Adonina Tardón

NAT2 slow acetylation, GSTM1 null genotype, and risk of bladder cancer: results from the Spanish Bladder Cancer Study and meta-analyses

BACKGROUND Many reported associations between common genetic polymorphisms and complex diseases have not been confirmed in subsequent studies. An exception could be the association between NAT2 slow acetylation, GSTM1 null genotype, and bladder-cancer risk. However, current evidence is based on meta-analyses of relatively small studies (range 23-374 cases) with some evidence of publication bias and study heterogeneity. Associations between polymorphisms in other NAT and GST genes and bladder-cancer risk have been inconsistent. METHODS We investigated polymorphisms in NAT2, GSTM1, NAT1, GSTT1, GSTM3, and GSTP1 in 1150 patients with transitional-cell carcinoma of the urinary bladder and 1149 controls in Spain; all the participants were white. We also carried out meta-analyses of NAT2, GSTM1, and bladder cancer that included more than twice as many cases as in previous reports. FINDINGS In our study, the odds ratios for bladder cancer for individuals with deletion of one or two copies of the GSTM1 gene were 1.2 (95% CI 0.8-1.7) and 1.9 (1.4-2.7) respectively (p for trend <0.0001). Compared with NAT2 rapid or intermediate acetylators, NAT2 slow acetylators had an increased overall risk of bladder cancer (1.4 [1.2-1.7]) that was stronger for cigarette smokers than for never smokers (p for interaction 0.008). No significant associations were found with the other polymorphisms. Meta-analyses showed that the overall association for NAT2 was robust (p<0.0001), and case-only meta-analyses provided support for an interaction between NAT2 and smoking (p for interaction 0.009). The overall association for GSTM1 was also robust (p<0.0001) and was not modified by smoking status (p=0.86). INTERPRETATION The GSTM1 null genotype increases the overall risk of bladder cancer, and the NAT2 slow-acetylator genotype increases risk particularly among cigarette smokers. These findings provide compelling evidence for the role of common polymorphisms in the aetiology of cancer. RELEVANCE TO PRACTICE Although the relative risks are modest, these polymorphisms could account for up to 31% of bladder cancers because of their high prevalence.

Prospective Study of FGFR3 Mutations As a Prognostic Factor in Nonmuscle Invasive Urothelial Bladder Carcinomas

PURPOSE To determine the frequency and the prognostic value of fibroblast growth factor receptor 3 (FGFR3) mutations in patients with nonmuscle invasive bladder tumors according to tumor stage and grade. PATIENTS AND METHODS Seven hundred seventy-two patients with newly diagnosed bladder tumors were recruited. Tumors were reviewed by expert pathologists. Patients were prospectively followed-up (median, 62.6 months for disease-free patients) through review of hospital records and telephone interviews. The sequence of exons 7 and 10 of FGFR3 was analyzed by polymerase chain reaction and direct sequencing. We assessed the association of mutations with stage and grade. The predictive value of mutations for recurrence, progression, and mortality were assessed using Kaplan-Meier and Cox multivariable models. RESULTS Mutations were more common among low malignant potential neoplasms (LMPN; 77%) and TaG1/TaG2 tumors (61%/58%) than among TaG3 tumors (34%) and T1G3 tumors (17%). The S249C, Y375C, S248C, and G372C mutations accounted for 91.5% of all sequence changes. The A393E substitution was associated with LMPN (P < .001). The F386L polymorphism was more frequent among patients with low-grade tumors (odds ratio, 6.97; 95%CI, 1.40 to 47.06; P = .009). In the multivariable analysis of all superficial tumors, mutations were associated with increased risk of recurrence. However, in the stratified analyses only patients with TaG1 tumors had a significantly higher risk of recurrence (hazard ratio, 2.12; 95%CI, 1.28 to 3.53; P = .004). CONCLUSION The findings of this large study strongly support the notion that FGFR3 mutations characterize a subgroup of bladder cancers with good prognosis; patients with mutant TaG1 tumors have a higher risk of recurrence; and the F386L variant is selectively associated with low-grade tumors.

Urinary concentrations of phthalates and phenols in a population of Spanish pregnant women and children

BACKGROUND Phthalate and phenol exposure is prevalent among the general population and of potential concern for pregnant women and children because of their suspected susceptibility to endocrine effects. OBJECTIVES To evaluate the extent of exposure to several phthalates and phenols in a sample of Spanish pregnant women - according to their individual characteristics (age, social class, education, and body mass index) - and children who participated in the INMA - Infancia y Medio Ambiente (Environment and Childhood) project. METHODS One spot urine sample was taken during the third trimester of pregnancy from 120 pregnant women and from 30 4-year old children belonging to 5 Spanish birth cohorts, and analyzed for 11 phthalate metabolites and 9 phenols. RESULTS Three metabolites of di(2-ethylhexyl) phthalate, mono-2-ethyl-5-carboxypentyl phthalate, mono-2-ethyl-5-hydroxyhexyl phthalate, and mono-2-ethyl-5-oxohexyl phthalate; two metabolites of dibutyl phthalates, mono-isobutyl phthalate and mono-n-butyl phthalate; monoethyl phthalate (MEP), the main metabolite of diethyl phthalate; and two phenols, methyl paraben (M-PB) and 2,5-dichlorophenol were detected in the urine samples of all women. The highest urinary concentrations were for MEP and M-PB. Urinary concentrations of all phthalate metabolites and of 2,4-dichlorophenol, 2,5-dichlorophenol, and bisphenol A were lower in the pregnant women than in the children. Among women, a positive relationship with social class and education was shown for most of the phthalate metabolites and phenols. Almost all phthalate metabolites varied by region even after adjusting for social class and education. CONCLUSIONS Phthalate and phenol exposures are prevalent in a group of pregnant women and young children, two susceptible populations, and these exposures might be positively related to social class.

Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study.

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 <0.0001. The lowest cancer risk was noted in never smokers in the highest methylation quartile (never smokers in Q4). By comparison with never smokers in the highest quartile, current smokers in the lowest methylation quartile had the highest risk of bladder cancer (Q1: OR 25.51 [9.61-67.76], p for interaction 0.06). In analyses stratified by smoking, hypomethylation was a strong risk factor in never smokers (OR 6.39 [2.37-17.22]). Amount of methylation in controls were not associated with baseline characteristics, micronutrients, or selected genotypes in folate metabolism pathways. INTERPRETATION For the first time, to our knowledge, we have shown in a large case-control study that leucocyte DNA hypomethylation is associated with increased risk of developing bladder cancer, and this association is independent of smoking and the other assessed risk factors. Amount of global methylation in genomic DNA could provide a useful biomarker of susceptibility to certain cancer types and further research is warranted.

PIK3CA Mutations Are an Early Genetic Alteration Associated with FGFR3 Mutations in Superficial Papillary Bladder Tumors

Bladder tumors constitute a very heterogeneous disease. Superficial tumors are characterized by a high prevalence of FGFR3 mutations and chromosome 9 alterations. High-grade and muscle-invasive tumors are characterized by Tp53 mutations and aneuploidy. We have analyzed the sequence of exons 9 and 20 of PIK3CA in a panel of bladder tumors covering the whole spectrum of the disease. DNA from formalin-fixed, paraffin-embedded tumor sections was amplified by PCR and products were sequenced. In an unselected panel of tumors representative of the disease, the PIK3CA mutation prevalence was 13% (11 of 87). Mutations occurred mainly at the previously identified hotspots (codons 542, 545, 1007, and 1047). The distribution according to stage was as follows: papillary urothelial neoplasms of uncertain malignant potential (PUNLMP; 11 of 43, 25.6%), T(a) (9 of 57, 16%), T(1) (2 of 10, 20%), and muscle-invasive tumors (0 of 20, 0%; P = 0.019). Mutations were associated with low-grade tumors: grade 1 (6 of 27, 22.2%), grade 2 (3 of 23, 13%), and grade 3 (2 of 37, 5.4%; P = 0.047). Overall, PIK3CA mutations were strongly associated with FGFR3 mutations: 18 of 69 (26%) FGFR3(mut) tumors were PIK3CA(mut), versus 4 of 58 (6.9%) FGFR3(wt) tumors (P = 0.005). Our findings indicate that PIK3CA mutations are a common event that can occur early in bladder carcinogenesis and support the notion that papillary and muscle-invasive tumors arise through different molecular pathways. PIK3CA may constitute a novel diagnostic and prognostic tool, as well as a therapeutic target, in bladder cancer.

Smoking and Bladder Cancer in Spain: Effects of Tobacco Type, Timing, Environmental Tobacco Smoke, and Gender

We examined the effects of dose, type of tobacco, cessation, inhalation, and environmental tobacco smoke exposure on bladder cancer risk among 1,219 patients with newly diagnosed bladder cancer and 1,271 controls recruited from 18 hospitals in Spain. We used unconditional logistic regression to estimate odds ratios (OR) and 95% confidence intervals (95% CI) for the association between bladder cancer risk and various characteristics of cigarette smoking. Current smokers (men: OR, 7.4; 95% CI, 5.3-10.4; women: OR, 5.1; 95% CI, 1.6-16.4) and former smokers (men: OR, 3.8; 95% CI, 2.8-5.3; women: OR, 1.8; 95% CI, 0.5-7.2) had significantly increased risks of bladder cancer compared with nonsmokers. We observed a significant positive trend in risk with increasing duration and amount smoked. After adjustment for duration, risk was only 40% higher in smokers of black tobacco than that in smokers of blond tobacco (OR, 1.4; 95% CI, 0.98-2.0). Compared with risk in current smokers, a significant inverse trend in risk with increasing time since quitting smoking blond tobacco was observed (≥20 years cessation: OR, 0.2; 95% CI, 0.1-0.9). No trend in risk with cessation of smoking black tobacco was apparent. Compared with men who inhaled into the mouth, risk increased for men who inhaled into the throat (OR, 1.7; 95% CI, 1.1-2.6) and chest (OR, 1.5; 95% CI, 1.1-2.1). Cumulative occupational exposure to environmental tobacco smoke seemed to confer increased risk among female nonsmokers but not among male nonsmokers. After eliminating the effect of cigarette smoking on bladder cancer risk in our study population, the male-to-female incidence ratio decreased from 8.2 to 1.7, suggesting that nearly the entire male excess of bladder cancer observed in Spain is explained by cigarette smoking rather than occupational/environmental exposures to other bladder carcinogens. (Cancer Epidemiol Biomarkers Prev 2006;15(7):1348–54)

Genetic Variation in the Nucleotide Excision Repair Pathway and Bladder Cancer Risk

Nucleotide excision repair (NER) is critical for protecting against damage from carcinogens in tobacco smoke. We evaluated the influence of common genetic variation in the NER pathway on bladder cancer risk by analyzing 22 single nucleotide polymorphisms (SNP) in seven NER genes (XPC, RAD23B, ERCC1, ERCC2, ERCC4, ERCC5, and ERCC6). Our study population included 1,150 patients with transitional cell carcinoma of the urinary bladder and 1,149 control subjects from Spain. Odds ratios (OR) and 95% confidence intervals (95% CI) were adjusted for age, gender, region, and smoking status. Subjects with the variant genotypes for SNPs in four of the seven genes evaluated had small increases in bladder cancer risk compared to subjects with the homozygous wild-type genotypes: RAD23B IVS5-15A>G (OR, 1.3; 95% CI, 1.1-1.5; P = 0.01), ERCC2 R156R (OR, 1.3; 95% CI, 1.1-1.6; P = 0.006), ERCC1 IVS5+33A>C (OR, 1.2; 95% CI, 1.0-1.5; P = 0.06; Ptrend = 0.04), and ERCC5 M254V (OR, 1.4; 95% CI, 1.0-2.0; P = 0.04). A global test for pathway effects indicated that genetic variation in NER characterized by the 22 SNPs analyzed in this study significantly predicts bladder cancer risk (P = 0.04). Pairwise comparisons suggested that carrying variants in two genes could result in substantial increases in risk. Classification tree analyses suggested the presence of subgroups of individuals defined by smoking and NER genotypes that could have substantial increases in risk. In conclusion, these findings provide support for the influence of genetic variation in NER on bladder cancer risk. A detailed characterization of genetic variation in key NER genes is warranted and might ultimately help identify multiple susceptibility variants that could be responsible for substantial joint increases in risk. (Cancer Epidemiol Biomarkers Prev 2006;15(3):536–42)

Maternal vitamin D status in pregnancy and risk of lower respiratory tract infections, wheezing, and asthma in offspring.

Background: Adequate vitamin D status in mothers during pregnancy may influence the health status of the child later in life. We assessed whether maternal circulating 25-hydroxyvitamin D (25[OH]D) concentrations in pregnancy are associated with risk of lower respiratory tract infections, wheezing, and asthma in the offspring. Methods: Data were obtained from 1724 children of the INfancia y Medio Ambiente (INMA) Project, a population-based birth cohort study. Maternal circulating 25(OH)D concentrations were measured in pregnancy (mean gestational age = 12.6 [SD = 2.5] weeks). When the child was age 1 year, parents were asked if their child had a physician-confirmed history of lower respiratory tract infections or a history of wheezing. The questions about wheezing were repeated annually thereafter. Asthma was defined as parental report of doctor diagnosis of asthma or receiving treatment at the age of 4–6 years or wheezing since the age of 4 years. Results: The median maternal circulating 25(OH)D concentration in pregnancy was 29.5 ng/mL (interquartile range, 22.5–37.1 ng/mL). After multivariable adjustment, there was a trend for an independent association between higher levels of maternal circulating 25(OH)D levels in pregnancy and decreased odds of lower respiratory tract infections in offspring (for cohort- and season-specific quartile Q4 vs. Q1, odds ratio = 0.67 [95% confidence interval = 0.50–0.90]; test for trend, P = 0.016). We found no association between 25(OH)D levels in pregnancy and risk of wheezing at age 1 year or 4 years, or asthma at age 4–6 years. Conclusions: Higher maternal circulating 25(OH)D concentrations in pregnancy were independently associated with lower risk of lower respiratory tract infections in offspring in the first year of life but not with wheezing or asthma in childhood.

Polymorphisms in XPC, XPD, XRCC1, and XRCC3 DNA repair genes and lung cancer risk in a population of Northern Spain

BackgroundPolymorphisms in DNA repair genes have been associated to repair DNA lesions, and might contribute to the individual susceptibility to develop different types of cancer. Nucleotide excision repair (NER), base excision repair (BER), and double-strand break repair (DSBR) are the main DNA repair pathways. We investigated the relationship between polymorphisms in two NER genes, XPC (poly (AT) insertion/deletion: PAT-/+) and XPD (Asp312Asn and Lys751Gln), the BER gene XRCC1 (Arg399Gln), and the DSBR gene XRCC3 (Thr241Met) and the risk of developing lung cancer.MethodsA hospital-based case-control study was designed with 516 lung cancer patients and 533 control subjects, matched on ethnicity, age, and gender. Genotypes were determined by PCR-RFLP and the results were analysed using multivariate unconditional logistic regression, adjusting for age, gender and pack-years.ResultsBorderline association was found for XPC and XPD NER genes polymorphisms, while no association was observed for polymorphisms in BER and DSBR genes. XPC PAT+/+ genotype was associated with no statistically significant increased risk among ever smokers (OR = 1.40; 95%CI = 0.94–2.08), squamous cell carcinoma (OR = 1.44; 95%CI = 0.85–2.44), and adenocarcinoma (OR = 1.72; 95%CI = 0.97–3.04). XPD variant genotypes (312Asn/Asn and 751Gln/Gln) presented a not statistically significant risk of developing lung cancer (OR = 1.52; 95%CI = 0.91–2.51; OR = 1.38; 95%CI = 0.85–2.25, respectively), especially among ever smokers (OR = 1.58; 95%CI = 0.96–2.60), heavy smokers (OR = 2.07; 95%CI = 0.74–5.75), and adenocarcinoma (OR = 1.88; 95%CI = 0.97–3.63). On the other hand, individuals homozygous for the XRCC1 399Gln allele presented no risk of developing lung cancer (OR = 0.87; 95%CI = 0.57–1.31) except for individuals carriers of 399Gln/Gln genotype and without family history of cancer (OR = 0.57; 95%CI = 0.33–0.98) and no association was found between XRCC3 Thr241Met polymorphism and lung cancer risk (OR = 0.92; 95%CI = 0.56–1.50), except for the 241Met/Met genotype and squamous cell carcinoma risk (OR = 0.47; 95%CI = 0.23–1.00).ConclusionIn conclusion, we analysed the association between XPC, XPD, XRCC1, and XRCC3 polymorphisms and the individual susceptibility to develop lung cancer in the Spanish population, specifically with a highly tobacco exposed population. We attempt to contribute to the discovery of which biomarkers of DNA repair capacity are useful for screening this high-risk population for primary preventing and early detection of lung cancer.

Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.