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Supplementary table 1. Correlations of sRANKL and the sRANKL/OPG ratio with select hormones among controls: EPIC cohort nested case-control study. Supplementary table 2. Distribution of covariates by quintiles of sRANKL. Supplementary table 3. Circulating concentrations of sRANKL and breast cancer risk by hormone-receptor subtype and menopausal status at blood collection. Supplementary table 4. The sRANKL/OPG ratio and breast cancer risk by hormone-receptor subtype and menopausal status at blood collection. Supplementary table 5. sRANKL additionally adjusted for continuous (log2) OPG and breast cancer risk by hormone-receptor subtype: EPIC nested case-control study. Supplementary table 6. sRANKL/OPG cross-classification based on median split of sRANKL and OPG and breast cancer risk by hormone-receptor subtype: EPIC nested case-control study.

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Perspective on This Article from Anthropometric Measures, Physical Activity, and Risk of Glioma and Meningioma in a Large Prospective Cohort Study

Prediagnostic endogenous sex steroid hormone levels have well established associations with overall risk of breast cancer. While evidence toward the existence of distinct subtypes of breast cancer accumulates, few studies have investigated the associations of sex steroid hormone levels with risk of hormone receptor [estrogen receptor (ER) and/or progesterone receptor (PR)] defined breast cancer. In a case–control study nested within the EPIC cohort (European Prospective Investigation into Cancer and Nutrition), estradiol, testosterone, and sex hormone–binding globulin levels were measured in prediagnostic serum samples from postmenopausal women not using hormone replacement therapy at blood donation. A total of 554 women who developed invasive breast cancer with information on receptor status were matched with 821 control subjects. Conditional logistic regression models estimated breast cancer risk with hormone concentrations according to hormone receptor status of the tumor. Sex steroid hormones were associated with risks of not only ER+PR+ breast cancer [estradiol OR for highest vs. lowest tertile = 2.91 (95% CI: 1.62–5.23), Ptrend = 0.002; testosterone OR = 2.27 (95% CI: 1.35–3.81), Ptrend = 0.002] but also of ER-PR- breast cancer [estradiol OR = 2.11 (95% CI: 1.00–4.46), Ptrend = 0.05; testosterone OR = 2.06 (95% CI: 0.95–4.46), Ptrend = 0.03], with associations appearing somewhat stronger in the receptor-positive disease. Serum androgens and estrogens are associated with risks of both hormone receptor–negative as well as receptor–positive breast tumors. Further research is needed to establish through which molecular pathways, and during which evolutionary stages of development, androgens and estrogens can promote the occurrence of both receptor-positive and -negative clinical breast tumors. Cancer Prev Res; 4(10); 1626–35. ©2011 AACR.

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Data on the role of dietary factors in endometrial cancer development are limited and inconsistent. We applied a “nutrient-wide association study” approach to systematically evaluate dietary risk associations for endometrial cancer while controlling for multiple hypothesis tests using the false discovery rate (FDR) and validating the results in an independent cohort. We evaluated endometrial cancer risk associations for dietary intake of 84 foods and nutrients based on dietary questionnaires in three prospective studies, the European Prospective Investigation into Cancer and Nutrition (EPIC; N = 1,303 cases) followed by validation of nine foods/nutrients (FDR ≤ 0.10) in the Nurses' Health Studies (NHS/NHSII; N = 1,531 cases). Cox regression models were used to estimate HRs and 95% confidence intervals (CI). In multivariate adjusted comparisons of the extreme categories of intake at baseline, coffee was inversely associated with endometrial cancer risk (EPIC, median intake 750 g/day vs. 8.6; HR, 0.81; 95% CI, 0.68–0.97, Ptrend = 0.09; NHS/NHSII, median intake 1067 g/day vs. none; HR, 0.82; 95% CI, 0.70–0.96, Ptrend = 0.04). Eight other dietary factors that were associated with endometrial cancer risk in the EPIC study (total fat, monounsaturated fat, carbohydrates, phosphorus, butter, yogurt, cheese, and potatoes) were not confirmed in the NHS/NHSII. Our findings suggest that coffee intake may be inversely associated with endometrial cancer risk. Further data are needed to confirm these findings and to examine the mechanisms linking coffee intake to endometrial cancer risk to develop improved prevention strategies. Cancer Epidemiol Biomarkers Prev; 24(2); 466–71. ©2015 AACR.

Background: Experimental and epidemiologic data suggest that higher circulating prolactin is associated with breast cancer risk; however, how various risk factors for breast cancer influence prolactin levels in healthy women is not clear.Methods: We analyzed cross-sectional associations between several suggested reproductive and lifestyle risk factors for breast cancer and circulating prolactin among pre- and postmenopausal women, taking into account the use of current postmenopausal hormone therapy, among 2,560 controls from a breast cancer nested case–control study within the EPIC cohort.Results: Adjusted geometric mean prolactin levels were significantly higher among premenopausal women, and among postmenopausal women using hormone therapy compared with nonusers (8.2, 7.0, and 6.3 ng/mL, respectively; Pcat = Pcat = 0.001). Prolactin levels were lower among parous women compared with nulliparous women (8.61 vs. 10.95 ng/mL; Pcat = 0.0002, premenopausal women); the magnitude of this difference depended on the number of full-term pregnancies (22.1% lower, ≥3 vs. 1 pregnancy, Ptrend = 0.01). Results for parity were similar but lower in magnitude among postmenopausal women. Prolactin did not vary by other studied factors, with the exception of lower levels among postmenopausal smokers compared with never smokers.Conclusions: Our study shows that current hormone therapy use, especially the use of combined hormone therapy, is associated with higher circulating prolactin levels in postmenopausal women, and confirms prior findings of lower circulating prolactin in parous women.Impact: Our study extends the knowledge linking various breast cancer risk factors with circulating prolactin. Cancer Epidemiol Biomarkers Prev; 23(11); 2532–42. ©2014 AACR.

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.

Increasing evidence points to a role for inflammation in lung carcinogenesis. A small number of circulating inflammatory proteins have been identified as showing elevated levels prior to lung cancer diagnosis, indicating the potential for prospective circulating protein concentration as a marker of early carcinogenesis. To identify novel markers of lung cancer risk, we measured a panel of 92 circulating inflammatory proteins in 648 prediagnostic blood samples from two prospective cohorts in Italy and Norway (women only). To preserve the comparability of results and protect against confounding factors, the main statistical analyses were conducted in women from both studies, with replication sought in men (Italian participants). Univariate and penalized regression models revealed for the first time higher blood levels of CDCP1 protein in cases that went on to develop lung cancer compared with controls, irrespective of time to diagnosis, smoking habits, and gender. This association was validated in an additional 450 samples. Associations were stronger for future cases of adenocarcinoma where CDCP1 showed better explanatory performance. Integrative analyses combining gene expression and protein levels of CDCP1 measured in the same individuals suggested a link between CDCP1 and the expression of transcripts of LRRN3 and SEM1. Enrichment analyses indicated a potential role for CDCP1 in pathways related to cell adhesion and mobility, such as the WNT/β-catenin pathway. Overall, this study identifies lung cancer–related dysregulation of CDCP1 expression years before diagnosis.Significance:Prospective proteomics analyses reveal an association between increased levels of circulating CDCP1 and lung carcinogenesis irrespective of smoking and years before diagnosis, and integrating gene expression indicates potential underlying mechanisms.See related commentary by Itzstein et al., p. 3441.

Supplemental tables 1 and 2. Supplementary Table 1. Odds ratios (95% CI) for ovarian cancer by tertile concentrations and for doubling in CRP and IL-6 by menopausal status, age at diagnosis and HRT use at blood donation. Supplementary Table 2. Odds ratios (95% CI) for ovarian cancer for doubling in CRP and IL-6 by BMI categories and waist circumference, mutual adjusted for IL-6 and CRP.

Supplementary Table S1. Adjusteda geometric mean prolactin levels, ng/ml (95% CI) by established correlates at the time of blood donation. Supplementary Table S2. Adjusteda geometric mean prolactin levels, ng/ml (95% CI) depending on the anthropometric factors. Supplementary Table S3. Adjusteda geometric mean prolactin levels, ng/ml (95% CI) depending on the lifestyle factors.

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Supplemental Material and Methods, Tables S1-S6, Figure S1 Supplemental Table S1. Hazard Ratiosa and 95% CIs from analyses of nutrient and food intakes (FDR≤0.10) reported in the baseline dietary assessment in relation to endometrial cancer risk in the EPIC study. Supplemental Table S2. Hazard Ratiosa and 95% CIs from analyses of intake of nutrients/foods at baseline (FDR > 0.10) in relation to endometrial cancer risk in the EPIC study. Supplemental Table S3. Age-standardized dietary intake of selected foods/nutrients (FDR≤0.10 in the EPIC studya) at the study baseline in the EPIC, NHS and NHSII study populations. Supplemental Table S4. Hazard Ratiosa and 95% CIs from analyses of intake of selected nutrients/foods at baseline in relation to endometrial cancer risk in the Nurses’ Health Study (NHS) and NHSII. Supplemental Table S5. Hazard Ratiosa and 95% CIs from analyses of the cumulative average intake of selected nutrients/foods in relation to endometrial cancer risk in the Nurses’ Health Study (NHS) and NHSII. Supplemental Table S6. Hazard Ratiosa and 95% CIs from analyses of the cumulative average intake of selected nutrients/foods in relation to invasive endometrial adenocarcinoma risk in the Nurses’ Health Study (NHS) and NHSII. Supplemental Figure S1. Summary of NWAS analytical method to test associations between food and nutrient intake and risk of endometrial cancer (EC.

Backgrounds: Multiple polymorphisms affecting smoking behavior have been identified through genome-wide association studies. Circulating levels of the nicotine metabolite cotinine is a marker of recent smoking exposure. Hence, genetic variants influencing smoking behavior are expected to be associated with cotinine levels.Methods: We conducted an analysis in a lung cancer case–control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. We investigated the effects of single-nucleotide polymorphisms (SNP) previously associated with smoking behavior on (i) circulating cotinine and (ii) lung cancer risk. A total of 894 cases and 1,805 controls were analyzed for cotinine and genotyped for 10 polymorphisms on 7p14, 8p11, 10q23, 15q25, and 19q13.Results: Two variants in the nicotinic acetylcholine receptor subunit genes CHRNA5 and CHRNA3 on 15q25, rs16969968 and rs578776, were associated with cotinine (P = 0.001 and 0.03, respectively) in current smokers and with lung cancer risk (P < 0.001 and P = 0.001, respectively). Two 19q13 variants, rs7937 and rs4105144, were associated with increased cotinine (P = 0.003 and P < 0.001, respectively) but decreased lung cancer risk (P = 0.01 for both, after adjusting for cotinine). Variants in 7p14, 8p11, and 10q23 were not associated with cotinine or lung cancer risk.Conclusions: 15q25 and 19q13 SNPs were associated with circulating cotinine. The directions of association for 15q25 variants with cotinine were in accordance with that expected of lung cancer risk, whereas SNPs on 19q13 displayed contrasting associations of cotinine and lung cancer that require further investigation.Impact: This study is the largest to date investigating the effects of polymorphisms affecting smoking behavior on lung cancer risk using circulating cotinine measures as proxies for recent smoking behavior. Cancer Epidemiol Biomarkers Prev; 20(10); 2250–61. ©2011 AACR.

Background: Circulating levels of insulin-like growth factor I (IGF-I) and its main binding protein, IGF binding protein 3 (IGFBP-3), have been associated with risk of several types of cancer. Heritable factors explain up to 60% of the variation in IGF-I and IGFBP-3 in studies of adult twins.Methods: We systematically examined common genetic variation in 18 genes in the IGF signaling pathway for associations with circulating levels of IGF-I and IGFBP-3. A total of 302 single nucleotide polymorphisms (SNP) were genotyped in >5,500 Caucasian men and 5,500 Caucasian women from the Breast and Prostate Cancer Cohort Consortium.Results: After adjusting for multiple testing, SNPs in the IGF1 and SSTR5 genes were significantly associated with circulating IGF-I (P < 2.1 × 10−4); SNPs in the IGFBP3 and IGFALS genes were significantly associated with circulating IGFBP-3. Multi-SNP models explained R2 = 0.62% of the variation in circulating IGF-I and 3.9% of the variation in circulating IGFBP-3. We saw no significant association between these multi-SNP predictors of circulating IGF-I or IGFBP-3 and risk of prostate or breast cancers.Conclusion: Common genetic variation in the IGF1 and SSTR5 genes seems to influence circulating IGF-I levels, and variation in IGFBP3 and IGFALS seems to influence circulating IGFBP-3. However, these variants explain only a small percentage of the variation in circulating IGF-I and IGFBP-3 in Caucasian men and women.Impact: Further studies are needed to explore contributions from other genetic factors such as rare variants in these genes and variation outside of these genes. Cancer Epidemiol Biomarkers Prev; 19(11); 2877–87. ©2010 AACR.

Supplementary Table S3. Multivariate Cox regression survival analysis, including TL, age, TG, BCG, radical cystectomy, radiotherapy and chemotherapy. All cause of death.

Supplementary Table S1. Description of populations included and not included in the TL study and comparative analysis.

Supplementary Methods, Figures and Tables.

Supplementary Table 1 from Eighteen Insulin-like Growth Factor Pathway Genes, Circulating Levels of IGF-I and Its Binding Protein, and Risk of Prostate and Breast Cancer

Background: Evidence suggests an etiologic role for inflammation in ovarian carcinogenesis and heterogeneity between tumor subtypes and anthropometric indices. Prospective studies on circulating inflammatory markers and epithelial invasive ovarian cancer (EOC) have predominantly investigated overall risk; data characterizing risk by tumor characteristics (histology, grade, stage, dualistic model of ovarian carcinogenesis) and anthropometric indices are sparse.Methods: We conducted a nested case–control study in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort to evaluate C-reactive protein (CRP), IL6, and EOC risk by tumor characteristics. A total of 754 eligible EOC cases were identified; two controls (n = 1,497) were matched per case. We used multivariable conditional logistic regression to assess associations.Results: CRP and IL6 were not associated with overall EOC risk. However, consistent with prior research, CRP >10 versus CRP ≤1 mg/L was associated with higher overall EOC risk [OR, 1.67 (1.03–2.70)]. We did not observe significant associations or heterogeneity in analyses by tumor characteristics. In analyses stratified by waist circumference, inflammatory markers were associated with higher risk among women with higher waist circumference; no association was observed for women with normal waist circumference [e.g., IL6: waist ≤80: ORlog2, 0.97 (0.81–1.16); waist >88: ORlog2, 1.78 (1.28–2.48), Pheterogeneity ≤ 0.01].Conclusions: Our data suggest that high CRP is associated with increased risk of overall EOC, and that IL6 and CRP may be associated with EOC risk among women with higher adiposity.Impact: Our data add to global evidence that ovarian carcinogenesis may be promoted by an inflammatory milieu. Cancer Epidemiol Biomarkers Prev; 24(6); 951–61. ©2015 AACR.

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

Background: Evidence is accumulating that immune cells play a prominent role in pancreatic cancer etiology but prospective investigations are missing. Methods: We conducted a nested case–control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) study with 502 pairs of incident pancreatic cancer cases and matched controls. Relative counts of circulating immune cells (neutrophils and lymphocyte sublineages: total CD3+, CD8+, CD4+, and FOXP3+ regulatory T cells (Tregs) relative to nucleated cells, (white blood cells) were measured by qRT-PCR. ORs with 95% confidence intervals were estimated using logistic regressions, modeling relative counts of immune cells on a continuous scale. Results: Neither relative counts of immune cell types taken individually, nor mutually adjusted for each other were associated with pancreatic cancer risks. However, in subgroup analyses by strata of lag-time, higher relative counts of Tregs and lower relative counts of CD8+ were significantly associated with an increased pancreatic cancer risks in participants diagnosed within the first 5 years of follow-up. Conclusions: These results might reflect reverse causation, due to higher relative counts of Tregs and lower counts of CD8+ cells among individuals with more advanced stages of latent pancreatic cancer, who are closer to the point of developing clinical manifest disease. Impact: We have shown, for the first time, that increased relative counts of regulatory T cells and lower relative counts of CD8+, cytotoxic T cells may be associated with pancreatic cancer risk or relatively late-stage tumor development. See related commentary by Michaud and Kelsey, p. 2176

Background: Experimental and epidemiologic data suggest that higher circulating prolactin is associated with breast cancer risk; however, how various risk factors for breast cancer influence prolactin levels in healthy women is not clear. Methods: We analyzed cross-sectional associations between several suggested reproductive and lifestyle risk factors for breast cancer and circulating prolactin among pre- and postmenopausal women, taking into account the use of current postmenopausal hormone therapy, among 2,560 controls from a breast cancer nested case–control study within the EPIC cohort. Results: Adjusted geometric mean prolactin levels were significantly higher among premenopausal women, and among postmenopausal women using hormone therapy compared with nonusers (8.2, 7.0, and 6.3 ng/mL, respectively; Pcat = Conclusions: Our study shows that current hormone therapy use, especially the use of combined hormone therapy, is associated with higher circulating prolactin levels in postmenopausal women, and confirms prior findings of lower circulating prolactin in parous women. Impact: Our study extends the knowledge linking various breast cancer risk factors with circulating prolactin. Cancer Epidemiol Biomarkers Prev; 23(11); 2532–42. ©2014 AACR.

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.

Background: Dietary and circulating carotenoids have been inversely associated with breast cancer risk, but observed associations may be due to confounding. Single-nucleotide polymorphisms (SNPs) in β-carotene 15,15′-monooxygenase 1 (BCMO1), a gene encoding the enzyme involved in the first step of synthesizing vitamin A from dietary carotenoids, have been associated with circulating carotenoid concentrations and may serve as unconfounded surrogates for those biomarkers. We determined associations between variants in BCMO1 and breast cancer risk in a large cohort consortium. Methods: We used unconditional logistic regression to test four SNPs in BCMO1 for associations with breast cancer risk in 9,226 cases and 10,420 controls from the National Cancer Institute Breast and Prostate Cancer Cohort Consortium (BPC3). We also tested weighted multi-SNP scores composed of the two SNPs with strong, confirmed associations with circulating carotenoid concentrations. Results: Neither the individual SNPs nor the weighted multi-SNP scores were associated with breast cancer risk [OR (95% confidence interval) comparing extreme quintiles of weighted multi-SNP scores = 1.04 (0.94–1.16) for β-carotene, 1.08 (0.98–1.20) for α-carotene, 1.04 (0.94–1.16) for β-cryptoxanthin, 0.95 (0.87–1.05) for lutein/zeaxanthin, and 0.92 (0.83–1.02) for retinol]. Furthermore, no associations were observed when stratifying by estrogen receptor status, but power was limited. Conclusions: Our results do not support an association between SNPs associated with circulating carotenoid concentrations and breast cancer risk. Impact: Future studies will need additional genetic surrogates and/or sample sizes at least three times larger to contribute evidence of a causal link between carotenoids and breast cancer. Cancer Epidemiol Biomarkers Prev; 22(5); 927–36. ©2013 AACR.

Risk models for lung cancer incidence would be useful for prioritizing individuals for screening and participation in clinical trials of chemoprevention. We present a risk model for lung cancer built using prospective cohort data from a general population which predicts individual incidence in a given time period. We build separate risk models for current and former smokers using 169,035 ever smokers from the multicenter European Prospective Investigation into Cancer and Nutrition (EPIC) and considered a model for never smokers. The data set was split into independent training and test sets. Lung cancer incidence was modeled using survival analysis, stratifying by age started smoking, and for former smokers, also smoking duration. Other risk factors considered were smoking intensity, 10 occupational/environmental exposures previously implicated with lung cancer, and single-nucleotide polymorphisms at two loci identified by genome-wide association studies of lung cancer. Individual risk in the test set was measured by the predicted probability of lung cancer incidence in the year preceding last follow-up time, predictive accuracy was measured by the area under the receiver operator characteristic curve (AUC). Using smoking information alone gave good predictive accuracy: the AUC and 95% confidence interval in ever smokers was 0.843 (0.810–0.875), the Bach model applied to the same data gave an AUC of 0.775 (0.737–0.813). Other risk factors had negligible effect on the AUC, including never smokers for whom prediction was poor. Our model is generalizable and straightforward to implement. Its accuracy can be attributed to its modeling of lifetime exposure to smoking. Cancer Prev Res; 5(6); 834–46. ©2012 AACR.

Background: The etiologies of glioma and meningioma tumors are largely unknown. Although reproductive hormones are thought to influence the risk of these tumors, epidemiologic data are not supportive of this hypothesis; however, few cohort studies have published on this topic. We examined the relation between reproductive factors and the risk of glioma and meningioma among women in the European Prospective Investigation into Cancer and Nutrition (EPIC). Methods: After a mean of 8.4 years of follow-up, 193 glioma and 194 meningioma cases were identified among 276,212 women. Information on reproductive factors and hormone use was collected at baseline. Cox proportional hazard regression was used to determine hazard ratios (HR) and 95% confidence intervals (95% CI). Results: No associations were observed between glioma or meningioma risk and reproductive factors, including age at menarche, parity, age at first birth, menopausal status, and age at menopause. A higher risk of meningioma was observed among postmenopausal women who were current users of hormone replacement therapy (HR, 1.79; 95% CI, 1.18-2.71) compared with never users. Similarly, current users of oral contraceptives were at higher risk of meningioma than never users (HR, 3.61; 95% CI, 1.75-7.46). Conclusion: Our results do not support a role for estrogens and glioma risk. Use of exogenous hormones, especially current use, seems to increase meningioma risk. However, these findings could be due to diagnostic bias and require confirmation. Impact: Elucidating the role of hormones in brain tumor development has important implications and needs to be further examined using biological measurements. Cancer Epidemiol Biomarkers Prev; 19(10); 2562–9. ©2010 AACR.

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.

Breast cancer is the most common female cancer worldwide and makes up 25% of all cancers diagnosed in women. There is a need for improved risk assessment methods to identify women at high risk before the disease develops. It is well established that estrogens are involved in estrogen receptor positive (ER+) breast cancer carcinogenesis, however the mechanisms are not fully understood. We hypothesize that lifetime estrogen exposure accumulates changes in DNA methylation detectable in blood as a surrogate for other tissues. Here, we have identified a methylation signature of estrogen exposure measured in blood DNA that could improve risk assessment for breast cancer. Our Estimated Lifetime Estrogen Exposure (ELEE) model, taking into account reproductive time (age at menopause minus age at menarche), number of pregnancies and breastfeeding duration, shows a 5% increased risk of developing breast cancer per unit increase in ELEE (range 18 – 44) in the European Prospective into Cancer and Nutrition (EPIC)-Italy study cohort (30671 healthy controls, 1193 cases, age at diagnosis ≥ 50). An Epigenome-Wide Association Study (EWAS) of ELEE was conducted on 450K methylation data in EPIC-Italy (199 controls, 97 cases) and the Generations Study (GS) cohort (n=65 controls). For validation targeted bisulfite sequencing using the Fluidigm 48.48 Access Array was performed on independent DNA samples from the GS cohort (440 matched ER+ case-control pairs). The Methylation Index (MI) of ELEE was developed on 450K data using ridge regression and includes DNA methylation levels at selected CpG sites. DNA methylation levels at 694 CpG sites show significant (false discovery rate q < 0.05) association with ELEE in the EWAS and of these 42 CpG sites from the top-probes were selected for validation. The MI was developed on 28 CpG sites passing quality control and shows high correlation with ELEE in 450K training data (r=0.69) and significant association with breast cancer risk in EPIC-Italy with an OR of 1.38 per unit MI (range 24 – 43, 95% CI: 1.23 – 1.57, P=2.0E-07). Validation step is yet to be finalized; initial analysis on the first 318 case-control pairs suggests a modest but significant association with ER+ breast cancer risk (unadjusted OR=1.04 per unit MI, 95% CI: 1.00 – 1.07, P=0.030, MI range 20 – 53). In summary, we show that changes in DNA methylation following estrogen exposure are detectable in blood and we have developed a Methylation Index of ELEE that is significantly increased in breast cancer cases compared to controls in EPIC-Italy, and modestly increased in the GS validation cohort. This molecular measure of estrogen exposure could potentially improve risk assessment methods and be used to identify women at high risk of developing breast cancer. Citation Format: Annelie Johansson, Domenico Palli, Giovanna Masala, Sara Grioni, Claudia Agnoli, Rosario Tumino, Maria Concetta Giurdanella, Francesca Fasanelli, Carlotta Sacerdote, Salvatore Panico, Amalia Mattiello, Anthony Swerdlow, Minouk Schoemaker, Michael Jones, Nick Orr, Olivia Fletcher, Nichola Johnson, Katarzyna Tomczyk, Paolo Vineis, James M. Flanagan. DNA methylation index of lifetime estrogen exposure in breast cancer [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 5316.

Bladder cancer (BC) is one of the leading causes of cancer-related death worldwide. BC is among the most expensive cancer per patient because it requires frequent surveillance and repeated treatments over many years. The identification of new biomarkers for early BC detection, recurrence/progression is urgently needed to both improve patient outcomes and decrease health care costs. MicroRNAs (miRNAs) are aberrantly expressed in many cancers, including BC, and may be isolated from various biological specimens, including urine. To investigate miRNA signatures in surrogate tissues may be a useful alternative to reduce invasiveness of biopsies, allowing repetitive samplings during follow-up and reducing health care costs for detection, monitoring of progression and treatment. We aim to identify specific miRNA signatures in urine samples from 66 BC male patients (10 muscle invasive BC (MIBC) and 56 non-muscle invasive BC (NMIBC)) and 48 healthy controls using a Next Generation Sequencing (NGS) approach able to accurately distinguish BC patients and predict disease outcome. The measurement of miRNA levels in urine could allow to measure the levels of promising biomarkers in one of the best and closest surrogate tissue for BC, since it is in direct contact with the tissue of tumor origin. A specific miRNA signatures that could distinguish the different types of BC patients from healthy controls was found in urine. For MIBC, a 18-miRNAs signature had over 80% predictive power (PP) to recognize patients from controls (data are under validation). For NMIBC we were able to stratify cases according to grade. In particular, 23 miRNAs resulted differentially expressed among G1-G2 cases and controls (5 of them had PP>0.70), while several miRNAs resulted differentially expressed among G3 patients and controls (a 10-miRNAs signature with PP>0.97). Interestingly, we found several differentially expressed miRNAs in common among cases and some miRNAs that were differentially expressed only in specific subcategories of BC cases. NGS data were also used to search for the most constant miRNAs in the set of samples to be used as reference genes in a validation step. Twenty-three miRNAs (21 target and 2 reference miRNAs) were validated by qPCR on 177 urine samples from 113 BC case and 64 controls . Interestingly, miRNAs differentially expressed among cases and controls were able to discriminate not only BC cases from controls, but also its subcategories. This data provide evidences of the possibility to use miRNAs specifically within the urine of patients with BC as diagnostic, prognostic and predictive biomarkers. Acknowledgements Work supported by Fondazione Umberto Veronesi (FUV) “Post-doctoral fellowship Year 2014, 2015 and 2016” (B.P. recipient), a FUV Grant 2013 (G.M. recipient), and by a HuGeF grant. Citation Format: Barbara Pardini, Francesca Cordero, Alessio Naccarati, Giulio Ferrero, Clara Viberti, Marco Oderda, Maddalena Arigoni, Raffaele Calogero, Carlotta Sacerdote, Paolo Gontero, Paolo Vineis, Giuseppe Matullo. Urine microRNA profiling in bladder cancer by next-generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4431. doi:10.1158/1538-7445.AM2017-4431

Bladder cancer (BC) is the sixth most commonly diagnosed tumor worldwide. DNA repair capacity (DRC) refers to the ability of a cell to protect the integrity of the genome and DNA repair pathways have been implicated in BC risk. It has been observed that individuals with low DRC tend to accumulate more damage than those with a more efficient DRC. This inter-individual variability is modulated by the genetic background, as well as differential gene expression and epigenetic regulation. We aimed at studying the relationship between DRC and DNA damage (evaluated by H2AX phosphorylation and micronucleus assays) and BC risk and clinical outcome, integrating with gene expression and epigenetic profile data in 159 BC cases and 159 matched controls, enrolled in the Turin Bladder Cancer Study (TBCS). We investigated ã-H2AX phosphorylation levels and MN frequencies in cryopreserved peripheral blood mononuclear cells. We found significant differences in micronuclei and nuclear buds frequencies, with higher number of these damages in cases compared to controls (p = 0.0002 and p = 0.002 respectively). On the other hand, we observed a significant association between ã-H2AX basal levels and risk of disease recurrence/progression in both BC patients as a whole and the subgroup of non-muscle invasive BC (NMIBC) (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): this suggests a protective effect of DNA double strand breaks signalling in terms of preventing BC recurrence or progression. In order to evaluate the genetic and epigenetic role in modulation of DRC we performed whole genome methylation and gene expression analyses on the same BC cases and controls. Preliminary analyses on methylation levels did not show any significant difference between cases and controls. Two metalloproteinases (MMP23A and MMP23B) resulted significantly under-expressed in BC compared to healthy controls (logFC = -0.23, p = 0.01; logFC = -0.37, p = 0.007, respectively). Interestingly, the expression levels of these genes were also significantly correlated with the relative CpGs methylation. Further analyses focusing on the integration of whole genome data with DRC assays are ongoing to unravel new prognostic biomarkers of disease. Citation Format: Giuseppe Matullo, Clara Viberti, Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Claudia Giachino, Giovanni Fiorito, Alessio Naccarati, Alessia Russo, Rossana Critelli, Paolo Destefanis, Marco Oderda, Paolo Gontero, Paolo Vineis, Carlotta Sacerdote. DNA repair capacity, chromosomal damage, methylation and gene expression levels in bladder cancer: An integrated analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 778.

Bladder cancer (BC) is one of the leading causes of cancer-related death worldwide. Most BCs are non-muscle invasive (NMIBC), which generally have a good prognosis but frequently recur or progress to muscle invasion (MIBC) within 5 years. By contrast, MIBC has a poor prognosis and treatment requires a multidisciplinary approach with radical surgery, radiotherapy, and chemotherapy. BC is among the most expensive cancer per patient because it requires frequent surveillance and repeated treatments over many years. Reliable predictors of disease and progression for BC are lacking so developing novel noninvasive diagnostics is imperative to both improve patient outcomes and decrease health costs. MicroRNAs (miRNAs) are aberrantly expressed in many cancers, including BC, and may be isolated from various biological specimens, including plasma and urine. Next generation sequencing (NGS) technology provides novel information about miRNA expression and is likely to become the gold standard method for comprehensive miRNA analysis in cancer genomics. So far, only few studies investigated miRNA signatures in BC by NGS and only on tissues. To investigate miRNA signatures in surrogate tissues may be a useful alternative to reduce invasiveness of biopsies, allowing repetitive samplings during follow-up and reducing health care costs for detection, monitoring of progression and treatment. We aim to identify specific miRNA signatures in urine and plasma samples from 20 NMIBC, 20 MIBC patients and 40 healthy controls using a NGS approach able to accurately distinguish BC patients and predict disease outcome. The measurement of miRNA levels in both urine and plasma samples from the same patients will allow us to compare the levels of promising biomarkers in two surrogate tissues. Urine is the best and closest surrogate tissue for BC, since it is in direct contact with the tissue of tumor origin; while plasma is one of the best surrogate tissue for distant metastasis and advanced cancer detection. In plasma samples, 5 miRNAs were differentially expressed among cases and controls (miR-222, miR-146b, miR-126, miR221 upregulated and miR-5096 downregulated). However, after repetition of the analyses stratifying cases for NMIBC (the most represented group in BC cases), only 2 of the previously described miRNAs (miR-222 and miR-126) resulted dysregulated among NMIBC and controls. In urine, 31 miRNAs were differentially expressed among BC cases and controls (adjusted p-value ranging from 0.0007 to 0.05). On the other hand, 26 and 31 miRNAs resulted differentially expressed among NMIBC and controls and MIBC and controls, respectively (for NMIBC: adjusted p-value from 0.0009 to 0.04; for MIBC adjusted p-value from 7.2×10-9 to 0.04). Interestingly, miRNAs differentially expressed among cases and controls were able to discriminate not only BC cases from controls, but also NMIBC and MIBC. Citation Format: Barbara Pardini, Francesca Cordero, Alessandra Allione, Clara Viberti, Alessio Naccarati, Marco Oderda, Mirko Preto, Marco Allasia, Maddalena Arigoni, Federica Riccardo, Raffaele Calogero, Carlotta Sacerdote, Giuseppe Matullo. MicroRNA profiling by a next generation sequencing approach in urine and plasma samples: from genomics to diagnostics and prognostics of bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1061.

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

The crosstalk between microRNAs (miRNAs) and other epigenetic factors may lead to novel hypotheses about carcinogenesis identifying new targets for research. Since a single miRNA can regulate multiple downstream target genes, its altered expression may potentially be a very sensitive biomarker to detect early malignant transformation and improve diagnosis and prognosis. In the current study, we used a genome-wide approach to test the hypothesis that altered methylation of miRNA encoding genes may be related to dietary and lifestyle factors and may be associated with deregulated mature miRNA expression ultimately leading to cancer. In a case-control study nested in a prospective cohort (EPIC-Italy), we analysed DNA methylation levels of miRNA encoding genes (2,191 CpG probes related to 517 genes) that are present in the Infinium Human Methylation450 BeadChip array in prediagnostic peripheral white blood cells of subjects who developed Colorectal Cancer (CRC, n = 159) and Breast Cancer (BC, n = 166) and matched subjects who remained clinically healthy. In the whole cohort, several differentially methylated miRNA genes were observed in association with age, sex, smoking habits and physical activity. Interestingly, in the case-control study, 8 differentially methylated miRNAs were identified in subjects who went on to develop BC (miR-328, miR-675, miR-1307, miR-1286, miR-1275, miR-1910, miR-24-1, and miR-548a-1; all Bonferroni-adjusted p-values < 0.05). No significant associations were found with CRC. Assuming that altered methylation of miRNAs may be present before diagnosis, it may represent a biomarker for early detection or risk of cancer and may help to understand the cascade of events preceding tumour onset. Citation Format: Alessio Naccarati, Silvia Polidoro, Giovanni Fiorito, Francesca Cordero, Giulio Ferrero, Gianluca Campanella, Carlotta Sacerdote, Amalia Mattiello, Salvatore Panico, Giovanna Masala, Domenico Palli, Claudia Agnoli, Vittorio Krogh, Graziella Frasca, Rosario Tumino, Paolo Vineis. Epigenome-wide study in prediagnostic samples from the European Prospective Investigation into Nutrition and Cancer (EPIC-Italy) cohort: Differentially methylated microRNAs in subjects who developed breast cancer. [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 LB-188. doi:10.1158/1538-7445.AM2015-LB-188

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

INTRODUCTION: The biguanide metformin may reduce cancer incidence according to recent meta-analyses of observational studies. Conversely, a meta-analysis of randomized controlled trials did not support the hypothesis that metformin lowers cancer risk. However, many of the published pharmaco-epidemiological studies suffer from severe biases and most randomized clinical trials are not designed or powered to look specifically at cancer safety outcomes. Therefore, we emulated the design and statistical analysis of a randomized trial in a large retrospective cohort study within the United Kingdom (UK) Clinical Practice Research Datalink (CPRD) to investigate associations comparing initiators of metformin to initiators of other oral first-line hypoglycemic agents among individuals newly diagnosed with diabetes in relation to risk of total cancer and by cancer site. METHODS: We retrieved data on all participants newly diagnosed with type 2 diabetes between January 1, 1987 and December 31, 2010. Eligible participants had no prior history of cancer and at least two years of follow-up in the CPRD after the diagnosis of diabetes. We also excluded the initial 12 months of follow-up after the first antidiabetic prescription. Treatment naive participants with diabetes at CPRD enrolment that first started using (initiators) metformin during follow-up were compared to initiators of other oral hypoglycemic agents. Exposure to certain antidiabetic drugs was classified only based on the initial twelve-month treatment period. We mimicked the statistical analysis of a randomized trial in an observational dataset using the intention-to-treat (ITT) principle. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI) after adjustment for known or suspected risk factors for cancer. We also calculated adherence-adjusted estimates using inverse probability weighting. RESULTS: A total of 95,820 participants were included. Of these, 51,484 (54%) individuals started taking metformin monotherapy and 18,264 (19%) started sulfonylurea monotherapy. A total of 3,805 first incident cancers were identified in a maximum follow-up of 24 years. Compared to initiators of sulfonylurea monotherapy, initiators of metformin had a similar incidence of total cancer (HR, 0.96; 95% CI, 0.89-1.04). There was no evidence for an association between use of metformin and cancers of the colorectum (HR, 0.92; 95% CI, 0.76-1.13), prostate (HR, 1.02; 95% CI, 0.83-1.25), lung (HR, 0.85; 95% CI, 0.68-1.07), postmenopausal breast (HR, 1.03; 95% CI, 0.82-1.31), or any other cancer. The estimates remained similar after adjustment for non-adherence. CONCLUSIONS: We found no evidence for a decreased or increased risk of any cancer among those exposed to metformin versus sulfonylurea. These findings need to be confirmed in large RCTs. Citation Format: Konstantinos K. Tsilidis, Despoina Capothanassi, Naomi Allen, Evangelos Rizos, David Lopez, Karin van Veldhoven, Carlotta Sacerdote, Deborah Ashby, Paolo Vineis, Ioanna Tzoulaki, John Ioannidis. Metformin and cancer risk: A cohort study in the UK Clinical Practice Research Datalink analyzed like a randomized trial. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2161. doi:10.1158/1538-7445.AM2014-2161

In Europe, bladder cancer (BC) is the sixth most commonly diagnosed tumor and the second most common cause of death among patients with genitourinary tract malignancies. The ability to repair DNA damage is strongly associated with the risk of cancer and inter-individual variation in DNA repair capacity (DRC) might account for different susceptibility of developing cancer. DRC represents a complex marker comprising the sum of several factors such as gene variants, gene expression, stability of gene products, and effect of inhibitors/stimulators. Individuals with low DRC will tend to accumulate more damage than those who have a better ability to repair such damage. This variability is modulated by the genetic background to which SNPs/haplotype combinations in DNA repair genes, as well as epigenetic regulation are likely to contribute. Phenotypic assays to evaluate DNA repair activity (in particular NER comet assay, H2AX phosphorylation and micronucleus assay) were performed on cryopreserved lymphocytes from 159 cases (collected before treatment) and 159 controls matched by age and smoking habits, enrolled in Turin Bladder Cancer Study (TBCS). We aimed at studying the relationship between DRC (evaluated by comet assay, micronuclei and H2AX phosphorylation assay) and bladder cancer integrating, gene expression and epigenetic profile data (methylation levels and microRNA expression). In particular, we performed an integrated analysis on the genotype/phenotype correlation in a population of bladder cancer cases and 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 combined biomarkers to be measured in blood cells as non-invasive predictive method. Citation Format: Barbara Pardini, Alessandra Allione, Simonetta Guarrera, Valentina Turinetto, Giovanni Fiorito, Fabio Rosa, Alessia Russo, Federica Modica, Claudia Giachino, Paolo Vineis, Carlotta Sacerdote, Giuseppe Matullo. DNA repair capacity, gene expression and epigenomic profiles in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5367. doi:10.1158/1538-7445.AM2014-5367

The protein MGMT (O6-methylguanine-DNA-methyltransferase) is responsible for the DNA repair from damage in O6 position of guanine induced by alkylating agent such as temozolomide, which represent the conventional treatment of gliomas. Silencing of the MGMT gene by epigenetic methylation of its promoter may disable this repair mechanism, thus increasing the cytotoxicity of chemotherapy. Previous studies showed that glioblastama patients, with a methylated MGMT gene promoter in tumour tissue, are more sensitive to the action of alkylating agents and experience higher survival. The aim of the study was to verify the feasibility and the utility of monitoring the state of methylation of MGMT in circulating DNA as a predictive marker of prognosis and response to treatment. Fiftyeight patients with glioma (12 with low and 46 with high grade) who underwent surgical resection and who were eligible for therapy with temozolomide, were enrolled in the study. Blood samples for the analysis of MGMT methylation status were collected before any treatment (T0) and during the treatment at the time of each magnetic resonance imaging (about every 3 months). The methylation status of the MGMT gene promoter in circulating DNA was investigated using PCR with a specific probe for the methylated sequence. The MGMT promoter methylation status in tumour tissue and in plasma was highly concordant: in the 79% of the patients the MGMT promoter was methylated both in tumour tissue and plasma, while in the 21% of patients MGMT promoter was methylated in tissue but not in plasma (Cohen's kappa coefficient = 0.75, p The risk of death increased in patients with unmethylated MGMT promoter both in tumour tissue (Adjusted HR=2.23; 95%CI 0.99-5.04) and in plasma (Adjusted HR1.73; 95%CI 0.92-3.22) Finally, in all patients after one year from diagnosis we noticed a decrease of the state of methylation of MGMT promoter in plasma. At the end of follow up in the 98% of patients who presented the methylation of the MGMT promoter at T0, a complete unmethylation was observed. In conclusion the results of this study confirm the feasibility of monitoring the state of methylation of MGMT in circulating DNA in glioma patients. MGMT promoter methylation status may be a prognostic marker of clinical interest in tissue and in plasma. Furthermore the complete unmethylation of MGMT promoter in the 98% of the glioma patients after one year from diagnosis, is a potentially important finding for future researches on biological history and treatment of gliomas. Citation Format: Valentina Fiano, Morena Trevisan, Carlotta Sacerdote, Anna Castiglione, Elisa Trevisan, Rebecca Senetta, Anna Gillio Tos, Laura De Marco, Paola Cassoni, Riccardo Soffietti, Franco Merletti. Gliomas: methylation status of MGMT gene monitoring in plasma DNA of patients receiving chemotherapy with alkylating agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2013-653

Aromatic amines are involved in the etiology of bladder cancer and these compounds are acetylated by N-acetyltransferase 1 (NAT1) and 2 (NAT2). Epidemiological studies have shown that the slow NAT2 acetylator phenotype is associated with increased risk of bladder cancer. Although several studies have shown an association between bladder cancer risk and SNPs in NAT2, the impact on survival has not been established. Because SNPs are innate characteristics of individuals, we hypothesize that their presence could have an effect even after tumor resection and, thus, be useful as prognostic markers. In order to investigate this possibility, we examined 5 SNPs in NAT2 gene (rs1041983, rs1801280, rs1799931, rs1495741, rs12545528) and investigated their impact on overall survival (OS). The study population comprised the cohort of new histologically confirmed cases of bladder cancers treated in the urology departments of the S. Giovanni Battista Hospital in Turin. Vital status ascertainment was carried out through linkage with the local town offices. Blood samples and questionnaires of 331 subjects were collected before therapy. White blood cell DNA was purified from stored blood samples. Polymorphic sites were genotyped by 5′Nuclease assay (TaqMan) with fluorogenic Minor Groove Binder probes. Subjects’ phenotype was estimated using a standard approach based on the combination of two polymorphisms: rs1041983 and rs1801280; we also estimated phenotype from SNP rs1495741 (Garcia-Closas et al. 2011). For each SNP we calculated crude and adjusted (by age, stage, grading and therapy) Hazard Ratios (HR) and the corresponding 95% Confidence Interval (95% CI) using the Cox proportional-hazard regression model for the co-dominant and per-allele model. Stratified analyses by kind of therapy and tumour type were also performed. We computed the phase of the haplotype using PHASE 2.1: for each haplotype we computed HR, comparing subjects carrying 2, 1 or 0 copies of the same haplotype. The rs1799931 and the rs1801280 variant alleles, which lead to reduced N-acetyltransferase activity and/or reduced protein stability, were significantly associated with a decreased OS (HR: 4.54; 95% CI: 1,78-11,61 and HR: 1.43; 95% CI: 1,04-1,96 adjusted per allele model, respectively); in contrast variant alleles of SNPs which do not seem to modify the protein activity (rs12545528 non-coding and rs1041983 silent) were significantly associated with an increased OS (HR: 0.68; 95% CI: 0,49-0,96 and HR: 0.58; 95% CI: 0,40-0,85 per allele model, respectively). Similar observations were made when the cases were stratified into papillary and non-papillary tumours or stratified by kind of therapy. Haplotype analysis confirmed the results described above not providing any other indication. In conclusion, NAT2 rs1799931 and rs1801280 functional SNPs can be considered as potential prognostic factors for the survival of patients with bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3582. doi:1538-7445.AM2012-3582

Excess body weight is a known risk factor for endometrial cancer but whether it differentially affects histological subtypes of endometrial cancer is unclear. The two proposed main subtypes are the “estrogen-dependent Type I” and “non estrogen-dependent and clinically aggressive Type II”. Little is known about risk factors for Type II tumors mainly because most epidemiologic studies lack sufficient cases to study these rare tumors separately. Here we examined the association between recent adult body mass index (BMI) and endometrial tumor subtypes in a pooled analysis of 25 studies in the Epidemiology of Endometrial Cancer Consortium (E2C2). Individual-level data from 10 cohort studies and 15 case-control studies provided a total of 14,409 endometrial cancer cases and 35,950 controls for this analysis. Cohort studies were analyzed using a nested case-control design. The majority of women were white (86%) and postmenopausal (83%). Endometrial cancer cases were classified into two subtypes: Type I (endometrioid adenocarcinomas, adenocarcinoma tubular, papillary adenocarcinomas, mucinous adenocarcinomas, adenocarcinomas with squamous metaplasia, n=13,286) and Type II (serous, squamous cell, small cell, mixed cell, n=1,123). The associations of BMI with the risk of tumor subtypes were evaluated by calculating odds ratios (OR) and 95% confidence intervals (95% CI) using polytomous logistic regression models. Potential confounders included in the analysis were age, race, parity, age at menarche, oral contraceptive (OC) use, menopausal hormone (PMH) use, and smoking status. BMI was positively associated with both Type I and Type II tumors, but the association was stronger for Type I than for Type II tumors (P value for the difference in OR between Type I and Type II was Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1029. doi:1538-7445.AM2012-1029

During the last decades, improvements in medical and surgical treatments have substantially increased the chances of surviving from a cancer. Cancer survivors now amount to more than 3.5% of population in the US, and about 3% in Western Europe. Cancer survivors face the problem of subsequent cancers possibly related to the late effects of treatments or to a common aetiology for the first and subsequent cancers. Cancer patients have a 20% higher risk of new primary cancer compared with the general population. Approximately one third of cancer survivors aged >60 years were diagnosed more than once with another cancer. Women with breast cancer as first primary cancer were the first largest group of multiple cancer prevalence reported in the United States in 2002, while the second and third groups were men and women with a diagnosis of primary colorectal cancer and men with prostate cancer, respectively. Multiple cancers arise in the same individual due to several causes: host factors, such as hormonal and/or genetic factors, lifestyle and environment, and treatments due to the first cancer (for example, radiotherapy) The aim of this study is to assess the incidence of second primary tumors in a cohort of women with a first breast cancer and to investigate the role of shared risk factors. The study was performed in the cohort of women with a primary breast cancer from the European Prospective Investigation into Cancer and Nutrition (EPIC) study. To correctly assess the incidence of second primary tumors, we applied a Markov model studying the transition intensities from first to second tumor with the Aalen-Johansen (AJ) estimators, as usually done in competing risk models. In the cohort of 11045 women, we observed 492 second primary tumors (not metachronous) after a median follow-up of 4.92 years. About 30% (140) of them were second primary breast cancers, 65 were colorectal cancers and 39 were endometrial cancers. In this cohort, there was a slight, not statistically significant increase of cancer incidence in women with breast cancer, compared to the general population; this was for all tumors together, except non-melanoma skin cancers. Analysis by subgroups identified a significant increase for second breast cancers and for endometrial cancers. Our findings suggest that second primaries may be an important problem for cancer survivors. We will now investigate the role played by lifestyle factors, genetics and possibly therapies in second primary occurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3585. doi:1538-7445.AM2012-3585

Bladder cancer (BC) survival is highly influenced by environmental and predisposing genetic factors. In the last decades growing evidence of the major role played by DNA repair systems in the survival after a BC has been provided. The objectives of the study are to assess the relationship between DNA Repair Genes polymorphisms and survival by treatment in bladder cancer patients. We conducted a hospital-based case-control investigation at S. Giovanni Battista hospital in Turin, where about half of the incident BC in the Turin metropolitan area are treated. Cases are histologically confirmed incident BC patients, male, aged 40 to 75 years and resident in the Turin metropolitan area. Each patient filled in a questionnaire on history of tobacco smoking, occupational history, 24-hours medication use recall and food habits, and provided a blood sample. We followed up BC patients for death and recurrences using municipal registry office, death's certificate, hospital discharge, medical records and pathologists’ reports. In the cohort of BC patients, we genotyped 36 Single Nucleotide Polymorphisms (SNPs) in 10 DNA Repair Genes. We analyzed survival on BC patients using a Cox proportional-hazards regression model for codominant and per-allele scenario, adjusted for age, stage and grading and stratified by therapy (chemotherapy vs no or other therapies). Chemotherapy group include patients tretated with systemic and local chemotherapy with different therapeutic plans (Carboplatinum doublets, Gencitabin, Epirubicin, etc). We identified 456 BC patients which were followed-up for mortality (median follow-up 62 months). In multivariate analysis both stage and grade predicted the outcome in a statistically significant way, after adjustment for age. Our preliminary analyses showed that the variant alleles in two SNPs in XRCC1 gene (rs 2854509 and rs 3213255) confer a significant lower risk of death and one SNP in ERCC2 gene (rs 171140) confers a significant higher risk of death compared to the wild type (analyses adjusted by age, stage and grading) only in the group of treated with chemotherapy. Our findings suggest a link between DNA repair genes variation and survival in BC patients that become evident only in patients treated with chemotherapy. We hypothesize that levels of DNA adducts or other DNA damages inducted by chemotherapy, are partially due to the individual DNA repair activity, resulting in a different response to chemotherapy and, consequentially, in different survival rates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4670. doi:10.1158/1538-7445.AM2011-4670

BACKGROUND Common sequence variants in 8q24 and 5p15 chromosomal regions have recently been associated with several cancer types and other pathologies. Notably, for most of them a strong environmental risk component has been described. The aim of this study is to investigate for the first time in a cohort study the association among SNPs in those regions and several diseases. METHODS We conducted the study on Turin EPIC (European Perspective Investigation in Cancer and Nutrition) cohort: 10,604 volunteers recruited from 1993 to 1998, aged 35 to 64 years old. Each subject compiled a detailed questionnaire on life style and diet, provided a blood sample and was followed-up for life status, cancer, cardiovascular and diabetes incidence. We selected from literature two SNPs in 5p15 region (rs401681, rs2736098) and five SNPs in 8q24 region (rs672888, rs1447295, rs9642880, rs16901979, rs6983267) which proved to be associated with several diseases in previous studies. We calculated Hazard Ratios for each cancer site and cause of mortality under different genetic models, and incident diabetes Odd Ratios. RESULTS We observed 830 incident cancers (737 excluding skin carcinomas) and 342 overall death in the cohort since 1993. Considering all cancers together, we observed a statistically significant increase in risk when the variant allele in SNP rs6983267 was present (HR 1.19, p We also found statistically significant associations among SNPs in 8q24 region and cancers of head and neck, colon-rectum, liver, lung, prostate, bladder, brain, thyroid, lymphomas, all cancers together and diabetes. We also observed statistically significant results for 5p15 region and melanoma. CONCLUSIONS Our analyses confirm for the first time in a cohort study the hypothesis of a pleiotropic effect on several disease susceptibility of 8q24 and 5p15 regions. We are also investigating if those associations are mediated by individual risk factors (diet, body mass index, smoking and blood pressure). Further functional analyses are needed to better elucidate the mechanisms underlying the risk increase conferred by genetic variation in 8q24 and 5p15 regions. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4736.

Background: Recent epidemiological studies suggest a reduced cancer incidence and mortality among metformin users. Some studies also assessed treatment with other anti-diabetic drugs, such as insulin, and found an increased risk of cancer. Our aim is to investigate in a large prospective study the possible association between metformin use and other anti-diabetic drugs and the risk of cancer. In order to disentangle the protective effect of metformin from the possible increased risk of other anti-diabetic drugs we will compare diabetic patients treated with metformin to diabetic patients treated with other anti-diabetic drugs and to non-diabetic subjects. Methods: We have requested data on diabetic patients and non-diabetic subjects from 5 different centers in Italy (about 40000 volunteers enrolled), participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. We expect to have approximately 1500 incident and 1000 prevalent diabetic patients. We will adjust our analyses for anthropometric, dietary, and lifestyle factors. Preliminary analyses have been performed on the Turin cohort, including 473 incident and 214 prevalent diabetic patients. We calculated odds ratios (ORs), adjusting for age and sex for diabetic patients developing cancer using metformin compared to diabetic patients not using metformin. Results: Among both incident and prevalent diabetic patients we found a decreased risk of cancer for metformin users versus non-metformin users: OR=0.89, 95%CI=0.25-3.14 and OR=0.61, 95%CI=0.22-1.69 respectively, however, these results were not statistically significant. Conclusion: The results seem to suggest a protective effect of metformin, although it is not statistically significant. Using data from all EPIC-Italy centers will increase our power. This will be the largest prospective cohort study investigating the possible association between the use of metformin and other anti-diabetic drugs, and cancer incidence, comparing diabetics to non diabetics and adjusting for different possible confounding factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-399.