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Supplementary Table S2. TL quartiles and OS association analysis (Crude Cox regression).

Supplementary Figure S1. Kaplan-Meier OS curves of bladder cancer patients stratified for TL quartiles. OS, overall survival; TL, telomere length.

Background: Shorter telomere length (TL) has been reported to be associated with increased risk of early death in elder individuals. Telomere shortening has been also related to chromosomal instability, which may possibly contribute to the development of several types of digestive or urogenital system cancers and smoking-related tumors. Therefore, we investigated the impact of TL on bladder cancer survival.Methods: TL was measured in leukocyte DNA from whole peripheral blood using quantitative real-time PCR in 463 patients with bladder cancer from a total 726 cases who were followed for up to 18 years.Results: Patients with muscle-invasive tumor/any grade had shorter telomere than patients with non–muscle-invasive tumor/high-grade and with non–muscle-invasive tumor/non–high-grade (TL reference 0.7 ± 0.2; vs. respectively, 0.8 ± 0.2, P = 3.4 × 10−2 and 0.8 ± 0.2, P = 3.6 × 10−2). Moreover, patients in the lowest quartiles of TL were associated with decreased survival after diagnosis (log-rank test, P = 3.9 × 10−4). A Cox regression adjusted by age, cancer aggressiveness, Bacillus Calmette-Guérin, radical cystectomy, radiotherapy, and chemotherapy showed an independent effect of TL on bladder cancer survival (HR, 3.9; 95% confidence interval, 1.7–9.1; P = 1.2 × 10−3).Conclusions: Our results suggest that leukocyte TL is only partly related to tumor aggressiveness and that shorter telomeres act as independent prognostic predictor of survival in patients with bladder cancer. TL information may allow to better select therapeutic approaches in patients with the same stage and grade.Impact: Blood leukocyte TL levels could provide an additional noninvasive prognostic marker to better predict survival and personalize therapies in patients with bladder cancer. Cancer Epidemiol Biomarkers Prev; 23(11); 2439–46. ©2014 AACR.

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. [Cancer Res 2009;69(17):6857–64]

Supplementary Tables 1-8 from Polymorphisms in DNA Repair Genes, Smoking, and Bladder Cancer Risk: Findings from the International Consortium of Bladder Cancer

Supplementary Figure Legend from Polymorphisms in DNA Repair Genes, Smoking, and Bladder Cancer Risk: Findings from the International Consortium of Bladder Cancer

Supplementary Figure 1 from Polymorphisms in DNA Repair Genes, Smoking, and Bladder Cancer Risk: Findings from the International Consortium of Bladder Cancer

Malignant pleural mesothelioma (MPM) is a tumour associated with asbestos exposure. Approximately, 10% of patients with MPM carry a germline pathogenic variant (PV), mostly in DNA repair genes, suggesting the occurrence of inherited predispositions.This article aimed to 1) search for new predisposing genes and assess the prevalence of PVs in DNA repair genes, by next-generation sequencing (NGS) analysis of germline DNA from 113 unselected patients with MPM and 2) evaluate whether these patients could be sensitive to tailored treatments.NGS was performed using a custom panel of 107 cancer-predisposing genes. To investigate the response to selected drugs in conditions of DNA repair insufficiency, we created a three-dimensional-MPM cell model that had a defect in ataxia telangiectasia mutated (ATM), the master regulator of DNA repair.We identified PVs in approximately 7% of patients with MPM (8/113) and a new PV in BAP1 in a further patient with familial MPM. Most of these PVs were in genes involved or supposedly involved in DNA repair (BRCA1, BRIP1, CHEK2, SLX4, FLCN and BAP1). In vitro studies showed apoptosis induction in ATM-silenced/inhibited MPM spheroids treated with an enhancer of zeste homologue 2 inhibitor (tazemetostat).Overall these data suggest that patients with MPM and DNA repair insufficiency may benefit from this treatment, which induces synthetic lethality.

Malignant pleural mesothelioma (MPM) is a rare and aggressive cancer mainly caused by asbestos exposure. Specific and sensitive non-invasive biomarkers may facilitate and enhance screening programs for the early detection of cancer. We investigated DNA methylation (DNAm) profiles in MPM pre-diagnostic blood samples in a case-control study nested in the European Prospective Investigation into Cancer and nutrition (EPIC) cohort, aiming to characterise DNAm biomarkers associated with MPM. From the EPIC cohort, we included samples from 135 participants who developed MPM during 20 years of follow up and from 135 matched, cancer-free, controls. For the discovery phase we selected EPIC participants who developed MPM within five years from enrolment (n=36) with matched controls. We identified nine differentially methylated CpGs, selected by 10-fold cross-validation and correlation analyses: cg25755428 (MRI1), cg20389709 (KLF11), cg23870316, cg13862711 (LHX6), cg06417478 (HOOK2), cg00667948, cg01879420 (AMD1), cg25317025 (RPL17) and cg06205333 (RAP1A). Receiver operating characteristic (ROC) analysis showed that the model including baseline characteristics (age, sex, PC1wbc) along with the nine MPM-related CpGs has a better predictive value for MPM occurrence than the baseline model alone, maintaining some performance also at more than five years before diagnosis [AUC (area under the curve) 10 years=0.75; baseline AUC range=0.63-0.67)]. DNAm changes as non-invasive biomarkers in pre-diagnostic blood samples of MPM cases were investigated for the first time. Their application can improve the identification of asbestos-exposed individuals at higher MPM risk in order to possibly adopt more intensive monitoring for early disease identification.

To reconstruct the phenotypical and clinical implications of the Italian genetic structure, we thoroughly analyzed a whole-exome sequencing data set comprised of 1686 healthy Italian individuals. We found six previously unreported variants with remarkable frequency differences between Northern and Southern Italy in the HERC2, OR52R1, ADH1B, and THBS4 genes. We reported 36 clinically relevant variants (submitted as pathogenic, risk factors, or drug response in ClinVar) with significant frequency differences between Italy and Europe. We then explored putatively pathogenic variants in the Italian exome. On average, our Italian individuals carried 16.6 protein-truncating variants (PTVs), with 2.5% of the population having a PTV in one of the 59 American College of Medical Genetics (ACMG) actionable genes. Lastly, we looked for PTVs that are likely to cause Mendelian diseases. We found four heterozygous PTVs in haploinsufficient genes (KAT6A, PTCH1, and STXBP1) and three homozygous PTVs in genes causing recessive diseases (DPYD, FLG, and PYGM). Comparing frequencies from our data set to other public databases, like gnomAD, we showed the importance of population-specific databases for a more accurate assessment of variant pathogenicity. For this reason, we made aggregated frequencies from our data set publicly available as a tool for both clinicians and researchers (http://nigdb.cineca.it; NIG-ExIT).

Simple Summary Malignant pleural mesothelioma (MPM) is an aggressive and still incurable cancer. There is an urgent need to identify effective and reliable tools for detecting and diagnosing the early onset of MPM. In our study, we investigated the whole miRNAs expression profile from serum extracellular vesicles to identify early changes related to MPM development. miR-11400, miR-148a-3p, and miR-409-3p levels were increased in pre-clinical MPM patients up to five years before their diagnosis. The three-miRNA pattern showed a good discrimination capacity to distinguish pre-clinical MPM from cancer-free controls. The three miRNAs also displayed high diagnostic capabilities for differentiating between MPM patients and controls. This study identified a potential EV miRNA signature in preclinical MPM up to five years before diagnosis and raises the possibility of early intervention. Malignant pleural mesothelioma (MPM) is an aggressive cancer with a dismal prognosis. Early therapeutic interventions could improve patient outcomes. We aimed to identify a pattern of microRNAs (miRNAs) as potential early non-invasive markers of MPM. In a case-control study nested in the European Prospective Investigation into Cancer and Nutrition cohort, we screened the whole miRNome in serum extracellular vesicles (EVs) of preclinical MPM cases. In a subgroup of 20 preclinical samples collected five years prior MPM diagnosis, we observed an upregulation of miR-11400 (fold change (FC) = 2.6, adjusted p-value = 0.01), miR-148a-3p (FC = 1.5, p-value = 0.001), and miR-409-3p (FC = 1.5, p-value = 0.04) relative to matched controls. The 3-miRNA panel showed a good classification capacity with an area under the receiver operating characteristic curve (AUC) of 0.81 (specificity = 0.75, sensitivity = 0.70). The diagnostic ability of the model was also evaluated in an independent retrospective cohort, yielding a higher predictive power (AUC = 0.86). A signature of EV miRNA can be detected up to five years before MPM; moreover, the identified miRNAs could provide functional insights into the molecular changes related to the late carcinogenic process, preceding MPM development.

Background Bladder cancer (BC) has the highest per-patient cost of all cancer types. Hence, we aim to develop a non-invasive, point-of-care tool for the diagnostic and molecular stratification of patients with BC based on combined microRNAs (miRNAs) and surface-enhanced Raman spectroscopy (SERS) profiling of urine. Methods Next-generation sequencing of the whole miRNome and SERS profiling were performed on urine samples collected from 15 patients with BC and 16 control subjects (CTRLs). A retrospective cohort (BC = 66 and CTRL = 50) and RT-qPCR were used to confirm the selected differently expressed miRNAs. Diagnostic accuracy was assessed using machine learning algorithms (logistic regression, naïve Bayes, and random forest), which were trained to discriminate between BC and CTRL, using as input either miRNAs, SERS, or both. The molecular stratification of BC based on miRNA and SERS profiling was performed to discriminate between high-grade and low-grade tumors and between luminal and basal types. Results Combining SERS data with three differentially expressed miRNAs (miR-34a-5p, miR-205-3p, miR-210-3p) yielded an Area Under the Curve (AUC) of 0.92 ± 0.06 in discriminating between BC and CTRL, an accuracy which was superior either to miRNAs (AUC = 0.84 ± 0.03) or SERS data (AUC = 0.84 ± 0.05) individually. When evaluating the classification accuracy for luminal and basal BC, the combination of miRNAs and SERS profiling averaged an AUC of 0.95 ± 0.03 across the three machine learning algorithms, again better than miRNA (AUC = 0.89 ± 0.04) or SERS (AUC = 0.92 ± 0.05) individually, although SERS alone performed better in terms of classification accuracy. Conclusion miRNA profiling synergizes with SERS profiling for point-of-care diagnostic and molecular stratification of BC. By combining the two liquid biopsy methods, a clinically relevant tool that can aid BC patients is envisaged.

Aim: To evaluate the performance of urinary fibrinogen β-chain (FBC) – either alone or associated with urinary tyrosine-phosphorylated proteins (UPY) – as bladder cancer (BCa) diagnostic biomarker. Materials & methods: 164 subjects were tested. Results: Significantly different FBC and UPY levels were found between BCa patients and controls, as well as between low-grade and high-grade cancers. The diagnostic accuracy was 0.84 for FBC and 0.87 for UPY. The combination of FBC and UPY improved the accuracy to 0.91. The addition of clinical variables (age, gender, and smoking habit) to FBC and UPY into a model for BCa prediction significantly improved the accuracy to 0.99. The combination of FBC and UPY adjusted for clinical variables associates with the highest sensitivity and good specificity. Conclusion: Urinary FBC and UPY could be used as biomarkers for BCa diagnosis., Lay abstract This research has developed and validated a highly accurate predictive model for BCa diagnosis based on the combination of two urinary biomarkers, fibrinogen β-chain (FBC), and urinary tyrosine-phosphorylated proteins (UPY), and some clinical variables (age, gender and smoking habit). If the preliminary promising results will be confirmed by external validations and prospective trials in selected clinical scenarios, our model could be transferred to clinical practice for screening of high-risk population., Graphical abstract

Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive tests aimed at an MPM risk assessment tool that might improve life expectancy. Three hundred asbestos-exposed subjects (163 MPM cases and 137 cancer-free controls), from the same geographical region in Italy, were recruited. The evaluation of asbestos exposure was conducted considering the frequency, the duration and the intensity of occupational, environmental and domestic exposure. A genome-wide methylation array was performed to identify novel blood DNA methylation (DNAm) markers of MPM. Multiple regression analyses adjusting for potential confounding factors and interaction between asbestos exposure and DNAm on the MPM odds ratio were applied. Epigenome-wide analysis (EWAS) revealed 12 single-CpGs associated with the disease. Two of these showed high statistical power (99%) and effect size (&gt, 0.05) after false discovery rate (FDR) multiple comparison corrections: (i) cg03546163 in FKBP5, significantly hypomethylated in cases (Mean Difference in beta values (MD) = −0.09, 95% CI = −0.12|−0.06, p = 1.2 × 10−7), and (ii) cg06633438 in MLLT1, statistically hypermethylated in cases (MD = 0.07, 95% CI = 0.04|0.10, p = 1.0 × 10−6). Based on the interaction analysis, asbestos exposure and epigenetic profile together may improve MPM risk assessment. Above-median asbestos exposure and hypomethylation of cg03546163 in FKBP5 (OR = 20.84, 95% CI = 8.71|53.96, p = 5.5 × 10−11) and hypermethylation of cg06633438 in MLLT1 (OR = 11.71, 95% CI = 4.97|29.64, p = 5.9 × 10−8) genes compared to below-median asbestos exposure and hyper/hypomethylation of single-CpG DNAm, respectively. Receiver Operation Characteristics (ROC) for Case-Control Discrimination showed a significant increase in MPM discrimination when DNAm information was added in the model (baseline model, BM: asbestos exposure, age, gender and white blood cells), area under the curve, AUC = 0.75, BM + cg03546163 at FKBP5. AUC = 0.89, 2.1 × 10−7, BM + cg06633438 at MLLT1. AUC = 0.89, 6.3 × 10−8. Validation and replication procedures, considering independent sample size and a different DNAm analysis technique, confirmed the observed associations. Our results suggest the potential application of DNAm profiles in blood to develop noninvasive tests for MPM risk assessment in asbestos-exposed subjects.

Simple Summary Our study is the first one to investigate DNA methylation changes in white blood cells (WBCs) from easily accessible peripheral blood as malignant pleural mesothelioma (MPM) survival biomarker. The Cox proportional hazards regression model highlighted that the methylation status of the CpG dinucleotide cg03546163 is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Biological validation and replication showed that epigenetic changes at the FKBP5 gene were robustly associated with overall survival (OS) in MPM cases. The identification of simple and valuable prognostic markers for MPM will enable clinicians to select patients who are most likely to benefit from aggressive therapies and avoid subjecting non-responder patients to ineffective treatment. Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor with median survival of 12 months and limited effective treatments. The scope of this study was to study the relationship between blood DNA methylation (DNAm) and overall survival (OS) aiming at a noninvasive prognostic test. We investigated a cohort of 159 incident asbestos exposed MPM cases enrolled in an Italian area with high incidence of mesothelioma. Considering 12 months as a cut-off for OS, epigenome-wide association study (EWAS) revealed statistically significant (p value = 7.7 × 10−9) OS-related differential methylation of a single-CpG (cg03546163), located in the 5′UTR region of the FKBP5 gene. This is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Cases with DNAm < 0.45 at the cg03546163 had significantly poor survival compared with those showing DNAm ≥ 0.45 (mean: 243 versus 534 days; p value< 0.001). Epigenetic changes at the FKBP5 gene were robustly associated with OS in MPM cases. Our results showed that blood DNA methylation levels could be promising and dynamic prognostic biomarkers in MPM.

Bladder cancer (BC) is the tenth most frequent cancer worldwide. Due to the need for recurrent cystoscopies and the lack of non-invasive biomarkers, BC is associated with a high management burden. In this respect, small non-coding RNAs (sncRNAs) have been investigated in urine as possible biomarkers for BC, but in plasma their potential has not yet been defined. The expression levels of sncRNAs contained in plasma extracellular vesicles (EVs) from 47 men with BC and 46 healthy controls were assessed by next-generation sequencing. The sncRNA profiles were compared with urinary profiles from the same subjects. miR-4508 resulted downregulated in plasma EVs of muscle-invasive BC patients, compared to controls (adj-p = 0.04). In World Health Organization (WHO) grade 3 (G3) BC, miR-126-3p was upregulated both in plasma EVs and urine, when compared to controls (for both, adj-p &lt, 0.05). Interestingly, two sncRNAs were associated with the risk class: miR-4508 with a downward trend going from controls to high risk BC, and piR-hsa-5936 with an upward trend (adj-p = 0.04 and adj-p = 0.05, respectively). Additionally, BC cases with low expression of miR-185-5p and miR-106a-5p or high expression of miR-10b-5p showed shorter survival (adj-p = 0.0013, adj-p = 0.039 and adj-p = 0.047, respectively). SncRNAs from plasma EVs could be diagnostic biomarkers for BC, especially in advanced grade.

Leukocyte telomere length (LTL) is a heritable biomarker of genomic aging. In this study, we perform a genome-wide meta-analysis of LTL by pooling densely genotyped and imputed association results across large-scale European-descent studies including up to 78,592 individuals. We identify 49 genomic regions at a false dicovery rate (FDR) < 0.05 threshold and prioritize genes at 31, with five highlighting nucleotide metabolism as an important regulator of LTL. We report six genome-wide significant loci in or near SENP7, MOB1B, CARMIL1, PRRC2A, TERF2, and RFWD3, and our results support recently identified PARP1, POT1, ATM, and MPHOSPH6 loci. Phenome-wide analyses in >350,000 UK Biobank participants suggest that genetically shorter telomere length increases the risk of hypothyroidism and decreases the risk of thyroid cancer, lymphoma, and a range of proliferative conditions. Our results replicate previously reported associations with increased risk of coronary artery disease and lower risk for multiple cancer types. Our findings substantially expand current knowledge on genes that regulate LTL and their impact on human health and disease.

Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter-individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co-twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co-twins were contrasted, selecting as variables the intra-twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke-induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551–559, 2017. © 2017 Wiley Periodicals, Inc.

Introduction Malignant pleural mesothelioma (MPM) is an aggressive tumor strongly associated with asbestos exposure. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive early diagnostic tests to monitor asbestos-exposed people. Methods We used a genome-wide methylation array to identify, in asbestos-exposed subjects, novel blood DNA methylation markers of MPM in 163 MPM cases and 137 cancer-free controls (82 MPM cases and 68 controls, training set; replication in 81 MPM cases and 69 controls, test set) sampled from the same areas. Results Evidence of differential methylation between MPM cases and controls was found (more than 800 cytosine-guanine dinucleotide sites, false discovery rate p value (pfdr) Conclusions We identified signatures of differential methylation in DNA from whole blood between asbestos exposed MPM cases and controls. Our results provide the rationale to further investigate, in prospective studies, the potential use of blood DNA methylation profiles for the identification of early changes related to the MPM carcinogenic process.

BACKGROUND One-carbon metabolism-important for DNA stability and integrity-may play a role in breast carcinogenesis. However, epidemiologic studies addressing this issue have yielded inconsistent results. OBJECTIVE We prospectively investigated associations between breast cancer and plasma folate, riboflavin, vitamin B-6, vitamin B-12, and homocysteine in women recruited to the Varese (Italy) cohort of the EPIC (European Prospective Investigation into Cancer and Nutrition) study. METHODS We performed a nested case-control study on women aged 35-65 y at recruitment with a median body mass index of 25.3 kg/m(2) who gave blood samples in 1987-1992 and again in 1993-1998. Breast cancer cases identified by 31 December 2009 were individually matched to controls. RRs of breast cancer (and subtypes defined by hormone receptor status) with 95% CIs were estimated by unconditional logistic regression, controlling for matching factors and breast cancer risk factors. RESULTS After a median of 14.9 y, 276 breast cancer cases were identified and matched to 276 controls. Increasing plasma vitamin B-6 was associated with decreased risk of overall (RR: 0.78; 95% CI: 0.63, 0.96 for 1-SD increase), premenopausal (RR: 0.66; 95% CI: 0.48, 0.92 for 1-SD increase), estrogen receptor-positive (RR: 0.79; 95% CI: 0.63, 1.00 for 1-SD increase), and progesterone receptor-positive (RR: 0.72; 95% CI: 0.55, 0.95 for 1-SD increase) breast cancers. Increased plasma vitamin B-6 was also associated with decreased breast cancer risk in alcohol consumers (≥7 g/d) compared with consumption of

Malignant pleural mesothelioma (MPM) is an aggressive tumor with median survival of 12 months and limited effective treatments. The scope of this study was to study the relationship between blood DNA methylation (DNAm) and overall survival (OS) aiming at a noninvasive prognostic test. We investigated a cohort of 159 incident asbestos exposed MPM cases enrolled in an Italian area with high incidence of mesothelioma. Considering 12 months as a cut-off for OS, epigenome-wide association study (EWAS) revealed statistically significant (p value = 7.7 &times, 10&minus, 9) OS-related differential methylation of a single-CpG (cg03546163), located in the 5&prime, UTR region of the FKBP5 gene. This is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Cases with DNAm &lt, 0.45 at the cg03546163 had significantly poor survival compared with those showing DNAm &ge, 0.45 (mean: 243 versus 534 days, p value&lt, 0.001). Epigenetic changes at the FKBP5 gene were robustly associated with OS in MPM cases. Our results showed that blood DNA methylation levels could be promising and dynamic prognostic biomarkers in MPM.

Urothelial bladder cancer (UBC) represents a public health problem because of its high incidence/relapse rates. At present, there are no suitable biomarkers for early diagnosis or relapse/progression prognosis. To improve diagnostic accuracy and overcome the disadvantages of current diagnostic strategies, the detection of UBC biomarkers in easily accessible biofluids, such as urine, represents a promising approach compared with painful biopsies. We investigated the levels of MMP23 genes (microarray and qPCR) and protein (western blot and enzyme-linked immunosorbent assay) in a set of samples (blood, plasma and urine) from patients with UBC and controls as biomarkers for this cancer. MMP23B and its pseudogene MMP23A resulted downregulated in blood cells from UBC compared with controls (66 cases, 70 controls; adjusted P-value = 0.02 and 0.03, respectively). In contrast, MMP23B protein levels in plasma (53 UBC, 49 controls) and urine (59 UBC, 57 controls) increased in cases, being statistically significant in urine. MMP23B dosage observed in urine samples was related to both tumor risk classification and grading. As the lack of correlation between mRNA and protein levels could be due to a posttranscriptional regulation mediated by microRNAs (miRNAs), we investigated the expression of urinary miRNAs targeting MMP23B. Five miRNAs resulted differentially expressed between cases and controls. We reported the first evidence of MMP23B secretion in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC as a potential non-invasive biomarker for this cancer. Further analyses are needed to elucidate the mechanism of regulation of MMP23B expression by miRNAs.

Occupational and environmental asbestos exposure is the main determinant of malignant pleural mesothelioma (MPM), however, the mechanisms by which its fibres contribute to cell toxicity and transformation are not completely clear. Aberrant DNA methylation is a common event in cancer but epigenetic modifications involved specifically in MPM carcinogenesis need to be better clarified. To investigate asbestos-induced DNA methylation and gene expression changes, we treated Met5A mesothelial cells with different concentrations of crocidolite and chrysotile asbestos (0.5 ÷ 5.0 µg/cm2, 72 h incubation). Overall, we observed 243 and 302 differentially methylated CpGs (≥ 10%) between the asbestos dose at 5 µg/cm2 and untreated control, in chrysotile and crocidolite treatment, respectively. To examine the dose–response effect, Spearman’s correlation test was performed and significant CpGs located in genes involved in migration/cell adhesion processes were identified in both treatments. Moreover, we found that both crocidolite and chrysotile exposure induced a significant up-regulation of CA9 and SRGN (log2 fold change > 1.5), previously reported as associated with a more aggressive MPM phenotype. However, we found no correlation between methylation and gene expression changes, except for a moderate significant inverse correlation at the promoter region of DKK1 (Spearman rho = − 1, P value = 0.02) after chrysotile exposure. These results describe for the first time the relationship between DNA methylation modifications and asbestos exposure. Our findings provide a basis to further explore and validate asbestos-induced DNA methylation changes, that could influence MPM carcinogenesis and possibly identifying new chemopreventive target.

Bladder cancer (BC) has a typical aetiology characterized by a multistep carcinogenesis due to environmental exposures, genetic susceptibility, and their interaction. Several lines of evidence suggest that DNA repair plays a role in the development and progression of BC. In particular, the study of individual susceptibility to DNA double strand breaks (DSBs) may provide valuable information on BC risk, and help to identify those patients at high-risk of either recurrence or progression of the disease, possibly personalizing both surveillance and treatment. Among the different DSB markers, the most well characterized is phosphorylation of the histone H2AX (γ-H2AX). We assessed any potential role of γ-H2AX as a molecular biomarker in a case-control study (146 cases and 146 controls) to identify individuals with increased BC risk and at high-risk of disease recurrence or progression. We investigated γ-H2AX levels in peripheral blood mononuclear cells before and after their exposure to ionizing radiation (IR). We did not find any significant difference among cases and controls. However, we observed a significant association between γ-H2AX basal levels and risk of disease recurrence or progression. In particular, both BC patients as a whole and the subgroup of non-muscle invasive BC (NMIBC) with high basal H2AX phosphorylation levels had a decreased risk of recurrence or progression (for all BC HR 0.70, 95%CI 0.52-0.94, P = 0.02; for NMIBC HR 0.68, 95%CI 0.50-0.92, P = 0.01), suggesting a protective effect of basal DSB signaling. Our data suggest that γ-H2AX can be considered as a potential molecular biomarker to identify patients with a higher risk of BC recurrence. © 2015 Wiley Periodicals, Inc.

Breast cancer (BC) is the leading cause of cancer death in women. Adipokines, and other inflammation molecules linked to adiposity, are suspected to be involved in breast carcinogenesis, however prospective findings are inconclusive. In a prospective nested case-control study within the EPIC-Varese cohort, we used conditional logistic regression to estimate rate ratios (RRs) for BC, with 95% confidence intervals (CI), in relation to plasma levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), interleukin-6, leptin, and adiponectin, controlling for BC risk factors. After a median 14.9 years, 351 BC cases were identified and matched to 351 controls. No marker was significantly associated with BC risk overall. Significant interactions between menopausal status and CRP, leptin, and adiponectin were found. Among postmenopausal women, high CRP was significantly associated with increased BC risk, and high adiponectin with significantly reduced risk. Among premenopausal women, high TNF-α was associated with significantly increased risk, and high leptin with reduced risk; interleukin-6 was associated with increased risk only in a continuous model. These findings constitute further evidence that inflammation plays a role in breast cancer. Interventions to lower CRP, TNF-α, and interleukin-6 and increase adiponectin levels may contribute to preventing BC.

Background Bladder cancer (BC) is one of the most aggressive malignancies of the urinary tract, with the highest lifetime treatment costs per patient of all cancers, due to the high rate of recurrences requiring continuous surveillance. An early diagnosis is essential to improve survival of patients with BC. Noninvasive and sensitive molecular biomarkers are needed to improve current strategies for the detection and monitoring of BC. Previous studies suggested that elevated DNA damage levels and suboptimal nucleotide excision DNA repair (NER) may be associated with BC. Methods In the present study, we investigated basal DNA damage and DNA repair capacity in peripheral blood mononuclear cells (PBMCs) from 146 newly diagnosed patients with BC and 155 controls using a modified comet assay able to evaluate NER activity after challenging cells by benzo(a)pyrene diolepoxide (BPDE). Results We found an association between DNA damage levels in PBMCs of BC cases and patients’ outcomes. Basal DNA damage at diagnosis was significantly increasing with tumor grades (trend test, P = 0.02) and risk classes (trend test, P = 0.02). The overall survival analysis showed that DNA damage in patients at BC diagnosis was significantly higher in subjects with a shorter survival time (hazard ratio = 3.7; 95% CI: 1.3–10.6; P = 0.02). Conclusions Based on these data, we suggest that DNA damage levels measured in PBMCs of patients with BC may potentially represent a prognostic marker associated with poor survival; further validation is needed to better stratify patients with BC for clinical trials.

Background: Bladder cancer (BC) is among the most common malignancies worldwide. The identification of new biomarkers for early BC detection, recurrence/progression is urgently needed. The cytokinesis-block micronucleus assay (CBMN) evaluates chromosome damage in cultured human lymphocytes and micronuclei (MN) provide a convenient and reliable index of both chromosome breakage and loss. Methods: Chromosomal damage (expressed as frequencies of MN, nucleoplasmic bridges and nuclear buds (NBUD)) was evaluated by CBMN assay in cryopreserved lymphocytes from 158 age/smoking-matched pairs of cases and controls in relation to BC risk, recurrence or progression. Moreover, non-muscle invasive BC (NMIBC) patients were characterised for 783 DNA repair gene polymorphisms for their possible association with the investigated cytogenetic end points. Results: MN and NBUD frequencies were significantly higher in cases than in controls (P=0.001 and P=0.006, respectively), with the associations being stronger in NMIBC. In a logistic regression model, for each increase of one unit in the MN frequency, a 1.12 increased risk of developing NMIBC was observed. In NMIBC cases, 10 polymorphisms were associated with different MN frequencies after genotype stratification. Conclusions: A model including traditional BC risk factors, MN frequency and the selected polymorphisms differentially distributed in cases and controls improved BC patient identification. Understanding the meaning of systemic chromosomal damage in BC patients with respect to the general population may help to adopt specific prevention strategies and therapeutic intervention.

Increased telomerase expression has been implicated in the pathogenesis of lung cancer and, since the primary cause of lung cancer is smoking, an association between telomerase reactivation and tobacco smoke has been proposed. In this work an investigation has been performed to assess the relationship between tobacco smoke exposure and telomerase activity (TA) in peripheral blood mononuclear cells of healthy smokers. The methylation status of the catalytic subunit of telomerase hTERT was concurrently investigated to assess the possible association between epigenetic modifications of hTERT and TA. Besides, the association between smoke and telomere length (TL) has been evaluated. Healthy monozygotic twins with discordant smoking habits were selected as study population to minimize inter-individual differences because of demographic characteristics and genetic heterogeneity. Statistically significant higher values of TA and TL were observed in smokers compared to nonsmoker co-twins. The multivariate analysis of data showed, besides smoking habits (P = 0.02), an influence of gender (P = 0.006) and BMI (P = 0.001) on TA and a borderline effect of gender (P = 0.05) on TL. DNA methylation analysis, focused on 100 CpG sites mapping in hTERT, highlighted nine CpG sites differentially methylated in smokers. When co-twins were contrasted, selecting as variables the intra-twin difference in TA and hTERT DNA methylation, a statistically significant inverse correlation (P = 0.003) was observed between TA and DNA methylation at the cg05521538 site. In conclusion, these results indicate an association of tobacco smoke with TA and TL and suggest a possible association between smoke-induced epigenetic effects and TA in healthy smokers. Environ. Mol. Mutagen. 58:551-559, 2017. © 2017 Wiley Periodicals, Inc.

Background: Shorter telomere length (TL) has been reported to be associated with increased risk of early death in elder individuals. Telomere shortening has been also related to chromosomal instability, which may possibly contribute to the development of several types of digestive or urogenital system cancers and smoking-related tumors. Therefore, we investigated the impact of TL on bladder cancer survival. Methods: TL was measured in leukocyte DNA from whole peripheral blood using quantitative real-time PCR in 463 patients with bladder cancer from a total 726 cases who were followed for up to 18 years. Results: Patients with muscle-invasive tumor/any grade had shorter telomere than patients with non–muscle-invasive tumor/high-grade and with non–muscle-invasive tumor/non–high-grade (TL reference 0.7 ± 0.2; vs. respectively, 0.8 ± 0.2, P = 3.4 × 10−2 and 0.8 ± 0.2, P = 3.6 × 10−2). Moreover, patients in the lowest quartiles of TL were associated with decreased survival after diagnosis (log-rank test, P = 3.9 × 10−4). A Cox regression adjusted by age, cancer aggressiveness, Bacillus Calmette-Guérin, radical cystectomy, radiotherapy, and chemotherapy showed an independent effect of TL on bladder cancer survival (HR, 3.9; 95% confidence interval, 1.7–9.1; P = 1.2 × 10−3). Conclusions: Our results suggest that leukocyte TL is only partly related to tumor aggressiveness and that shorter telomeres act as independent prognostic predictor of survival in patients with bladder cancer. TL information may allow to better select therapeutic approaches in patients with the same stage and grade. Impact: Blood leukocyte TL levels could provide an additional noninvasive prognostic marker to better predict survival and personalize therapies in patients with bladder cancer. Cancer Epidemiol Biomarkers Prev; 23(11); 2439–46. ©2014 AACR.

Survival of bladder cancer patients depends on several factors including disease stage and grade at diagnosis, age, health status of the patient and the applied treatment. Several studies investigated the role of DNA repair genetic variants in cancer susceptibility, but only few studies investigated their role in survival and response to chemotherapy for bladder cancer. We genotyped 28 single nucleotide polymorphisms (SNP) in DNA repair genes in 456 bladder cancer patients, reconstructed haplotypes and calculated a score for combinations of the SNPs. We estimated Hazard Ratios (adjHR) for time to death. Among patients treated with chemotherapy, variant alleles of five SNPs in the XRCC1 gene conferred better survival (rs915927 adjHR 0.55 (95%CI 0.32–0.94); rs76507 adjHR 0.48 (95%CI 0.27–0.84); rs2854501 adjHR 0.25 (95%CI 0.12–0.52); rs2854509 adjHR 0.21 (95%CI 0.09–0.46); rs3213255 adjHR 0.46 (95%CI 0.26–0.80). In this group of patients, an increasing number of variant alleles in a XRCC1 gene score were associated with a better survival (26% decrease of risk of death for each additional variant allele in XRCC1). By functional analyses we demonstrated that the previous XRCC1 SNPs confer lower DNA repair capacity. This may support the hypothesis that survival in these patients may be modulated by the different DNA repair capacity determined by genetic variants. Chemotherapy treated cancer patients bearing an increasing number of “risky” alleles in XRCC1 gene had a better survival, suggesting that a proficient DNA repair may result in resistance to therapy and shorter survival. This finding may have clinical implications for the choice of therapy.

Due to the great number of genes involved in DNA repair and the interactions among the pathways responsible for the repair of different types of DNA damage, there is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacity (DRC). The use of peripheral blood mononuclear cells (PBMCs) in DRC assays is particularly useful for human monitoring studies. However, in such studies it is not always possible to collect and process samples on the same day as the blood is taken. We performed a genotype-phenotype correlation study on DRC on 225 healthy subjects. Due to the large number of blood samples to be processed, PBMCs were either isolated and cryopreserved on the same day of blood collection (day 1) or on the following day after 24h blood storage at room temperature (day 2-RT). Samples processed in different days showed a significant difference in the DRC evaluated as 8-oxoguanine glycosylase activity (OGG assay) in cell extracts (p

Obesity is an important health problem worldwide. Adipose tissue acts as an endocrine organ that secretes various bioactive substances, called adipokines, including pro-inflammatory biomarkers such as TNF-α, IL-6, leptin and C-reactive protein (CRP) and anti-inflammatory molecules such as adiponectin. The deregulated production of adipokines in obesity is linked to the pathogenesis of various disease processes and monitoring their variation is critical to understand metabolic diseases. The aim of this study was to determine the plasma concentration of adipokines in healthy subjects by multiplexed measurements and the effect of anticoagulants on their levels. Plasma samples from 10 healthy donors were collected in two different anticoagulants (sodium citrate or heparin). All markers, excluding TNF-α, showed significantly higher concentrations in heparinized compared to citrate plasma. However, levels of adipokines in different plasma samples were highly correlated for most of these markers. We reported that different anticoagulants used in the preparation of the plasma samples affected the measurements of some adipokines. The importance of the present results in epidemiology is relevant when comparing different studies in which blood samples were collected with different anticoagulants.

Inter-individual susceptibility to mutagens/carcinogens can be assessed by either genotyping DNA repair genes in different pathways or phenotyping DNA repair capacity (DRC) at the molecular or cellular level. Due to the large number of known DNA repair genes, and the interactions between repair pathways, phenotyping is becoming the preferred approach to measure DRC, and reliable assays are therefore increasingly needed. The use of a cellular phenotype comet assay for the nucleotide excision repair (NER) pathway using benzo[a]pyrene diol epoxide (BPDE) has been described in previous papers, but no thorough evaluation of its applicability in large genotype-phenotype studies has been presented. Our aim was to evaluate the possibility of using cryopreserved instead of fresh peripheral blood mononuclear cells (PBMCs) to evaluate intra- and inter-assay variation, and inter-individual variation, for the aphidicolin (APC)-block NER comet assay. Moreover, we measured the variation for the designated internal standard (K562 erythroleukaemia cell line) and we evaluated the feasibility to use lymphoblastoid cell lines (LCLs) as surrogate of PBMCs. Our results showed a low intra-assay [coefficient of variation (CV) 19.9%] and inter-assay (CV 32.3%) variation, with a good inter-individual variation (122 subjects, mean ± standard deviation 7.38 ± 4.99; range 0.66-26.14; CV 67.63%). A significant correlation between results derived from cryopreserved and fresh PBMCs from the same individuals was found (10 subjects, r = 0.62, P = 0.05). Results from LCLs and cryopreserved PBMCs from the same subjects showed an inverse significant correlation (10 subjects, r = -0.712, P = 0.02). K562 cells as internal standard showed low intra-assay variation. In the present study the APC-block NER comet assay on cryopreserved PBMCs seemed to be a reliable method to measure DRC variation in epidemiological studies; LCLs were not a good surrogate in this assay. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (Italy; G.M.), the Progetto Integrato Oncologia, Regione Toscana—Ministero della Salute ‘Identification of population risk profiles as an approach to cancer prevention’ and the Environmental Cancer Risk Nutrition and Individual Susceptibility project (G.M., M.K.V.), a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943).

The 8q24 region is a gene desert, although chromosomal aberrations and somatic amplification involving this region, including translocations involving the protooncogene c-MYC, have been frequently reported in people with cancer. To investigate the role of variants in 8q24 region, the authors analyzed data from a prospective study (n = 10,372 participants who were followed for 11 years) in which a large number of health events (>1,500) occurred (1993-1998). They genotyped all subjects for 5 candidate single nucleotide polymorphisms (rs672888, rs1447295, rs9642880, rs16901979, and rs6983267) that were identified in previous genome-wide scans. Although significant associations with individual single nucleotide polymorphisms were small in magnitude, the authors observed higher increases in the risks of different types of cancer with specific haplotypes, particularly when subjects were homozygous for the haplotype: for breast cancer and homozygotes for haplotype CAGCT, hazard ratio = 3.40, 95% confidence interval: 1.24, 9.21; for prostate cancer and grouped rare haplotypes, hazard ratio = 7.43, 95% confidence interval: 3.00, 18.37; and for brain cancer and homozygotes for haplotype CGGCT, hazard ratio = 13.48, 95% confidence interval: 3.00, 59.53. Significant associations were also observed between haplotypes and deaths from cardiovascular diseases and cerebrovascular diseases; the most stable association was between homozygotes for haplotypes CGTCG and CAGCT and total deaths in men (hazard ratio = 3.5, 95% confidence interval: 1.8, 6.9, and hazard ratio = 2.8, 95% confidence interval: 1.3, 6.4, respectively). In conclusion, the authors have observed a strong pleiotropic effect of the 8q24 region in a large prospective study. This observation can shed light on the mechanisms underlying reported associations between 8q24 variants and disparate chronic diseases. This work was supported by grants from the Associazione Italiana per le Ricerche sul Cancro, Italy (to GiuseppeMatullo) and the Environmental Cancer Risk Nutrition and Individual Susceptibility project (to Giuseppe Matullo and Paolo Vineis), a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘‘Food Quality and Safety’’ (contract No.513943). This work was also supported by grants from the Compagnia di San Paolo, Turin, Italy (to Paolo Vineis and Giuseppe Matullo) and the Progetto Integrato Oncologia of the Italian government (to Paolo Vineis and Giuseppe Matullo).

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratory's own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories. This work was supported by 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).

Background DNA methylation profiles are responsive to environmental stimuli and metabolic shifts. This makes DNA methylation a potential biomarker of environmental-related and lifestyle-driven diseases of adulthood. Therefore, we investigated if white blood cells’ (WBCs) DNA methylation profiles are associated with myocardial infarction (MI) occurrence. Whole-genome DNA methylation was investigated by microarray analysis in 292 MI cases and 292 matched controls from the large prospective Italian European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (EPICOR study). Significant signals (false discovery rate (FDR) adjusted P

Several lines of evidence suggest an association between oxidative DNA-damage repair capacity and cancer risk. In particular, a DNA-glycosylase assay for removal of 8-oxoguanine (8-oxoG) in peripheral blood mononuclear cells (PBMC) has been successfully applied to identify populations with increased risk for lung cancer and squamous cell carcinomas of head and neck. In order to verify whether EBV-transformed lymphoblastoid cell lines (LCL) are a suitable surrogate for PBMC in specific DNA-repair phenotypic assays, a validation trial was conducted. PBMC from 20 healthy subjects were collected and an aliquot was transformed with EBV to obtain LCL. The ability of cell-free extracts from both cell types to incise a 3'-fluorescently labelled duplex oligonucleotide containing a single 8-oxoG (OGG assay) was evaluated. Since this activity is mediated predominantly by OGG1, the OGG1 gene expression was also measured. 8-oxoG DNA-glycosylase activity and OGG1 expression were significantly higher (p

DNA double-strand breaks (DSB) are the most lethal form of ionizing radiation-induced DNA damage, and failure to repair them results in cell death. In order to see if any associations exist between DNA repair gene polymorphisms and phenotypic profiles of DSB repair (DSBR) we performed a genotype-phenotype correlation study in 118 young healthy subjects (mean age 25.8±6.7years). Subjects were genotyped for 768 single nucleotide polymorphisms (SNPs) with a custom Illumina Golden Gate Assay, and an H2AX histone phosphorylation assay was done to test DSBR capacity. We found that H2AX phosphorylation at 1h was significantly lower in subjects heterozygous (no variant homozygotes were observed) for the XPA gene SNP rs3176683 (p-value=0.005), while dephosphorylation was significantly higher in subjects carrying the variant allele in three MRE11A gene SNPs: rs1014666, rs476137 and rs2508784 (p-value=0.003, 0.003 and 0.008, respectively). An additive effect of low-activity DNA repair alleles was associated with altered DSBR activity, as demonstrated by both H2AX phosphorylation at 1 h (p-trend

Risk factors for gastric cancer (GC) include inter-individual variability in the inflammatory response to Helicobacter pylori infection, in the ability of detoxifying DNA reactive species and repairing DNA damage generated by oxidative stress and dietary carcinogens. To evaluate the association between polymorphic DNA repair genes and GC risk, a case-control study including 314 histologically confirmed GC patients and 548 healthy controls was conducted in a GC high-risk area in Tuscany, Italy. Polymorphic variants of base excision repair (APE1-D148E, XRCC1-R194W, XRCC1-R399Q and OGG1-S326C), nucleotide excision repair (XPC-PAT, XPA-23G> A, ERCC1-19007T> C and XPD-L751Q), recombination (XRCC3-T241M) and alkylation damage reversal (MGMT-L84F) were tested for their potential role in the development of GC by using logistic regression models. The same population was also characterised for GSTT1 and GSTM1 variant alleles to search for possible functional interactions between metabolic and DNA repair genotypes by two-way interactions using multivariate logistic models. No significant association between any single DNA repair genotype and GC risk was detected with a borderline association with the XPC-PAT homozygous genotype [odds ratio (OR) = 1.42; 95% confidence interval (CI) 0.94-2.17]. Gene-gene interaction analysis revealed combinations of unfavourable genotypes involving either multiple DNA repair polymorphisms or DNA repair and GST-specific genotypes. The combination of the XPC-PAT and the XPA variant alleles significantly increased GC risk (OR = 2.15; 95% CI 1.17-3.93, P = 0.0092). A significant interaction was also found between the APE1 wild-type genotype and either the single GSTT1 (OR = 4.90; 95% CI 2.38-10.11, P = 0.0079) or double GSTM1-GSTT1 null (OR = 7.84; 95% CI 3.19-19.22, P = 0.0169) genotypes or the XPA-mutant allele (OR = 3.56; 95% CI 1.53-8.25, P = 0.0012). These findings indicate that a complex interaction between host factors such as oxidative stress, antioxidant capacity and efficiency of multiple DNA repair pathways underlies the inter-individual variability in GC risk.

Urothelial bladder cancer (UBC) represents a public health problem because of its elevated incidence and relapse rate. After prostate cancer, UBC is the most frequent malignancy of the urinary tract with a higher incidence in men and smokers. To date, there are no suitable biomarkers for an early diagnosis or detection of relapse/progression. In the attempt to improve diagnostic accuracy and to overcome the disadvantages of current diagnostic strategies, biomarkers found in easily accessible biofluids, such as blood or urine, represent a non-invasive and promising approach. The first aim of this study was the identification of reliable biomarkers of UBC risk starting with whole gene expression profile in white blood cells from 66 UBC cases and 70 controls. We observed a lower expression of MMP23B and its relative pseudogene MMP23A in UBC compared to controls (LogFC=-0.37, FDR adjusted p-value=0.02 and LogFC=-0.23, FDR adjusted p-value=0.03, respectively, adjusted for age, smoking, and batch). Thus, we investigated MMP23B protein levels both in plasma (49 controls and 53 UBC) and in urine (57 controls and 59 UBC) samples, to confirm gene expression observations. Unexpectedly, both western blot evidences and ELISA quantification showed increased MMP23B levels in UBC cases respect to controls, reaching a statistical significance in urine (mean UBC=1797.70pg/ml, mean controls=1075.73pg/ml, p-value=0.02). A positive trend of association of MMP23 levels was observed also for grade and risk. As the lack of correlation between mRNA and protein levels could be due to a post-transcriptional crosstalk mediated by microRNAs (miRNAs), we investigated the expression levels of miRNAs targeting MMP23B gene in urine of UBC patients and controls. We identified 5 differentially expressed miRNAs targeting MMP23B 3'UTR in UBC cases and controls. The present outcomes indicate a potential role of MMP23B as non-invasive UBC biomarkers. Moreover, we reported the first evidence of MMP23B secretion both in plasma and urine, suggesting a role of this poorly characterized metalloproteinase in UBC. Further analyses are needed to better elucidate the mechanism of regulation of MMP23B expression by miRNAs present in UBC urine. Citation Format: Barbara Pardini, Alessandra Allione, Clara Viberti, Giuliana Giribaldi, Stefano Turini, Cornelia Di Gaetano, Simonetta Guarrera, Francesca Cordero, Marco Oderda, Marco Allasia, Paolo Gontero, Carlotta Sacerdote, Alessio Naccarati, Paolo Vineis, Giuseppe Matullo. MMP23B expression and protein levels in blood and urine are associated with bladder cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1258.

Formaldehyde is an ubiquitous pollutant to which humans are exposed. Pathologists can experience high formaldehyde exposure levels. Formaldehyde-among other properties-induce oxidative stress and free radicals, which react with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. We measured the levels of air-formaldehyde exposure in a group of Italian pathologists and controls. We analyzed the effect of formaldehyde exposure on leukocyte malondialdehyde-deoxyguanosine adducts (M(1)-dG), a biomarker of oxidative stress and lipid peroxidation. We studied the relationship between air-formaldehyde and M(1)-dG adducts. Air-formaldehyde levels were measured by personal air samplers. M(1)-dG adducts were analyzed by a (32)P-postlabeling assay. Reduction room pathologists were significantly exposed to air-formaldehyde with respect to controls and to the pathologists working in other laboratory areas (p0.001). A significant difference for M(1)-dG adducts between exposed pathologists and controls was found (p = 0.045). The effect becomes stronger when the evaluation of air-formaldehyde exposure was based on personal samplers (p = 0.018). Increased M(1)dG adduct levels were only found in individuals exposed to air-formaldehyde concentrations higher than 66 microg/m(3). When the exposed workers and controls were subgrouped according to smoking, M(1)-dG tended to increase in all of the subjects, but a significant association between M(1)-dG and air-formaldehyde was only found in nonsmokers (p = 0.009). Air-formaldehyde played a role positive but not significant (r = 0.355, p = 0.075, Pearson correlation) in the formation of M(1)-dG, only in nonsmokers. Working in the reduction rooms and exposure to air-formaldehyde concentrations higher than 66 microg/m(3) are associated with increased levels of M(1)-dG adducts.

As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by gamma-rays and DNA repair capacity were evaluated in unstimulated (G(0)) and mitogen-simulated (G(2)) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G(0)-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G(2)-treated PBMC compared to LCL. Concerning gamma-H2AX measurements, phosphorylation levels 1h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of gamma-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype-phenotype correlations with phenotypic DNA repair assays, especially when low impact functional genetic variants are involved. This work was supported by a grant of the Associazione Italiana per le Ricerche sul Cancro (Italy; G.M., A.Z., C.G.), of the Environmental Cancer Risk Nutrition and Individual Susceptibility project (G.M.), a network of excellence operating within the European Union Sixth Framework Program, Priority 5: ‘Food Quality and Safety’ (Contract No. 513943) and of the Agenzia Spaziale Italiana (A.Z.). A grant contribution has also been provided by the Programma Integrato Oncologia (PIO) (G.M.) and by Regione Piemonte Ricerca Sanitaria Finalizzata 2008 and 2009 (G.M. and C.G.). A grant contribution has been given by the Progetto Ricerca Sanitaria Finalizzata Regione Piemonte 2008 (A.F.).

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N ≤ 71,225 European ancestry, N ≤ 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10(-24)), CYP1A2 (P = 1 × 10(-23)), FGF5 (P = 1 × 10(-21)), SH2B3 (P = 3 × 10(-18)), MTHFR (P = 2 × 10(-13)), c10orf107 (P = 1 × 10(-9)), ZNF652 (P = 5 × 10(-9)) and PLCD3 (P = 1 × 10(-8)) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

The (32)P-post-labelling assay has emerged as a major tool for detecting bulky DNA adducts in subjects exposed to carcinogens, especially aromatic compounds. However, the (32)P-post-labelling protocol still requires the use of high amounts of radioactivity, i.e. 25-50 muCi per sample, an obstacle that limits its use in large studies. The characterization of the DNA adducts measured is also limited. Methodological improvements and increased DNA adduct characterization are necessary to make this assay capable of achieving higher throughput. A new protocol was tested to ensure efficient hydrolysis to reduce the use of radioactive material and to obtain higher chromatography resolution. Different chromatography systems based on high-urea or ammonium hydroxide systems were also employed to characterize the adducts being measured. Improvements were tested by re-analysing DNA adducts in a group of police officers and urban residents in Genoa, Italy. The analysis of carcinogen-modified DNA standards was also included in the study for qualitative and quantitative comparison. An efficient DNA digestion was obtained using a method involving hydrolysis by micrococcal nuclease and a mixture of two spleen phosphodiesterases at fixed concentrations. A 72% reduction of the amount of radioactivity used for labelling was achieved in respect to the non-modified protocol without loss of DNA adduct sensitivity. An improved chromatography resolution was obtained by reducing the volume of sample to be spotted on the chromatogram. Lower volume of spotting sample can decrease sample diffusion and the formation of unresolved spots on the thin-layer chromatography plate. The amount of output produced using a single batch of carrier-free [gamma-(32)P]ATP was increased by about 3.5-fold. A complex pattern of DNA adducts was observed in leukocytes using both high-urea or isopropanol-ammonium hydroxide systems, two techniques effective in the detection of aromatic DNA adducts. The above observations indicate that DNA adducts being measured are likely to have been induced by aromatic compounds.