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This chapter discusses water contaminants that may contribute to the human cancer burden. Specifically, it addresses the epidemiologic evidence for several contaminants and includes information on their levels and environmental distribution, as well as individual susceptibility, where data exist. The three categories of drinking water contaminants that may be carcinogenic and that have been studied most systematically are arsenic, disinfection by-products, and nitrate. In addition, radionuclides, microbiological agents, organic compounds from human commerce, and asbestiform particles have been reported to cause cancer, either as they occur in drinking water or in other media, giving rise to suspicion about their carcinogenicity when ingested. Future research priorities and prevention strategies are discussed.

Biomarkers are increasingly being incorporated into epidemiologic studies of cancer etiology, often referred to as molecular epidemiology. This chapter focuses on the use of biomarkers in the context of etiologic research. It begins with a discussion of the development and characterization of biomarkers for use in epidemiologic studies, with a particular emphasis on understanding the components of variance and their impact on estimates of disease risk. It then discusses key issues in the use of exposure, intermediate end points, genetic susceptibility, and tumor biomarkers; and reviews the main epidemiologic study designs that use biomarkers. The chapter concludes with comments on collecting and processing biologic samples for use in molecular epidemiology studies.

In the United States, an estimated 57,400 cases of cancer of the urinary bladder are diagnosed and 12,500 deaths from the disease occur each year. These account for 6% of all new cases of cancer among men and 2% of cases among women, as well as 3% of cancer deaths among men and 1% among women. This chapter reviews the epidemiology of bladder cancer. Topics covered include demographic patterns, risk factors, biological markers of exposure, tumor markers, and preventive measures.

Background: Although overall 5-year survival rates for ovarian cancer are poor (10-30%), stage I/IIa patients have a 95% 5-year survival. New biomarkers that improve the diagnostic performance of existing tumor markers are critically needed. A previous study by Zhang et al. reported identification and validation of three biomarkers using proteomic profiling that together improved early-stage ovarian cancer detection. Methods: To evaluate these markers in an independent study population, postdiagnostic/pretreatment serum samples were collected from women hospitalized at the Mayo Clinic from 1980 to 1989 as part of the National Cancer Institute Immunodiagnostic Serum Bank. Sera from 42 women with ovarian cancer, 65 with benign tumors, and 76 with digestive diseases were included in this study. Levels of various posttranslationally forms of transthyretin and apolipoprotein A1 were measured in addition to CA125. Results: Mean levels of five of the six forms of transthyretin were significantly lower in cases than in controls. The specificity of a model including transthyretin and apolipoprotein A1 alone was high [96.5%; 95% confidence interval (95% CI), 91.9-98.8%] but sensitivity was low (52.4%; 95% CI, 36.4-68.0%). A class prediction algorithm using all seven markers, CA125, and age maintained high specificity (94.3%; 95% CI, 89.1-97.5%) but had higher sensitivity (78.6%; 95% CI, 63.2-89.7%). Conclusions: We were able to replicate the findings reported by Zhang et al. in an independently conducted blinded study. These results provide some evidence that including age of patient and these markers in a model may improve specificity, especially when CA125 levels are ≥35 units/mL. Influences of sample handling, subject characteristics, and other covariates on biomarker levels require further consideration in discovery and replication or validation studies. (Cancer Epidemiol Biomarkers Prev 2006;15(9):1641–6)

Methods for efficiently identifying subjects with constantly acidic pH in epidemiological and clinical studies have not been assessed. We recruited 30 volunteers to estimate the minimum number of urine pH measurements using pH strips needed to identify subjects with “constantly acidic urine pH” Spearman’s correlation coefficients between urine pH measured with a pH meter and with the four pH strips ranged from 0.94 to 0.95 (p < 0.001 for all four strips). Overall agreement within ±0.5 pH units between the four strips and the pH meter ranged from 62.2% to 74.4%. When using a spot urine sample from a single morning to classify participants with respect to their urine pH, 80% of individuals fell into the acidic urine pH (pH equal to or lower than 6.0) group. When we required subjects to have urine pH equal to or lower than 6.0 in six consecutive AM spot urine samples and seven spot PM urine samples, only 20% of participants fulfilled this criterion. Measuring urine pH twice a day (early in the morning and early in the evening) during four consecutive days classified individuals in the same way as two daily measurements for one week. A single pH measurement from a spot urine sample is not reliable to identify individuals with constantly acidic pH. Morning and evening urine pH measurements with pH strips during four consecutive days identify individuals with constantly acidic urine pH individuals as well as one week of measurements, and thus might be useful to identify subjects with constantly acidic urine pH in epidemiological and clinical studies.

Objective: We sought to assess whether the metabolism of arsenic impacts a person’s susceptibility to bladder cancer. Methods: Urinary methylation products were measured in subjects from Argentina (114 cases and 114 controls) and the United States (23 cases and 49 controls). Results: In Argentina, the adjusted odds ratio (OR) for subjects with a high proportion of ingested arsenic excreted as monomethylarsonate (%MMA) was 2.17 (95% confidence interval [CI] 1.02–4.63) in smokers and 0.48 (95% CI 0.17–1.33) in nonsmokers. In the United States, the adjusted ORs for high %MMA in subjects with arsenic intakes less than and greater than 100 g/d were 1.20 (95% CI 0.27–5.38) and 2.70 (95% CI 0.39–18.6). Conclusions: Overall, these results are consistent with data from Taiwan suggesting that some individuals who excrete a higher proportion of ingested arsenic as MMA are more susceptible to arsenic-related cancer. (J Occup Environ Med. 2006;48: 478–488)

Mate is a ‘tea’, made from Ilex paraguariensis, widely consumed in South America, as mate con bombilla and mate cocido. Mate consumption has been associated with esophageal, oral, lung, and bladder cancers. This bladder cancer case-control study involved 114 Argentinean case-control pairs. Mate consumption was recorded for time of interview, and 20 and 40 years previously. Mate con bombilla consumed 20 years ago was associated with bladder cancer in ever-smokers (odds ratio=3.77, 95% confidence interval: 1.17–12.1), but not in never-smokers. Mate cocido was not associated with bladder cancer. These results are consistent with a previous study in Uruguay.

With the rapid development of biomarkers and new technologies, large-scale biologically-based cohort studies present expanding opportunities for population-based research on disease etiology and early detection markers. The prostate, lung, colorectal and ovarian cancer (PLCO) screening trial is a large randomized trial designed to determine if screening for these cancers leads to mortality reduction for these diseases. Within the Trial, the PLCO etiology and early marker study (EEMS) identifies risk factors for cancer and other diseases and evaluates biologic markers for the early detection of disease. EEMS includes 155,000 volunteers who provide basic risk factor information. Serial blood samples are collected at each of six screening rounds (including one collection for cryopreserved whole blood) from screening arm participants (77,000 subjects) and buccal cells are collected from those in the control arm of the trial. Etiologic studies consider environmental (e.g., diet), biochemical, and genetic factors. Early detection studies focus on blood-based biologic markers of early disease. Clinical epidemiology is also an important component of the PLCO trial.

Cigarette smoking is a risk factor for colon adenoma. The glutathione S-transferase enzymes are involved in the detoxification of carcinogenic compounds including those found in tobacco smoke, and thus, may be important modifiers of individual risk of developing this disease. We examined the prevalence of GSTM1 and GSTT1 gene deletions, and two GSTP1 polymorphisms in 772 cases with advanced colorectal adenomas (>1 cm, villous elements or high-grade dysplasia) of the distal colon (descending or sigmoid colon or rectum) and 777 sigmoidoscopy negative controls enrolled in the screening arm of the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Epidemiologic data on smoking was collected by self-administered questionnaire and DNA was extracted from whole blood or buffy coat. For GSTM1 and GSTT1, we used a newly developed TaqMan-based assay capable of discriminating heterozygous (+/−) individuals from those with two active alleles (+/+) and homozygous deletions (−/−). For GSTP1, the I105V and the A114V substitutions were identified using end point 5′ nuclease assays (TaqMan). Adjusted odds ratios (OR) and 95% confidence intervals (95% CI) were determined using unconditional logistic regression, controlling for age, race, and gender. Advanced adenoma risk was increased in current/former smokers (OR, 1.4; 95% CI, 1.2-1.8). Risks were decreased in subjects with ≥1 inactive GSTM1 alleles (OR, 0.6; 95% CI, 0.4-0.9); and the association was independent of smoking status (P interaction = 0.59). Having ≥1 inactive GSTT1 allele was associated with increased risk among smokers (OR, 1.4; 95% CI, 1.1-1.9; Ptrend = 0.02) but not among never smokers (OR, 0.9; 95% CI, 0.6-1.3) and a significant interaction between smoking and genotype was observed (P interaction = 0.05). In summary, this is the first study to report associations between colorectal adenomas and GSTM1 wild-type and GSTT1 null allele among smokers. These findings only became apparent using a newly developed assay able to distinguish heterozygous from wild-type individuals. Our data provide evidence that phenotypic differences between these two groups exist.

Previous studies have suggested that susceptibility to arsenic toxicity could be influenced by micronutrients, in particular selenium, methionine, and beta-carotene. A case-control study was conducted in West Bengal, India, in a region known to have groundwater arsenic contamination, to determine whether differences in micronutrient status contribute to susceptibility to arsenic-induced skin lesions. Micronutrient status was assessed by blood levels of specific micronutrients and metabolic indicators. Blood was obtained from 180 cases with skin lesions and 192 controls. Blood assays measured micronutrients and carotenoids (folate, selenium, vitamin B12, vitamin B6, retinol, alpha-tocopherol, lutein/zeaxanthin, beta-carotene, lycopene, beta-cryptoxanthin) and metabolic indicators such as glucose, cholesterol, transthyretin, amino acids, and proteins potentially associated with methylation (cysteine, homocysteine, methionine, glutathione). The distributions of nutrient concentrations were similar in cases and controls. The median selenium concentrations in cases and controls were both 1.15 micromol/L, and there was little evidence of differences in other micronutrients. Odds ratios (ORs) for arsenic-induced skin lesions were estimated for each quartile of nutrient concentrations, using the quartile with the highest nutrient level as the referent group. There were no clear trends associated with deficiencies of any micronutrient or metabolic indicator. For decreasing quartiles of selenium, the OR estimates were 1.00, 0.67, 0.99, 0.80; P=0.81; for methionine, the OR estimates were 1.00, 0.83, 0.78, 0.72; P=0.29. For beta-carotene, the ORs were 1.00, 0.53, 0.51, 0.96, demonstrating no increased risk at the lower quartiles. The measured micronutrients and metabolic indicators investigated do not appear to modify the risk of developing arsenic-induced skin lesions. The lack of any trend of increasing risk with lower selenium, vitamin E, and beta-carotene concentrations has important implications for proposed therapeutic interventions. The emphasis of interventions should be on reducing arsenic exposure.

We conducted a large death certificate-based case-control study to assess occupational risks for salivary gland cancer. African American (168 cases, 672 controls) and white (2237 cases, 8748 controls) cases from 24 states (1984-1989) were matched to controls by age, sex, race, and region. Race- and sex-stratified multiple logistic regression models calculated adjusted odds ratios. The proportion of young cases (

Inorganic arsenic exposure through drinking water causes cancer in adults; however, the carcinogenic potential in children remains unknown. A recent leukemia cluster in Churchill County, Nevada, where arsenic levels in water supplies are relatively high, has prompted concern. The authors investigated the incidence of childhood cancer between 1979 and 1999 in all 17 Nevada counties, grouped by low (i.e., < 10 microg/l), medium (10-25 microg/l), and high (35-90 microg/l) population-weighted arsenic levels in public drinking water supplies. The standardized incidence ratios (SIRs) for all childhood cancers combined were 1.00 (95% confidence interval [CI] = 0.94, 1.06), 0.72 (95% CI = 0.43, 1.12), and 1.25 (95% CI = 0.91, 1.69) for low-, medium-, and high-exposure counties, respectively. There was no relationship between arsenic levels in water and childhood leukemia (SIRs = 1.02, 0.61, and 0.86, respectively [95% CIIs = 0.90, 1.15; 0.12, 1.79; and 0.37, 1.70, respectively]). For all childhood cancers, excluding leukemias, the SIRs were 0.99 (95% CI = 0.92, 1.07), 0.82 (95% CI = 0.42, 1.22), and 1.37 (0.92, 1.83), respectively. The excess in 5- to 9-yr-old children and 10- to 14-yr-old children was in bone cancers, and the excess in 15- to 19-yr-old young adults was primarily in lymphomas. The findings in this study are reassuring in that leukemia risks were not increased at the concentrations of arsenic in water found in this study. Nonetheless, the results raise the possibility that there are increased risks for nonleukemic childhood cancers that require confirmation in other studies, particularly those in which higher exposures are addressed.

Background: A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell cycle protein. Methods: Genetic fine mapping analysis of the CCNE1 region was performed using data from two bladder cancer GWAS, which included a total of 5,942 cases and 10,857 controls. Cyclin E protein expression was evaluated by immunohistochemistry analysis in 265 bladder tumors in relation to CCNE1 genetic variants and tumor characteristics. Functional effects of cyclin E over-expression were evaluated with cell cycle assays. Findings: The original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r2≥0.7) associated with increased bladder cancer risk. From this group we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWAS, rs7257330 was associated only with aggressive bladder cancer (muscle-invasive or high-grade non-muscle-invasive cancer), with a per-allele odds ratio (OR) =1.18 (95%CI=1.09-1.27, p=4.67×10-5). Cyclin E protein expression was increased in aggressive bladder tumors (p=0.013) and, independently, with each rs7257330-A risk allele (ptrend=0.024). Over-expression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. Conclusions: Molecular mechanisms linking the CCNE1 GWAS signal and aggressive bladder cancer risk are related to cyclin E over-expression and cell cycle regulation. In combination with other genetic and clinical markers, CCNE1 genetic variants may be translationally useful for early prediction of aggressive bladder cancer. Citation Format: Yi-Ping Fu, Indu Kohaar, Lee Moore, Petra Lenz, Jonine D. Figueroa, Wei Tang, Patricia Porter-Gill, Stephen Chanock, Stephen M. Hewitt, Debra T. Silverman, Nathaniel Rothman, NCI-GWAS Bladder Cancer Consortium, Ludmila Prokunina-Olsson. Translational implications of the 19q12 bladder cancer GWAS signal for aggressive bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 944. doi:10.1158/1538-7445.AM2014-944

Chromosome 8q24 has emerged as an important region for genetic susceptibility to several cancers, but little is known about the contribution of DNA methylation in this region. To explore the extent of variation in DNA methylation at 8q24 in peripheral blood and its relationship with genetic variation in the region, we conducted a cross-sectional study using blood samples from 80 non-Hispanic Caucasian males in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Specifically, we aimed to evaluate between-individual variation in DNA methylation levels at specific CpG sites at 8q24 and to investigate the underlying genetic structure in the region by examining correlations for DNA methylation levels with each other and with the established cancer susceptibility SNPs at 8q24. We quantified DNA methylation levels at 145 CpG sites nearby cancer susceptibility single nucleotide polymorphisms (SNPs) at 8q24 or the MYC oncogene using pyrosequencing of bisulfite-treated DNA, which is considered a highly sensitive method to detect differences in DNA methylation levels between individuals. We calculated pairwise Spearman correlations (rho), adjusting for multiple testing using the False Discovery Rate (FDR) method. We identified a large number of CpG sites that were reproducible and demonstrated moderate to high between-individual variation in our study population. Among these CpG sites, some sites within or nearby MYC and POU5F1B were strongly correlated with one another (highest rho=0.74), suggesting a coordination of DNA methylation levels in gene regions. Moreover, we observed strong correlations between several CpG sites and some of the known cancer susceptibility SNPs at 8q24. Some of the correlations remained statistically significant after adjustment for multiple comparisons, including a CpG site and an established prostate cancer SNP in the long non-coding RNA PRNCR1 (Chr8:128167809 and rs1456315, rho=0.52; p-value=1.4x10-6; FDR-adjusted p-value=0.002) and a CpG site (Chr8:128498051) in POU5F1B and the known prostate/colorectal cancer SNP rs6983267 (rho=0.46; p-value=2.0x10-5; FDR-adjusted p-value=0.01). This is the first study to report associations between DNA methylation levels at 8q24 in peripheral blood and 8q24 cancer susceptibility SNPs, suggesting that DNA methylation at this important susceptibility locus may contribute to risk. Additional studies are needed to clarify the relationship between genetic and epigenetic variation at 8q24 and cancer risk. Citation Format: Kathryn Hughes Barry, Lee Moore, Joshua Sampson, Liying Yan, Ann Meyer, Charles C. Chung, Meredith Yeager, Laufey Amundadottir, Sonja I. Berndt. DNA methylation patterns in peripheral blood and the relationship with cancer susceptibility loci at chromosome 8q24. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 288. doi:10.1158/1538-7445.AM2014-288

Objectives: Esophageal cancer is the sixth leading cause of cancer death worldwide. The prognosis for esophageal cancer is dismal because most patients are diagnosed at a late stage. Patients can be successfully treated if diagnosed early, but current early detection screening tests are inadequate. Balloon cytology is a simple and inexpensive method of retrieving esophageal cells, but traditional cytologic examination of these cells has poor sensitivity and specificity for detecting precursor lesions. Thus, a biomarker such as DNA hypomethylation, which predisposes cells to chromosomal defects and genetic instability, may be used to improve the early detection of precursor lesions. In this study, we examined whether overall methylation levels of transposable elements Alu and long-interspersed nuclear elements (LINE-1) in the DNA extracted from balloon cytology-collected esophageal cells are associated with precursor lesions of esophageal squamous cell carcinoma (ESCC). Methods: The Cytology Sampling Study 2, a population-based early detection screening study of ESCC, recruited over 700 healthy subjects from a high-risk population in Linxian, China. The screening study used a mesh-covered balloon to collect esophageal cells. All subjects underwent endoscopy and Lugol's iodine, and precursor lesions were diagnosed by biopsy. Using a nested case-control study design, we examined the association between Alu and LINE-1 methylation levels and esophageal health in 50 esophagitis cases, 38 low-grade dysplasia cases, 55 high-grade dysplasia cases, and 94 matched normal controls. DNA methylation assessment of Alu and LINE-1 repetitive elements was conducted using quantitative PCR pyrosequencing on bisulphate-treated DNA extracted from cells collected by balloon cytology. Receiver operating characteristic (ROC) curves were plotted, and the area under the ROC curve (AUC) was calculated for methylation levels as a diagnostic marker for high-grade dysplasia. Logistic regression models adjusted for age, sex, smoking tobacco, alcohol intake, and hypertension were used to examine the association between Alu and LINE-1 methylation levels and risk of high-grade dysplasia compared with the other diagnoses. Results: Median Alu methylation levels were comparable among the esophagitis cases (24.5%), low-grade dysplasia cases (24.6%), high-grade dysplasia cases (24.6%), and normal controls (24.6%). Median LINE-1 methylation levels were also comparable among the esophagitis cases (71.8%), low-grade dysplasia cases (72.2%), high-grade dysplasia cases (71.6%), and controls (72.2%). The AUCs for using Alu and LINE-1 methylation levels as a diagnostic marker for high-grade dysplasia were 0.62 and 0.59, respectively. Overall, Alu and LINE-1 methylation levels were not associated with risk of high-grade dysplasia, with odds ratios (95% confidence interval) of 1.1 (0.9-1.4) and 1.1 (0.9-1.3), respectively. Conclusions: Given that esophageal cancers have very poor prognoses, it is of utmost importance to improve early detection methods. In this study, esophageal cell DNA methylation levels of Alu and LINE-1 were not associated with esophageal precursor lesions, and thus do not appear useful as an early detection marker. Future early detection studies may examine DNA hypomethylation in general or loci-specific methylation levels. Citation Format: Shih-Wen Lin, Christian C. Abnet, Neal D. Freedman, Lee Moore, Liying Yan, Ann Meyer, Qin-Jing Pan, Mark Roth, Guo-Qing Wang, Wen-Qiang Wei, Ning Lu, Philip R. Taylor, You-Lin Qiao, Sanford M. Dawsey. Measuring Alu and LINE-1 methylation for the early detection of precursor lesions of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C25.

Background: Alteration of DNA methylation is thought to promote carcinogenesis by weakening chromosomal stability and changing normal gene expression patterns. These epigenetic alterations that occur throughout the genome are considered early events in the carcinogenic process. Lately, long interspersed nucleotide element (LINE-1) methylation levels in blood DNA have been examined in relation to risk of several cancers. One previous renal cell cancer (RCC) case-control study reported higher LINE-1 methylation levels in blood DNA for cases than controls. Since DNA samples from that study were collected post-diagnostically, it remains unclear if the observed difference occurred prior to, or as a result of carcinogenesis. Thus, we examined the association between global methylation and RCC risk using prospectively collected blood samples. Methods: A nested RCC case-control study was conducted amid subjects in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) cohort, a study of Finnish male Caucasian smokers, 50 to 69 years old. LINE-1 percent 5-methylcytosine (LINE-1 %5-MeC) levels from 191 cases and 575 controls, matched on age at randomization (+/-5 years), were quantified using bisulfite treated blood DNA and pyrosequencing. Cancer risk was assessed in quartiles (Q1:79.5) of methylation based on control levels. Unconditional logistic regression was used to compute odds ratios (ORs) and 95% confidence intervals (CIs) adjusting for pack-years of smoking, age at randomization, hypertension, family history of cancer, body mass index (BMI) and intervention group. Results: RCC risk was significantly increased with increasing LINE-1 %5-MeC levels (Q1REF. vs. Q2OR= 1.12 (0.63-1.92), Q3OR= 1.78 (1.06-3.00), Q4OR= 1.78 (1.00-3.03); p-trend= 0.01). Associations appeared more pronounced for males with a higher BMI: (BMI Conclusion: Our study findings that increasing LINE-1 %5-MeC levels were associated with higher RCC risk using pre-diagnostically collected blood DNA are consistent with results reported in a previous case-control study. Yet, stratified results for BMI and smoking observed post-diagnostically in that case-control study are not supported in pre-diagnostic samples used here. Our results suggest that higher methylation levels are reflective of elevated RCC risk prior to diagnosis. Additional studies are being conducted in a second cohort to replicate these findings. Citation Format: Sara Karami, Gabriella Andreotti, Ruth M. Pfeiffer, Linda M. Liao, Stephanie Weinstein, Demetrius Albanes, Jarmo Virtamo, Nathaniel Rothman, Lee E. Moore. LINE-1 %5-Methylcytosine levels in pre-diagnostic leukocyte DNA and subsequent renal cell cancer risk . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 686. doi:10.1158/1538-7445.AM2013-686

Inorganic arsenic (In-As), an occupational and environmental human carcinogen, undergoes biomethylation to monomethylarsonate (MMA) and dimethylarsinate (DMA). It has been proposed that saturation of methylation capacity at high exposure levels may lead to a threshold for the carcinogenicity of In-As. The relative distribution of urinary In-As, MMA, and DMA is used as a measure of human methylation capacity. The most common pathway for elevated environmental exposure to In-As worldwide is through drinking water. We conducted a biomarker study in northern Chile of a population chronically exposed to water naturally contaminated with high arsenic content (600 micrograms/l). In this paper we present the results of a prospective follow-up of 73 exposed individuals, who were provided with water of lower arsenic content (45 micrograms/l) for 2 months. The proportions of In-As, MMA, and DMA in urine were compared before and after intervention, and the effect of other factors on the distribution of arsenic metabolites was also analyzed. The findings of this study indicate that the decrease in arsenic exposure was associated with a small decrease in the percent In-As in urine (from 17.8% to 14.6%) and in the MMA/DMA ratio (from 0.23 to 0.18). Other factors such as smoking, gender, age, years of residence, and ethnicity were associated mainly with changes in the MMA/DMA ratio, with smoking having the strongest effect. Nevertheless, the factors investigated accounted for only about 20% of the large interindividual variability observed. Genetic polymorphisms in As-methylating enzymes and other co-factors are likely to contribute to some of the unexplained variation. The changes observed in the percent In-As and in the MMA/DMA ratio do not support an exposure-based threshold for arsenic methylation in humans. Images Figure 1. A Figure 1. B Figure 2. A Figure 2. B

Methylation is considered the detoxification pathway for inorganic arsenic (InAs), an established human carcinogen. Urinary speciation analysis is used to assess the distribution of metabolites [monomethylarsonate (MMA), dimethylarsinate (DMA), and unmethylated arsenic (InAs)], as indicators of methylation capacity. We conducted a large biomarker study in northern Chile of a population chronically exposed to high levels of arsenic in drinking water. We report the results of the methylation study, which focused on the effects of exposure and other variables on the percent InAs, MMA, DMA, and the ratio of MMA to DMA in urine. The study consisted of 122 people in a town with arsenic water levels around 600 micrograms/l and 98 participants in a neighboring town with arsenic levels in water of about 15 micrograms/l. The corresponding mean urinary arsenic levels were 580 micrograms/l and 60 micrograms/l, of which 18.4% and 14.9% were InAs, respectively. The main differences were found for MMA:DMA; exposure, smoking, and being male were associated with higher MMA:DMA, while longer residence, Atacameño ethnicity, and being female were associated with lower MMA:DMA. Together, these variables explained about 30% of the variability in MMA:DMA. Overall, there was no evidence of a threshold for methylation capacity, even at very high exposures, and the interindividual differences were within a much wider range than those attributed to the variables investigated. The differences in percent InAs were small and within the ranges of other studies of background exposure levels. The biological significance of MMA:DMA, which was more than 1.5 times greater in the exposed group, and its relationship to sex, length of exposure, and ethnicity need further investigation because its relevance to health risk is not clear. Images p620-a Figure 1. Figure 2. Figure 2. Figure 2. Figure 2.

Background: The family of long interspersed nuclear elements (LINE-1) retrotransposons is reportedly hypomethylated in many cancers and reflects global methylation status in the genome. Global hypomethylation in peripheral blood leukocyte DNA has been increasingly studied and associated with increased risk of cancer, including colorectal cancer and adenoma in cross-sectional studies. Methods: To minimize disease- and/or treatment-related effects, we studied LINE-1 methylation levels in prospectively collected blood specimens from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial in relation to subsequent colorectal cancer risk. In a nested study of 398 cases and 519 controls who were cancer-free at blood collection, the extent of LINE-1 hypomethylation was measured by using the McrBC methylation specific restriction enzyme to cut methylated but not unmethylated DNA, followed by real-time PCR of the undigested/unmethylated LINE1 promoter sequences. We estimated the association between colorectal cancer risk and LINE-1 methylation categories by computing odds ratios (ORs) and 95% confidence intervals (CIs) through multivariable logistic regression modeling. Results: Overall, we observed no clear association between colorectal cancer and LINE-1 methylation categories (lowest tertile vs. highest: OR=1.06, 95% CI=0.74-1.52; P trend=0.72). However, we observed a nearly significant trend of decreasing LINE-1 methylation (hypomethylation) associated with increasing colorectal cancer risk, after excluding cases diagnosed within 1 year (lowest tertile vs. highest: OR=1.40, 95% CI=0.87-2.27; P trend=0.14) and 5 years (OR=2.65, 95% CI=0.96-7.36; P trend=0.07) following the blood collection. We noted no significant risk modification by folate intake or other suspected risk factors of colorectal cancer, such as smoking, obesity, and aspirin/ ibuprofen use in analyses including all subjects or excluding cases diagnosed within 5 years following blood collection (P interaction ranged between = 0.12 to 0.98). Conclusion: Findings from this prospective investigation offer further support for a potential dose-dependent relationship between LINE-1 hypomethylation in leukocyte DNA and subsequent colorectal cancer risk. Citation Format: Wen-Yi Huang, L. Joseph Su, Sonja I. Berndt, Lee E. Moore, Hormuzd A. Katki, Srinivasan Yegnasubramanian, Mark P. Purdue. Prospective study of LINE-1 hypomethylation in peripheral leukocyte DNA and colorectal cancer risk. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 51.

Background: Low global methylation levels measured in post-diagnostic case compared to healthy control blood DNA have been associated with elevated bladder cancer risk. Objective: To examine this association using blood DNA collected prior to bladder cancer diagnosis. Methods: Cases (n=437) and controls (N=847) were selected from the Prostate, Lung, Colorectal, Ovarian Cancer Screening Trial (PLCO). A replication case-control study was conducted among males from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) cohort (391 cases/778 controls). LINE-1 percent 5-methyl-cytosine (LINE-1 %5-MeC) levels were quantified using bisulfite treated blood DNA and pyrosequencing. Cancer risk was assessed in deciles (D1-D10: 10-100%) and quartiles (Q1-Q4:25-100%) of methylation levels based on controls. Logistic regression was used to evaluate individual and pooled study data. Findings: In PLCO, significantly lower bladder cancer risk was associated with the lowest LINE-1 %5-MeC decile (D1) compared to D2-D10 subjects adjusted for sex and pack-years of smoking (OR=0.35; 95% CI: 0.18-0.68, p=0.002). Findings were corroborated in the ATBC study, (D1 vs. D2-D10: OR=0.31;95% CI:0.17-0.57, p Interpretation: Findings observed in two independent studies using pre-diagnostically collected bladder cancer case/control subject DNA support that higher methylation levels may reflect higher bladder cancer risk prior to diagnosis. Associations using pre-diagnostically collected blood DNA were opposite to those from previous case-control studies using post-diagnostic DNA, suggesting changes in methylation levels associated with the carcinogenic process. Additional prospective studies evaluating LINE1 and gene-specific CpG methylation levels are needed to replicate and extend these findings. This proffered talk is also presented as Poster 11. Citation Format: Gabriella Andreotti, Sara Karami, Ruth Pfeiffer, Lauren Hurwitz, Linda Liao, Stephanie Weinstein, Demetrius Albanes, Jarmo Virtamo, Debra Silverman, Nathaniel Rothman, Lee Moore. LINE-1 %5-methyl cytosine levels in prediagnostic leukocyte DNA and future bladder cancer risk among PLCO and ATBC cohort subjects. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr PR1.

BACKGROUND: Renal cell carcinoma (RCC) is made up of several histological subtypes, the most common of which include clear cell, papillary and chromophobe RCC. These subtypes possess distinct genetic and clinical characteristics, and may also differ in etiology. To better understand whether the associations with established RCC risk factors [body mass index (BMI), smoking, hypertension, family history of kidney cancer] differ across subtypes, we conducted analyses in two large case-control studies conducted in the U.S. (1,217 cases, 1,235 controls) and Central and Eastern Europe (1,097 cases, 1,476 controls). METHODS: Histology was ascertained for 706 U.S. cases (58% of participating cases) and 891 European cases (81%) through a central pathology review conducted by a single pathologist. For cases not included in the central review, histology was abstracted from the original diagnostic pathology report. Case-only analyses were performed to compute odds ratios (OR) and 95% confidence intervals (CI) summarizing subtype differences by age, sex, and race. Case-control analyses were performed to compute subtype-specific ORs for other risk factors using polytomous regression. RESULTS: In case-only analyses, papillary cases (N=237) were older (OR=1.2, 95% CI=1.1-1.4 per 10-year increase), less likely to be female (OR=0.5, 95 % CI=0.4-0.8) and more likely to be black (OR=2.6, 95% CI=1.8-3.9) compared to clear cell cases (N=1,524). In case-control analyses, BMI was associated with clear cell (OR=1.5, 95% CI=1.3-1.7 per 10 kg/m2 increase) and chromophobe RCC (N=80; OR=1.5, 95% CI=1.1-2.0), but not papillary RCC (OR=1.1, 95% CI 0.9-1.5; heterogeneity test vs. clear cell, P=0.006). No differences across subtypes were observed for other risk factors. Analyses stratified by study and restricted to cases included in the central pathology review yielded virtually identical findings. CONCLUSIONS: Our results from this analysis, to our knowledge the most comprehensive investigation of etiologic heterogeneity across RCC histologic subtypes conducted to date, support the existence of distinct age, sex and racial distributions for RCC subtypes, and suggest that the obesity-RCC association differs by histology. These findings underscore the importance of accounting for histologic subtype in investigations of RCC etiology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5515. doi:1538-7445.AM2012-5515

Background: Mitochondrial DNA (mtDNA) is vulnerable to mutations, and the number of copies of mtDNA per cell may increase to compensate for DNA damage. Recent studies have evaluated blood leukocyte mtDNA copy number and risk of various types of cancer, including renal cell carcinoma (RCC). Although alterations in mtDNA copy number have been associated with cancer risk, the direction of this association has been inconsistent between studies with prospective and retrospective blood collection. One case-control study found that low mtDNA copy number was associated with an increased risk of RCC; however, this association has not been investigated prospectively. Methods: We conducted a nested case-control study of RCC risk in relation to mtDNA copy number in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. A total of 230 cases and 469 controls matched on age, sex, race, date of blood collection and specimen type were included. Measurements of mtDNA copy number were performed in triplicate using a fluorescence-based QPCR assay. Assay results were highly reproducible, with a coefficient of variation of 5.3% for replicate QC samples from a single individual and an intraclass correlation coefficient of 0.69 for duplicate samples collected an average of 3.6 years apart from 45 controls. To evaluate risk of RCC in relation to mtDNA copy number, we calculated odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression models adjusted for all matching variables as well as history of hypertension, body mass index, smoking history, and family history of kidney cancer. Subjects were assigned to quartiles of mtDNA copy number based on the distribution among controls. Results: Median mtDNA copy number was higher for cases than for controls (131 vs. 121; P=0.001, Wilcoxon rank-sum test). Relative to subjects in the lowest quartile of mtDNA copy number, the ORs for subjects in the 2nd, 3rd, and 4th quartiles were 1.0 (95% CI 0.6-1.6), 1.5 (95% 0.9-2.3), and 1.9 (95% CI 1.2-3.1), respectively (Ptrend = 0.003). The association between mtDNA copy number and RCC was stronger among men (highest vs. lowest quartile, OR 2.4, 95% CI 1.3-4.4) than among women (OR 1.3, 95% CI 0.5-3.2; Pint = 0.24). Results were similar in conditional logistic regression analyses of matched sets, and when we restricted to subjects with mtDNA from buffy coat specimens and to cases diagnosed more than two years after sample collection. Conclusions: Results of this study, to our knowledge the first prospective investigation of mtDNA copy number and RCC risk, suggest that high mtDNA copy number is associated with an increased risk of RCC, particularly among men. Although our findings were inconsistent with prior case-control evidence, most prospective studies of other cancers (e.g., lung, pancreas, non-Hodgkin lymphoma) have reported positive associations. Replication of these findings in other prospective cohorts is needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4461. doi:1538-7445.AM2012-4461

Chromosome 9p21 has been implicated in the pathogenesis of cutaneous malignant melanoma (CMM), glioma, non-small cell lung cancer, and leukemia. This region includes cyclin-dependent kinase inhibitor 2A (CDKN2A), the major known high-risk susceptibility gene for CMM; CDKN2B; antisense noncoding RNA in the INK4 locus (ANRIL), a GWAS hotspot for multiple phenotypes including cancers; methylthioadenosine phosphorylase (MTAP), a gene with tumor suppressor function and a cluster of type I interferon (IFN) genes. The goal of this study was to examine the association between genetic variation in the 9p21 region and multiple cancer outcomes using genotyping data from studies participating in the National Cancer Institute's (NCI) iSelect project. We evaluated up to 202 tagSNPs in 22 genes in the 9p21 region (19.9-32.8 Mb), in case-control studies of thyroid cancer (THC), endometrial cancer (EC), testicular cancer (TC), renal cell carcinoma (RCC), colorectal cancer (CRC), colorectal adenoma (CA), esophageal squamous cell carcinoma (ESCC), gastric cardia cancer (GCC) and osteosarcoma (OS). The number of cases in each of the nine studies ranged from 96 to 1234. We first performed single SNP-based analyses for each study separately, using logistic regression adjusted for age and study-specific covariates. To combine SNP based analyses, we used a newly developed subset-based statistical approach (ASSET), which allow for heterogeneity of SNP effect on different outcomes. This approach exhaustively explores subsets of studies for the presence of association signals, and then evaluates the significance from subsets with the strongest association signal, meanwhile accounting for multiple tests required by the subset search. Gene based P-values were computed using an adaptive rank truncated product (ARTP) statistic and a permutation-based sampling procedure (10,000 permutations) that took into account the numbers of SNPs in each gene and their linkage disequilibrium structure. When analyzing each study separately, we found that a SNP (rs3731257) in CDKN2A was significantly associated with the risk of ESCC after Bonferroni correction for number of SNPs and studies (P=7 x 10-6). Meta-analyses by ASSET found that this SNP was significantly associated with both ESCC and EC (PASSET Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-330. doi:1538-7445.AM2012-LB-330

Introduction: Vitamin D, obtained primarily from ultraviolet (UV) exposure, is hydroxylated to its biologically active form within the kidneys. Epidemiological evidence supporting a relationship between vitamin D and kidney cancer risk has been inconsistent despite experimental data indicating that vitamin D and its metabolites may inhibit carcinogenesis. Previously we reported an inverse association between renal cell carcinoma (RCC) risk and occupational UV exposure among men in Europe. In the current study, we examined the association between occupational UV exposure and RCC risk among U.S. residents and investigated whether this association varies by race and sex. Methods: Lifetime occupational data for 1,217 RCC cases and 1,235 controls in a large population-based case-control study were blindly assessed by an industrial hygiene expert to create a job exposure matrix that categorized frequency, duration, confidence, and intensity of occupational UV exposure. Categorical exposure metrics used to evaluate exposure-response relationships with occupational exposure were based on tertiles of exposure levels among controls. Unconditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to examine the association between RCC risk and UV exposure by sex and race. Results: Across all exposure-response matrices, women in the highest tertile of occupational UV exposure had a 40% reduction in RCC risk compared to those in the lowest tertile; risk decreased significantly as occupational UV exposure increased (p-trend=0.01 to 0.004). A marginally significant, non-linear inverse association was observed among men. Further stratification by race revealed similar findings for black and white women. Significantly decreased associations between RCC risk and occupational UV exposure were observed among black men (p-trend=0.05), but not among white men, although results for blacks were based on small numbers. Our findings suggest that occupational UV exposure may protect against RCC. We caution any inferences made using race, given power limitations for stratified analyses, until additional well-powered studies can replicate and extend these findings. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A72.

Background: Systematic genome-wide (global) reductions of methylated cytosine levels have been observed in most cancer tissues, including colorectal cancer, and are hypothesized to contribute to carcinogenesis through genomic instability and protooncogene activation. Recently, genomic DNA hypomethylation has been measured in peripheral blood leukocytes and associated with colorectal tumors. However, as these studies used post-diagnostic specimens, it is possible that the observed findings reflect disease- and/or treatment-induced effects on leukocyte DNA methylation. Methods: Using prospectively collected blood specimens and questionnaire data from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial, we studied genomic leukocyte DNA methylation status in relation to subsequent colorectal cancer risk in a nested case-control study of 370 cases and 493 controls with no cancer diagnosis prior to the blood draw. Genomic leukocyte DNA methylation status was determined by an HPLC/Tandem Mass Spectrometry method and expressed as the relative amount of methyl-cytosine to total cytosine residues, or %5-mC. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed using unconditional logistic regression models, adjusting for age, gender, race, time since initial sigmoidoscopy screening, and year of randomization. Inclusion of other suspected colorectal cancer risk factors in the models did not materially change the results. Results: We observed a relatively narrow range of genomic leukocyte DNA methylation levels among cases and controls, with %5-mC ranging from 3.57 to 4.60 (median = 4.03, standard deviation = 0.19). Categorizing subjects by quartiles of %5-mC content in controls, using the lowest quartile as the reference category (Q1), we observed no trend in risk with adjusted ORs (95% CIs) for Q2, 1.44 (1.01-2.08); Q3, 1.06 (0.75-1.51); and Q4, 1.01 (0.70-1.47) (P-trend = 0.49). Dichotomizing subjects by median of %5-mC in controls with the lower %5-mC group as the reference, we found a borderline-significantly decreased risk for subjects with %5-mC above the median (OR = 0.81, CI = 0.61-1.06), particularly among subjects whose natural folate intake levels were above the median (OR = 0.60, CI = 0.39-0.95; P-interaction = 0.06). Conclusion: Preliminary findings from our study, to our knowledge the first prospective investigation of DNA methylation and colorectal cancer, show no clear dose-dependent association between genomic leukocyte DNA methylation status and subsequent cancer risk. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3755. doi:10.1158/1538-7445.AM2011-3755

Here we describe new techniques that employ fluorescence in situ hybridization (FISH) with centromeric and chromosome-specific DNA probes to detect aneuploidy and micronucleus formation in exfoliated human epithelial cells. Micronuclei arise from chromosome breakage or lagging, which results in chromosome fragments and whole chromosomes being left outside of the main nucleus at telophase. By using a centromeric DNA probe to detect the presence of whole chromosomes in micronuclei and propidium iodide as a general DNA stain in exfoliated nasal, buccal, and bladder cells, we have developed a new fluorescent method that can detect micronuclei and determine the mechanism of formation. The new fluorescent technique gave results that were very similar to those obtained with the standard Feulgen-fast green method. The spontaneous levels of micronuclei in healthy volunteers were buccal, 0.13%, nasal, 0.21%, and urothelial, 0.07%, in approximately 1500 cells per data point. These values are lower than that found in cultured lymphocytes, 0.4-0.8%. Approximately 50% of the exfoliated cell micronuclei contained whole chromosomes (centromeric DNA). FISH was also used to detect aneuploidy in exfoliated buccal and bladder cells. A DNA probe specific for chromosome 9 was used. Average frequencies for 0, 1, 2, 3, and 4 hybridization regions were 4.8, 9.3, 84.8, 0.8, and 0.3% for urothelial cells and 8.2, 9.9, 80.1, 1.4, and 0.4% for buccal cells. The estimated frequency of aneuploidy in exfoliated cells is similar to that found in human lymphocytes analyzed by FISH with the same probe for chromosome 9. These techniques are potentially useful for epidemiological studies of exposed populations and are currently being applied in our laboratory for studies of arsenic- and formaldehyde-exposed populations.

Change in chromosome number, numerical aneuploidy, has been consistently linked with cancer development. Since 90% of cancers arise in epithelial tissues, techniques that measure aneuploidy in these tissues would be very useful. Here we describe methods of optimization and suggest use of fluorescent in situ hybridization (FISH) to detect aneuploidy in exfoliated epithelial cells collected from the mouth and bladder. A total of 10,383 urothelial cells and 4,691 buccal cells were scored in order to determine a baseline frequency of aneuploidy in human volunteers using a classical satellite probe for chromosome 9. Protein digestion with pepsin was found to be more efficient at removing the keratinized cell membrane and optimizing probe penetration than acid washes, detergent washes, or hypotonic treatments. A 20 min cellular digestion with 200 μg/ml and a 30 min digestion with 300 μg/ml of pepsin in 0.01 M HCI optimized probe penetration in urothelial and buccal cells, respectively. Average frequencies for 0, 1, 2, 3, and 4 hybridization regions were 10.3, 10.1, 78.4, 1.0, and 0.3% for urothelial cells and 8.8, 9.8, 79.4, 1.3, and 0.3% for buccal cells, respectively. These results are very similar to those previously described in lymphocytes. The urothelial cells of males had a lower frequency of diploid cells and a higher frequency of cells without hybridization regions than females (P < 0.02). No statistically significant variability was found between individuals or sex groups in buccal cells. Our data show that FISH is a useful tool to detect changes in frequency of aneuploidy in exfoliated epithelial cells and has good potential for monitoring human populations exposed to genotoxic agents. © 1993 Wiley-Liss, Inc.

Objective. Ecological studies conducted in the United States and globally have shown an inverse association between levels of solar ultraviolet irradiance and kidney cancer incidence, suggesting that vitamin D, which is obtained from exposure to ultraviolet light (sunlight), may be associated with a reduced risk of kidney cancer. However, results from two prospective cohort and two case-control studies focusing on dietary vitamin D reported no statistically significant association with the development of kidney cancer. Thus, the purpose of this study was to examine the association between circulating 25-hydroxyvitamin D (25(OH)D), the accepted biomarker for vitamin D status, and the development of kidney cancer within a large pooled nested case-control study carried out as part of the Cohort Consortium Vitamin D Pooling Project of Rarer Cancers (VDPP). Methods. Kidney cancer cases (n = 775) and age-, sex-, race-, and season-matched controls (n = 775) were identified from eight prospective cohort studies participating in the VDPP. Data on potential confounders were obtained and harmonized by a central data coordinating center. Serum/plasma samples were assayed for 25(OH)D at Heartland Assays, Inc using the DiaSorin LIAISON 25(OH)D TOTAL assay, a direct, competitive chemiluminescence immunoassay. Clinically-defined cutpoints suggesting deficient, insufficient, and sufficient 25(OH)D concentrations were used to analyze the association between high and low circulating 25(OH)D and kidney cancer, with 50 to Results. Neither low nor high concentrations of circulating 25(OH)D were significantly associated with kidney cancer risk overall or the renal cell carcinoma subtype. Sex-specific analyses showed, however, that high concentrations of 25(OH)D (>75 nmol/l) were associated with a non-significant increased risk of kidney cancer among males (reference: 50 to Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2800.

Insight into the cancer genome has recently increased in an unprecedented way due to high throughput (HT) sequencing technology to examine the genome, exome, and transcriptome. We sought new genetic biomarkers for cancer by using HT sequencing to examine highly characterized primary sporadic tumors and tumor-derived cell lines. Several tumors/cell lines represented the most common subtype of kidney cancer, clear cell renal cell carcinoma, as well as NCI-60 panel cell lines from breast (MCF7) and prostate (PC3). Incorporation of NCI-60 panel cell lines was advantageous due to extensive prior characterization of these reagents, including gene expression, comparative genome hybridization, SNPs, and drug cytotoxicity. The study was performed using three major platforms (454, Illumina, ABI) to compare sequencing quality, cost, quantity, and computational amenability. Different methodologies were examined including exome, transcriptome, and whole genome sequencing. Alignment of sequence to a consensus genome (hg 18) allowed identification of novel and previously documented genomic variants. Various filters were implemented to sort the large number of variants detected, including dbSNP, personal genome variants, and 1000 genomes data. An average of 65 million 35-50 base pair sequences were generated from transcriptomes of two primary clear cell RCC tumors. Over 2 million 375 base pair sequences were generated from each of four cell line exomes. Differences were observed in extent and depth of coverage of a given target region by various experimental approaches. SNP array genotyping showed 1% discordance with sequencing. Previously characterized mutations were confirmed in HT sequences. We identified multiple regions of homozygosity at an unprecedented resolution. For example, in the clear cell renal cancer cell line, 786-0, a known mutation in VHL (311delG) was identified, and LOH at SNP positions across the entire length of chromosome 3 was confirmed. Alignment to a reference genome (hg 18) permitted identification of 2583-5307 genomic sequence variants, including 92-297 novel non-synonymous variants in the exomes of each cell line. Variants include frameshift and nonsense mutations. Novel non-synonymous variants were identified in cancer-related genes and these are being examined in additional tumors. Potential functions of newly identified mutated genetic loci suggest perturbation of signaling pathways and are candidates for targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2198.

The countries of Central and Eastern Europe have among the highest worldwide rates of renal cell cancer. Few studies have examined whether genetic variation in xenobiotic metabolic pathway genes may modify risk for this cancer. The Central and Eastern Europe Renal Cell Cancer study was a hospital-based case-control study conducted between 1998 and 2003 across seven centers in central and eastern Europe. The study centers were in the Czech Republic (Ceske, Prague, Olomouc, Brno), Poland (Lodz), Romania (Bucharest), and Russia (Moscow). Incident cases of primary kidney cancer were recruited and underwent an in-person interview in which detailed data were collected on demographics as well as work history and occupational exposure to chemical agents. Genes (cytochrome P-450 family, N-acetyltransferases, NAD(P)H:quinone oxidoreductase I (NQO1), microsomal epoxide hydrolase (mEH), catechol-O-methyltransferase (COMT)) were selected for the present analysis based on their putative role in xenobiotic metabolism. Genotyping (874 cases and 2396 controls) was conducted with a GoldenGate® Oligo Pool All (OPA) assay by Illumina® and the 5’ nuclease assay (Taqman, Applied Biosystems). Haplotypes were calculated using fastPhase. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by unconditional logistic regression adjusted for country of residence, age, and sex. We observed an increased risk of renal cell cancer (RCC) with two NAT1 SNPs (NAT1A40T, OR=1.36, CI 1.00, 1.86; NAT1R187Q, OR=1.65, CI 1.01, 2.71) and with CYP1B1V432L (OR=1.14, CI 1.01, 1.28). The slow (NAT1*14) phenotype was associated with increased risk of RCC (OR=1.69, CI 1.00-2.86) in comparison to normal phenotype (NAT1*4, *3, *11). Among persons with NAT1*14 phenotype, occupational exposure to trichloroethylene (OR=9.94, CI 1.15, 86.1) and chlorinated solvents (OR=9.79, CI 1.13, 84.8) conferred an increased risk of RCC. Among those with the CYP1B1V432L variant, occupational exposure to trichloroethylene (OR=1.68, CI 1.21, 2.34) and chlorinated solvents (OR=1.58, CI 1.14, 2.19) also had increased risk of RCC. These results require replication, but provide evidence that the relationship between certain agents and RCC may be modified by particular variants in xenobiotic metabolism genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 915.

Objectives We used data from a large, population-based case–control study in Maine, New Hampshire, and Vermont to examine relationships between occupation, industry and bladder cancer risk. Methods Lifetime occupational histories were obtained by personal interview from 1158 patients newly diagnosed with urothelial carcinoma of the bladder in 2001–2004, and from 1402 population controls. Unconditional logistic regression was used to calculate ORs and 95% CIs, adjusted for demographic factors, smoking and employment in other high-risk occupations. Results Male precision metalworkers and metalworking/plasticworking machine operators had significantly elevated risks and significant trends in risk with duration of employment (precision metalworkers: OR 2.2, 95% CI 1.4 to 3.4, p trend =0.0065; metalworking/plasticworking machine operators: OR 1.6, 95% CI 1.01 to 2.6, p trend =0.047). Other occupations/industries for which risk increased significantly with duration of employment included: for men, textile machine operators, mechanics/repairers, automobile mechanics, plumbers, computer systems analysts, information clerks, and landscape industry workers; for women, service occupations, health services, cleaning and building services, management-related occupations, electronic components manufacturing and transportation equipment manufacturing. Men reporting use of metalworking fluids (MWF) had a significantly elevated bladder cancer risk (OR 1.7, 95% CI 1.1 to 2.5). Conclusions Our findings support the hypothesis that some component(s) of MWF may be carcinogenic to the bladder. Our results also corroborate many other previously reported associations between bladder cancer risk and various occupations. More detailed analyses using information from the study9s job-specific questionnaires may help to identify MWF components that may be carcinogenic, and other bladder carcinogens associated with a variety of occupations.

In the kidney vitamin D is converted to its active form. Since vitamin D exerts its activity through binding to the nuclear vitamin D receptor (VDR), most genetic studies have primarily focused on variation within this gene. Therefore, analysis of genetic variation in VDR and other vitamin D pathway genes may provide insight into the role of vitamin D in renal cell carcinoma (RCC) etiology. RCC cases (N = 777) and controls (N = 1,035) were genotyped to investigate the relationship between RCC risk and variation in eight target genes. Minimum-p-value permutation (Min-P) tests were used to identify genes associated with risk. A three single nucleotide polymorphism (SNP) sliding window was used to identify chromosomal regions with a False Discovery Rate of

© 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:176–176, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20074

It has been suggested that the indigenous Atacameño people in Northern Chile might be protected from the health effects of arsenic in drinking water because of many centuries of exposure. Here we report on the first intensive investigation of arsenic-induced skin lesions in this population. We selected 11 families (44 participants) from the village of Chiu Chiu, which is supplied with water containing between 750 and 800 microg/L inorganic arsenic. For comparison, 8 families (31 participants) were also selected from a village where the water contains approximately 10 microg/L inorganic arsenic. After being transported to the nearest city for blind assessment, participants were examined by four physicians with experience in studying arsenic-induced lesions. Four of the six men from the exposed village, who had been drinking the contaminated water for more than 20 years, were diagnosed with skin lesions due to arsenic, but none of the women had definite lesions. A 13-year-old girl had definite skin pigmentation changes due to arsenic, and a 19-year-old boy had both pigmentation changes and keratoses on the palms of his hands and the soles of his feet. Family interviews identified a wide range of fruits and vegetables consumed daily by the affected participants, as well as the weekly intake of red meat and chicken. However, the prevalence of skin lesions among men and children in the small population studied was similar to that reported with corresponding arsenic drinking water concentrations in both Taiwan and West Bengal, India--populations in which extensive malnutrition has been thought to increase susceptibility.

BACKGROUND: DNA hypomethylation has been suggested to cause genomic instability and increase cancer risk. We aimed to test the hypothesis that DNA hypomethylation is associated with increased risk of bladder cancer. METHODS: We measured cytosine methylation (5-mC) content in genomic DNA from blood cells from patients with bladder cancer enrolled in a large case-control study in Spain between Jan 1, 1998, and Dec 31, 2001. Cases were men and women with newly diagnosed and histologically confirmed urothelial carcinoma of the bladder. Controls were selected from patients admitted to the same hospital for diseases or conditions unrelated to smoking or other known risk factors for bladder cancer. Controls were individually matched to cases on age (within 5 years), sex, race, and area of hospital referral. 5-mC content was measured in leucocyte DNA by use of a combination of high-performance capillary electrophoresis, Hpa II digestion, and densitometry. Data on demographics, 34 polymorphisms in nine folate metabolism genes, and nutritional intake of six B vitamins (including folate), alcohol, and smoking were assessed as potential confounders. Relative 5-mC content was expressed as a percentage (%5-mC) with respect to the total cytosine content (the sum of methylated and non-methylated cytosines). The primary endpoint was median %5-mC DNA content. FINDINGS: %5-mC was measured in leucocyte DNA from 775 cases and 397 controls. Median %5-mC DNA was significantly lower in cases (3.03% [IQR 2.17-3.56]) than in controls (3.19% [2.46-3.68], p=0.0002). All participants were subsequently categorised into quartiles by %5-mC content in controls. When the highest quartile of %5-mC content was used as the reference category (Q4), the following adjusted odds ratios (OR) and 95% CI were recorded for decreasing methylation quartiles: OR(Q3) 2.05 (95% CI 1.37-3.06); OR(Q2) 1.62 (1.07-2.44); and OR(Q1) 2.67 (1.77-4.03), p for trend