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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.

Smoke constituents can induce DNA adducts that cause mutations and lead to lung cancer. We have analyzed DNA adducts and polymorphisms in two DNA repair genes, for example, XRCC1 Arg194Trp and Arg399Gln genes and XRCC3 Thr241Met gene, in 34 lung cancer cases in respect to 30 subjects with benign lung cancer disease and 40 healthy controls. When the study population was categorized in base to the number of risk alleles, adducts were significantly increased in individuals bearing 3-4 risk alleles (OR=4.195% C.I. 1.28–13.09,P=.009). A significant association with smoking was noticed in smokers for more than 40 years carrying 3-4 risk alleles (OR=36.38, 95% C.I. 1.17–1132.84,P=.040). A not statistically significant increment of lung cancer risk was observed in the same group (OR=4.54, 95% C.I. 0.33–62.93,P=.259). Our results suggest that the analysis of the number of risk alleles predicts the interindividual variation in DNA adducts of smokers and lung cancer cases.

Malignant pleural mesothelioma (MPM) is a rare aggressive tumor associated with asbestos exposure. The possible role of genetic factors has also been suggested and MPM has been associated with single nucleotide polymorphisms (SNPs) of xenobiotic and oxidative metabolism enzymes. We have identified an association of the DNA repair gene XRCC1 with MPM in the population of Casale Monferrato, a town exposed to high asbestos pollution. To extend this observation we examined 35 SNPs in 15 genes that could be involved in MPM carcinogenicity in 220 MPM patients and 296 controls from two case-control studies conducted in Casale (151 patients, 252 controls) and Turin (69 patients, 44 controls), respectively. Unconditional multivariate logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Two DNA repair genes were associated with MPM, i.e. XRCC1 and ERCC1. Considering asbestos-exposed only, the risk increased with the increasing number of XRCC1-399Q alleles (Casale: OR=1.44, 95%CI 1.02-2.03; Casale+Turin: OR=1.34, 95%CI 0.98-1.84) or XRCC1 -77T alleles (Casale+Turin: OR=1.33, 95%CI 0.97-1.81). The XRCC1-TGGGGGAACAGA haplotype was significantly associated with MPM (Casale: OR=1.76, 95%CI 1.04-2.96). Patients heterozygotes for ERCC1 N118N showed an increased OR in all subjects (OR=1.66, 95%CI 1.06-2.60) and in asbestos-exposed only (OR=1.59, 95%CI 1.01-2.50). When the dominant model was considered (i.e. ERCC1 heterozygotes CT plus homozygotes CC versus homozygotes TT) the risk was statistically significant both in all subjects (OR=1.61, 95%CI 1.06-2.47) and in asbestos-exposed only (OR=1.56, 95%CI 1.02-2.40). The combination of ERCC1 N118N and XRCC1 R399Q was statistically significant (Casale: OR=2.02, 95%CI 1.01-4.05; Casale+Turin: OR=2.39, 95%CI 1.29-4.43). The association of MPM with DNA repair genes support the hypothesis that an increased susceptibility to DNA damage may favour asbestos carcinogenicity.

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/10(6) bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/10(6) bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose-response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories. Environmental Cancer Risk, Nutrition and Individual Susceptibility, a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No 513943); the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); the European Union 6th Framework Program strategic targeted research project ‘COMICS’ (Contract No 037575); Swedish Research Council (Vetenskapsra°det).

OBJECTIVES: There is some evidence that dietary components that are rich in antioxidant and vitamins are inversely associated with DNA adduct levels induced by environmental carcinogens such as polycyclic aromatic hydrocarbons, although the epidemiologic data are inconsistent. This study addresses the association between vitamins, DNA adducts and smoking. METHODS: A combined analysis of individual data on the association between bulky DNA adducts and dietary vitamins was conducted. A Medline search was performed to identify studies on healthy subjects in which smoking and vitamins intake information were available, and bulky DNA adducts were measured in peripheral blood with 32P-postlabelling. Eight published studies met the eligibility criteria, and individual data from 7 data sets including 2758 subjects were obtained. GSTM1 and GSTT1 were also available on all the subjects. RESULTS: Vitamin E was inversely significantly associated with DNA adducts after adjustment for possible confounding factors. Vitamins A and C were not independent predictors of DNA adducts. A stratified analysis showed that vitamin A had a significant inverse association with DNA adducts in ever smokers only. CONCLUSIONS: This result is relevant to planning any future chemo-preventive interventions directed to high risk subgroups of the population, for cancer prevention. This study was partially supported by grants from ‘‘Associazione Italiana per la Ricerca sul Cancro’’, and by the Environmental Cancer Risk, Nutrition and Individual Susceptibility European Union Network of Excellence (ECNIS, Contract No. FOOD-CT-2005- 513943) and a grant from NIH 5P20CA132385-03.

The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levene's test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay. Environmental Cancer Risk, Nutrition and Individual Susceptibility, a Network of Excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (contract no. 513943); the Danish Research Council (to L.F., S.L. and P.M.); the Italian Association for Cancer Research (to A.A. and G.M.); FP6 COMICS Project (contract no. 037575 to A.R.C. and A.A.).

Tobacco smoking is the most important and well-established bladder cancer risk factor and a rich source of chemical carcinogens and reactive oxygen species that can induce damage to DNA in urothelial cells. Therefore, common variation in DNA repair genes might modify bladder cancer risk. In this study, we present results from meta-analyses and pooled analyses conducted as part of the International Consortium of Bladder Cancer. We included data on 10 single nucleotide polymorphisms corresponding to seven DNA repair genes from 13 studies. Pooled analyses and meta-analyses included 5,282 cases and 5,954 controls of non-Latino white origin. We found evidence for weak but consistent associations with ERCC2 D312N [rs1799793; per-allele odds ratio (OR), 1.10; 95% confidence interval (95% CI), 1.01-1.19; P = 0.021], NBN E185Q (rs1805794; per-allele OR, 1.09; 95% CI, 1.01-1.18; P = 0.028), and XPC A499V (rs2228000; per-allele OR, 1.10; 95% CI, 1.00-1.21; P = 0.044). The association with NBN E185Q was limited to ever smokers (interaction P = 0.002) and was strongest for the highest levels of smoking dose and smoking duration. Overall, our study provides the strongest evidence to date for a role of common variants in DNA repair genes in bladder carcinogenesis. The TBCS was supported by a grant to ECNIS, a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘‘Food Quality and Safety’’ (contract no. 513943), by a grant of the Italian Association for Cancer Research, of the Piedmont Region Progetti di Ricerca Sanitaria Finalizzata, and of the Ministry of Health, Oncology Integrated Project ‘‘Cancer Primary Prevention in Italy: a research-based approach,’’ Italy.

Bladder cancer risk is highly influenced by environmental and/or predisposing genetic factors. In the last decades growing evidence of the major role played by DNA repair systems in the developing of bladder cancer has been provided. To better investigate the involvement of DNA repair genes previously reported to be significantly associated with bladder cancer risk, we examined in a case-control study (456 cases and 376 hospital controls) 36 single nucleotide polymorphisms (SNPs) in 10 DNA repair genes, through a better gene coverage and a deep investigation of the haplotype role. A single SNP analysis showed a significantly increased risk given by XRCC1-rs915927 G allele (OR=1.55, CI 95% 1.02-2.37 for dominant model) and a protective effect of the rare alleles of 3 ERCC1 SNPs: rs967591 (OR=0.66, CI 95% 0.46-0.95), rs735482 (OR=0.62, CI 95% 0.42-0.90) and rs2336219 (OR=0.63, CI 95% 0.43-0.93). Haplotype analysis revealed that cases had a statistically significant excess of XRCC3-TAGT and ERCC1-GAT haplotypes, whereas ERCC1-AAC, MGMT-TA, XRCC1-TGCC and ERCC2-TGAA haplotypes were significantly underrepresented. Together with other published data on large case-control studies, our findings provide epidemiological evidence supporting a link between DNA repair gene variants and bladder cancer development, and suggest that the effects of high-order interactions should be taken into account as modulating factors affecting bladder cancer risk. A detailed characterization of DNA repair genetic variation is warranted and might ultimately help to identify multiple susceptibility variants that could be responsible for joint effects on the risk. This paper was made possible by a grant from the Compagnia di San Paolo (Turin, Italy; P.V.) and of the Associazione Italiana per le Ricerche sul Cancro (G.M.). F.R. was partially founded by the Master in Epidemiology, University of Turin and San Paolo Foundation. C.S. was partially founded by Piedmont Region and University of Turin, “Brain Drain” project. P.V. and G.M. were partially founded by Environmental Cancer Risk Nutrition and Individual Susceptibility, a network of excellence operating within the European Union sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943) and Piedmont Region Grants.

BACKGROUND: Several genes encoding for DNA repair molecules implicated in maintaining genomic integrity have been proposed as cancer-susceptibility genes. Although efforts have been made to create synopses for specific fields that summarize the data from genetic association studies, such an overview is not available for genes involved in DNA repair. METHODS: We have created a regularly updated database of studies addressing associations between DNA repair gene variants (excluding highly penetrant mutations) and different types of cancer. Using 1087 datasets and publicly available data from genome-wide association platforms, meta-analyses using dominant and recessive models were performed on 241 associations between individual variants and specific cancer types that had been tested in two or more independent studies. The epidemiological strength of each association was graded with Venice criteria that assess amount of evidence, replication, and protection from bias. All statistical tests were two-sided. RESULTS: Thirty-one nominally statistically significant (ie, P < .05 without adjustment for multiple comparisons) associations were recorded for 16 genes in dominant and/or recessive model analyses (BRCA2, CCND1, ERCC1, ERCC2, ERCC4, ERCC5, MGMT, NBN, PARP1, POLI, TP53, XPA, XRCC1, XRCC2, XRCC3, and XRCC4). XRCC1, XRCC2, TP53, and ERCC2 variants were each nominally associated with several types of cancer. Three associations were graded as having "strong" credibility, another four had modest credibility, and 24 had weak credibility based on Venice criteria. Requiring more stringent P values to account for multiplicity of comparisons, only the associations of ERCC2 codon 751 (recessive model) and of XRCC1 -77 T>C (dominant model) with lung cancer had P This work was made possible by a grant to ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (contract no. 513943), and by a grant of the compagnia di San Paolo, of the Italian Association for Cancer Research, Italy, and of the Piedmont Region Progetti di Ricerca Sanitaria Finalizzata. F. K. Kavvoura is supported by a PENED training grant cofi nanced by EU — European Social Fund (75%) and the Greek Ministry of Development – General Secretariat of Research and Technology (25%).

Udgivelsesdato: 2009-Jan-1 We followed-up for mortality and cancer incidence 1088 healthy non-smokers from a population-based study, who were characterized for 22 variants in 16 genes involved in DNA repair pathways. Follow-up was 100% complete. The association between polymorphism and mortality or cancer incidence was analyzed using Cox Proportional Hazard regression models. Ninety-five subjects had died in a median follow-up time of 78 months (inter-quartile range 59-93 months). None of the genotypes was clearly associated with total mortality, except variants for two Double-Strand Break DNA repair genes, XRCC3 18067 C>T (rs#861539) and XRCC2 31479 G>A (rs#3218536). Adjusted hazard ratios were 2.25 (1.32-3.83) for the XRCC3 C/T genotype and 2.04 (1.00-4.13) for the T/T genotype (reference C/C), and 2.12 (1.14-3.97) for the XRCC2 G/A genotype (reference G/G). For total cancer mortality, the adjusted hazard ratios were 3.29 (1.23-7.82) for XRCC3 C/T, 2.84 (0.81-9.90) for XRCC3 T/T and 3.17 (1.21-8.30) for XRCC2 G/A. With combinations of three or more adverse alleles, the adjusted hazard ratio for all cause mortality was 17.29 (95% C.I. 8.13-36.74), and for all incident cancers the HR was 5.28 (95% C.I. 2.17-12.85). Observations from this prospective study suggest that polymorphisms of genes involved in the repair of DNA double-strand breaks significantly influence the risk of cancer and non-cancer disease, and can influence mortality.

Contains fulltext : 80627.pdf (Publisher’s version ) (Closed access) We conducted a genome-wide association study on 969 bladder cancer cases and 957 controls from Texas. For fast-track validation, we evaluated 60 SNPs in three additional US populations and validated the top SNP in nine European populations. A missense variant (rs2294008) in the PSCA gene showed consistent association with bladder cancer in US and European populations. Combining all subjects (6,667 cases, 39,590 controls), the overall P-value was 2.14 x 10(-10) and the allelic odds ratio was 1.15 (95% confidence interval 1.10-1.20). rs2294008 alters the start codon and is predicted to cause truncation of nine amino acids from the N-terminal signal sequence of the primary PSCA translation product. In vitro reporter gene assay showed that the variant allele significantly reduced promoter activity. Resequencing of the PSCA genomic region showed that rs2294008 is the only common missense SNP in PSCA. Our data identify rs2294008 as a new bladder cancer susceptibility locus.

The presence or absence of genetic heterogeneity in Sicily has long been debated. Through the analysis of the variation of Y-chromosome lineages, using the combination of haplogroups and short tandem repeats from several areas of Sicily, we show that traces of genetic flows occurred in the island, due to ancient Greek colonization and to northern African contributions, are still visible on the basis of the distribution of some lineages. The genetic contribution of Greek chromosomes to the Sicilian gene pool is estimated to be about 37% whereas the contribution of North African populations is estimated to be around 6%. In particular, the presence of a modal haplotype coming from the southern Balkan Peninsula and of its one-step derivates associated to E3b1a2-V13, supports a common genetic heritage between Sicilians and Greeks. The estimate of Time to Most Recent Common Ancestor is about 2380 years before present, which broadly agrees with the archaeological traces of the Greek classic era. The Eastern and Western part of Sicily appear to be significantly different by the χ2-analysis, although the extent of such differentiation is not very high according to an analysis of molecular variance. The presence of a high number of different haplogroups in the island makes its gene diversity to reach about 0.9. The general heterogeneous composition of haplogroups in our Sicilian data is similar to the patterns observed in other major islands of the Mediterranean, reflecting the complex histories of settlements in Sicily.

Udgivelsesdato: 2008-Feb-14 In contrast to some extensively examined food mutagens, for example, aflatoxins, N-nitrosamines and heterocyclic amines, some other food contaminants, in particular polycyclic aromatic hydrocarbons (PAH) and other aromatic compounds, have received less attention. Therefore, exploring the relationships between dietary habits and the levels of biomarkers related to exposure to aromatic compounds is highly relevant. We have investigated in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort the association between dietary items (food groups and nutrients) and aromatic DNA adducts and 4-aminobiphenyl-Hb adducts. Both types of adducts are biomarkers of carcinogen exposure and possibly of cancer risk, and were measured, respectively, in leucocytes and erythrocytes of 1086 (DNA adducts) and 190 (Hb adducts) non-smokers. An inverse, statistically significant, association has been found between DNA adduct levels and dietary fibre intake (P = 0.02), vitamin E (P = 0.04) and alcohol (P = 0.03) but not with other nutrients or food groups. Also, an inverse association between fibre and fruit intake, and BMI and 4-aminobiphenyl-Hb adducts (P = 0.03, 0.04, and 0.03 respectively) was observed. After multivariate regression analysis these inverse correlations remained statistically significant, except for the correlation adducts v. fruit intake. The present study suggests that fibre intake in the usual range can modify the level of DNA or Hb aromatic adducts, but such role seems to be quantitatively modest. Fibres could reduce the formation of DNA adducts in different manners, by diluting potential food mutagens and carcinogens in the gastrointestinal tract, by speeding their transit through the colon and by binding carcinogenic substances.

Contains fulltext : 71044.pdf (Publisher’s version ) (Closed access) We conducted a genome-wide SNP association study on 1,803 urinary bladder cancer (UBC) cases and 34,336 controls from Iceland and The Netherlands and follow up studies in seven additional case-control groups (2,165 cases and 3,800 controls). The strongest association was observed with allele T of rs9642880 on chromosome 8q24, 30 kb upstream of MYC (allele-specific odds ratio (OR) = 1.22; P = 9.34 x 10(-12)). Approximately 20% of individuals of European ancestry are homozygous for rs9642880[T], and their estimated risk of developing UBC is 1.49 times that of noncarriers. No association was observed between UBC and the four 8q24 variants previously associated with prostate, colorectal and breast cancers, nor did rs9642880 associate with any of these three cancers. A weaker signal, but nonetheless of genome-wide significance, was captured by rs710521[A] located near TP63 on chromosome 3q28 (allele-specific OR = 1.19; P = 1. 15 x 10(-7)).

BACKGROUND: We chose a set of candidate single nucleotide polymorphisms (SNPs) to investigate gene-environment interactions in three types of cancer that have been related to air pollution (lung, bladder and myeloid leukemia). PATIENTS AND METHODS: The study has been conducted as a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (409 cancer cases and 757 matched controls). We included never and ex-smokers. SNPs were in genes involved in oxidative stress, phase I metabolizing genes, phase II metabolizing genes and methylenetetrahydrofolate reductase (MTHFR). RESULTS: The most notable findings are: GSTM1 deletion and bladder cancer risk [odds ratio (OR) = 1.60; 95% confidence interval 1.00-2.56]; CYP1A1 and leukemia (2.22, 1.33-3.70; heterozygotes); CYP1B1 and leukemia (0.47, 0.27-0.84; homozygotes); MnSOD and leukemia (1.91, 1.08-3.38; homozygotes) and NQO1 and lung cancer (8.03, 1.73-37.3; homozygotes). Other statistically significant associations were found in subgroups defined by smoking habits (never or ex-smokers), environmental tobacco smoke or gender, with no obvious pattern. When gene variants were organized according to the three main pathways, the emerging picture was of a strong involvement of combined phase I enzymes in leukemia, with an OR of 5 (1.63-15.4) for those having three or more variant alleles. The association was considerably stronger for leukemias arising before the age of 55. Mortality data for The Netherlands were obtained from ‘Statistics Netherlands’. This paper was made possible by a grant of the European Community (5th Framework Programme) to PV (grant QLK4CT199900927) and a grant of the Compagnia di San Paolo to the ISI Foundation, and a grant of the EC to the ECNIS Network of Excellence (grant FOOD-CT-2005-513943)(WP4-8). All authors are independent from funders. Also, the work described in the paper was carried out with the financial support of: Europe Against Cancer Program of the European Commission (SANCO); ISCIII, Red de Centros RCESP, C03/09; Deutsche Krebshilfe; Deutsches Krebsforschungszentrum; German Federal Ministry of Education and Research; Danish Cancer Society; Health Research Fund (FIS) of the Spanish Ministry of Health; Spanish Regional Governments of Andalucia, Asturias, Basque Country, Murcia and Navarra; Cancer Research, UK; Medical Research Council, UK; Stroke Association, UK; British Heart Foundation; Department of Health, UK; Food Standards Agency, UK; Wellcome Trust, UK; Greek Ministry of Education; Italian Association for Research on Cancer; Italian National Research Council; Dutch Ministry of Public Health, Welfare and Sports; World Cancer Research Fund; Swedish Cancer Society; Swedish Scientific Council; Regional Government of Skåne, Sweden; Norwegian Cancer Society.

Consumption of dairy products seems to increase the risk of cancer at several sites, while intake of cruciferous vegetables could have protective effects. However, these dietary intakes are subject to measurement error, and associations with cancer could be due to confounders. Mendelian randomization has been suggested as a way to overcome confounding by exploiting the random allocation of alleles from parents to offspring. In mid-2006, the authors conducted a study of allele frequencies for the lactase (LCT) and taste receptor, type 2, member 38 (TAS2R38) genes, including 634 volunteers recruited (1992-1998) from the Italian branch of the European Prospective Investigation into Cancer and Nutrition. The authors hypothesized that there would be a lower milk intake among carriers of the LCT CC genotype and a different intake of cruciferous vegetables among carriers of the TAS2R38 variant. Overall, the frequency of the LCT T allele was higher in northern Italy than in southern Italy. Food intake was associated with gene variants. An association was evident for ice cream and LCT variants (p = 0.004); less so for milk intake. In addition, the TAS2R38 variant showed a geographic gradient and an association with cruciferous vegetable intake. These results suggest that the LCT and TAS2R38 variants are good candidates for Mendelian randomization studies of cancer and other health outcomes.

The objective is to investigate the relationships between fruit and vegetable intake, DNA repair gene polymorphisms and the risk of bladder cancer. We have analyzed a hospital-based case-control study of 266 individuals with incident, histologically confirmed bladder cancer diagnosed between 1994 and 2003. Controls (n = 193) were patients treated for benign diseases recruited daily in a random fashion from the same hospital as the cases. All cases and controls were interviewed face-to-face for major risk factors, along fruit and vegetable consumption. Odds ratios (ORs) for fruit and vegetable intake and DNA repair gene polymorphisms were adjusted for age and smoking status, using unconditional logistic regression. A statistically significant decreased risk was observed for fruit and vegetable intake above median (versus below the median) [unadjusted OR 0.61, confidence interval (CI) 95% 0.50-0.96 and OR 0.54, CI 95% 0.39-0.80, respectively]; the decreased risk persisted after adjustment for age and cigarette smoking (OR 0.73, CI 95% 0.49-1.01 and OR 0.86, CI 95% 0.56-1.08, respectively). The fruits and vegetables associated with decreased risks included leafy green vegetables, cruciferous vegetables, apples and citrus fruits. We did not find any interactions between DNA repair gene polymorphisms and fruit and vegetable intake. This study found a reduced risk associated with fruit and vegetable intake. No interaction was observed between fruit and vegetable consumption and DNA repair gene polymorphisms. This paper was made possible by a grant from the Compagnia di San Paolo (Turin, Italy; P.V.) and of the Associazione Italiana per le Ricerche sul Cancro (G.M.). P.V. and G.M. are partially funded by Environmental Cancer Risk Nutrition and Individual Suspectibility, a network of excellence operating within the European Union sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943).

A diet rich in fruit and vegetables can be effective in the reduction of oxidative stress, through the antioxidant effects of phytochemicals and other mechanisms. Protection against the carcinogenic effects of chemicals may also be exerted by an enhancement of detoxification and DNA damage repair mechanisms. To investigate a putative effect of flavonoids, a class of polyphenols, on the regulation of the gene expression of DNA repair and metabolic genes, a 1-month flavonoid-rich diet was administered to thirty healthy male smokers, nine of whom underwent gene expression analysis. We postulated that tobacco smoke is a powerful source of reactive oxygen species. The expression level of twelve genes (APEX, ERCC1, ERCC2, ERCC4, MGMT, OGG1, XPA, XPC, XRCC1, XRCC3, AHR, CYP1A1) was investigated. We found a significant increase (P AHR levels were significantly reduced after the diet (P = 0·038), whereas the other genes showed a generalized up regulation, significant for XRCC3 gene (P = 0·038). Also in the context of a generalized up regulation of DNA repair genes, we found a non-significant negative correlation between flavonoid intake and the expression of all the DNA repair genes. Larger studies are needed to clarify the possible effects of flavonoids in vivo; our preliminary results could help to better plan new studies on gene expression and diet.

Objectives: A number of common non-synonymous single nucleotide polymorphisms (SNPs) in DNA repair genes have been reported to modify bladder cancer risk. These include: APE1-Asn148Gln, XRCC1-Arg399Gln and XRCC1-Arg194Trp in the BER pathway, XPD-Gln751Lys in the NER pathway and XRCC3-Thr241Met in the DSB repair pathway. Methods: To examine the independent and interacting effects of these SNPs in a large study group, we analyzed these genotypes in 1,029 cases and 1,281 controls enrolled in two case-control studies of incident bladder cancer, one conducted in New Hampshire, USA and the other in Turin, Italy. Results: The odds ratio among current smokers with the variant XRCC3-241 (TT) genotype was 1.7 (95% CI 1.0–2.7) compared to wild-type. We evaluated gene-environment and gene-gene interactions using four analytic approaches: logistic regression, Multifactor Dimensionality Reduction (MDR), hierarchical interaction graphs, classification and regression trees (CART), and logic regression analyses. All five methods supported a gene-gene interaction between XRCC1-399/XRCC3-241 (p = 0.001) (adjusted OR for XRCC1-399 GG, XRCC3-241 TT vs. wild-type 2.0 (95% CI 1.4–3.0)). Three methods predicted an interaction between XRCC1-399/XPD-751 (p = 0.008) (adjusted OR for XRCC1-399 GA or AA, XRCC3-241 AA vs. wild-type 1.4 (95% CI 1.1–2.0)). Conclusions: These results support the hypothesis that common polymorphisms in DNA repair genes modify bladder cancer risk and highlight the value of using multiple complementary analytic approaches to identify multi-factor interactions.

The origin of the Etruscan people has been a source of major controversy for the past 2,500 years, and several hypotheses have been proposed to explain their language and sophisticated culture, including an Aegean/Anatolian origin. To address this issue, we analyzed the mitochondrial DNA (mtDNA) of 322 subjects from three well-defined areas of Tuscany and compared their sequence variation with that of 55 western Eurasian populations. Interpopulation comparisons reveal that the modern population of Murlo, a small town of Etruscan origin, is characterized by an unusually high frequency (17.5%) of Near Eastern mtDNA haplogroups. Each of these haplogroups is represented by different haplotypes, thus dismissing the possibility that the genetic allocation of the Murlo people is due to drift. Other Tuscan populations do not show the same striking feature; however, overall, ~5% of mtDNA haplotypes in Tuscany are shared exclusively between Tuscans and Near Easterners and occupy terminal positions in the phylogeny. These findings support a direct and rather recent genetic input from the Near East--a scenario in agreement with the Lydian origin of Etruscans. Such a genetic contribution has been extensively diluted by admixture, but it appears that there are still locations in Tuscany, such as Murlo, where traces of its arrival are easily detectable.

The glutathione S-transferase P1 genotype (GSTP1) is involved in the inactivation of cigarette smoke carcinogens, and sequence variation in the gene may alter bladder cancer susceptibility. To examine the association between GSTP1Ile 105Val and bladder cancer, the authors undertook a meta- and pooled analysis. Summary crude and adjusted odds ratios and corresponding 95% confidence intervals were pooled by using a random-effects model. In the meta-analysis (16 studies, 4,273 cases and 5,081 controls), the unadjusted summary odds ratios for GSTP1 Ile/Val and Val/Val compared with GSTP1 Ile/Ile were 1.54 (95% confidence interval: 1.21, 1.99; p < 0.001) and 2.17 (95% confidence interval: 1.27, 3.71; p = 0.005). The association appeared to be the strongest in Asian countries. When the analysis was limited to European descendents (nine studies), the summary odds ratio decreased (odds ratio = 1.24, 95% confidence interval: 1.00, 1.52) (Q = 17.50; p = 0.02). All relevant data previously contributed to the International Study on Genetic Susceptibility to Environmental Carcinogens were pooled (eight studies, 1,305 cases and 1,558 controls). The summary odds ratios were similar to the ones from the meta-analysis. Case-only analyses did not detect an interaction between the GSTP1 genotype and smoking status (never/ever). GSTP1 Ile 105Val appears to be associated with a modest increase in the risk of bladder cancer. The work of W. A. Schulz was funded by the Deutsche Forschungsgemeinschaft. E. Taioli and G. Matullo are partially funded by ECNIS (Environmental Cancer Risk Nutrition and Individual Suspectibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: "Food Quality and Safety" (contract 513943).

Background The renin-angiotensin-aldosterone system (RAAS) is involved in the cardiovascular homeostasis as shown by previous studies reporting a positive association between specific RAAS genotypes and an increased risk of myocardial infarction. Anyhow the prognostic role in a long-term follow-up has not been yet investigated. Aim of the study was to evaluate the influence of the most studied RAAS genetic Single Nucleotide Polymorphisms (SNPs) on the occurrence and the long-term prognosis of acute myocardial infarction (AMI) at young age in an Italian population. Methods The study population consisted of 201 patients and 201 controls, matched for age and sex (mean age 40 ± 4 years; 90.5% males). The most frequent conventional risk factors were smoke (p < 0.001), family history for coronary artery diseases (p < 0.001), hypercholesterolemia (p = 0.001) and hypertension (p = 0.002). The tested genetic polymorphisms were angiotensin converting enzyme insertion/deletion (ACE I/D), angiotensin II type 1 receptor (AGTR1) A1166C and aldosterone synthase (CYP11B2) C-344T. Considering a long-term follow-up (9 ± 4 years) we compared genetic polymorphisms of patients with and without events (cardiac death, myocardial infarction, revascularization procedures). Results We found a borderline significant association of occurrence of AMI with the ACE D/I polymorphism (DD genotype, 42% in cases vs 31% in controls; p = 0.056). DD genotype remained statistically involved in the incidence of AMI also after adjustment for clinical confounders. On the other hand, during the 9-year follow-up (65 events, including 13 deaths) we found a role concerning the AGTR1: the AC heterozygous resulted more represented in the event group (p = 0.016) even if not independent from clinical confounders. Anyhow the Kaplan-Meier event free curves seem to confirm the unfavourable role of this polymorphism. Conclusion Polymorphisms in RAAS genes can be important in the onset of a first AMI in young patients (ACE, CYP11B2 polymorphisms), but not in the disease progression after a long follow-up period. Larger collaborative studies are needed to confirm these results.

Bladder cancer (BC), which is the fourth most common malignancy among men in the Western world, has a typical aetiology characterized by a multistep carcinogenesis, reflecting that multiple lesions in the DNA are required for tumour development. DNA repair capacity (DRC) is a complex marker comprising the sum of several factors such as gene variants, gene expression, stability of gene products, and effect of inhibitors/stimulators and might account for different susceptibility of developing this cancer. Individuals with low DRC will tend to accumulate more damage than those who have a better ability to repair such damage. The identification of patients with particular DRC and at high-risk of BC and/or recurrence of the disease could help in personalizing both surveillance and treatment. We aimed at studying the relationship between DRC evaluated by H2AX phosphorylation assays and BC, integrating gene expression and epigenetic profile data (methylation levels and microRNA expression) in cryopreserved lymphocytes from 159 BC cases and 159 controls matched by age and smoking habits. We observed a significant association between γ-H2AX basal levels and H2AX dephosphorylation capacity and risk of BC recurrence. Patients with high basal DSB signaling had a better event-free survival (HR 0.35, 95% CI 0. 19-0.63, p = 0.0005). Additionally, there was an association when considering the percentage of dephosphorylation after 3h divided into two categories (above and under the calculated best cut-off). Interestingly, when compared with the lower category (“slow repair”), the other group (“fast repair”) had a better event-free survival (HR 0.41, 95%CI 0.23-0.72, p = 0.002). Our data suggest that γ-H2AX can be considered as a possible molecular biomarker to identify patients with a higher risk of BC recurrence. A subgroup of cases and controls (39 and 44, respectively) was also analyzed for evaluating their gene expression (Illumina HumanHT-12 Expression BeadChip array) and methylation profile data (Illumina HumanMethylation450 BeadChip array). We found a set of CpG islands that were differentially methylated among cases and controls and a set of genes whose expression was deregulated among different subgroups (e.g. cases vs healthy controls, controls vs BC patients with muscle invasive tumor, etc). We performed an integrated analysis on the genotype/phenotype correlation in the analysed BC cases and healthy controls provided with detailed description of the follow-up for response to therapy/recurrence/survival. This integrated approach will help in elucidating the role of DRC (and its determinants) as predictive and prognostic marker and to identify DNA repair phenotypic assays to be performed in blood cells as non-invasive predictive method. Results will be presented at the AACR Annual Meeting. Citation Format: Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Giovanni Fiorito, Clara Viberti, Alessia Russo, Paolo Vineis, Carlotta Sacerdote, Claudia Giachino, Giuseppe Matullo. H2AX phosphorylation assays, gene expression and epigenomic profiles as markers in bladder cancer: an integrated approach. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4615. doi:10.1158/1538-7445.AM2015-4615

In Europe, bladder cancer (BC) is the second most common cause of death among patients with genitourinary tract malignancies. Patients with non-muscle invasive bladder cancer (NMIBC) have excellent survival; however two-thirds develop recurrences. For the diagnosis of BC, we mainly rely on urine cytology and cystoscopy. Although cystoscopy is the mainstay of the diagnosis, it is an invasive and costly procedure. Cytology has high specificity but has low sensitivity especially in low-grade, low-stage tumors. Urine sediment cells are a source of tumor DNA, they are easily accessible, and their sampling is not invasive. The identification of a urine-based biomarker could partly replace cystoscopies, thereby reducing the frequency and associated costs, improving patient's quality of life and even leading to earlier detection of recurrences. Tumor-specific mutations can be used to detect recurrences in urine, presenting a non-invasive diagnostic procedure respect to cystoscopy. The present study involves 252 male subjects diagnosed with BC recruited between 1994 and 2012 from the Turin Bladder Cancer Study. Urine samples have been collected for the first time at the time of diagnosis, and exfoliated cells have been extracted. A subgroup of 83 patients has been followed-up for three years, collecting a urine sample every six months. We have investigated the diagnostic and prognostic potentiality of a panel of 40 mutations in hTERT, FGFR3, PIK3CA, and Ras genes detected in DNA from exfoliated cells by SNApShot assay. For each gene we calculated crude and adjusted (by age, smoking status, chemotherapy, low/intermediate/high risk, grade) Hazard Ratios (HR) and the corresponding 95% Confidence Intervals (95% CI) using the Cox proportional-hazard regression model. The results were correlated with stage and tumor grade, with response to therapy and the occurrence of recurrences, in order to assess the validity of exfoliated cells as a source of DNA for the development of a non-invasive test for the follow-up of patients. Mutations in hTERT gene promoter and FGFR3 gene are the most frequent somatic mutations in BC. In particular, hTERT mutations were significantly correlated with high risk, high grade and muscle invasive tumors (p Citation Format: Rossana Critelli, Francesca Fasanelli, Manuela B. Assumma, Ellen C. Zwarthoff, Marco Oderda, Silvia Polidoro, Carlotta Sacerdote, Paolo Vineis, Giuseppe Matullo, Alessio Naccarati. Mutation detection in urine from bladder cancer patients as non-invasive prognostic tool. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4614. doi:10.1158/1538-7445.AM2015-4614

To estimate the relationship between air pollution and lung cancer, a nested case-control study was set up within EPIC (European Prospective Investigation on Cancer and Nutrition). Cases had newly diagnosed lung cancer, accrued after a median follow-up of 7 years among the EPIC exsmokers (since at least 10 years) and never smokers. Three controls per case were matched. Matching criteria were gender, age (±5 years), smoking status, country of recruitment and time elapsed between recruitment and diagnosis. We studied residence in proximity of heavy traffic roads as an indicator of exposure to air pollution. In addition, exposure to air pollutants (NO2, PM10, SO2) was assessed using concentration data from monitoring stations in routine air quality monitoring networks. Cotinine was measured in plasma. We found a nonsignificant association between lung cancer and residence nearby heavy traffic roads (odds ratio = 1.46, 95% confidence interval, CI, 0.89-2.40). Exposure data for single pollutants were available for 197 cases and 556 matched controls. For NO2 we found an odds ratio of 1.14 (95% CI, 0.78-1.67) for each increment of 10 μg/m3, and an odds ratio of 1.30 (1.02-1.66) for concentrations greater than 30 μg/m3. The association with NO2 did not change after adjustment by cotinine and additional potential confounders, including occupational exposures. No clear association was found with other pollutants. © 2006 Wiley-Liss, Inc.

Hundreds of polymorphisms in DNA repair genes have been identified; however, for many of these polymorphisms, the impact on repair phenotype and cancer susceptibility remains uncertain. In this review, the authors focused on the x-ray repair cross-complementing protein group 3 (XRCC3) and xeroderma pigmentosum group D (XPD)/excision repair cross-complementing rodent repair deficiency (ERCC2) genes, because they are among the most extensively studied but no final conclusion has yet been drawn about their role in cancer occurrence. XRCC3 participates in DNA double-strand break/recombinational repair through homologous recombination to maintain chromosome stability. XPD/ERCC2 is a helicase involved in the nucleotide excision repair pathway, which recognizes and repairs many structurally unrelated lesions, such as bulky adducts and thymidine dimers. The authors identified a sufficient number of epidemiologic studies on cancer to perform meta-analyses for XPD/ERCC2 variants in codons 156, 312, and 751 and XRCC3 variants in codon 241. The authors evaluated all cancer sites to investigate whether DNA repair is likely to take place in a rather nonspecific manner for different carcinogens and different cancers. For the most part, the authors found no association between these genes and the cancer sites investigated, except for some statistically significant associations between XPD/ERCC2 single nucleotide polymorphisms and skin, breast, and lung cancers.

Eight Y-chromosomal short tandem repeats (STRs)-DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393 and DYS385 - were typed in a population sample (n = 255) of unrelated Sicilian males from nine different towns on the main island and from the island of Pantelleria. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Eight Y-chromosome STR loci (DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393 and DYS385) were analysed in a sample of 236 unrelated males from four towns of Piedmont (Trino, Biella, Cavaglia, Postua). One hundred and fifty six different haplotypes were observed and 55 of them were not previously observed in the Y-STR Haplotype Reference Database (http://www.ystr.org/).

Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic mutations and cancer. Our study investigated the relationships between 22 polymorphisms (and their haplotypes) in 16 DNA repair genes belonging to different repair pathways in 1094 controls and 567 cancer cases (bladder cancer, 131; lung cancer, 134; oral-pharyngeal cancer, 41; laryngeal cancer, 47; leukaemia, 179; death from emphysema and chronic obstructive pulmonary disease, 84). The design was a case-control study nested within a prospective investigation. Among the many comparisons, few polymorphisms were associated with the diseases at the univariate analysis: XRCC1-399 Gln/Gln variant homozygotes [odds ratios (OR) = 2.20, 95% confidence intervals (CI) = 1.16-4.17] and XRCC3-241 Met/Met homozygotes (OR = 0.51, 95% CI = 0.27-0.96) and leukaemia. The recessive model in the stepwise multivariate analysis revealed a possible protective effect of XRCC1-399Gln/Gln in lung cancer (OR = 0.22, 95% CI = 0.05-0.98), and confirmed an opposite effect (OR = 2.47, 95% CI = 1.02-6.02) in the leukaemia group. Our results also suggest that the XPD/ERCC1-GAT haplotype may modulate leukaemia (OR = 1.28, 95% CI = 1.02-1.61), bladder cancer (OR = 1.38, 95% CI = 1.06-1.79) and possibly other cancer risks. Further investigations of the combined effects of polymorphisms within these DNA repair genes, smoking and other risk factors may help to clarify the influence of genetic variation in the carcinogenic process. © 2006 Oxford University Press.

Background This study evaluated vascular brachytherapy (VBT) as a potent antiproliferative treatment to prevent in-stent restenosis (ISR) after coronary angioplasty of de novo lesions in patients carrying the D allele of the I/D polymorphism of the ACE gene and high ACE plasma levels (>34 U/l). Methods and materials A prospective randomized trial was designed to detect a 30% improvement in the minimal lumen diameter (MLD) of the stenotic artery, as measured by quantitative coronary analysis (QCA), 6 months following VBT at the time of stented angioplasty. All patients were carriers of the D allele of the ACE gene, with plasma ACE levels >34 U/l. Results Thirty-one patients (33 stenoses) were allocated to stent implantation (control group) and 30 patients (31 stenoses) to VBT and stented angioplasty. After angioplasty, in-stent MLD was similar in the two groups. At 6 months in the control group, in-stent MLD had decreased to 1.74±0.8 versus 2.25±1.05 mm in the VBT group ( P =.04). The mean in-stent diameter was 2.3±0.8 mm in the control group versus 2.9±1.05 mm after VBT ( P =.02), and the restenosis rate was 37.5% versus 17.9%, respectively ( P =.08). At 6 months, a higher need for target vessel revascularization (TVR) was observed in the control group: 35.5% versus 13.3% ( P =.04). Conclusions This randomized study confirms that patients with high plasma ACE concentrations are exposed to an increased risk for ISR after coronary stenting. The preventive use of VBT in these patients reduced neointimal formation by 65% such that the MLD at follow-up was increased by 29% compared with the control group.

One of the many commercial technologies for genotyping single nucleotide polymorphisms (SNPs) is template direct dye-terminator incorporation with fluorescence-polarization (TDI-FP assay). It is a single-base extension assay followed by reading the fluorescence polarization values in an appropriate instrument. We have evaluated the suitability of the TDI-FP technique to detect haploid uniparentally inherited DNA polymorphisms on the nonrecombining portion of the Y chromosome. A sample of 47 individuals has been genotyped for 8 Y chromosome biallelic markers. The SNP typing was blindly duplicated by the denaturing high-performance liquid chromatography (DHPLC) technique for comparison. In the cases under examination the TDI-FP assay was able to resolve an allelic state fully. Such a result showed 100% concordance indicating how efficiently the TDI assay can be used to genotype Y chromosome DNA SNPs. However, a percentage of indeterminate genotypes remained unresolved by simple visual inspection: it varied from 0% to 11% depending on the SNP locus and on the success of amplification. This is consistent with previous findings. A maximum likelihood classificatory analysis allowed some of the indeterminate genotypes to be assigned and some potentially misclassified samples to be identified. Their percentage remains relatively high despite retyping and therefore alternative techniques for these noncompliant situations are required.

The aim of this study was to evaluate whether biomarkers of environmental tobacco smoke exposure [i.e., 4-aminobiphenyl-hemoglobin (4-ABP-Hb) adducts] were predictive of the risk of tobacco-related cancers and diseases. We did a case-control study nested within the European Prospective Investigation into Cancer and Nutrition, involving 190 controls and 149 cases (incident cancer of the lung, bladder, pharynx, larynx, oral cavity, leukemias, and chronic obstructive pulmonary disease or emphysema deaths). All individuals were never smokers or ex smokers for >10 years. 4-ABP-Hb adducts were analyzed in peripheral blood collected before the onset of the disease (median, 7 years). Overall, 4-ABP-Hb adducts were higher, although not statistically significantly so, in cases (as a whole) than controls. In the control population, high fruit and vegetable consumption significantly lowered the frequency of detectable adducts (Fisher's exact test, P = 0.025). Restricting the analysis to women, 4-ABP-Hb adducts were higher in cases than controls (Mann-Whitney P = 0.036) and the odds ratio (OR) for the presence/absence of adducts was 2.42 [95% confidence interval (95% CI), 1.18-4.98]. Moreover, the association of adducts with the individual cancer types was stronger in women than in the whole study population, although statistically significant only for leukemias (OR, 2.77; 95% CI, 1.06-7.20). The results provide some evidence that women may be more susceptible to environmental tobacco smoke, as suggested by their higher adduct levels. The most important finding of this prospective study is that, at least in women, 4-ABP-Hb adducts may help identify subjects at high risk of cancers related to environmental tobacco smoke exposure.

Bladder cancer is associated with tobacco smoking and occupational exposure. The repair of DNA damage has a key role in protecting the genome from the insults of cancer-causing agents. We analyzed 13 polymorphisms in seven DNA repair genes belonging to different repair pathways [X-ray repair cross-complementing group 1 (XRCC1): 26304C>T, 26651A>G, 28152A>G; xeroderma pigmentosum-D (XPD): 23591A>G, 35931A>C; excision repair complementing defective in Chinese hamster, group 1 (ERCC1): 19007C>T; XRCC3: 4541T>C, 17893A>G, 18067C>T; proliferating cell nuclear antigen (PCNA): 6084G>C; ERCC4: 30028C>T, 30147A>G; and XRCC2-31479A>G] in 317 incident bladder cancer patients and 317 controls. After adjustment for age and smoking, the PCNA-6084C variant was significantly associated with an increased risk of bladder cancer [CC + CG versus GG, odds ratio (OR), 1.61; 95% confidence interval (95% CI), 1.00-2.61], as well as the XRCC1-26651G variant (GG+AG versus AA: OR, 1.73; 95% CI, 1.17-2.56). After stratifying by smoking habits, an elevated risk for carriers of the XRCC3-18067T allele was detected both in current (TT versus CC: OR, 2.65; 95% CI, 1.21-5.80; CT versus CC: OR, 1.96; 95% CI, 1.09-3.52) and never smokers (TT versus CC: OR, 4.34; 95% CI, 1.14-16.46; CT versus CC: OR, 2.02; 95% CI, 0.72-5.66), whereas an opposite and slightly weaker effect was associated to the XRCC3-17893G allele in current smokers (GG versus AA: OR, 0.30; 95%CI, 0.11-0.82; AG versus AA: OR, 0.73; 95% CI, 0.42-1.27). XRCC3,XRCC1, ERCC4, and XPD-ERCC1 haplotype frequencies were estimated by the maximum likelihood method. The XRCC3-TAT haplotype was associated with an enhanced risk in the current smokers group (OR, 1.62; 95% CI, 1.15-2.29), whereas a reduction of the risk in the overall sample was observed in the presence of the XRCC3-TAC (OR, 0.69; 95% CI, 0.50-0.97). A significant protective effect of the XPD-ERCC1-ACC haplotype was observed among never smokers (OR, 0.16; 95% CI, 0.03-0.81). Our results suggest that polymorphisms and/or haplotypes in XRCC3, XRCC1, and PCNA genes and spanning XPD-ERCC1 region may modulate bladder cancer risk and that some of these effects may preferentially affect current smokers.

OBJECTIVES: Host genetic factors, including the IL1 gene cluster, play a key role in determining the long-term outcome of Helicobacter pylori infection. The aim of the study was to investigate the relationship between selected IL1 loci polymorphisms and gastric cancer risk in an Italian population.METHODS: In a case-control study we compared the IL1B-31 and IL1B+3954 biallelic and IL1RN pentaallelic variable number of tandem repeats (VNTR) polymorphisms in 185 gastric cancer patients and 546 controls randomly sampled from the general population of an area at high gastric cancer risk (Tuscany, Central Italy).RESULTS: Genotype frequencies of the IL1B-31 T/C, IL1B+3954 C/T, and IL1RN polymorphisms among our population controls were in Hardy-Weinberg equilibrium. In multivariate analyses, no increase in gastric cancer risk was observed for the IL1B-31*C- and IL1B+3954*T- carriers; a significant 50% increase emerged for IL1RN*2 allele carriers (OR = 1.49; 95% Cl: 1.01-2.21). Analyses based on combined genotypes showed also that the association with IL1RN*2 allele was limited to two-variant allele carriers who were also homozygous for the IL1B-31*T allele (OR = 2.23; 95% Cl: 1.18-4.23) with a statistically significant interaction between these two genotypes (p = 0.043). Haplotype analysis showed an increased risk for the haplotype IL1RN*2/IL1B-31*T.CONCLUSIONS: Our results suggest that host genetic factors (such as the IL1RN and the IL1B-31 polymorphisms) interact in the complex process of gastric carcinogenesis in this high-risk Italian population. Overall, this effect appears more modest than previously reported in other populations, supporting the hypothesis that other still-to-be-defined factors are important in gastric carcinogenesis. These findings might be due to a haplotype effect.

Glutathione S-Transferases (GSTs) are a family of phase II enzymes involved in the detoxification of potential carcinogens and provided of a strong antioxidant function by neutralizing electrophiles and free radicals. The GSTM1 and GSTT1 isoenzymes exhibit deletion polymorphisms, resulting in a lack of activity, and the null genotypes have been associated with increased cancer risk at several sites, including the stomach, although with contrasting results. We carried out a case-control study to evaluate whether these polymorphisms modulate the risk of developing gastric cancer (GC). Genotypes for GSTM1 and GSTT1 were obtained from a series of 175 histologically confirmed GC patients and a large series of 546 healthy controls randomly sampled from the general population of Tuscany, an area at high GC risk. No difference in the frequency of GSTM1 null genotype was observed between cases and controls, whereas the GSTT1 null genotype was more frequent among cases (p = 0.04). Multivariate single-gene analyses adjusted for possible confounders showed that the GSTT1 null genotype, but not the GSTM1 null genotype, was associated with an increased GC risk. Combined-genotype analyses showed a significantly increased GC risk only for the double null (GSTM1-GSTT1) genotype (OR = 2.27; 95% CI: 1.14-4.53). A statistically significant positive interaction between the 2 null genotypes was observed (p = 0.02). Our findings suggest that only subjects lacking both GSTM1 and GSTT1 activity are at increased GC risk. This study provides further support to the hypothesis that the risk of developing GC is influenced by inter-individual variation in both carcinogen detoxification and antioxidant capacity. (c) 2005 Wiley-Liss, Inc.

In this issue of the Journal, Hung et al. ( 1 ) describe the results of a large study on the association between base excision repair gene polymorphisms and lung cancer risk. Their study demonstrates problems typical of investigations of gene – environment interactions, in particular, the fact that among the generally negative results, some seemingly noteworthy associations are identifi ed in subgroups of subjects who are defi ned on the basis of their tumor histology or smoking habits. The unique aspect of this study is that the authors have estimated the probability that the associations found are attributable to chance (i.e., false positives). The idea of evaluating new observations in the light of existing evidence is not novel and belongs to at least two traditions: Bayesian statistics and clinical epidemiology. In clinical epidemiology, formal treatment of “proof ” from prior evidence is updated into posterior probability according to Bayes’ theorem. This approach has proved extremely useful because ( a ) it has revealed that diagnosis (and, by extension, clinical and scientifi c reasoning) develops in a sequence of steps that incorporate explicitly or implicitly previous knowledge ( 2 ) and ( b ) it has trained physicians to assign probabilities to the different components of such reasoning, namely, prior knowledge and “likelihood,” or the probability, of a positive test result in the presence of disease. Wacholder et al. ( 3 ) have proposed these same approaches to assess the validity of the increasing number of associations that are being identifi ed among genetic variants, environmental exposures, and disease (i.e., gene – environment and gene – gene interactions). A large number of such associations have been reported and even more are expected to emerge in the future. Tens of thousands of single nucleotide polymorphisms (SNPs) are or will be investigated for their association with cancer, and many of the observed results will be false positives. The challenge is to distinguish the false-positive associations from the true positives. Wacholder et al. ( 3 ) proposed a simple Bayesian approach that is based on the estimation of a prior probability and the calculation of posterior probability. The prior probability can be estimated from results of previous studies, from biochemical or molecular information (e.g., gene expression data) that supports the function of a SNP, or from other types of evidence such as sequence homology ( 4 ) . The general idea is to weight a new observation with the available prior evidence to derive a posterior probability. Recently, in an extension of the model proposed by Wacholder et al. ( 3 ) , Ioannidis ( 5 ) has incorporated selective reporting and other biases and the fact that specifi c hypotheses, including gene – disease associations and gene – environment interactions, are usually tested by many teams worldwide. By taking these parameters into account when scrutinizing all the evidence, one can show that very few identifi ed gene – disease associations and gene – environment interactions are probably real. The approach proposed by Wacholder et al. is ingenious and more convincing than other approaches, such as those based on Bonferroni’s correction for multiple comparisons or similar statistical methods ( 6 , 7 ) . Although the study by Hung et al. is a clever application of the proposal by Wacholder et al., execution of that proposal is not free from problems. The main problem is how best to calculate the prior probabilities. First, there is often no information available to use. Second, any available information may not be easy to evaluate, either because it is indirect (for example, from sequence homology) or because it is contradictory. The latter is very often the case with the epidemiologic evidence of gene – environment interactions (see below). Third, methods to quantify prior probabilities are not available, even when we have good evidence. The attempt made by Hung et al. is a good step forward, but it is still imperfect. The authors have considered and applied fi ve categories of prior probability (from 50% to 0.1%); however, the basis for these numbers and categories is unclear. A further step forward might consist of using results of welldone meta-analyses or pooled analyses of the available studies. In this case, we have good examples from the clinical literature: a good meta-analysis can allow clinicians to judge how much a new study changes the estimate of effi cacy of a drug. For example, if a meta-analysis provides an odds ratio of 0.9 and a new study reports an odds ratio of 0.2, then the real contribution of the new study to the advancement of knowledge will be judged by its ability to change the odds ratio provided by the meta-analysis. This ability will depend on both qualitative considerations (such as potential biases and the response rate) and the size of the study. Metaanalyses can be extremely useful for obtaining prior estimates against which the new evidence can be challenged because they include ( a ) an overall odds ratio from virtually all the available studies, ( b ) an evaluation of the quality of the studies (a quality score is usually assigned to each study included in a metaanalysis), ( c ) a weight that depends on the study size, and ( d ) a measure of heterogeneity. In a meta-analysis, a large and well-conducted positive study can thus outweigh several small negative studies. Figure 1 shows an example of a meta-analysis of some of the genes included in the Hung et al. article that we performed using data on gene–environment interactions, with a specifi c focus on DNA repair polymorphisms (summarized at http://perseus.isi.it/ huge [last accessed: March 24, 2005]). A network of investigative groups involved in human genome epidemiology research has been developed to combine data from many studies to overcome the defi ciencies of currently available, relatively small datasets (Ioannidis JPA, Altman R, Boffetta P, Danesh J, Hartge P, Little J, et al.: unpublished data). Overall, the results shown in the fi gure support the choices made by Hung et al. in assigning prior

Objectives To investigate the association between environmental tobacco smoke, plasma cotinine concentration, and respiratory cancer or death. Design Nested case-control study within the European prospective investigation into cancer and nutrition (EPIC). Participants 303 020 people from the EPIC cohort (total 500 000) who had never smoked or who had stopped smoking for at least 10 years, 123 479 of whom provided information on exposure to environmental tobacco smoke. Cases were people who developed respiratory cancers or died from respiratory conditions. Controls were matched for sex, age (plus or minus 5 years), smoking status, country of recruitment, and time elapsed since recruitment. Main outcome measures Newly diagnosed cancer of lung, pharynx, and larynx; deaths from chronic obstructive pulmonary disease or emphysema. Plasma cotinine concentration was measured in 1574 people. Results Over seven years of follow up, 97 people had newly diagnosed lung cancer, 20 had upper respiratory cancers (pharynx, larynx), and 14 died from chronic obstructive pulmonary disease or emphysema. In the whole cohort exposure to environmental tobacco smoke was associated with increased risks (hazard ratio 1.30, 95% confidence interval 0.87 to 1.95, for all respiratory diseases; 1.34, 0.85 to 2.13, for lung cancer alone). Higher results were found in the nested case-control study (odds ratio 1.70, 1.02 to 2.82, for respiratory diseases; 1.76, 0.96 to 3.23, for lung cancer alone). Odds ratios were consistently higher in former smokers than in those who had never smoked; the association was limited to exposure related to work. Cotinine concentration was clearly associated with self reported exposure (3.30, 2.07 to 5.23, for detectable/non-detectable cotinine), but it was not associated with the risk of respiratory diseases or lung cancer. Frequent exposure to environmental tobacco smoke during childhood was associated with lung cancer in adulthood (hazard ratio 3.63, 1.19 to 11.11, for daily exposure for many hours). Conclusions This large prospective study, in which the smoking status was supported by cotinine measurements, confirms that environmental tobacco smoke is a risk factor for lung cancer and other respiratory diseases, particularly in ex-smokers.

Background. The renin–angiotensin system (RAS) and nitric oxide synthase (NOS) play a key role in the progression of primary glomerulonephritis (GN). Although previous studies have examined genetic risk associated with single gene variations, experiments assessing risk conferred by multiple gene variations are still scanty. Methods. The effect of combination of variant alleles of four genes encoding for three components of the RAS [angiotensin converting enzyme insertion/ deletion (ACE I/D), angiotensin II receptor 1 (AT1R 1166A/C), angiotensinogen (AGT M235T)] and for NOS (ecNOS4b/a) on the development and progression of membranous GN (MGN) were evaluated in a longitudinal study comparing 117 patients with serum creatinine (s-Cr)

Levels of plasma DNA concentrations in cancer patients have been shown to be higher than the plasma DNA concentrations found in healthy subjects. The value of plasma DNA levels for development of neoplastic or pulmonary disease was evaluated in a large prospective study. Plasma samples (n = I,184) were analyzed from 776 controls, 359 cases of cancer (lung, bladder, oral cavity, pharynx, larynx, leukemia) and 49 deaths from chronic obstructive pulmonary disease (COPD), including never smokers and ex-smokers, from 9 countries across Europe. The amount of plasma DNA was variable across the European Prospective Investigation into Cancer and Nutrition (EPIC) centers. High DNA concentrations in some centers might be due to the type of population recruited and/or the treatment of the samples. An elevated and statistically significant odds ratio (OR) was found for COPD deaths (OR = 2.53; 95% CI = 1.06-6.02), while nonsignificant increased ORs were present for oral cancers, cancers of the pharynx and larynx and leukemia. When the analyses were stratified by time since recruitment (below or above 36 months), the increased ORs were limited to the more recent period of recruitment, i.e., a time elapsed between blood drawing and disease onset lower than 36 months. This was particularly true for COPD deaths (OR = 12.7; 95% CI = 1.57-103) and leukemia (OR = 2.37; 95% CI = 1.20-4.67). © 2004 Wiley-Liss, Inc.

This study tested the combination of vascular brachytherapy (VBT) and self-expanding Wallstent implantation in coronary lesions of patients at high risk for restenosis as assessed angiographically by quantitative coronary analysis and by 3-dimensional intravascular ultrasound analysis. Twenty-nine "de novo" lesions were managed with a self-expanding stent alone (n = 19) or with a self-expanding stent after beta-VBT (n = 10) in 27 patients who had been identified by high levels of plasma angiotensin-converting enzyme as being prone to myointimal growth after stent implantation. At 6 months, the increase in stent strut diameter was similar in the 2 groups by quantitative coronary analysis and 3-dimensional intravascular ultrasound (Delta mean stent strut diameter -0.33 +/- 0.3 vs -0.40 +/- 0.3 mm, p = 0.5; Delta stent area -11.8 +/- 6.1 vs -12.0 +/- 6.1 mm(2), p = 0.9; Delta stent volume -96.9 +/- 112 vs -83.5 +/- 73 mm(3), p = 0.7; for groups treated with VBT and self-expanding stents and only self-expanding stents, respectively). In-stent neointimal proliferation was decreased in the group treated with VBT and self-expanding stents (minimal luminal diameter 2.5 +/- 0.8 vs 1.88 +/- 0.8 mm, p = 0.04) by quantitative coronary analysis (minimal luminal area 6.7 +/- 2.5 vs 4.1 +/- 1.9 mm(2), p = 0.01), by intravascular ultrasound, and proliferation volume (84.6 +/- 66.4 vs 159.2 +/- 103.5 mm(3), p = 0.05) by 3-dimensional intravascular ultrasound. Positive vessel and luminal remodelings were observed in 50% of the group treated with VBT and self-expanding stents and in 11% of the group treated only with self-expanding stents (p = 0.02). The combined use of VBT and self-expanding stents is a novel approach that enlarges vascular lumen by preventing vessel constriction and neointimal proliferation. The feasibility and good results of this experimental approach suggest that the simultaneous use of these 2 technologies may be an interesting alternative for difficult vascular districts with high restenosis rates, such as peripheral circulation in the lower limbs.

Transforming growth factor beta1 (TGF-beta1) plays an important role in the modulation of cellular growth and differentiation in a wide variety of cell types and in the production/degradation of the extracellular matrix (ECM). We investigated whether G-800A, C-509T and Leu10--Pro polymorphisms in the TGF-beta1 gene could be involved in the development and progression of immunoglobulin A nephropathy (IgAN).DNA samples were obtained from 101 patients with biopsy proven IgA mesangial nephropathy and 118 healthy controls. The genotypes of G-800A, C-509T and Leu10--Pro polymorphisms in the TGF-beta1 gene were determined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) with MaeIII, Eco 81I and Pst I, respectively.No significant differences were observed in the genotype distribution of the three TGF-beta1 polymorphisms between patients and controls. The TAC haplotype (T=Leu10, A-800 and C-509 alleles, respectively) was significantly associated with IgAN (p=0.043; odds ratio (OR) =2.334, 95 % confidence interval (95%CI) 1.01-5.41).Our study suggests that the haplotype reconstruction of TGF-beta1 gene polymorphisms could be more informative than the investigation of single nucleotide polymorphisms for defining the associated risk of developing IgAN. Further research is needed on larger cohorts to confirm TGF-beta1 involvement and test other TGF-beta1 variants with possible additive or synergistic effects.

Purpose Accumulating evidence shows that germline polymorphisms may affect survival in cancer. The purpose of this study was to investigate the association between polymorphisms in a group of candidate genes and survival with soft tissue sarcoma. Patients and Methods We measured single nucleotide polymorphisms in the metabolizing, detoxifying, and DNA repair pathways in 120 newly diagnosed patients with soft tissue sarcoma. We assessed polymorphisms in the aryl hydrocarbon receptor (AhR-Arg554Lys), null variants of the glutathione S-transferase superfamily (GSTM1 and GSTT1), x-ray repair cross-complementing 1 and 3, and Xeroderma pigmentosum, group D (XRCC1-Arg399Gln, XRCC3-Thr241Met, XPD-Lys751Gln). We followed the patients for survival for a median of 7.6 years. Results Cox proportional hazards models demonstrated that a polymorphism at codon 554 in exon 10 of the AhR was significantly and adversely associated with survival (hazard ratio, 2.2; 95% CI, 1.3 to 3.9; P < .01), even while accounting for major clinical characteristics such as tumor grade, tumor size, anatomic site, and patient age. Conclusion Further study of the role of the AhR polymorphism is warranted.

The ID (insertion/deletion) polymorphism of the ACE (angiotensin-converting enzyme) gene controls plasma ACE levels. Both have been correlated with ISR (in-stent restenosis) in preliminary analyses, but not confirmed in larger studies. In the present study, baseline and 6-month quantitative coronary analysis were performed in 897 patients who had stent implantation and the ID polymorphism genotyped. Plasma ACE levels were measured in 848 patients (95 %). Restenosis rates among genotypes were 31.2 % DD, 25.5 % ID and 28.8 % II (not significant). Plasma ACE levels were significantly higher in restenotic patients compared with patients without restenosis (30.7 + 18.6 units/l compared with 22.8 + 12.8 units/l; P = 0.0001) and a strong independent predictor of ISR [OR (odds ratio) = 3.70; 95 % CI (confidence interval), 2.40–5.71; P < 0.0001], except in diabetics. In the subgroup of diabetics and patients with AMI (acute myocardial infarction), the DD genotypes actually had a lower risk of ISR than the II genotypes (diabetics, OR = 0.16; 95 % CI, 0.04–0.69; P = 0.014; and patients with AMI, OR = 0.21; 95 % CI, 0.061–0.749; P = 0.016). After exclusion of diabetics and patients with AMI, ISR rates for genotypes in 632 patients were 31.7 % DD, 24.3 % ID and 17.6 % II (P = 0.02; DD compared with non-DD OR = 1.57; 95 % CI, 1.09–2.25). The association between the D allele and ISR observed in selected populations does not hold with a larger sample size. Other than sample size, clinical variables can modulate the association between ID polymorphism and ISR. Plasma ACE level is a risk factor for ISR, independently of the ID genotype.

Mutations of the N- and K-ras genes occur in approximately 15-30% of acute myeloid leukaemia patients. The role of the oncogenic ras in leukaemogenesis remains unclear. Few studies have revealed that mutations in the ras oncogene family are more probably found in acute myeloid leukaemia patients with previous exposure to toxic agents. A case-case study was conducted in the areas of Florence and Turin, Italy, to investigate whether the presence of N- and K-ras mutations in acute myeloid leukaemia patients was related to a higher frequency of exposure to chemicals. During a 3-year period, 111 acute myeloid leukaemia patients were enrolled. All the patients were interviewed using a semi-structured questionnaire collecting data on residential history, occupation, personal habits and pathological history. The presence of N- and K-ras mutations was analysed by amplification and synthetic oligonucleotide probes and by the so-called polymerase chain reaction amplification for specific alleles technique. A total of 34 (30.6%) patients were found to harbour ras mutations in N-ras and/or K-ras. Fourteen patients (12.6%) had a single ras mutation and 20 patients (18%) had two ras mutations. A positive association between a priori at risk jobs and ras mutations was found, based on nine exposed cases; the odds ratio, adjusted by age, sex and previous X-ray and/or chemotherapy was 2.8 (95% confidence intervals: 0.9-9.0). When considering only subjects with two ras mutations the odds ratio was 4.8 (95% confidence intervals: 1.2-18.8). The odds ratio for a previous X-ray and/or chemotherapy was 16.2 (95% confidence intervals: 1.8-755.9); when only subjects with two ras mutations were considered, the odds ratio was 26.1 (95% confidence intervals: 2.5-1248.9). In conclusion, our data suggest that ras oncogene mutations might identify a group of leukaemia in people with previous X-ray/chemotherapy or with exposure to chemical agents in the work environment.

Objective— Because of the receptor-mediated antiproliferative effects of estradiol on vascular smooth muscle cells, our study aimed at identifying a role of PvuII and XbaI polymorphisms of the α-estrogen receptor (αER) gene in the occurrence of restenosis after coronary stent implantation (in-stent restenosis [ISR]). Methods and Results— In 858 patients (148 women), 955 lesions were treated with stent implantation, and the PvuII C/T and XbaI G/A polymorphisms of the αER gene were determined. Quantitative angiography was performed before and after stenting and at 6-month follow-up. The allelic frequencies were similar between sexes (C/T allele, 0.43/0.57 and 0.44/0.56; P =0.9; G/A allele, 0.35/0.65 and 0.38/0.62; P =0.8; in women and men, respectively). A significantly higher ISR rate in women than in men homozygous for the T-allele of the PvuII polymorphism (42.6% versus 26.9%, P =0.03) or the G-allele of the XbaI polymorphism (41.2% versus 19.4%, P =0.04) was observed. At multivariate analysis, T/T genotype was the only independent predictor of ISR in women but not in men (odds ratio, 1.5; 95% CI, 1.0 to 2.1; P =0.03). XbaI polymorphism was no longer associated with ISR in both sexes. Conclusions— Women homozygous for the T-allele of the PvuII polymorphism of the αER gene treated with coronary stent implantation have a higher risk of ISR than men.

DNA samples from 465 blood donors living in 7 towns of Sicily, the largest island of Italy, have been collected according to well defined criteria, and their genetic heterogeneity tested on the basis of 9 autosomal microsatellite and mitochondrial DNA polymorphisms for a total of 85 microsatellite allele and 10 mtDNA haplogroup frequencies. A preliminary account of the results shows that: a) the samples are genetically heterogeneous; b) the first principal coordinates of the samples are correlated more with their longitude than with their latitude, and this result is even more remarkable when one outlier sample (Butera) is not considered; c) distances among samples calculated from allele and haplogroup frequencies and from the isonymy matrix are weakly correlated (r = 0.43, P = 0.06) but such correlation disappears (r = 0.16) if the mtDNA haplogroups alone are taken into account; d) mtDNA haplogroups and microsatellite distances suggest settlements of people occurred at different times: divergence times inferred from microsatellite data seem to describe a genetic composition of the town of Sciacca mainly derived from settlements after the Roman conquest of Sicily (First Punic war, 246 BC), while all other divergence times take root from the second to the first millennium BC, and therefore seem to backdate to the pre-Hellenistic period. A more reliable association of these diachronic genetic strata to different historical populations (e.g. Sicani, Elymi, Siculi), if possible, must be postponed to the analysis of more samples and hopefully more informative uniparental DNA markers such as the recently available DHPLC-SNP polymorphisms of the Y chromosome.

The Plasma level of angiotensin-converting enzyme (ACE) has been identified as a major risk factor for restenosis after coronary stent implantation in selected patients; ACE inhibition may therefore contribute to prevent its occurrence. The effect of oral ACE inhibition at conventional doses was analyzed retrospectively in a series of 897 patients with ischemia who receivedor=1 coronary stent on 998 lesions and underwent angiographic follow-up; no exclusion criteria were introduced in this analysis. The restenosis rate in 282 patients (31.4%) taking ACE inhibitors was 36.6% compared with 22.9% in 615 non-ACE-inhibited patients (p = 0.00001, odds ratio [OR] 1.94, 95% confidence interval [CI] 1.45 to 2.59), and the late loss in minimum lumen diameter was 1.25 +/- 0.8 versus 0.96 +/- 0.8 mm, respectively (p = 0.0001). During univariate analysis, a negative effect of the drug on restenosis was observed in all subgroups of patients (i.e., hypertensives, diabetics, women, and patients with previous myocardial infarction). Similar effects were observed independently of the ACE gene insertion/deletion polymorphism. During multivariate analysis, ACE inhibition was confirmed as an independent risk factor for restenosis (OR 1.84, 95% CI 1.35 to 2.51, p = 0.0001). Other predictors were the implantation of multiple stents (OR 2.41, 95% CI 1.60 to 3.64, p0.0001), diabetes (OR 2.34, 95% CI 1.61 to 3.41, p0.0001), and vessel reference diameter before angioplasty (OR 0.51, 95% CI 0.38 to 0.69, p0.0001). Although unexplained and apparently contradictory, our data suggest that the use of conventional oral doses of ACE inhibitors in a "real-world" population who underwent coronary stent implantation increases the incidence of in-stent restenosis. Such a finding does not negate the known clinical benefits of ACE inhibitors, but it may deserve attention when a patient treated with ACE inhibitors becomes a candidate for stent implantation.

Mutations seem to be only one of the mechanisms involved in carcinogenesis; selection of mutated clones is a second crucial mechanism. An evolutionary (darwinian) theory of carcinogenesis can be useful to explain some contradictory observations of epidemiology, and to provide a common theoretical framework for carcinogenesis. In both the selection of species and in carcinogenesis (selection of mutated cells), mutation and selection can be interpreted as necessary and insufficient causes. Selection presupposes competition among clones-that is, survival advantage of the mutated species; without selective forces a mutation is mute, while the lack of mutations makes selective advantage impossible. The identification of carcinogen related fingerprints is ambiguous: it can suggest both a genuine mutational hotspot left by the carcinogenic stimulus (like in tobacco related p53 mutations), and selective advantage of clones whose mutations seem to be not exposure specific (like in the case of aflatoxin). We present several examples of exposures that can increase the risk of cancer in humans not via mutations but through a putative mechanism of clone selection.