MGMT Gene Promoter Methylation Research Articles

Computational Predictions of MGMT Promoter Methylation in Gliomas: A Mathematical Radiogenomics Approach

In the treatment of glioblastomas, non-invasive methods for determining MGMT gene promoter methylation status are crucial due to their implications for chemotherapy responsiveness. This study utilizes a mathematical and computational framework to extract and analyze radiogenomic data from MRI images to predict the methylation status. The first step in our framework involves extracting radiogenomic data from MRI images. This process requires sophisticated image processing techniques to convert MRI scans into a format suitable for machine learning analysis. The features extracted include textural patterns, intensity distributions, and other relevant radiomic characteristics.To identify the most significant features, we employ a Random Forest (RF) algorithm. Mathematically, RF is an ensemble learning method that operates by constructing multiple decision trees during training and outputting the mode of the classes for classification tasks. The importance of each feature is evaluated based on its contribution to the accuracy of the model, quantified by metrics such as Gini impurity or information gain. Employing advanced machine learning models like VGG19, ResNet50, and Sequential FCNet Artificial Neural Networks, alongside traditional classifiers such as Naive Bayes and Logistic Regression, we analyze features identified by a Random Forest algorithm. Our mathematical approach ensures rigorous evaluation of model accuracy through sensitivity, specificity, and other performance metrics, presenting the Sequential FCNet ANN combined with ResNet50 as the superior model. This research contributes to the field of precision healthcare by enhancing the mathematical methods used in the non-invasive diagnosis of glioblastomas.

Abstract 7395: Histology-based prognosis prediction using deep learning outperforms and is independent of the MGMT methylation status in patients with glioblastoma

Abstract Introduction: MGMT gene promoter methylation status is today the only biomarker of survival for GBM patients and predicts favorable response to temozolomide (TMZ). Here we propose a deep learning-based model using histology data (H&E slides) to predict the prognosis of patients with Glioblastoma (GBM) treated by standard of care (SOC). Methods: We included TCGA GBM patients and selected only patients treated after 2005 (Stupp protocol start); compliant to WHO 2021 classification; treated by surgery and adjuvant therapy; with a follow-up superior to median overall survival (OS) (14.5 months). Short and long survivor labels were defined based on median OS cut-off. A deep learning pipeline was trained to predict these labels from the H&E slides (baseline surgery samples). First, the slides were automatically segmented into tissue and background, divided into a regular grid of tiles (112 × 112µm), and tiles were vectorized using a vision transformer-based feature extractor trained on H&E slides of 5,500 patients with 16 cancer subtypes (GBM excluded). In the second step, ABMIL deep learning model was trained to predict the survival category using the bag of tile features available from step 1 for each patient. The cohort was divided into five folds with even numbers of short and long survivors. For each fold, 50 ABMIL models were trained on the remaining folds and ensembled to predict the category of the patients in the fold. Training hyperparameters were tuned on a separated cohort excluding GBM patients. Results: After filtering, we analyzed n = 192 TCGA GBM patients including 154 patients with a known MGMT promoter methylation status. Our histology-based model had an accuracy of 0.661 and a ROC AUC of 0.706. It stratified the population into short and long survivors with an Hazard Ratio (HR) of 0.48 (95% CI 0.34-0.69). Our model outperformed the MGMT methylation status that had an HR of 0.57 (95% CI 0.40-0.83). Moreover, our model was independent of MGMT methylation status and the combination of both was able to further improve prognosis prediction: we applied our model independently to the 91 patients with an unmethylated MGMT status (predicted: 43 low, 48 high) and to the 63 patients with a methylated MGMT status (predicted: 30 low, 33 high), and obtained HR of 0.42 (95% CI 0.27-0.67) and 0.49 (95% CI 0.28-0.86) respectively. Conclusion: Having selected patients treated by the latest SOC and classified as GBM by the latest WHO criteria, we propose a model predicting survival based on histological specimens available in routine care, which outperformed the current unique biomarker MGMT methylation status. The combination of this status and our model improves prognosis predictions compared to each strategy alone. Citation Format: Lucas Fidon, Celine Thiriez, Alexandre Grimaldi, Omar Darwiche Domingues, Charles Maussion, Caroline Hoffmann. Histology-based prognosis prediction using deep learning outperforms and is independent of the MGMT methylation status in patients with glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7395.

Hypermethylation of MGMT Gene Promoter in Peripheral Blood Mononuclear Cells as a Noninvasive Biomarker for Colorectal Cancer Diagnosis.

Early colorectal cancer (CRC) diagnosis can drastically reduce CRC-related morbidity and mortality. In this regard, increasing attention is now being directed to DNA-based tests, especially the evaluation of methylation levels, to prioritize high-risk suspected persons for colonoscopy examination. Therefore, we aimed to assess the accuracy of MGMT gene promoter methylation levels in peripheral blood mononuclear cells (PBMCs) for distinguishing CRC patients from healthy people. For this study, a total of seventy individuals with CRC and 75 healthy individuals from Iran were included. The methylation level of MGMT in the DNA isolated from PBMCs was evaluated using the methylation quantification endonuclease-resistant DNA technique. To assess the diagnostic capability of the MGMT promoter methylation level, a receiver operating characteristic (ROC) curve was generated. The mean promoter methylation level of MGMT in the CRC and control groups was, respectively, 27.83 ± 22.80 vs. 12.36 ± 14.48. The average percentage of methylation of the MGMT promoter between the CRC and control groups was significantly different (P < 0.001). Also, the MGMT promoter was more hypermethylated in female patients than in males. ROC analyses indicated that the diagnostic power of the MGMT promoter methylation level for CRC was 0.754, with a sensitivity of 81.43% and a specificity of 75.71%, indicating a good biomarker for CRC diagnosis. Methylation evaluation of MGMT in PBMCs could be utilized as a diagnostic biomarker with high accuracy for prioritizing suspected CRC patients before colonoscopy.

P11.23.A GLIOBLASTOMA MOLECULAR CORRELATES OF SEIZURE OCCURRENCE/RECURRENCE AFTER SURGERY: THE ROLE OF P53 MUTATION

Abstract BACKGROUND The integration of molecular data and histology changed the diagnostic approach and prognostic characterization for patients with brain tumors. The aim of this study is identify molecular risk factors associated with seizure occurrence and/or recurrence after surgery in patients with brain glioblastoma. MATERIAL AND METHODS Patients with brain glioblastoma were consecutively enrolled and underwent brain surgery, followed by histopathological and molecular evaluation. Immuno-histochemical, molecular (synaptophysin, GFAP, ATRX, p53, ki67 index and MGMT gene promoter methylation) and clinical (age, sex and seizure occurrence/recurrence before and/or after surgery) data were collected. Histopathological and molecular features were compared between patients with and without seizure occurrence/recurrence after surgery. A step-backward logistic regression was performed to investigate the association between molecular features and seizure occurrence/recurrence. RESULTS 100 patients (age 59.51±1.23, 65 males) were enrolled. Forty-nine patients had seizures before surgery. After surgery, 40 patients (age 59.43±1.64, 27 males) had seizure occurrence/recurrence, while 60 (age 59.57±1.75, 38 males) had not. Patients with epilepsy (i.e., seizures before and/or after surgery) had lower synaptophysin (33.33%, p=0.008) and higher p53 (85%, p=0.038) mutation rate compared with patients without epilepsy. In the whole sample, seizure occurrence/recurrence after surgery was significantly associated with p53 mutation (OR: 3.9, CI 1.12-13.50, p=0.032) and seizure occurrence before surgery (OR: 5.3, CI 2.15-13.08, p&amp;lt;0.001). In patients without seizures before surgery, p53 mutation perfectly predicted seizure occurrence, therefore the OR was not computable. In fact, all patients without p53 mutation didn’t have seizure after surgery, while all patients that had seizure after surgery, also had p53 mutation (p=0.028). However, to note 71% patients with p53 mutation didn’t have seizure after surgery. CONCLUSION The p53 mutation is related with a high risk of seizure occurrence/recurrence after surgery in brain glioblastoma, even in patients that didn’t have seizures before surgery. Conversely, the absence of p53 mutation appears to protect against the occurrence of seizures. If confirmed, this result may help in the management of anti-seizure treatment after surgery in brain glioblastoma patients.

Growth and Molecular Characteristics of Temozolomide-Resistant Human A172 and R1 Glioblastoma Cells

Glioblastoma recurrence is caused by initial and acquired as a result of therapy resistance of tumor cells. Studies searching the markers that would allow predicting the level of glioblastoma cell resistance to therapy are in progress. The complexity of the problem is related to the high heterogeneity of individual tumors and the cellular content of each tumor. In present work, a comparative study of the influence of single temozolomide (in Temodal® form) ex-posure on the well-known glioblastoma cell line A172 and a new one R1 was performed. In A172 (highly tem-ozolomide-sensitive cell line) after treatment with 0.1 mM of this drug only individual cells persisted and resumed proliferation. In R1 glioblastoma cell line single cells survived and resumed proliferation after treatment with 1 mM temozolomide. The populations resulting from the proliferation of these cells were designated as resistant. The expression of MGMT, as well as genes responsible for resistance to chemotherapy and tumor progression (MGMT, ABCB1, ABCC1, ABCG2), growth factor genes (VEGF, HGF), cytokines IL-6 and IL-8, and their encoding genes was examined in resistant A172 and R1 cells. In A172 cells, the methylated status of MGMT gene promoter was confirmed, as well as the absence of the corresponding gene expression. It was shown for the first time that glioblastoma R1 is heterogeneous by the methylation status of MGMT gene promoter and expression of the relevant enzyme. In A172 and R1 resistant cell populations, the level of MGMT gene promoter methylation was lower than in the intact cells, and MGMT gene expression was enhanced. We suspect that this may be the reason for greater resistance of such cells to chemotherapy. The expression of most genes associated with resistance to chemotherapy and a more aggressive course of the disease, genes of growth factors, and interleukins in resistant A172 cells was higher than in intact cells. In contrast, in resistant R1 cells, the expression of most of the same genes (with the exception of ABCC1 and VEGF, for which the expression level changed insignificantly) was lower than in the intact cells. These results confirm the significance of MGMT in the formation of glioblastoma cell resistance to temozolomide. The prognostic value of the other studied parameters is still ambiguous.

Genetic Profile of Primary Brain Tumours and the Degree of Their Resection Versus Survival of Patients Undergoing Surgery and Complementary Oncological Treatment

Introduction. Recently, the incidence of brain glial tumours has increased significantly worldwide. Gliomas are among the most malignant types of tumours of the central nervous system. Patients diagnosed with glial tumours have a very unfavourable prognosis leading to death, and the course of the disease itself has a negative impact on their physical, mental and social condition. Aim. The aim of this study was to determine the effect of the results of histological and genetic tests as well as the degree of resection of primary CNS tumours on the survival of patients undergoing neurosurgery and complementary oncological treatment — pharmacology and radiotherapy. Material and Methods. The research was carried out on the basis of an analysis of medical records (2019–2021) of 63 adult patients from the Department of Neurosurgery of the Specialist Municipal Hospital of Nicolaus Copernicus in Toruń. The study group included patients who had been diagnosed with a primary brain tumour and had undergone neurosurgery and complementary oncological treatment. The analysis of medical documentation consisted in comparing the results of histopathological tests with the results of specialized molecular tests and correlating them with the survival time of patients. The statistical analysis was carried out using the Pearson correlation, statistical inference was made at the significance level of p=0.05. Results. Based on the obtained results, it was found that the presence of MGMT gene promoter methylation (r=0.30, p=0.018) and IDH mutation (r=0.38, p=0.002) correlated positively with survival. The extent of resection also had a significant impact on patient survival (r=0.55, p&lt;0.001). Patients who underwent complete tumour resection survived an average of 19.34 months, while those who underwent biopsy survived for 7.94 months. Conclusions. The data collected during the conducted analyses may be important for the prognosis perspective and the selection of the optimal treatment strategy for both current and future patients. (JNNN 2022;11(4):162–166)

Deep DNA sequencing of MGMT, TP53 and AGT in Mexican astrocytoma patients identifies an excess of genetic variants in women and a predictive biomarker.

Astrocytomas are a type of malignant brain tumor with an unfavorable clinical course. The impact of AGT and MGMT somatic variants in the prognosis of astrocytoma is unknown, and it is controversial for TP53. Moreover, there is a lack of knowledge regarding the molecular characteristics of astrocytomas in Mexican patients. We studied 48 Mexican patients, men and women, with astrocytoma (discovery cohort). We performed DNA deep sequencing in tumorsamples, targeting AGT, MGMT and TP53, and we studied MGMT gene promoter methylation status. Then we compared our findings to a cohort which included data from patients with astrocytoma from The Cancer Genome Atlas (validation cohort). In the discovery cohort, we found a higher number of somatic variants in AGT and MGMT than in the validation cohort (10.4% vs < 1%, p < 0.001), and, in both cohorts, we observed only women carried variants AGT variants. We also found that the presence of either MGMT variant or promoter methylation was associated to better survival and response to chemotherapy, and, in conjunction with TP53 variants, to progression-free survival. The occurrence of AGT variants only in women expands our knowledge about the molecular differences in astrocytoma between men and women. The increased prevalence of AGT and MGMT variants in the discovery cohort also points towards possible distinctions in the molecular landscape of astrocytoma among populations. Our findings warrant further study.

MGMT gene promoter methylation by pyrosequencing method correlates volumetric response and neurological status in IDH wild-type glioblastomas.

PurposeAlthough the usefulness of O6-methylguanine DNA methyltransferase (MGMT) promoter methylation analysis for predicting response to chemoradiotherapy and the prognosis of patients with glioblastoma has been widely reported, there is still no consensus regarding how to define MGMT promoter methylation percentage (MGMTpm%) cutoffs by pyrosequencing method. The aim of this study was to determine the optimal cutoff value of MGMT promoter methylation status using volumetric analysis focused on the tumor volume ratio (TVR) measured by MRI.MethodsThis retrospective study included newly diagnosed IDH wild-type glioblastoma patients with residual tumor after surgery, followed by local radiotherapy with temozolomide. TVR was defined as the tumor volume at 6 months after the initial chemoradiotherapy administration divided by the tumor volume before the start of therapy. The mean MGMTpm% of 16 CpG islands (74–89) was analyzed using pyrosequencing. We statistically analyzed the correlation between MGMTpm%, TVR, and change in Karnofsky performance status.ResultsThe study included 44 patients with residual tumors. Thirteen (92.9%) of 14 patients with MGMTpm% ≥ 23.9% showed 50% or more volumetric response, leading to prolonged survival, and 17 (70.8%) of 24 patients with MGMTpm% < 8.2% had progressive disease after initial chemoradiotherapy administration. Three (50.0%) of six patients with MGMTpm% 8.2% to < 23.9% had stable disease or partial response.ConclusionEvaluation of MGMTpm% by pyrosequencing is important in predicting the volumetric response and prognosis of glioblastoma patients with residual tumors.

O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation.

BACKGROUNDO6-methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the mispairing base O6-methyl-guanine induced by environmental and experimental carcinogens. It can transfer the alkyl group to a cysteine residue in its active site and became inactive. The chemical carcinogen N-nitroso compounds (NOCs) can directly bind to the DNA and induce the O6-methylguanine adducts, which is an important cause of gene mutation and tumorigenesis. However, the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis, especially in the initiation phase, remains largely unclear.AIMTo investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODSWe established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitroso-urea (MNU) treatment. Cell proliferation, colony formation, soft agar, cell migration, and xenograft assays were used to verify the malignant phenotype. By using quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis, we detected the MGMT expression in malignant transformed cells. We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry. MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR. The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays. RESULTSWe observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment. Moreover, we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells. Inhibition of the MGMT expression by O6-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes. Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU. Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues, but downregulated in the gastric cancer tissues.CONCLUSIONOur finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation. The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis, which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.

MGMT gene promoter methylation in humoral tissue as biomarker for lung cancer diagnosis: An update meta-analysis.

ObjectiveTo investigate O‐6‐methylguanine‐DNA methyltransferase (MGMT) gene promoter methylation in humoral tissue as biomarker for lung cancer diagnosis by pooling relevant open published data.MethodsClinical studies relevant to MGMT gene promoter methylation and lung cancer were systematic electronic searched in the databases of Medline, EMBASE, Ovid, Web of Science, and CNKI. Data of true positive (tp), false positive (fp), false negative (fn), and true negative (tn) were extracted from the included studies and made combination. The diagnostic sensitivity, specificity, diagnostic odds ratio (DOR) and summary receiver operating characteristic (SROC) of MGMT gene methylation for lung cancer diagnosis were pooled.ResultsTwelve studies were included in the meta‐analysis. The diagnostic sensitivity, specificity, DOR were 0.39 (95% CI = 0.31–0.49) 0.92 (95% CI = 0.77–0.97), and 4.20 (95% CI = 2.09–8.44), respectively under random effect model. The SROC of MGMT gene methylation for lung cancer diagnosis was 0.58 (95% CI = 0.53–0.62).ConclusionMGMT methylation rate was higher in plasma and bronchoalveolar lavage fluid (BLAF) of lung cancer cases compared to controls. High diagnostic specificity indicated that MGMT methylation in plasma and BLAF can be applied as lung cancer confirmation test.

A Prospective Clinical Study on MGMT Protein Expression and the Effect of Gene Promoter Methylation on Sensitivity to Chemotherapeutics in Spinal Glioma.

ObjectiveThe present study discusses the O6-methylguanine-DNA methyltransferase (MGMT) protein expression of spinal glioma cells and the correlation between the sensitivity of promoter methylation of the MGMT gene to chemotherapy drugs, establishes a prediction method for the sensitivity of chemotherapy drugs on spinal gliomas, providing a theoretical basis for determining the best chemotherapy regimens for clinical patients after a spinal glioma operation.MethodsA total of 67 patients, who received microsurgical resection for spinal glioma from October 2010 to June 2016, were selected for the present study. Immunohistochemistry and methylation were performed after the operation. Among these patients, 47 patients with postoperative chemotherapy were assigned as the experimental group, while 20 patients without chemotherapy were designated as the control group.ResultsAmong the 47 patients in the experimental group, 39 patients had no tumor recurrence after two years, while tumors increased and symptoms were aggravated in eight patients. The progression-free survival rate of chemotherapy was 82.9%, and the two-year survival rate was 100%. The adverse reactions of patients during chemotherapy were slight. Among the 20 patients in the control group, seven patients had no tumor recurrence, while 13 patients had increased tumor size, and the progression-free survival rate was 35.0%.ConclusionUnder the guidance of MGMT immunohistochemical detection and MGMT gene promoter methylation detection after surgery, chemotherapy can effectively delay tumor recurrence, prevent a reoperation, and have good safety and tolerability. This chemotherapy regimen has good prospects.

A Meta-Analysis of the Prognostic Value of Abnormally Expressed lncRNAs and Correlation with Clinicopathological Characteristics in Glioma

Objectives: Abnormal expression of lncRNAs are involved in the occurrence and development of glioma. Thus, we conducted a meta-analysis to clarify the prognostic value of lncRNAs in glioma integrally. Methods: Pubmed, Web of Science, Embase, Cochrane Library, Clinical Trial.gov and Trip database were searched. Hazard ratio (HR) and 95% confidence intervals (CI) were calculated in the analysis of prognostic indicators. Odds ratio (OR) and 95%CI were applied to estimate the association between lncRNAs and clinicopathological features. Results: The expression level of lncRNA was significantly correlated with the OS rate of glioma patients (HR=1.29, 95%CI: 1.03-1.61, p=0.03). The low expression of CASC2 (HR=0.42, 95%CI: 0.27-0.66, p=0.0001) showed a poor prognosis of glioma patients. MALAT1 (HR=1.42, 95%CI: 0.38-5.29, p=0.60), HOTAIR (HR=1.70, 95%CI: 0.69-4.21, p=0.25) and TUSC7 (HR=1.81, 95%CI: 0.87-3.75, p=0.11) have no statistical significance for the prognosis of glioma patients. The high expression of lncRNA was related to WHO staging (OR=0.31, 95%CI: 0.19-0.50, p<0.00001), EGFR expression (OR=3.14, 95%CI: 1.70-5.77, p=0.0002) and IDH1 status (OR=0.33, 95% CI: 0.12-0.93, p=0.04), while unrelated to age (OR=1.05, 95%CI: 0.78-1.42, p=0.75), gender (OR=1.01, 95%CI: 0.85-1.20, p=0.92), tumor size (OR=0.67, 95%CI: 0.35-1.27, p=0.22), PTEN (OR=0.26, 95%CI: 0.06-1.10, p=0.07), KPS score (OR=1.06, 95%CI: 0.44-2.59, p=0.90), MGMT promoter methylation (OR=2.05, 95%CI: 0.94-4.50, p=0.07) and the recurrence (OR=0.87, 95%CI: 0.42-1.82, p=0.72). Conclusion: Abnormal expression of lncRNAs were related to survival of glioma patients and may serve as potential prognostic indicators in glioma. The higher expression level of lncRNAs, the worse prognosis of glioma. The expression of lncRNA CASC2 is correlated with the prognosis of glioma patients. The higher the expression level of CASC2, the better prognosis. LncRNAs are related to WHO staging, EGFR expression and IDH1 mutation, while unrelated to age, gender, tumor size, PTEN, KPS score, MGMT gene promoter methylation or tumor recurrence. Keywords: LncRNAs, clinicopathological features, glioma, prognosis

The Potential Effect of the IDH1 Mutation and MGMT Gene Promoter Methylation on the Control of Glioblastoma-Associated Epilepsy in Patients Receiving Anti-Epileptic Agents and Chemotherapies

The Potential Effect of the IDH1 Mutation and MGMT Gene Promoter Methylation on the Control of Glioblastoma-Associated Epilepsy in Patients Receiving Anti-Epileptic Agents and Chemotherapies

Favorable role of IDH1/2 mutations aided with MGMT promoter gene methylation in the outcome of patients with malignant glioma.

Aim:The implications of molecular biomarkers IDH1/2 mutations and MGMT gene promoter methylation were evaluated for prognostic outcome of glioma patients.Materials & methods:Glioma cases were analyzed for IDH1/2 mutations and MGMT promoter methylation by DNA sequencing and methylation-specific PCR, respectively.Results:Mutations found in IDH1/2 genes totaled 63.4% (N = 40) wherein IDH1 mutations were significantly associated with oligidendrioglioma (p = 0.005) and astrocytoma (p = 0.0002). IDH1 mutants presented more, 60.5% in MGMT promoter-methylated cases (p = 0.03). IDH1 mutant cases had better survival for glioblastoma and oligodendrioglioma (log-rank p = 0.01). Multivariate analysis confirmed better survival in MGMT methylation carriers (hazard ratio [HR]: 0.59; p = 0.031). Combination of both biomarkers showed better prognosis on temozolomide (p < 0.05).Conclusion:IDH1/2 mutations proved independent prognostic factors in glioma and associated with MGMT methylation for better survival.

Influence of Intratumor Heterogeneity on the Predictivity of MGMT Gene Promoter Methylation Status in Glioblastoma.

Glioblastoma is the most aggressive tumor of the central nervous system. Prognosis is poor, even in the presence of a methylated state of MGMT gene promoter, which represents the biomarker with the highest prognostic/predictive value for the standard treatment of patients. Among patients with a methylated MGMT status, we identified an intermediate range of methylation above the standard 9% cut-off (gray zone) in which the predictive strength of the marker was lost. In an effort to improve the evaluation of the biomarker in clinical decision-making, we are carrying out a retrospective study, performing an in-depth analysis of samples used for diagnosis to understand how molecular heterogeneity, a hallmark of glioblastoma, impacts the evaluation of MGMT gene promoter methylation. Preliminary data from samples belonging to the “gray zone” tend to confirm the hypothesis of a mismatch between methylation values used for clinical decision-making and those included in our in-depth analysis. Confirmation of these data would help to better define the predictive power of MGMT promoter methylation status and greatly facilitate clinical decision-making.

370MO FOLAGLI: A phase I study of folinic acid combined with temozolomide and radiotherapy to modulate MGMT gene promoter methylation in newly diagnosed MGMT non-methytated glioblastoma

Glioblastoma is a very aggressive disease with a poor prognosis. The MGMT gene encodes an enzyme involved in DNA repair, the epigenetic silencing (by methylation) of which confers a better prognostic for GBM patients. We hypothesized that administration of folic acid could restore a methylation of MGMT promoter gene in non-methylated GBM and thus increase sensitivity to temozolomide combined with radiotherapy.

Abstract 3186: Methylation MGMT gene promoter analysis based on a high throughput method combines bisulfite conversion with amplicon sequencing

Abstract Background: Methylation of the MGMT gene promoter was observed in approximately 50% of glioblastoma multiforme (GBM). Epigenetic silencing of the MGMT gene by promoter methylation results in decreased MGMT protein expression, reduced DNA repair activity, and potential increased sensitivity to alkylating agent-based chemotherapy. Although MGMT promoter methylation status has been widely evaluated by methylation-specific PCR and bisulfite pyrosequencing. The limitations to these methods include low quantitative accuracy and low sample throughput. Here, we developed a high throughput method (in-house One-Step Seq Method, Genetron) which combines bisulfite conversion with amplicon sequencing of MGMT gene promoter. Methods: DNA was extracted from 144 FFPE or fresh frozen glioma tissues. For bisulfite pyrosequencing, MGMT promoter methylation status was analyzed using the MGMT Pyro Kit (Qiagen 970061) following the manufacturer's protocol. At the same time,the MGMT promoter methylation status was also analyzed by in-house One-Step Seq Methodfollowing bisulfite conversion. In briefly, the DNA was pre-treated by sodium bisulfite. Then Exon 1 of human MGMT gene was amplified from bisulfite conversed DNA. And library was constructed at the same time. High-throughput sequencing was performed on Ion GeneStudio S5. Reads mapping and methylation calling was handled by Bismark. The DNA sample was identified as MGMT methylation, if average methylation level of all CpG sites in exon 1 of MGMT was more than 21 %. Results: Currently, bisulfite pyrosequencing was considered as the gold standard for DNA methylation analysis. Comparing the result of bisulfite pyrosequencing analysis with the result of in-house methylation status analysis, 94.4% of DNA samples from 144 glioma tissues showed consistent methylation status of MGMT gene promotor. 5 DNA samples were identified as non-methylation in bisulfite pyrosequencing, but with methylation in in-house methylation status analysis. While 3 DNA samples were identified as methylation in bisulfite pyrosequencing, but not in in-house methylation status analysis. Meanwhile, by in-house One-Step Seq Method following bisulfite conversion, both the bisulfite conversion and library construction could be completed injust 4 hours using 10 ng DNA, and MGMT gene promoter methylation status could be estimated in two days. Conclusions: We developed a High-throughput sequencing based method to analyze MGMT status accurately combines bisulfite conversion with amplicon sequencing. This method shows high accuracy, high throughput, and easy manipulation for molecular classification of brain cancer. Citation Format: Yukun Zhang, Min Shi, Qiaosong Zheng, Xiao Shi, Min Chen, Le Li, Huiming Zhu, Liping Jiang, Tonghui Ma, Sumin Geng. Methylation MGMT gene promoter analysis based on a high throughput method combines bisulfite conversion with amplicon sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3186.

Subventricular zone contacting glioblastoma: tumor size, molecular biological factors and patient survival

Background Several studies show that subventricular zone (SVZ) contact of glioblastoma at diagnosis is a negative prognosticator of survival. In this report, we study glioblastoma patient survival, molecular biological and MRI-based volumetric findings according to SVZ contact. Patients and methods We conducted a retrospective study of adult patients diagnosed with supratentorial glioblastoma and uniformly treated with temozolomide-based chemoradiotherapy after surgery. The patient cohort was dichotomized according to tumor contact with the SVZ at diagnosis as determined on preoperative MR imaging. Tumor volume was measured using semi-automated segmentation technique. MGMT-gene promoter methylation and IDH mutation status were determined on stored tumor tissue. Kaplan-Meier survival curves were constructed. Cox regression analysis was used to adjust for known confounding factors of glioblastoma patient survival. Results A total of 214 patients were included in the study of whom 68% belonged to the SVZpos group. Median tumor volume was significantly larger in the SVZpos group (33,8 mL vs 15,6 mL; p < .001). MGMT-unmethylated glioblastoma was more frequent in the SVZpos group (61.4% vs 44.9%; p = .028). The overall survival and progression-free survival were 12.2 months and 5.9 months for the SVZpos patient group but 16.9 months and 10.3 months for the SVZneg group (log-rank p = .016 and .007 respectively). In multivariate Cox survival analysis, SVZ contact proved a negative prognostic parameter, independent from age, KPS, extent of resection, MGMT-methylation and IDH mutation status. Conclusions This study confirms SVZ contact at diagnosis as an independent negative prognostic factor for glioblastoma patient survival. SVZpos glioblastoma had larger tumor size and a larger proportion of unmethylated tumors than SVZneg glioblastoma. Further research is needed to establish whether the observed differences are solely explained by a different molecular profile of SVZpos glioblastoma or by interaction of glioblastoma with the unique SVZ microenvironment.

Granulocyte-macrophage colony-stimulating factor enhances effect of temozolomide on high-grade glioma cells.

In the present study, to delve into the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism, six cases of high-grade glioma cells from patient's tumor tissues were cultured. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed to detect cell proliferation and toxicity. Flow cytometry was performed to ascertain cell cycle and apoptosis rate. To detect the expressions of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein, respectively, specific PCR and immunofluorescence were performed. According to the results of MTT assay, compared with the results of control group, GM-CSF group exhibited enhanced cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the combination group [(67.67 ± 1.16), (68.13 ± 1.06), (68.42 ± 1.73)] had noticeably lower cell viability than the corresponding TMZ group [(90.00 ± 1.73), (82.33 ± 1.53), (82.67 ± 2.11)] (P < 0.01). Nevertheless, the two groups showed no significant difference in another three cases (MGMT gene unmethylated) (P > 0.05). In combination group, the apoptosis rate of the MGMT methylation cells was higher than that in the corresponding TMZ group (P < 0.01), which is consistent with MTT assay results. In all six cases of primary glioma cells, the fraction of cells in G1 phase of GM-CSF-treated group was noticeably down-regulated and was up-regulated in S phase (P < 0.01). GM-CSF could induce high-grade glioma cells to rapidly enter the cell cycle, thereby enhancing the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation. However, this effect is not ideal on glioma cells with MGMT unmethylation.

Clinicopathological associations and prognostic values of IDH1 gene mutation, MGMT gene promoter methylation, and PD-L1 expressions in high-grade glioma treated with standard treatment

Introductionthe objective was to evaluate the impact of IDH1 R132H mutation, MGMT methylation and PD-L1 expression in high grade glioma that received standard therapy (surgery, radiation and chemotherapy) to overall survival (OS).Methodsthis is a retrospective study of 35 high grade glioma cases. Genotyping of IDH1 gene alteration on the mutation hotspot R132 (Sanger sequencing method with Applied Biosystems 3500 Genetic Analyzer), EZ DNA Methylation-Gold kit (Zymo Research) is used to study the methylation, Cell line BT549 (ATCC HTB-122) and HCT-116 (ATCC CCL-247) were used as unmethylated control and partially methylated control respectively. Anti-human PD-L1 antibody clone E1L3N®from Cell Signalling Technology (USA) and Rabbit XP®were used to see PDL-1 expression.Resultsanaplastic astrocytoma cases had more MGMT promoter methylation (50%) than glioblastoma multiforme (GBM) (20%), more IDH1 R132H mutation (42%) than GBM (4.3%). Immunohistochemistry tumor proportion score method (TPS) identified 17% and 8.7% were PD-L1 positive in AA and GBM groups, respectively. Cases with IDH1 R132H mutation and MGMT methylation still showed better OS although with high PD-L1 expression.ConclusionIDH1 R132H mutation and MGMT methylation were good prognostic markers. High expression of PD-L1 apparently might not indicate poor overall survival in the presence of IDH1 R132 mutation and MGMT methylation.