Gene expression signature of cigarette smoking and its role in lung adenocarcinoma development and survival

Background

Tobacco smoking is responsible for over 90% of lung cancer cases, and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure.

Methodology/Principal Findings

We performed gene expression analysis using HG-U133A Affymetrix chips on 135 fresh frozen tissue samples of adenocarcinoma and paired noninvolved lung tissue from current, former and never smokers, with biochemically validated smoking information. ANOVA analysis adjusted for potential confounders, multiple testing procedure, Gene Set Enrichment Analysis, and GO-functional classification were conducted for gene selection. Results were confirmed in independent adenocarcinoma and non-tumor tissues from two studies. We identified a gene expression signature characteristic of smoking that includes cell cycle genes, particularly those involved in the mitotic spindle formation (e.g., NEK2, TTK, PRC1). Expression of these genes strongly differentiated both smokers from non-smokers in lung tumors and early stage tumor tissue from non-tumor tissue (p<0.001 and fold-change >1.5, for each comparison), consistent with an important role for this pathway in lung carcinogenesis induced by smoking. These changes persisted many years after smoking cessation. NEK2 (p<0.001) and TTK (p = 0.002) expression in the noninvolved lung tissue was also associated with a 3-fold increased risk of mortality from lung adenocarcinoma in smokers.

Conclusions/Significance

Our work provides insight into the smoking-related mechanisms of lung neoplasia, and shows that the very mitotic genes known to be involved in cancer development are induced by smoking and affect survival. These genes are candidate targets for chemoprevention and treatment of lung cancer in smokers.     

Fecapentaene concentration and mutagenicity in 718 North American stool samples

The fecapentaenes (FP) are the predominant fecal mutagens identified to date, but they have not been shown to be carcinogenic. Epidemiologists looking for other fecal mutagens that may be related to colorectal cancer must disentangle from their investigations the pervasive mutagenic effect of the fecapentaenes. As a first step to studying the epidemiology of fecal mutagenicity independent of fecapentaenes, we compared FP measurements and Salmonella mutagenicity assay results for 718 acetone-extracted stool samples collected from a variety of subjects in the Washington DC metropolitan areas. In this large group, 50% of mutagenic samples contained elevated fecapentaenes. Specifically, three-quarters of the samples mutagenic in TA100 contained high FP levels. In contrast, mutagenicity in TA98 was not generally explainable by fecapentaenes, suggesting that non-fecapentaene TA98 mutagenicity should be one focus of future efforts to uncover colorectal carcinogens of etiologic importance.     

Children's exposure to magnetic fields produced by U.S. television sets used for viewing programs and playing video games

Two epidemiologic studies have reported increased risk of childhood leukemia associated with the length of time children watched television (TV) programs or played video games connected to TV sets. To evaluate magnetic field exposures resulting from these activities, the static, ELF, and VLF magnetic fields produced by 72 TV sets used by children to watch TV programs and 34 TV sets used to play video games were characterized in a field study conducted in Washington DC and its Maryland suburbs. The resulting TV-specific magnetic field data were combined with information collected through questionnaires to estimate the magnetic field exposure levels associated with TV watching and video game playing. The geometric means of the ELF and VLF exposure levels so calculated were 0.0091 and 0.0016 microT, respectively, for children watching TV programs and 0.023 and 0.0038 microT, respectively, for children playing video games. Geometric means of ambient ELF and VLF levels with TV sets turned off were 0.10 and 0.0027 microT, respectively. Summed over the ELF frequency range (6-3066 Hz), the exposure levels were small compared to ambient levels. However, in restricted ELF frequency ranges (120 Hz and 606-3066 Hz) and in the VLF band, TV exposure levels were comparable to or larger than normal ambient levels. Even so, the strengths of the 120 Hz or 606-3066 Hz components of TV fields were small relative to the overall ambient levels. Consequently, our results provide little support for a linkage between childhood leukemia and exposure to the ELF magnetic fields produced by TV sets. Our results do suggest that any future research on possible health effects of magnetic fields from television sets might focus on the VLF electric and magnetic fields produced by TV sets because of their enhanced ability relative to ELF fields to induce electric currents.     

Flexible design for following up positive findings

As more population-based studies suggest associations between genetic variants and disease risk, there is a need to improve the design of follow-up studies (stage II) in independent samples to confirm evidence of association observed at the initial stage (stage I). We propose to use flexible designs developed for randomized clinical trials in the calculation of sample size for follow-up studies. We apply a bootstrap procedure to correct the effect of regression to the mean, also called "winner's curse," resulting from choosing to follow up the markers with the strongest associations. We show how the results from stage I can improve sample size calculations for stage II adaptively. Despite the adaptive use of stage I data, the proposed method maintains the nominal global type I error for final analyses on the basis of either pure replication with the stage II data only or a joint analysis using information from both stages. Simulation studies show that sample-size calculations accounting for the impact of regression to the mean with the bootstrap procedure are more appropriate than is the conventional method. We also find that, in the context of flexible design, the joint analysis is generally more powerful than the replication analysis.     

Time to First Morning Cigarette and Risk of Chronic Obstructive Pulmonary Disease: Smokers in the PLCO Cancer Screening Trial

BackgroundTime to first cigarette (TTFC) after waking is an indicator of nicotine dependence. The association between TTFC and chronic obstructive pulmonary disease (COPD), the third leading cause of death in the United States, has not yet been reported.MethodsWe investigated the cross-sectional association between TTFC and prevalent COPD among 6,108 current smokers in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. COPD was defined as a self-reported diagnosis of emphysema, chronic bronchitis, or both. Current smokers in PLCO reported TTFC, the amount of time they typically waited before smoking their first cigarette of the day after waking, in four categories: ≤5, 6-30, 31-60, or >60 minutes. We used logistic regression models to investigate the association between TTFC and prevalent COPD with adjustments for age, gender, race, education, and smoking (cigarettes/day, years smoked during lifetime, pack-years, age at smoking initiation), and prior lung cancer diagnosis.ResultsCOPD was reported by 19% of these 6,108 smokers. Individuals with the shortest TTFC had the greatest risk of COPD; compared to those with the longest TTFC (>60 minutes) the adjusted odds ratios (OR) and 95% confidence intervals (CI) for COPD were 1.48 (95% CI, 1.15-1.91), 1.64 (95% CI, 1.29-2.08), 2.18 (95% CI, 1.65-2.87) for those with TTFC 31-60 minutes, 6-30 minutes, and ≤5 minutes, respectively (P-trend <0.0001). The association between TTFC and emphysema was similar to that for bronchitis, albeit the ORs were slightly stronger for chronic bronchitis; comparing TTFC ≤5 minutes to >60 minutes, the adjusted OR (95% CI) was 2.29 (1.69-3.12) for emphysema and 2.99 (1.95-4.59) for chronic bronchitis.ConclusionsCurrent smokers with shorter TTFC have increased risk of COPD compared to those with longer TTFC, even after comprehensive adjustment for established smoking covariates. Future epidemiologic studies, including prospective designs, should incorporate TTFC to better assess disease risk and evaluate the potential utility of TTFC as a COPD screening tool for smokers in the clinical setting.     

Theoretical models of the influence of genomic architecture on the dynamics of speciation

A long‐standing problem in evolutionary biology has been determining whether and how gradual, incremental changes at the gene level can account for rapid speciation and bursts of adaptive radiation. Using genome‐scale computer simulations, we extend previous theory showing how gradual adaptive change can generate nonlinear population transitions, resulting in the rapid formation of new, reproductively isolated species. We show that these transitions occur via a mechanism rooted in a basic property of biological heredity: the organization of genes in genomes. Genomic organization of genes facilitates two processes: (i) the build‐up of statistical associations among large numbers of genes and (ii) the action of divergent selection on persistent combinations of alleles. When a population has accumulated a critical amount of standing, divergently selected variation, the combination of these two processes allows many mutations of small effect to act synergistically and precipitously split one population into two discontinuous, reproductively isolated groups. Periods of allopatry, chromosomal linkage among loci, and large‐effect alleles can facilitate this process under some conditions, but are not required for it. Our results complement and extend existing theory on alternative stable states during population divergence, distinct phases of speciation and the rapid emergence of multilocus barriers to gene flow. The results are thus a step towards aligning population genomic theory with modern empirical studies.     

Y chromosome haplogroups and prostate cancer in populations of European and Ashkenazi Jewish ancestry

Genetic variation on the Y chromosome has not been convincingly implicated in prostate cancer risk. To comprehensively analyze the role of inherited Y chromosome variation in prostate cancer risk in individuals of European ancestry, we genotyped 34 binary Y chromosome markers in 3,995 prostate cancer cases and 3,815 control subjects drawn from four studies. In this set, we identified nominally significant association between a rare haplogroup, E1b1b1c, and prostate cancer in stage I (P = 0.012, OR = 0.51; 95% confidence interval 0.30-0.87). Population substructure of E1b1b1c carriers suggested Ashkenazi Jewish ancestry, prompting a replication phase in individuals of both European and Ashkenazi Jewish ancestry. The association was not significant for prostate cancer overall in studies of either Ashkenazi Jewish (1,686 cases and 1,597 control subjects) or European (686 cases and 734 control subjects) ancestry (P(meta) = 0.078), but a meta-analysis of stage I and II studies revealed a nominally significant association with prostate cancer risk (P(meta) = 0.010, OR = 0.77; 95% confidence interval 0.62-0.94). Comparing haplogroup frequencies between studies, we noted strong similarities between those conducted in the US and France, in which the majority of men carried R1 haplogroups, resembling Northwestern European populations. On the other hand, Finns had a remarkably different haplogroup distribution with a preponderance of N1c and I1 haplogroups. In summary, our results suggest that inherited Y chromosome variation plays a limited role in prostate cancer etiology in European populations but warrant follow-up in additional large and well characterized studies of multiple ethnic backgrounds.     

A case-control study of peripheral blood mitochondrial DNA copy number and risk of renal cell carcinoma

Background

Low mitochondrial DNA (mtDNA) copy number is a common feature of renal cell carcinoma (RCC), and may influence tumor development. Results from a recent case-control study suggest that low mtDNA copy number in peripheral blood may be a marker for increased RCC risk. In an attempt to replicate that finding, we measured mtDNA copy number in peripheral blood DNA from a U.S. population-based case-control study of RCC.

Methodology/Principal Findings

Relative mtDNA copy number was measured in triplicate by a quantitative real-time PCR assay using DNA extracted from peripheral whole blood. Cases (n = 603) had significantly lower mtDNA copy number than controls (n = 603; medians 0.85, 0.91 respectively; P = 0.0001). In multiple logistic regression analyses, the lowest quartile of mtDNA copy number was associated with a 60% increase in RCC risk relative to the highest quartile (OR = 1.6, 95% CI = 1.1–2.2; P _trend = 0.009). This association remained in analyses restricted to cases treated by surgery alone (OR _Q1 = 1.4, 95% CI = 1.0–2.1) and to localized tumors (2.0, 1.3–2.8).

Conclusions/Significance

Our findings from this investigation, to our knowledge the largest of its kind, offer important confirmatory evidence that low mtDNA copy number is associated with increased RCC risk. Additional research is needed to assess whether the association is replicable in prospective studies.     

HPV16 Seropositivity and Subsequent HPV16 Infection Risk in a Naturally Infected Population: Comparison of Serological Assays

Background

Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays.

Methodology/Principal Findings

Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27–0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28–0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection.

Conclusions/Significance

Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity.