MGMT Hypermethylation Research Articles

Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma

ENWEndNote BIBJabRef, Mendeley RISPapers, Reference Manager, RefWorks, Zotero AMA Adamek D, Zazula M, Radwańska E, Grzywna E, Sińczak-Kuta A, Moskała M. Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma. Contemporary Oncology/Współczesna Onkologia. 2011;15(6):362-366. doi:10.5114/wo.2011.26436. APA Adamek, D., Zazula, M., Radwańska, E., Grzywna, E., Sińczak-Kuta, A., & Moskała, M. (2011). Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma. Contemporary Oncology/Współczesna Onkologia, 15(6), 362-366. https://doi.org/10.5114/wo.2011.26436 Chicago Adamek, Dariusz, Monika Zazula, Edyta Radwańska, Ewelina Grzywna, Anna Sińczak-Kuta, and Marek Moskała. 2011. "Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma". Contemporary Oncology/Współczesna Onkologia 15 (6): 362-366. doi:10.5114/wo.2011.26436. Harvard Adamek, D., Zazula, M., Radwańska, E., Grzywna, E., Sińczak-Kuta, A., and Moskała, M. (2011). Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma. Contemporary Oncology/Współczesna Onkologia, 15(6), pp.362-366. https://doi.org/10.5114/wo.2011.26436 MLA Adamek, Dariusz et al. "Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma." Contemporary Oncology/Współczesna Onkologia, vol. 15, no. 6, 2011, pp. 362-366. doi:10.5114/wo.2011.26436. Vancouver Adamek D, Zazula M, Radwańska E, Grzywna E, Sińczak-Kuta A, Moskała M. Co-occurrence of MGMT gene promoter methylation and amplification of EGFR in glioblastoma. Contemporary Oncology/Współczesna Onkologia. 2011;15(6):362-366. doi:10.5114/wo.2011.26436.

Frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT in laryngeal squamous cell carcinomas and adjacent normal mucosa

Laryngeal cancers are the most frequent cancers of the head and neck region. While recent observations indicate the occurrence of an epigenetic field defect in head and neck cancer patients, a detailed exploration of the characteristic changes in the DNA methylation profile in laryngeal cancer patients was lacking. The aim of this study was to assess the methylation frequency of seven genes in a group of patients with primary laryngeal squamous cell carcinoma. Along tumor sections, matching samples of normal mucosa from epiglottis and trachea were analyzed. Gene methylation was assessed using the methylation-specific polymerase chain reaction. We found frequent gene hypermethylation in both the tumor and normal mucosa samples. The methylation of MGMT in tumor cells was associated with lymph node involvement. We report that laryngeal squamous cell carcinomas are characterized by frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT. Moreover, evidence is shown for the occurrence of a large epigenetically changed field of epithelial cells in laryngeal cancer patients. Our findings indicate the high prevalence of epigenetic abnormalities in laryngeal tumors.

MGMT promoter methylation, loss of expression and prognosis in 855 colorectal cancers

O⁶-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme. MGMT promoter hypermethylation and epigenetic silencing often occur as early events in carcinogenesis. However, prognostic significance of MGMT alterations in colorectal cancer remains uncertain. Utilizing a database of 855 colon and rectal cancers in two prospective cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we detected MGMT promoter hypermethylation in 325 tumors (38%) by MethyLight and loss of MGMT expression in 37% (247/672) of tumors by immunohistochemistry. We assessed the CpG island methylator phenotype (CIMP) using eight methylation markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and LINE-1 (L1) hypomethylation, TP53 (p53), and microsatellite instability (MSI). MGMT hypermethylation was not associated with colorectal cancer-specific mortality in univariate or multivariate Cox regression analysis [adjusted hazard ratio (HR) = 1.03; 95% confidence interval (CI), 0.79-1.36] that adjusted for clinical and tumor features, including CIMP, MSI, and BRAF mutation. Similarly, MGMT loss was not associated with patient survival. MGMT loss was associated with G>A mutations in KRAS (p = 0.019) and PIK3CA (p = 0.0031). Despite a well-established role of MGMT aberrations in carcinogenesis, neither MGMT promoter methylation nor MGMT loss serves as a prognostic biomarker in colorectal cancer.

Differential hypermethylation of genes in vulvar cancer and lichen sclerosus coexisting or not with vulvar cancer

Squamous cell carcinoma (SCC) of the vulva is a heterogeneous disease, associated or not with vulvar lichen sclerosus (LS). The precursor role of LS in vulvar cancer is unclear. We studied the epigenetic alterations of RASSF1A, RASSF2A, p16, TSP-1 and MGMT genes in vulvar SCCs, LS associated with SCC, isolated LS and normal vulvar skin. Gene hypermethylation and human papillomavirus presence were evaluated by methylation-specific PCR and PCR/reverse line blot, respectively. High-risk human papillomavirus types were present in 16.7% of the patients with vulvar SCC. There were increasing percentages of hypermethylation of genes from isolated LS to LS associated with vulvar SCC and vulvar SCC. The genes were hypermethylated more frequently in vulvar SCC associated with LS than in those not associated with LS, MGMT and RASSF2A being unmethylated in LS not associated with vulvar SCC. TSP-1 hypermethylation was related to recurrence in patients with vulvar cancer. Conclusions are as follows: (i) the epigenetic inactivation of genes is a common event in vulvar SCC and is also present in adjacent lesions, implying a possible precursor role for these alterations; (ii) MGMT and RASSF2A hypermethylation are present exclusively in vulvar SCC and LS associated with SCC, and absent from isolated LS; and (iii) TSP-1 hypermethylation is a bad prognosis factor in vulvar SCC.

Frequent gene hypermethylation in laryngeal cancer cell lines and the resistance to demethylation induction by plant polyphenols

Promoter hypermethylation is one of the mechanisms in the transcriptional inactivation of certain carcinoma – associated genes. In laryngeal cancers hypermethylation of tumor suppressor genes is related to their major risk factors- cigarette smoking and drinking strong alcohols. Since DNA methylation is reversible, modulation of the activity of DNA methyltransferases is an established therapeutic strategy, which can be also applied in cancer chemoprevention. Here, using the MSP procedure, we evaluated the frequency of hypermethylation of RARbeta, RASSF1A, HIN-1, GSTP1, MGMT, VHL and DAPK genes in several laryngeal and other head and neck squamous cell carcinoma cell lines and the effect of various polyphenols on the methylation of RARbeta and MGMT genes in the UT-SCC 42B cell line. Most of the cell lines tested were characterized by the hypermethylation of at least one of the genes analyzed. The most frequently hypermethylated genes were RARbeta and MGMT, while GSTP1 and VHL were not methylated in any of the cell lines. None of the tested compounds, including decitabine used as a reference compound, changed the methylation of RARbeta and MGMT genes. These findings suggest that although hypermethylation of RARbeta and MGMT may be considered as potential epigenetic biomarker, their application as therapeutic/chemopreventive targets requires further studies.

Gene promoter hypermethylation in leukoplakia of the oral mucosa

Gene promoter hypermethylation in leukoplakia of the oral mucosa Mingli Liu1, Lei Feng2, Ximing Tang3, Shanchun Guo41Department of Physics, Tufts University School of Medicine, Boston, Massachussetts; 2Department of Thoracic/Head and Neck Medical Oncology, 3Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 4Sylvester Cancer Center, University of Miami School of Medicine, Florida, USAAbstract: To examine whether aberrant DNA methylation in the promoter region might occur earlier in tumorigenesis, particularly in premalignant lesions, we examined biopsies from 111 participants in a chemoprevention trial aimed at reversal of oral leukoplakia, using methylation-specific polymerase chain reaction for the promoter regions of the tumor suppressor gene CDKN2A (p16), the putative metastasis suppressor gene for death-associated protein kinase (DAP-K), the DNA repair gene O6-methyguanine-DNA-methyltransferase (MGMT), and the detoxification gene glutathione S-transferase p1(GSTP1). p16 promoter hypermethylation was detected in 21 of 82 (25.6%), DAP-K hypermethylation in 28 of 87 (32.2%), and MGMT hypermethylation in 32 of 106 (30.2%) oral leukoplakia lesions analyzed. No aberrant methylation was found at the GSTP1 gene in 110 lesions examined. Among 68 biopsies analyzed for all three genes (p16, DAP-K, MGMT), 17 biopsies were detected with an abnormal methylation pattern at only one gene, 15 at two genes, and 8 at all three genes. Among clinical characteristics and their correlation with methylation, only alcohol consumption was correlated with DAP-K methylation (P = 0.027), while MGMT methylation was more frequent in females (P = 0.003) and nonsmokers (P = 0.0005). A significant correlation was found between p16 and DAP-K hypermethylation; p16 promoter was methylated in 14 (56%) of 25 lesions with DAP-K methylation, and only 5 (11.1%) of 45 DAP-K methylation-negative lesions (P = 0.0001). DAP-K aberrant methylation was also significantly correlated with MGMT methylation (16 of 31 in MGMT methylation-positive lesions versus 12 of 52 MGMT methylation-negative lesions, P = 0.0016). Our results suggest that epigenetic mechanisms of inactivation, such as aberrant methylation of p16, DAP-K, and MGMT genes, occur early in head and neck tumorigenesis, and might play a role in the progression of these lesions.Keywords: p16, DAP-K, MGMT, GSTP1 genes, methylation, leukoplakia

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Abstract SY28-04: Rational incorporation of novel agents into multimodality treatment of glioma and neuroblastoma

Detecting methylation patterns of p16, MGMT, DAPK and E‐cadherin genes in multiple myeloma patients

Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16, O6-methyl guanine DNA methyl transferase (MGMT), death-associated protein kinase (DAPK) and E-cadherin (ECAD); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16, MGMT, DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

Epigenetic alterations in disseminated neuroblastoma tumour cells: influence of TMS1 gene hypermethylation in relapse risk in NB patients

Most neuroblastoma patients over 18 months of age at diagnosis present disseminated disease. The presence of neuroblastoma cells in bone marrow can be used to evaluate the response to treatment. It is possible that alterations in certain tumour cells might confer a selective advantage over tumour dissemination process, and probably be helpful in the clonal selection of tumour-specific cells that could originate metastasis. We performed real-time quantitative PCR to identify the presence of disseminated tumour cells in bone marrow samples, and we used MSP to analyse the methylation profile of 20 genes putatively implied in dissemination. We described epigenetic alterations in the methylated status of certain genes in disseminated tumour cells from bone marrow. Those cases with high rate of hypermethylation showed an increased probability of relapse during or after treatment. We found significantly poor prognosis in event-free survival in cases with hypermethylation of TMS1, MGMT and RARbeta2 genes. We could not confirm the presence of a specific methylation profile in disseminated neuroblastoma tumour cells, but a high accumulation of epigenetic events in those cells is associated with a high risk of relapse, independently of MYCN amplification.

CpG Methylation and Reduced Expression of O6-Methylguanine DNA Methyltransferase Is Associated With Helicobacter pylori Infection

The gastric epithelium genome undergoes extensive epigenetic alterations during Helicobacter pylori-induced gastritis. Expression of the gene encoding the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) might be reduced via hypermethylation of its promoter in patients with H pylori gastritis. We characterized expression of MGMT and its epigenetic regulation via CpG methylation in gastric tissue from patients with H pylori gastritis and investigated the effects of H pylori infection eradication on MGMT expression. Gastric biopsy samples were collected from patients with H pylori gastritis before and after eradication and from H pylori-negative control subjects. AGS cells were cocultured with H pylori to study the effects of H pylori infection on MGMT RNA, protein expression, and CpG methylation. CpG methylation of MGMT was more frequent in the gastric mucosa of patients with H pylori gastritis (69.7%) than in those without (28.6%, P = .022). MGMT methylation was significantly reduced after H pylori eradication (from 70% to 48% of cases, P = .039), and mean levels of CpG methylation decreased from 12.6% to 5.7% (P = .025), increasing MGMT expression. MGMT methylation was significantly associated with CagA-positive H pylori (P = .035). H pylori reduced MGMT protein and RNA levels and induced MGMT CpG methylation in gastric AGS cells. H pylori gastritis, particularly in patients infected with H pylori CagA-positive strains, is associated with hypermethylation of MGMT and reduced levels of MGMT in the gastric epithelium. MGMT promoter methylation is partially reversible after eradication of H pylori infection. These data indicate that DNA repair is disrupted during H pylori gastritis, increasing mutagenesis in H pylori-infected gastric mucosa.

Frequent MGMT (06-methylguanine-DNA methyltransferase) hypermethylation in long-term survivors of glioblastoma: a single institution experience

BackgroundThe aim of this retrospective study was to analyse the MGMT (06-methylguanine-DNA methyltransferase) promoter methylation status in long-term surviving (≥ 3 years) patients with glioblastoma multiforme (GBM).MethodsThe methylation status of the MGMT promoter was determined by bisulfite modification of the DNA and subsequent methylation-specific polymerase-chain-reaction (MSP). DNA was extracted from routinely formalin-fixed and paraffin-embedded tumour tissue samples.ResultsMSP yielded interpretable results in only 14 of 33 (42%) long-term surviving patients with GBM. A methylated band was seen in 3 of 14, methylated as well as unmethylated bands in 8 of 14 and an only unmethylated band in 3 of 14 patients, thus, yielding MGMT promoter methylation in 11 of 14 patients. The two groups of patients with methylated and unmethylated MGMT promoter status were too small to draw any firm statistical conclusions.ConclusionsLong-term surviving patients with GBM have very frequently intratumoural MGMT promoter methylation. This phenomenon discriminates long-term survivors from a non-selected group of patients with GBM. The standardization of the MSP for the determination of the MGMT promoter methylation status seems to be necessary in order to make this methodology a more reliable one.

Abstract A123: MGMT and MMR testing: NSCLC susceptibility to temozolomide

Abstract Background: The functional status of DNA repair systems impacts the sensitivity of tumors to chemotherapeutics. The anti-neoplastic activity of alkylating agents, such as temozolomide, is affected by the direct repair enzyme, O6-Methylguanine-DNA methyltransferase (MGMT), and by the mismatch repair system (MMR). Lack of MGMT makes a tumor susceptible to temozolomide, while a non-functional MMR renders the tumor resistant to this agent. We assayed these repair systems in a group of patients affected by Non-small cell lung cancer (NSCLC) in order to investigate the potential benefit of temozolomide to these patients, a group with one of the highest mortality rate and for which alkylating agents are not currently used. Method: The status of MGMT and MMR were determined by MGMT promoter hypermethylation and by Microsatellite Instability (MSI) testing on 96 paraffin-embedded NSCLC samples. MGMT hypermethylation results in gene silencing and low levels of the protein. This test was conducted at OncoMethylome by methylation-specific PCR of the gene's promoter sequences. MSI testing was conducted at LabCorp by multiplex PCR of six microsatellite regions. Samples with a normal MMR system show genomic stability in the microsatellite regions, while samples with an impaired MMR system show instability in one or more of these regions. Results: Ninety three (93) samples yielded interpretable results. Most of them (80) had no detected defects in the two repair mechanisms assessed. Nine (9) samples (10%) had hypermethylation of the MGMT promoter and four (4) samples (4%) had microsatellite instability. The defects were not overlapping. These results indicate that NSCLC has a low rate of MGMT silencing by promoter hypermethylation and also a very low rate of mutations in the MMR proteins Conclusions: MGMT silencing by promoter hypermethylation sensitizes cells to apoptosis by temozolomide, but only in the presence of a functional MMR system. If MMR is disabled, apoptosis is not triggered. Our results indicate that although NSCLC is not particularly susceptible to alkylating agents, there are no indications that it has active mechanisms of resistance to them. Our understanding of the molecular characteristics of NSCLC suggests that alkylating agents such as temozolomide, in combination with inhibitors that artificially lower the expression of MGMT, may be viable option to treat NSCLC. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A123.

Prognostic Significance of Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia (GCH) and Lymphomas of Germinal Center Origin.

Abstract 3950Poster Board III-886 INTRODUCTIONGerminal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. GC have been known to be a major source of B-cell lymphomas, including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCBLs. Much less is known about the role of methylation in GCH. We determined the methylation status of 5 tumor-suppressor genes in 50 patients with lymphoma of GC origin and 50 GCH in order to find any differences between the pathological and the physiological state as well as its prognostic significance. MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 30 DLCL, 20 FL and 50 GCH were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14 and MGMT. Methylation status of each gene was correlated with clinicopathological status. Overall survival (OS) rates were calculated by the Kaplan-Meier method and differences were compared with the log-rank test. RESULTS. Median age was 65 in patients with lymphoma and 19 for GCH. Sex distribution was similar in all entities (60% females). Both lymphoma groups were balanced with respect to the presence of B symptoms, bulky disease, bone marrow infiltration, advanced stage and high IPI/FLIPI. DAP-k promoter methylation was present in more patients with lymphoma (89 and 87%) than with BFH (37%) p<0.0001. RaRB was methylated with higher frequency in FL (60%) than in DLCL (23%) and FH (12%) p<0.0001. SHP1 was more frequently methylated in FL (67%) and GCH (58%) than in DLCL (20%) p=0.01. Promoter hypermethylation of SHP1 was significantly associated with longer OS (p=0.021). Methylation of RaRB, p14 and MGMT were associated with shortened OS but the differences were not statistically significant. Those patients with DAPK methylated live longer but not significantly. In multivariate analysis hypermethylation of none of the genes studied remained an independent prognostic factor. CONCLUSIONS. Inactivation of DAP-K, and Rarβ is present in GC lymphomas with significantly higher frequency than in BFH. Thus, it may have pathogenic significance. SHP1 is methylated more frequently if FL and BFH than in DLCL, therefore that gene may be associated with aggressive disease. Methylation of DAP-k, SHP1, Rarβ, p14 and MGMT has no significant impact on overall survival. Markers for aberrant methylation may represent a promising way to monitor the onset and progression of malignancies but more extensive and prospective trials are needed to precisely define its role. Disclosures:No relevant conflicts of interest to declare.

Promoter hypermethylation of multiple genes in early gastric adenocarcinoma and precancerous lesions

Promoter hypermethylation is an alternative mechanism of gene silencing in human cancers including gastric cancer. To date, several reports on methylation of various genes in gastric cancer have been published. However, most of these studies have focused on cancer tissues or only a single gene. In this study, we determined the methylation frequency of 5 genes, including p16, Runx3, MGMT, DAPK, and RASSF1A, by methylation-specific polymerase chain reaction, in a series of formalin-fixed paraffin-embedded tissues including normal gastric mucosa (n = 20), intestinal metaplasia (n = 14), gastric epithelial dysplasia (n = 27), and early gastric adenocarcinoma (n = 16). Immunohistochemistry was used to determine expression of MGMT and RASSF1A protein. All 20 histologically normal gastric biopsy specimens were methylation-free for all 5 genes. Aberrant hypermethylation of RASSF1A was not detected in any case from intestinal metaplasia to early gastric adenocarcinoma. The methylation rate of the other 4 genes increased with the histological progression from intestinal metaplasia to gastric epithelial dysplasia, to early gastric adenocarcinoma. Methylation was detected in 28.6% of intestinal metaplasia (4/14), in 77.8% of gastric epithelial dysplasia (21/27), and in 87.5% of early gastric adenocarcinoma (14/16). The average number of methylated genes in intestinal metaplasia, gastric epithelial dysplasia, and early gastric adenocarcinoma was 0.43, 1.3, and 1.8, respectively. Concurrent methylation in 3 or more genes was found in 7.1% of intestinal metaplasia, 11.1% of gastric epithelial dysplasia, and 31.3% of early gastric adenocarcinoma. No correlation was found between hypermethylation and other clinicopathologic parameters such as age, sex, Helicobacter pylori infection, and location of lesions. However, we observed a significant association between hypermethylation of p16 and MGMT and elevated serum carcinoembryonic antigen level. No reduction or loss of RASSF1A expression was observed in our study. Weak or loss of MGMT expression was found in 20 lesions and was significantly associated with promoter hypermethylation (P < .01). Our results suggest that promoter hypermethylation of the p16, Runx3, MGMT, and DAPK genes may play an important role in the pathogenesis of gastric precancerous lesions and early gastric adenocarcinoma. Hypermethylation and inactivation of RASSF1A, however, could be a later event in malignant transformation. Further studies are warranted to determine whether the presence of promoter hypermethylation in gastric precancerous lesions is associated with higher risk of subsequent cancer development and how to interrupt the malignant transition from intestinal metaplasia and gastric epithelial dysplasia to early gastric adenocarcinoma by developing some gene-targeting therapies that may reverse aberrant methylation.

Promoter methylation of TIMP3 and CDH1 predicts better outcome in head and neck squamous cell carcinoma treated by radiotherapy only

As with other solid tumor types, head and neck squamous cell carcinoma (HNSCC) has been identified as an epigenetic, as well as genetic, disease. Consequently, promoter hypermethylation, being the most important aberrant epigenetic characteristic, has been intensively investigated for its biomarker potential in this cancer type. As many of these evaluations are obscured by a heterogeneity of treatments, the current study aimed to evaluate the incidence and prognostic value of the promoter hypermethylation of TIMP3, CDH1, DAPK, RASSF1A, p16INK4A and MGMT in HNSCC treated solely by radiotherapy. In 46 patients with advanced HNSCC treated with a hybrid accelerated fractionation radiotherapy schedule, DNA extracted from pretreatment paraffin-embedded tumor biopsies was used to determine the methylation status of the genes of interest by methylation-specific PCR (MSP). The detected epigenetic silencing was related with outcome in terms of locoregional control (LRC), and overall (OS), disease-free (DFS) and disease-specific survival (DSS). Tumor biopsies revealed the epigenetic silencing of MGMT in 42.5% (17 of 40) of patients and of TIMP3 in 40.5% (17 of 42) of cases. For the remaining investigated genes, a lower methylation percentage was detected: 13.2% (5 of 38) for CDH1, 11.4% (4 of 44) for DAPK, 4.8% (2 of 42) for p16INK4A and 2.4% (1 of 41) for RASSF1A. The promoter hypermethylation of TIMP3 and CDH1 was significantly related with better LRC (p=0.009 and p=0.02, respectively), OS (p=0.005 and p=0.002, respectively), DFS (p=0.02 and p=0.004, respectively) and DSS (p=0.12 and p=0.007, respectively). In conclusion, in this representative group of 46 patients with advanced HNSCC treated by radiotherapy only, the epigenetic silencing of TIMP3 and CDH1 predicted a better outcome.

MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events

Background: To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients.Methods: We compared MGMT hypermethylation with G:C>A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations.Results: Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C>A:T KRAS mutations (55%) compared with those without these mutations (38%, P<0.001). No such difference was observed for G:C>A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P=0.006) or KRAS mutations (P=0.001), but was positively associated with BRAF mutations (P<0.001).Conclusions:MGMT hypermethylation is associated with G:C>A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.

P1.113. Hypermethylation of genes p14ARF, p16INK4a and MGMT on oral scrapings of patients with oral lichenoid disease

P1.113. Hypermethylation of genes p14ARF, p16INK4a and MGMT on oral scrapings of patients with oral lichenoid disease

Correlation of promoter hypermethylation in hTERT, DAPK and MGMT genes with cervical oncogenesis progression

DNA hypermethylation occurs during the multistep process of cervical carcinogenesis. We investigated whether the methylation status in the promoter region of a potential oncogene, the human telomerase reverse transcriptase (hTERT), and the tumor suppressor genes death-associated protein kinase (DAPK) and O6-methylguanine DNA methyltransferase (MGMT), were able to distinguish the early from late stages of cervical oncogenesis. The methylation status in the promoter of these genes was analyzed using real-time MethyLight analysis in 115 cervical specimens, including normal, premalignant [atypical squamous epithelial cells (ASCUS), low-grade squamous intraepithelial lesions (LGSIL), high-grade squamous intraepithelial lesions (HGSIL)] and cancer specimens. Clinicopathological parameters (cytology, histology, grade, stage) were compared to the levels of promoter hypermethylation. We found that hTERT, MGMT and DAPK hypermethylation levels were increased during cervical oncogenesis progression. hTERT promoter hypermethylation was able to distinguish normal from cancer (p=0.008), normal from premalignant (p=0.036), as well as premalignant from cervical cancer cases (p=0.003). A significant association was also observed between all three genes and the grade of cervical cancer, with hTERT showing a better association (p<0.0001). Our data suggest that the combination of hTERT, MGMT, DAPK promoter hypermethylation could have a potential function as molecular biomarker of cervical oncogenesis progression.

Silencing of MGMT expression by promoter hypermethylation in the metaplasia–dysplasia–carcinoma sequence of Barrett’s esophagus

To determine the relevance of MGMT in Barrett’s carcinogenesis, we analyzed promotor hypermethylation and expression of MGMT in Barrett’s adenocarcinomas and its paired precursor lesions from 133 patients using a methylation-specific PCR, real-time RT-PCR and immunohistochemistry. Hypermethylation was detected in 78.9% of esophageal adenocarcinomas, in 100% of Barrett’s intraepithelial neoplasia, in 88.9% of Barrett’s metaplasia, but only in 21.4% of normal esophageal mucosa samples ( P < 0.001) and correlated significantly with downregulation of MGMT transcripts ( P = 0.048) and protein expression ( P = 0.02). Decrease of protein expression was significantly correlated with progressed stage of disease, lymph node invasion and tumor size. We conclude, that aberrant promoter methylation of MGMT is a frequent and early event during tumorigenesis of Barrett’s esophagus. High prevalence of MGMT hypermethylation may represent a candidate marker for improved diagnosis and targeted therapy in Barrett’s adenocarcinoma.

Hypermethylation of O6-Methylguanine-DNA Methyltransferase (MGMT) Predicts Response to Laromustine (Cloretazine®) in Patients with Acute Myelogenous Leukemia (AML) or High-Risk Myelodysplastic Syndrome (MDS)

Laromustine (Cloretazine®) (1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl] hydrazine), a sulfonylhydrazine prodrug producing chlorethylating and carbamoylating subspecies, has demonstrated clinical activity in patients with hematologic disorders (Giles et al., J Clin Oncol, 2007). The effect of laromustine is modulated primarily through the formation of hard chloroethylating electrophiles with preferential alkylating activity for the O6 position of guanine, ultimately resulting in the formation of interstrand cross-links which prevent DNA replication and transcription, leading to cell death. The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) plays a major role in repairing O6-chloroethylguanine alkylations, critical to the formation of interstrand crosslinks. Epigenetic silencing of the MGMT encoding gene related to DNA hypermethylation has been shown to participate in the pathogenesis of neoplastic disease (Hegi et al., NEJM 2005). Since the alkylating properties of laromustine target DNA sites normally repaired by MGMT, the absence of the enzyme may represent a unique cellular environment for specific susceptibility to laromustine (Ishiguro et al., Mol Cancer Ther, 2005). In vitro findings have supported the hypothesis that cellular content of MGMT may predict response. In vivo correlation between clinical response in patients treated with laromustine and the cellular evidence of epigenetic silencing of the encoding MGMT gene has yet to be demonstrated (Giles et al., Clin Cacner Res, 2004). Our research aimed to determine the DNA methylation status of MGMT isolated from the peripheral blood or bone marrow of patients with AML or high-risk MDS enrolled to a phase II, single-agent study of laromustine (600 mg/m2 IV over 30 minutes) (Giles et al. J Clin Oncol, 2007). We also aimed to establish a correlation between hypermethylation of MGMT and clinical response to laromustine. Combined bisulfite restriction analysis (COBRA) was used to determine the MGMT gene methylation status of patients treated with laromustine. Bone marrow or peripheral blood leukocyte samples from 76 patients enrolled in a phase II, single agent study of laromustine were coded and blinded to investigators. DNA from each sample was extracted and bisulfite treated. PCR was used to amplify the MGMT CpG Island promoter region (REF/NT_008818.15/Hs10_8975) from 58 patients, prior to methylation specific restriction enzyme digestion. Results were correlated with clinical data of response to laromustine. The DNA methylation status of MGMT was determined in 58 of the enrolled patients. DNA hypermethylation was found in 3 of the 58 patients (5%). Two of these 3 patients achieved a complete response (CR) (66%), compared to 11 of the 55 patients who achieved a CR, CR with platelet recovery < 100,000/mm3 (CRp) or a partial response (PR) and did not exhibit MGMT hypermethylation (20%). Hypermethylation of the MGMT gene promoter is a rare event in AML (5%), however, epigenetic inactivation of MGMT may predict a subgroup of patients with a higher likelihood of response to laromustine (Odds Ratio=8). Clinical investigation of laromustine in patients with AML, high-risk MDS and other cancers, specifically those with a higher methylation frequency of the MGMT encoding gene is warranted.