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Summary

Aberrant methylation in the promoter region of cancer-related genes leads to gene transcriptional inactivation and plays an integral role in lung tumorigenesis. Recent studies demonstrated that promoter methylation was detected not only in lung tumors from patients with lung cancer but also in sputum of smokers without the disease, suggesting the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. In the present study, we investigated promoter methylation of 4 genes frequently detected in lung tumors, including p16, MGMT, RASSF1A and DAPK genes, in sputum samples obtained from 107 individuals, including 34 never-smoking females and 73 mostly smoking males, who had no evidence of lung cancer but who were exposed to smoky coal emission in Xuan Wei County, China, where lung cancer rate is more than 6 times the Chinese national average rate. Forty nine of the individuals showed evidence of chronic bronchitis while the remaining 58 individuals showed no such a symptom. Promoter methylation of p16, MGMT, RASSF1A and DAPK was detected in 51.4% (55/107), 17.8% (19/107), 29.9% (32/107), and 15.9% (17/107) of the sputum samples from these individuals, respectively. There were no differences in promoter methylation frequencies of any of these genes according to smoking status or gender of the subjects or between individuals with chronic bronchitis and those without evidence of such a symptom. Therefore, individuals exposed to smoky coal emissions in this region harbored in their sputum frequent promoter methylation of these genes that have been previously found in lung tumors and implicated in lung cancer development.

Background

Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors.

Methods

Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies.

Results

The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene.

Conclusion

Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of these events may represent independent event in non-small lung tumorigenesis.

Background

Gene silencing of O6-methylguanine–DNA methyltransferase (MGMT) by promoter methylation improves the outcome of glioblastoma patients after combined therapy of alkylating chemotherapeutic agents and radiation. The purpose of this study was to assess the frequency of MGMT promoter methylation in soft tissue sarcoma to identify patients eligible for alkylating agent chemotherapy such as temozolomide.

Findings

Paraffin tumor blocks of 75 patients with representative STS subtypes were evaluated. The methylation status of the MGMT promoter was assessed by methylation-specific polymerase-chain-reaction analysis (PCR). Furthermore, immunohistochemistry was applied to verify expression of MGMT. MGMT gene silencing was assumed if MGMT promoter methylation was present and the fraction of tumor cells expressing MGMT was 20% or less. Methylation specific PCR detected methylated MGMT promoter in 10/75 cases. Immunohistochemical staining of nuclear MGMT was negative in 15/75 cases. 6/75 tumor samples showed MGMT promoter methylation and negative immunohistochemical nuclear staining of MGMT. In none of the tested STS subtypes we found a fraction of tumors with MGMT silencing exceeding 22%.

Conclusion

MGMT gene silencing is a rare event in soft tissue sarcoma and cannot be recommended as a selection criterion for the therapy of STS patients with alkylating agents such as temozolomide.

Background

Chronic obstructive pulmonary disease (COPD) is a disorder associated to cigarette smoke and lung cancer (LC). Since epigenetic changes in oncogenes and tumor suppressor genes (TSGs) are clearly important in the development of LC. In this study, we hypothesize that tobacco smokers are susceptible for methylation in the promoter region of TSGs in airway epithelial cells when compared with non-smoker subjects. The purpose of this study was to investigate the usefulness of detection of genes promoter methylation in sputum specimens, as a complementary tool to identify LC biomarkers among smokers with early COPD.

Methods

We determined the amount of DNA in induced sputum from patients with COPD (n = 23), LC (n = 26), as well as in healthy subjects (CTR) (n = 33), using a commercial kit for DNA purification, followed by absorbance measurement at 260 nm. The frequency of CDKN2A, CDH1 and MGMT promoter methylation in the same groups was determined by methylation-specific polymerase chain reaction (MSP). The Fisher’s exact test was employed to compare frequency of results between different groups.

Results

DNA concentration was 7.4 and 5.8 times higher in LC and COPD compared to the (CTR) (p < 0.0001), respectively. Methylation status of CDKN2A and MGMT was significantly higher in COPD and LC patients compared with CTR group (p < 0.0001). Frequency of CDH1 methylation only showed a statistically significant difference between LC patients and CTR group (p < 0.05).

Conclusions

We provide evidence that aberrant methylation of TSGs in samples of induced sputum is a useful tool for early diagnostic of lung diseases (LC and COPD) in smoker subjects.

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Purpose

To address the association between sequence variants within the MGMT promoter-enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy.

Experimental Design

SNPs identified through sequencing a 1.9kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide was assessed.

Results

The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20–41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to temozolamide was strongly correlated with levels of MGMT methylation and expression.

Conclusions

These studies provide strong evidence that the A allele of a MGMT promoter-enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, temozolamide treatment may benefit a subset of lung cancer patients methylated for MGMT.

Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11–81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16–19 other genes, thus predicting for a widespread methylation phenotype. Kaplan–Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event.

Objective

O6 –methylguanine-DNA methyltransferase (MGMT) acts to repair DNA damaged by alkylation of guanine residues. MGMT promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes [1-3]. The loss of MGMT activity and promoter methylation is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas [4-6]. We sought to determine if MGMT promoter methylation plays a role in endometrial cancer.

Methods

One hundred and twenty primary endometrial cancers were analyzed for MGMT promoter methylation by combined bisulfite restriction analysis (COBRA). The cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. Twenty-one endometrioid and mixed endometrioid ovarian cancers were also analyzed. A subset of the primary tumors was analyzed for MGMT expression by immunohistochemistry.

Results

No MGMT promoter methylation was seen in the 120 endometrial cancers evaluated or the 6 endometrial cancer cell lines. One of the 21 endometrioid ovarian cancers showed methylation. Immunohistochemistry revealed moderate to high level expression of MGMT in the primary endometrial tumors.

Conclusion

MGMT promoter methylation is an infrequent event in endometrial cancer. MGMT expression and the ability to repair damaged alkylguanine residues could in part explain the limited response of endometrial tumors to alkylating chemotherapy.

O6-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.

DNA methylation and epigenetic inactivation of the O6-methylguanine methyltransferase (MGMT) gene induces MGMT deficiency, reducing the tumor cell’s DNA repair capacity and increasing its susceptibility to alkylating chemotherapeutic agents. Consequently, adult patients whose tumors are deficient in MGMT have better outcomes with alkylator chemotherapy, and MGMT methylation has been proposed as a screening marker of deficient tumors. In order to test the feasibility of this approach for medulloblastoma, a common brain tumor in children, we determined the methylation status, mRNA expression pattern, and protein expression of MGMT in a panel of clinical specimens. Methylation-specific polymerase chain reaction analysis revealed methylation of MGMT in 28 of 37 tumor samples. Quantitative real-time reverse transcriptase–polymerase chain reaction showed a range of expression of MGMT mRNA varying more than 20-fold. However, there was no correlation found between MGMT methylation and mRNA expression. Immunohistochemistry demonstrated that all tumors were immunoreactive for MGMT in the nucleus of the medulloblastoma cells in a heterogeneous pattern. The intercell variability of MGMT complement explained the discordance between methylation and expression. Therefore, MGMT methylation as determined by methylation-specific polymerase chain reaction cannot be used as a marker for MGMT deficiency in medulloblastoma. Further, these findings support the use of pharmacological MGMT depletion as a rational approach for intensification of alkylator chemotherapy in the treatment of medulloblastoma.

The O6-methylguanine-methyltransferase (MGMT) promoter methylation status is a predictive parameter for the response of malignant gliomas to alkylating agents such as temozolomide. First clinical reports on treating brain metastases with temozolomide describe varying effects. This may be due to the fact that MGMT promoter methylation of brain metastases has not yet been explored in depth. Therefore, we assessed MGMT promoter methylation of various brain metastases including those derived from lung (n = 91), breast (n = 72) kidney (n = 49) and from malignant melanomas (n = 113) by methylation-specific polymerase chain reaction (MS-PCR) and MGMT immunoreactivity. Fifty-nine of 199 brain metastases (29.6%) revealed a methylated MGMT promoter. The methylation rate was the highest in brain metastases derived from lung carcinomas (46.5%) followed by those from breast carcinoma (28.8%), malignant melanoma (24.7%) and from renal carcinoma (20%). A significant correlation of homogeneous MGMT-immunoreactivity (>95% MGMT positive tumor cells) and an unmethylated MGMT promoter was found. Promoter methylation was detected in 26 of 61 (43%) tumors lacking MGMT immunoreactivity, in 17 of 63 (27%) metastases with heterogeneous MGMT expression, but only in 5 of 54 brain metastases (9%) showing a homogeneous MGMT immunoreactivity. Our results demonstrate that a significant number of brain metastases reveal a methylated MGMT-promoter. Based on an obvious correlation between homogeneous MGMT immunoreactivity and unmethylated MGMT promoter, we hypothesize that immunohistochemistry for MGMT may be a helpful diagnostic tool to identify those tumors that probably will not benefit from the use of alkylating agents. The discrepancy between promoter methylation and a lack of MGMT immunoreactivity argues for assessing MGMT promoter methylation both by immunohistochemical as well as by molecular approaches for diagnostic purposes.

O6-Methylguanine DNA methyltransferase (MGMT) is implicated as a major predictive factor for treatment response to alkylating agents including temozolomide (TMZ) of glioblastoma multiforme (GBM) patients. However, whether the MGMT status in GBM patients should be detected at the level of promoter methylation or protein expression is still a matter of debate. Here, we compared promoter methylation (by methylation-specific polymerase chain reaction) and protein expression (by Western blot) in tumor cell explants with respect to prediction of TMZ response and survival of GBM patients (n = 71). Methylated MGMT gene promoter sequences were detected in 47 of 71 (66%) cases, whereas 37 of 71 (52%) samples were scored positive for MGMT protein expression. Although overall promoter methylation correlated significantly with protein expression (χ2 test, P < .001), a small subgroup of samples did not follow this association. In the multivariate Cox regression model, a significant interaction between MGMT protein expression, but not promoter methylation, and TMZ therapy was observed (test for interaction, P = .015). In patients treated with TMZ (n = 42), MGMT protein expression predicted a significantly shorter overall survival (OS; hazard ratio [HR] for death 5.53, 95% confidence interval [CI] 1.76–17.37; P = .003), whereas in patients without TMZ therapy (n = 29), no differences in OS were observed (HR for death 1.00, 95% CI 0.45–2.20; P = .99). These data suggest that lack of MGMT protein expression is superior to promoter methylation as a predictive marker for TMZ response in GBM patients.

Large tumor suppressor (LATS) 1 and 2 are tumor suppressor genes implicated in the regulation of the cell cycle. The methylation statuses of the promoter regions of these genes were studied in Japanese lung cancers. The methylation statuses of the promoter regions of LATS1 and LATS2 were investigated by methylation-specific PCR. The findings were compared to clinicopathological features of lung cancer. Methylation-specific PCR showed that the LATS1 promoter region was hypermethylated in 95 out of 119 (79.8%) lung cancers. The methylation status of LATS1 was significantly associated with squamous histology (p=0.0267) and smoking status (never smoker vs. smoker; p=0.0399). LATS1-ummethylated patients harbored more EGFR mutations (p=0.0143). The LATS2 promoter region was hypermethylated in 160 out of 203 (78.8%) lung cancers. However, the methylation status had no association with the clinicopathological characteristics of the lung cancers cases. Both the LATS1 and LATS2 methylation statuses did not correlate with survival of lung cancer patients. Thus, the EGFR methylation status of the LATS genes has limited value in Japanese lung cancers.

Background and aims: O6-methylguanine methyltransferase (MGMT) repairs inappropriately methylated guanine in DNA. MGMT mutations have not previously been reported in cancers, but in colorectal tumours MGMT promoter methylation is common and has been associated with increased G:C>A:T transitions, a high frequency of K-ras mutations, and low level microsatellite instability (MSI low). However, some have suggested that MGMT changes are background or secondary events, with little importance for tumorigenesis.

Methods: We have analysed fresh frozen colorectal cancers and colorectal cancer cell lines for MGMT changes: mutations, allelic loss, and protein expression.

Results: Six of 113 cancers harboured somatic missense MGMT mutations, at least three of which probably caused reduced MGMT function and were accompanied by silencing or loss of the wild-type allele. Cancers with pathogenic MGMT mutations tended to harbour G:C>A:T somatic mutations at other loci. Overall, MGMT expression was reduced or lost in more than half of the cancers. We found no association between MGMT expression and the somatic mutation spectrum at APC, beta-catenin, K-ras, or p53, but decreased MGMT expression was weakly associated with the presence of a G:C>A:T change at any one of these loci. Reduced MGMT expression was not however associated with an increased frequency of K-ras mutations or with MSI low.

Conclusion: In summary, we found that mutation of MGMT contributes to decreased protein function. Our findings provide good evidence to show that MGMT changes, including methylation, are selected rather than background events, at least in some cases. Decreased MGMT expression or function probably has a weak or moderate effect on the mutation spectrum in colorectal cancers.

OBJECTIVES:

1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma.

BACKGROUND:

The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response.

METHODS:

Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry.

RESULTS:

MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis.

DISCUSSION AND CONCLUSION:

MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

Purpose

O6-methylguanine-methyltransferase (MGMT) promoter methylation has been shown to predict survival of patients with glioblastomas if temozolomide is added to radiotherapy (RT). It is unknown if MGMT promoter methylation is also predictive to outcome to RT followed by adjuvant procarbazine, lomustine, and vincristine (PCV) chemotherapy in patients with anaplastic oligodendroglial tumors (AOT).

Patients and Methods

In the European Organisation for the Research and Treatment of Cancer study 26951, 368 patients with AOT were randomly assigned to either RT alone or to RT followed by adjuvant PCV. From 165 patients of this study, formalin-fixed, paraffin-embedded tumor tissue was available for MGMT promoter methylation analysis. This was investigated with methylation specific multiplex ligation-dependent probe amplification.

Results

In 152 cases, an MGMT result was obtained, in 121 (80%) cases MGMT promoter methylation was observed. Methylation strongly correlated with combined loss of chromosome 1p and 19q loss (P = .00043). In multivariate analysis, MGMT promoter methylation, 1p/19q codeletion, tumor necrosis, and extent of resection were independent prognostic factors. The prognostic significance of MGMT promoter methylation was equally strong in the RT arm and the RT/PCV arm for both progression-free survival and overall survival. In tumors diagnosed at central pathology review as glioblastoma, no prognostic effect of MGMT promoter methylation was observed.

Conclusion

In this study, on patients with AOT MGMT promoter methylation was of prognostic significance and did not have predictive significance for outcome to adjuvant PCV chemotherapy. The biologic effect of MGMT promoter methylation or pathogenetic features associated with MGMT promoter methylation may be different for AOT compared with glioblastoma.

Hypermethylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene has been shown to be associated with improved outcome in glioblastoma (GBM) and may be a predictive marker of sensitivity to alkylating agents. However, the predictive utility of this marker has not been rigorously tested with regard to sensitivity to other therapies, namely radiation. To address this issue, we assessed MGMT methylation status in a cohort of patients with GBM who underwent radiation treatment but did not receive chemotherapy as a component of adjuvant treatment. Formalin-fixed, paraffin-embedded tumor samples from 225 patients with newly diagnosed GBM were analyzed via methylation-specific, quantitative real-time polymerase chain reaction following bisulfite treatment on isolated DNA to assess MGMT promoter methylation status. In patients who received radiotherapy alone following resection, methylation of the MGMT promoter correlated with an improved response to radiotherapy. Unmethylated tumors were twice as likely to progress during radiation treatment. The median time interval between resection and tumor progression of unmethylated tumors was also nearly half that of methylated tumors. Promoter methylation was also found to confer improved overall survival in patients who did not receive adjuvant alkylating chemotherapy. Multivariable analysis demonstrated that methylation status was independent of age, Karnofsky performance score, and extent of resection as a predictor of time to progression and overall survival. Our data suggest that MGMT promoter methylation appears to be a predictive biomarker of radiation response. Since this biomarker has also been shown to predict response to alkylating agents, perhaps MGMT promoter methylation represents a general, favorable prognostic factor in GBM.

Pancreatic cancer consists of an accumulation of genetic and epigenetic alterations. Recently, aberrant methylation of CpG islands of cancer-related genes has emerged as an important epigenetic mechanism of their transcriptional dysregulation during tumour development [1]. Therefore, new diagnostic methods, for early detection based on a better understanding of the molecular biology of pancreatic cancer, are required. We examined the methylation status of p16INK4A, RASSF 1A and methylguanine methyltransferase (MGMT) genes considered to be inactivated by promoter methylation in several tumours.

The p16INK4A is an important G1/S cell cycle regulator gene [2]. RASSF 1A gene is involved in apoptotic signalling, microtubule stabilization and cell cycle progression [3]. The MGMT gene removes mutagenic and cytotoxic alkyl-adducts from the O6-position of guanine in DNA. Hypermethylation of the gene leads to the inactivation of DNA repair and to microsatellite instability [4].

To date, little is known about the exact role of hypermethylation of these genes in pancreatic adenocarcinoma, as the molecular mechanisms underlying these neoplasms remain poorly understood.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the malignant transformation in gliomas. We hypothesized that quantitative analysis of methylated genes will provide prognostic values in malignant glioma patients. We used an immunocapturing approach followed by real-time polymerase chain reaction analysis to detect altered patterns of promoter methylation in O-6-methylguanine-DNA methyltransferase (MGMT), p16INK4a, tissue inhibitor of metalloproteinase-3 (TIMP-3), and thrombospondin 1 (THBS1). The tumor tissue and paired serum as well as cerebrospinal fluid (CSF) from 66 patients with malignant gliomas were studied. Serum and CSF from 20 age-matched noncancer individuals were used as control. Promoter hypermethylation in MGMT, p16INK4a, TIMP-3, and THBS1 was detected at high frequencies in tumor tissue, serum, and CSF. None of the control serum or CSF showed aberrant methylation. Hypermethylation in serum and CSF DNA was all accompanied with methylation in the corresponding tumor tissues with 100% specificity. Highly elevated MGMT, p16INK4a, and THBS1 methylation levels in gliomas serum were the sole independent factors predicting inferior overall survival in this cohort. For progression-free survival, hypermethylation of MGMT and THBS1 in CSF were the independent prognostic factors. Multiple gene promoter hypermethylation analysis appears to be promising as a prognostic factor in glioma and as a mini-invasive tumor marker in serum and/or CSF DNA. Evaluation of these changes may help in selecting glioma patients for optimal adjuvant treatments and modifying chemotherapy.

O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.

Methylating agents are involved in carcinogenesis, and the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) removes methyl group from O6-methylguanine. Genetic variation in DNA repair genes has been shown to contribute to susceptibility to squamous cell carcinoma of the head and neck (SCCHN). We hypothesize that MGMT polymorphisms are associated with risk of SCCHN. In a hospital-based case-control study of 721 patients with SCCHN and 1,234 cancer-free controls frequency-matched by age, sex and ethnicity, we genotyped four MGMT polymorphisms, two in exon 3, 16196C>T and 16286C>T and two in the promoter region, 45996G>T and 46346C>A. We found that none of these polymorphisms alone had a significant effect on risk of SCCHN. However, when these four polymorphisms were evaluated together by the number of putative risk genotypes (i.e. 16195CC, 16286CC, 45996GT+TT, and 46346CA+AA), a statistically significantly increased risk of SCCHN was associated with the combined genotypes with three to four risk genotypes, compared with those with zero to two risk genotypes [adjusted odds ratio (OR) = 1.27; 95% confidence interval (CI) = 1.05-1.53]. This increased risk was also more pronounced among young subjects (OR = 1.81; 95% CI = 1.11-2.96), men (OR = 1.24; 95% CI = 1.00-1.55), ever smokers (OR = 1.25; 95% = 1.01-1.56), ever drinkers (OR = 1.29; 95% CI = 1.04-1.60), patients with oropharyngeal cancer (OR = 1.45; 95% CI = 1.12-1.87), and oropharyngeal cancer with regional lymph node metastasis (OR = 1.52; 95% CI = 1.16-2.01). In conclusion, our results suggest that any one of MGMT variants may not have a substantial effect on SCCHN risk, but a joint effect of several MGMT variants may contribute to risk and progression of SCCHN, particularly for oropharyngeal cancer, in non-Hispanic whites.

Background:

Despite limited clinical efficacy, treatment with dacarbazine or temozolomide (TMZ) remains the standard therapy for metastatic melanoma. In glioblastoma, promoter methylation of the counteracting DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) correlates with survival of patients exposed to TMZ in combination with radiotherapy. For melanoma, data are limited and controversial.

Methods:

Biopsy samples from 122 patients with metastatic melanoma being treated with TMZ in two multicenter studies of the Dermatologic Cooperative Oncology Group were investigated for MGMT promoter methylation. We used the COBRA (combined bisulphite restriction analysis) technique to determine aberrant methylation of CpG islands in small amounts of genomic DNA isolated from paraffin-embedded tissue sections. To detect aberrant methylation, bisulphite-treated DNA was amplified by PCR, enzyme restricted, and visualised by gel electrophoresis.

Results:

Correlation with clinical data from 117 evaluable patients in a best-response evaluation indicated no statistically significant association between MGMT promoter methylation status and response. A methylated MGMT promoter was observed in 34.8% of responders and 23.4% of non-responders (P=0.29). In addition, no survival advantage for patients with a methylated MGMT promoter was detectable (P=0.79). Interestingly, we found a significant correlation between MGMT methylation and tolerance of therapy. Patients with a methylated MGMT promoter had more severe adverse events, requiring more TMZ dose reductions or discontinuations (P=0.007; OR 2.7 (95% CI: 1.32–5.7)). Analysis of MGMT promoter methylation comparing primaries and different metastases over the clinical course revealed no statistical difference (P=0.49).

Conclusions:

In advanced melanoma MGMT promoter, methylation correlates with tolerance of therapy, but not with clinical outcome.

To evaluate the mechanism of the development of therapeutic resistance after temozolomide treatment, we focused on changes in O6-methylguanine DNA methyltransferase (MGMT) and mismatch repair (MMR) between initial and recurrent glioblastomas. Tissue samples obtained from 24 paired histologically confirmed initial and recurrent adult glioblastoma patients who were initially treated with temozolomide were used for MGMT and MMR gene promoter methylation status and protein expression analysis using methylation-specific multiplex ligation probe amplification (MS-MLPA), methylation-specific polymerase chain reaction (MSP), and immunohistochemical staining. There was a significant decrease in the methylation ratio of the MGMT promoter determined by MS-MLPA, which was not detectable with MSP, and MGMT protein expression changes were not remarkable. However, there was no epigenetic variability in MMR genes, and a relatively homogeneous expression of MMR proteins was observed in initial and recurrent tumors. We conclude that the development of reduced methylation in the MGMT promoter is one of the mechanisms for acquiring therapeutic resistance after temozolomide treatment in glioblastomas.

Background

Lung cancer is the worldwide leading cause of death from cancer. Tobacco usage is the major pathogenic factor, but all lung cancers are not attributable to smoking. Specifically, lung cancer in never-smokers has been suggested to represent a distinct disease entity compared to lung cancer arising in smokers due to differences in etiology, natural history and response to specific treatment regimes. However, the genetic aberrations that differ between smokers and never-smokers’ lung carcinomas remain to a large extent unclear.

Methods

Unsupervised gene expression analysis of 39 primary lung adenocarcinomas was performed using Illumina HT-12 microarrays. Results from unsupervised analysis were validated in six external adenocarcinoma data sets (n=687), and six data sets comprising normal airway epithelial or normal lung tissue specimens (n=467). Supervised gene expression analysis between smokers and never-smokers were performed in seven adenocarcinoma data sets, and results validated in the six normal data sets.

Results

Initial unsupervised analysis of 39 adenocarcinomas identified two subgroups of which one harbored all never-smokers. A generated gene expression signature could subsequently identify never-smokers with 79-100% sensitivity in external adenocarcinoma data sets and with 76-88% sensitivity in the normal materials. A notable fraction of current/former smokers were grouped with never-smokers. Intriguingly, supervised analysis of never-smokers versus smokers in seven adenocarcinoma data sets generated similar results. Overlap in classification between the two approaches was high, indicating that both approaches identify a common set of samples from current/former smokers as potential never-smokers. The gene signature from unsupervised analysis included several genes implicated in lung tumorigenesis, immune-response associated pathways, genes previously associated with smoking, as well as marker genes for alveolar type II pneumocytes, while the best classifier from supervised analysis comprised genes strongly associated with proliferation, but also genes previously associated with smoking.

Conclusions

Based on gene expression profiling, we demonstrate that never-smokers can be identified with high sensitivity in both tumor material and normal airway epithelial specimens. Our results indicate that tumors arising in never-smokers, together with a subset of tumors from smokers, represent a distinct entity of lung adenocarcinomas. Taken together, these analyses provide further insight into the transcriptional patterns occurring in lung adenocarcinoma stratified by smoking history.

PURPOSE

We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.

EXPERIMENTAL DESIGN

To determine the analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen gene promoters from 10 patients with primary lung tumors by using Quantitative methylation specific PCR. We then tested this 15 gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A and AIM) for further elucidation of the diagnostic application of this panel of markers.

RESULTS

Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI 25%, 47%) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64%, 84%) but decreased the specificity from 100% to 73% (95% CI:54%, 88%).

CONCLUSION

This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.

AIM: To investigate aberrant DNA methylation of CpG islands and subsequent low- or high-level DNA microsatellite instability (MSI) which is assumed to drive colon carcinogenesis.

METHODS: DNA of healthy individuals, adenoma (tubular or villous/tubulovillous) patients, and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1 (hMLH1), Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), and O-6-methylguanine DNA methyltransferase (MGMT), as well as their relation to MSI.

RESULTS: The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma. MGMT showed the highest frequency in each group. MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas (tubular vs tubullovillous and villous adenomas). All patients with tubulovillous/villous adenomas, as well as all colorectal cancer patients, showed promoter methylation in at least one of the examined loci. These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progression in colorectal carcinogenesis. MSI and methylation seem to be interdependent, as simultaneous hMLH1, CDKN2A/p16, and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype.

CONCLUSION: Methylation analysis of hMLH1, CDKN2A/p16, and MGMT revealed specific methylation profiles for tubular adenomas, tubulovillous/villous adenomas, and colorectal cancers, supporting the use of these alterations in assessment of colorectal tumorigenesis.