methylguanine-DNA Methyltransferase Promoter Methylation Research Articles

Low O6‐methylguanine‐DNA methyltransferase (MGMT) expression and response to temozolomide in aggressive pituitary tumours

Recent case reports detail the successful use of temozolomide in the management of aggressive pituitary tumours. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that counteracts the effect of temozolomide. To study MGMT expression in pituitary tumours and consider whether MGMT expression is associated with response to temozolomide therapy in aggressive pituitary tumours. We report two patients with aggressive pituitary tumours treated with temozolomide, one who responded to temozolomide and the other who did not. MGMT expression was assessed in a further 88 archived pituitary tumour samples. MGMT expression was assessed by immunohistochemistry. MGMT promoter methylation was studied by methylation-specific polymerase chain reaction (MSP), sequencing of MGMT was performed and loss of heterozygosity (LOH) analysis undertaken. Low MGMT expression and MGMT promoter methylation were found in the pituitary tumour of the patient who responded to temozolomide. Conversely, high MGMT expression was seen in the patient demonstrating a poor response to temozolomide. Eleven out of 88 archived tumour samples (13%) had low MGMT expression. Prolactinomas were more likely to have low MGMT expression compared with other pituitary tumour subtypes (P < 0.001). There was no significant difference in MGMT expression between invasive and noninvasive tumours, or between recurrent and nonrecurrent tumours. A significant inverse correlation was found between MGMT expression and promoter methylation (P = 0.012). MGMT expression as assessed by immunohistochemistry may predict response to temozolomide therapy in patients with aggressive pituitary tumours. MGMT promoter methylation is likely to explain low MGMT expression in some, but not all, pituitary tumours.

Temozolomide concomitant and adjuvant to radiotherapy in elderly patients with glioblastoma

A recent randomized study conducted on newly diagnosed glioblastoma (GBM) patients demonstrated that concomitant and adjuvant temozolomide added to standard radiotherapy had a survival advantage compared with radiotherapy alone. The overall survival benefit of this aggressive treatment, however, was attenuated in older or poor performance status patients. The aim of the present study was to verify the activity and the toxicity of temozolomide administration concurrent and adjuvant to radiotherapy as first-line treatment for elderly GBM patients, and to explore correlations between clinical outcome and O6 methylguanine-DNA methyltransferase (MGMT) promoter methylation status. Newly diagnosed GBM patients>or=65 years were considered eligible. Treatment comprised radiotherapy (60 Gy in 30 fractions over 6 weeks) plus continuous daily temozolomide (75 mg/m2/day), followed by 12 maintenance temozolomide cycles (150 mg/m2 once a day for 5 consecutive days every 28 days) if MRI showed no enhancement suggesting a tumor; otherwise, chemotherapy was delivered until complete response or unequivocal progression. A total of 58 patients (34 males; median age, 68 years; range, 65-82 years) were enrolled. Sixteen patients (43%) presented MGMT promoter methylated and 21 unmethylated (57%) status. The median progression-free survival and median survival time (MST) were 9.5 months (95% confidence interval [CI], 8.6-10.5) and 13.7 months (95% CI, 10-17.3 months), respectively. Mental status deterioration grade 3-4 was detected in 25% of patients. Leukoencephalopathy was diagnosed in 10% of patients. The overall and progression-free survival of patients given concomitant and adjuvant temozolomide are greater than in those given radiotherapy alone; however, this regimen incurs a greater deterioration in mental status. Further randomized trials should, therefore, be conducted to investigate the efficacy and against the toxicity of this regimen as first-line therapy in patients with GBM.

Quantitative analysis of 06-methylguanine-DNA methyltransferase (MGMT) promoter methylation in patients with low-grade gliomas

2069 Background: Loss of heterozygosity (LOH) on the chromosomes 1p and 19q is associated with sensitivity to alkylating agents like temozolomide (TMZ) in patients with low-grade gliomas; whether methylation of the MGMT-promoter, a predictive factor in glioblastoma patients, also correlates with tumor response to TMZ in low-grade gliomas is unclear. Methods: We performed a retrospective analysis of patients with histologically verified low-grade gliomas (WHO Grade II) who were treated with TMZ for tumor progression at our hospital between November 1999 and November 2007. Objective tumor response was assessed by MRI at 6-month intervals. LOH of microsatellite markers on chromosomes 1p and 19q was determined by polymerase chain reaction (PCR) amplification of the matched pairs of blood and tumor DNA. A methylation-specific, primer extension based PCR method was developed to quanitatively assess the MGMT methylation status in the tumour tissue. Results: Twenty-two patients with a median age of 53 years (range 27–72) were included in the study; 59% were male. Seven patients had prior surgical resection of the tumor. Histological classification revealed 10 oligodendrogliomas, 7 oligoastrocytomas, and 5 astrocytomas. All patients were treated with TMZ 200mg/m2 day1–5 in a 4 wk cycle. Grade 3–4 hematological toxicity occurred in 32% of the patients (9% leucopenia, 23% thrombocytopenia). The progression free survival was 32 months. Combined LOH 1p and 19q was found in 14 pts; 1 patient had LOH 1p alone and 1 patient LOH 19q alone. The LOH status could not be determined in two patients and was normal in the remaining four. MGMT promoter methylation was detectable in 20 pts by conventional PCR and quantitative analysis revealed a methylation status between 12%-100%. The volumetric response to chemotherapy analyzed after 6 months by MRI correlated with the level of MGMT promoter methylation (p = 0.012). Conclusions: Quantitative methylation-specific PCR of the MGMT promoter correlates with radiological response to chemotherapy with temozolomide in WHO grade II gliomas. No significant financial relationships to disclose.

Interferon‐beta, MCNU, and conventional radiotherapy for pediatric patients with brainstem glioma

Most children with brainstem glioma die within 2 years of diagnosis, and the median survival time for patients with this condition is less than 1 year. The role of chemotherapy in the treatment of children with brainstem glioma is not well defined. The primary aim of this study is to evaluate the effects of treatment with interferon-beta (IFN-beta), ranimustine (MCNU), and radiotherapy (IMR therapy) administered to brainstem glioma patients treated at our institution. We also determined patient response to IMR therapy by evaluating O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serum DNA. We retrospectively reviewed 15 patients who were newly diagnosed to have brainstem tumors and were administered IFN-beta (1-2 MIU/day, days 1-7; 0.5-1 MIU/day, days 8-14) and MCNU (80 mg/m(2) on day 2) concurrently with conventional radiotherapy. Responses were assessed by MRI scan, and data on clinical course and toxicity were obtained from the medical records. The MGMT promoter methylation in serum DNA of five patients was assayed by methylation-specific PCR. Of the 15 patients, partial response, stable disease, and progressive disease were noted in 5 patients each. The median overall survival time and the median progression-free survival time were 14.7 and 4.6 months, respectively. The protocol was not terminated in any of the patients because of hematological toxicity, nephrotoxicity, or neurotoxicity. The MGMT promoter methylation status in the serum appeared to correlate with a positive response to IMR therapy. The IMR combination therapy is well tolerated and may be a promising treatment for brainstem glioma.

High Quality Assessment of DNA Methylation in Archival Tissues from Colorectal Cancer Patients Using Quantitative High-Resolution Melting Analysis

High-resolution melting (HRM) analysis is a novel tool for analysis of promoter methylation. The aim of the present study was to establish and validate HRM analysis for detection of promoter methylation on archival formalin-fixed paraffin-embedded tissues from colorectal cancer patients. We first evaluated HRM assays for O(6)-methylguanine-DNA methyltransferase (MGMT) and adenomatous polyposis coli (APC) promoter methylation on a methylated DNA dilution matrix and DNA extracted from eight fresh or formalin-fixed paraffin-embedded human cancer cell lines. Then we used these assays for the analysis of MGMT and APC promoter methylation in a subset of archival formalin-fixed paraffin-embedded colorectal tumor specimens. All samples with promoter methylation of MGMT or APC and randomly selected samples without promoter methylation were analyzed twice. All results generated by HRM were validated with MGMT and APC MethyLight assays. APC and MGMT promoter methylation data were consistent and reproducible throughout the dilutions and all three replicates in the methylated DNA dilution matrix and between two experiments in clinical samples. There was high concordance between HRM and MethyLight results. HRM for APC promoter methylation revealed consistent results between fresh and formalin-fixed paraffin-embedded human cancer cell line DNA. The methylation status in archival tumor specimens from patients with colorectal cancer can therefore be determined with high quality by HRM. The ability to analyze archival tissues greatly facilitates further research and its clinical implementation.

The Association of Methylation in the Promoter of APC and MGMT and the Prognosis of Taiwanese CRC Patients

The purpose of this study was to investigate the association of methylation in the promoter regions of adenomatous polyposis coli (APC) and O(6)-methylguanine-DNA methyltransferase (MGMT) and the survival of Taiwanese colorectal cancer (CRC) subjects who received 5-fluorouracil (5-FU) adjuvant chemotherapy. DNA isolated from tumor tissue of 117 CRC subjects was analyzed for the existence of methylation in the promoter regions of APC and MGMT by methylation-specific PCR. Various characteristics of the 117 subjects were recorded and used in the Cox proportional-hazard model analyses. Methylation in the promoter region is 62.4% (73/117) for APC and 60.7% (71/117) for MGMT in our CRC patients. Subjects presenting methylation in the APC promoter demonstrate significantly lower hazards for all causes of death (hazard ratios=0.378, p=0.011) or CRC deaths (hazard ratios=0.426, p=0.039). However, no significant correlation is found between the methylation of MGMT promoter and the prognosis of CRC subjects. In addition, no interaction between 5-FU adjuvant chemotherapy and methylation of the two genes are observed. Methylation in the APC promoter may serve as a predictor for the prognosis of Taiwanese CRC patients.

High incidence of MGMT promoter methylation in primary glioblastomas without correlation with TP53 gene mutations

O 6 -methylguanine DNA methyltransferase ( MGMT) reduces cytotoxicity of therapeutic or environmental alkylating agents. MGMT promoter methylation has been associated with TP53 G: C to A:T transition mutations in various types of cancers, and with poor prognosis in patients who did not receive chemotherapy. Mutations of TP53 are more frequent in secondary than in primary glioblastoma, thus the expected MGMT promoter methylation was low in primary glioblastoma. Glioblastoma patients with MGMT promoter methylation showed better response to chemotherapy based on alkylating agents and longer survival than patients without MGMT methylation. We examined 32 primary glioblastomas, treated with radiotherapy and surgery, for TP53 mutation by direct sequencing and MGMT promoter methylation by methylation-specific PCR. MGMT promoter methylation and TP53 mutations were detected in 72% and 31% of primary glioblastoma, respectively. Although not statistically significant, the frequency of TP53 G:C to A:T mutations were higher in cases with (26%) than without (11%) MGMT promoter methylation ( p=0.376). MGMT promoter methylation had no impact on patient survival. Our data indicate that MGMT promoter methylation occurs frequently in primary glioblastoma, but does not lead to G:C to A:T TP53 mutations, has no independent prognostic value and is not a predictive marker unless glioblastoma patients are treated with chemotherapy.

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.

Promoter Methylation and Polymorphisms of the MGMT Gene in Glioblastomas: A Population-Based Study

O⁶-methylguanine-DNA methyltransferase (MGMT) is a repair enzyme that removes promutagenic O⁶-methylguanine adducts in DNA, to protect cells from acquisition of G:C→ A:T mutations. MGMT promoter methylation and polymorphisms may affect MGMT expression and activity. In the present study, we assessed MGMT promoter methylation and polymorphisms (Leu84Phe, Ile143Val, c.–56C>T) in 371 glioblastomas diagnosed at the population level. MGMT methylation was observed in 165 (44%) glioblastomas, with a higher frequency in females than males (53 vs. 39%; p = 0.0106) and in secondary than primary glioblastomas (73 vs. 43%; p = 0.0074). The frequency of TP53 G:C→A:T mutations in glioblastomas with MGMT methylation was 25%, which was significantly higher than that in glioblastomas with MGMT methylation (16%; Fisher exact test; p = 0.0385). MGMT 143 Val allele in glioblastomas was significantly less frequent than in a healthy European Caucasian population, and was associated with longer survival than those with the MGMT 143 Ile allele (hazard ratio 0.70; 95% CI 0.48–1.01). These results suggest that MGMT methylation may be associated with susceptibility to acquire TP53 G:C→A:T mutations, and that MGMT polymorphisms may affect the risk and prognosis of glioblastomas.

Association of O6-Methylguanine-DNA Methyltransferase (MGMT) Promoter Methylation with p53 Mutation Occurrence in Non-Small Cell Lung Cancer with Different Histology, Gender, and Smoking Status

O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been demonstrated to associate with the G:C-->A:T transition mutation in the p53 gene of lung tumors. The purpose of this study is to clarify whether MGMT promoter methylation is not only associated with the shift from the G:C-->A:T mutation in the p53 gene but also whether MGMT increases other mutation patterns in lung tumors. To further verify whether a different prevalence of MGMT promoter methylation is observed in lung tumors with a different tumor histology, gender, and smoking status, 220 lung tumors were collected to evaluate the status of MGMT promoter methylation and p53 mutation using methylation-specific PCR (MSP) and direct sequencing, respectively. The data shows that a higher prevalence of MGMT promoter methylation was observed in tumors with the G:C-->A:T transition or other p53 mutation patterns compared with those with p53 wild-type (P < 0.001 for G:C-->A:T; P = 0.015 for other mutation patterns), and this prevalence was more pronounced in tumors from male than from female patients. MGMT promoter methylation in p53 mutation patterns had a different effect on squamous cell carcinomas (SCC) and adenocarcinomas (ADC). Interestingly, the highest prevalence of MGMT promoter methylation was found in male nonsmokers followed by male smokers and female nonsmokers. This may be a partial explanation for the reason why male nonsmokers had a higher p53 mutation occurrence than female nonsmokers. MGMT promoter methylation may associate with increased occurrence of p53 mutation including the G:C-->A:T transition and other p53 mutation patterns in lung cancer, especially among male nonsmokers.

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 temozolomide, thalidomide, and celecoxib for newly diagnosed glioblastoma in adults

We conducted a phase II study of the combination of temozolomide and angiogenesis inhibitors for treating adult patients with newly diagnosed glioblastoma. Patients who had stable disease following standard radiation therapy received temozolomide for 5 days in 28-day cycles, in combination with daily thalidomide and celecoxib. Patients were treated until tumor progression or development of unacceptable toxicity. Four-month progression-free survival (PFS) from study enrollment was the primary end point, and overall survival (OS) was the secondary end point. In addition, we sought to correlate response with O(6)-methylguanine-DNA methyltransferase promoter methylation status and serum levels of angiogenic peptides. Fifty patients with glioblastoma were enrolled (18 women, 32 men). Median age was 54 years (range, 29-78) and median KPS score was 90 (range, 70-100). From study enrollment, median PFS was 5.9 months (95% confidence interval [CI]: 4.2-8.0) and 4-month PFS was 63% (95% CI: 46%-75%). Median OS was 12.6 months (95% CI: 8.5-16.4) and 1-year OS was 47%. Of the 47 patients evaluable for best response, none had a complete response, five (11%) had partial response, four (9%) had minor response, 22 (47%) had stable disease, and 16 (34%) had progressive disease. Analysis of serial serum samples obtained from 47 patients for four angiogenic peptides failed to show a significant correlation with response or survival for three of the peptides; higher vascular endothelial growth factor levels showed a trend toward correlation with decreased OS (p=0.07) and PFS (p=0.09). The addition of celecoxib and thalidomide to adjuvant temozolomide was well tolerated but did not meet the primary end point of improvement of 4-month PFS from study enrollment.

Methylation pattern of the O6‐methylguanine‐DNA methyltransferase gene in colon during progressive colorectal tumorigenesis

O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous polyps (AP), 220 normal colonic mucosa from CRC patients (N-C) and 36 normal colonic mucosa specimens obtained from subjects without colorectal neoplasia (N-N) by combined bisulfite restriction analysis (COBRA). The region-specific methylation data were compared to the MGMT protein expression, spectrum of KRAS mutations and other clinical features. Extensive (including both Mp and Eh) and partial (either Mp or Eh) MGMT methylation were found in 24.5% and 11.6% of CRCs, 3.8% and 27.9% of APs, 0.5% and 7.7% of C-Ns and 2.8% and 2.8% of N-Ns, respectively. Extensive methylation of MGMT promoter was primarily present in CRCs while partial methylation was common in APs. Extensive methylation of MGMT promoter was associated with loss/reduced protein expression (p < 0.0001), as well as with G to A mutations in KRAS (p = 0.0017). We herein provide first evidence that extensive methylation of MGMT promoter region is essential for methylation-induced silencing of this gene. Our data suggest that MGMT methylation may evolve and spread throughout the promoter in a stepwise manner as the colonic epithelial cells progress through the classical-adenoma-cancer multistep cascade.

Variation of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation in serial samples in glioblastoma

Methylation of the promoter region of the O ( 6 ) -methylguanine-DNA methyltransferase (MGMT) gene is known to be predictive of response to temozolomide treatment in patients with glioblastoma. Contrastingly, little is known about variation in the methylation status of the MGMT promoter after treatment or across different regions of the same tumor. About 22 samples from 10 patients who had undergone multiple resections of a glioblastoma were examined with promoter sequencing. Of these, 20 were also analyzed using Methylation Specific PCR (MSP). The methylation status of the MGMT promoter was altered in the specimens obtained pre and post treatment in 2 of 9 samples as assessed by MSP and 7 out of 10 patients as assessed by promoter sequencing. In four patients, the MGMT promoter was unmethylated at primary surgery, but displayed some methylation (32, 44, 12, and 4%) on post-treatment sampling. Alteration in MSP status from unmethylated to methylated was also observed in 2 of these 4 patients. In another patient, methylation increased from 40% on initial sampling to 68% on the second sample. The remaining two patients initially demonstrated some degree of methylation (72% and 12%); subsequent sampling showed no methylation of the MGMT promoter. To ensure variable methylation status was not due to intra-tumoral variability, three to four specimens were sampled from different regions of large glioblastomas (n = 7). Promoter sequencing revealed minimal variation in methylation in all but two sites examined. Immunohistochemistry also demonstrated minimal change in MGMT expression across the tumors. This suggests that variation in MGMT promoter methylation can occur within the same tumor after treatment, necessitating caution in clinical decision-making based on this analysis.

New developments in the treatment of malignant gliomas

Malignant gliomas represent an heterogeneous group of brain tumors both in terms of natural history and response to treatment. The standard therapeutic approach for treating glioblastomas is a combination of radiotherapy and concomitant/adjuvant temozolomide, and methylguanine-DNA methyltransferase promoter methylation is now recognized as an important factor for predicting both prognosis and response to alkylating agents. In the future, the discovery of targeted therapies will increasingly allow personalized medical treatments. Anaplastic oligodendroglial tumors display a better prognosis and are more chemosensitive than glioblastomas; the discovery of molecular factors of prognostic significance, such as 1p/19q codeletion, will lead to different treatment strategies for different subgroups of patients. Gliomatosis cerebri is a rare diffuse glioma, and upfront chemotherapy is increasingly being employed instead of whole-brain radiotherapy to avoid/delay cognitive defects in long surviving patients, despite the lack of data to support this.

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.

Efficacy of Temozolomide Is Correlated With 1p Loss and Methylation of the Deoxyribonucleic Acid Repair Gene MGMT in Malignant Gliomas

Promoter methylation of the deoxyribonucleic acid (DNA) repair gene, O(6)-methylguanine-DNA methyltransferase (MGMT), is associated with improved outcome of patients with glioblastoma multiforme and anaplastic astrocytoma treated with temozolomide (TMZ). Molecular genetic analysis of loss of heterozygosity (LOH) of 1p, 19q, or 10q, p53 mutation, and MGMT promoter methylation was performed in 44 assessable tumor specimens obtained from 46 patients with recurrent malignant gliomas, including 21 with glioblastoma multiforme, 17 with anaplastic astrocytoma, and eight with anaplastic oligoastrocytoma, which have heterogeneous features and variable histological diagnosis, to assess the correlation with the response to TMZ. LOHs of 1p and 19q, and MGMT promoter methylation showed positive correlations with the clinical response to TMZ therapy (p < 0.005, 0.05, and 0.05, respectively; Fisher's exact test). In addition, LOH of 1p and MGMT promoter methylation were associated with longer progression-free survival (p < 0.05 and 0.05, respectively; Cox regression analysis). LOH of 1p in the heterogeneous population of malignant gliomas may be one of the important factors besides MGMT methylation that predict better outcome in patients treated with TMZ.

Comethylation ofp16 andMGMTgenes in colorectal carcinoma: Correlation with clinicopathological features and prognostic value

To investigate the significance of p16 and O(6)-methylguanine-DNA methyltransferase (MGMT) genes promoter hypermethylation and K-ras mutations on colorectal tumorigenesis and progression. p16 and MGMT methylation status was examined on 47 tumor samples, and K-ras mutational status was examined on 85 tumor samples. For methylation analysis, a methylation specific PCR (MS-PCR) method was used. p16 and MGMT promoter methylation was found in 51% (24/47) and 43% (20/47) of CRCs, respectively, and the K-ras mutation was found in 44% (37/85) of CRCs. Comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease within a two-year period of observation. Only 27% of patients with simultaneous p16 and MGMT methylation showed the detectable occurrence of metastasis and/or death, compared to 67% of patients without double methylation or with no methylation (3/11 vs 22/33, P < 0.05, chi(2)-test). In addition, p16 and MGMT comethylation showed a trend toward an association with longer survival in patients with CRCs (35.5 +/- 6.0 mo vs 23.1 +/- 3.2 mo, P = 0.072, Log-rank test). Progression of the disease within a two-year period was observed in 66% of patients carrying the K-ras mutation, compared to only 19% of patients with wild type K-ras (29/44 vs 7/37, P < 0.001, chi(2)-test). The presence of the K-ras mutation significantly correlated to shortened overall survival (20.0 +/- 1.9 mo vs 37.0 +/- 1.8 mo, P < 0.001, Log-rank test). The comethylation of p16 and MGMT genes was significantly associated with lower aggressiveness of the disease even when K-ras mutations were included in the analysis as an independent variable. Our data suggest that comethylation of promoters of p16 and MGMT genes could have a prognostic value in patients with CRC. Specifically, concurrent methylation of both genes correlates with better prognosis.

Pattern of expression of genes linked to epigenetic silencing in human breast cancer

Epigenetic mechanisms such as DNA methylation are now recognized to play an important role in neoplasia. The aim of this study is to relate the pattern of expression of multiple cancer genes known to undergo epigenetic inactivation by promoter hypermethylation in breast cancer with histologic and outcome data. We used immunohistochemistry to study expression of the tumor suppressor gene p16, estrogen receptor (ER) alpha, ERbeta, progesterone receptor (PR), and the DNA repair gene MGMT (O6 -methylguanine-DNA methyltransferase) in a panel of 200 breast cancers. Methylation-specific polymerase chain reaction was used to confirm MGMT promoter methylation. Loss of expression of MGMT, ERalpha, ERbeta, PR, and p16 was observed in 19%, 24%, 13%, 40%, and 50% of cases, respectively. A significant correlation was seen between grade III tumor and loss of expression of ERalpha, ERbeta, PR (all P < .0001), and MGMT (P = .04), whereas loss of expression of p16 was associated with grades I and II tumors (P < .001). Cases that expressed 3 or less of the 5 proteins studied had significantly reduced survival (P = .0016). Methylation-specific polymerase chain reaction in a subset of 20 cancers showed DNA methylation associated with the loss of MGMT expression (P < .001). In conclusion, there is silencing of several key genes in breast cancer affecting molecular pathways involved in cell immortalization, DNA repair, and hormonal regulation, and this correlates significantly with risk of cancer-specific death. This expression profile could be linked to epigenetic events, and if so, these pathways have potential as targets for therapeutic strategies based on reversal of epigenetic silencing.

Frequent Promoter Hypermethylation of the O6- Methylguanine-DNA Methyltransferase (MGMT) Gene in Testicular Cancer

Testicular germ cell tumours are classified into two major histological subgroups, seminomas and nonseminomas. All tumours display several recurrent chromosomal aberrations, but few target genes have been identified. Previous studies have shown that genome-wide hypermethylation of CpG islands is significantly more prevalent in nonseminomas than in seminomas. We have studied two potential target genes in testicular cancer. A series of 70 tumours were analysed for methylation of CpG sites in the O 6 methylguanine-DNA methyltransferase (MGMT) gene promoter, and in exon 1a of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A). In addition, eight microsatellite markers within and flanking these genes at chromosome arms 10q and 9p, respectively, were analysed for allelic imbalances. Allele alterations were frequently seen at 9p loci (47 out of 70, 67%), but none of the tumours (none out of 55) showed methylation of CDKN2A. On the other hand, a high frequency of MGMT promoter methylation (32 out of 69, 46%) was found, as well as allelic imbalances at 10q markers (50 out of 70, 71%). A significantly higher methylation frequency was found in nonseminomas (24 out of 35, 69%) compared to seminomas (eight out of 33, 24%) (P=0.0003, Fisher’s exact test). Immunohistochemical analysis of the MGMT protein in a subgroup (n=20) of the testicular tumours supported the hypothesis of gene silencing being the functional consequence of the promoter methylation. In summary, our data suggest that inactivation of MGMT contributes to development of nonseminomatous testicular cancer. Oncogene (2002) 21, 8878‐8884. doi:10.1038/sj.onc. 1205978