Exploiting the role of O6-methylguanine-DNA-methyltransferase (MGMT) in gastrointestinal cancers.

Gastrointestinal (GI) cancer is a prevalent disease and is recognized as the primary cause of cancer-related mortality globally. Therefore, there is an urgent need for novel diagnostic and treatment approaches for GC. The methylation of the O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter is a significant factor in the development of colorectal cancer (CRC), namely in roughly 30-40% of cases where the cancer has spread. MGMT plays a role in the repair of DNA damage caused by methylating drugs like temozolomide (TMZ) and chloroethylating compounds like carmustine. As a result, it contributes to the resistance of chemotherapy when these agents are utilized. Although MGMT's role in the development of CRC is well established, its prognostic significance remains a subject of debate. Only a limited number of research have been conducted to examine the prognostic significance of MGMT methylation, yielding varying outcomes. This review explores the structural functions and repair processes of MGMT, focusing on the putative structural and functional significance of the N-terminal domain of MGMT. It also investigates the advancement of cancer treatment techniques that specifically target MGMT.

Dose escalation with simultaneous integrated boost for un-methylated multiple glioblastoma.

Background: Simultaneous involvement of multiple distinct brain regions occurs in 2-5% of all high-grade gliomas (HGG) and is associated with poor prognosis. Whereas radiotherapy (RT) is an important and well-established treatment for high-grade glioma, the role of dose-escalated radiotherapy has yet to be established. In this case series, we report upon the dosimetry, adverse effects, and response in patients with multiple un-methylated high-grade gliomas receiving dose-escalated radiation. Materials and methods: We reviewed charts of patients with multifocal high grade glioma treated at our institution since January 2022. All patients had stereotactic biopsies after an magnetic resonance imaging (MRI) contrast-enhanced with T1, T2, FLAIR sequences and were discussed in a multidisciplinary oncology team. MGMT-positive patients received either TMZ alone or RT with TMZ and were excluded from this analysis. Un-methylated patients received dose-escalated RT without temezolamide (TMZ). Following computed tomography (CT) and MR simulation, the gros tumor volume (GTV) was delineated and prescribed 52.5 Gy in 15 fractions within the standard 40.05 Gy planning treatment volume (PTV). Treatment planning was volumetric modulated arc therapy. Results: A total of 20 patients with multiple un-methylated MGMT glioblastoma multiforme were treated with dose-escalated radiation therapy between January 2022 and June 2023. All patients completed dose escalated radiotherapy without acute adverse effects. Progression-free survival at six months was 85%, as defined by the RANO criteria. Conclusion: In this case series, we showed that un-methylated multiple high-grade glioma could be safely treated with dose escalation. Results of progression-free survival should be validated in a larger prospective clinical trial.

Unveiling hierarchy and spatial distribution of O6-methylguanine-DNA methyltransferase promoter methylation in World Health Organization grade 2-3 gliomas.

This study investigated the role of O6-methylguanine-DNA methyltransferase promoter (MGMTp) methylation hierarchy and heterogeneity in grade 2-3 gliomas, focusing on variations in chemotherapy benefits and resection dependency. A cohort of 668 newly diagnosed grade 2-3 gliomas, with comprehensive clinical, radiological, and molecular data, formed the basis of this analysis. The extent of resection was categorized into gross total resection (GTR ≥100%), subtotal resection (STR >90%), and partial resection (PR ≤90%). MGMTp methylation levels were examined using quantitative pyrosequencing. Our findings highlighted the critical role of GTR in improving the prognosis for astrocytomas (IDH1/2-mutant and 1p/19q non-codeleted), contrasting with its lesser significance for oligodendrogliomas (IDH1/2 mutation and 1p/19q codeletion). Oligodendrogliomas demonstrated the highest average MGMTp methylation levels (median: 28%), with a predominant percentage of methylated cases (average methylation levels >20%). Astrocytomas were more common in the low-methylated group (10%-20%), while IDH wild-type gliomas were mostly unmethylated (<10%). Spatial distribution analysis revealed a decrement in frontal lobe involvement from methylated, low-methylated to unmethylated cases (72.8%, 59.3%, and 47.8%, respectively). In contrast, low-methylated and unmethylated cases were more likely to invade the temporal-insular region (19.7%, 34.3%, and 40.4%, respectively). Astrocytomas with intermediate MGMTp methylation were notably associated with temporal-insular involvement, potentially indicating a moderate response to temozolomide and underscoring the importance of aggressive resection strategies. In conclusion, our study elucidates the complex interplay of MGMTp methylation hierarchy and heterogeneity among grade 2-3 gliomas, providing insights into why astrocytomas and IDH wild-type lower-grade glioma might derive less benefit from chemotherapy.

A simple and rapid in vitro assay for identification of direct inhibitors of O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that is overexpressed in certain tumors and is associated with resistance to the DNA alkylating agent temozolomide. MGMT inhibitors show potential in combating temozolomide resistance, but current assays for MGMT enzyme activity and inhibition, primarily oligonucleotide-based and fluorescent probe-based, are laborious and costly. The clinical relevance of temozolomide therapy calls for more convenient methodologies to study MGMT inhibition. Here, we extended the application of SNAP-Capture magnetic beads to develop a novel MGMT inhibition assay that demonstrated efficacy not only with known MGMT inhibitors, but also with the aldehyde dehydrogenase inhibitor, disulfiram. The assay uses standard fluorescence microscopy as a simple and reliable detection method, and is translationally applicable in drug discovery programs.

A cell line expressing MGMT-GFP fusion protein was generated. After harvesting the cells, the cell lysate was prepared and combined with SNAP-Capture magnetic beads and incubated at room temperature. Successful immobilization of MGMT-GFP on SNAP-Capture magnetic beads was verified by fluorescence microscopy. For the MGMT inhibition assay, the cell lysate underwent pre-treatment with established MGMT inhibitors before interaction with SNAP-capture magnetic beads and then underwent immobilization and fluorescence microscopy.

Tricarboxylic Acid Cycle Relationships with Non-Metabolic Processes: A Short Story with DNA Repair and Its Consequences on Cancer Therapy Resistance.

Metabolic changes involving the tricarboxylic acid (TCA) cycle have been linked to different non-metabolic cell processes. Among them, apart from cancer and immunity, emerges the DNA damage response (DDR) and specifically DNA damage repair. The oncometabolites succinate, fumarate and 2-hydroxyglutarate (2HG) increase reactive oxygen species levels and create pseudohypoxia conditions that induce DNA damage and/or inhibit DNA repair. Additionally, by influencing DDR modulation, they establish direct relationships with DNA repair on at least four different pathways. The AlkB pathway deals with the removal of N-alkylation DNA and RNA damage that is inhibited by fumarate and 2HG. The MGMT pathway acts in the removal of O-alkylation DNA damage, and it is inhibited by the silencing of the MGMT gene promoter by 2HG and succinate. The other two pathways deal with the repair of double-strand breaks (DSBs) but with opposite effects: the FH pathway, which uses fumarate to help with the repair of this damage, and the chromatin remodeling pathway, in which oncometabolites inhibit its repair by impairing the homologous recombination repair (HRR) system. Since oncometabolites inhibit DNA repair, their removal from tumor cells will not always generate a positive response in cancer therapy. In fact, their presence contributes to longer survival and/or sensitization against tumor therapy in some cancer patients.

Stanniocalcin-1 promotes temozolomide resistance of glioblastoma through regulation of MGMT.

Temozolomide (TMZ) resistance is a major challenge in the treatment of glioblastoma (GBM). Tumour reproductive cells (TRCs) have been implicated in the development of chemotherapy resistance. By culturing DBTRG cells in three-dimensional soft fibrin gels to enrich GBM TRCs and performing RNA-seq analysis, the expression of stanniocalcin-1 (STC), a gene encoding a secreted glycoprotein, was found to be upregulated in TRCs. Meanwhile, the viability of TMZ-treated TRC cells was significantly higher than that of TMZ-treated 2D cells. Analysis of clinical data from CGGA (Chinese Glioma Genome Atlas) database showed that high expression of STC1 was closely associated with poor prognosis, glioma grade and resistance to TMZ treatment, suggesting that STC1 may be involved in TMZ drug resistance. The expression of STC1 in tissues and cells was examined, as well as the effect of STC1 on GBM cell proliferation and TMZ-induced DNA damage. The results showed that overexpression of STC1 promoted and knockdown of STC1 inhibited TMZ-induced DNA damage. These results were validated in an intracranial tumour model. These data revealed that STC1 exerts regulatory functions on MGMT expression in GBM, and provides a rationale for targeting STC1 to overcome TMZ resistance.

Current chemotherapy strategies for adults with IDH-wildtype glioblastoma.

Introduction: Glioblastoma, despite advancements in molecular evolution, remains incurable and has low survival rates. Currently, two of the most commonly used chemotherapy regimens are temozolomide and CCNU. This review aims to provide a comprehensive analysis of the current status of chemotherapy strategies for GBM. Methods: We reviewed the published literature describing the chemotherapy regimen differences in system treatment of GBM reported in the last ten years and summarised the available information that may reveal the latest changes in chemotherapy. Results: In patients with adequate functioning, temozolomide and radiation are the primary treatments for newly diagnosed GBM. We recommend postoperative radiation therapy with concurrent and adjuvant temozolomide for patients with MGMT-methylated GBM who are less than 70 years old. Combining temozolomide and lomustine with radiation therapy may be an option for younger, fit patients, but efficacy data is inconclusive. For patients with unknown MGMT methylation status, radiation therapy combined with temozolomide remains the standard of care. We recommend hypofractionated radiation and concurrent temozolomide treatment for elderly patients over 70 years old who have satisfactory performance and no significant underlying health conditions. We should tailor treatment choices to each patient's personal preferences, previous treatments, function, quality of life, and overall care objectives. Conclusion: Radiation therapy, along with temozolomide, is still the standard of care for most people with MGMT-unmethylated GBMs because there aren't any better options, and it's generally safe and well-tolerated. These patients have a lower overall survival rate and less benefit from temozolomide, but there are no better alternatives. Clinical trial participation is encouraged.

Glioma Grade and Molecular Markers: Comparing Machine-Learning Approaches Using VASARI (Visually AcceSAble Rembrandt Images) Radiological Assessment.

OBJECTIVES: This study aimed to leverage Visually AcceSAble Rembrandt Images (VASARI) radiological features, extracted from magnetic resonance imaging (MRI) scans, and machine-learning techniques to predict glioma grade, isocitrate dehydrogenase (IDH) mutation status, and O6-methylguanine-DNA methyltransferase (MGMT) methylation. METHODOLOGY: A retrospective evaluation was undertaken, analyzing MRI and molecular data from 107 glioma patients treated at a tertiary hospital. Patients underwent MRI scans using established protocols and were evaluated based on VASARI criteria. Tissue samples were assessed for glioma grade and underwent molecular testing for IDH mutations and MGMT methylation. Four machine learning models, namely, Random Forest, Elastic-Net, multivariate adaptive regression spline (MARS), and eXtreme Gradient Boosting (XGBoost), were trained on 27 VASARI features using fivefold internal cross-validation. The models' predictive performances were assessed using the area under the curve (AUC), sensitivity, and specificity. RESULTS: For glioma grade prediction, XGBoost exhibited the highest AUC (0.978), sensitivity (0.879), and specificity (0.964), with f6 (proportion of non-enhancing) and f12 (definition of enhancing margin) as the most important predictors. In predicting IDH mutation status, XGBoost achieved an AUC of 0.806, sensitivity of 0.364, and specificity of 0.880, with f1 (tumor location), f12, and f30 (perpendicular diameter to f29) as primary predictors. For MGMT methylation, XGBoost displayed an AUC of 0.580, sensitivity of 0.372, and specificity of 0.759, highlighting f29 (longest diameter) as the key predictor. CONCLUSIONS: This study underscores the robust potential of combining VASARI radiological features with machine learning models in predicting glioma grade, IDH mutation status, and MGMT methylation. The best and most balanced performance was achieved using the XGBoost model. While the prediction of glioma grade showed promising results, the sensitivity in discerning IDH mutations and MGMT methylation still leaves room for improvement. Follow-up studies with larger datasets and more advanced artificial intelligence techniques can further refine our understanding and management of gliomas.

Unveiling the role of O(6)-methylguanine-DNA methyltransferase in cancer therapy: insights into alkylators, pharmacogenomics, and others.

Cancer chemotherapy is advancing as we understand how cellular mechanisms and drugs interact, particularly involving the enzyme MGMT, which repairs DNA damage that can cause cancer. This review examines MGMT's role in DNA repair, its impact on chemotherapy, and its complex interaction with radiation therapy. MGMT activity can both protect against mutations and cause drug resistance. Modulating MGMT could improve treatment efficacy and tailoring therapy to MGMT status may enhance patient outcomes. Understanding MGMT is crucial for developing precise cancer treatments and advancing patient care.

MGMT promoter methylation is a strong prognostic factor for survival after progression in high-grade gliomas.

BACKGROUND: High-grade gliomas (HGGs) have a rapid relapse and short survival. Studies have identified many clinical characteristics and biomarkers associated with progression-free survival (PFS) and over-survival (OS). However, there has not yet a comprehensive study on survival after the first progression (SAP). METHODS: From CGGA and TCGA, 319 and 308 HGGs were confirmed as the first progression. The data on clinical characteristics and biomarkers were analyzed in accordance with OS, PFS, and SAP. RESULTS: Analysis of 319 patients from CGGA, significant predictors of improved OS/PFS/SAP were WHO grade, MGMT promoter methylation, and Ki-67 expression in univariate analysis. Further multivariate analysis showed MGMT promoter methylation and Ki-67 expression were independent predictors. However, an analysis of 308 patients from TCGA found MGMT promoter methylation is the only prognostic marker. A longer SAP was observed in patients with methylated MGMT promoter after standard chemoradiotherapy. In our data, HGGs could be divided into low, intermediate, and high-risk groups for SAP by MGMT methylation and Ki-67 expression. CONCLUSIONS: Patients with MGMT promoter methylation have a prolonger SAP after standard chemoradiotherapy. HGGs could be divided into low, intermediate, and high-risk groups for SAP according to MGMT status and Ki-67 expression.

Challenges and future perspectives for the use of temozolomide in the treatment of SCLC.

Small-cell lung cancer (SCLC), accounting for 10-20 % of all lung tumors, represents the most aggressive high-grade neuroendocrine carcinoma. Most patients are diagnosed with extensive-stage SCLC (ES-SCLC), with brian metastases identified in âˆ¼ 80 % of cases during the disease cours, and the prognosis is dismal, with a 5-year survival rate of less than 5 %. Current available treatments in the second-line setting are limited, and topotecan has long been the only FDA-approved drug in relapsed or refractory ES-SCLC, until the recent approval of lurbinectedin, a selective inhibitor of RNA polymerase II. Temozolomide (TMZ) is an oral alkylating agent, which showed single-agent activity in SCLC, particularly among patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. Several studies have revealed the synergistic activity of temozolomide with poly-ADP-ribose polymerase (PARP) inhibitors, that prevent repair of TMZ-induced DNA damage. This review focuses on the rationale for the use of TMZ in ES-SCLC and provides an overview of the main trials that have evaluated and are currently investigating its role, both as a single-agent and in combinations, in relapse or refractory disease.

Gender differences in gliomas: From epidemiological trends to changes at the hormonal and molecular levels.

Gender plays a crucial role in the occurrence and development of cancer, as well as in the metabolism of nutrients and energy. Men and women display significant differences in the incidence, prognosis, and treatment response across various types of cancer, including certain sex-specific tumors. It has been observed that male glioma patients have a higher incidence and worse prognosis than female patients, but there is currently a limited systematic evaluation of sex differences in gliomas. The purpose of this study is to provide an overview of the association between fluctuations in sex hormone levels and changes in their receptor expression with the incidence, progression, treatment, and prognosis of gliomas. Estrogen may have a protective effect on glioma patients, while exposure to androgens increases the risk of glioma. We also discussed the specific genetic and molecular differences between genders in terms of the malignant nature and prognosis of gliomas. Factors such as TP53, MGMT methylation status may play a crucial role. Therefore, it is essential to consider the gender of patients while treating glioma, particularly the differences at the hormonal and molecular levels. This approach can help in the adoption of an individualized treatment strategy.

Adaptive fine-tuning based transfer learning for the identification of MGMT promoter methylation status.

Background.Glioblastoma Multiforme (GBM) is an aggressive form of malignant brain tumor with a generally poor prognosis.O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been shown to be a predictive bio-marker for resistance to treatment of GBM, but it is invasive and time-consuming to determine methylation status. There has been effort to predict the MGMT methylation status through analyzing MRI scans using machine learning, which only requires pre-operative scans that are already part of standard-of-care for GBM patients.Purpose.To improve the performance of conventional transfer learning in the identification of MGMT promoter methylation status, we developed a 3D SpotTune network with adaptive fine-tuning capability. Using the pretrained weights of MedicalNet with the SpotTune network, we compared its performance with a randomly initialized network for different combinations of MR modalities.Methods.Using a ResNet50 as the base network, three categories of networks are created: (1) A 3D SpotTune network to process volumetric MR images, (2) a network with randomly initialized weights, and (3) a network pre-trained on MedicalNet. These three networks are trained and evaluated using a public GBM dataset provided by the University of Pennsylvania. The MRI scans from 240 patients are used, with 11 different modalities corresponding to a set of perfusion, diffusion, and structural scans. The performance is evaluated using 5-fold cross validation with a hold-out testing dataset.Results.The SpotTune network showed better performance than the randomly initialized network. The best performing SpotTune model achieved an area under the Receiver Operating Characteristic curve (AUC), average precision of the precision-recall curve (AP), sensitivity, and specificity values of 0.6604, 0.6179, 0.6667, and 0.6061 respectively.Conclusions.SpotTune enables transfer learning to be adaptive to individual patients, resulting in improved performance in predicting MGMT promoter methylation status in GBM using equivalent MRI modalities as compared to a randomly initialized network.

Unveiling the role of TAGLN2 in glioblastoma: From proneural-mesenchymal transition to Temozolomide resistance.

Glioblastoma (GBM) presents a daunting challenge due to its resistance to temozolomide (TMZ), a hurdle exacerbated by the proneural-to-mesenchymal transition (PMT) from a proneural (PN) to a mesenchymal (MES) phenotype. TAGLN2 is prominently expressed in GBM, particularly in the MES subtype compared to low-grade glioma (LGG) and the PN subtype. Our research reveals TAGLN2's involvement in PMT and TMZ resistance through a series of in vitro and in vivo experiments. TAGLN2 knockdown can restrain proliferation and invasion, trigger DNA damage and apoptosis, and heighten TMZ sensitivity in GBM cells. Conversely, elevating TAGLN2 levels amplifies resistance to TMZ in cellular and intracranial xenograft mouse models. We demonstrate the interaction relationship between TAGLN2 and ERK1/2 through co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrometry analysis. Knockdown of TAGLN2 results in a decrease in the expression of p-ERK1/2, whereas overexpression of TAGLN2 leads to an increase in p-ERK1/2 expression within the nucleus. Subsequently, the regulatory role of TAGLN2 in the expression and control of MGMT has been demonstrated. Finally, the regulation of TAGLN2 by NF-κB has been validated through chromatin immunoprecipitation and ChIP-PCR assays. In conclusion, our results confirm that TAGLN2 exerts its biological functions by interacting with the ERK/MGMT axis and being regulated by NF-κB, thereby facilitating the acquisition of promoting PMT and increased resistance to TMZ therapy in glioblastoma. These results provide valuable insights for the advancement of targeted therapeutic approaches to overcome TMZ resistance in clinical treatments.

MGMT activated by Wnt pathway promotes cisplatin tolerance through inducing slow-cycling cells and nonhomologous end joining in colorectal cancer.

Chemotherapy resistance plays a pivotal role in the prognosis and therapeutic failure of patients with colorectal cancer (CRC). Cisplatin (DDP)-resistant cells exhibit an inherent ability to evade the toxic chemotherapeutic drug effects which are characterized by the activation of slow-cycle programs and DNA repair. Among the elements that lead to DDP resistance, O 6-methylguanine (O 6-MG)-DNA-methyltransferase (MGMT), a DNA-repair enzyme, performs a quintessential role. In this study, we clarify the significant involvement of MGMT in conferring DDP resistance in CRC, elucidating the underlying mechanism of the regulatory actions of MGMT. A notable upregulation of MGMT in DDP-resistant cancer cells was found in our study, and MGMT repression amplifies the sensitivity of these cells to DDP treatment in vitro and in vivo. Conversely, in cancer cells, MGMT overexpression abolishes their sensitivity to DDP treatment. Mechanistically, the interaction between MGMT and cyclin dependent kinase 1 (CDK1) inducing slow-cycling cells is attainted via the promotion of ubiquitination degradation of CDK1. Meanwhile, to achieve nonhomologous end joining, MGMT interacts with XRCC6 to resist chemotherapy drugs. Our transcriptome data from samples of 88 patients with CRC suggest that MGMT expression is co-related with the Wnt signaling pathway activation, and several Wnt inhibitors can repress drug-resistant cells. In summary, our results point out that MGMT is a potential therapeutic target and predictive marker of chemoresistance in CRC.

MEN1 Deficiency-Driven Activation of the ß-Catenin-MGMT Axis Promotes Pancreatic Neuroendocrine Tumor Growth and Confers Temozolomide Resistance.

O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the guanine O6 position (O6-MG) and repairs DNA damage. High MGMT expression results in poor response to temozolomide (TMZ). However, the biological importance of MGMT and the mechanism underlying its high expression in pancreatic neuroendocrine tumors (PanNETs) remain elusive. Here, it is found that MGMT expression is highly elevated in PanNET tissues compared with paired normal tissues and negatively associated with progression-free survival (PFS) time in patients with PanNETs. Knocking out MGMT inhibits cancer cell growth in vitro and in vivo. Ectopic MEN1 expression suppresses MGMT transcription in a manner that depends on ß-Catenin nuclear export and degradation. The Leucine 267 residue of MEN1 is crucial for regulating ß-Catenin-MGMT axis activation and chemosensitivity to TMZ. Interference with ß-Catenin re-sensitizes tumor cells to TMZ and significantly reduces the cytotoxic effects of high-dose TMZ treatment, and MGMT overexpression counteracts the effects of ß-Catenin deficiency. This study reveals the biological importance of MGMT and a new mechanism by which MEN1 deficiency regulates its expression, thus providing a potential combinational strategy for treating patients with TMZ-resistant PanNETs.

Prognosis of MGMT -Positive Gene in Patients with Brain Tumors of Grade III and Grade IV.

Sumit GoyalObjective To evaluate MGMT gene positivity is associated with better survival in patients diagnosed with brain tumor World Health Organization (WHO) grades III and IV Material and Methods Single-institute restrospective study. A total of 80 patients were enrolled, all underwent surgery either total or subtotal excision of the tumor and MGMT gene testing on tumor tissue by RT-PCR. All received adjuvant radiation (60 Gy/30 fractions, 5 fractions/week) with concurrent temozolomide (75 mg/m 2 ), followed by 12 cycles of adjuvant temozolomide (150 mg/m 2 1st cycle followed by 200 mg/m 2 ) with regular follow-up. Results A total of 80 patients, 75 underwent subtotal excision, 27 were WHO grade III vs. 48 WHO grade IV. Five underwent total excision 1 was WHO grade III vs. 4 WHO grade IV. The median PFS and OS in five patients in total excision in grade III patient was 9.0 and 20 compared with Grade IV, where the median PFS and OS was 8.8 and 17.8 months. Out of 75 patients in the subtotal group median PFS and OS, respectively, in Grade III group was 9.1 and 19.3 and, WHO grade IV with median PFS of 8.8 and OS of 18.8. Conclusion MGMT gene positivity is a prognostic factor in grade III and IV brain tumor.

No benefit from TMZ treatment in GB with truly unmethylated MGMT promoter: Reanalysis of the CE.6 and the pooled Nordic/NOA-08 trials in elderly GB patients.

BACKGROUND: The treatment of elderly/ frail patients with glioblastoma is a balance between avoiding undue toxicity, while not withholding effective treatment. It remains debated, whether these patients should receive combined chemo-radiotherapy with temozolomide (RT/TMZ➜TMZ) regardless of the O6-methylguanine DNA methyltransferase gene promoter (MGMTp) methylation status. MGMT is a well-known resistance factor blunting the treatment effect of TMZ, by repairing the most genotoxic lesion. Epigenetic silencing of the MGMTp sensitizes glioblastoma to TMZ. For risk adapted treatment, it is of utmost importance to accurately identify patients, who will not benefit from TMZ treatment. METHODS: Here, we present a reanalysis of the clinical trials CE.6 and the pooled NOA-08 and Nordic trials in elderly glioblastoma patients that compared RT to RT/TMZ➜TMZ, or RT to TMZ, respectively. For 687 patients with available MGMTp methylation data, we applied a cutoff discerning truly unmethylated glioblastoma, established in a pooled analysis of four clinical trials for glioblastoma, with RT/TMZ➜TMZ treatment, using the same quantitative methylation specific MGMTp PCR assay. RESULTS: When applying this restricted cutoff to the elderly patient population, we confirmed that glioblastoma with truly unmethylated MGMTp derived no benefit from TMZ treatment. In the Nordic/NOA-08 trials RT was better than TMZ, suggesting little or no benefit from TMZ. CONCLUSION: For evidence-based treatment of glioblastoma patients validated MGMTp methylation assays should be used that accurately identify truly unmethylated patients. Respective stratified management of patients will reduce toxicity without compromising outcome and allow testing of more promising treatment options.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory.

Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.