O6-methylguanine DNA Methyltransferase Methylation Research Articles

Principles in the Management of Glioblastoma.

Glioblastoma, the most aggressive and common malignant primary brain tumour, is characterized by infiltrative growth, abundant vascularization, and aggressive clinical evolution. Patients with glioblastoma often face poor prognoses, with a median survival of approximately 15 months. Technological progress and the subsequent improvement in understanding the pathophysiology of these tumours have not translated into significant achievements in therapies or survival outcomes for patients. Progress in molecular profiling has yielded new omics data for a more refined classification of glioblastoma. Several typical genetic and epigenetic alterations in glioblastoma include mutations in genes regulating receptor tyrosine kinase (RTK)/rat sarcoma (RAS)/phosphoinositide 3-kinase (PI3K), p53, and retinoblastoma protein (RB) signalling, as well as mutation of isocitrate dehydrogenase (IDH), methylation of O6-methylguanine-DNA methyltransferase (MGMT), amplification of epidermal growth factor receptor vIII, and codeletion of 1p/19q. Certain microRNAs, such as miR-10b and miR-21, have also been identified as prognostic biomarkers. Effective treatment options for glioblastoma are limited. Surgery, radiotherapy, and alkylating agent chemotherapy remain the primary pillars of treatment. Only promoter methylation of the gene MGMT predicts the benefit from alkylating chemotherapy with temozolomide and it guides the choice of first-line treatment in elderly patients. Several targeted strategies based on tumour-intrinsic dominant signalling pathways and antigenic tumour profiles are under investigation in clinical trials. This review explores the potential genetic and epigenetic biomarkers that could be deployed as analytical tools in the diagnosis and prognostication of glioblastoma. Recent clinical advancements in treating glioblastoma are also discussed, along with the potential of liquid biopsies to advance personalized medicine in the field of glioblastoma, highlighting the challenges and promises for the future.

Abstract CT230: Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments

Abstract CheckMate 548 (NCT02667587, CM548) is a phase III, randomized trial evaluating first line nivolumab (NIVO) in combination with standard of care (SOC) [surgical resection followed by radiotherapy and temozolomide (TMZ)] versus placebo plus SOC in patients with newly diagnosed glioblastoma (GBM) positive for promoter methylation of O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation status and PD-L1 expression predict better outcomes in GBM patients treated with TMZ and in patients treated with NIVO in other indications, respectively. However, the impact of varying levels of MGMT promoter methylation and PD-L1 expression on patient outcome in GBM remains unclear. In CM548, MGMT promoter methylation levels were quantified through a central lab assay (LapCorp) and PD-L1 expression was determined by percentage of detectable membrane staining of tumor cells. MGMT methylation and PD-L1 data were prospectively generated at the time of enrolment. Our analyses showed that increasing levels of MGMT promoter methylation level were associated with longer overall survival in patients treated with SOC alone (n = 349). However, no survival benefit was observed with increasing levels of MGMT methylation in patients treated with NIVO in combination with SOC (n = 353). In addition, our data showed a non-linear relationship between PD-L1 expression and overall survival across both arms. In patients with intermediate PD-L1 expression (≥5%, <20%), longer survival was observed in the nivolumab + SOC arm compared to SOC alone. This benefit was not observed in patients with low (≥1%, <5%) and high (>20%) PD-L1 expression. Potential mechanisms driving these observed associations are currently explored using genomic data generated from patient samples. MGMT promoter methylation and PD-L1 expression are potential clinically relevant predictive biomarkers of response to treatments administered in the CM-548; however, the binary classification of positive/negative status of these markers does not fully capture their relationship with efficacy in CM548. Data generated from our exploratory analyses suggests that new, refined criteria are needed for better patient outcomes prediction. Citation Format: Yu-Han Hung, Aparna Chhibber, Michael Lim, Michael Weller, David A. Reardon, Antonio Omuro, Keith L. Ligon, Ana Lako, Ann Forslund. Exploratory analyses of MGMT promoter methylation and PD-L1 expression from newly diagnosed glioblastoma patients in CheckMate 548 study suggest refinement in thresholds for predicting response to standard of care and nivolumab treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT230.

Practice Patterns for Managing Recurrent Glioblastoma Multiforme

Abstract Introduction Glioblastoma multiforme (GBM) is a devastating form of cancer with a poor prognosis despite available treatments. Managing recurrent GBM remains challenging and lacks guidelines. This study aims to provide practice patterns for managing recurrent GBMs in India. Methods A panel of experts was assembled to develop practice patterns using the Delphi technique. Their responses were analyzed anonymously to ensure impartiality and generate recommendations. The statements were intended to be nonbinding and focused on promoting best practices in the field, without legal or regulatory authority. Results A total of 23 experts participated in the study, providing their opinions on various aspects of managing recurrent GBM. Consensus was achieved on individualized and multidisciplinary management as the preferred approach. Surgery in combination with other treatments was found to impact survival in patients older than 65 years, with re-surgery and adjuvant radiation and chemotherapy being the preferred options. Gadolinium-enhanced magnetic resonance imaging (MRI) brain with spectroscopy and diffusion-weighted imaging was favored. Molecular profiling was considered significant, with O6-methylguanine DNA methyltransferase methylation being most relevant. Surgery was recommended for recurrent GBMs, primarily based on Karnofsky's performance score (KPS). Surgical adjuncts such as neuronavigation and intraoperative MRI were considered valuable. Radiation therapy, specifically stereotactic radiosurgery, was recommended for selected cases, while opinions on re-chemotherapy were divided. Palliative care was deemed important. Conclusion This study presents practice patterns for managing recurrent GBM in India, providing standardized recommendations for practice. By implementing these, clinicians can make informed decisions, leading to improved patient outcomes and reduced variability in the management of recurrent GBM.

Assessment of The Utility of The Sarcoma DNA Methylation Classifier In Surgical Pathology.

Diagnostic classification of soft tissue tumors is based on histology, immunohistochemistry, genetic findings, and radiologic and clinical correlations. Recently, a sarcoma DNA methylation classifier was developed, covering 62 soft tissue and bone tumor entities. The classifier is based on large-scale analysis of methylation sites across the genome. It includes DNA copy number analysis and determines O 6 methylguanine DNA methyl-transferase methylation status. In this study, we evaluated 619 well-studied soft tissue and bone tumors with the sarcoma classifier. Problem cases and typical examples of different entities were included. The classifier had high sensitivity and specificity for fusion sarcomas: Ewing, synovial, CIC -rearranged, and BCOR -rearranged. It also performed well for leiomyosarcoma, malignant peripheral nerve sheath tumors (MPNST), and malignant vascular tumors. There was low sensitivity for diagnoses of desmoid fibromatosis, neurofibroma, and schwannoma. Low specificity of matches was observed for angiomatoid fibrous histiocytoma, inflammatory myofibroblastic tumor, Langerhans histiocytosis, schwannoma, undifferentiated sarcoma, and well-differentiated/dedifferentiated liposarcoma. Diagnosis of lipomatous tumors was greatly assisted by the detection of MDM2 amplification and RB1 loss in the copy plot. The classifier helped to establish diagnoses for KIT-negative gastrointestinal stromal tumors, MPNSTs with unusual immunophenotypes, and undifferentiated melanomas. O 6 methylguanine DNA methyl-transferase methylation was infrequent and most common in melanomas (35%), MPNSTs (11%), and undifferentiated sarcomas (11%). The Sarcoma Methylation Classifier will likely evolve with the addition of new entities and refinement of the present methylation classes. The classifier may also help to define new entities and give new insight into the interrelationships of sarcomas.

Label-Free Raman Spectromicroscopy Unravels the Relationship between MGMT Methylation and Intracellular Lipid Accumulation in Glioblastoma.

Temozolomide (TMZ) is considered a first line chemotherapy drug for glioblastoma (GBM). Unfortunately, the GBM without methylation of O6-methylguanine-DNA methyltransferase (MGMT), accounting for about 70% of all GBM, shows an inherent resistance to TMZ treatment. Aberrant accumulation of neutral lipids, primarily triglycerides (TGs) and cholesteryl esters (CEs), in lipid droplets (LDs) has been recognized as metabolic vulnerability for GBM therapy. However, it is not known whether MGMT methylation affects lipid accumulation in GBM. Herein, we employed label-free Raman spectromicroscopy, which integrated stimulated Raman scattering (SRS) microscopy and confocal Raman spectroscopy, to quantitatively analyze both the amount and composition of intracellular LDs in intact GBM tissues obtained from patients who had undergone resection surgery. Our results showed significant reductions in both the LD amount and the CE percentage in MGMT unmethylated GBMs (MGMT methylation < 15%) compared to MGMT methylated ones (MGMT methylation ≥ 15%). Due to a big variation of lipid accumulation in the MGMT methylated GBMs, these patients were further divided into hypermethylated group (MGMT methylation ≥ 50%) and intermediate-methylated group (MGMT methylation 15∼50%), according to the significantly different median survival rates of these two groups. Remarkable differences in LD amount, CE percentage, and also lipid saturation degree were found between the hypermethylated group and the other two groups, but not between the unmethylated and intermediate-methylated groups. To elucidate the possible underlying mechanism, we analyzed the differential expression of lipid metabolism-related genes in GBM with different levels of MGMT methylation using The Cancer Genome Atlas Program (TCGA) dataset. It was shown that the genes related to lipid oxidation and lipid efflux were upregulated, and the genes related to lipid synthesis were downregulated in unmethylated group. These findings unravel the relationship between MGMT methylation and lipid accumulation in GBM, which may offer new opportunities for the diagnosis and treatment of TMZ-resistant GBM.

Survival in a consecutive series of 467 glioblastoma patients: Association with prognostic factors and treatment at recurrence at two independent institutions.

Therapy of recurrent glioblastoma (GBM) is challenging due to lack of standard treatment. We investigated physicians' treatment choice at recurrence and prognostic and predictive factors for survival in GBM patients from Norway's two largest regional hospitals. Clinicopathological data from n = 467 patients treated at Haukeland and Oslo university hospitals from January 2015 to December 2017 was collected. Data included tumour location, promoter methylation of O6 methylguanine-DNA methyltransferase (MGMT) and mutation of isocitrate dehydrogenase (IDH), patient age, sex, extent of resection at primary diagnosis and treatment at successive tumour recurrences. Cox-proportional hazards regression adjusting for multiple risk factors was used. Median overall survival (OS) was 12.1 months and 21.4% and 6.8% of patients were alive at 2 and 5 years, respectively. Median progression-free survival was 8.1 months. Treatment at recurrence varied but was not associated with difference in overall survival (OS) (p = 0.201). Age, MGMT hypermethylation, tumour location and extent of resection were independent prognostic factors. Patients who received 60 Gray radiotherapy with concomitant and adjuvant temozolomide at primary diagnosis had 16.1 months median OS and 9.3% were alive at 5 years. Patients eligible for gamma knife/stereotactic radiosurgery alone or combined with chemotherapy at first recurrence had superior survival compared to chemotherapy alone (p<0.001). At second recurrence, combination chemotherapy with or without bevacizumab were both superior to no treatment. Treatment at recurrence differed between the institutions but there was no difference in median OS, indicating that it is the disease biology that dictates patient outcome.

Lack of Benefit of Extending Temozolomide Treatment in Patients with High Vascular Glioblastoma with Methylated MGMT.

Simple SummaryDespite the complete treatment with surgery, chemotherapy and radiotherapy, patients with glioblastoma have a devasting prognosis. Although the role of extending temozolomide treatment has been explored, the results are inconclusive. Recent evidence suggested that tumor vascularity may be a modulating factor in combination with methylation of O6-methylguanine-DNA methyltransferase (MGMT) promotor gene on the effect of temozolomide-based therapies, opening new possibilities for personalized treatments. Before proposing a prospective interventional clinical study, it is necessary to confirm the beneficial effect of the combined effect of MGMT methylation and moderate tumor vascularity, as well as the lack of benefit of temozolomide in patients with a highly vascular tumor.In this study, we evaluated the benefit on survival of the combination of methylation of O6-methylguanine-DNA methyltransferase (MGMT) promotor gene and moderate vascularity in glioblastoma using a retrospective dataset of 123 patients from a multicenter cohort. MRI processing and calculation of relative cerebral blood volume (rCBV), used to define moderate- and high-vascular groups, were performed with the automatic ONCOhabitats method. We assessed the previously proposed rCBV threshold (10.7) and the new calculated ones (9.1 and 9.8) to analyze the association with survival for different populations according to vascularity and MGMT methylation status. We found that patients included in the moderate-vascular group had longer survival when MGMT is methylated (significant median survival difference of 174 days, p = 0.0129*). However, we did not find significant differences depending on the MGMT methylation status for the high-vascular group (p = 0.9119). In addition, we investigated the combined correlation of MGMT methylation status and rCBV with the prognostic effect of the number of temozolomide cycles, and only significant results were found for the moderate-vascular group. In conclusion, there is a lack of benefit of extending temozolomide treatment for patients with high vascular glioblastomas, even presenting MGMT methylation. Preliminary results suggest that patients with moderate vascularity and methylated MGMT glioblastomas would benefit more from prolonged adjuvant chemotherapy.

Prognostic Impact of the Combination of MGMT Methylation and TERT Promoter Mutation in Glioblastoma.

Background  The concept of combinational analysis between the methylation of O 6 -methylguanine-DNA methyltransferase ( MGMT ) and telomerase reverse transcriptase promoter ( pTERT ) mutation in glioblastoma (GBM) has been reported. The main study objective was to determine the prognosis of patients with GBM based on MGMT/pTERT classification, while the secondary objective was to estimate the temozolomide effect on the survival time of GBM with MGMT/pTERT classification. Methods  A total of 50 GBM specimens were collected after tumor resection and were selected for investigating MGMT methylation and pTERT mutation. Clinical imaging and pathological characteristics were retrospectively analyzed. Patients with MGMT/pTERT classification were analyzed using survival analysis to develop the nomogram for forecasting and individual prognosis. Results  All patients underwent resection (total resection: 28%, partial resection: 64%, biopsy: 8%). Thirty-two percent of all cases received adjuvant temozolomide with radiotherapy. Sixty-four percent of the case was found methylated MGMT , and 56% of the present cohort found pTERT mutation. Following combinational analysis of biomarkers, results showed that the GBMs with methylated MGMT and wild-type pTERT had a superior prognosis compared with other subtypes. Using Cox regression analysis with multivariable analysis, the extent of resection, postoperative chemoradiotherapy, MGMT/pTERT classification were associated with a favorable prognosis. Hence, a web-based nomogram was developed for deploying individual prognostication. Conclusions  The interaction of MGMT methylation and pTERT mutation was confirmed for predicting prognosis. The results from the present study could help physicians create treatment strategies for GBM patients in real-world situations.

Organochlorine pesticides induce promoter hypermethylation of MGMT in papillary thyroid carcinoma

BackgroundEnvironmental factors namely pesticides may contribute to the development of cancer through epigenetic mechanisms. In order to evaluate the impact of organochlorine pesticides (OCPs) on promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT) in thyroid cancer, this research was designed. MethodsIn this study, DNA methylation profile of 61 papillary thyroid carcinoma (PTC) and 70 benign thyroid nodules (BTN) subjects was carried out. In order to measure MGMT gene promoter methylation, methylation-specific PCR (MSP) was used. ResultsHypermethylation of MGMT marker were found in PTC compare with BTN. Also, OCPs increased the odds ratio of methylation of MGMT (OR = 3.66, P = 0.001). Logistic regression analysis showed that OCPs enhance the methylation of MGMT. ConclusionThe results indicated that OCPs lead to PTC development through MGMT gene promoter hypermethylation. Furthermore, MGMT promoter methylation assessment could forecast PTC incidence.

Narrative review of palliative hypofractionated radiotherapy for high grade glioma.

High grade gliomas (HGG) include World Health Organization (WHO) grade III anaplastic astrocytoma (AA) and WHO grade IV glioblastoma (GBM). As genomic alterations are prognostic, even WHO grade II, IDH-wildtype gliomas may be considered as HGG. Current management of HGG include best supportive care (BSC), surgery, radiation therapy (RT), chemotherapy, and a combination. Elderly patients (defined here as age ≥65) with GBM have significantly worse survival compared to younger patients. Similarly, patients with poor performance status [defined as Karnofsky performance status (KPS) <60 or ECOG performance status (PS) >2], regardless of age have worse outcomes. The standard of care for treatment of HGG involves surgery and chemoradiation. However, the optimal treatment in terms of efficacy, safety and maintaining quality of life (QoL), remains a matter of debate in the elderly and/or poor performing patients due to their worse prognosis. Less aggressive interventions are usually reserved for these patients despite surgery providing a survival and neurologic benefit. Improved survival has been noted in elderly patients treated with RT in comparison with those receiving best supportive care (BSC) alone, with similar survival for patients undergoing standard RT (60 Gy/30 fractions) and hypofractionated RT (25-40 Gy in 5-15 daily fractions). An alkylating agent, temozolomide (TMZ), represents a safe and effective option in select patients with promoter methylation of O6-methylguanine-DNA-methyltransferase (MGMT) gene. A recent phase III randomized trial for GBM patients (age ≥65 years, ECOG PS 0-2) demonstrated a significant improvement in progression-free survival (PFS) and overall survival (OS) with hypofractionated RT (40 Gy/15 fractions) with concurrent and adjuvant TMZ vs. RT alone, without adversely impacting either QoL or functional status. Despite chemoradiation becoming the recommended treatment in GBM patients who are elderly but fit, several questions remain unanswered. This includes the survival impact of chemoradiation in patients with severe comorbidities or with ECOG PS >2 or a combination of poor prognostic features such as male gender, poor neurocognition, biopsy only and lack of MGMT methylation. Personalized management of patients with HGG is warranted in the modern era as we attempt to balance the benefit of efficacious treatment with potential toxicity while appreciating the many nuances associated with multiple prognostic factors on anticipated survival. Here, we aim to review the palliative management options available for HGG patients with an emphasis on the role of RT.

Activity and safety of temozolomide in advanced adrenocortical carcinoma patients.

Temozolomide has shown a significant anti-proliferative activity on adrenocortical cancer (ACC) cells in vitro. On the basis of these results the drug was prescribed as second/third line in advanced metastatic ACC patients in four referral centers in Italy. We retrospectively collected anagraphic, clinical and pathological data of patients with advanced ACC with disease progression to standard chemotherapy plus mitotane who were treated with temozolomide at the dose of 200 mg/m2/die given for 5 consecutive days every 28 days. The primary endpoint was the disease control rate, defined as objective response or disease stabilization after 3 months. Secondary endpoints were overall survival (OS), progression-free survival (PFS) and drug safety. Twenty-eight patients have been included in the study. Ten patients (35.8%, 95% CI: 17.8-53.8) obtained a disease control from temozolomide treatment. In particular, 1 patient had a complete response, 5 patients a partial response and 4 patients stable disease. Median PFS was 3.5 months and median OS was 7.2 months. Disease response was more frequently observed in patients with methylation of O6-methylguanine-DNA methyltransferase (MGMT) gene. Temozolomide therapy was well tolerated and most toxicities were limited to grade G1-2 according to WHO criteria. Temozolomide was found active in the management of advanced ACC patients. The disease control rate obtained, however, was short-lived and the prognosis of treated patients was poor.

Glioblastoma evolution pattern under surgery and radio(chemo)therapy (RCHT) to identify novel methylome based glioma subtypes.

2012 Background: Identification of isocitrate dehydrogenase mutations (IDHm) and glioma CpG island methylator phenotype (G-CIMP) as well as methylation of O6-methylguanine DNA methyltransferase (MGMT) promotor has substantially improved stratification of glioma patients into prognostic subgroups. In extension of static pre-therapy diagnostic, we sought to investigate the impact of glioblastoma evolution under selection pressure of standard therapy on methylome level. Methods: For the training cohort (T), methylome (450k Illumina) data of paired samples from 50 patients with glioblastoma (GBM, 11 G-CIMP+) were analyzed, i.e., primary (P) and at the time point of recurrence (R, re-surgery) after standard therapy at NCT. For 39 pairs matching RNASeq data was analyzed. Validation cohorts consisted of Heidelberg (V1) total n = 650, GBM (n:585, 8 G-CIMP+), grade III (n:65, CIMP+ 65), Austrian GBM (V2, n = 499, 36 IDHm, pyrosequencing data) and the TCGA (V3) Lower-grade-glioma cohort (LGG, n = 477, grade III n: 247, 178 G-CIMP+, grade II n: 228, 206 G-CIMP+). Results: Limited number of consensus differentially methylated probes (DMP) were found across all P vs. R samples (nCpG = 411 CpGs, FDR &lt; 0.05). In contrast, heterogeneity in GBM evolution was found by similarity analysis of delta-methylome data of 50 PR pairs resulting in two distinct clinical subgroups and one “intermediate” group. Intriguingly, n = 114.652 DMP (FDR &lt; 0.05) was found by comparing the evolutionary “poor” (n = 15) vs. “good” (n = 13) GBM phenotypes. A random forest classifier was built to identify the evolutionary subgroups in P samples. The performance of “good” prognosis classifier was in T cohort HR: 0.54 [0.30-0.97], p = 0.04; V1: 0.57 [0.43-0.76], p &lt; 0.001, V2: 0.62 [0.47-0.82], p &lt; 0.001, LGG: 0.16 [0.08-0.32], p &lt; 0.001. In “good” prognosis group (T), neither G-CIMP+ (n = 3) nor MGMT-STP27 (oddsratio, OR: 0.56, p = 0.47) was enriched. MGMT-STP27 OR was 0.47 (V1, p = 0.47) or 1.28 (V2, p = 0.45), respectively. The evolutionary subgroups remain prognostic independent of GCIMP status in LGG (V3). “Poor” glioma are enriched for RTKI/II methylome subtypes, and contain less frequently the mesenchymal subtype. Bevacizumab treatment showed a survival benefits only in “poor” subtype (V1+2). Conclusions: Discovery of a methylome based classifier of glioma evolution informs on “good” and “poor” prognosis subtypes and may have ramification for stratifying patients for therapy such as e.g., antiangiogenesis.

Glioblastoma in Elderly Patients: Current Management and Future Perspectives.

The incidence of glioblastoma (GBM) in the elderly population is slowly increasing in Western countries. Current management includes surgery, radiation therapy (RT) and chemotherapy; however, survival is significantly worse than that observed in younger patients and the optimal treatment in terms of efficacy and safety remains a matter of debate. Surgical resection is often employed as initial treatment for elderly patients with GBM, although the survival benefit is modest. Better survival has been reported in elderly patients treated with RT compared with those receiving supportive care alone, with similar survival outcome for patients undergoing standard RT (60 Gy over 6 weeks) and hypofractionated RT (25–40 Gy in 5–15 daily fractions). Temozolomide, an alkylating agent, may represent an effective and safe therapy in patients with promoter methylation of O6-methylguanine-DNA-methyltransferase (MGMT) gene which is predictor of responsiveness to alkylating agents. An abbreviated course of RT, 40 Gy in 15 daily fractions in combination with adjuvant and concomitant temozolomide has emerged as an effective treatment for patients aged 65 years old or over with GBM. Results of the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG CE6) and European Organization for Research and Treatment of Cancer (EORTC 26062/22061) randomized study of short-course RT with or without concurrent and adjuvant temozolomide have demonstrated a significant improvement in progression-free survival and overall survival for patients receiving RT and temozolomide over RT alone, without impairing either quality of life or functional status. Although combined chemoradiation has become the recommended treatment in fit elderly patients with GBM, several questions remain unanswered, including the survival impact of chemoradiation in patients with impaired neurological status, advanced age (>75–80 years old), or for those with severe comorbidities. In addition, the efficacy and safety of alternative therapeutic approaches according to the methylation status of the O6-methylguanine-DNA methyl-transferase (MGMT) gene promoter need to be explored in future trials.

Functional Blockade of Small GTPase RAN Inhibits Glioblastoma Cell Viability.

Glioblastoma, the most common malignant tumor in the brain, lacks effective treatments and is currently incurable. To identify novel drug targets for this deadly cancer, the publicly available results of RNA interference screens from the Project Achilles database were analyzed. Ten candidate genes were identified as survival genes in 15 glioblastoma cell lines. RAN, member RAS oncogene family (RAN) was expressed in glioblastoma at the highest level among all candidates based upon cDNA microarray data. However, Kaplan-Meier survival analysis did not show any correlation between RAN mRNA levels and patient survival. Because RAN is a small GTPase that regulates nuclear transport controlled by karyopherin subunit beta 1 (KPNB1), RAN was further analyzed together with KPNB1. Indeed, GBM patients with high levels of RAN also had more KPNB1 and levels of KPNB1 alone did not relate to patient prognosis. Through a Cox multivariate analysis, GBM patients with high levels of RAN and KPNB1 showed significantly shorter life expectancy when temozolomide and promoter methylation of O6-methylguanine DNA methyltransferase were used as covariates. These results indicate that RAN and KPNB1 together are associated with drug resistance and GBM poor prognosis. Furthermore, the functional blockade of RAN and KPNB1 by importazole remarkably suppressed cell viability and activated apoptosis in GBM cells expressing high levels of RAN, while having a limited effect on astrocytes and GBM cells with undetectable RAN. Together, our results demonstrate that RAN activity is important for GBM survival and the functional blockade of RAN/KPNB1 is an appealing therapeutic approach.

FoxD2-AS1 is a prognostic factor in glioma and promotes temozolomide resistance in a O6-methylguanine-DNA methyltransferase-dependent manner.

Glioma is the most common brain tumor with a dismal prognosis. While temozolomide (TMZ) based chemotherapy significantly improves survival in glioma patients, resistance against this compound commonly leads to glioma treatment failure. Overexpression of long-noncoding RNA (LncRNA) FoxD2 adjacent opposite strand RNA 1 (FoxD2-AS1) was identified to promote glioma development, but the role in TMZ resistance remains unclear. In this paper, we found that FoxD2-AS1 was overexpressed in recurrent glioma, high FoxD2-AS1 expression was significantly correlated with poor patient outcome. Methylation of O6-methylguanine-DNA methyltransferase (MGMT) is significantly less frequent in high FoxD2-AS1 expression patients. Knockdown of FoxD2-AS1 decreased the proliferation, metastatic ability of glioma cells and promote the sensitivity to TMZ in glioma cells. Furthermore, knockdown of FoxD2-AS1 induced hypermethylation of the promoter region of MGMT. Our data suggested that FoxD2-AS1 is a clinical relevance LncRNA and mediates TMZ resistance by regulating the methylation status of the MGMT promoter region.

Ten genes associated with MGMT promoter methylation predict the prognosis of patients with glioma.

Glioma originates from the glial cells of the spine or brain, and promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT) can promote the chemosensitivity of glioma. The present study aimed to reveal the key genes implicated in MGMT promoter methylation in patients with glioma. RNA-sequencing data and methylation data for glioma were extracted from The Cancer Genome Atlas database. Following expression characteristic analysis and differential expression analysis using unsupervised hierarchical clustering and a rank sum test, the feature genes were identified between high and low methylation groups. Furthermore, multivariate survival analysis for the feature genes was performed using the survival package in R. Additionally, the independent glioma RNA expression datasets GSE7696 and GSE42669 were used to validate the prognostic efficiency of the gene combination. The results indicated that the prognosis of the low methylation group was significantly worse than that of the high methylation group. The ten genes corresponding to the cut-off value of 0.56 (Rho GTPase-activating protein 21, CECR2, histone acetyl-lysine reader, endosulfine α, G-patch domain-containing 8, KIAA1109, MGMT, protocadherin β 13, selenoprotein M, sperm-associated antigen 9 and WD repeat domain 6) were able to significantly predict prognosis and were differentially expressed between the two groups. Multivariate survival analysis suggested that the ten genes were effective for sample classification and prognostic prediction. Furthermore, the validation datasets confirmed the correlation of the ten genes with prognosis. In conclusion, these 10 genes may be mediated by MGMT promoter methylation in glioma. In addition, the ten-gene combination may be associated with the prognosis of patients with glioma.

Association between MGMT Promoter Methylation and Risk of Breast and Gynecologic Cancers: A Systematic Review and Meta-Analysis

The role of the promoter methylation of O6-methylguanine-DNA methyltransferase (MGMT) remains controversial for breast and gynecologic cancers. We conducted a meta-analysis to assess the association between hypermethylation of MGMT promoter and the risk of breast and gynecologic cancers. A comprehensive search was conducted in PubMed and Embase electronic databases up to 19th August 2017 for studies about the association between MGMT promoter hypermethylation and breast and gynecologic cancers. A total of 28 articles including 2,171 tumor tissues and 1,191 controls were involved in the meta-analysis. The pooled results showed that MGMT promoter methylation status was significantly associated with an increased risk of breast and gynecologic cancers (OR = 4.37, 95% CI: 2.68–7.13, P < 0.05). The associations were robust in subgroup analysis based on ethnicity, cancer type, methylation detection method, and control source. This meta-analysis indicated that MGMT hypermethylation was significantly associated with the risk of breast and gynecological cancers, and it may be utilized as a valuable biomarker in early diagnostics and prognostication of these cancers. Further efforts are needed to identify and validate this finding in prospective studies, especially in situation with new methylation testing methods and samples from plasma circulating DNA.

Promoter methylation of MGMT in oral carcinoma: A population-based study and meta-analysis

IntroductionThe relevance of DNA methylation of O6-methylguanine-DNA methyltransferase (MGMT) in relation to several cancers and other disorders has been extensively explored in several cancer types. AimsTo ascertain the significance of DNA methylation of MGMT promoter in oral squamous cell carcinoma (OSCC), we undertook a study to a) analyse the methylation patterns of MGMT gene promoter in afflicted and normal population of coastal Karnataka, b) determine the expression status of MGMT in oral cancer cell lines (CAL-27 and SCC-4) and its relationship to DNA methylation and c) performed a meta-analysis of the published data. MethodsBisulfite sequencing of MGMT promoter region was performed on non-malignant/malignant oral samples, and oral cancer cell lines, followed by gene expression studies. Further, using a systematic search, 1024 publications were retrieved from PubMed, Scopus, Google Scholar and Web of Science and 23 relevant articles were reviewed. ResultsSignificant association of MGMT promoter methylation with OSCC (p<0.0001) was observed in the case-control study. The studies chosen for meta-analysis showed predictive significance of MGMT gene promoter. Overall, we obtained a statistically significant (p<0.0001) association for both sensitivity and specificity of MGMT DNA promoter methylation in oral cancer cases without publication bias. Gene expression was significantly elevated in both oral cancer cell lines (p<0.03) after treatment with a demethylating agent (5-Aza-2′-deoxycytidine). ConclusionDNA promoter hypermethylation and gene expression of MGMT may associate with recursive mutagenesis and is a promising biomarker for OSCC prediction. Studies suggest further validation in large distinct cohorts to facilitate translation to clinics.

MPTH-07GLIOBLASTOMAS: CORRELATION OF MGMT GENE PROMOTER METHYLATION WITH CLINICO-PATHOLOGICAL AND MOLECULAR PARAMETERS

Methylation of O6-methylguanine DNA methyltransferase (MGMT) is one of the predictive and prognostic markers of glioblastomas (GBMs). In this study, GBMs have been evaluated for the same and correlated with other clinicopathological and molecular parameters. MATERIALS AND METHODS: Diagnosed cases of GBM, during the period of June 2014 to December 2014 in the Department of Pathology of our Institute, wherein they have been evaluated for MGMT methylation by methylation specific polymerase chain reaction, IDH1R132H and p53 protein expression by immunohistochemistry and EGFR gene amplification by fluorescence in-situ hybridisation formed the study sample. Statistical correlation was done by Chi-square and Fischer's exact, using IBM SPSS Software 16 version. RESULTS: A total of 97 cases of GBM formed the study sample with age range of 20-75 years (mean age:49.76 years) and male: female ratio of 3.2:1 (M:74;F:23).Cerebral hemispheric location was commonest (frontal:35%; temporal:43 & parietal:19%). 46 (47.4%) cases were MGMT methylated (low methylation: 10) and 51 were unmethylated. 23 out of 84 cases (27.4%) were EGFR amplified; 10 cases (10.3%) were positive for IDH1R132H; 16 showed loss of ATRX protein expression (of which 7 were IDH1R132H positive; while the other 3 IDHR132H positive cases showed retained ATRX protein). Of the 46 MGMT methylated cases, 10 were EGFR amplified (10 out of 23 EGFR amplified cases were MGMT methylated); 5 were IDH1R132H positive and 6 showed loss of ATRX expression. None of the molecular markers showed any correlation for the histological features of calcification and necrosis (neither for pseudopalisading or confluent). CONCLUSIONS: MGMT was methylated in 47.4%of GBMs and had no significant statistical correlation with EGFR gene amplification, IDH1R132H mutation, ATRX protein expression and other histomorphological features. This study did not identify any surrogate marker for MGMT promoter methylation.

Combined IDH1 mutation and MGMT methylation status on long-term survival of patients with cerebral low-grade glioma

ObjectiveThe management of low-grade glioma (LGG) still remains controversial because the effectiveness of early and extensive resection is unclear, and the use of radiation therapy or chemotherapy is not well-defined. In particular, the importance of prognostic factors for survival remains a matter of discussion. The purpose of this study was to validate prognostic factors for survival in patients with LGG. Materials and methodsA consecutive series of 55 patients with WHO grade II LGG treated in our institute between 1983 and 2013 were retrospectively reviewed to determine the prognostic factors for survival. All data were retrospectively analyzed from the aspect of baseline characteristics, pathological findings, genetic change, surgical treatments, adjuvant therapies, and survival time. Cox multivariate analysis was performed to determine the prognostic factors for survival. ResultsThere were 28 patients with diffuse astrocytoma (DA), 21 patients with oligodendroglioma (OG), and 6 patients with oligoastrocytoma (OA) diagnosed on initial surgery. The median overall survival was 193 months and fifteen patients (27.3%) died. A mutation in isocitrate dehydrogenase-1 (IDH1) was found in 72.9% of LGG, and this mutation was positively correlated with methylation of O6-methylguanine-DNA methyltransferase (MGMT) (p=0.02). A better prognosis was significantly associated with combined IDH1 mutation and MGMT methylation status (both positive vs both negative, HR 0.079 [95% CI 0.008–0.579], p=0.012), as well as histology (OG vs DA and OA, HR 0.158 [95% CI 0.022–0.674], p=0.011) and tumor size (<6cm vs ≥6cm, HR 0.120 [95% CI 0.017–0.595], p=0.008). ConclusionsTumor histology, size and IDH-mutation status are important predictors for prolonged overall survival in patients with LGG and may provide a reliable tool for standardizing future treatment strategies.