methylguanine-DNA Methyltransferase Status Research Articles

O6-Methylguanine DNA methyltransferase protein expression in tumor cells predicts outcome of temozolomide therapy in glioblastoma patients

O(6)-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (chi(2) test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76-17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45-2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.

Prognostic impact of O6‐methylguanine‐DNA methyltransferase silencing in patients with recurrent glioblastoma multiforme who undergo surgery and carmustine wafer implantation

O(6)-methylguanine-DNA methyltransferase (MGMT) is a key enzyme in the DNA repair process after alkylating agent action. Epigenetic silencing of the MGMT gene by promoter methylation has been associated with longer survival in patients with newly diagnosed glioblastoma multiforme (GBM) who receive alkylating agents. In this study, the authors evaluated the prognostic value of different biomarkers in recurrent GBM and analyzed the changes in MGMT status between primary tumors and recurrent tumors. Twenty-two patients who had recurrent GBM and who underwent surgery with carmustine wafer implantation were enrolled prospectively between 2005 and 2007. The authors investigated the correlation between MGMT silencing in the tumor at recurrence and survival taking into account other clinically recognized prognostic factors. MGMT status was determined by using methylation-specific polymerase chain reaction analysis, a high-throughput quantitative methylation assay, and immunohistochemistry. In addition, expression analyses of human mutL homolog 1, human mutS homolog 2, and tumor necrosis factor alpha-induced protein 3 at recurrence were conducted with regard to their prognostic impact. The median progression-free survival (PFS) and overall survival (OS) rates after recurrence were 3.6 months and 9.9 months, respectively, and the 6-month PFS rate after recurrence was 27.2%. On multivariate analysis, only age (P=.04) and MGMT promoter hypermethylation at recurrence, as determined by MethyLight technology (P=.0012) and methylation-specific polymerase chain reaction (MSP) analysis (P=.004), were correlated with better PFS. On multivariate analysis, only MGMT promoter hypermethylation at recurrence, as determined by using MethyLight technology (P=.019) and MSP analysis (P=.046), was associated with better OS. MGMT methylation status was an important prognostic factor in patients with recurrent GBM who underwent surgery plus carmustine wafer implantation; therefore, it was useful in predicting the outcome of GBM therapy at recurrence.

Prolonged and severe thrombocytopenia with pancytopenia induced by radiation-combined temozolomide therapy in a patient with newly diagnosed glioblastoma—analysis of O 6-methylguanine-DNA methyltransferase status

We report a case of a 51-year-old woman with newly diagnosed glioblastoma multiforme (GBM) who was treated with surgery followed by the standard concomitant temozolomide (TMZ) and radiotherapy (RT). Although TMZ is generally safe and well-tolerated, she developed a sudden onset of prolonged and severe thrombocytopenia as the most prominent event of pancytopenia during the combined treatment, leading to discontinuation of the combined therapy. Thrombocytopenia lasted for more than 2 months with intensive, intermittent platelet transfusions. A bone marrow aspiration and biopsy performed after recovery of severe suppression still revealed reduced number of megakaryocytes. O(6)-methylguanine-DNA methyltransferase (MGMT) analyses showed methylated MGMT promoter in GBM, but unmethylated promoters in both peripheral blood leukocytes and bone marrow cells. This is the first report suggesting the irrelevance of MGMT status of normal hematopoietic cells to TMZ-induced severe thrombocytopenia and pancytopenia.

Validation of Real-Time Methylation-Specific PCR to Determine O6-Methylguanine-DNA Methyltransferase Gene Promoter Methylation in Glioma

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

Phase II study of combination BCNU and temozolomide (TMZ) prior to radiotherapy in newly diagnosed anaplastic oligodendroglial tumors

2068 Background: Chemosensitivity of oligodendroglial tumors has been widely demonstrated also no standard treatment has been defined to date. Combination of BCNU and TMZ offers the potential advantage of combining the two most efficient drugs in high grade glioma, and have been described to overcome chemoresistance in part by depleting O-6 methylguanine-DNA methyl transferase (MGMT). Our objective was to assess the clinical activity of the combination of BCNU and TMZ administered prior to radiotherapy, and to determine the predictive value of 1p/19q codeletion and MGMT status on response for patients (pts) with newly diagnosed anaplastic oligodendroglioma (AO) and anaplastic oligoastrocytoma (AOA). Methods: To be eligible in this ongoing multicentric phase II study, pts must present a newly diagnosed AO or AOA, with measurable disease after initial surgery. Pts are given BCNU 150 mg/m2 on day 1 and TMZ 110 mg/m2 on day one to day 5 of every 42-day cycle, up to 6 cycles before radiotherapy unless progression. MRI is performed every two cycles. Primary endpoint is objective response rate (Macdonald criteria). Secondary endpoints include progression-free survival, overall survival and predictive value of response for 1p and 19q deletions and MGMT status. Results: To date, 36 pts have been enrolled out of a planned accrual of 50 pts. For interim analysis which included centralised review of histology (WHO 2007) and imaging of the first 23 patients, 21 pts are evaluable and meet inclusion criterias: median age is 55 years; median KPS is 80; histology (WHO 2007) is AO grade III (6 pts), AOA grade III (9 pts), AOA grade IV (6). Median PFS is 16 months [CI 2.6 - 29.4]. Response include CR: 2(10%) and PR: 5(24%), SD: 5(24%), PD: 9(43%). Responses (RC+RP) were of 3/6 (50%) in AO grade III, 3/9 (33%) in AOA grade III, and 1/6 (17%) in AOA grade IV. Toxicity is acceptable. Molecular analysis is in progress. Conclusions: Interim analysis of this phase II study suggests that BCNU and TMZ is active in anaplastic oligodendroglial tumors and confirms heterogeneity of this population. Impact of molecular markers will be presented. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration AstraZeneca Oncology, Lilly Oncology, Schering-Plough

Intratumoral homogeneity of MGMT promoter hypermethylation as demonstrated in serial stereotactic specimens from anaplastic astrocytomas and glioblastomas

Hypermethylation of the DNA repair gene O(6)-methyl-guanine DNA methyltransferase (MGMT) has been linked to prolonged survival in glioblastoma patients treated with alkylating agents. It was aimed to analyze prospectively whether the MGMT status of malignant gliomas could be determined from small-sized stereotactic biopsies (maximum volume: 1 mm(3)). Special attention was directed towards the intratumoral distribution of the MGMT promoter methylation, the MGMT protein expression and potential correlations between both. Twenty-five adult patients were included (20 patients with primary World Health Organisation (WHO) Grade III or IV malignant gliomas, 5 patients with secondary malignant gliomas). About 2-4 biopsy specimens per tumor were collected from different sites within the tumor. Promoter methylation of the MGMT gene was assessed by methylation-specific PCR (MSP) and sodium bisulfite sequencing in each of the collected specimens (overall number of specimens: 69). Both methods were validated for application in small-sized tissue samples (1 mm(3)). The MGMT protein expression was analyzed by immunohistochemistry. The overall MGMT promoter methylation rate was 30% in the de novo group and 80% in the tumor progression group. The success rates of MSP and sequencing were 100% and 80%, respectively. Sequence analysis and MSP exhibited 100% concordant findings. No differences in MGMT promoter methylation were detected between the different samples of each individual tumor in 24 of 25 patients. One false negative result was obtained due to the contamination of the biopsy specimen by necrotic tissue. Tissue samples taken from different sites of each individual tumor (13 tumors investigated) exhibited equal or highly similar MGMT protein expression. No correlation between MGMT protein expression and MGMT promoter methylation was observed. The MGMT promoter methylation status of malignant gliomas can be reliably determined from small-sized stereotactic biopsies. The methylation profile, as defined by MSP and sodium bisulfite sequencing, constitutes a homogeneous marker throughout malignant gliomas. The lack of correlation between MGMT status and MGMT protein expression needs further evaluation.

A randomized phase II trial of concurrent temozolomide (TMZ) and radiotherapy (RT) followed by dose dense compared to metronomic TMZ and maintenance cis-retinoic acid for patients with newly diagnosed glioblastoma multiforme (GBM)

2031 Background: Metronomic and dose dense scheduling are alternatives to conventional TMZ regimens to overcome drug resistance in part by depleting O-6 methylguanine-DNA methyltransferase (MGMT). Furthermore, metronomic TMZ may inhibit endothelial recovery and act as an anti-angiogenic therapy; dose dense TMZ increases the intensity of drug delivery. Objective: To determine the overall (OS) and progression free survival (PFS) of patients with newly diagnosed GBM treated with concurrent TMZ and RT followed by dose dense or metronomic TMZ and maintenance cis-retinoic acid. Methods: Patients with newly diagnosed, histologically confirmed GBM underwent standard RT with TMZ. Upon completion of this treatment, patients were randomized to receive dose-dense TMZ (150mg/m2, days 1–7 and 15–21 of a 28 day cycle) or metronomic TMZ (50mg/m2 daily in 28 day cycles), for 6 cycles. Maintenance cis-retinoic acid was prescribed following the 6 cycles of adjuvant TMZ. OS and PFS were calculated from date of diagnosis. Prospective correlative tissue analysis of MGMT status is planned. A Simon minimax 2-stage design was used for each cohort. If either group has 70% survival probability at 1 year, further evaluation in a phase III trial will be recommended. Results: 51 patients were randomized: 24 to metronomic, and 27 to dose dense. Median age is 57, and median KPS 90. 26 patients have progression of disease (POD), with a median follow up of 5 months. Grade 3/4 hematologic toxicity occurred in 7 patients (14%), 3 in the metronomic and 4 in the dose dense arm. Conclusions: Our patient population is comparable to that of other upfront GBM treatment trials. Metronomic and dose dense TMZ appear to be well tolerated with equivalent toxicities. Early analysis suggests that patients on the dose dense regimen may have better PFS than those on the metronomic arm. [Table: see text] No significant financial relationships to disclose.

Apoptosis triggered by DNA damage O6-methylguanine in human lymphocytes requires DNA replication and is mediated by p53 and Fas/CD95/Apo-1.

Various tumor-therapeutic drugs and environmental carcinogens alkylate DNA inducing O(6)-methylguanine (O(6)MeG) that provokes cell death by apoptosis. In rodent fibroblasts, apoptosis triggered by O(6)MeG is executed via the mitochondrial damage pathway. Conversion of O(6)MeG into critical downstream lesions requires mismatch repair (MMR). This is thought to signal apoptosis upon binding to O(6)MeG lesions mispaired with thymine. Alternatively, O(6)MeG lesions might be processed by MMR giving rise to DNA double-strand breaks (DSBs) during replication that finally provoke apoptosis. To test this, we examined apoptosis triggered by O(6)MeG in human peripheral lymphocytes in which O(6)-methylguanine-DNA methyltransferase (MGMT) had been inactivated by O(6)-benzylguanine (O(6)BG) and which were not proliferating or proliferating upon CD3/CD28 stimulation. Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the anticancer drug temozolomide induced apoptosis only in proliferating, but not resting cells. With exceptional high alkylation doses (>/=15 microM of MNNG), apoptosis was also observed in resting lymphocytes, albeit at a lower level than in proliferating cells. This response was not affected by O(6)BG, suggesting that replication-independent apoptosis at high dose levels is caused by lesions other than O(6)MeG. O(6)MeG-triggered apoptosis in proliferating lymphocytes was preceded by a wave of DSBs, which coincided with p53 and Fas receptor upregulation, while Fas ligand, Bax and Bcl-2 expression was not altered. Treatment with anti-Fas neutralizing antibody attenuated MNNG-induced apoptosis in MGMT-depleted proliferating lymphocytes. The data suggest that O(6)MeG is converted by MMR and DNA replication into DSBs that trigger apoptosis by p53 stabilization and Fas/CD95/Apo-1 upregulation. This is supported by the finding that ionizing radiation, inducing DSBs on its own, provokes apoptosis in lymphocytes in a replication-independent way. The strict proliferation dependence of apoptosis triggered by O(6)MeG may explain the specific killing response of MGMT-deficient proliferating cells, including tumors, to O(6)MeG generating anticancer drugs and suggests that tumor proliferation rate, Fas responsiveness, MGMT and MMR status are important prognosis parameters.

Deficient expression of O(6)-methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and hMSH2 is related to poor prognosis in human biliary tract carcinoma.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O(6)-methylguanine to itself. Alkylation of DNA at the O(6) position of guanine is an important step in the induction of mutations in the organism by alkylating agents. The O(6)-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway. The biliary duct is highly exposed to alkylating agents because of its anatomical location. We examined 39 surgically resected gallbladder carcinomas and 35 extrahepatic bile duct carcinomas and evaluated the expression of MGMT and MMR protein (hMLH1 and hMSH2) by immunohistochemical staining. MGMT-negative staining was detected in 59.0% of gallbladder carcinoma specimens and 60.0% of extrahepatic bile duct carcinoma specimens. In gallbladder carcinoma, hMLH1- and hMSH2-negative staining was observed in 51.3% and 59.0%, respectively, whereas in extrahepatic bile duct carcinoma, the respective values were 57.1% and 65.7%. MGMT-negative staining correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, a combined MGMT and MMR status was shown to be a more significant prognostic biomarker in both tumor types. Combined MGMT and MMR is a possible prognostic marker that probably reflects an accumulation of genetic mutations.