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Background: Genomic regions identified by genome-wide association studies (GWAS) for bladder cancer risk provide new insights into etiology., Objective: To identify new susceptibility variants for bladder cancer in a meta-analysis of new and existing genome-wide genotype data., Design, Setting, and Participants: Data from 32 studies that includes 13,790 bladder cancer cases and 343,502 controls of European ancestry were used for meta-analysis., Outcome Measurements and Statistical Analyses: Log-additive associations of genetic variants were assessed using logistic regression models. A fixed-effects model was used for meta-analysis of the results. Stratified analyses were conducted to evaluate effect modification by sex and smoking status. A polygenic risk score (PRS) was generated on the basis of known and novel susceptibility variants and tested for interaction with smoking., Results and Limitations: Multiple novel bladder cancer susceptibility loci (6p.22.3, 7q36.3, 8q21.13, 9p21.3, 10q22.1, 19q13.33) as well as improved signals in three known regions (4p16.3, 5p15.33, 11p15.5) were identified, bringing the number of independent markers at genome-wide significance (p < 5 × 10 -8 ) to 24. The 4p16.3 (FGFR3/TACC3) locus was associated with a stronger risk for women than for men (p-interaction = 0.002). Bladder cancer risk was increased by interactions between smoking status and genetic variants at 8p22 (NAT2; multiplicative p value for interaction [p M-I ] = 0.004), 8q21.13 (PAG1; p M-I  = 0.01), and 9p21.3 (LOC107987026/MTAP/CDKN2A; p M-I  = 0.02). The PRS based on the 24 independent GWAS markers (odds ratio per standard deviation increase 1.49, 95% confidence interval 1.44-1.53), which also showed comparable results in two prospective cohorts (UK Biobank, PLCO trial), revealed an approximately fourfold difference in the lifetime risk of bladder cancer according to the PRS (e.g., 1st vs 10th decile) for both smokers and nonsmokers., Conclusions: We report novel loci associated with risk of bladder cancer that provide clues to its biological underpinnings. Using 24 independent markers, we constructed a PRS to stratify lifetime risk. The PRS combined with smoking history, and other established risk factors, has the potential to inform future screening efforts for bladder cancer., Patient Summary: We identified new genetic markers that provide biological insights into the genetic causes of bladder cancer. These genetic risk factors combined with lifestyle risk factors, such as smoking, may inform future preventive and screening strategies for bladder cancer., (Published by Elsevier B.V.)

Background: Bladder cancer mortality rates have been elevated in northern New England for at least five decades. Incidence rates in Maine, New Hampshire, and Vermont are about 20% higher than the United States overall. We explored reasons for this excess, focusing on arsenic in drinking water from private wells, which are particularly prevalent in the region., Methods: In a population-based case-control study in these three states, 1213 bladder cancer case patients and 1418 control subjects provided information on suspected risk factors. Log transformed arsenic concentrations were estimated by linear regression based on measurements in water samples from current and past homes. All statistical tests were two-sided., Results: Bladder cancer risk increased with increasing water intake (Ptrend = .003). This trend was statistically significant among participants with a history of private well use (Ptrend = .01). Among private well users, this trend was apparent if well water was derived exclusively from shallow dug wells (which are vulnerable to contamination from manmade sources, Ptrend = .002) but not if well water was supplied only by deeper drilled wells (Ptrend = .48). If dug wells were used pre-1960, when arsenical pesticides were widely used in the region, heavier water consumers (>2.2 L/day) had double the risk of light users (<1.1 L/day, Ptrend = .01). Among all participants, cumulative arsenic exposure from all water sources, lagged 40 years, yielded a positive risk gradient (Ptrend = .004); among the highest-exposed participants (97.5th percentile), risk was twice that of the lowest-exposure quartile (odds ratio = 2.24, 95% confidence interval = 1.29 to 3.89)., Conclusions: Our findings support an association between low-to-moderate levels of arsenic in drinking water and bladder cancer risk in New England. In addition, historical consumption of water from private wells, particularly dug wells in an era when arsenical pesticides were widely used, was associated with increased bladder cancer risk and may have contributed to the New England excess., (Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the United States.)

Candidate gene and genome-wide association studies (GWAS) have identified 15 independent genomic regions associated with bladder cancer risk. In search for additional susceptibility variants, we followed up on four promising single-nucleotide polymorphisms (SNPs) that had not achieved genome-wide significance in 6911 cases and 11 814 controls (rs6104690, rs4510656, rs5003154 and rs4907479, P < 1 × 10(-6)), using additional data from existing GWAS datasets and targeted genotyping for studies that did not have GWAS data. In a combined analysis, which included data on up to 15 058 cases and 286 270 controls, two SNPs achieved genome-wide statistical significance: rs6104690 in a gene desert at 20p12.2 (P = 2.19 × 10(-11)) and rs4907479 within the MCF2L gene at 13q34 (P = 3.3 × 10(-10)). Imputation and fine-mapping analyses were performed in these two regions for a subset of 5551 bladder cancer cases and 10 242 controls. Analyses at the 13q34 region suggest a single signal marked by rs4907479. In contrast, we detected two signals in the 20p12.2 region-the first signal is marked by rs6104690, and the second signal is marked by two moderately correlated SNPs (r(2) = 0.53), rs6108803 and the previously reported rs62185668. The second 20p12.2 signal is more strongly associated with the risk of muscle-invasive (T2-T4 stage) compared with non-muscle-invasive (Ta, T1 stage) bladder cancer (case-case P ≤ 0.02 for both rs62185668 and rs6108803). Functional analyses are needed to explore the biological mechanisms underlying these novel genetic associations with risk for bladder cancer., (Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Few studies have demonstrated gene/environment interactions in cancer research. Using data on high-risk occupations for 2258 case patients and 2410 control patients from two bladder cancer studies, we observed that three of 16 known or candidate bladder cancer susceptibility variants displayed statistically significant and consistent evidence of additive interactions; specifically, the GSTM1 deletion polymorphism (P interaction ≤ .001), rs11892031 (UGT1A, P interaction = .01), and rs798766 (TMEM129-TACC3-FGFR3, P interaction = .03). There was limited evidence for multiplicative interactions. When we examined detailed data on a prevalent occupational exposure associated with increased bladder cancer risk, straight metalworking fluids, we also observed statistically significant additive interaction for rs798766 (TMEM129-TACC3-FGFR3, P interaction = .02), with the interaction more apparent in patients with tumors positive for FGFR3 expression. All statistical tests were two-sided. The interaction we observed for rs798766 (TMEM129-TACC3-FGFR3) with specific exposure to straight metalworking fluids illustrates the value of integrating germline genetic variation, environmental exposures, and tumor marker data to provide insight into the mechanisms of bladder carcinogenesis., (Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

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. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 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 GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models., (©2014 American Association for Cancer Research.)

Bladder cancer is a complex disease with known environmental and genetic risk factors. We performed a genome-wide interaction study (GWAS) of smoking and bladder cancer risk based on primary scan data from 3002 cases and 4411 controls from the National Cancer Institute Bladder Cancer GWAS. Alternative methods were used to evaluate both additive and multiplicative interactions between individual single nucleotide polymorphisms (SNPs) and smoking exposure. SNPs with interaction P values < 5 × 10(-) (5) were evaluated further in an independent dataset of 2422 bladder cancer cases and 5751 controls. We identified 10 SNPs that showed association in a consistent manner with the initial dataset and in the combined dataset, providing evidence of interaction with tobacco use. Further, two of these novel SNPs showed strong evidence of association with bladder cancer in tobacco use subgroups that approached genome-wide significance. Specifically, rs1711973 (FOXF2) on 6p25.3 was a susceptibility SNP for never smokers [combined odds ratio (OR) = 1.34, 95% confidence interval (CI) = 1.20-1.50, P value = 5.18 × 10(-) (7)]; and rs12216499 (RSPH3-TAGAP-EZR) on 6q25.3 was a susceptibility SNP for ever smokers (combined OR = 0.75, 95% CI = 0.67-0.84, P value = 6.35 × 10(-) (7)). In our analysis of smoking and bladder cancer, the tests for multiplicative interaction seemed to more commonly identify susceptibility loci with associations in never smokers, whereas the additive interaction analysis identified more loci with associations among smokers-including the known smoking and NAT2 acetylation interaction. Our findings provide additional evidence of gene-environment interactions for tobacco and bladder cancer., (Published by Oxford University Press 2014.)

Introduction: Dysregulation of the hedgehog signaling pathway has been linked to the development and progression of a variety of different human tumors including cancers of the skin, brain, colon, prostate, blood, and pancreas. We assessed the clinicopathological factors that are potentially related to expression of Gli1, the transcription factor that is thought to be the most reliable marker of hedgehog pathway activation in bladder cancer., Methods: Bladder cancer cases were identified from the New Hampshire State Cancer Registry as histologically confirmed primary bladder cancer diagnosed between January 1, 2002, and July 31, 2004. Immunohistochemical analysis was performed on a tissue microarray to detect Gli1 and p53 expression in these bladder tumors. We computed odds ratios (ORs) and their 95% CIs for Gli1 positivity for pathological category using T category (from TNM), invasiveness, and grade with both the World Health Organization 1973 and World Health Organization International Society of Urological Pathology criteria. We calculated hazard ratios and their 95% CI for Gli1 positivity and recurrence for both Ta-category and invasive bladder tumors (T1+)., Results: A total of 194 men and 67 women, whose tumors were assessable for Gli1 staining, were included in the study. No appreciable differences in Gli1 staining were noted by sex, age, smoking status, or high-risk occupation. Ta-category tumors were more likely to stain for Gli1 as compared with T1-category tumors (adjusted OR = 0.38, CI: 0.17-0.87). Similarly, low-grade (grades 1-2) tumors were more likely to stain for Gli1 as compared with high-grade tumors (grade 3) (adjusted OR = 0.44, CI: 0.21-0.93). In a Cox proportional hazards regression analysis, non-muscle-invasive bladder tumors expressing Gli1 were less likely to recur (adjusted hazard ratio = 0.48; CI: 0.28-0.82; P<0.05) than those in which Gli1 was absent., Conclusion: Our findings indicate that Gli1 expression may be a marker of low-stage, low-grade bladder tumors and an indicator of a reduced risk of recurrence in this group., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Candidate gene and genome-wide association studies (GWAS) have identified 11 independent susceptibility loci associated with bladder cancer risk. To discover additional risk variants, we conducted a new GWAS of 2422 bladder cancer cases and 5751 controls, followed by a meta-analysis with two independently published bladder cancer GWAS, resulting in a combined analysis of 6911 cases and 11 814 controls of European descent. TaqMan genotyping of 13 promising single nucleotide polymorphisms with P < 1 × 10(-5) was pursued in a follow-up set of 801 cases and 1307 controls. Two new loci achieved genome-wide statistical significance: rs10936599 on 3q26.2 (P = 4.53 × 10(-9)) and rs907611 on 11p15.5 (P = 4.11 × 10(-8)). Two notable loci were also identified that approached genome-wide statistical significance: rs6104690 on 20p12.2 (P = 7.13 × 10(-7)) and rs4510656 on 6p22.3 (P = 6.98 × 10(-7)); these require further studies for confirmation. In conclusion, our study has identified new susceptibility alleles for bladder cancer risk that require fine-mapping and laboratory investigation, which could further understanding into the biological underpinnings of bladder carcinogenesis.

Bladder cancer results from the combined effects of environmental and genetic factors, smoking being the strongest risk factor. Evaluating absolute risks resulting from the joint effects of smoking and genetic factors is critical to assess the public health relevance of genetic information. Analyses included up to 3,942 cases and 5,680 controls of European background in seven studies. We tested for multiplicative and additive interactions between smoking and 12 susceptibility loci, individually and combined as a polygenic risk score (PRS). Thirty-year absolute risks and risk differences by levels of the PRS were estimated for U.S. males aged 50 years. Six of 12 variants showed significant additive gene-environment interactions, most notably NAT2 (P = 7 × 10(-4)) and UGT1A6 (P = 8 × 10(-4)). The 30-year absolute risk of bladder cancer in U.S. males was 6.2% for all current smokers. This risk ranged from 2.9% for current smokers in the lowest quartile of the PRS to 9.9% for current smokers in the upper quartile. Risk difference estimates indicated that 8,200 cases would be prevented if elimination of smoking occurred in 100,000 men in the upper PRS quartile compared with 2,000 cases prevented by a similar effort in the lowest PRS quartile (P(additive) = 1 × 10(-4)). Thus, the potential impact of eliminating smoking on the number of bladder cancer cases prevented is larger for individuals at higher than lower genetic risk. Our findings could have implications for targeted prevention strategies. However, other smoking-related diseases, as well as practical and ethical considerations, need to be considered before any recommendations could be made., (©2012 AACR.)

A recent genome-wide association study of bladder cancer identified the UGT1A gene cluster on chromosome 2q37.1 as a novel susceptibility locus. The UGT1A cluster encodes a family of UDP-glucuronosyltransferases (UGTs), which facilitate cellular detoxification and removal of aromatic amines. Bioactivated forms of aromatic amines found in tobacco smoke and industrial chemicals are the main risk factors for bladder cancer. The association within the UGT1A locus was detected by a single nucleotide polymorphism (SNP) rs11892031. Now, we performed detailed resequencing, imputation and genotyping in this region. We clarified the original genetic association detected by rs11892031 and identified an uncommon SNP rs17863783 that explained and strengthened the association in this region (allele frequency 0.014 in 4035 cases and 0.025 in 5284 controls, OR = 0.55, 95%CI = 0.44-0.69, P = 3.3 × 10(-7)). Rs17863783 is a synonymous coding variant Val209Val within the functional UGT1A6.1 splicing form, strongly expressed in the liver, kidney and bladder. We found the protective T allele of rs17863783 to be associated with increased mRNA expression of UGT1A6.1 in in-vitro exontrap assays and in human liver tissue samples. We suggest that rs17863783 may protect from bladder cancer by increasing the removal of carcinogens from bladder epithelium by the UGT1A6.1 protein. Our study shows an example of genetic and functional role of an uncommon protective genetic variant in a complex human disease, such as bladder cancer.

Genome-wide association studies have identified a SNP, rs2294008, on 8q24.3 within the prostate stem cell antigen (PSCA) gene, as a risk factor for bladder cancer. To fine-map this region, we imputed 642 SNPs within 100 Kb of rs2294008 in addition to 33 markers genotyped in one of the reported genome-wide association study in 8,652 subjects. A multivariable logistic regression model adjusted for rs2294008 revealed a unique signal, rs2978974 (r(2) = 0.02, D' = 0.19 with rs2294008). In the combined analysis of 5,393 cases and 7,324 controls, we detected a per-allele odds ratio (OR) = 1.11 [95% confidence interval (CI) = 1.06-1.17, P = 5.8 × 10(-5)] for rs2294008 and OR = 1.07 (95% CI = 1.02-1.13, P = 9.7 × 10(-3)) for rs2978974. The effect was stronger in carriers of both risk variants (OR = 1.24, 95% CI = 1.08-1.41, P = 1.8 × 10(-3)) and there was a significant multiplicative interaction (P = 0.035) between these two SNPs, which requires replication in future studies. The T risk allele of rs2294008 was associated with increased PSCA mRNA expression in two sets of bladder tumor samples (n = 36, P = 0.0007 and n = 34, P = 0.0054) and in normal bladder samples (n = 35, P = 0.0155), but rs2978974 was not associated with PSCA expression. SNP rs2978974 is located 10 Kb upstream of rs2294008, within an alternative untranslated first exon of PSCA. The non-risk allele G of rs2978974 showed strong interaction with nuclear proteins from five cell lines tested, implying a regulatory function. In conclusion, a joint effect of two PSCA SNPs, rs2294008 and rs2978974, suggests that both variants may be important for bladder cancer susceptibility, possibly through different mechanisms that influence the control of mRNA expression and interaction with regulatory factors.

Arsenic is a carcinogen that contaminates drinking water worldwide. Accumulating evidence suggests that both exposure and genetic factors may influence susceptibility to arsenic-induced malignancies. We sought to identify novel susceptibility loci for arsenic-related bladder cancer in a US population with low to moderate drinking water levels of arsenic. We first screened a subset of bladder cancer cases using a panel of approximately 10,000 non-synonymous single nucleotide polymorphisms (SNPs). Top ranking hits on the SNP array then were considered for further analysis in our population-based case-control study (n = 832 cases and 1,191 controls). SNPs in the fibrous sheath interacting protein 1 (FSIP1) gene (rs10152640) and the solute carrier family 39, member 2 (SLC39A2) in the ZIP gene family of metal transporters (rs2234636) were detected as potential hits in the initial scan and validated in the full case-control study. The adjusted odds ratio (OR) for the FSIP1 polymorphism was 2.57 [95% confidence interval (CI) 1.13, 5.85] for heterozygote variants (AG) and 12.20 (95% CI 2.51, 59.30) for homozygote variants (GG) compared to homozygote wild types (AA) in the high arsenic group (greater than the 90th percentile), and unrelated in the low arsenic group (equal to or below the 90th percentile) (P for interaction = 0.002). For the SLC39A2 polymorphism, the adjusted ORs were 2.96 (95% CI 1.23, 7.15) and 2.91 (95% CI 1.00, 8.52) for heterozygote (TC) and homozygote (CC) variants compared to homozygote wild types (TT), respectively, and close to one in the low arsenic group (P for interaction = 0.03). Our findings suggest novel variants that may influence risk of arsenic-associated bladder cancer and those who may be at greatest risk from this widespread exposure.

Pathway analysis of genome-wide association studies (GWAS) offer a unique opportunity to collectively evaluate genetic variants with effects that are too small to be detected individually. We applied a pathway analysis to a bladder cancer GWAS containing data from 3,532 cases and 5,120 controls of European background (n = 5 studies). Thirteen hundred and ninety-nine pathways were drawn from five publicly available resources (Biocarta, Kegg, NCI-PID, HumanCyc, and Reactome), and we constructed 22 additional candidate pathways previously hypothesized to be related to bladder cancer. In total, 1421 pathways, 5647 genes and ∼90,000 SNPs were included in our study. Logistic regression model adjusting for age, sex, study, DNA source, and smoking status was used to assess the marginal trend effect of SNPs on bladder cancer risk. Two complementary pathway-based methods (gene-set enrichment analysis [GSEA], and adapted rank-truncated product [ARTP]) were used to assess the enrichment of association signals within each pathway. Eighteen pathways were detected by either GSEA or ARTP at P≤0.01. To minimize false positives, we used the I(2) statistic to identify SNPs displaying heterogeneous effects across the five studies. After removing these SNPs, seven pathways ('Aromatic amine metabolism' [P(GSEA) = 0.0100, P(ARTP) = 0.0020], 'NAD biosynthesis' [P(GSEA) = 0.0018, P(ARTP) = 0.0086], 'NAD salvage' [P(ARTP) = 0.0068], 'Clathrin derived vesicle budding' [P(ARTP) = 0.0018], 'Lysosome vesicle biogenesis' [P(GSEA) = 0.0023, P(ARTP)<0.00012], 'Retrograde neurotrophin signaling' [P(GSEA) = 0.00840], and 'Mitotic metaphase/anaphase transition' [P(GSEA) = 0.0040]) remained. These pathways seem to belong to three fundamental cellular processes (metabolic detoxification, mitosis, and clathrin-mediated vesicles). Identification of the aromatic amine metabolism pathway provides support for the ability of this approach to identify pathways with established relevance to bladder carcinogenesis.

Aromatic amine components in hair dyes and polymorphisms in genes that encode enzymes responsible for hair dye metabolism may be related to bladder cancer risk. We evaluated the association between hair dye use and bladder cancer risk and effect modification by N-acetyltransferase-1 (NAT1), NAT2, glutathione S-transferase Mu-1 (GSTM1) and glutathione S-transferase theta-1 (GSTT1) genotypes in a population-based case-control study of 1193 incident cases and 1418 controls from Maine, Vermont and New Hampshire enrolled between 2001 and 2004. Individuals were interviewed in person using a computer-assisted personal interview to assess hair dye use and information on potential confounders and effect modifiers. No overall association between age at first use, year of first use, type of product, color, duration or number of applications of hair dyes and bladder cancer among women or men was apparent, but increased risks were observed in certain subgroups. Women who used permanent dyes and had a college degree, a marker of socioeconomic status, had an increased risk of bladder cancer [odds ratio (OR) = 3.3, 95% confidence interval (CI): 1.2-8.9]. Among these women, we found an increased risk of bladder cancer among exclusive users of permanent hair dyes who had NAT2 slow acetylation phenotype (OR = 7.3, 95% CI: 1.6-32.6) compared to never users of dye with NAT2 rapid/intermediate acetylation phenotype. Although we found no relation between hair dye use and bladder cancer risk in women overall, we detected evidence of associations and gene-environment interaction with permanent hair dye use; however, this was limited to educated women. These results need confirmation with larger numbers, requiring pooling data from multiple studies., (Copyright © 2011 UICC.)

Genome-wide and candidate-gene association studies of bladder cancer have identified 10 susceptibility loci thus far. We conducted a meta-analysis of two previously published genome-wide scans (4501 cases and 6076 controls of European background) and followed up the most significant association signals [17 single nucleotide polymorphisms (SNPs) in 10 genomic regions] in 1382 cases and 2201 controls from four studies. A combined analysis adjusted for study center, age, sex, and smoking status identified a novel susceptibility locus that mapped to a region of 18q12.3, marked by rs7238033 (P = 8.7 × 10(-9); allelic odds ratio 1.20 with 95% CI: 1.13-1.28) and two highly correlated SNPs, rs10775480/rs10853535 (r(2)= 1.00; P = 8.9 × 10(-9); allelic odds ratio 1.16 with 95% CI: 1.10-1.22). The signal localizes to the solute carrier family 14 member 1 gene, SLC14A1, a urea transporter that regulates cellular osmotic pressure. In the kidney, SLC14A1 regulates urine volume and concentration whereas in erythrocytes it determines the Kidd blood groups. Our findings suggest that genetic variation in SLC14A1 could provide new etiological insights into bladder carcinogenesis.

Background: Diabetes is an emerging public health issue in the US, affecting 11% of Americans over the age of 20, with long-term complications that include cardiovascular disease, retinopathy, neuropathy, and nephropathy. A recent meta-analysis found that bladder cancer incidence was approximately 40% higher in individuals with diabetes; however, few studies considered duration or type of therapy and had limited adjustment for potentially confounding factors., Methods: To further investigate the potential role of diabetes in risk of bladder cancer, the authors analyzed data from a case-control study conducted in New Hampshire in which patients with bladder cancer, and controls sampled from the population, completed an interview regarding history of diabetes, cigarette smoking history, height, weight, and history of urinary tract infections., Results: The study consisted of 331 cases and 263 controls on whom information regarding diabetes was ascertained. History of diabetes was related to an increased bladder cancer risk (adjusted odds ratio = 2.2, 95% CI, 1.3 to 3.8). The association was strongest in those who had diabetes for the longest duration (OR for 16 or more years = 3.6, 1.1 to 11.2) and in those taking oral hypoglycemic medications (OR = 3.3, 1.5 to 7.1)., Conclusions: Our findings support an association between bladder cancer incidence and diabetes, and further suggest that the risk may be greater among patients taking oral hypoglycemics and those with diabetes of longer duration., (Copyright © 2010 American Cancer Society.)

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 study's job-specific questionnaires may help to identify MWF components that may be carcinogenic, and other bladder carcinogens associated with a variety of occupations.

Introduction: Incidence rates of bladder cancer are notably higher in men than in women. While there is evidence that reproductive and hormonal risk factors may influence risk of bladder cancer, data are inconclusive., Materials and Methods: We examined reproductive, menstrual and hormonal use history in our population-based case-control study of bladder cancer in New Hampshire (NH), USA (n=207 women cases and n=463 women controls). Additionally, we performed a meta-analysis of the published literature. We used unconditional logistic regression analysis to compute adjusted odds ratios associated with each risk factor in the NH study. We combined these estimates with those from the published literature using inverse variance effects models., Results: In the NH study, a slightly decreased odds ratio was found among women who had ever had a birth compared to nulliparous women and an elevated odds ratio among women who underwent surgical menopause (bilateral oophorectomy), especially at an early age. No overall associations were found with oral contraceptive use or hormone replacement therapy. These findings were generally in agreement with the meta-analytic results for which the combined relative risk (RR) estimate was reduced among ever parous women (combined RR estimate for ever parous versus nulliparous=0.66, 95% confidence intervals [95% CI] 0.55-0.79) and elevated among those undergoing an early menopause (combined RR estimate for early versus late menopause=1.59, 95% CI 1.31-1.92). No consistent risk was observed for the other factors., Discussion: Some reproductive and menstrual factors appear to be related to the incidence of bladder cancer among women; but whether effects are due to female hormones is uncertain., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case-control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the 'null' genotype) were 1.26 (0.85-1.88) and 1.54 (1.05-2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71-1.51), former (0.95; 0.75-1.20) or current smokers (1.33; 0.91-1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14-2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22-8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

We conducted a multi-stage, genome-wide association study of bladder cancer with a primary scan of 591,637 SNPs in 3,532 affected individuals (cases) and 5,120 controls of European descent from five studies followed by a replication strategy, which included 8,382 cases and 48,275 controls from 16 studies. In a combined analysis, we identified three new regions associated with bladder cancer on chromosomes 22q13.1, 19q12 and 2q37.1: rs1014971, (P = 8 × 10⁻¹²) maps to a non-genic region of chromosome 22q13.1, rs8102137 (P = 2 × 10⁻¹¹) on 19q12 maps to CCNE1 and rs11892031 (P = 1 × 10⁻⁷) maps to the UGT1A cluster on 2q37.1. We confirmed four previously identified genome-wide associations on chromosomes 3q28, 4p16.3, 8q24.21 and 8q24.3, validated previous candidate associations for the GSTM1 deletion (P = 4 × 10⁻¹¹) and a tag SNP for NAT2 acetylation status (P = 4 × 10⁻¹¹), and found interactions with smoking in both regions. Our findings on common variants associated with bladder cancer risk should provide new insights into the mechanisms of carcinogenesis.

DNA methylation profiles can be used to define molecular cancer subtypes that may better inform disease etiology and clinical decision-making. This investigation aimed to create DNA methylation profiles of bladder cancer based on CpG methylation from almost 800 cancer-related genes and to then examine the relationship of those profiles with exposures related to risk and clinical characteristics. DNA, derived from formalin-fixed paraffin-embedded tumor samples obtained from incident cases involved in a population-based case-control study of bladder cancer in New Hampshire, was used for methylation profiling on the Illumina GoldenGate Methylation Bead Array. Unsupervised clustering of those loci with the greatest change in methylation between tumor and non-diseased tissue was performed to defined molecular subgroups of disease, and univariate tests of association followed by multinomial logistic regression was used to examine the association between these classes, bladder cancer risk factors and clinical phenotypes. Membership in the two most methylated classes was significantly associated with invasive disease (P < 0.001 for both class 3 and 4). Male gender (P = 0.04) and age >70 years (P = 0.05) was associated with membership in one of the most methylated classes. Finally, average water arsenic levels in the highest percentile predicted membership in an intermediately methylated class of tumors (P = 0.02 for both classes). Exposures and demographic associated with increased risk of bladder cancer specifically associate with particular subgroups of tumors defined by DNA methylation profiling and these subgroups may define more aggressive disease.

Objective: Although the effect of fruit and vegetables on the risk of bladder cancer has been widely studied, little is known about their micronutrient components. Our aim was to investigate associations between minerals and vitamins and bladder cancer., Methods: A case-control study was conducted in New Hampshire, USA. Dietary data were collected from 322 cases and 239 controls using a 121-item food frequency questionnaire. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression adjusting for sex, age, smoking characteristics, and energy intake., Results: The ORs (95% CI) for highest quartile versus lowest quartile for total intake of vitamin E was 0.66 (0.36-1.20; p trend = 0.09) and 0.49 (0.21-1.17; p trend = 0.13) for dietary phosphorus. The odds of bladder cancer for heavy smokers with the highest total intake of vitamin E, carotenoids, and niacin were 0.58 (0.34-0.99), 0.62 (0.36-1.09), and 0.66 (0.39-1.14), respectively. Higher total intakes of carotenoids, vitamin D, thiamin, niacin, and vitamin E were inversely related to bladder cancer risk among older individuals., Conclusion: Our findings suggest further investigation of the effect of vitamin E, carotenoids, vitamin D, thiamin, and niacin on bladder cancer risk may be warranted. Future studies should focus on high risk groups such as heavy smokers and older individuals.

Background: Cigarette smoking is a well-established risk factor for bladder cancer. The effects of smoking duration, intensity (cigarettes per day), and total exposure (pack-years); smoking cessation; exposure to environmental tobacco smoke; and changes in the composition of tobacco and cigarette design over time on risk of bladder cancer are unclear., Methods: We examined bladder cancer risk in relation to smoking practices based on interview data from a large, population-based case-control study conducted in Maine, New Hampshire, and Vermont from 2001 to 2004 (N = 1170 urothelial carcinoma case patients and 1413 control subjects). We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression. To examine changes in smoking-induced bladder cancer risk over time, we compared odds ratios from New Hampshire residents in this study (305 case patients and 335 control subjects) with those from two case-control studies conducted in New Hampshire in 1994-1998 and in 1998-2001 (843 case patients and 1183 control subjects)., Results: Regular and current cigarette smokers had higher risks of bladder cancer than never-smokers (for regular smokers, OR = 3.0, 95% CI = 2.4 to 3.6; for current smokers, OR = 5.2, 95% CI = 4.0 to 6.6). In New Hampshire, there was a statistically significant increasing trend in smoking-related bladder cancer risk over three consecutive periods (1994-1998, 1998-2001, and 2002-2004) among former smokers (OR = 1.4, 95% CI = 1.0 to 2.0; OR = 2.0, 95% CI = 1.4 to 2.9; and OR = 2.6, 95% CI = 1.7 to 4.0, respectively) and current smokers (OR = 2.9, 95% CI = 2.0 to 4.2; OR = 4.2, 95% CI = 2.8 to 6.3; OR = 5.5, 95% CI = 3.5 to 8.9, respectively) (P for homogeneity of trends over time periods = .04). We also observed that within categories of intensity, odds ratios increased approximately linearly with increasing pack-years smoked, but the slope of the increasing trend declined with increasing intensity., Conclusions: Smoking-related risks of bladder cancer appear to have increased in New Hampshire since the mid-1990s. Based on our modeling of pack-years and intensity, smoking fewer cigarettes over a long time appears more harmful than smoking more cigarettes over a shorter time, for equal total pack-years of cigarettes smoked.

Background: Use of immunosuppressive drugs post organ transplantation, and prolonged use of glucorticoids for other conditions have been associated with subsequent risk of certain malignancies, that is, skin cancers and lymphoma. There is evidence that the incidence of bladder cancer is also elevated among organ transplant recipients, however, it is unknown whether other groups of patients, that is, those taking oral glucocorticoids, likewise are at an increased risk., Methods: In a population-based case-control study in New Hampshire, USA, we compared the use of glucocorticoids in 786 bladder cancer cases and in 1083 controls. We used unconditional logistic regression analysis to compute adjusted odds ratios (ORs) associated with oral glucocorticoid use., Results: In our analysis, the risk of bladder cancer was related to a history of prolonged oral glucocorticoid use (OR=1.85, 95% CI=1.24-2.76, adjusted for age, gender and smoking). Associations with oral glucocorticoid use were stronger for invasive tumours (OR=2.12, 95% CI=1.17-3.85) and tumours with high (3+) p53 staining intensity (OR=2.35, 95% CI=1.26-4.36)., Conclusion: Our results raise the possibility of an increased risk of bladder cancer from systemic use of glucocorticoids, and a potential role of immune surveillance in bladder cancer aetiology.

Emerging evidence indicates a potential role of selenium in the prevention of several types of cancer, including bladder cancer. We investigated the association between toenail selenium concentrations and bladder cancer risk in a population-based case-control study in New Hampshire. We analyzed data from 857 incidence cases diagnosed between July 1, 1994 and June 30, 2001 and 1,191 general population controls. Newly diagnosed cases of bladder cancer were identified from the New Hampshire State Cancer Registry, which operates a rapid reporting system. Controls were selected from population lists (driver's license and Medicare enrollment). We used logistic regression analyses to generate odds ratios (OR) and 95% confidence intervals (95% CI), controlling for age, sex, and pack-years of smoking and conducted separate analyses according to the intensity of p53 immunohistochemical staining of the tumor. Overall, toenail selenium concentrations were not significantly related to bladder cancer [OR Q4 versus Q1, 0.90 (95% CI, 0.68-1.19); P(trend) = 0.15]. However, within specific subgroups there were inverse associations, i.e., among moderate smokers [OR, 0.61 (95% CI, 0.39-0.96); P(trend) = 0.004], women [OR, 0.66 (95% CI, 0.40-1.10); P(trend) = 0.11], and those with p53-positive cancers [OR Q4 versus Q1, 0.57 (95% CI, 0.34-0.94); P(trend) = 0.01]. Our results indicate that selenium is not inversely related to risk of bladder cancer overall; however, they raise the possibility that selenium may be preventive in certain molecular phenotypes of tumors (e.g., p53 positive) or within certain subsets of a population (e.g., women or moderate smokers).

Background: Use of phenacetin and other analgesic and non-steroidal anti-inflammatory drugs (NSAIDs) potentially influences bladder cancer incidence, but epidemiologic evidence is limited., Methods: We analyzed data from 376 incident bladder cancer cases and 463 controls from a population-based case-control study in New Hampshire on whom regular use of analgesic drugs and NSAIDs was obtained. Odds ratios and 95% confidence intervals were computed using logistic regression with adjustment for potentially confounding factors. Separate models by tumor stage, grade and TP53 status were conducted., Results: We found an elevated odds ratio (OR) associated with reported use of phenacetin-containing medications, especially with longer duration of use (OR >8 years = 3.00, 95% confidence interval (CI) = 1.4-6.5). In contrast, use of paracetamol did not relate overall to risk of bladder cancer. We also found that regular use of any NSAID was associated with a statistically significant decrease in bladder cancer risk (OR = 0.6, 95% CI = 0.4-0.9), and specifically use of aspirin. Further, the association with NSAID use was largely among invasive, high grade and TP53 positive tumors., Conclusion: While these agents have been investigated in several studies, a number of questions remain regarding the effects of analgesic and NSAID use on risk of bladder cancer.

Tobacco smoking, certain occupational exposures, and exposure to inorganic arsenic in drinking water have been associated with the occurrence of bladder cancer. However, in these tumors the exposure-associated pattern of somatic alterations in genes in the causal pathway for disease has been poorly characterized. Animal and in vitro studies have suggested that arsenic, tobacco carcinogens, and other exposures may act through epigenetic mechanisms. We, therefore, examined, in a population-based study of human bladder cancer (n = 351), the relationship between epigenetic silencing of the tumor-suppressor genes, p16(INK4A), RASSF1A, PRSS3, and the four SFRP genes and exposure to both tobacco and arsenic in bladder cancer. Promotor methylation silencing of each of these genes occurred in approximately 30-50% of bladder cancers. Epigenetic silencing of RASSF1A and PRSS3 and any of the SFRP genes were each significantly associated with advanced tumor stage (P < 0.001, P < 0.04, and P < 0.005, respectively). Arsenic exposure, measured as toenail arsenic, was associated with RASSF1A (P < 0.02) and PRSS3 (P < 0.1) but not p16(INK4A) or SFRP promotor methylation, in models adjusted for stage and other risk factors. Cigarette smoking was associated with a greater than twofold increased risk of promotor methylation of the p16(INK4A) gene, with greater risk seen in patients with exposures more recent to disease diagnosis, and smoking was also significantly associated with any SFRP gene methylation (P < 0.01). These results from human bladder tumors, add to the body of animal and in vitro evidence that suggests bladder carcinogens play a crucial role in the induction of important epigenetic alterations.

Tobacco smoking, certain occupational exposures, and exposure to inorganic arsenic in drinking water have been associated with the occurrence of bladder cancer. However, in these tumors the exposure-associated pattern of somatic alterations in genes in the causal pathway for disease has been poorly characterized. In particular, the mechanism by which arsenic induces bladder cancer and the effects of lower environmental levels of exposure remain uncertain. Animal and in-vitro studies have suggested that arsenic and other exposures may act through epigenetic mechanisms. We, therefore, examined, in a population-based study of human bladder cancer, the relationship between epigenetic silencing of three tumor suppressor genes, p16(INK4A), RASSF1A and PRSS3, and exposure to both tobacco and arsenic in bladder cancer. Promoter methylation of each of these genes occurred in approximately 30% of bladder cancers, and both RASSF1A and PRSS3 promoter methylation were associated with advanced tumor stage (P<0.001 and P<0.04, respectively). Arsenic exposure, measured as toenail arsenic, was associated with RASSF1A (P<0.02) and PRSS3 (P<0.1) but not p16INK4A promoter methylation, in models adjusted for stage and other factors. Cigarette smoking was associated with a >2-fold increased risk of promoter methylation of the p16INK4A gene only, with greater risk seen in patients with exposures more recent to disease diagnosis. These results, from human bladder tumors, add to the body of animal and in vitro evidence that suggests a role in epigenetic alterations for bladder carcinogens.

The molecular pathology of bladder cancer has been the subject of considerable interest, and current efforts are targeted toward elucidating the interrelationships between individual somatic gene loss and both etiologic and prognostic factors. Mutation of the TP53 gene has been associated with more invasive bladder cancer, and evidence suggests that TP53 mutation, independent of stage, may be predictive of outcome in this disease. However, there is no consensus in the literature that bladder carcinogen exposure is associated with inactivation of the TP53 gene. Work to date has been primarily hospital based and, as such, subject to possible bias associated with selection of more advanced cases for study. We examined exposure relationships with both TP53 gene mutation and TP53 protein alterations in a population-based study of 330 bladder cancer cases in New Hampshire. Tobacco smoking was not associated with TP53 alterations. We found a higher prevalence of TP53 inactivation (i.e., mutation and nuclear accumulation) among hair dye users (odd ratio [OR] = 4.1; 95% confidence interval [CI] 1.2-14.7), and the majority of these mutations were transversions. Men who had "at risk" occupations were more likely to have mutated TP53 tumors (OR = 2.9; 95% CI 1.1-7.6). There also was a relative absence of TP53 mutation (OR = 0.4; 95% CI 0.0-2.9) and TP53 protein alterations (OR = 0.6; 95% CI 0.3-1.4) in bladder cancers from individuals with higher arsenic exposure. Our data suggest that there is exposure-specific heterogeneity in inactivation of the TP53 pathway in bladder cancers and that integration of the spectrum of pathway alterations in population-based approaches (capturing the full range of exposures to bladder carcinogens) may provide important insights into bladder tumorigenesis., ((c) 2005 Wiley-Liss, Inc.)

Background: Somatic loss of the 9q allele as well as alteration of the tumor suppressor p53 occurs commonly in bladder cancers. Although alteration of p53 has been strongly associated with invasive stage disease, the prognostic significance of 9q loss of heterozygosity (LOH) and the relations between these alterations are less well defined., Methods: The 9q LOH was examined at five microsatellites and p53 alterations (mutation and persistent immunohistochemical staining) in a population-based case series of 271 newly diagnosed bladder cancer patients. Loss of heterozygosity was scored quantitatively and p53 mutation completed using single-strand conformation polymorphism screening followed by sequencing., Results: Overall, allelic loss at 9q was detected in 74.5% (202/271) of cases and allele loss was associated with invasive disease (P < 0.05). Although based on small numbers, all nine in situ lesions contained 9q LOH. Age, gender, and smoking were not significantly associated with chromosome 9q allele loss. Both intense persistent p53 staining and LOH at 9q were independently associated with invasive disease (P < 10(-14) and P < 0.05, respectively)., Conclusions: These data, using a population-based sample, suggest a relation between 9q LOH and invasive stage bladder cancer, and thereby suggests that a tumor suppressor gene at this loci, in addition to p53, may be important in the development of this more aggressive form of the disease., ((c) 2005 American Cancer Society.)

Functional variants in the methylenetetrahydrofolate reductase (MTHFR) gene, including the 677C>T and 1298A>C polymorphisms, have been associated with a moderately reduced risk of several cancers, including colorectal cancers. While recent studies have investigated the role of these polymorphisms on bladder cancer susceptibility, results have been mixed. To clarify the role of MTHFR polymorphisms on bladder cancer risk, we genotyped MTHFR 677C > T and MTHFR 1298A > C in a population-based study of bladder cancer of 352 patients and 551 controls from New Hampshire, USA. The allelic frequency was 35.6% for MTHFR 677C>T and 40.4% for MTHFR 1298A > C among controls. We found no evidence of a main gene effect for either polymorphism (adjusted OR for MTHFR 677C>T variants versus the reference genotype = 1.1; 95% CI, 0.8-1.4 and adjusted OR for MTHFR 1298A>C variants versus the reference genotype = 1.0; 95% CI, 0.7-1.4). Odds ratios did not appear to differ by smoking status or gender. We observed differences in the risk estimates for the MTHFR polymorphisms by arsenic exposure, but they were not statistically significant (P = 0.67 for MTHFR 677C > T and P = 0.12 for MTHFR 1298A>C). Thus, our findings do not support the presence of a main gene effect. The possibility that MTHFR polymorphism affects susceptibility to environmental exposures warrants further consideration.

Objective: Arsenic is a known bladder carcinogen and populations exposed to high arsenic levels in their water supply have reported elevated bladder cancer mortality and incidence rates. To examine the effects of lower levels of arsenic exposure on bladder cancer incidence, we conducted a case-control study in New Hampshire, USA where levels above 10 micro/l are commonly found in private wells., Methods: We studied 383 cases of transitional cell carcinoma of the bladder cancer, newly diagnosed between July 1, 1994 and June 30, 1998 and 641 general population controls. Individual exposure to arsenic was determined in toenail clippings using instrumental neutron activation analysis., Results: Among smokers, an elevated odds ratio (OR) for bladder cancer was observed for the uppermost category of arsenic (OR: 2.17, 95% CI: 0.92-5.11 for greater than 0.330 mcg/g compared to less than 0.06 micro/g). Among never smokers, there was no association between arsenic and bladder cancer risk., Conclusions: These, and other data, suggest that ingestion of low to moderate arsenic levels may affect bladder cancer incidence, and that cigarette smoking may act as a co-carcinogen., (Copyright 2004 Kluwer Academic Publishers)

The molecular pathology of bladder cancer has been the subject of considerable interest and mutation of the p53 gene, which has been associated with more invasive bladder cancer, has been widely studied. Further, there is evidence that p53 inactivation (either mutation or protein dysregulation), independent of stage, may be predictive of bladder cancer progression. In an effort to avoid possible biases associated with selection of more advanced cases, we examined p53 inactivation in a population-based study of bladder cancer in New Hampshire, using both mutation and immunohistochemical methods. We found the overall prevalence of mutation to be approximately 10%, while immunohistochemical analysis suggests that approximately 66% of the tumours have dysregulated p53 at the protein level. There was a significant association of mutation with persistent p53 staining, but there remained a marked number of tumours discordant for mutation and aberrant p53 immunohistochemistry. Based upon immunohistochemical staining alone, intensity rather than extent of p53 staining was more strongly related to tumour invasiveness. Additionally, all tumours with a mutation in exon 8 stained intensely. Taken together, this suggests that intense staining represents a distinct phenotype of dysfunctional protein. Our data indicate that population-based approaches to somatic alteration of p53 in bladder cancer are crucial to understanding the relationship of p53 changes to aetiology and the outcome of this disease, and further suggest that the pattern of immunohistochemical staining may represent distinct, discernible phenotypes. British Journal of Cancer (2004) 90, 1572-1576. doi:10.1038/sj.bjc.6601748 www.bjcancer.com Published online 6 April 2004

Verrucous carcinoma of the bladder unassociated with bilharzial cystitis is rare with only 6 cases reported to date. Verrucous carcinoma of the bladder, like that involving the cervix, vagina, oral cavity, perineum and lower extremities, may invade surrounding structures but remain surgically curable. Although it is similar to condyloma acuminatum, verrucous carcinoma is considered malignant because of its invasive growth pattern and it should be treated accordingly. However, no evidence of metastases has been noted to date. To our knowledge we report the first case of extravesical extension of verrucous carcinoma of the bladder that was treated successfully by surgical removal.

Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively). We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245) through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.