Radiation-induced lung adenocarcinoma is associated with increased frequency of genes inactivated by promoter hypermethylation.

Epigenetic inactivation of genes by promoter hypermethylation, a major mechanism in the initiation and progression of tobacco-induced cancer, has also been associated with lung cancer induced through environmental and occupational exposures. Our previous study of gene methylation in workers from the MAYAK nuclear enterprise identified a significantly higher prevalence for methylation of the p16 gene (CDKN2A) in adenocarcinomas from workers compared to tumors from non-worker controls. The purpose of this investigation was to determine whether genes in addition to p16 are "targeted" for silencing and whether overall gene methylation was more common in radiation-induced adenocarcinoma. A significant increase in the prevalence of methylation of GATA5 was seen in tumors from workers compared to tumors from controls. The prevalence for methylation of PAX5 beta and H-cadherin did not differ in tumors from workers and controls. Evaluating the frequency for methylation of a five-gene panel revealed that 93% of adenocarcinomas from workers compared to 66% of tumors from controls were methylated for at least one gene. Moreover, a twofold increase was seen in the number of tumors methylated for three or more genes for tumors from workers compared to controls. Increased frequency for inactivation of genes by promoter hypermethylation and targeting of tumor suppressor genes such as GATA5 may be factors that contribute to the increased risk for lung cancer associated with radiation exposure.

Multiplicity of abnormal promoter methylation in lung adenocarcinomas from smokers and never smokers.

The prevalence of methylation of the p16, DAPK and RASSF1A genes was investigated in lung adenocarcinoma from smokers, former uranium miners and never smokers. The association between a common genetic alteration in adenocarcinoma, mutation of the K-ras gene and methylation of these genes, as well as survival was examined. Adenocarcinomas from 157 smokers, 46 never smokers and 34 former uranium miners were evaluated for methylation of the p16, DAPK and RASSF1A genes using the methylation-specific PCR assay. Comparisons were also made to prevalences of methylation of the MGMT gene and mutation of the K-ras gene previously examined in these tumors. The prevalence of methylation for all genes was similar between adenocarcinomas from smokers and never smokers, although the prevalence for methylation of the p16 gene tended to be higher in smokers compared to never smokers. A significantly higher prevalence for p16 methylation was seen in central vs. peripheral lung tumors. At least 1 gene was methylated in 35% of stage I tumors, whereas 2 and >/=3 genes were methylated in 40% and 16% of tumors, respectively. Methylation of all genes was independent of K-ras mutation, whereas methylation of the DAPK and RASSF1A genes was positively associated. Environmental tobacco smoke, the strongest lung cancer risk factor among never smokers, induces adenocarcinoma in part through inactivation of the p16, DAPK and RASSF1A genes. Adenocarcinomas may develop through 2 distinct processes: multiple gene inactivations through promoter hypermethylation and activation of the K-ras gene.

Plutonium targets the p16 gene for inactivation by promoter hypermethylation in human lung adenocarcinoma.

Lung cancer from radon or (239)plutonium exposure has been linked to alpha-particles that damage DNA through large deletions and point mutations. We investigated the involvement of an epigenetic mechanism, gene inactivation by promoter hypermethylation in adenocarcinomas from plutonium-exposed workers at MAYAK, the first Russian nuclear enterprise established to manufacture weapons plutonium. Adenocarcinomas were collected retrospectively from 71 workers and 69 non-worker controls. Lung adenocarcinomas were examined from workers and non-worker controls for methylation of the CDKN2A (p16), O(6)-methylguanine-DNA methyltransferase (MGMT), death associated protein kinase (DAP-K), and Ras effector homolog 1 genes (RASSF1A). The prevalence for methylation of the MGMT or DAP-K genes did not differ between workers and controls, while a higher prevalence for methylation of the RASSF1A gene was seen in tumors from controls. In marked contrast, the prevalence for methylation of p16, a key regulator of the cell cycle, was increased significantly (P = 0.03) in tumors from workers compared with non-worker controls. Stratification of plutonium exposure into tertiles also revealed a striking dose response for methylation of the p16 gene (P = 0.008). Workers in the plutonium plant where exposure to internal radiation was highest had a 3.5 times (C.I. 1.5, 8.5; P = 0.001) greater risk for p16 methylation in their tumors than controls. This increased probability for methylation approximated the 4-fold increase in relative risk for adenocarcinoma in this group of workers exposed to plutonium. In addition, a trend (P = 0.08) was seen for an increase in the number of genes methylated (> or =2 genes) with plutonium dose. Here we demonstrate that exposure to plutonium may elevate the risk for adenocarcinoma through specifically targeting the p16 gene for inactivation by promoter methylation.

Promoter hypermethylation of the O6-methylguanine-DNA methyltransferase gene: more common in lung adenocarcinomas from never-smokers than smokers and associated with tumor progression.

Adenocarcinoma (AC) is the most common type of lung cancer diagnosed in the United States, comprising up to 40% of tumors in smokers and 50-80% of tumors in never-smokers. Exposures to cigarette smoke, direct or second-hand, and radiation in the form of radon progeny are the major risk factors for lung AC in both smokers and never-smokers. The goal of the current study was to determine the prevalence for O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation in a large sample of central or peripheral ACs from smokers (n = 157), former uranium miners (n = 34), and never-smokers (n = 46). The mutation rate at codon 12 of the K-ras gene was determined to assess whether activation of this oncogene was associated with MGMT methylation. The overall prevalence for MGMT methylation was 51%. Significantly more tumors from never-smokers than smokers exhibited MGMT methylation (66 versus 47%, respectively). In contrast, exposure to radon through uranium mining did not affect the prevalence for methylation. The frequency of MGMT methylation was increased significantly in association with tumor stage. K-ras mutations were detected in 24% of all ACs and 22, 24, and 28% of tumors from never-smokers, smokers, and miners, respectively. Alterations in both the K-ras and MGMT genes were seen in only 11% of ACs. Kaplan-Meier survival estimates did not reveal any difference between patient survival with or without MGMT methylation. In contrast, survival was significantly reduced over the initial 60 months after diagnosis for patients with a transition mutation in the K-ras gene compared with those with a transversion mutation. This investigation demonstrates that MGMT promoter hypermethylation is a common event in the progression of early stage AC of the lung. We have shown that the incidence of MGMT methylation was significantly higher in never-smokers than smokers and have detected a higher frequency of mutations within the K-ras gene than previously reported in never-smokers. This study also suggests that K-ras activation is independent of MGMT methylation.

Multiple imputation methods for longitudinal blood pressure measurements from the Framingham Heart Study.

Missing data are a great concern in longitudinal studies, because few subjects will have complete data and missingness could be an indicator of an adverse outcome. Analyses that exclude potentially informative observations due to missing data can be inefficient or biased. To assess the extent of these problems in the context of genetic analyses, we compared case-wise deletion to two multiple imputation methods available in the popular SAS package, the propensity score and regression methods. For both the real and simulated data sets, the propensity score and regression methods produced results similar to case-wise deletion. However, for the simulated data, the estimates of heritability for case-wise deletion and the two multiple imputation methods were much lower than for the complete data. This suggests that if missingness patterns are correlated within families, then imputation methods that do not allow this correlation can yield biased results.