methylguanine-DNA Methyltransferase Promoter Hypermethylation Research Articles

Prognostic effects of O6-Methylguanine DNA methyltransferase promoter hypermethylation in high-grade glioma patients with carmustine wafer implants

Background: Local chemotherapy with carmustine (BCNU) wafer implantation has survival benefits for malignant glioma patients. However, available data regarding its association with O6-methylguanine-DNA-methyltransferase (MGMT) are scant. Purpose: To evaluate whether MGMT hypermethylation has prognostic effects in malignant glioma patients with interstitial BCNU wafer implants. Methods: From September 2004 to August 2007, 32 patients with malignant gliomas underwent surgical resection plus interstitial BCNU wafer implantation at our hospital. Results and Conclusion: BCNU wafer implantation was performed in 18 patients with newly diagnosed gliomas and in 14 with recurrent gliomas. All patients had a Karnofsky performance status of ≥70. The median age was 51 years. At a median follow-up of 31 months, the 1- and 2-year overall survival (OS) rate was 43% and 22%, respectively. OS rates did not significantly differ between the newly diagnosed and recurrent patients. Gross total tumor resection was achieved in 19 (59%) patients, and MGMT hypermethylation was noted in 13 (41%) tumor specimens. Multivariate analysis demonstrated that patients with MGMT hypermethylation in their tumors and gross total tumor removal have more favorable survival rates (P = 0.03).

A potential role of karyopherin a2 in the impaired maturation of dendritic cells observed in glioblastoma patients

Aim: Patients with glioblastomas demonstrate well-documented immunological impairments including decreased numbers of mature dendritic cells (DCs). Recent data identified karyopherin a2 (KPNA2), a nucleocytoplasmic shuttling receptor, as diagnostic and prognostic biomarker for gliomas. The aim of this ongoing study is to correlate parameters of immunity and nucleocytoplasmic transport in glioblastoma patients. Methods: We preoperatively collected serum from 17 patients with glioblastomas and determined DC subsets (HLA DR+ Lin-, CD34-, CD45+, CD123+, CD11+ were analyzed) using a 6-color flow cytometry panel. Expression levels of KPNA2 and nuclear accumulation of p53 were evaluated semi-quantitatively by immunohistochemistry. O 6 -methylguanine DNA methyltransferase (MGMT) and isocitrate dehydrogenase-1 (IDH-1) status were assessed by pyrosequencing and immunohistochemistry, respectively. Results: Median expression levels for both KPNA2 and p53 were 5-10%. IDH-1-R132H mutation and MGMT promoter hypermethylation was detected in 3/16 and 1/9 patients, respectively. Mean counts of total mature DCs, myeloid DCs and plasmacytoid DCs were 9.6, 2.1, 3.4 cells/μL. A preliminary analysis suggests an association between low KPNA2 nuclear expression and increased numbers of mature DCs. However, this correlation did not reach statistical significance so far (P = 0.077). Conclusion: Our preliminary data may indicate a role of KPNA2 in the impaired maturation of DCs observed in glioblastoma patients.

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.

SDHB mutations are associated with response to temozolomide in patients with metastatic pheochromocytoma or paraganglioma.

Cyclophosphamide-dacarbazine-vincristine regimen is recommended for the treatment of malignant pheochromocytoma and paraganglioma (MPP); however, dacarbazine is the only recognized active drug in neuroendocrine tumours. We investigated the therapeutic benefit of temozolomide (TMZ), an oral alternative to dacarbazine, in patients with MPP. This is a retrospective study of consecutive patients with documented progressive MPP. We examined the correlation between Succinate dehydrogenase B (SDHB) mutation and O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation and MGMT expression in the French nation-wide independent cohort of 190 pheochromocytomas or paragangliomas (PP). Progression-free survival (PFS) according to RECIST 1.1 and PERCIST 1.0 criteria was the primary end point. Fifteen consecutive patients with MPP were enrolled; ten (67%) carried a mutation in SDHB. The mean dose intensity of TMZ was 172 mg/m(2) /d for 5 days every 28 days. Median PFS was 13.3 months after a median follow-up of 35 months. There were five partial responses (33%), seven stable (47%) and three progressive diseases (20%). Grade 3 toxicities were lymphopenia in two patients and hypertension in one. Partial responses were observed only in patients with mutation in SDHB. MGMT immunohistochemistry was negative in tumour samples from four patients who responded to treatment. SDHB germline mutation was associated with hypermethylation of the MGMT promoter and low expression of MGMT in 190 samples of the French nation-wide independent cohort. This study demonstrates that TMZ is an effective antitumour agent in patients with SDHB-related MPP. The silencing of MGMT expression as a consequence of MGMT promoter hypermethylation in SDHB-mutated tumours may explain this finding.

Loss expression of O6-methylguanine DNA methyltransferase by promoter hypermethylation and its relationship to betel quid chewing in oral squamous cell carcinoma

O(6)-methylguanine-DNA methyltransferase (MGMT) ameliorates mutagenic, carcinogenic, and cytotoxic adducts from O(6)-methylguanine in DNA through a direct reversal mechanism. Decreased expression of MGMT has been reported in a variety of human malignant tumors. The purpose of this study was to clarify the correlation of MGMT expression levels in oral squamous cell carcinoma (OSCC) with promoter hypermethylation and with betel quid chewing and cigarette smoking. MGMT protein expression in 63 cases of oral squamous cell carcinoma by immunohistochemistry was investigated. Methylation status of the MGMT was analyzed by methylation-specific PCR. Correlation with clinicopathologic parameters was then tested by statistical analysis. MGMT immunohistochemistry revealed nuclear staining in normal epithelium, whereas 47 (75%) of 63 OSCC tumors were devoid of MGMT expression and this was related to tumor cell differentiation. Furthermore, the association between loss of MGMT expression and promoter hypermethylation was significant. Lacking protein expression for MGMT in OSCC was also associated with the use of betel quid. The results suggest that the absence of MGMT expression, which would seem to be associated with promoter hypermethylation, is related to betel quid chewing and, thus, in turn, might be a significant event in oral carcinogenesis.

O 6-Methylguanine DNA methyltransferase determined by promoter hypermethylation and immunohistochemical expression is correlated with progression-free survival in patients with glioblastoma

The prognostic significance of O(6)-methylguanine DNA methyltransferase (MGMT) was evaluated by analysis of both MGMT promoter methylation and protein expression in a series of patients with newly diagnosed glioblastoma. Seventy-three patients with glioblastomas treated with alkylating agents were analyzed for MGMT expression by immunohistochemistry. Genomic DNA was isolated from frozen surgical specimens obtained from 62 of 73 patients. MGMT promoter methylation was determined by methylation-specific polymerase chain reaction. The prognostic significance of MGMT was evaluated together with other well-known prognostic factors. MGMT promoter hypermethylation was detected in 35 of 62 patients (56.4%). MGMT immunoreactivity was low in 26 (35.6%) tumors, moderate in 24 (32.9%), and high in 23 (31.5%). Significant correlation was observed between MGMT expression and MGMT promoter methylation (P < 0.001). Both MGMT promoter methylation and low MGMT expression were independently associated with better progression-free survival but not with longer overall survival. However, in the subgroup analysis, MGMT promoter hypermethylation was significantly associated with longer overall survival in patients treated with temozolomide (TMZ) after nimustine hydrochloride (ACNU) treatment. Low MGMT expression and MGMT promoter methylation are both predictive markers for slower tumor progression in patients with glioblastoma.

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.

Influence of expression of EGFR and PTEN on outcome in patients with primary glioblastoma treated with standard radiochemotherapy and cetuximab: Interim analysis from the GERT-Protocol

2050 Background: The epidermal growth factor receptor (EGFR) is commonly amplified, overexpressed, and mutated in glioblastoma (GBM). Anti-EGFR treatments have shown to be associated with EGFR deletion mutant variant III (EGFRvIII) expression in presence of PTEN expression, suggesting a prognostic role of EGFRvIII expression. We determined molecular biomarkers and correlated these with outcome in the GERT trial. Methods: To date, 39 patients were treated within the GERT protocol (Combs SE et al., 2006) evaluating radiochemotherapy (RCHT) with temozolomide (TMZ) and weekly CTX. Pretreatment paraffin-embedded tumor tissue of 32 patients was available for molecular analysis. Twenty-three patients were male, 9 were female. Median age was 49 years. We analyzed amplification of EGFR, expression of EGFR, EGFRvIII, the tumor-suppressor PTEN and O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Results: Median follow-up was 12 months. Overall survival (OS) at 12 and 24 months was 89% and 42%. Progression-free survival (PFS) was 76% and 45% at 6 and 12 months, respectively. MGMT promoter hypermethylation was detected in 16/32 tumours. Methylated MGMT did not impact PFS or OS (p = 0.48 and p = 0.08). Data on EGFR copy number of 31/32 tumors showed EGFR gene amplification in 11 tumors. EGFR protein expression was found in 23/32 patients. EGFR-amplification did not impact PFS or OS (p = 0.56, p = 0.3). Patients with EGFR expression showed longer PFS (p = 0.05), but unaltered OS (p = 0.06). EGFRvIII was seen in 5/32 patients, only in tumors with EGFR amplification. Expression of EGFRvIII did not influence PFS (p = 0.26) or OS (p = 0.09). Reduced PTEN (22/32) did not influence PFS or OS (p = 0.27, p = 0.85). Outcome was not associated with coexpression of EGFRvIII and PTEN (n = 5); coexpression of EGFR and PTEN (n = 13) significantly influenced PFS (p = 0.005), but not OS (p = 0.10). Conclusions: Expression of EGFR, and coexpression of EGFR/PTEN is associated with significant increase in PFS after RCHT with TMZ and CTX. EGFR-amplification, reduction of PTEN expression and expression of EGFRvIII did not impact PFS nor OS. Randomized data in the primary treatment of GBM might help identify patients for anti-EGFR therapies. [Table: see text]

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.

Promoter hypermethylation and inactivation of O6-methylguanine-DNA methyltransferase in esophageal squamous cell carcinomas and its reactivation in cell lines

The purpose of this study was to characterize the role of DNA hypermethylation in the loss of expression of O(6)-methylguanine-DNA methyltransferase (MGMT) during the development of esophageal squamous cell carcinoma (ESCC) and to investigate its reactivation in cell lines. Samples were collected from Linzhou City of the Henan province in northern China, a high-risk area of this disease. The hypermethylation of promoter CpG islands of the MGMT gene was examined by methylation-specific PCR in ESCC and neighboring non-tumorous tissues, as well as in laser capture microdissected samples with normal epithelium, basal cell hyperplasia (BCH), and dysplasia (DYS). The MGMT mRNA and protein expression were determined with RT-PCR and immunohistochemistry, respectively. Five of 17 (29%) normal, 10 of 20 (50%) BCH, 8 of 12 (67%) DYS, and 13 of 18 (72%) ESCC samples had DNA hypermethylation in the MGMT promoter region, showing a gradual increase with the progression of carcinogenesis. The frequency of the loss of MGMT mRNA and protein expression progressively decreased from normal to BCH, DYS, and ESCC, and it was highly correlated with MGMT promoter hypermethylation according to Fisher's exact tests. When each individual sample was considered, good concordance between DNA hypermethylation and the loss of expression of MGMT was also observed. In samples from the same patient, if hypermethylation was detected in earlier lesions, it was usually observed in more severe lesions. In the ESCC cell line KYSE 510, treatment with a DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine partially reversed the CpG hypermethylation status and restored mRNA expression of the MGMT gene. Similar reactivation of MGMT gene by dietary polyphenols, (-)-epigallocatechin-3-gallate and genistein, has also been observed. The results suggest that the DNA hypermethylation of MGMT is an important mechanism for MGMT gene silencing in the development of ESCC, and this epigenetic event may be prevented or reversed by these polyphenols for the prevention of carcinogenesis.

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.

Inactivation of DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relation to p53 mutations in esophageal squamous cell carcinoma.

The development of esophageal squamous cell carcinoma (ESCC) has been linked to exposure to carcinogens such as nitrosamines that cause various alkyl DNA damages and O6-methylguanine-DNA methyltransferase (MGMT) is a primary defence against alkylation-induced mutagenesis and carcinogenesis. This study was to investigate the role of inactivation of MGMT by promoter hypermethylation and its relation to p53 mutations in ESCC. Methylation of MGMT promoter was determined by methylation-specific polymerase chain reaction in 119 ESCC specimens, 22 corresponding tissue samples adjacent to the tumors, and 21 normal epithelial specimens of the esophagus. The levels of MGMT protein in ESCC with methylated or unmethylated MGMT were analyzed by quantitative immunohistochemistry. Mutations of p53 in 119 ESCC were detected by denaturing high-performance liquid chromatography and sequencing. We found that all 21 normal esophageal tissues had unmethylated MGMT; however, among 119 ESCC, 46 (38.7%) had hypermethylated MGMT. This epigenetic change also occurred in some normal tissues adjacent to the tumors. The level of MGMT protein in MGMT-methylated ESCC was significantly lower than that in MGMT-unmethylated ESCC, whereas great inter-individual variation and poor expression was also observed among MGMT-unmethylated ESCC. Fifty-one percent (61/119) ESCC showed p53 mutations but the distribution of the mutations did not differ significantly between MGMT-methylated ESCC (44%) and MGMT-unmethylated ESCC (56.2%; P = 0.18). MGMT promoter hypermethylation was neither associated with overall G:C to A:T mutations nor associated with this type of mutations in non-CpG dinucleotides in p53. Our results demonstrate that inactivation of MGMT by aberrant promoter methylation is a frequent molecular event in ESCC. This epigenetic alteration is an important, but may not be the sole, mechanism leading to the impaired expression of MGMT. Aberrant MGMT methylation seemed not to be associated with overall frequency and spectrum of p53 mutations in ESCC.

Alteration of O6-methylguanine-DNA methyltransferase in colorectal neoplasms in sporadic and familial adenomatous polyposis patients.

DNA repair failure is known to be a critical event during carcinogenesis of colorectal cancers. To investigate whether O(6)-methylguanine-DNA methyltransferase (MGMT) is altered during colorectal carcinogenesis, we performed immunohistochemical staining on 265 sporadic colorectal cancers, 113 sporadic adenomas, 33 familial adenomatous polyposis (FAP) colorectal cancers, and 93 FAP adenomas. Sixty-seven of 265 sporadic colorectal cancer cases and five of 113 sporadic adenoma cases showed loss of MGMT expression (P < 0.001). Among FAP patients, four of 33 cancers and six of 93 adenomas showed loss of MGMT protein expression. When we compared the association between MGMT promoter hypermethylation and protein expression, almost all cases without a methylated allele were positive for the expression of MGMT. In contrast, cases with promoter methylation frequently showed loss of MGMT expression (P < 0.01). Loss of MGMT was correlated with some clinicopathological characteristics, i.e., tumor invasion (P = 0.013) and stage (P = 0.035) in sporadic colorectal cancer, and degree of atypism (P = 0.042) in sporadic adenoma. Our results show that loss of expression of MGMT occurs more frequently in cancer than in adenoma in both sporadic and FAP patients, and that loss of expression of MGMT is associated with hypermethylation of the promoter area of MGMT gene.

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma.

O(6)-methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes promutagenic alkyl groups from the O(6) position of guanine in DNA. MGMT is transcriptionally silenced by promoter hypermethylation in several human cancers. Methylation-specific PCR (MSP) was used to analyze the MGMT promoter methylation status of 83 hepatocellular carcinomas (HCC) and 2 HCC cell lines (HepG2 and Hep3B). Hypermethylation was detected in 32 of 83 (39%) HCC tissues, but it was not found in either HCC cell line. We also analyzed MGMT expression by immunohistochemical analysis of HCC tissue samples. The presence of aberrant hypermethylation was associated with loss of MGMT protein. The relationship between methylation status and risk factors and tumor markers including environmental exposure to aflatoxin B(1) (AFB(1)), measured as DNA adducts, and status of tumor suppressor gene p53 was also investigated. A statistically significant association was found between MGMT promoter hypermethylation and high level of AFB(1)-DNA adducts in tumor tissues (OR = 5.05, 95% CI = 1.29-19.73). A significant association was also found between methylation and p53 mutation status (OR = 2.97, 95% CI = 1.09-8.11). These results suggest that epigenetic inactivation of MGMT plays an important role in the development of HCC and exposure to environmental carcinogens may be related to altered methylation of genes involved in cancer development. The role of chemical carcinogens in hypermethylation needs further investigation.

Promoter Methylation Status of the DNA Repair Genes hMLH1 and MGMT in Gastric Carcinoma and Metaplastic Mucosa

Hypermethylation of CpG islands in the promoter region is associated with the silencing of a variety of tumor suppressor genes. DNA repair genes human Mut L homologue 1 (hMLH1) and O⁶-methylguanine-DNA methyltransferase (MGMT) have been shown to be hypermethylated in certain carcinomas. We studied DNA methylation of CpG islands in hMLH1 and MGMT in 50 gastric carcinomas and 10 intestinal metaplastic mucosa samples. We analyzed the methylation status of hMLH1 and MGMT using methylation-specific polymerase chain reaction and DNA sequencing analysis. We measured protein levels of hMLH1 using Western blot and immunohistochemical analysis. CpG island hypermethylation of hMLH1 and MGMT was detected in 11 (22%) and 8 (16%) of the 50 gastric tumors, respectively. Hypermethylation of the promoter was more common in intestinal-type gastric carcinomas than in poorly diffuse-type gastric carcinomas (p = 0.016 and 0.021, respectively; Fisher’s exact test). However, hMLH1 promoter hypermethylation did not coincide with MGMT promoter hypermethylation except in 1 patient. Hypermethylation of the hMLH1 promoter but not the MGMT promoter occurred in intestinal metaplastic mucosae. Immunohistochemical analysis revealed a corresponding reduction in hMLH1 protein expression in some of the intestinal metaplastic mucosae. Our results suggest that at least two types of promoter methylation participate in the development of gastric carcinoma. Tumor-specific promoter hypermethylation of hMLH1 may be an early event in carcinogenesis in the stomach.

Hypermethylation of the DNA repair gene O(6)-methylguanine DNA methyltransferase and survival of patients with diffuse large B-cell lymphoma.

The gene encoding the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is transcriptionally silenced by promoter hypermethylation in several human cancers, including diffuse large B-cell lymphoma (B-DLCL). MGMT promoter hypermethylation is a favorable prognostic marker in patients with brain tumors treated with alkylating agents. In a retrospective cohort study, we used methylation-specific polymerase chain reaction to analyze the MGMT promoter methylation status in tumor DNA of B-DLCL patients receiving cyclophosphamide as part of multidrug regimens. Molecular data were compared with patient response with the use of Student's t test. Disease-free survival and overall survival were estimated by the Kaplan-Meier method and compared with the use of the log-rank test. Multivariable survival analyses were performed with the Cox proportional hazards model. All statistical tests were two-sided. Thirty (36%) of 84 B-DLCL patients showed MGMT promoter hypermethylation in their lymphomas. The presence of MGMT methylation was associated with a statistically significant increase in overall survival (hazard ratio for time to death for nonmethylation versus methylation = 2.8; 95% confidence interval (CI) = 1.2 to 7.5; P =.01) and progression-free survival (hazard ratio for time to progression for nonmethylation versus methylation = 2.6; 95% CI = 1.3 to 5.8; P =.02). MGMT promoter hypermethylation was both independent of and stronger than established prognostic factors, such as age, disease stage, serum lactic dehydrogenase level, and performance status. MGMT promoter hypermethylation appears to be a useful marker for predicting survival in patients with B-DLCL treated with multidrug regimens including cyclophosphamide.