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1.  Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers 
BMC Cancer  2007;7:74.
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.
doi:10.1186/1471-2407-7-74
PMCID: PMC1877812  PMID: 17477876
2.  Aberrant promoter methylation of multiple genes in sputum from individuals exposed to smoky coal emissions 
Anticancer research  2008;28(4B):2061-2066.
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.
PMCID: PMC2974317  PMID: 18751376
Smoky coal emissions; Gene promoter methylation; Lung cancer
3.  Association between MGMT Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis 
PLoS ONE  2013;8(9):e72633.
Background
O6-methylguanine-DNA methyltransferase (MGMT) is one of most important DNA repair enzyme against common carcinogens such as alkylate and tobacco. Aberrant promoter methylation of the gene is frequently observed in non-small cell lung cancer (NSCLC). However, the importance of epigenetic inactivation of the gene in NSCLC published in the literature showed inconsistence. We quantified the association between MGMT promoter methylation and NSCLC using a meta-analysis method.
Methods
We systematically reviewed studies of MGMT promoter methylation and NSCLC in PubMed, EMBASE, Ovid, ISI Web of Science, Elsevier and CNKI databases and quantified the association between MGMT promoter methylation and NSCLC using meta-analysis method. Odds ratio (OR) and corresponding 95% confidence interval (CI) were calculated to evaluate the strength of association. Potential sources of heterogeneity were assessed by subgroup analysis and meta-regression.
Results
A total of 18 studies from 2001 to 2011, with 1, 160 tumor tissues and 970 controls, were involved in the meta-analysis. The frequencies of MGMT promote methylation ranged from 1.5% to 70.0% (median, 26.1%) in NSCLC tissue and 0.0% to 55.0% (median, 2.4%) in non-cancerous control, respectively. The summary of OR was 4.43 (95% CI: 2.85, 6.89) in the random-effects model. With stratification by potential source of heterogeneity, the OR was 20.45 (95% CI: 5.83, 71.73) in heterogeneous control subgroup, while it was 4.16 (95% CI: 3.02, 5.72) in the autologous control subgroup. The OR was 5.31 (95% CI: 3.00, 9.41) in MSP subgroup and 3.06 (95% CI: 1.75, 5.33) in Q-MSP subgroup.
Conclusion
This meta-analysis identified a strong association between methylation of MGMT gene and NSCLC. Prospective studies should be required to confirm the results in the future.
doi:10.1371/journal.pone.0072633
PMCID: PMC3784462  PMID: 24086261
4.  Analysis of aberrant methylation on promoter sequences of tumor suppressor genes and total DNA in sputum samples: a promising tool for early detection of COPD and lung cancer in smokers 
Diagnostic Pathology  2012;7:87.
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.
Virtual slides
The abstract MUST finish with the following text: Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1127865005664160
doi:10.1186/1746-1596-7-87
PMCID: PMC3424112  PMID: 22818553
DNA methylation; Sputum; Lung cancer; COPD
5.  The A/G Allele of Rs16906252 Predicts for MGMT Methylation and Is Selectively Silenced in Premalignant Lesions from Smokers and in Lung Adenocarcinomas 
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.
doi:10.1158/1078-0432.CCR-10-3026
PMCID: PMC3070839  PMID: 21355081
MGMT; allele specific methylation; single nucleotide polymorphism; sputum; lung cancer
6.  Lung Cancer Occurrence in Never-Smokers: An Analysis of 13 Cohorts and 22 Cancer Registry Studies  
PLoS Medicine  2008;5(9):e185.
Background
Better information on lung cancer occurrence in lifelong nonsmokers is needed to understand gender and racial disparities and to examine how factors other than active smoking influence risk in different time periods and geographic regions.
Methods and Findings
We pooled information on lung cancer incidence and/or death rates among self-reported never-smokers from 13 large cohort studies, representing over 630,000 and 1.8 million persons for incidence and mortality, respectively. We also abstracted population-based data for women from 22 cancer registries and ten countries in time periods and geographic regions where few women smoked. Our main findings were: (1) Men had higher death rates from lung cancer than women in all age and racial groups studied; (2) male and female incidence rates were similar when standardized across all ages 40+ y, albeit with some variation by age; (3) African Americans and Asians living in Korea and Japan (but not in the US) had higher death rates from lung cancer than individuals of European descent; (4) no temporal trends were seen when comparing incidence and death rates among US women age 40–69 y during the 1930s to contemporary populations where few women smoke, or in temporal comparisons of never-smokers in two large American Cancer Society cohorts from 1959 to 2004; and (5) lung cancer incidence rates were higher and more variable among women in East Asia than in other geographic areas with low female smoking.
Conclusions
These comprehensive analyses support claims that the death rate from lung cancer among never-smokers is higher in men than in women, and in African Americans and Asians residing in Asia than in individuals of European descent, but contradict assertions that risk is increasing or that women have a higher incidence rate than men. Further research is needed on the high and variable lung cancer rates among women in Pacific Rim countries.
Michael Thun and colleagues pooled and analyzed comprehensive data on lung cancer incidence and death rates among never-smokers to examine what factors other than active smoking affect lung cancer risk.
Editors' Summary
Background.
Every year, more than 1.4 million people die from lung cancer, a leading cause of cancer deaths worldwide. In the US alone, more than 161,000 people will die from lung cancer this year. Like all cancers, lung cancer occurs when cells begin to divide uncontrollably because of changes in their genes. The main trigger for these changes in lung cancer is exposure to the chemicals in cigarette smoke—either directly through smoking cigarettes or indirectly through exposure to secondhand smoke. Eighty-five to 90% of lung cancer deaths are caused by exposure to cigarette smoke and, on average, current smokers are 15 times more likely to die from lung cancer than lifelong nonsmokers (never smokers). Furthermore, a person's cumulative lifetime risk of developing lung cancer is related to how much they smoke, to how many years they are a smoker, and—if they give up smoking—to the age at which they stop smoking.
Why Was This Study Done?
Because lung cancer is so common, even the small fraction of lung cancer that occurs in lifelong nonsmokers represents a large number of people. For example, about 20,000 of this year's US lung cancer deaths will be in never-smokers. However, very little is known about how age, sex, or race affects the incidence (the annual number of new cases of diseases in a population) or death rates from lung cancer among never-smokers. A better understanding of the patterns of lung cancer incidence and death rates among never-smokers could provide useful information about the factors other than cigarette smoke that increase the likelihood of not only never-smokers, but also former smokers and current smokers developing lung cancer. In this study, therefore, the researchers pooled and analyzed a large amount of information about lung cancer incidence and death rates among never smokers to examine what factors other than active smoking affect lung cancer risk.
What Did the Researchers Do and Find?
The researchers analyzed information on lung cancer incidence and/or death rates among nearly 2.5 million self-reported never smokers (men and women) from 13 large studies investigating the health of people in North America, Europe, and Asia. They also analyzed similar information for women taken from cancer registries in ten countries at times when very few women were smokers (for example, the US in the late 1930s). The researchers' detailed statistical analyses reveal, for example, that lung cancer death rates in African Americans and in Asians living in Korea and Japan (but not among Asians living in the US) are higher than those in people of the European continental ancestry group. They also show that men have higher death rates from lung cancer than women irrespective of racial group, but that women aged 40–59 years have a slightly higher incidence of lung cancer than men of a similar age. This difference disappears at older ages. Finally, an analysis of lung cancer incidence and death rates at different times during the past 70 years shows no evidence of an increase in the lung cancer burden among never smokers over time.
What Do These Findings Mean?
Although some of the findings described above have been hinted at in previous, smaller studies, these and other findings provide a much more accurate picture of lung cancer incidence and death rates among never smokers. Most importantly the underlying data used in these analyses are now freely available and should provide an excellent resource for future studies of lung cancer in never smokers.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050185.
The US National Cancer Institute provides detailed information for patients and health professionals about all aspects of lung cancer and information on smoking and cancer (in English and Spanish)
Links to other US-based resources dealing with lung cancer are provided by MedlinePlus (in English and Spanish)
Cancer Research UK provides key facts about the link between lung cancer and smoking and information about all other aspects of lung cancer
doi:10.1371/journal.pmed.0050185
PMCID: PMC2531137  PMID: 18788891
7.  O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation 
PLoS ONE  2011;6(2):e17156.
Background
We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression.
Methodology/Principal Findings
Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression.
Conclusions/Significance
MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined.
doi:10.1371/journal.pone.0017156
PMCID: PMC3041820  PMID: 21365007
8.  MGMT gene promoter methylation correlates with tolerance of temozolomide treatment in melanoma but not with clinical outcome 
British Journal of Cancer  2010;103(6):820-826.
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.
doi:10.1038/sj.bjc.6605796
PMCID: PMC2966614  PMID: 20736948
MGMT; gene methylation; melanoma; therapy toxicity; temozolomide
9.  Expression of O6-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue 
Journal of cancer therapy  2013;4(4):919-931.
The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included (a) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, (b) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and (c) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor-selective reduction in MGMT activity exist in human tissue.
doi:10.4236/jct.2013.44103
PMCID: PMC3740405  PMID: 23946891
O6-Methylguanine-DNA Methyltransferase (MGMT, O6-Alkylguanine-DNA Alkyltransferase, AGT); [Benzene-3H]O6-Benzylguanine; Methylation-Specific PCR (MSP); Laromustine (Onrigin, Cloretazine, VNP40101M, 101M); Temozolomide
10.  O6-methylguanine-DNA methyltransferase (MGMT) Promoter methylation is a rare event in soft tissue sarcoma 
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.
doi:10.1186/1748-717X-7-180
PMCID: PMC3549846  PMID: 23110891
Soft tissue sarcoma; O6-methylguanine–DNA methyltransferase; Promoter methylation; Temozolomide; Epigenetic gene silencing; Radiation therapy
11.  Homogeneous MGMT Immunoreactivity Correlates with an Unmethylated MGMT Promoter Status in Brain Metastases of Various Solid Tumors 
PLoS ONE  2009;4(3):e4775.
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.
doi:10.1371/journal.pone.0004775
PMCID: PMC2652028  PMID: 19274096
12.  Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor 
AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.
METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-ras G to A point mutation in codon 12.
RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-ras G to A point mutation in codon 12 had methylated MGMT (P = 0.011). The frequencies of K-ras G to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.
CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.
doi:10.3748/wjg.v11.i13.2022
PMCID: PMC4305730  PMID: 15800999
O6-methylguanine-DNA methyltransferase; CpG island; DNA methylation; Epigenetic change; K-ras mutation
13.  MGMT Promoter Methylation Is Prognostic but Not Predictive for Outcome to Adjuvant PCV Chemotherapy in Anaplastic Oligodendroglial Tumors: A Report From EORTC Brain Tumor Group Study 26951 
Journal of Clinical Oncology  2009;27(35):5881-5886.
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.
doi:10.1200/JCO.2009.24.1034
PMCID: PMC2793037  PMID: 19901104
14.  Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: Pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells 
Oncology Reports  2012;27(4):1135-1141.
Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.
doi:10.3892/or.2012.1624
PMCID: PMC3583513  PMID: 22246327
O-6-methylguanine-DNA methyltransferase; mutL homolog 1; Ras association domain family member 1; tumor suppressor gene; head and neck squamous cell carcinoma; 5-azacytidine
15.  MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status 
Acta Neuropathologica  2012;124(4):547-560.
The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg1243587 and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p < 0.001) using a training-set of 63 glioblastomas from homogenously treated patients, for whom MGMT methylation was previously shown to be predictive for outcome based on classification by methylation-specific PCR. MGMT-STP27 was successfully validated in an independent cohort of chemo-radiotherapy-treated glioblastoma patients (n = 50; kappa = 0.88; outcome, log-rank p < 0.001). Lower prevalence of MGMT methylation among CpG island methylator phenotype (CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM-450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-012-1016-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s00401-012-1016-2
PMCID: PMC3444709  PMID: 22810491
MGMT; DNA methylation; MSP; Infinium methylation platform; Prediction model
16.  The Global DNA Methylation Surrogate LINE-1 Methylation Is Correlated with MGMT Promoter Methylation and Is a Better Prognostic Factor for Glioma 
PLoS ONE  2011;6(8):e23332.
Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.
doi:10.1371/journal.pone.0023332
PMCID: PMC3150434  PMID: 21829728
17.  Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene. 
Molecular and Cellular Biology  1997;17(9):5612-5619.
O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.
PMCID: PMC232409  PMID: 9271436
18.  MGMT promoter methylation status and MGMT and CD133 immunohistochemical expression as prognostic markers in glioblastoma patients treated with temozolomide plus radiotherapy 
Background
The CD133 antigen is a marker of radio- and chemo-resistant stem cell populations in glioblastoma (GBM). The O6-methylguanine DNA methyltransferase (MGMT) enzyme is related with temozolomide (TMZ) resistance. Our propose is to analyze the prognostic significance of the CD133 antigen and promoter methylation and protein expression of MGMT in a homogenous group of GBM patients uniformly treated with radiotherapy and TMZ. The possible connection between these GBM markers was also investigated.
Methods
Seventy-eight patients with GBM treated with radiotherapy combined with concomitant and adjuvant TMZ were analyzed for MGMT and CD133. MGMT gene promoter methylation was determined by methylation-specific polymerase chain reaction after bisulfite treatment. MGMT and CD133 expression was assessed immunohistochemically using an automatic quantification system. Overall and progression-free survival was calculated according to the Kaplan–Meier method.
Results
The MGMT gene promoter was found to be methylated in 34 patients (44.7%) and unmethylated in 42 patients (55.3%). A significant correlation was observed between MGMT promoter methylation and patients’ survival. Among the unmethylated tumors, 52.4% showed low expression of MGMT and 47.6% showed high-expression. Among methylated tumors, 58.8% showed low-expression of MGMT and 41.2% showed high-expression. No correlation was found between MGMT promoter methylation and MGMT expression, or MGMT expression and survival. In contrast with recent results, CD133 expression was not a predictive marker in GBM patients. Analyses of possible correlation between CD133 expression and MGMT protein expression or MGMT promoter methylation were negative.
Conclusions
Our results support the hypothesis that MGMT promoter methylation status but not MGMT expression may be a predictive biomarker in the treatment of patients with GBM. In addition, CD133 should not be used for prognostic evaluation of these patients. Future studies will be necessary to determine its clinical utility.
doi:10.1186/1479-5876-10-250
PMCID: PMC3551841  PMID: 23245659
Glioblastoma; Radiotherapy; Temozolomide; MGMT; Methylation; CD133
19.  Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis 
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.
doi:10.1002/ijc.23398
PMCID: PMC2851179  PMID: 18240147
O6-methylguanine-DNA methyltransferase; KRAS mutations; promoter methylation; colorectal cancer; adenomatous polyps
20.  Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients 
Oncology Letters  2013;6(5):1370-1376.
CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 (MLH1) and DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman’s rank test. Agreement was determined by generalized κ-statistics. Spearman’s rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.
doi:10.3892/ol.2013.1543
PMCID: PMC3813787  PMID: 24179526
colorectal cancers; MLH1; MGMT; methylation
21.  DNA Hypermethylation of Tumor-Related Genes in Gastric Carcinoma 
Journal of Korean Medical Science  2005;20(2):236-241.
The hypermethylation of the CpG islands is a common mechanism for the inactivation of tumor-related genes. In the present study, we analyzed the methylation status of genes for cell repair such as hMLH1, MGMT, and GSTP1, and a gastric cancer-specifically methylated DNA fragment, MINT 25 in gastric cancer cases and control groups. The study population consisted of 100 gastric cancer patients (50 distal and 50 proximal carcinomas), and 238 healthy controls. All genes showed more frequent hypermethylation in the cases than in the control group (p<0.0001). We investigated the association between promoter hypermethylation and relevant parameters including age, gender, alcohol consumption, smoking, and family history. There was a common hypermethylation of hMLH1 (p=0.008), MGMT (p=0.0001), and GSTP1 (p=0.0003) in females. This study also demonstrates that hypermethylation was strongly associated with non-drinkers (MGMT, p=0.046 and MINT 25, p=0.049) and non-smokers (hMLH1, p=0.044; MGMT, p=0.0003; MINT 25, p=0.029). Moreover, the frequency of MINT 25 hypermethylation increased with age (p=0.037), and MGMT methylation was frequently detected in distal gastric cancer than in proximal type (p=0.038). Our study suggested that promoter hypermethylation of the genes involved in cell repair system and MINT 25 is associated strongly with some subgroups of primary gastric carcinoma.
doi:10.3346/jkms.2005.20.2.236
PMCID: PMC2808599  PMID: 15831994
Stomach Neoplasms; DNA Methylation; MLH1 Protein, mammalian; O(6)-Methylguanine-DNA Methyltransferase; Glutathione S-transferase pi; MINT 25
22.  Polymorphisms of the DNA repair gene MGMT and risk and progression of head and neck cancer 
DNA repair  2010;9(5):558-566.
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.
doi:10.1016/j.dnarep.2010.02.006
PMCID: PMC2883263  PMID: 20206583
oral cancer; DNA repair; methylation; genetic susceptibility; molecular epidemiology
23.  Absence of MGMT promoter methylation in endometrial cancer 
Gynecologic oncology  2008;112(1):224-228.
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.
doi:10.1016/j.ygyno.2008.08.038
PMCID: PMC2949277  PMID: 18973931
MGMT; endometrial neoplasms; methylation
24.  Relation between smoking history and gene expression profiles in lung adenocarcinomas 
BMC Medical Genomics  2012;5:22.
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.
doi:10.1186/1755-8794-5-22
PMCID: PMC3447685  PMID: 22676229
Lung cancer; Smoking; Gene expression analysis; Adenocarcinoma; EGFR; Never-smokers; Immune response
25.  Promoter methylation status of hMLH1, MGMT, and CDKN2A/p16 in colorectal adenomas 
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.
doi:10.3748/wjg.v16.i28.3553
PMCID: PMC2909555  PMID: 20653064
Promoter methylation; Microsatellite instability; Human DNA mismatch repair gene mutator L homologue 1; O-6-methylguanine DNA methyltransferase; Cyclin-dependent kinase inhibitor 2A

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