Methylation Of The O6-methylguanine-DNA Methyltransferase Research Articles

MGMT methylation and its prognostic significance in inoperable IDH-wildtype glioblastoma: the MGMT-GBM study

IntroductionThe methylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) promoter is a valid biomarker for predicting response to therapy with alkylating agents and, independently, prognosis in IDH-wildtype(IDH-w) glioblastoma. We aim to study the impact of its methylation in overall survival of the unresectable IDH-w glioblastoma undergoing biopsy and systemic treatment.MethodsWe collected six-year retrospective (2017–2023) data at a quaternary neurosurgery center for patients undergoing biopsy as the only surgical procedure for an unresectable IDH wildtype glioblastoma. Data was collected from patient records including neuro-oncology multidisciplinary team meeting (MDT) documentation. Patients were grouped into categories according to different types of treatment received after biopsy (no treatment, chemotherapy (CT), radiotherapy (RT), chemoradiotherapy (CRT), chemoradiotherapy with adjuvant temozolomide (CRT with adjuvant TMZ), EORTC-NCIC protocol followed by second line treatment) and according to methylation status (unmethylated (< 5%), borderline methylated (5–15%) and strongly methylated (> 15%)). Survival analysis was performed.Results166 glioblastoma IDH wildtype patients were included in the study with mean age of 62.5 years (M: F = 1.5: 1). 70 (49.3%) patients had unmethylated MGMT status (< 5%), 29 (20.4%) patients had borderline methylated MGMT status (5–15%) and 43 (30.2%) patients had methylated MGMT status (> 15%). 36 (25.3%) patients did not receive any treatment post biopsy, 13 (9.1%) received CT only, 27 (19%) RT only, 12 (8.4%) CRT, 33 (23.2%) CRT with adjuvant TMZ, whereas 21 (14.7%) received EORTC-NCIC protocol along with second line treatment.In biopsy only group, there was no notable difference in survival outcomes among the different methylation statuses. For biopsy and any-other-form-of-treatment methylated groups showed a distinct trend of better survival compared to the borderline or unmethylated groups. Overall, methylated patients had better survival as compared to unmethylated or borderline groups.ConclusionMethylated MGMT status are predictors for better overall survival in unresectable IDH wildtype glioblastoma patients undergoing biopsy and treatment regardless of the treatment modality.

Full kinetic modeling analysis of [18F]fluorocholine Positron Emission Tomography (PET) at initial diagnosis of high-grade glioma

PurposeThe main objective was to characterize the tracer uptake kinetics of [18F]fluoromethylcholine ([18F]F-CHO) in high-grade gliomas (HGG) through a full PET kinetic modeling approach. Secondarily, we aimed to explore the relationship between the PET uptake measures and the HGG molecular features. Materials and methodsTwenty-four patients with a suspected diagnosis of HGG were prospectively included. They underwent a dynamic brain [18F]F-CHO-PET/CT, from which a tumoral time-activity curve was extracted. The plasma input function was obtained through arterial blood sampling with metabolite correction. These data were fitted to 1- and 2-tissue-compartment models, the best of which was selected through the Akaike information criterion. We assessed the correlation between the kinetic parameters and the conventional static PET metrics (SUVmax, SUVmean and tumor-to-background ratio TBR). We explored the association between the [18F]F-CHO-PET quantitative parameters and relevant molecular biomarkers in HGG. ResultsTumoral time-activity curves in all patients showed a rapid rise of [18F]F-CHO uptake followed by a plateau-like shape. Best fits were obtained with near-irreversible 2-tissue-compartment models. The perfusion-transport constant K1 and the net influx rate Ki showed strong correlation with SUVmax (r = 0.808–0.861), SUVmean (r = 0.794–0.851) and TBR (r = 0.643–0.784), p < 0.002. HGG was confirmed in 21 patients, of which those with methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) gene promoter showed higher mean Ki (p = 0.020), K1 (p = 0.025) and TBR (p = 0.001) than the unmethylated ones. Conclusion[18F]F-CHO uptake kinetics in HGG is best explained by a 2-tissue-compartment model. The conventional static [18F]F-CHO-PET measures have been validated against the perfusion-transport constant (K1) and the net influx rate (Ki) derived from kinetic modeling. A relationship between [18F]F-CHO uptake rate and MGMT methylation is suggested but needs further confirmation.

Development of A Radiomic Model for MGMT Promoter Methylation Detection in Glioblastoma Using Conventional MRI.

The methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter is a molecular marker associated with a better response to chemotherapy in patients with glioblastoma (GB). Standard pre-operative magnetic resonance imaging (MRI) analysis is not adequate to detect MGMT promoter methylation. This study aims to evaluate whether the radiomic features extracted from multiple tumor subregions using multiparametric MRI can predict MGMT promoter methylation status in GB patients. This retrospective single-institution study included a cohort of 277 GB patients whose 3D post-contrast T1-weighted images and 3D fluid-attenuated inversion recovery (FLAIR) images were acquired using two MRI scanners. Three separate regions of interest (ROIs) showing tumor enhancement, necrosis, and FLAIR hyperintensities were manually segmented for each patient. Two machine learning algorithms (support vector machine (SVM) and random forest) were built for MGMT promoter methylation prediction from a training cohort (196 patients) and tested on a separate validation cohort (81 patients), based on a set of automatically selected radiomic features, with and without demographic variables (i.e., patients' age and sex). In the training set, SVM based on the selected radiomic features of the three separate ROIs achieved the best performances, with an average of 83.0% (standard deviation: 5.7%) for accuracy and 0.894 (0.056) for the area under the curve (AUC) computed through cross-validation. In the test set, all classification performances dropped: the best was obtained by SVM based on the selected features extracted from the whole tumor lesion constructed by merging the three ROIs, with 64.2% (95% confidence interval: 52.8-74.6%) accuracy and 0.572 (0.439-0.705) for AUC. The performances did not change when the patients' age and sex were included with the radiomic features into the models. Our study confirms the presence of a subtle association between imaging characteristics and MGMT promoter methylation status. However, further verification of the strength of this association is needed, as the low diagnostic performance obtained in this validation cohort is not sufficiently robust to allow clinically meaningful predictions.

The LUMIERE dataset: Longitudinal Glioblastoma MRI with expert RANO evaluation

Publicly available Glioblastoma (GBM) datasets predominantly include pre-operative Magnetic Resonance Imaging (MRI) or contain few follow-up images for each patient. Access to fully longitudinal datasets is critical to advance the refinement of treatment response assessment. We release a single-center longitudinal GBM MRI dataset with expert ratings of selected follow-up studies according to the response assessment in neuro-oncology criteria (RANO). The expert rating includes details about the rationale of the ratings. For a subset of patients, we provide pathology information regarding methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter status and isocitrate dehydrogenase 1 (IDH1), as well as the overall survival time. The data includes T1-weighted pre- and post-contrast, T2-weighted, and fluid-attenuated inversion recovery (FLAIR) MRI. Segmentations from state-of-the-art automated segmentation tools, as well as radiomic features, complement the data. Possible applications of this dataset are radiomics research, the development and validation of automated segmentation methods, and studies on response assessment. This collection includes MRI data of 91 GBM patients with a total of 638 study dates and 2487 images.

MGMT and Whole-Genome DNA Methylation Impacts on Diagnosis, Prognosis and Therapy of Glioblastoma Multiforme.

Epigenetic changes in DNA methylation contribute to the development of many diseases, including cancer. In glioblastoma multiforme, the most prevalent primary brain cancer and an incurable tumor with a median survival time of 15 months, a single epigenetic modification, the methylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene, is a valid biomarker for predicting response to therapy with alkylating agents and also, independently, prognosis. More recently, the progress from single gene to whole-genome analysis of DNA methylation has allowed a better subclassification of glioblastomas. Here, we review the clinically relevant information that can be obtained by studying MGMT gene and whole-genome DNA methylation changes in glioblastomas, also highlighting benefits, including those of liquid biopsy, and pitfalls of the different detection methods. Finally, we discuss how changes in DNA methylation, especially in glioblastomas bearing mutations in the Isocitrate Dehydrogenase (IDH) 1 and 2 genes, can be exploited as targets for tailoring therapy.

Using variant allelic fraction of driver mutations to determine the reliability of MGMT promoter methylation testing in adult-type diffuse gliomas.

e14034 Background: Methylation of the O6-methylguanine-DNA methyltransferase ( MGMT) promoter predicts response to temozolomide (TMZ) in adult-type diffuse gliomas. MGMT testing is thus standard practice. However, accurate results depend on adequate tumor cellularity in the analyzed sample. If the sample contains too many admixed nonneoplastic cells (which are consistently MGMT unmethylated), a false-negative result may be generated. Currently, ̃70% tumor cells are considered the minimum for reliable testing, but that is based on visual estimation by light microscopy; such estimation varies between observers and is often inaccurate. Since next-generation sequencing (NGS) is also frequently done on adult-type diffuse gliomas, and such tumors usually have heterozygous driver mutations known to be present in nearly 100% of tumor cells (IDH mutations in astrocytomas and oligodendrogliomas; TERT promoter mutations in ̃85% of IDH wild-type GBM), we sought to determine whether the variant allelic fraction (VAF) of these mutations could improve quality assurance in MGMT promoter methylation testing. Methods: We used an in-house cohort of 175 WHO gliomas (112 IDHwt, TERTmut GBM, 34 WHO grade 2-4 IDHmut astrocytomas, 29 WHO grade 2-3 IDHmut oligodendrogliomas), all with MGMT pyrosequencing and NGS data obtained from the same tissue blocks. Gliomas with average 10% methylation across all four CpG sites in the MGMT promoter were considered positive, as per standard cutoffs for that assay. Results: Overall, there was a positive correlation between driver VAF and amount of MGMT promoter methylation (slope = 0.25, R2= 0.33, P = 0.016). The largest dropoff in average methylation was when VAF was below 13% (7.4% average CpG methylation when VAF &lt; 13% vs. 19.0% when VAF≥13%, P= 0.029). Based on our prior published study (PMID: 33830235), 36.5% of IDHwt GBMs, 86.7% of IDHmut astrocytomas, and 98.6% of IDHmut oligodendrogliomas should have MGMT promoter methylation. Among our GBMs, only 1/7 (14.0%) below a TERT mutant VAF of 13% reached the cutoff for MGMT promoter methylation, whereas 39/105 (37.0%) of GBMs VAF≥13% did. However, this did not reach statistical significance, likely because our cohort was underpowered for GBMs with TERTmut VAF &lt; 13%. Among 34 IDHmut astrocytomas, only 1 had a VAF below 13%, and was reported as MGMT promoter unmethylated; all others had VAF between 21-56%, and showed consistent rates of MGMT methylation positivity across that entire range. All 29 oligodendrogliomas had VAFs of at least 26%; 2/29 were MGMT-negative, and both had VAFs at or above 36%. Conclusions: Additional cases are being prospectively collected to boost statistical power. Still, our existing data suggest that, when driver mutation VAF falls below ̃13% (i.e., tumor cellularity ̃26%), the risk of false-negative MGMT methylation results increases, and caution is warranted in such cases.

Complete and Incomplete Resection for Progressive Glioblastoma Prolongs Post-Progression Survival.

ObjectiveThe role of resection in progressive glioblastoma (GBM) to prolong survival is still controversial. The aim of this study was to determine 1) the predictors of post-progression survival (PPS) in progressive GBM and 2) which subgroups of patients would benefit from recurrent resection.MethodsWe have conducted a retrospective bicentric cohort study on isocitrate dehydrogenase (IDH) wild-type GBM treated in our hospitals between 2006 and 2015. Kaplan-Maier analyses and univariable and multivariable Cox regressions were performed to identify predictors and their influence on PPS.ResultsOf 589 patients with progressive IDH wild-type GBM, 355 patients were included in analyses. Median PPS of all patients was 9 months (95% CI 8.0-10.0), with complete resection 12 months (95% CI 9.7-14.3, n=81), incomplete resection 11 months (95% CI 8.9-13.1, n=70) and without resection 7 months (95% CI 06-08, n=204). Multivariable Cox regression demonstrated a benefit for PPS with complete (HR 0.67, CI 0.49-0.90) and incomplete resection (HR 0.73, 95% CI 0.51-1.04) and confirmed methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene promoter, lower age at diagnosis, absence of deep brain and multilocular localization, higher Karnofsky Performance Status (KPS) and recurrent therapies to be associated with longer PPS. In contrast, traditional eloquence and duration of progression-free survival had no effect on PPS. Subgroup analyses showed that all subgroups of confirmed predictors benefited from resection, except for patients in poor condition with a KPS <70.ConclusionsOut data suggest a role for complete and incomplete recurrent resection in progressive GBM patients regardless of methylation of MGMT, age, or adjuvant therapy but not in patients with a poor clinical condition with a KPS <70.

Federated Learning Approach for Measuring the Response of Brain Tumors to Chemotherapy

Brain tumor is a fatal disease and one of the major causes of rising death rates in adults. Predicting methylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene status utilizing Magnetic resonance imaging (MRI) imaging is highly important since it is a predictor of brain tumor responses to chemotherapy, which reduces the number of needed surgeries. Deep Learning (DL) approaches became powerful in extracting meaningful relationships and making accurate predictions. DL-based models require a large database and accessing or transferring patient data to train the model. Federated machine learning has recently gained popularity, as it offers practical solutions for data privacy, centralized computation, and high computing power. This study aims to investigate the feasibility of federated learning (FL) by developing a FL-based approach to predict MGMT promoter methylation status using the BraTs2021 dataset for the four sequence types, (Fluid Attenuated Inversion Recovery (FLAIR), T1-weighted (T1w), T1-weighted Gadolinium Post Contrast (T1wCE/T1Gd), and T2-weighted (T2w)) MRI images. The FL model compared to the DL-based and the experimental results show that even with imbalanced and heterogeneous datasets, the FL approach reached the training model to 99.99% of the model quality achieved with centralized data after 300 communication rounds between 10 institutions using OpenFl framework and the improved EfficentNet-B3 neural network architecture.

Cost-effectiveness of concomitant and adjuvant temozolomide for glioblastoma patients with unmethylated O6-methylguanine-DNA methyltransferase promoter regions in the United States.

2058 Background: Promoter region methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene has emerged as a predictive factor for longer overall (OS) and progression-free (PFS) survival response to concomitant and adjuvant temozolomide (TMZ) chemotherapy in glioblastoma. Recently, several investigators have suggested the small survival benefit of TMZ for unmethylated patients is insufficient to warrant the increased risk of adverse events and that these patients should instead be enrolled in clinical trials. This study informs the debate by estimating the incremental costs and benefits associated with TMZ use in methylated and unmethylated patients based on available published evidence. Methods: Published OS and PFS Kaplan-Meier curves were digitized, parameterized, and extrapolated to ten years. Partitioned Survival Analysis trees were constructed in TreeAge Pro 2020. Costs of TMZ, RT, and inpatient hospitalization were included, among others. It was assumed that all patients receive surgery prior to beginning radiation, and that RT and TMZ (six adjuvant cycles) dosing was standard. Published utilities associated with the progression-free and post-progression health states, subject to each treatment, were used. A 3% annual discount rate and a willingness-to-pay of $150,000/quality-adjusted life year (QALY) were assumed. Probabilistic sensitivity analysis (PSA) was conducted. Results: In methylated patients, RT + TMZ versus RT is associated with $67,622 in additional costs and 10.91 additional quality-adjusted life months (QALMs). In unmethylated patients, RT + TMZ versus RT is associated with $9,203 in additional costs and 2.24 additional QALMs. The implied incremental cost effectiveness ratios (ICERs) support TMZ use in both populations and suggest it is more favorable for unmethylated patients ($74,378/QALY and $49,302/QALY for methylated and unmethylated patients, respectively). However, the incremental net monetary benefit (INMB) associated with TMZ use is substantially smaller for unmethylated patients ($18,797 versus $68,753) due to the smaller survival gains for this population. PSA results indicate that TMZ is more likely to be cost-effective for methylated (89.3%) than unmethylated (77.4%) patients. Conclusions: Post-radiation clinical trial enrollment should be considered instead of TMZ for unmethylated glioblastoma patients. While the estimated ICER justifies the use of TMZ for unmethylated patients – in fact, does so more strongly than for methylated patients – the relatively small incremental health benefit is plausibly outweighed by the opportunity cost of not enrolling these patients in clinical trials that offer the possibility of more substantive gains to current and future glioblastoma patients.

Clinical and histopathological analyses of VEGF receptors peptide vaccine in patients with primary glioblastoma - a case series

BackgroundThe expression of vascular endothelial growth factor (VEGF)-A/ VAGF receptors (VEGFRs) signaling plays a pivotal role in the tumor angiogenesis and the development of the immunosuppressive tumor microenvironment in glioblastomas. We have previously conducted exploratory clinical studies investigating VEGFRs peptide vaccination with and without multiple glioma oncoantigens in patients with recurrent high-grade gliomas. Recently, an exploratory clinical investigation of VEGFRs peptide vaccination was conducted in patients with progressive neurofibromatosis type 2. Those studies suggested that cytotoxic T lymphocytes (CTLs) induced by the vaccination can directly kill a wide variety of cells associated with tumor growth, including tumor vessels, tumor cells, and immunosuppressive cells expressing VEGFR1 and/or 2. In the present study, synergistic activity of the combination of VEGFRs peptide vaccination with chemotherapy was evaluated.MethodsWe performed the first clinical trial to assess VEGFR1 and 2 vaccination along with temozolomide (TMZ) -based chemoradiotherapy for the patients with primary glioblastomas. Furthermore, histopathological changes after the vaccination were evaluated using paired pre- and post- vaccination specimens.ResultsThe disappearance of radiographically enhanced lesion was observed in 2 patients after the vaccination, including one in which the methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter was not observed. The histopathological findings of pre- and post-vaccination specimens demonstrated that tumor vessels showed negative or slight VEGFRs expressions after the vaccination and most endothelial cells were covered with PDGFR-β-positive pericytes. Notably, CTLs induced by VEGFRs peptide vaccination attacked not only tumor vessels but also tumor cells and regulatory T cells expressing VEGFRs even in recurrent tumors.ConclusionsVEGFR1 and 2 vaccination may have a preliminary synergistic effect when administered with TMZ. The limitation of the present study was the paucity of the number of the samples. Further studies involving more patients are warranted to confirm the findings of this study.Trial registrationThis study was registered as UMIN000013381 (University Hospital Medical Information Network-Clinical Trial Registry: UMIN-CTR) on 5 March, 2014 and with the Japan Registry of Clinical Trials (jRCT) as jRCTs031180170 on 1 March, 2019.

Changes of O6-Methylguanine DNA Methyltransferase (MGMT) Promoter Methylation in Glioblastoma Relapse-A Meta-Analysis Type Literature Review.

Methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter has emerged as strong prognostic factor in the therapy of glioblastoma multiforme. It is associated with an improved response to chemotherapy with temozolomide and longer overall survival. MGMT promoter methylation has implications for the clinical course of patients. In recent years, there have been observations of patients changing their MGMT promoter methylation from primary tumor to relapse. Still, data on this topic are scarce. Studies often consist of only few patients and provide rather contrasting results, making it hard to draw a clear conclusion on clinical implications. Here, we summarize the previous publications on this topic, add new cases of changing MGMT status in relapse and finally combine all reports of more than ten patients in a statistical analysis based on the Wilson score interval. MGMT promoter methylation changes are seen in 115 of 476 analyzed patients (24%; CI: 0.21–0.28). We discuss potential reasons like technical issues, intratumoral heterogeneity and selective pressure of therapy. The clinical implications are still ambiguous and do not yet support a change in clinical practice. However, retesting MGMT methylation might be useful for future treatment decisions and we encourage clinical studies to address this topic.

P14.02 Influence of individual CpG methylation status on outcome in adult patients with glioblastoma multiforme receiving alkylating agent treatment

Abstract Background: Methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promotor causes gene silencing and has been associated with a favourable prognosis in patients with glioblastoma multiforme (GBM) receiving alkylating chemotherapy. However, analysis of MGMT promotor methylation is usually reported as a cut-off depending on the results of the correspondent CpG site testing. This approach disregards a possible heterogeneity concerning the methylation status within the individual CpG sites and its possible association with prognosis in GBM patients. The current study aimed at elucidating the association between methylation of CpG sites 74–98 within the MGMT promotor region and outcome in GBM patients receiving alkylating agents. Material and Methods: Individual methylation status of 230 patients with histologically proven GBM following concomitant radio-chemotherapy with TMZ after stereotactic biopsy or open tumor resection (OTR) was assessed by the Sanger sequencing (Sseq) approach. Methylation of CpG sites 74–98 within the MGMT promotor region was defined according to a ratio of cytosine /thymine peak &gt;50%. The total number of methylated CpG sites as well as clinical factors such as age, Karnofsky Performance Score (KPS) and mode of surgical procedure were correlated with outcome using proportional hazards models. In a subset of 34 patients, a correlation between individual CpG methylation and MGMT mRNA expression was performed. Results: Median progression-free (PFS) and overall survival (OS) were 7.8 and 14.6 months, respectively. Alongside younger age, KPS&gt; 80 and OTR, the cumulative total number of methylated loci within the CpG sites 74–98 was strongly associated with both PFS and OS and retained its prognostic influence on outcome in multivariate models (p &lt;0.001). Furthermore, a linear coherence between the total number of methylated CpG sites 74–98 and survival parameters could be observed. Moreover, low number of methylated CpG sites was observed in tumor specimen with a high mRNA expression and vice versa (Spearman correlation coefficient: -0.62). Conclusion: In contrast to the concept of dichotomizing the MGMT promotor status into ‘methylated’ and ‘non-methylated’, our approach shows a clear heterogeneity within the methylation status of the CpG sites 74–98 within the GBM tumor specimens. Our data suggest a strong correlation between outcome and the total number of methylated CpG sites, thus an up-front analysis of the individual GpC site methylation status prior to initiation of alkylating chemotherapy might help to improve treatment response in GBM patients.

Pyrosequencing Analysis of MGMT Promoter Methylation in Meningioma.

Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a well-established predictor of response to the DNA-alkylating agent temozolomide in patients with glioblastoma. Pyrosequencing analysis was used to determine the MGMT promoter methylation status in 61 meningiomas, to clarify whether it might have a predictive role. Only two tumors (3%) had a mean methylation frequency higher than the cut-off value of 10% for the four CpG sites examined. The methylation of the MGMT promoter is uncommon, or occurs at a low frequency in meningiomas. There is no convincing rationale to test such tumors for their MGMT methylation status in a clinical setting.

Basic principles of mathematical growth modeling applied to high-grade gliomas: A brief clinical review for clinicians.

The battle against cancer has intensified in the last decade. New experimental techniques and theoretical models have been been proposed to understand the behavior, growth, and evolution of different types of brain tumors. Unfortunately, for glioblastoma multiforme (GBM), except for methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter that has some benefit in the local control of tumors using alkylating agents such as temozolomide, to date personalized treatments do not exist. In this article, we present a comprehensive review of different aspects intertwined in the mathematical growth modeling applied to high-grade gliomas. We briefly cover the following fundamental aspects related to the conventional imaging in GBM: defining the tumor regions in GBM, segmentation of the tumor regions using magnetic resonance imaging (MRI) of the brain, response assessment using the neuro-oncology response criteria versus the Macdonald criteria, availability of software for the segmentation of MRI of the brain, mathematical modeling applied to tumor growth, principles of mathematical modeling, factors involved in tumor growth models, mathematical modeling based on imaging data, most common equations used in high-grade glioma growth modeling, integration of mathematical growth models in computer simulators, tumor growth modeling as a part of brain's complex system, and challenges in mathematical growth modeling. We conclude by saying that it is the combination of biomedical imaging and mathematical modeling that allows the assembling of clinically relevant models of tumor growth and treatment response; the most appropriate model will depend on the premise and findings of each experiment.

Clinical and Molecular Prognostic Factors for Long-Term Survival of Patients with Glioblastomas in Single-Institutional Consecutive Cohort.

The purpose of this study was to clarify the clinical and molecular characteristics associated with long-term survival in patients with glioblastoma. We analyzed the characteristics of 96 glioblastoma patients. Long-term survivors (LTSs) were classified into moderate LTSs (mLTSs), who survived >3 years, and LTSs, who survived >5 years, and compared with short-term survivors (STSs). Clinical and molecular factors were investigated. Younger age, better recursive partitioning analysis class, lack of subventricular zone (SVZ) involvement, promoter methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene, and loss of 19q were associated with mLTSs as compared with STSs. After adjustment for these factors, younger age and MGMT methylation remained independently associated with mLTSs. Younger age, better recursive partitioning analysis class, lack of SVZ involvement, and loss of 19q were associated with LTSs as compared with STSs. After adjustment, younger age and better preoperative Karnofsky performance scale (KPS) score remained independently associated with LTSs. Kaplan-Meier analyses revealed that younger age (<50 years), better preoperative KPS score (≥70), lack of SVZ involvement, and loss of 19q were associated with longer overall survival. In the multivariate analysis, only age was significantly associated with overall survival. Younger age and better preoperative KPS score were the characteristics associated with LTSs as compared with STSs. MGMT promoter methylation was associated with mLTSs, but not with LTSs. In addition, lack of SVZ involvement and loss of 19q might be prognostic for longer survival.

A novel truncated form of HMGA2 in tumors of the ovaries.

Neoplasms of the ovary are the second most common tumor of the female reproductive system, and the most lethal of the gynecological malignancies. Ovarian tumors are divided into a copious number of different groups reflecting their different features. The present study analyzed 187 ovarian tumors (39 sex-cord stromal tumors, 22 borderline tumors and 126 carcinomas) for the expression of the high-mobility group AT-hook 2 (HMGA2) gene, for mutations in the isocitrate dehydrogenase (NADP(+)) 1, cytosolic (IDH1), isocitrate dehydrogenase (NADP(+)) 2, mitochondrial (IDH2) and telomerase reverse transcriptase (TERT) genes, and for methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. Reverse transcription-polymerase chain reaction analysis showed that HMGA2 was expressed in 74.5% of the samples (120/161). A truncated transcript of HMGA2 was identified in 11 cases. A novel truncated form of HMGA2 was found in 4 serous high-grade carcinomas. Only 4 tumors (4/185) showed the TERT C228T mutation. No IDH1 or IDH2 mutations were found. Methylation of the promoter of MGMT was found in 2 borderline tumors (2/185). HMGA2 was expressed, in its truncated and native form, in different ovarian tumors, even the less aggressive types, underscoring the general importance of this gene in ovarian tumorigenesis. Mutations involving TERT, as well as MGMT promoter methylation, are rare events in ovarian tumors.

Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

New insights into acquired temozolomide resistance in glioblastoma?

This scientific commentary refers to ‘c-Myc–miR-29c–REV3L signalling pathway drives the acquisition of temozolomide resistance in glioblastoma’ by Luo et al. (doi:10.1093/brain/awv287) The emergence of tumour cell resistance to chemotherapy represents a major challenge for the development of durable therapeutic strategies across most solid cancers including glioblastoma, the most malignant primary brain tumour in adults. The standard of care for glioblastoma is maximum surgery as safely feasible followed by radiotherapy, with concomitant and maintenance chemotherapy with the alkylating agent temozolomide (TMZ/RT→TMZ). This results in a median overall survival in the range of 12 months on a population level (Weller et al. , 2014). Radiotherapy alone doubled median survival in early studies, but the best radiological response is commonly stable disease, progression is inevitable, and the efficacy of radiotherapy may be partly related to anti-angiogenic rather than intrinsic tumour cell cytotoxic effects. Mechanisms underlying resistance to radiotherapy remain poorly understood, but extensive hypoxia may be an important factor. TMZ was approved for the treatment of newly diagnosed glioblastoma because of a moderate prolongation of median survival (Stupp et al. , 2005). Subgroup analyses revealed that the benefit from TMZ was largely restricted to patients with glioblastomas that exhibit a particular epigenetic alteration, promoter methylation of the O6-methylguanine DNA methyltransferase ( MGMT ) gene (Hegi et al. , 2005). However, even these patients eventually all progress and succumb to their disease, in the absence of changes in MGMT promoter methylation …

P108: Participation of the genes MVP/LRP and YB-1 in the determination of the rate of sensitivity of glioblastoma cells to temodal

Background Glioblastomas (GBL) are most aggressive brain tumors in adults. They are often radio- and chemotherapy resistant. The standard therapy of GBL includes surgery followed by radiotherapy with concomitant chemotherapy with temozolomide named temodal (imidazotetrazine derivative). Methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene promoter is considered as predictor of better outcome of temodal therapy in comparison with the cases without MGMT promoter methylation (Stupp et al., 2009). The aim of this study is finding of additional predictors of temozolomide therapy effectiveness. Materials and methods We obtained primary cultures of GBL. The rate of GBL sensitivity to temozolomide was revealed by MTT test. Expression of multidrug resistance genes and genes which mediate temozolomide sensitivity (YB1, MDR1, MRP1, LRP, BCRP and MGMT) was determined by real time PCR. Protein localization and expression was revealed by immunohistochemical technique. Results We developed 14 primary GBL cultures. Staining for nestin was used for demonstration of neuronal origin of cell cultures. Analysis of gene expression and the rate of cell proliferation of the cultures showed that the rate of YB1 gene expression in slow proliferating cultures is significantly lower than in quickly proliferating cell populations. Cell cultures differed in temodal sensitivity 2–2.5-fold. We analyzed whether GBL cell sensitivity to temodal and the rate of several genes’ expression are correlated. We did not find the correlation between cell temodal sensitivity and the rate of MGMT gene expression. These data are in accordance with some results of another authors (Mullins et al., 2013; Brennan et al., 2013). However, we revealed positive correlation of the temodal sensitivity of GBL cultures and MVP/LRP expression (r = 0.5592, p = 0.04). We found also the trend to negative correlation between GBL temodal sensitivity and YB1 gene expression (r = −0.43602, p = 0.02) as well as MDR1 expression (r = −0.4195, p = 0.02). Conclusion The rate of temodal sensitivity of GBL primary cultures is connected with the rate of the expression of MVP/LRP, YB1 and MDR1genes. It is likely that YB-1 protein affects temodal sensitivity by influence on DNA reparation. YB1 could also have an effect on MDR1 expression. MVP/LRP expression is independent factor of GBL prognosis. This paper was completed with partial support from the Russian Foundation for Basic Research – Russia (Grant No. 23-04-40204 ).

Personalized therapy for gliomas

Current therapies for patients with malignant gliomas are starting to integrate molecular factors and age. Nonetheless, these therapies are still not sufficiently individualized. Some positive examples of transfer from basic science to clinical application are currently integrated into the standard treatment and guidelines. These are mainly genetic and other molecular factors that improve diagnosis and classification of gliomas and markers supporting prognostication. Examples for predictive biomarkers are methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter and the codeletion of chromosome arms 1p and 19q (1p/19q codel). The autoactive, truncated form of epidermal growth factor receptor (EGFRvIII) and the R132H mutation of isocitrate dehydrogenase 1 (IDH-1) are used as targets in currently running immunotherapeutic, targeted trials. Integration of functional imaging parameters into the monitoring and development of uniform assessment criteria improve the ability to evaluate therapy response and implement imaging biomarkers to guide therapies. As a result of the current efforts there are better classified prognostic groups and improved survival times with maintained functional and quality of life parameters in some glioma subgroups. Given the current dynamics, an improved, better differentiated classification of brain tumors including molecular parameters as well as more rational precise guiding of therapies with early, uniform response assessment is expected in the near future.