Methylguanine DNA Methyltransferase Hypermethylation Research Articles

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.

Phase 1 Study of Preoperative Chemoradiation Therapy With Temozolomide and Capecitabine in Patients With Locally Advanced Rectal Cancer

Preoperative chemoradiation therapy (CRT) with capecitabine is a standard treatment strategy in patients with locally advanced rectal cancer (LARC). Temozolomide improves the survival of patients with glioblastoma with hypermethylated O(6)-methylguanine DNA methyltransferase (MGMT); MGMT hypermethylation is one of the colorectal carcinogenesis pathways. We aimed to determine the dose-limiting toxicity (DLT) and recommended dose (RD) of temolozomide in combination with capecitabine-based preoperative CRT for LARC. Radiation therapy was delivered with 45Gy/25 daily fractions with coned-down boost of 5.4Gy/3 fractions. Concurrent chemotherapy comprised fixed and escalated doses of capecitabine and temozolomide, respectively. The MGMT hypermethylation was evaluated in pretreatment tumor samples. This trial is registered with ClinicalTrials.gov with the number NCT01781403. Twenty-two patients with LARC of cT3-4N0 or cTanyN1-2 were accrued. Dose level 3 was chosen as the RD because DLT was noticeably absent in 10 patients treated up to dose level 3. An additional 12 patients were recruited in this group. Grade III adverse events were noted, and pathologic complete response (pCR) was observed in 7 patients (31.8%); MGMT hypermethylation was detected in 16. The pCR rate was 37.5% and 16.7% in the hypermethylated and unmethylated MGMT groups, respectively (P=.616). There was a tendency toward higher pCR rates in patients with hypermethylated MGMT. Future randomized studies are therefore warranted.

Clinicopathological significance and potential drug target of O6-methylguanine-DNA methyltransferase in colorectal cancer: a meta-analysis.

Emerging evidence indicates that O(6)-methylguanine-DNA methyltransferase (MGMT) is a candidate for tumor suppression in several types of human tumors including colorectal cancer (CRC). However, the correlation between MGMT hypermethylation and clinicopathological characteristics of CRC remains unclear. In this study, we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of MGMT hypermethylation on the incidence of CRC and clinicopathological characteristics. A comprehensive literature search was done from Web of Science, the Cochrane Library Database, PubMed, EMBASE, CINAHL, and the Chinese Biomedical Database for related research publications written in English and Chinese. Methodological quality of the studies was also evaluated. Analyses of pooled data were performed with Review Manager 5.2. Odds ratio (OR) and hazard ratio (HR) were calculated and summarized, respectively. Final analysis from 28 eligible studies was performed. MGMT hypermethylation is found to be significantly higher in CRC than in normal colorectal mucosa, the pooled OR from 13 studies including 1085 CRC and 899 normal colorectal mucosa, OR = 6.04, 95 % confidence interval (CI) = 4.69-7.77, p < 0.00001. MGMT hypermethylation is also significantly higher in colorectal adenoma than in normal colorectal mucosa, but it is significantly less compared to that in CRC patients. Interestingly, MGMT hypermethylation is correlated with sex status and is significantly higher in female than in male. MGMT hypermethylation is also associated with high levels of microsatellite instability (MSI). The pooled HR for overall survival (OS) shows that MGMT hypermethylation is not associated with worse survival in CRC patients. The results of this meta-analysis suggest that MGMT hypermethylation is associated with an increased risk and high levels of MSI and may play an important role in CRC initiation. However, MGMT hypermethylation may play an important role in the early stage of CRC progression and development, as well as having limited value in prediction of prognosis in CRC patients. We also discussed that MGMT may serve as a potential drug target of CRC.

MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area.

Inactivation of tumor suppressor and DNA repair genes by promoter hypermethylation does commonly occur in human cancers. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes methyl groups as well as larger adducts at the O(6) position of guanine. In the absence of MGMT activity, O(6)-methylguanine mispairs with thymine during DNA replication, resulting in G:C to A:T transitions. Promoter hypermethylation of the MGMT gene has been observed in various cancers, including gastric cancer. Here, we aimed at assessing the promoter hypermethylation, mutation and expression status of the MGMT gene in patients from a geographic region with a high incidence of gastric cancer (Kashmir, North India) and to investigate their association with various clinicopathological characteristics. In this study 82 gastric cancer samples and adjacent normal tissues were included. Mutations in the MGMT gene were detected by single stranded conformational polymorphism (SSCP) analysis and direct sequencing. Methylation-specific polymerase chain reaction (MS-PCR) and Western blot analyses were performed to detect promoter hypermethylation and concomitant (loss of) expression of the MGMT gene. Promoter hypermethylation of the MGMT gene was found in 52.44% (43 of 82) of the tumor samples and loss of MGMT protein expression was detected in 45.12% (37 of 82) of the tumor samples. Hypermethylation and loss of expression were significantly associated with higher tumor grades (moderately/poorly differentiated) (P < 0.05) and higher tumor stages (III/IV) (P < 0.05). In addition, MGMT hypermethylation and loss of expression were found to be significantly associated with high salt tea consumption (P < 0.05). Our results indicate that MGMT promoter hypermethylation and concomitant loss of MGMT protein expression may play an important role in the development of gastric cancer in the Kashmiri population. High salt tea consumption may be a risk factor.

MGMT Hydroxymethylation in glioblastoma

O⁶ -methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme which is commonly hypermethylated in glioblas-tomas. Methylation of the MGMT promoter is associated with an improved response to alkylating agent chemotherapy and patient survival. Hence MGMT promoter methylation is often assessed as a prognostic marker for determining the course of treatment. Cytosine hydroxymethylation is another DNA modification whose significance is only now becoming clear, and in mammals it is known to occur at high levels in the brain and pluripotent stem cells. Importantly, 5'hydroxymethylcytosine is indistinguishable from 5'methylcytosine by bisulphite-based techniques. We hypothesised that hydroxymethylation may confound MGMT methylation assessment in a subset of glioblastomas, and may account in part for those glioblastomas that are classified as having MGMT hypermethylation but who fail to respond to chemotherapy. In this study the levels of both hydroxymethylcytosine and methyl-cytosine were assessed in a cohort of gliomas. Hydroxymethylation was found to be present in most samples, and ranged from 0.7% to 80% of the total ‘methylation' signature of this region. These results demonstrate that hydroxymethylation can comprise a substantial amount of the ‘methylation' levels detected at the MGMT promoter in glioblastomas, and may potentially confound the classification of patients for treatment with alkylating agents.

Long-term survival with unmethylated MGMT glioblastoma multiforme

Introduction: Glioblastoma multiforme is the most aggressive tumor of the central nervous system. Despite advances in its management, overall prognosis remains poor, with a median survival time of less than one year. Good response to chemotherapy with temozolomide (TMZ) is usually associated with methylation of the promoter of the O(6)-methylguanine-DNA methyltransferase ( MGMT ) gene. Case Report: We describe here a patient with glioblastoma multiforme, who had good prognostic clinical features, age and performance status, but unmethylated MGMT promoter and who survived for eight years while treated with a multi-modal approach. Conclusion: Good response to treatment, despite the absence of MGMT hypermethylation, suggests that other genetic or molecular factors may predict prognosis and therapeutic response in patients with glioblastoma multiforme.

Epigenetic silencing of O6 -methylguanine DNA methyltransferase gene in NiS-transformed cells

Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O(6)-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.

Aberrant Hypermethylation of p14ARF and O6‐methylguanine‐DNA Methyltransferase Genes in Astrocytoma Progression

The aim of the present study was to elucidate genetic alterations that are critically involved in astrocytoma progression. We characterized 27 World Health Organization grade II fibrillary astrocytomas which later underwent recurrence or progression, paying specific attention to the CpG island methylation status of critical growth regulatory genes. p14(ARF) and O(6)-methylguanine-DNA methyltransferase (MGMT) hypermethylation represented frequent events (26% and 63%, respectively), which were mutually exclusive except in one case, with alternate or simultaneous methylation of these two genes occurring in 85% of our tumor series. Seventeen tumors (63%) contained TP53 mutations, which were closely related to the presence of MGMT methylation. Methylation of the p21(Waf1/Cip1), p27(Kip1) and p73 genes and homozygous deletion of the p16(INK4a), p15(INK4b) and p14(ARF) genes were not detected in any of the primary low-grade tumors. The presence of p14(ARF) methylation at first biopsy was associated with shorter patient survival, whereas the presence of MGMT methylation carried a better clinical outcome after salvage therapy. Examination of 20 cases whose histological data for recurrent tumors were available revealed that malignant progression occurred in all of the tumors with p14(ARF) methylation but less frequently (50%) in the lesions with MGMT methylation. On analysis of their respective recurrent tumors, five of six patients whose primary low-grade tumors carried p14(ARF) methylation exhibited homozygous co-deletions of the p14(ARF), p15(INK4b) and p16(INK4a) genes, which were restricted to glioblastoma as the most malignant end point. Our findings suggest that p14(ARF) hypermethylation and MGMT hypermethylation constitute distinct molecular pathways of astrocytoma progression, which could differ in biological behavior and clinical outcome.

High frequency and strong prognostic relevance of O 6-methylguanine DNA methyltransferase silencing in diffuse large B-cell lymphomas from the Middle East

Several clinically relevant molecular classifiers of diffuse large B-cell lymphoma (DLBCL) have recently been demonstrated in Western populations. However, substantial molecular differences have recently been shown between tumors derived from different ethnic groups. To investigate prevalence and interrelationship of recently suggested molecular prognostic markers in Middle East DLBCL, we analyzed coexpression of CD10/Bcl6 (by immunohistochemistry), t(14;18) translocations (by fluorescence in situ hybridization), and methylation of the gene encoding the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) in a series of 190 DLBCL patients from Saudi Arabia with clinical follow-up data. Coexpression of CD10/Bcl6 (germinal center-like immunophenotype) was found in 13%, t(14;18) translocations in 17.9%, and MGMT methylation in 75.9% of cases. There was a trend toward better prognosis (although statistically insignificant) in tumors with coexpression of CD10/Bcl6. MGMT methylation were significantly related to good prognosis. The combined analysis of both parameters revealed that MGMT methylation was independent of immunophenotype and remained a significant predictor of prognosis in nongerminal center-like DLBCL subgroup. t(14;18) was significantly associated with CD10/Bcl6 coexpression (46.7%) but infrequent in CD10-/Bcl6-negative lymphomas (9.4%; P = .0073). However, t(14;18) was unrelated to clinical outcome. In summary, our data suggest a strong prognostic importance of MGMT methylation independent of DLBCL immunophenotype. Based on previous data from Western patients, the rate of MGMT hypermethylation was higher, and the portion of germinal center-like DLBCL was lower than expected. These results provide evidence for molecular differences between Saudi Arabian and Western DLBCL.

The prognostic impact of O6‐Methylguanine‐DNA Methyltransferase (MGMT) promotor hypermethylation in esophageal adenocarcinoma

Promotor hypermethylation is a common event in human cancer. O6-Methylguanine-DNA Methyltransferase (MGMT) is a gene involved in DNA repair, which is methylated in a variety of cancer types. In colorectal cancer and lung cancer, hypermethylation of MGMT has been correlated with p53 mutation. In the present study, 132 samples of esophageal adenocarcinoma and 58 samples of normal esophageal tissue were investigated for MGMT hypermethylation status by methylation-specific real-time PCR and results were correlated to clinicopathological parameters, patient's survival, p53 mutation and expression of p53 protein and MGMT protein. In the carcinomas, hypermethylation of MGMT was found in 63.6% of cases and loss of MGMT protein expression in 48.5% of cases. Furthermore, MGMT hypermethylation was found in 5.7% of normal esophageal smooth muscle tissue, in 20.0% of esophageal squamous epithelium and in 61.5% of nonneoplastic Barrett's mucosa. In the carcinomas, hypermethylation of the MGMT gene was correlated with loss MGMT protein expression (p < 0.0001) and with high tumor differentiation (p = 0.0079). In contrast, no correlation between MGMT hypermethylation, Lauren's classification, WHO classification, tumor size, gender, age, pT category and pN category, and p53 status was found. Neither MGMT hypermethylation nor loss of MGMT protein expression was correlated with patient's survival. In conclusion, MGMT hypermethylation in esophageal adenocarcinoma is a frequent event that is associated with loss of MGMT protein expression but not with patient's outcome.

Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumors.

Allelic losses on the chromosome arms 1p and 19q have been associated with favorable response to chemotherapy and good prognosis in anaplastic oligodendroglioma patients, but the molecular mechanisms responsible for this relationship are as yet unknown. The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) may cause resistance to DNA-alkylating drugs commonly used in the treatment of anaplastic oligodendrogliomas and other malignant gliomas. We report on the analysis of 52 oligodendroglial tumors for MGMT promoter methylation, as well as mRNA and protein expression. Using sequencing of sodium bisulfite-modified DNA, we determined the methylation status of 25 CpG sites within the MGMT promoter. In 46 of 52 tumors (88%), we detected MGMT promoter hypermethylation as defined by methylation of more than 50% of the sequenced CpG sites. Real-time reverse transcription-PCR showed reduced MGMT mRNA levels relative to non-neoplastic brain tissue in the majority of tumors with hypermethylation. Similarly, immunohistochemical analysis showed either no or only small fractions of MGMT positive tumor cells. MGMT promoter hypermethylation was significantly more frequent and the percentage of methylated CpG sites in the investigated MGMT promoter fragment was significantly higher in tumors with loss of heterozygosity on chromosome arms 1p and 19q as compared to tumors without allelic losses on these chromosomes arms. Taken together, our data suggest that MGMT hypermethylation and low or absent expression are frequent in oligodendroglial tumors and likely contribute to the chemosensitivity of these tumors.

CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas.

The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents, and its loss in cancer cells is associated with hypermethylation of the MGMT CpG island. Thus, methylation of MGMT has been correlated with the clinical response to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in primary gliomas. Here, we investigate whether the presence of MGMT methylation in gliomas is also a good predictor of response to another emergent alkylating agent, temozolomide. Using a methylation-specific PCR approach, we assessed the methylation status of the CpG island of MGMT in 92 glioma patients who received temozolomide as first-line chemotherapy or as treatment for relapses. Methylation of the MGMT promoter positively correlated with the clinical response in the glioma patients receiving temozolomide as first-line chemotherapy (n = 40). Eight of 12 patients with MGMT-methylated tumors (66.7%) had a partial or complete response, compared with 7 of 28 patients with unmethylated tumors (25.0%; P = 0.030). We also found a positive association between MGMT methylation and clinical response in those patients receiving BCNU (n = 35, P = 0.041) or procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (n = 17, P = 0.043) as first-line chemotherapy. Overall, if we analyze the clinical response of all of the first-line chemotherapy treatments with temozolomide, BCNU, and procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea as a group in relation to the MGMT methylation status, MGMT hypermethylation was strongly associated with the presence of partial or complete clinical response (P < 0.001). Finally, the MGMT methylation status determined in the initial glioma tumor did not correlate with the clinical response to temozolomide when this drug was administered as treatment for relapses (P = 0.729). MGMT methylation predicts the clinical response of primary gliomas to first-line chemotherapy with the alkylating agent temozolomide. These results may open up possibilities for more customized treatments of human brain tumors.

Generating mutations but providing chemosensitivity: the role of O6-methylguanine DNA methyltransferase in human cancer.

O(6)-methylguanine DNA methyltransferase (MGMT) is a key enzyme in the DNA repair network. MGMT removes mutagenic and cytotoxic adducts from O(6)-guanine in DNA, the preferred point of attack of many carcinogens (i.e. methylnitrosourea) and alkylating chemotherapeutic agents (i.e. BCNU, temozolamide, etc.). Hypermethylation of the CpG island located in the promoter region of MGMT is primarily responsible for the loss of MGMT function in many tumor types. The methylation-mediated silencing of MGMT has two consequences for cancer. First, tumors with MGMT methylation have a new mutator phenotype characterized by the generation of transition point mutations in genes involved in cancer etiology, such as the tumor suppressor p53 and the oncogene K-ras. Second, MGMT hypermethylation demonstrates the possibility of pharmacoepigenomics: methylated tumors are more sensitive to the killing effects of alkylating drugs used in chemotherapy. These recent results underscore the importance of MGMT in basic and translational cancer research.

Increased Sensitivity of Transforming Growth Factor (TGF) β1 Null Cells to Alkylating Agents Reveals a Novel Link between TGFβ Signaling and O6-Methylguanine Methyltransferase Promoter Hypermethylation

Inactivation of the transforming growth factor β (TGFβ)-signaling pathway and gene silencing through hypermethylation of promoter CpG islands are two frequent alterations in human and experimental cancers. Here we report that nonneoplastic TGFβ1−/− keratinocyte cell lines exhibit increased sensitivity to cell killing by alkylating agents, and this is due to lack of expression of the DNA repair enzyme<i>O</i> ⁶-methylguanine DNA methyltransferase (MGMT). In TGFβ1−/− but not TGFβ1+/− cell lines, the CpG dinucleotides in the MGMT promoter are hypermethylated, as measured by restriction enzyme analysis and methylation specific polymerase chain reaction. In one unstable TGFβ1+/− cell line, loss of the wild type TGFβ1 allele correlates with the appearance of methylation in the MGMT promoter. Bisulfite sequencing shows that in the KO3 TGFβ1−/− cell line nearly all of the 28 CpG sites in the MGMT promoter 475 base pairs upstream of the start site of transcription are methylated, whereas most are unmethylated in the H1 TGFβ1+/− line. Treatment of the TGFβ1−/− cell lines with 5-azacytidine causes reexpression of MGMT mRNA and demethylation of CpG islands in the promoter. Analysis of the time course of methylation using methylation-specific polymerase chain reaction shows a lack of methylation in primary TGFβ1−/− keratinocytes and increasing methylation with passage number of immortalized clones. Subcloning of early passage clones reveals a remarkable heterogeneity and instability of the methylation state in the TGFβ1−/− keratinocytes. Thus, the TGFβ1−/− genotype does not directly regulate MGMT methylation but predisposes cells to immortalization-associated MGMT hypermethylation.