The high lung cancer rate in Xuan Wei, China, is associated with smoky coal use in unvented homes, but not with wood or smokeless coal use. Smoky coal combustion emits higher polycyclic aromatic hydrocarbon (PAH) concentrations than wood combustion. This study used DNA adducts as biomarkers for human exposure to PAH from combustion emissions. DNA adducts were determined by enzyme-linked immunosorbent assays (ELISA) in placentas and peripheral and cord white blood cells (WBC) from Xuan Wei women burning smoky coal or wood and from Beijing women using natural gas. Color ELISA gave positive results in 58, 47, and 5% of the placentas from Xuan Wei women burning smoky coal without and with chimneys, and from Beijing women, respectively. Fluorescence ELISA indicated that 46, 65, 56, and 25% of placentas were positive from Xuan Wei women who lived in houses without and with chimneys, Xuan Wei women burning wood, and Beijing controls, respectively. Peripheral WBC samples were positive in 7/9, 8/9, and 3/9 for the Xuan Wei women who lived in houses without and with chimneys and Beijing women, respectively. PAH-DNA adducts were detected in a higher percentage of placentas from Xuan Wei women living in houses exposed to smoky coal or wood emissions than from those of the Beijing controls. No dose-response relationship was observed between the air benzo[alpha]pyrene concentrations and DNA adduct levels or percentage of detectable samples. The results suggest that DNA adducts can be used as a qualitative biomarker to assess human exposure to combustion emissions.
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.
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.
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.
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.
In Xuan Wei County, Yunnan Province, lung cancer mortality rates are among China's highest in males and females. Previous studies have shown a strong association of lung cancer mortality with air pollution from "smoky" coal combustion. In the present quantitative risk assessment of indoor air pollution study, the result strongly shows an obvious on-site exposure-response relationship between benzo[a]pyrene concentration in indoor air and lung cancer mortality and strongly supports the hypothesis that indoor air pollution is the main risk factor in inducing lung cancer in Xuan Wei County. In the present case-control study, the result shows that in females, the presence of lung cancer is statistically significantly associated with chronic bronchitis and family history of lung cancer. The results also suggest an association of lung cancer with duration of cooking food, but not with passive smoking. In males, the presence of lung cancer is associated with smoking, bronchitis, family history of lung cancer, and personal history of cooking food.
Smoky coal contains polycyclic aromatic hydrocarbons (PAHs) and has been strongly implicated in etiology of lung cancer in Xuan Wei, China. While PAHs have been demonstrated to form bulky adducts in nuclear DNA, they have a 90-fold greater affinity for mitochondrial DNA (mtDNA). To compensate for mitochondrial dysfunction or damage, mtDNA content is thought to increase. We conducted a population-based case-control study of lung cancer in Xuan Wei, China hypothesizing that mtDNA content is associated with lung cancer risk. Cases (n = 122) and controls (n = 121) were individually matched on age (±2yrs), sex, village of residence, and type of heating/cooking fuel currently used. Lifetime smoky coal use and potential confounders were determined with questionnaires. mtDNA was extracted from sputum and content was determined with quantitative RT-PCR. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated with unconditional logistic regression. mtDNA content was dichotomized at the median based on the distribution among the controls. mtDNA content > 157 was associated with a 2-fold increase in lung cancer risk (OR = 1.8; 95% CI = 1.0–3.2) compared with those with ≤157 copies. Risk was higher among those >57 years of age compared with those ≤ 57 years (p interaction = 0.01). In summary, mtDNA content was positively associated with lung cancer risk. Furthermore, there was some evidence that mtDNA content was more strongly associated with lung cancer risk among older individuals. However, due to the small sample size, additional studies are needed to evaluate these associations.
Lung cancer rates in Xuanwei County have been among the highest in China for both males and females, and have been causally associated with exposure to indoor smoky (bituminous) coal emissions that contain very high levels of polycyclic aromatic hydrocarbons. There are numerous coal mines across the County. Although lung cancer risk is strongly associated with use of smoky coal as a whole, variation in risk by smoky coal subtype has not been characterized as yet. We conducted a population-based case-control study of 498 lung cancer cases and 498 controls, individually matched to case subjects on age (±2 years) and sex, to examine risk by coal subtype. Odds ratios (ORs) and 95% confidence intervals (CIs) for coal subtype were calculated by conditional logistic regression, adjusting for potential confounders. Overall, smoky coal use was statistically significantly associated with lung cancer risk, as compared to use of smokeless coal or wood (OR=7.7, 95% CI=4.5 to 13.3). Furthermore, there was marked heterogeneity in risk estimates for specific subtypes of smoky coal (test for heterogeneity: p=5.17 × 10−10). Estimates were highest for coal from the Laibin (OR=24.8, 95% CI=12.4 to 49.6) and Longtan (OR=11.6, 95% CI = 5.0 to 27.2) coal types, and lower for coal from other types. These findings strongly suggest that in Xuanwei and elsewhere, the carcinogenic potential of coal combustion products can exhibit substantial local variation by specific coal source.
Coal; lung cancer; indoor air pollution; Xuanwei; China
The high incidence of lung cancer in Xuanwei County, China has been attributed to exposure to indoor smoky coal emissions that contain polycyclic aromatic hydrocarbons. The inflammatory response induced by coal smoke components may promote lung tumor development. We studied the association between single nucleotide polymorphisms (SNP) in genes involved in innate immunity and lung cancer risk in a population-based case-control study (122 cases and 122 controls) in Xuanwei. A total of 1,360 tag SNPs in 149 gene regions were included in the analysis. FCER2 rs7249320 was the most significant SNP (OR: 0.30; 95% CI: 0.16–0.55; P, 0.0001; false discovery rate value, 0.13) for variant carriers. The gene regions ALOX12B/ALOX15B and KLK2 were associated with increased lung cancer risk globally (false discovery rate value < 0.15). In addition, there were positive interactions between KLK15 rs3745523 and smoky coal use (OR: 9.40; P interaction = 0.07), and between FCER2 rs7249320 and KLK2 rs2739476 (OR: 10.77; P interaction = 0.003). Our results suggest that genetic polymorphisms in innate immunity genes may play a role in the carcinogenesis of lung cancer caused by polycyclic aromatic hydrocarbon-containing coal smoke. Integrin/receptor and complement pathways as well as IgE regulation are particular noteworthy.
lung cancer; innate immunity; single nucleotide polymorphism; polycyclic aromatic hydrocarbon; coal; FERC2; KLK
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.
Lung tumors; p16; MGMT; promoter methylation; never-smokers
We used MethyLight assays to analyze DNA methylation status of 27 genes on 49 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients who underwent surgical resection. Seven genes (RARB, BVES, CDKN2A, KCNH5, RASSF1, CDH13, and RUNX) were found to be methylated significantly more frequently in tumor tissues than in noncancerous tissues. Only methylation of CCND2 and APC was frequently detected in both cancerous and noncancerous tissues, supporting the hypothesis that the methylation of these two genes is a preneoplastic change and may be associated with tobacco smoking exposure. Methylation of any one of eight genes (RASSF1, DAPK1, BVES, CDH13, MGMT, KCNH5, RARB, or CDH1) was present in 80% of NSCLC tissues but only in 14% of noncancerous tissues. Detection of methylation of these genes in blood might have utility in monitoring and detecting tumor recurrence in early-stage NSCLC after curative surgical resection.
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.
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.
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).
We provide evidence that aberrant methylation of TSGs in samples of induced sputum is a useful tool for early diagnostic of lung diseases (LC and COPD) in smoker subjects.
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DNA methylation; Sputum; Lung cancer; COPD
We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.
To determine the analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen gene promoters from 10 patients with primary lung tumors by using Quantitative methylation specific PCR. We then tested this 15 gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A and AIM) for further elucidation of the diagnostic application of this panel of markers.
Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI 25%, 47%) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64%, 84%) but decreased the specificity from 100% to 73% (95% CI:54%, 88%).
This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.
DNA methylation/epigenetics; serum; lung cancer
Systemic methylation changes may be a diagnostic marker for tumor development or prognosis. Here, we investigate the relationship between gene methylation in lung tumors relative to normal lung tissue, and whether DNA methylation changes can be detected in paired blood samples.
Material and methods
Sixty five patients were enrolled in a surgical case series of non-small cell lung cancer (NSCLC) at a single institution. Using bisulfite pyrosequencing, CpG methylation was quantified at five genes (RASSF1A, CDH13, MGMT, ESR1 and DAPK) in lung tumor, pathologically normal lung tissue, and circulating blood from enrolled cases.
The analyses of methylation in tumors compared to normal lung tissue identified higher methylation of CDH13, RASSF1A, and DAPK genes, while ESR1 and MGMT methylation did not differ significantly between these tissue types. We then examined whether the three aberrantly methylated genes could be detected in blood. The difference in methylation observed in tumors was not reflected in methylation status of matching blood samples, indicating a low feasibility of detecting lung cancer by analyzing these genes in a blood-based test. Lastly we probed whether tumor methylation was associatied with clinical and demographic characteristics. Histology and gender were associated with methylation at the CDH13 gene, while stage was associated with methylation at MGMT.
Our results show higher methylation of RASSF1A, CDH13, and DAPK genes in lung tumors compared to normal lung. The lack of reflection of these methylation changes in blood samples from patients with NSCLC indicate their poorly suitability for a screening test.
methylation; non-small cell lung cancer; CDH13; MGMT; clinicopathological characteristics
Promoter methylation of the RASSF1A and RARβ genes has been associated with susceptibility to different types of cancer. In addition, RASSF1A and RARβ methylation plays an important role in the pathogenesis of lung cancer. We investigated the aberrant promoter methylation of RASSF1A and RARβ in lung cancer patients using methylation-specific polymerase chain reaction (MSP). Aberrant promoter methylation of the RASSF1A gene was detected in 45 of 56 (80.36%) cancer patients and aberrant promoter methylation of the RARβ gene was found in 48 of 56 (85.71%) cases; promoter methylation of both genes was found in 42 of 56 (75%) lung cancer cases. None of the 52 samples from controls exhibited DNA methylation in these two target genes. Methylation was significantly associated with the lung cancer cases compared to controls for the RASSF1A gene (adjusted OR=7.50; 95% CI, 3.935–14.296; p<0.001); similar results were obtained for methylation of the RARβ gene (adjusted OR=5.727; 95% CI, 3.348–9.797; p<0.001). In addition, the association remained significant in these two target genes (adjusted OR=8.429; 95% CI, 4.205–16.896; p<0.001). Our results indicated that the high percentage of promoter methylation in the RARβ and RASSF1A genes indicate their important role in the development of lung cancer in the population studied, and that risk of lung cancer for carriers positive for both genes is higher than in single-gene positive carriers, which may serve as a useful marker for prognosis and a target for the treatment of lung cancer.
methylation; Ras association domain family 1 A gene; RARβ gene; lung cancer; methylation-specific polymerase chain reaction
DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture.
Objective To estimate the risk of lung cancer associated with the use of different types of coal for household cooking and heating.
Setting Xuanwei County, Yunnan Province, China.
Design Retrospective cohort study (follow-up 1976-96) comparing mortality from lung cancer between lifelong users of “smoky coal” (bituminous) and “smokeless coal” (anthracite).
Participants 27 310 individuals using smoky coal and 9962 individuals using smokeless coal during their entire life.
Main outcome measures Primary outcomes were absolute and relative risk of death from lung cancer among users of different types of coal. Unadjusted survival analysis was used to estimate the absolute risk of lung cancer, while Cox regression models compared mortality hazards for lung cancer between smoky and smokeless coal users.
Results Lung cancer mortality was substantially higher among users of smoky coal than users of smokeless coal. The absolute risks of lung cancer death before 70 years of age for men and women using smoky coal were 18% and 20%, respectively, compared with less than 0.5% among smokeless coal users of both sexes. Lung cancer alone accounted for about 40% of all deaths before age 60 among individuals using smoky coal. Compared with smokeless coal, use of smoky coal was associated with an increased risk of lung cancer death (for men, hazard ratio 36 (95% confidence interval 20 to 65); for women, 99 (37 to 266)).
Conclusions In Xuanwei, the domestic use of smoky coal is associated with a substantial increase in the absolute lifetime risk of developing lung cancer and is likely to represent one of the strongest effects of environmental pollution reported for cancer risk. Use of less carcinogenic types of coal could translate to a substantial reduction of lung cancer risk.
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.
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.
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.
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.
MGMT; allele specific methylation; single nucleotide polymorphism; sputum; lung cancer
Rationale: Wood smoke–associated chronic obstructive pulmonary disease (COPD) is common in women in developing countries but has not been adequately described in developed countries.
Objectives: Our objective was to determine whether wood smoke exposure was a risk factor for COPD in a population of smokers in the United States and whether aberrant gene promoter methylation in sputum may modify this association.
Methods: For this cross-sectional study, 1,827 subjects were drawn from the Lovelace Smokers' Cohort, a predominantly female cohort of smokers. Wood smoke exposure was self-reported. Postbronchodilator spirometry was obtained, and COPD outcomes studied included percent predicted FEV1, airflow obstruction, and chronic bronchitis. Effect modification of wood smoke exposure with current cigarette smoke, ethnicity, sex, and promoter methylation of lung cancer-related genes in sputum on COPD outcomes were separately explored. Multivariable logistic and poisson regression models were used for binary and rate-based outcomes, respectively.
Measurements and Main Results: Self-reported wood smoke exposure was independently associated with a lower percent predicted FEV1 (point estimate [± SE] −0.03 ± 0.01) and a higher prevalence of airflow obstruction and chronic bronchitis (odds ratio, 1.96; 95% confidence interval, 1.52–2.52 and 1.64 (95% confidence interval, 1.31–2.06, respectively). These associations were stronger among current cigarette smokers, non-Hispanic whites, and men. Wood smoke exposure interacted in a multiplicative manner with aberrant promoter methylation of the p16 or GATA4 genes on lower percent predicted FEV1.
Conclusions: These studies identify a novel link between wood smoke exposure and gene promoter methylation that synergistically increases the risk for reduced lung function in cigarette smokers.
wood smoke; cigarette smokers; airflow obstruction; gene promoter methylation in sputum DNA
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.
gene methylation; folate; multi-vitamins; green vegetables; smokers
In Xuanwei County, Yunnan Province, China, lung cancer mortality rates in both males and females are among the highest in China.
We evaluated differential effects of smoking on lung cancer mortality before and after household stove improvement with chimney to reduce exposure to smoky coal emissions in the unique cohort in Xuanwei, China. Effects of independent variables on lung cancer mortality were measured as hazard ratios and 95% confidence intervals using a multivariable Cox regression model that included separate time-dependent variables for smoking duration (years) before and after stove improvement.
Results and conclusion:
We found that the effect of smoking on lung cancer risk becomes considerably stronger after chimney installation and consequent reduction of indoor coal smoke exposure.
smoky coal; smoking; lung cancer; mortality; Xuanwei cohort; time-dependent variable
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.
promoter methylation; DNA double strand break; single nucleotide polymorphism; DNA repair capacity; association study
Detection of cancer cells at early stages could potentially increase survival rates in cancer patients. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. A recent report from our laboratory described the use of quantitative methylation-specific PCR assays for discriminating patients with lung cancer from those without lung cancer using lung biopsies as well as sputum samples. TCF21 is known to be essential for differentiation of epithelial cells adjacent to mesenchyme. Using restriction landmark genomic scanning, a recent study identified TCF21 as candidate tumor suppressor at 6q23-q24 that is epigenetically inactivated in lung and head and neck cancers. Using DNA sequencing technique, we narrowed down a short CpG-rich segment (eight specific CpG sites in the CpG island within exon 1) of the TCF21 gene, which was unmethylated in normal lung epithelial cells but predominantly methylated in lung cancer cell lines. We specifically targeted this short CpG-rich sequence and developed a quantitative methylation-specific PCR assay suitable for high-throughput analysis. We showed the usefulness of this assay in discriminating patients with lung cancer from those without lung cancer using biopsies and sputum samples. We further showed similar applications with multiple other malignancies. Our assay might have important implications in early detection and surveillance of multiple malignancies.
Lung cancer is the leading cause of cancer deaths, with an overall survival of 15% at five years. Biomarkers that can sensitively and specifically detect lung cancer at early stage are crucial for improving this poor survival rate. Sputum has been the target for the discovery of non-invasive biomarkers for lung cancer because it contains airway epithelial cells, and molecular alterations identified in sputum are most likely to reflect tumor-associated changes or field cancerization caused by smoking in the lung. Sputum-based molecular biomarkers include morphology, allelic imbalance, promoter hypermethylation, gene mutations and, recently, differential miRNA expression. To improve the sensitivity and reproducibility of sputum-based biomarkers, we recommend standardization of processing protocols, bronchial epithelial cell enrichment, and identification of field cancerization biomarkers.
lung cancer; non-small cell lung cancer; sputum; biomarker; early detection
Aberrant promoter hypermethylation is one of the major mechanisms in carcinogenesis and some critical growth regulatory genes have shown commonality in methylation across solid tumors. Twenty-six genes, 14 identified through methylation in colon and breast cancers, were evaluated using primary lung adenocarcinomas (n = 175) from current, former and never smokers. Tumor specificity of methylation was validated through comparison of 14 lung cancer cell lines to normal human bronchial epithelial cells derived from bronchoscopy of 20 cancer-free smokers. Twenty-five genes were methylated in 11–81% of primary tumors. Prevalence for methylation of TNFRSF10C, BHLHB5 and BOLL was significantly higher in adenocarcinomas from never smokers than smokers. The relation between methylation of individual genes was examined using pairwise comparisons. A significant association was seen between 138 (42%) of the possible 325 pairwise comparisons. Most notably, methylation of MMP2, BHLHB4 or p16 was significantly associated with methylation of 16–19 other genes, thus predicting for a widespread methylation phenotype. Kaplan–Meier log-rank test and proportional hazard models identified a significant association between methylation of SULF2 (a pro-growth, -angiogenesis and -migration gene) and better patient survival (hazard ratio = 0.23). These results demonstrate a high degree of commonality for targeted silencing of genes between lung and other solid tumors and suggest that promoter hypermethylation in cancer is a highly co-ordinated event.
Domestic fuel combustion from cooking and heating, to which about 3 billion people worldwide are exposed, is associated with increased lung cancer risk. Lung cancer incidence in Xuanwei is the highest in China, and the attributable risk of lung cancer from unvented smoky coal burning is greater than 90%. To evaluate any lung cancer mortality reduction after changing from unvented stoves to portable stoves, we used lifetime smoky coal users in a retrospective cohort of all farmers born during 1917–1951 and residing in Xuanwei in 1976. Of the 42 422 enrolled farmers, 4054 lifetime smoky coal users changed to portable stoves, 4364 did not change, and 1074 died of lung cancer. Lung cancer morality associated with stove change was assessed by product-limit survival curves and multivariate Cox regression models. Both men (P<0.0001) and women (P<0.0001) who changed to portable stoves had a significantly increased probability of survival compared with those who did not change. Portable stoves were associated with decreased risk of lung cancer mortality in male participants (hazard ratio (HR)=0.62, 95% confidence interval (CI)=0.46–0.82) and female participants (HR=0.41, 95% CI=0.29–0.57). Portable stove use is associated with reduced lung cancer mortality risk, highlighting a cost-effective intervention that could substantially benefit health in developing countries.
lung cancer; stove; mortality; fuel; home
There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.
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)].
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.
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.
Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hyper-methylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-β2 (RAR-β2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methy-latransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n = 20) compared to metastatic melanomas. However, hypermethylation of RAR-β2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had ≥1 gene(s) and 59% had ≥2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5′-aza 2′-deoxycytidine (5Aza-dC). Analysis of melanoma patients’ plasma (preoperative blood; n = 31) demonstrated circulating hypermethylated MGMT, RAR-β2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.
MGMT; RAR-β2; RASSF1A; methylation; melanoma