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1.  Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients 
Respiratory Research  2009;10(1):86.
Background
There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.
Methods
To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5β promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)].
Results
We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5β and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5β, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5β promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (padj = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1)~-215~-113 and (2) -84 ~+26); while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci.
Conclusion
Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.
doi:10.1186/1465-9921-10-86
PMCID: PMC2759916  PMID: 19781081
2.  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
3.  Increased methylation of lung cancer-associated genes in sputum DNA of former smokers with chronic mucous hypersecretion 
Respiratory Research  2014;15(1):2.
Background
Chronic mucous hypersecretion (CMH) contributes to COPD exacerbations and increased risk for lung cancer. Because methylation of gene promoters in sputum has been shown to be associated with lung cancer risk, we tested whether such methylation was more common in persons with CMH.
Methods
Eleven genes commonly silenced by promoter methylation in lung cancer and associated with cancer risk were selected. Methylation specific PCR (MSP) was used to profile the sputum of 900 individuals in the Lovelace Smokers Cohort (LSC). Replication was performed in 490 individuals from the Pittsburgh Lung Screening Study (PLuSS).
Results
CMH was significantly associated with an overall increased number of methylated genes, with SULF2 methylation demonstrating the most consistent association. The association between SULF2 methylation and CMH was significantly increased in males but not in females both in the LSC and PLuSS (OR = 2.72, 95% CI = 1.51-4.91, p = 0.001 and OR = 2.97, 95% CI = 1.48-5.95, p = 0.002, respectively). Further, the association between methylation and CMH was more pronounced among 139 male former smokers with persistent CMH compared to current smokers (SULF2; OR = 3.65, 95% CI = 1.59-8.37, p = 0.002).
Conclusions
These findings demonstrate that especially male former smokers with persistent CMH have markedly increased promoter methylation of lung cancer risk genes and potentially could be at increased risk for lung cancer.
doi:10.1186/1465-9921-15-2
PMCID: PMC3893562  PMID: 24405663
Methylation of gene promoters; Persistent cough and phlegm; Sputum DNA; Former smoker; Lung cancer genes
4.  Driver Mutations Determine Survival in Smokers and Never Smokers with Stage IIIB/IV Lung Adenocarcinomas 
Cancer  2012;118(23):5840-5847.
Background
We previously demonstrated that stage IIIB/IV non-small cell lung cancer (NSCLC) never smokers lived 50% longer than former/current smokers. This observation persisted after adjusting for age, performance status, and gender. We hypothesized that smoking-dependent differences in the distribution of driver mutations might explain differences in prognosis between these subgroups.
Methods
We reviewed 293 never smokers and 382 former/current smokers with lung adenocarcinoma who underwent testing for EGFR and KRAS mutations and rearrangements in ALK between 2009 and 2010. Clinical outcomes and patient characteristics were collected. Survival probabilities were estimated using the Kaplan-Meier method. Group comparison was performed with log-rank tests and Cox proportional hazards methods.
Results
While the overall incidence of these mutations was nearly identical (55% never smokers vs. 57% current/former smokers, p=0.48), there were significant differences in the distribution of mutations between these groups: EGFR mutations- 37% never smokers vs. 14% former/current smokers (p<0.0001); KRAS mutations- 4% never smokers vs. 43% former/current smokers (p<0.0001); ALK rearrangements- 12% never smokers vs. 2% former/current smokers (p<0.0001). Among never smokers and former/current smokers, prognosis differed significantly by genotype. Patients harboring KRAS mutations demonstrated the poorest survival. Smoking status, however, had no influence on survival within each genotype.
Conclusion
Never smokers and former/current smokers with lung adenocarcinomas are not homogeneous subgroups. Each is made up of individuals whose tumors have a unique distribution of driver mutations which are associated with different prognoses, irrespective of smoking history.
doi:10.1002/cncr.27637
PMCID: PMC3424296  PMID: 22605530
non-small cell lung cancer; adenocarcinoma; EGFR; KRAS; ALK; never smoker
5.  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
6.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Background
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
Conclusions
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Background.
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030486.
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
doi:10.1371/journal.pmed.0030486
PMCID: PMC1716188  PMID: 17194187
7.  Aberrant DNA Methylation of OLIG1, a Novel Prognostic Factor in Non-Small Cell Lung Cancer 
PLoS Medicine  2007;4(3):e108.
Background
Lung cancer is the leading cause of cancer-related death worldwide. Currently, tumor, node, metastasis (TNM) staging provides the most accurate prognostic parameter for patients with non-small cell lung cancer (NSCLC). However, the overall survival of patients with resectable tumors varies significantly, indicating the need for additional prognostic factors to better predict the outcome of the disease, particularly within a given TNM subset.
Methods and Findings
In this study, we investigated whether adenocarcinomas and squamous cell carcinomas could be differentiated based on their global aberrant DNA methylation patterns. We performed restriction landmark genomic scanning on 40 patient samples and identified 47 DNA methylation targets that together could distinguish the two lung cancer subgroups. The protein expression of one of those targets, oligodendrocyte transcription factor 1 (OLIG1), significantly correlated with survival in NSCLC patients, as shown by univariate and multivariate analyses. Furthermore, the hazard ratio for patients negative for OLIG1 protein was significantly higher than the one for those patients expressing the protein, even at low levels.
Conclusions
Multivariate analyses of our data confirmed that OLIG1 protein expression significantly correlates with overall survival in NSCLC patients, with a relative risk of 0.84 (95% confidence interval 0.77–0.91, p < 0.001) along with T and N stages, as indicated by a Cox proportional hazard model. Taken together, our results suggests that OLIG1 protein expression could be utilized as a novel prognostic factor, which could aid in deciding which NSCLC patients might benefit from more aggressive therapy. This is potentially of great significance, as the addition of postoperative adjuvant chemotherapy in T2N0 NSCLC patients is still controversial.
Christopher Plass and colleagues find thatOLIG1 expression correlates with survival in lung cancer patients and suggest that it could be used in deciding which patients are likely to benefit from more aggressive therapy.
Editors' Summary
Background.
Lung cancer is the commonest cause of cancer-related death worldwide. Most cases are of a type called non-small cell lung cancer (NSCLC). Like other cancers, treatment of NCSLC depends on the “TNM stage” at which the cancer is detected. Staging takes into account the size and local spread of the tumor (its T classification), whether nearby lymph nodes contain tumor cells (its N classification), and whether tumor cells have spread (metastasized) throughout the body (its M classification). Stage I tumors are confined to the lung and are removed surgically. Stage II tumors have spread to nearby lymph nodes and are treated with a combination of surgery and chemotherapy. Stage III tumors have spread throughout the chest, and stage IV tumors have metastasized around the body; patients with both of these stages are treated with chemotherapy alone. About 70% of patients with stage I or II lung cancer, but only 2% of patients with stage IV lung cancer, survive for five years after diagnosis.
Why Was This Study Done?
TNM staging is the best way to predict the likely outcome (prognosis) for patients with NSCLC, but survival times for patients with stage I and II tumors vary widely. Another prognostic marker—maybe a “molecular signature”—that could distinguish patients who are likely to respond to treatment from those whose cancer will inevitably progress would be very useful. Unlike normal cells, cancer cells divide uncontrollably and can move around the body. These behavioral changes are caused by alterations in the pattern of proteins expressed by the cells. But what causes these alterations? The answer in some cases is “epigenetic changes” or chemical modifications of genes. In cancer cells, methyl groups are aberrantly added to GC-rich gene regions. These so-called “CpG islands” lie near gene promoters (sequences that control the transcription of DNA into mRNA, the template for protein production), and their methylation stops the promoters working and silences the gene. In this study, the researchers have investigated whether aberrant methylation patterns vary between NSCLC subtypes and whether specific aberrant methylations are associated with survival and can, therefore, be used prognostically.
What Did the Researchers Do and Find?
The researchers used “restriction landmark genomic scanning” (RLGS) to catalog global aberrant DNA methylation patterns in human lung tumor samples. In RLGS, DNA is cut into fragments with a restriction enzyme (a protein that cuts at specific DNA sequences), end-labeled, and separated using two-dimensional gel electrophoresis to give a pattern of spots. Because methylation stops some restriction enzymes cutting their target sequence, normal lung tissue and lung tumor samples yield different patterns of spots. The researchers used these patterns to identify 47 DNA methylation targets (many in CpG islands) that together distinguished between adenocarcinomas and squamous cell carcinomas, two major types of NSCLCs. Next, they measured mRNA production from the genes with the greatest difference in methylation between adenocarcinomas and squamous cell carcinomas. OLIG1 (the gene that encodes a protein involved in nerve cell development) had one of the highest differences in mRNA production between these tumor types. Furthermore, three-quarters of NSCLCs had reduced or no expression of OLIG1 protein and, when the researchers analyzed the association between OLIG1 protein expression and overall survival in patients with NSCLC, reduced OLIG1 protein expression was associated with reduced survival.
What Do These Findings Mean?
These findings indicate that different types of NSCLC can be distinguished by examining their aberrant methylation patterns. This suggests that the establishment of different DNA methylation patterns might be related to the cell type from which the tumors developed. Alternatively, the different aberrant methylation patterns might reflect the different routes that these cells take to becoming tumor cells. This research identifies a potential new prognostic marker for NSCLC by showing that OLIG1 protein expression correlates with overall survival in patients with NSCLC. This correlation needs to be tested in a clinical setting to see if adding OLIG1 expression to the current prognostic parameters can lead to better treatment choices for early-stage lung cancer patients and ultimately improve these patients' overall survival.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040108.
Patient and professional information on lung cancer, including staging (in English and Spanish), is available from the US National Cancer Institute
The MedlinePlus encyclopedia has pages on non-small cell lung cancer (in English and Spanish)
Cancerbackup provides patient information on lung cancer
CancerQuest, provided by Emory University, has information about how cancer develops (in English, Spanish, Chinese and Russian)
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence gives background information and the latest news about epigenetics (in several European languages)
doi:10.1371/journal.pmed.0040108
PMCID: PMC1831740  PMID: 17388669
8.  Aberrant Promoter Methylation of p16 and MGMT Genes in Lung Tumors from Smoking and Never-Smoking Lung Cancer Patients1 
Neoplasia (New York, N.Y.)  2006;8(1):46-51.
Abstract
Aberrant methylation in gene promoter regions leads to transcriptional inactivation of cancer-related genes and plays an integral role in tumorigenesis. This alteration has been investigated in lung tumors primarily from smokers, whereas only a few studies involved never-smokers. Here, we applied methylation-specific polymerase chain reaction to compare the frequencies of the methylated promoter of p16 and O6-methylguanine-DNA methyltransferase (MGMT) genes in lung tumors from 122 patients with non-small cell lung cancer, including 81 smokers and 41 never-smokers. Overall, promoter methylation was detected in 52.5% (64 of 122) and 30.3% (37 of 122) of the p16 and MGMT genes, respectively. Furthermore, the frequency of promoter methylation was significantly higher among smokers, compared with never-smokers, for both the p16 [odds ratio (OR) = 3.28; 95% confidence interval (CI) = 1.28-8.39; P = .013] and MGMT (OR = 3.93; 95% CI = 1.27-12.21; P = .018) genes. The trend for a higher promoter methylation frequency of these genes was also observed among female smokers compared with female never-smokers. Our results suggest an association between tobacco smoking and an increased incidence of aberrant promoter methylation of the p16 and MGMT genes in non-small cell lung cancer.
PMCID: PMC1584289  PMID: 16533425
Lung tumors; p16; MGMT; promoter methylation; never-smokers
9.  Methylation of the DLEC1 gene correlates with poor prognosis in Japanese lung cancer patients 
Oncology Letters  2010;1(2):283-287.
The incidence of chromosome 3p gene alterations is one of the most frequent and earliest documented events in lung cancer. This study aimed to investigate promoter methylation in the deleted in lung and esophageal cancer 1 (DLEC1) gene, as well as the p16 and CDH1 genes in Japanese lung cancer cases. The methylation status of the promoter regions of DLEC1, p16 and CDH1 was investigated using methylation-specific PCR. The findings were compared to the clinicopathological features of lung cancer. Methylation-specific PCR showed that the DLEC1 promoter region was methylated in 65 out of 116 (56%) lung cancers. Patients with DLEC1-methylated cancer were associated with a significantly worse prognosis than those with unmethylated cancer (p=0.0368; hazard ratio=1.83). The p16 methylation status correlated with squamous histology (p=0.03) and smoking status (never smoker vs. smoker; p=0.0122). Patients with p16 ummethylated cancer harbored more EGFR mutations (p=0.0071). The CDH1 promoter region was hypermethylated in 65 out of 118 (55.1%) lung cancer cases. However, the CDH1 methylation status was not associated with the clinicopathological characteristics of the lung cancer types. p16 and CDH1 methylation status did not correlate with survival in the lung cancer patients. Thus, in our Japanese cohort, the methylation status of the DLEC1 gene was a marker of poor prognosis independent of stage.
doi:10.3892/ol_00000050
PMCID: PMC3436471  PMID: 22966295
methylation; DLEC1 gene; lung cancer
10.  SULF2 Methylation is Prognostic for Lung Cancer Survival and Increases Sensitivity to Topoisomerase-I inhibitors via Induction of ISG15 
Oncogene  2011;31(37):4107-4116.
The heparan sulfate 6-O-endosulfatase (SULF2) promotes growth and metastasis of solid tumors. We recently identified that cytosine methylation of the SULF2 promoter is associated with better survival of resected lung adenocarcinoma patients and now also demonstrate a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (HR = 0.63, p = 0.07). Subsequent studies focused on investigating the effect of methylation on SULF2 expression and its genome-wide impact. The genes and pathways modulated by epigenetic inactivation of SULF2 and the effects on sensitivity to chemotherapy were characterized in vitro and in vivo. Silencing SULF2 through siRNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin. NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared to SULF2 unmethylated (SULF2U) NSCLC cell lines and normal human bronchial epithelial cells. In vitro, SULF2M and high ISG15 (ISG15H) expressing NSCLC cell lines were 134-fold more sensitive to camptothecin than SULF2U and low ISG15 (ISG15L) expressing cell lines. Topotecan, a soluble analogue of camptothecin and FDA approved anti-cancer drug, dramatically arrested the growth of SULF2M-ISG15H, but not SULF2U-ISG15L lung tumors in nude mice (p < 0.002). Similarly, high ISG15 expression that is comparable to the topotecan sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared to normal lung indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized topotecan therapy.
doi:10.1038/onc.2011.577
PMCID: PMC3307938  PMID: 22158045
NSCLC; Camptothecin; SULF-2; Oncogene; Topotecan
11.  Hypermethylation of the large tumor suppressor genes in Japanese lung cancer 
Oncology Letters  2010;1(2):303-307.
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.
doi:10.3892/ol_00000054
PMCID: PMC3436364  PMID: 22966299
hypermethylation; large tumor suppressor gene; lung cancer
12.  Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker 
BMC Cancer  2009;9:24.
Background
Squamous cell carcinoma of esophagus (SCCE) occurs at a high incidence rate in certain parts of the world. This feature necessitates that different aspects of the disease and in particular genetic characteristics be investigated in such regions. In addition, such investigations might lead to achievement of molecular markers helpful for early detection, successful treatment and follow up of the disease. Adenomatous Polyposis Coli (APC) promoter hypermethylation has been shown to be a suitable marker for both serum and solid tumors of adenocarcinoma of esophagus. We investigated the status of APC promoter hypermethylation in Iranian patients, compared the results with the former studies, and evaluated its applicability as a candidate molecular marker by examining association between survival of SCCE patients and APC promoter methylation.
Methods
For evaluating the status of APC promoter hypermethylation and its association with SCCE, a qualitative methylation specific PCR (MSP) was used. DNA was extracted and digested with an appropriate restriction enzyme, treated with sodium bisulfite in agarose beads and amplified in two-step PCR reaction by applying either methylated or unmethylated promoter specific primers. Universally methylated DNA and methylase treated blood DNA of healthy donors were used as positive controls as well. Survival of patients was followed up for two years after treatment and survival rate of patients with methylated APC promoter was compared with that of unmethylated patients.
Results
Assessment of APC promoter methylation revealed that normal tissues were unmethylated, while twenty out of forty five (44.4%) tumor tissues were hypermethylated either in one or both alleles of APC. Among the tissues in which methylation was detected, seven were hypermethylated in both alleles while the other thirteen were hypermethylated in one of the two alleles of APC. Analyzing two-year survival rate of patients with respect to promoter hypermethylation showed a lower rate of survival for patients with methylated APC promoter following their treatment. Further investigation into the association between promoter hypermethylation and tumor differentiation status indicated that patients with well differentiated tumors were more likely to develop promoter hypermethylation.
Conclusion
Observing similar level of APC promoter hypermethylation in patients with SCCE in this high risk region and comparing it with other parts of the world could support the hypothesis that a common molecular mechanism might be involved in tumorigenesis of SCCE. In addition, the higher rate of two-year survival for patients with unmethylated APC promoter as well as its relationship with tumor differentiation would suggest that this tumor suppressor could be an appropriate candidate molecular marker for evaluating tumor malignancy and predicting survival of patients subsequent to treatment.
doi:10.1186/1471-2407-9-24
PMCID: PMC2637891  PMID: 19149902
13.  Multi-Vitamins, Folate, and Green Vegetables Protect Against Gene Promoter Methylation in the Aerodigestive Tract of Smokers 
Cancer research  2010;70(2):568-574.
The detection of gene promoter hypermethylation in sputum is a promising molecular marker for early lung cancer detection. Epidemiologic studies suggest that dietary fruits and vegetables and the micronutrients they contain may reduce risk of lung cancer. This investigation evaluated whether diet and multi-vitamin use influence the prevalence for gene methylation in the cells exfoliated from the aerodigestive tract of current and former smokers. Members (n = 1101) of the Lovelace Smokers Cohort completed the Harvard Food Frequency Questionnaire and provided a sputum sample that was assessed for promoter methylation of eight genes commonly silenced in lung cancer and associated with risk for this disease. Methylation status was categorized as low (< 2 genes methylated) or high (≥2 genes methylated). Logistic regression models were used to identify associations between methylation status and 21 dietary variables hypothesized to affect the acquisition of gene methylation. Significant protection against methylation was observed for leafy green vegetables (OR = 0.83 per 12 monthly servings, CI: 0.74, 0.93) and folate (OR = 0.84 per 750 mcg/day, CI: 0.72, 0.99). Protection against gene methylation was also seen with current use of multi-vitamins (OR = 0.57, CI: 0.40, 0.83). This is the first cohort-based study to identify dietary factors associated with reduced promoter methylation in cells exfoliated from the airway epithelium of smokers. Novel interventions to prevent lung cancer should be developed based on the ability of diet and dietary supplements to affect reprogramming of the epigenome.
doi:10.1158/0008-5472.CAN-09-3410
PMCID: PMC3076796  PMID: 20068159
gene methylation; folate; multi-vitamins; green vegetables; smokers
14.  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
15.  Cigarette smoke induces methylation of the tumor suppressor gene NISCH 
Epigenetics  2013;8(4):383-388.
We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (< 20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients’ plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer.
doi:10.4161/epi.24195
PMCID: PMC3674047  PMID: 23503203
lung cancer; Nisch; methylation; smoking; tobacco
16.  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
17.  Prognostic significance of CDH13 hypermethylation and mRNA in NSCLC 
OncoTargets and therapy  2014;7:1987-1996.
Aberrant methylation of CpG dinucleotides is a commonly observed epigenetic modification in human cancer. Thus, detection of aberrant gene promoter methylation as a tool for diagnosis of tumors or as a prognostic marker has been widely described for many types of cancers, including nonsmall cell lung cancer (NSCLC). Emerging evidence indicates that CDH13 is a candidate tumor suppressor in several types of human tumors, including NSCLC. However, the correlation between CDH13 hypermethylation and clinicopathological characteristics of NSCLC remains unclear. In the current study, we conducted a systematic review and meta-analysis to quantitatively evaluate the effects of CDH13 hypermethylation on the incidence of NSCLC and clinicopathological characteristics. Final analysis of 803 NSCLC patients from eleven eligible studies was performed. CDH13 hypermethylation was observed to be significantly higher in NSCLC than in normal lung tissue, with the pooled odds ratio (OR) from seven studies including 448 NSCLC and 345 normal lung tissue (OR, 7.85; 95% confidence interval, 5.12–12.03; P<0.00001). CDH13 hypermethylation was also associated with pathological types. The pooled OR was obtained from four studies, including 111 squamous cell carcinoma and 106 adenocarcinoma (OR, 0.35; 95% confidence interval, 0.19–0.66; P=0.001), which indicated that CDH13 hypermethylation plays a more important role in the pathogenesis of adenocarcinoma. NSCLC with CDH13 hypermethylation was found more frequently in poorly differentiated NSCLC patients. NSCLC patients with CDH13 hypermethylation had a lower survival rate than those without CDH13 hypermethylation. In addition, CDH13 mRNA high expression was found to correlate with better overall survival for all NSCLC patients followed for 20 years (hazard ratio, 0.81; P=0.0056). Interestingly, CDH13 mRNA overexpression was found to correlate with better overall survival only in adenocarcinoma patients (hazard ratio, 0.42; P=9.6e–09), not in squamous cell carcinoma patients (hazard ratio, 0.93; P=0.59). The results of this meta-analysis suggest that CDH13 hypermethylation is associated with an increased risk and worse survival in NSCLC. CDH13 hypermethylation and mRNA expression play an important role in carcinogenesis, progression, and development, as well as clinical outcomes.
doi:10.2147/OTT.S67355
PMCID: PMC4222896  PMID: 25382980
prognosis; methylation; lung cancer; tumor suppressor gene; meta-analysis; odds ratio; hazard ratio
18.  Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation 
Cancer research  2008;68(8):3049-3056.
Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer.
doi:10.1158/0008-5472.CAN-07-6344
PMCID: PMC2483467  PMID: 18413776
promoter methylation; DNA double strand break; single nucleotide polymorphism; DNA repair capacity; association study
19.  Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma 
Molecular Cancer  2007;6:70.
Background
Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients.
Results
We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value << 0.0001). Using the current tissue collection and 5-fold cross validation, the four most significant loci (CDKN2A EX2, CDX2, HOXA1 and OPCML) individually distinguish lung adenocarcinoma from non-cancer lung with a sensitivity of 67–86% and specificity of 74–82%. DNA methylation of these loci did not differ significantly based on gender, race, age or tumor stage, indicating their wide applicability as potential lung adenocarcinoma markers. We applied random forests to determine a good classifier based on a subset of our loci and determined that combined use of the same four top markers allows identification of lung cancer tissue from non-lung cancer tissue with 94% sensitivity and 90% specificity.
Conclusion
The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.
doi:10.1186/1476-4598-6-70
PMCID: PMC2206053  PMID: 17967182
20.  Genetic and Epigenetic Inactivation of TNFRSF10C in Human Prostate Cancer 
The Prostate  2009;69(3):327-335.
BACKGROUND
TNFRSF10C, is located on 8p21.3, one of the most frequently deleted loci in the genome of prostate cancer (PCa). Hypermethylation of TNFRSF10C promoter CpG island (CGI) had been reported in many tumors including PCa. However, the interplay between somatic deletion and promoter hypermethylation of TNFRSF10C on PCa development has not been investigated.
METHODS
Methylation status of promoter CGI and deletion status of the TNFRSF10C locus was investigated by bisulfite sequencing and Affymetrix SNP array, respectively in 59 pairs of PCa tumor and matched normal samples with 3 PCa cell lines. TNFRSF10C gene expression changes in relation to cancer-associated genetic/epigenetic changes in clinical specimens, and change of TNFRSF10C expression before and after 5-aza-2’-deoxycytidine treatment in the PC3 PCa cell line were assessed by real-time RT-PCR.
RESULTS
We found that TNFRSF10C promoter CGI was differentially methylated in 46 of 59 primary cancers (78.0%). Hemizygous deletion at TNFRSF10C was found in 44 of the 59 prostate tumors (74.5%). Interestingly, in 94.9% of the tumors (56 out of 59), TNFRSF10C was either hemizygously deleted or its promoter CGI hypermethylated. Deletion and/or methylation of the TNFRSF10C gene was correlated with decreased mRNA expression of the gene in clinical specimens. Demethylation of the TNFRSF10C promoter CGI was accompanied by transcriptional re-activation of TNFRSF10C in the prostate cancer cell line PC3.
CONCLUSION
We found a notably high frequency of promoter CGI methylation and deletion of TNFRSF10C in PCa tissues. Our results indicated that inactivation of TNFRSF10C by chromosomal deletion and promoter methylation may play an important role in PCa development.
doi:10.1002/pros.20882
PMCID: PMC2795318  PMID: 19035483
promoter methylation; CpG islands; somatic copy number alterations
21.  Non-small cell lung cancer in never smokers: a clinical entity to be identified 
Clinics  2011;66(11):1873-1877.
OBJECTIVES:
It has been recognized that patients with non-small cell lung cancer who are lifelong never-smokers constitute a distinct clinical entity. The aim of this study was to assess clinical risk factors for survival among never-smokers with non-small cell lung cancer.
METHODS:
All consecutive non-small cell lung cancer patients diagnosed (n = 285) between May 2005 and May 2009 were included. The clinical characteristics of never-smokers and ever-smokers (former and current) were compared using chi-squared or Student's t tests. Survival curves were calculated using the Kaplan-Meier method, and log-rank tests were used for survival comparisons. A Cox proportional hazards regression analysis was evaluated by adjusting for age (continuous variable), gender (female vs. male), smoking status (never- vs. ever-smoker), the Karnofsky Performance Status Scale (continuous variable), histological type (adenocarcinoma vs. non-adenocarcinoma), AJCC staging (early vs. advanced staging), and treatment (chemotherapy and/or radiotherapy vs. the best treatment support).
RESULTS:
Of the 285 non-small cell lung cancer patients, 56 patients were never-smokers. Univariate analyses indicated that the never-smoker patients were more likely to be female (68% vs. 32%) and have adenocarcinoma (70% vs. 51%). Overall median survival was 15.7 months (95% CI: 13.2 to 18.2). The never-smoker patients had a better survival rate than their counterpart, the ever-smokers. Never-smoker status, higher Karnofsky Performance Status, early staging, and treatment were independent and favorable prognostic factors for survival after adjusting for age, gender, and adenocarcinoma in multivariate analysis.
CONCLUSIONS:
Epidemiological differences exist between never- and ever-smokers with lung cancer. Overall survival among never-smokers was found to be higher and independent of gender and histological type.
doi:10.1590/S1807-59322011001100005
PMCID: PMC3203958  PMID: 22086516
Lung neoplasm; Non-small cell lung cancer; Adenocarcinoma; Never-smoker; Smoking
22.  Methylation of TMEFF2 Gene in Tissue and Serum DNA from Patients with Non-Small Cell Lung Cancer 
Molecules and Cells  2012;34(2):171-176.
Lung cancer remains a global health problem with a high mortality rate. CpG island methylation is a common aberration frequently associated with gene silencing in multiple tumor types, emerging as a highly promising biomarker. The transmembrane protein with a single EGF-like and two follistatin domains (TMEFF2) is epigenetically silenced in numerous tumor types, suggesting a potential role as a potential tumor suppressor. However, the role of TMEFF2 in lung cancer remains to be fully elucidated. We explored the methylation status of TMEFF2 gene in 139 patients with non-small cell lung cancer (NSCLC) and the feasibility of detecting circulating methylated DNA as a screening tool for NSCLC using methylation-specific PCR in 316 patients and 50 age-matched health controls. TMEFF2 methylation in tumor tissues was found in 73 of the 139 NSCLCs (52.5%) and was related to gene expression. The frequency of TMEFF2 methylation was higher in females and never-smokers than in males and smokers with borderline significance (65.8% vs 47.8%, p = 0.06; 65.7% vs 48.1%, p = 0.07). Notably, in adenocarcinomas, TMEFF2 methylation was significantly more frequent in tumors without EGFR mutation than those with EGFR mutation (adjusted odds ratio = 7.13, 95% confidence interval = 2.05–24.83, P = 0.002). Furthermore, TMEFF2 methylation was exclusively detected in the serum of NSCLC patients at a frequency of 9.2% (29/316). These findings suggest that methylation-associated down-regulation of TMEFF2 gene may be involved in lung tumorigenesis and TMEFF2 methylation can serve as a specific blood-based biomarker for NSCLC.
doi:10.1007/s10059-012-0083-5
PMCID: PMC3887809  PMID: 22814847
methylation; methylation-specific PCR; non-small cell lung cancer; serum; TMEFF2
23.  DNA Methylation Changes in Atypical Adenomatous Hyperplasia, Adenocarcinoma In Situ, and Lung Adenocarcinoma 
PLoS ONE  2011;6(6):e21443.
Background
Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation.
Methodology/Principal Findings
We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma), and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer.
Conclusions/Significance
The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA methylation events in tumor development. In addition, it identifies which DNA methylation changes might be used as molecular markers for the detection of preinvasive lesions.
doi:10.1371/journal.pone.0021443
PMCID: PMC3121768  PMID: 21731750
24.  Epigenomic analysis of lung adenocarcinoma reveals novel DNA methylation patterns associated with smoking 
OncoTargets and therapy  2013;6:1471-1479.
The importance of epigenetic regulation has been increasingly recognized in the development of cancer. In this study, we investigated the impact of smoking, a major risk factor of lung cancer, on DNA methylation by comparing the genome-wide DNA methylation patterns between lung adenocarcinoma samples from six smokers and six nonsmokers. We identified that smoking-induced DNA methylations were enriched in the calcium signaling and neuroactive ligand receptor signaling pathways, which are closely related to smoking-induced lung cancers. Interestingly, we discovered that two genes in the mitogen-activated protein kinase signaling pathway (RPS6KA3 and ARAF) were hypomethylated in smokers but not in nonsmokers. In addition, we found that the smoking-induced lung cancer-specific DNA methylations were mostly enriched in nuclear activities, including regulation of gene expression and chromatin remodeling. Moreover, the smoking-induced hypermethylation could only be seen in lung adenocarcinoma tissue but not in adjacent normal lung tissue. We also used differentially methylated DNA loci to construct a diagnostic model to distinguish smoking-associated lung cancer from nonsmoking lung cancer with a sensitivity of 88.9% and specificity of 83.2%. Our results provided novel evidence to support that smoking can cause dramatic changes in the DNA methylation landscape of lung cancer, suggesting that epigenetic regulation of specific oncogenic signaling pathways plays an important role in the development of lung cancer.
doi:10.2147/OTT.S51041
PMCID: PMC3818101  PMID: 24204162
lung cancer; epigenome; methylation; tumor suppressor gene; smoking
25.  Cigarette Smoking and p16INK4α Gene Promoter Hypermethylation in Non-Small Cell Lung Carcinoma Patients: A Meta-Analysis 
PLoS ONE  2011;6(12):e28882.
Background
Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes play an important role in carcinogenesis. Recently, many studies have investigated the association between cigarette smoking and p16INK4α gene hypermethylation in lung cancer, but could not reach a unanimous conclusion.
Methods and Findings
Nineteen cross-sectional studies on the association between cigarette smoking and p16INK4α methylation in surgically resected tumor tissues from non-small cell lung carcinoma (NSCLC) patients were identified in PubMed database until June 2011. For each study, a 2×2 cross-table was extracted. In total, 2,037 smoker and 765 nonsmoker patients were pooled with a fixed-effects model weighting for the inverse of the variance. Overall, the frequency of p16INK4α hypermethylation was higher in NSCLC patients with smoking habits than that in non-smoking patients (OR = 2.25, 95% CI = 1.81–2.80). The positive association between cigarette smoking and p16INK4α hypermethylation was similar in adenocarcinoma and squamous-cell carcinoma. In the stratified analyses, the association was stronger in Asian patients and in the studies with larger sample sizes.
Conclusion
Cigarette smoking is positively correlated to p16INK4α gene hypermethylation in NSCLC patients.
doi:10.1371/journal.pone.0028882
PMCID: PMC3236763  PMID: 22174919

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