult and pediatric brain tumors where diagnosis, grade and/or molecular subtype were not conclusive by morphology, immunohistochemistry or standard molecular studies alone. There was 100% concordance with concurrently performed molecular tests such as 1p/19q, EGFR/BRAF CNV, MGMT promoter methylation or IDH1 status testing when these tests were performed for clinical care. Methylation profiling provided additional, relevant information in 30 of 60 (50%) cases, leading to a change of diagnosis in 9 (15%), clarification of the diagnosis in 7 (12%) cases, and further molecular subgroup refinement in 14 (23%) of cases, helping to direct further molecular testing and clinical management. Conclusion: The 450k methylation array platform represents a cost-efficient method to obtain molecular profiles of brain tumors to identify biologically relevant diagnostic subgroups, thereby improving diagnostic accuracy, and helping inform appropriate clinical management decisions. Citation Format: Kasthuri S. Kannan, Aristotelis Tsirigos, Jonathan Serrano, Lynn Ann Forrester, Arline Faustin, Cheddhi Thomas, David Capper, Volker Hovestadt, Stefan M. Pfister, David T. W Jones, Martin Sill, Daniel Schrimpf, Andreas von Deimling, Adriana Heguy, Sharon L. Gardner, Jeffrey Allen, Cyrus Hedvat, David Zagzag, Matija Snuderl, Matthias A. Karajannis. Advancing methylation profiling in neuropathology: Diagnosis and clinical management. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 11.

Assessing MGMT methylation status and its current impact on treatment in glioblastoma.

MGMT promoter methylation status is a strong and independent prognostic factor in patients with newly diagnosed glioblastoma and a clinically relevant predictive marker in the subpopulation of elderly glioblastoma patients. However, there is still lack of consensus on the optimal assay for reliable MGMT promoter methylation testing and a variety of test are being used in different laboratories. Pyrosequencing is the only method for which an adequately high analytical performance (high intra- and interlaboratory repeatability and reproducibility) has been demonstrated in a fully published ring trial. For clinical decision-making MGMT promoter methylation testing should be performed only in experienced laboratories using meticulous validation of assay accuracy. Ideally, such laboratories should undergo regular accreditation through a quality control consortium.

Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing.

Use of personalized molecular biomarkers in the clinical care of adults with glioblastomas

This study was conducted to assess the current pattern of use and the impact of available molecular predictive and prognostic biomarkers on clinical care in patients with glioblastoma (GBM). An online questionnaire consisting of 15 questions about the frequency of use and clinical utility of tissue-based molecular tests was distributed to 1,053 members of the Neuro-Oncology Community in the United States. A total of 320 responses (30.4 %) were collected. 73 respondents who did not see GBM patients were excluded from analysis. MGMT promoter methylation testing (MGMT-meth) was the most commonly requested (37.2; 95 % CI, 31-44), followed by EGFR amplification (22.7; 95 % CI, 18-28), co-deletion of 1p/19q (22.3 %), EGFR expression (21.5 %), P53 mutation (19.8 %), PTEN mutation or deletion (17.4 %), EGFRvIII mutation (12.1 %), IDH1/2 mutation (12.1 %), PDGFR (4.5 %), and PIK3CA (0.8 %). The perceived utility of these studies was variable between participants. A small percentage of respondents felt that any of the studies were "always" or "almost always" helpful in clinical decision making (MGMT-meth 10.9 %; range, 0-13.8 %), but more frequently "never" or "almost never" helpful (MGMT-meth 25.9 %; range, 25-54.7 %). 26.7 % reported not to routinely order any of these studies. Although molecular markers are frequently ordered for patients with GBM, only a minority of clinicians ordering these tests report that the results influence clinical decision-making. Molecular markers that are likely to affect patient care should be ordered with the goal to maximize benefit for patients and to avoid non-actionable results and additional costs.

Clinical use of personalized molecular biomarkers in patients with glioblastoma.

2022 Background: The development of predictive and prognostic biomarkers in malignant gliomas is the subject of intense research designed to provide personalized therapies for patients with these cancers. The availability, ordering, and clinical influence of molecular tests are not well described. This study was conducted to assess the current use of available biomarkers in patients with glioblastoma (GBM). Methods: A questionnaire consisting of 13 multiple-choice and 2 open questions was distributed online to members of the neuro-oncology community in the United States using email addresses published in the 2010 meeting program of the Society for Neuro-Oncology. All answers were collected between January 5 and 16, 2011. Results: The response rate to this survey was 30.4% (320/1053). Participants who did not see GBM patients were excluded from analysis (n=73). The 247 qualified respondents saw 0-2 (22%), 3-10 (34%), 11-30 (30%), or >30 (15%) patients with GBM per month. When seeing patients with newly diagnosed GBM, participants (n=201) reported routinely ordering no molecular tests (33%), MGMT promoter methylation (46%), EGFR amplification (28%), 1p19q co-deletion (27%), EGFR expression (26%), p53 mutation (24%), PTEN mutation or deletion (21%), IDH1/IDH2 mutation (15%), EGFRvIII mutation (15%), PDGFR expression (6%), or PIK3CA mutation (1%). The following percentages of clinicians seeing >3 patients with GBM/month noted that molecular tests “always” or “almost all the time” influenced their management of patients with GBM: 1p/19q co-deletion 20% (28/142), MGMT promoter methylation 11% (17/158), EGFR expression 3% (4/134), EGFR amplification 3% (4/143), EGFRvIII mutation 3% (4/139), IDH1/IDH2 mutation 2% (3/132), p53 mutation 1% (1/133), PDGFR expression 0% (0/131), PIK3CA mutation 0% (0/126), or PTEN mutation or deletion 0% (0/133). Conclusions: An estimated two thirds of clinicians routinely order one or more molecular biomarkers for patients with newly diagnosed GBM. The most commonly ordered tests were MGMT promoter methylation, EGFR amplification, and 1p19q testing. However, the impact of the molecular test results on clinical decision making remains modest.

MGMT methylation testing in RTOG 0525: A phase III trial of newly diagnosed glioblastoma

2051 Background: MGMT promoter methylation has been described as a prognostic marker in glioblastoma (GBM) and may be associated with chemosensitivity to alkylating agents. This study determined the feasibility of real-time determination of MGMT promoter methylation testing in a large international phase III clinical trial. Methods: Paraffin tumor blocks were obtained from patients registered onto RTOG 0525 then distributed to one of two central pathology labs: MD Anderson (Houston TX, K. Aldape) or CHUV (Lausanne, CH, R. Janzer). After histologic confirmation of GBM, unstained slides (40 microns of tumor tissue) were sent to the testing laboratories. Results were used for randomization into a treatment arm. MGMT methylation was assessed using methylation specific real-time PCR (MSP). The assay determined the number of copies of both methylated MGMT and of beta-actin (ACTB) in the sample. The ACTB copy number was used to assess the quality and quantity of the sample DNA. Results: Results were available for 995 samples. Following MSP samples were categorized into one of five possible results: failed (2, 0.2%), methylated (302, 30.2%), non-methylated (602, 60.2%), indeterminate (62, 6.2%), and invalid (27, 2.7%). Among cases that were evaluable as either methylated or unmethylated (n = 904) the MGMT promoter methylation rate in newly diagnosed GBM was approximately 1/3 (33.4%), a rate that is somewhat lower than prior reports. The average time from receipt of sample at the central pathology laboratory to reporting of results was 9.3 days. The time required decreased over the course of the clinical trial. This was due, in part, to training of the sites to deliver samples just before the start of runs. Conclusions: The results demonstrate the feasibility of performing real-time MGMT methylation testing, a tumor based assay, as a stratification factor in a multinational clinical trial. This study confirms that treatment decisions based on the molecular characteristics of the tumor are feasible, thereby providing opportunities to develop more molecularly-based tumor stratification or selection, a major advance in developing personalized treatment regimens. No significant financial relationships to disclose.