Predictive MGMT status in a homogeneous cohort of IDH wildtype glioblastoma patients

Methylation of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter is predictive for treatment response in glioblastoma patients. However, precise predictive cutoff values to distinguish "MGMT methylated" from "MGMT unmethylated" patients remain highly debated in terms of pyrosequencing (PSQ) analysis. We retrospectively analyzed a clinically and molecularly very well-characterized cohort of 111 IDH wildtype glioblastoma patients, who underwent gross total tumor resection and received standard Stupp treatment. Detailed clinical parameters were obtained. Predictive cutoff values for MGMT promoter methylation were determined using ROC curve analysis and survival curve comparison using Log-rank (Mantel-Cox) test. MGMT status was analyzed using pyrosequencing (PSQ), semi-quantitative methylation specific PCR (sqMSP) and direct bisulfite sequencing (dBiSeq). Highly methylated (> 20%) MGMT correlated with significantly improved progression-free survival (PFS) and overall survival (OS) in our cohort. Median PFS was 7.2 months in the unmethylated group (UM, 20% mean methylation). Median OS was 13.4 months for UM, 17.9 months for LM and 29.93 months for HM. Within the LM group, correlation of PSQ and sqMSP or dBiSeq was only conclusive in 51.5% of our cases. ROC curve analysis revealed superior test precision for survival if additional sqMSP results were considered (AUC = 0.76) compared to PSQ (cutoff 10%) alone (AUC = 0.67). We therefore challenge the widely used, strict PSQ cutoff at 10% which might not fully reflect the clinical response to alkylating agents and suggest applying a second method for MGMT testing (e.g. MSP) to confirm PSQ results for patients with LM MGMT levels if therapeutically relevant

Synthesis and Quantitative Structure–Activity Relationship of Imidazotetrazine Prodrugs with Activity Independent of O6-Methylguanine-DNA-methyltransferase, DNA Mismatch Repair and p53.

The antitumor prodrug Temozolomide is compromised by its dependence for activity on DNA mismatch repair (MMR) and the repair of the chemosensitive DNA lesion, O6-methylguanine (O6-MeG), by O6-methylguanine-DNA-methyltransferase (EC 2.1.1.63, MGMT). Tumor response is also dependent on wild-type p53. Novel 3-(2-anilinoethyl)-substituted imidazotetrazines are reported that have activity independent of MGMT, MMR and p53. This is achieved through a switch of mechanism so that bioactivity derives from imidazotetrazine-generated arylaziridinium ions that principally modify guanine-N7 sites on DNA. Mono- and bi-functional analogs are reported and a quantitative structure-activity relationship (QSAR) study identified the p-tolyl-substituted bi-functional congener as optimized for potency, MGMT-independence and MMR-independence. NCI60 data show the tumor cell response is distinct from other imidazotetrazines and DNA-guanine-N7 active agents such as nitrogen mustards and cisplatin. The new imidazotetrazine compounds are promising agents for further development and their improved in vitro activity validates the principles on which they were designed

O6-methylguanine-DNA methyltransferase (MGMT) Promoter methylation is a rare event in soft tissue sarcoma

BACKGROUND: Gene silencing of O6-methylguanine–DNA methyltransferase (MGMT) by promoter methylation improves the outcome of glioblastoma patients after combined therapy of alkylating chemotherapeutic agents and radiation. The purpose of this study was to assess the frequency of MGMT promoter methylation in soft tissue sarcoma to identify patients eligible for alkylating agent chemotherapy such as temozolomide. FINDINGS: Paraffin tumor blocks of 75 patients with representative STS subtypes were evaluated. The methylation status of the MGMT promoter was assessed by methylation-specific polymerase-chain-reaction analysis (PCR). Furthermore, immunohistochemistry was applied to verify expression of MGMT. MGMT gene silencing was assumed if MGMT promoter methylation was present and the fraction of tumor cells expressing MGMT was 20% or less. Methylation specific PCR detected methylated MGMT promoter in 10/75 cases. Immunohistochemical staining of nuclear MGMT was negative in 15/75 cases. 6/75 tumor samples showed MGMT promoter methylation and negative immunohistochemical nuclear staining of MGMT. In none of the tested STS subtypes we found a fraction of tumors with MGMT silencing exceeding 22%. CONCLUSION: MGMT gene silencing is a rare event in soft tissue sarcoma and cannot be recommended as a selection criterion for the therapy of STS patients with alkylating agents such as temozolomide

Homogeneous MGMT Immunoreactivity Correlates with an Unmethylated MGMT Promoter Status in Brain Metastases of Various Solid Tumors

The O6-methylguanine-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical reports on treating brain metastases with temozolomide describe varying effects. This may be due to the fact that MGMT promoter methylation of brain metastases has not yet been explored in depth. Therefore, we assessed MGMT promoter methylation of various brain metastases including those derived from lung (n = 91), breast (n = 72) kidney (n = 49) and from malignant melanomas (n = 113) by methylation-specific polymerase chain reaction (MS-PCR) and MGMT immunoreactivity. Fifty-nine of 199 brain metastases (29.6%) revealed a methylated MGMT promoter. The methylation rate was the highest in brain metastases derived from lung carcinomas (46.5%) followed by those from breast carcinoma (28.8%), malignant melanoma (24.7%) and from renal carcinoma (20%). A significant correlation of homogeneous MGMT-immunoreactivity (>95% MGMT positive tumor cells) and an unmethylated MGMT promoter was found. Promoter methylation was detected in 26 of 61 (43%) tumors lacking MGMT immunoreactivity, in 17 of 63 (27%) metastases with heterogeneous MGMT expression, but only in 5 of 54 brain metastases (9%) showing a homogeneous MGMT immunoreactivity. Our results demonstrate that a significant number of brain metastases reveal a methylated MGMT-promoter. Based on an obvious correlation between homogeneous MGMT immunoreactivity and unmethylated MGMT promoter, we hypothesize that immunohistochemistry for MGMT may be a helpful diagnostic tool to identify those tumors that probably will not benefit from the use of alkylating agents. The discrepancy between promoter methylation and a lack of MGMT immunoreactivity argues for assessing MGMT promoter methylation both by immunohistochemical as well as by molecular approaches for diagnostic purposes

The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma

Background: The use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6- methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity. Methods: Tinostamustine has been tested in models of GBM by using 13 GBM cell lines and seven patient-derived GBM proliferating/stem cell lines in vitro. U87MG and U251MG (MGMT negative), as well as T98G (MGMT positive), were subcutaneously injected in nude mice, whereas luciferase positive U251MG cells and patient-derived GBM stem cell line (CSCs-5) were evaluated the orthotopic intra-brain in vivo experiments. Results: We demonstrated that tinostamustine possesses stronger antiproliferative and pro-apoptotic effects than those observed for vorinostat and bendamustine alone and similar to their combination and irrespective of MGMT expression. In addition, we observed a stronger radio-sensitization of single treatment and temozolomide used as control due to reduced expression and increased time of disappearance of γH2AX indicative of reduced signal and DNA repair. This was associated with higher caspase-3 activation and reduction of RT-mediated autophagy. In vivo, tinostamustine increased time-to-progression (TTP) and this was additive/synergistic to RT. Tinostamustine had significant therapeutic activity with suppression of tumor growth and prolongation of DFS (disease-free survival) and OS (overall survival) in orthotopic intra-brain models that was superior to bendamustine, RT and temozolomide and showing stronger radio sensitivity. Conclusions: Our data suggest that tinostamustine deserves further investigation in patients with glioblastoma

Expression of O6-methylguanine-DNA methyltransferase in childhood medulloblastoma

Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O6-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In sevenMB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7-92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely (n=46; Pearson correlation r 2=0.14, P=0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT'91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should no

Изменение экспрессии гена MGMT под действием экзогенных цитокинов в клетках человека in vitro

Исследовано влияние цитокинов LIF, SCF, IL-3, EMAP II и препарата Лаферобион (IFN-a2b) на экспрессию гена репаративного энзима MGMT в культурах клеток человека. Показано, что экзогенные цитокины модулируют экспрессию гена MGMT на уровне белка. EMAP II способен повышать или понижать уровень экспрессии гена MGMT в зависимости от условий эксперимента. Цитокины LIF, SCF, IL-3 и Лаферобион, как правило, вызывали снижение экспрессии гена MGMT в исследуемых клетках человека. Определены условия, которые способствуют разрушению белкового комплекса MGMT.Досліджено вплив цитокінів LIF, SCF, IL-3, EMAP II і препарату Лаферобіон (IFN-a2b) на експресію гена репаративного ензиму MGMT в культурах клітин людини. Встановлено, що екзогенні цитокіни здатні модулювати експресію гена MGMT на рівні білка. EMAP II підвищував або знижував рівень експресії гена MGMT в залежності від умов експерименту. Цитокіни LIF, SCF, IL-3 і Лаферобіон, як правило, спричиняли зниження експресії гена MGMT в досліджуваних клітинах людини. Визначено умови, які сприяють руйнуванню білкового комплексу MGMT.The influence of cytokines LIF, SCF, IL-3, and EMAP II and the Laferobion (IFN-a2b) drug on the MGMT gene expression in human cell cultures has been studied. It was shown that exogenous cytokines can modulate the MGMT gene expression at the protein level. EMAP II is able to increase or decrease the MGMT level, depending on the experimental conditions. Cytokines LIF, SCF, IL-3 and Laferobion decreased the MGMT expression level in human cells in vitro. Some conditions leading to the destruction of the MGMT protein complex were identified

Definition of the Prognostic Role of MGMT Promoter Methylation Value by Pyrosequencing in Newly Diagnosed IDH Wild‐Type Glioblastoma Patients Treated with Radiochemotherapy: A Large Multicenter Study

Background. O6‐methylguanine (O6‐MeG)‐DNA methyltransferase (MGMT) methylation status is a predictive factor for alkylating treatment efficacy in glioblastoma patients, but its prognostic role is still unclear. We performed a large, multicenter study to evaluate the association between MGMT methylation value and survival. Methods. We evaluated glioblastoma patients with an assessment of MGMT methylation status by pyrosequencing from nine Italian centers. The inclusion criteria were histological diagnosis of IDH wild‐type glioblastoma, Eastern Cooperative Oncology Group Performance Status (ECOG‐PS) ≤2, and radio‐chemotherapy treatment with temozolomide. The relationship between OS and MGMT was investigated with a time‐dependent Receiver Operating Characteristics (ROC) curve and Cox regression models. Results. In total, 591 newly diagnosed glioblastoma patients were analyzed. The median OS was 16.2 months. The ROC analysis suggested a cut‐off of 15% for MGMT methylation. The 2‐year Overall Survival (OS) was 18.3% and 51.8% for MGMT methylation <15% and ≥15% (p < 0.0001). In the multivariable analysis, MGMT methylation <15% was associated with impaired survival (p <0.00001). However, we also found a non‐linear association between MGMT methylation and OS (p = 0.002): median OS was 14.8 months for MGMT in 0–4%, 18.9 months for MGMT in 4–40%, and 29.9 months for MGMT in 40– 100%. Conclusions. Our findings suggested a non‐linear relationship between OS and MGMT promoter methylation, which implies a varying magnitude of prognostic effect across values of MGMT promoter methylation by pyrosequencing in newly diagnosed IDH wild‐type glioblastoma patients treated with chemoradiotherapy

Sensitivity Analysis of the MGMT-STP27 Model and Impact of Genetic and Epigenetic Context to Predict the MGMT Methylation Status in Gliomas and Other Tumors.

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Our model MGMT-STP27 allows prediction of the methylation status of the MGMT promoter using data from the Illumina's Human Methylation BeadChips (HM-27K and HM-450K) that is publically available for many cancer data sets. Here, we investigate the impact of the context of genetic and epigenetic alterations and tumor type on the classification and report on technical aspects, such as robustness of cutoff definition and preprocessing of the data. The association between gene copy number variation, predicted MGMT methylation, and MGMT expression revealed a gene dosage effect on MGMT expression in lower grade glioma (World Health Organization grade II/III) that in contrast to glioblastoma usually carry two copies of chromosome 10 on which MGMT resides (10q26.3). This implies some MGMT expression, potentially conferring residual repair function blunting the therapeutic effect of alkylating agents. A sensitivity analyses corroborated the performance of the original cutoff for various optimization criteria and for most data preprocessing methods. Finally, we propose an R package mgmtstp27 that allows prediction of the methylation status of the MGMT promoter and calculation of appropriate confidence and/or prediction intervals. Overall, MGMT-STP27 is a robust model for MGMT classification that is independent of tumor type and is adapted for single sample prediction