Genetic loci associated with circulating phospholipid trans fatty acids: a meta-analysis of genome-wide association studies from the CHARGE Consortium.

BACKGROUND: Circulating trans fatty acids (TFAs), which cannot be synthesized by humans, are linked to adverse health outcomes. Although TFAs are obtained from diet, little is known about subsequent influences (e.g., relating to incorporation, metabolism, or intercompetition with other fatty acids) that could alter circulating concentrations and possibly modulate or mediate impacts on health. OBJECTIVE: The objective was to elucidate novel biologic pathways that may influence circulating TFAs by evaluating associations between common genetic variation and TFA biomarkers. DESIGN: We performed meta-analyses using 7 cohorts of European-ancestry participants (n = 8013) having measured genome-wide variation in single-nucleotide polymorphisms (SNPs) and circulating TFA biomarkers (erythrocyte or plasma phospholipids), including trans-16:1n-7, total trans-18:1, trans/cis-18:2, cis/trans-18:2, and trans/trans-18:2. We further evaluated SNPs with genome-wide significant associations among African Americans (n = 1082), Chinese Americans (n = 669), and Hispanic Americans (n = 657) from 2 of these cohorts. RESULTS: Among European-ancestry participants, 31 SNPs in or near the fatty acid desaturase (FADS) 1 and 2 cluster were associated with cis/trans-18:2; a top hit was rs174548 (ß = 0.0035, P = 4.90 × 10(-15)), an SNP previously associated with circulating n-3 and n-6 polyunsaturated fatty acid concentrations. No significant association was identified for other TFAs. rs174548 in FADS1/2 was also associated with cis/trans-18:2 in Hispanic Americans (ß = 0.0053, P = 1.05 × 10(-6)) and Chinese Americans (ß = 0.0028, P = 0.002) but not African Americans (ß = 0.0009, P = 0.34); however, in African Americans, fine mapping identified a top hit in FADS2 associated with cis/trans-18:2 (rs174579: ß = 0.0118, P = 4.05 × 10(-5)). The association between rs174548 and cis/trans-18:2 remained significant after further adjustment for individual circulating n-3 and n-6 fatty acids, except arachidonic acid. After adjustment for arachidonic acid concentrations, the association between rs174548 and cis/trans-18:2 was nearly eliminated in European-ancestry participants (ß-coefficient reduced by 86%), with similar reductions in Hispanic Americans and Chinese Americans. CONCLUSIONS: Our findings provide novel evidence for genetic regulation of cis/trans-18:2 by the FADS1/2 cluster and suggest that this regulation may be influenced/mediated by concentrations of arachidonic acid, an n-6 polyunsaturated fat.

Dietary fatty acids modulate associations between genetic variants and circulating fatty acids in plasma and erythrocyte membranes: Meta-analysis of nine studies in the CHARGE consortium.

SCOPE: Tissue concentrations of omega-3 fatty acids may reduce cardiovascular disease risk, and genetic variants are associated with circulating fatty acids concentrations. Whether dietary fatty acids interact with genetic variants to modify circulating omega-3 fatty acids is unclear. We evaluated interactions between genetic variants and fatty acid intakes for circulating alpha-linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid. METHODS AND RESULTS: We conducted meta-analyses (N = 11 668) evaluating interactions between dietary fatty acids and genetic variants (rs174538 and rs174548 in FADS1 (fatty acid desaturase 1), rs7435 in AGPAT3 (1-acyl-sn-glycerol-3-phosphate), rs4985167 in PDXDC1 (pyridoxal-dependent decarboxylase domain-containing 1), rs780094 in GCKR (glucokinase regulatory protein), and rs3734398 in ELOVL2 (fatty acid elongase 2)). Stratification by measurement compartment (plasma versus erthyrocyte) revealed compartment-specific interactions between FADS1 rs174538 and rs174548 and dietary alpha-linolenic acid and linoleic acid for docosahexaenoic acid and docosapentaenoic acid. CONCLUSION: Our findings reinforce earlier reports that genetically based differences in circulating fatty acids may be partially due to differences in the conversion of fatty acid precursors. Further, fatty acids measurement compartment may modify gene-diet relationships, and considering compartment may improve the detection of gene-fatty acids interactions for circulating fatty acid outcomes.

Genome-wide association study of selenium concentrations.

Selenium (Se) is an essential trace element in human nutrition, but its role in certain health conditions, particularly among Se sufficient populations, is controversial. A genome-wide association study (GWAS) of blood Se concentrations previously identified a locus at 5q14 near BHMT. We performed a GW meta-analysis of toenail Se concentrations, which reflect a longer duration of exposure than blood Se concentrations, including 4162 European descendants from four US cohorts. Toenail Se was measured using neutron activation analysis. We identified a GW-significant locus at 5q14 (P < 1 × 10(-16)), the same locus identified in the published GWAS of blood Se based on independent cohorts. The lead single-nucleotide polymorphism (SNP) explained ∼1% of the variance in toenail Se concentrations. Using GW-summary statistics from both toenail and blood Se, we observed statistical evidence of polygenic overlap (P < 0.001) and meta-analysis of results from studies of either trait (n = 9639) yielded a second GW-significant locus at 21q22.3, harboring CBS (P < 4 × 10(-8)). Proteins encoded by genes at 5q14 and 21q22.3 function in homocysteine (Hcy) metabolism, and index SNPs for each have previously been associated with betaine and Hcy levels in GWAS. Our findings show evidence of a genetic link between Se and Hcy pathways, both involved in cardiometabolic disease.

Genetic loci associated with circulating levels of very long-chain saturated fatty acids.

Very long-chain saturated fatty acids (VLSFAs) are saturated fatty acids with 20 or more carbons. In contrast to the more abundant saturated fatty acids, such as palmitic acid, there is growing evidence that circulating VLSFAs may have beneficial biological properties. Whether genetic factors influence circulating levels of VLSFAs is not known. We investigated the association of common genetic variation with plasma phospholipid/erythrocyte levels of three VLSFAs by performing genome-wide association studies in seven population-based cohorts comprising 10,129 subjects of European ancestry. We observed associations of circulating VLSFA concentrations with common variants in two genes, serine palmitoyl-transferase long-chain base subunit 3 (SPTLC3), a gene involved in the rate-limiting step of de novo sphingolipid synthesis, and ceramide synthase 4 (CERS4). The SPTLC3 variant at rs680379 was associated with higher arachidic acid (20:0 , P = 5.81 × 10(-13)). The CERS4 variant at rs2100944 was associated with higher levels of 20:0 (P = 2.65 × 10(-40)) and in analyses that adjusted for 20:0, with lower levels of behenic acid (P = 4.22 × 10(-26)) and lignoceric acid (P = 3.20 × 10(-21)). These novel associations suggest an inter-relationship of circulating VLSFAs and sphingolipid synthesis.

Large-scale genome-wide association studies and meta-analyses of longitudinal change in adult lung function.

BACKGROUND: Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function. METHODS: We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis. RESULTS: The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P  =  5.71 × 10(-7)). In addition, meta-analysis using the five cohorts with ≥3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P  =  2.18 × 10(-8)) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively. CONCLUSIONS: In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function.

Relationship between carbon dioxide levels and reported headaches on the international space station.

OBJECTIVE: Because of anecdotal reports of CO(2)-related symptoms onboard the International Space Station (ISS), the relationship between CO(2) and in-flight headaches was analyzed. METHODS: Headache reports and CO(2) measurements were obtained, and arithmetic means and single-point maxima were determined for 24-hour and 7-day periods. Multiple imputation addressed missing data, and logistic regression modeled the relationship between CO(2), headache probability, and covariates. RESULTS: CO(2) level, age at launch, time in-flight, and data source were significantly associated with headache. For each 1-mm Hg increase in CO(2), the odds of a crew member reporting a headache doubled. To keep the risk of headache below 1%, average 7-day CO(2) would need to be maintained below 2.5 mm Hg (current ISS range: 1 to 9 mm Hg). CONCLUSIONS: Although headache incidence was not high, results suggest an increased susceptibility to physiological effects of CO(2) in-flight.

Genome-wide association study of plasma N6 polyunsaturated fatty acids within the cohorts for heart and aging research in genomic epidemiology consortium.

BACKGROUND: Omega6 (n6) polyunsaturated fatty acids (PUFAs) and their metabolites are involved in cell signaling, inflammation, clot formation, and other crucial biological processes. Genetic components, such as variants of fatty acid desaturase (FADS) genes, determine the composition of n6 PUFAs. METHODS AND RESULTS: To elucidate undiscovered biological pathways that may influence n6 PUFA composition, we conducted genome-wide association studies and meta-analyses of associations of common genetic variants with 6 plasma n6 PUFAs in 8631 white adults (55% women) across 5 prospective studies. Plasma phospholipid or total plasma fatty acids were analyzed by similar gas chromatography techniques. The n6 fatty acids linoleic acid (LA), γ-linolenic acid (GLA), dihomo-GLA, arachidonic acid, and adrenic acid were expressed as percentage of total fatty acids. We performed linear regression with robust SEs to test for single-nucleotide polymorphism-fatty acid associations, with pooling using inverse-variance-weighted meta-analysis. Novel regions were identified on chromosome 10 associated with LA (rs10740118; P=8.1×10(-9); near NRBF2), on chromosome 16 with LA, GLA, dihomo-GLA, and arachidonic acid (rs16966952; P=1.2×10(-15), 5.0×10(-11), 7.6×10(-65), and 2.4×10(-10), respectively; NTAN1), and on chromosome 6 with adrenic acid after adjustment for arachidonic acid (rs3134950; P=2.1×10(-10); AGPAT1). We confirmed previous findings of the FADS cluster on chromosome 11 with LA and arachidonic acid, and further observed novel genome-wide significant association of this cluster with GLA, dihomo-GLA, and adrenic acid (P=2.3×10(-72), 2.6×10(-151), and 6.3×10(-140), respectively). CONCLUSIONS: Our findings suggest that along with the FADS gene cluster, additional genes may influence n6 PUFA composition.

Modeling the natural history and detection of lung cancer based on smoking behavior.

In this study, we developed a method for modeling the progression and detection of lung cancer based on the smoking behavior at an individual level. The model allows obtaining the characteristics of lung cancer in a population at the time of diagnosis. Lung cancer data from Surveillance, Epidemiology and End Results (SEER) database collected between 2004 and 2008 were used to fit the lung cancer progression and detection model. The fitted model combined with a smoking based carcinogenesis model was used to predict the distribution of age, gender, tumor size, disease stage and smoking status at diagnosis and the results were validated against independent data from the SEER database collected from 1988 to 1999. The model accurately predicted the gender distribution and median age of LC patients of diagnosis, and reasonably predicted the joint tumor size and disease stage distribution.

Doppler ultrasound of the central retinal artery in microgravity.

BACKGROUND: Ocular changes have been noted during long-duration spaceflight; we studied central retinal artery (CRA) blood flow using Doppler before, during, and after long-term microgravity exposure in astronauts compared with data from a control group of nonastronauts subjected to head-down tilt (HDT). METHODS: Available Doppler spectra of International Space Station (ISS) crewmembers were obtained from the NASA Lifetime Surveillance of Astronaut Health database, along with 2D ultrasound-derived measurements of the optic nerve sheath diameter (ONSD). CRA Doppler spectra and optic nerve sheath images were also obtained from healthy test subjects in an acute HDT experiment at 20 min of exposure (the ground-based analogue). RESULTS: HDT CRA peak systolic velocity in the ground-based analogue group increased by an average of 3 cm -s(-1) (33%) relative to seated values. ONSD at 300 of HDT increased by 0.5 mm relative to supine values. CRA Doppler spectra obtained on orbit were of excellent quality and demonstrated in-flight changes of +5 cm x s(-1) (50%) compared to preflight. ONSD increased in ISS crewmembers during flight relative to before flight, with some reversal postflight. DISCUSSION: A significant ONSD response to acute postural change and to spaceflight was demonstrated in this preliminary study. Increases in Doppler peak flow velocities correlated with increases in ONSD. Further investigations are warranted to corroborate the relationship between ONSD, intracranial pressure, and central retinal blood flow for occupational surveillance and research purposes.

Genome-wide association study identifies novel loci associated with concentrations of four plasma phospholipid fatty acids in the de novo lipogenesis pathway: results from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium.

BACKGROUND- Palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1n-7), and oleic acid (18:1n-9) are major saturated and monounsaturated fatty acids that affect cellular signaling and metabolic pathways. They are synthesized via de novo lipogenesis and are the main saturated and monounsaturated fatty acids in the diet. Levels of these fatty acids have been linked to diseases including type 2 diabetes mellitus and coronary heart disease. METHODS AND RESULTS- Genome-wide association studies were conducted in 5 population-based cohorts comprising 8961 participants of European ancestry to investigate the association of common genetic variation with plasma levels of these 4 fatty acids. We identified polymorphisms in 7 novel loci associated with circulating levels of ≥1 of these fatty acids. ALG14 (asparagine-linked glycosylation 14 homolog) polymorphisms were associated with higher 16:0 (P=2.7×10(-11)) and lower 18:0 (P=2.2×10(-18)). FADS1 and FADS2 (desaturases) polymorphisms were associated with higher 16:1n-7 (P=6.6×10(-13)) and 18:1n-9 (P=2.2×10(-32)) and lower 18:0 (P=1.3×10(-20)). LPGAT1 (lysophosphatidylglycerol acyltransferase) polymorphisms were associated with lower 18:0 (P=2.8×10(-9)). GCKR (glucokinase regulator; P=9.8×10(-10)) and HIF1AN (factor inhibiting hypoxia-inducible factor-1; P=5.7×10(-9)) polymorphisms were associated with higher 16:1n-7, whereas PKD2L1 (polycystic kidney disease 2-like 1; P=5.7×10(-15)) and a locus on chromosome 2 (not near known genes) were associated with lower 16:1n-7 (P=4.1×10(-8)). CONCLUSIONS- Our findings provide novel evidence that common variations in genes with diverse functions, including protein-glycosylation, polyunsaturated fatty acid metabolism, phospholipid modeling, and glucose- and oxygen-sensing pathways, are associated with circulating levels of 4 fatty acids in the de novo lipogenesis pathway. These results expand our knowledge of genetic factors relevant to de novo lipogenesis and fatty acid biology.

Chapter 11: Rice-MD Anderson lung cancer model.

The Rice-MD Anderson group uses a two-stage clonal expansion (TSCE) model of lung cancer mortality calibrated to a combination of MD Anderson case-control data on smoking histories and lung cancer mortality/incidence rate data collected from prospective cohorts in order to predict risk of lung cancer. This model is used to simulate lung cancer mortality in the U.S. population under the three scenarios of CISNET lung group's smoking base case project in order to estimate the effect of tobacco control policy on lung cancer mortality rates. Simulation results show that tobacco control policies have achieved 35% of the reduction in lung cancer mortality that would have resulted from cessation of all smoking in 1965.

Genetic loci associated with plasma phospholipid n-3 fatty acids: a meta-analysis of genome-wide association studies from the CHARGE Consortium.

Long-chain n-3 polyunsaturated fatty acids (PUFAs) can derive from diet or from α-linolenic acid (ALA) by elongation and desaturation. We investigated the association of common genetic variation with plasma phospholipid levels of the four major n-3 PUFAs by performing genome-wide association studies in five population-based cohorts comprising 8,866 subjects of European ancestry. Minor alleles of SNPs in FADS1 and FADS2 (desaturases) were associated with higher levels of ALA (p = 3 x 10⁻64) and lower levels of eicosapentaenoic acid (EPA, p = 5 x 10⁻58) and docosapentaenoic acid (DPA, p = 4 x 10⁻¹54). Minor alleles of SNPs in ELOVL2 (elongase) were associated with higher EPA (p = 2 x 10⁻¹²) and DPA (p = 1 x 10⁻4³) and lower docosahexaenoic acid (DHA, p = 1 x 10⁻¹5). In addition to genes in the n-3 pathway, we identified a novel association of DPA with several SNPs in GCKR (glucokinase regulator, p = 1 x 10⁻8). We observed a weaker association between ALA and EPA among carriers of the minor allele of a representative SNP in FADS2 (rs1535), suggesting a lower rate of ALA-to-EPA conversion in these subjects. In samples of African, Chinese, and Hispanic ancestry, associations of n-3 PUFAs were similar with a representative SNP in FADS1 but less consistent with a representative SNP in ELOVL2. Our findings show that common variation in n-3 metabolic pathway genes and in GCKR influences plasma phospholipid levels of n-3 PUFAs in populations of European ancestry and, for FADS1, in other ancestries.

Modeling the mortality reduction due to computed tomography screening for lung cancer.

BACKGROUND: The efficacy of computed tomography (CT) screening for lung cancer remains controversial because results from the National Lung Screening Trial are not yet available. In this study, the authors used data from a single-arm CT screening trial to estimate the mortality reduction using a modeling-based approach to construct a control comparison arm. METHODS: To estimate the potential lung cancer mortality reduction because of CT screening, a previously developed and validated model was applied to the screening trial to predict the number of lung cancer deaths in the absence of screening. By using age, gender, and smoking characteristics matching those of the trial participants, the model was used to simulate 5000 trials in the absence of CT screening to produce the expected number of lung cancer deaths along with 95% confidence intervals (95% CIs), while adjusting for healthy volunteer bias. RESULTS: There were 64 observed lung cancer deaths in the screening cohort (n = 7995), whereas the model predicted 117.7 deaths (95% CI, 98 deaths-139 deaths), indicating a mortality reduction of 45.6% (P < .001). When a more conservative healthy volunteer adjustment was applied, 111.3 lung cancer deaths were predicted (95% CI, 91 deaths-132 deaths), for a lung cancer-specific mortality reduction of 42.5% (P < .001). CONCLUSIONS: The results of the current study indicate that CT screening along with early stage treatment can reduce lung cancer-specific mortality. This mortality reduction is greatly influenced by the protocol of nodule follow-up and treatment, and the length of follow-up.

Association of smoking with tumor size at diagnosis in non-small cell lung cancer.

Tumor size at diagnosis (TSD) indirectly reflects tumor growth rate. The relationship between TSD and smoking is poorly understood. The aim of the study was to determine the relationship between smoking and TSD. We reviewed 1712 newly diagnosed and previously untreated non-small cell lung cancer (NSCLC) patients' electronic medical records and collected tumor characteristics. Demographic and epidemiologic characteristics were derived from questionnaires administered during personal interviews. Univariate and multivariate linear regression models were used to evaluate the relationship between TSD and smoking controlling for demographic and clinical factors. We also investigated the relationship between the rs1051730 SNP in an intron of the CHRNA3 gene (the polymorphism most significantly associated with lung cancer risk and smoking behavior) and TSD. We found a strong dose dependent relationship between TSD and smoking. Current smokers had largest and never smokers smallest TSD with former smokers having intermediate TSD. In the multivariate linear regression model, smoking status (never, former, and current), histological type (adenocarcinoma versus SqCC), and gender were significant predictors of TSD. Smoking duration and intensity may explain the gender effect in predicting TSD. We found that the variant allele of rs1051730 in CHRNA3 gene was associated with larger TSD of squamous cell carcinoma. In the multivariate linear regression model, both rs1051730 and smoking were significant predictors for the size of squamous carcinomas. We conclude that smoking is positively associated with lung tumor size at the moment of diagnosis.

Adjusting a cancer mortality-prediction model for disease status-related eligibility criteria.

BACKGROUND: Volunteering participants in disease studies tend to be healthier than the general population partially due to specific enrollment criteria. Using modeling to accurately predict outcomes of cohort studies enrolling volunteers requires adjusting for the bias introduced in this way. Here we propose a new method to account for the effect of a specific form of healthy volunteer bias resulting from imposing disease status-related eligibility criteria, on disease-specific mortality, by explicitly modeling the length of the time interval between the moment when the subject becomes ineligible for the study, and the outcome. METHODS: Using survival time data from 1190 newly diagnosed lung cancer patients at MD Anderson Cancer Center, we model the time from clinical lung cancer diagnosis to death using an exponential distribution to approximate the length of this interval for a study where lung cancer death serves as the outcome. Incorporating this interval into our previously developed lung cancer risk model, we adjust for the effect of disease status-related eligibility criteria in predicting the number of lung cancer deaths in the control arm of CARET. The effect of the adjustment using the MD Anderson-derived approximation is compared to that based on SEER data. RESULTS: Using the adjustment developed in conjunction with our existing lung cancer model, we are able to accurately predict the number of lung cancer deaths observed in the control arm of CARET. CONCLUSIONS: The resulting adjustment was accurate in predicting the lower rates of disease observed in the early years while still maintaining reasonable prediction ability in the later years of the trial. This method could be used to adjust for, or predict the duration and relative effect of any possible biases related to disease-specific eligibility criteria in modeling studies of volunteer-based cohorts.

Assessment of lung-cancer mortality reduction from CT Screening.

BACKGROUND: CT screening has been shown to increase lung cancer curability and we now assess the corresponding reduction in lung cancer mortality. METHODS: Lung-cancer mortality in a cohort of 7995 smokers who underwent CT screening for lung cancer in New York State (NYS) was compared with two unscreened cohorts (CPS-II and CARET). The standardized mortality ratio (SMR) of observed to expected lung cancer deaths for NYS was jointly adjusted for age, sex, and smoking history. As more current NYS smokers might have quit as a result of the screening, thus reducing deaths from lung cancer, another analysis was restricted to those participants smoking at entry and still smoking 6 years later. RESULTS: The SMR was 64/99.8=0.64 (P = 0.84 × 10⁻4) and 28/77.6=0.36 (P = 0.83 × 10⁻¹°), showing a significant reduction in deaths from lung cancer of 36% and 64% for CPS-II and CARET, respectively. Considering participants who were smoking at entry and still smoking 6 years later, the SMR using CPS-II rates was 29/49.1 = 0.59 (P = 0.001) and using CARET rates it was 21/57.4 = 0.37 (P = 0.31 × 10⁻7). CONCLUSIONS: CT screening significantly reduces lung-cancer mortality.

A smoking-based carcinogenesis model for lung cancer risk prediction.

Lung cancer is the leading cancer killer for both men and women worldwide. Over 80% of lung cancers are attributed to smoking. In this analysis, the authors propose to use a two-stage clonal expansion (TSCE) model to predict an individual's lung cancer risk based on gender and smoking history. The TSCE model is traditionally fitted to prospective cohort data. Here, the authors describe a new method that allows for the reconstruction of cohort data from the combination of risk factor data obtained from a case-control study, and tabled incidence/mortality rate data, and discuss alternative approaches. The method is applied to fit a TSCE model based on smoking. The fitted model is validated against independent data from the control arm of a lung cancer chemoprevention trial, CARET, where it accurately predicted the number of lung cancer deaths observed.

Estimation of the effects of smoking and DNA repair capacity on coefficients of a carcinogenesis model for lung cancer.

Numerous prospective and retrospective studies have clearly demonstrated a dose-related increased lung cancer risk associated with cigarette smoking, with evidence also for a genetic component to risk. In this study, using the two-stage clonal expansion stochastic model framework, for the first time we investigated the roles of both genetic susceptibility and smoking history in the initiation, clonal expansion, and malignant transformation processes in lung carcinogenesis, integrating information collected by a case-control study and a large-scale prospective cohort study. Our results show that individuals with suboptimal DNA repair capacity have enhanced transition rates of key events in carcinogenesis.