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original articles Annals of Oncology Annals of Oncology 23: 1488–1493, 2012 doi:10.1093/annonc/mdr475 Published online 2 November 2011 Proanthocyanidins and other flavonoids in relation to pancreatic cancer: a case–control study in Italy M. Rossi1,2*, A. Lugo1, P. Lagiou3,4, A. Zucchetto5, J. Polesel5, D. Serraino5, E. Negri1, D. Trichopoulos3,6 & C. La Vecchia1,2,7 1 Department of Epidemiology, ‘‘Mario Negri’’ Institute for Pharmacological Research, Milan; 2Department of Occupational Medicine, University of Milan, Milan, Italy; Department of Epidemiology, Harvard School of Public Health, Boston, USA; 4Department of Hygiene, Epidemiology and Medical Statistics, University of Athens Medical School, Athens, Greece; 5Unit of Epidemiology and Biostatistics, IRCCS Centro di Riferimento Oncologico, Aviano, Italy; 6Bureau of Epidemiologic Research, Academy of Athens, Athens, Greece; 7International Prevention Research Institute (IPRI), Lyon, France 3 Received 6 June 2011; accepted 13 September 2011 Background: Four cohort studies have examined the relation between flavonoids and pancreatic cancer risk providing inconsistent results. Patients and methods: We conducted a case–control study between 1991 and 2008 in Northern Italy. Subjects were 326 cases with incident pancreatic cancer and 652 frequency-matched controls (admitted to the same hospitals as cases for acute non-neoplastic conditions) who answered a reproducible and valid food-frequency questionnaire. We computed odds ratios (ORs) using logistic regression models conditioned on gender, age and study center, and adjusted for education, history of diabetes, tobacco smoking, alcohol drinking and energy intake. Results: Proanthocyanidins with three or more mers were inversely related to pancreatic cancer risk. The ORs were similar in all classes of polymers with three or more mers and in their combination (OR for the highest versus the lowest quintile of intake, 0.41; 95% confidence interval 0.24–0.69), and did not substantially change after adjustment for fruit and vegetable consumption, and for vitamin C and folate intakes. Eating an additional portion of fruits rich in proanthocyanidins every day reduced the risk of pancreatic cancer by 25%. Conclusion: Dietary proanthocyanidins—mostly present in apples, pears and pulses—may convey some protection against pancreatic cancer risk. Key words: diet, flavonoids, pancreatic cancer, proanthocyanidins, risk introduction Flavonoids are a large group of polyphenolic compounds that are naturally occurring in plants [1]. They are the substances responsible for the pigmentation of plants and the foods of plant origin such as red, yellow, orange and dark green vegetables and fruits. Flavonoids are generally grouped in six major classes, namely flavanols, flavanones, flavonols, anthocyanidins, flavones and isoflavones, and a family of polymers of flavanols without added sugar called proanthocyanidins [1]. Due to their antioxidant, antimutagenic and antiproliferative properties in vitro, flavonoids have been hypothesized to be responsible for the favorable effects of fruits and vegetables against various chronic diseases, including some common neoplasms [2, 3]. The availability of reliable food composition data for the six classes of flavonoids and, more recently, for proanthocyanidins has allowed epidemiological studies to investigate the role of flavonoids in cancer risk [4, 5]. Intake of various flavonoids has been inversely, though inconsistently with respect to specific classes, related to the risk *Correspondence to: Dr M. Rossi, Department of Epidemiology, Istituto di Ricerche Farmacologiche ‘‘Mario Negri’’, Via La Masa, 19, Milano 20156, Italy. Tel: +39-02-39014541; Fax: +39-02-33200231; E-mail: [email protected] of cancers of the upper aerodigestive tract [6–10], stomach [11], colorectum [12–14], urogenital tract [15–17] and breast [18]. Of these studies, only three investigations included data also on proanthocyanidins reporting inverse associations with the risk of stomach, colorectal and lung cancers [10, 13, 19]. With respect to pancreatic cancer, it is still unclear whether diet may affect the risk of this tumor. There is a some evidence suggesting that the consumption of fruits and vegetables, in particular of those containing folate, protects against pancreatic carcinoma [20]. The pancreas is involved in digestion, and it seems reasonable to consider diet among the possible causal factors for this tumor [21]. Unlike other parts of the gastrointestinal tract, however, the pancreas is never exposed to ingested foods or their modified products. The effects of diet on pancreatic carcinogenesis must be via changes in the internal metabolic environment of that organ [21]. In a murine xenograft study, it has been shown that flavonoids were absorbed in the circulatory system and were accumulated in pancreatic cancer cells in vivo [22]. Moreover, studies in vitro and in vivo showed that flavonoids can act as cancer inhibitors at various molecular targets of the pancreas (cell cycle, nuclear factor kB and others) [23, 24]. Flavonoids have also been suggested for treatment of pancreatic cancer or as an adjuvant ª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected] original articles Annals of Oncology to conventional therapeutic regimens, by increasing the action of cytostatic drugs and thus allowing decrease of dosages and reduction of side-effects of the treatment [25, 26]. Four observational studies, all of cohort design, have examined the relation between flavonoids and pancreatic cancer risk [27–30]. The Finnish Alpha-Tocopherol, BetaCarotene Cancer Prevention (ATBC) study [27], a cohort of 27 111 male smokers with 306 pancreatic cancers, found an inverse association between intake of flavonoids (computed as the sum of selected flavanols, flavones and flavonols) and pancreatic cancer incidence [hazard ratio (HR) 0.36, 95% confidence interval (CI) 0.17–0.78 for the highest versus the lowest quartile, P for trend 0.009] in subjects not consuming supplemental a-tocopherol and/or b-carotene. In the multiethnic cohort of 183 518 participants (529 cases of pancreatic cancer) from Hawaii and California [28], intake of flavonols was inversely related to pancreatic cancer risk among current smokers (HR 0.41, 95% CI 0.22–0.74 for the highest versus the lowest quartile, P for trend 0.002). Results from the Iowa Women’s Health Study cohort of 34 651 postmenopausal women (130 cases of pancreatic cancer) [29] point to a nonsignificant inverse trend in risk for pancreatic cancer with increasing intake of catechins (belonging to flavanols) (HRs across quartiles: 1.00, 0.56, 0.53 and 0.74). A Finnish cohort of 62 440 subjects (29 cases of pancreatic cancer) reported fewer cases among those with higher intake of flavonoids (computed as the sum of selected flavones and flavonols), although the inverse association was not significant [30]. To our knowledge, no study has evaluated proanthocyanidins in relation to the risk of pancreatic cancer. We investigated whether flavonoids, including proanthocyanidins, were associated with pancreatic cancer risk in an Italian multicentric case–control study, in which the consumption of fruits and vegetables has been found to be inversely related to pancreatic cancer risk [31]. A reproducible [33] and validated [34] food-frequency questionnaire (FFQ) was used to assess the usual diet during the 2 years before diagnosis (for cases) or hospital admission (for controls). The FFQ included 78 foods, food groups or recipes. An additional section collected data on history of consumption of five alcoholic beverages. Subjects were asked to indicate the average weekly frequency of consumption for each dietary item; intakes less than once a week, but at least once a month, were coded as 0.5 per week. We obtained food and beverage content of six classes of flavonoids (flavanols, flavanones, flavonols, anthocyanidins, flavones and isoflavones) and six classes of proanthocyanidins (monomers, dimers, trimers, 4–6 mers, 7–10 mers, >10 mers) from databases developed by the USA Department of Agriculture [5, 35], supplemented with others sources when needed [36]. Nutrient and energy intakes were computed using an Italian food composition database, integrated with other published data [37]. Major flavonoids included in the six classes are epicatechin and catechin for flavanols; hesperetin and naringenin for flavanones; quercetin for flavonols; malvidin for anthocyanidins; cyanidin, apigenin and luteolin for flavones and genistein for isoflavones. In our population, the major sources of flavonoids in order of importance were tea, apples, pears and wine for flavanols; citrus fruits and juices for flavanones; apples, pears and various common vegetables for flavonols; wine, strawberries, cherries and onions for anthocyanidins; cooked vegetables and tea for flavones; and soy, soy products and pulses for isoflavones. Given the low consumption of soybeans in our population, intake of isoflavones could not be reliably estimated and we excluded this class from the analysis. Procyanidins (polymers of [epi]-catechin) are the most common proanthocyanidins in foods, and prodelphinidins (derived from [epi]gallocatechin) and propelargonidins (derived from afzelechin) have been also identified [5]. Analytical technology does not allow quantification of proanthocyanidins according to their type linkage but only according to their degree of polymerization. The major food sources of combined monomer and dimer proanthocyanidins were apples, pears, chocolate, peaches, apricots, prunes and tea besides wine and those of proanthocyanidins with three or more mers were apples, pears, pulses, peaches, apricots and prunes besides wine. materials and methods statistical analysis We conducted a case–control study of pancreatic cancer between 1991 and 2008 in the province of Pordenone and in the greater Milan area, Northern Italy [32]. Cases were 326 patients (174 men and 152 women; median age 63 years, range 34–80) with incident pancreatic cancer, admitted to major teaching and general hospitals in the study areas, diagnosed no longer than 1 year before the interview and with no previous diagnosis of cancer. Controls were 652 patients (348 men and 304 women; median age 63 years, range 34–80) admitted to the same hospitals as cases for a wide spectrum of acute non-neoplastic conditions, unrelated to smoking, alcohol consumption or long-term modifications of diet. They were admitted for traumatic orthopedic disorders (31%), other orthopedic disorders (31%), acute surgical conditions (28%) and miscellaneous other illnesses, including eye, nose, ear, skin or dental disorders (10%). Controls were frequency matched to cases by gender, age and study center with a control-to-case ratio of 2:1. Less than 5% of cases and controls approached refused to participate. Trained interviewers administered a structured questionnaire to cases and controls during their hospital stay. The questionnaire included information on sociodemographic characteristics, anthropometric measures, selected lifestyle habits (e.g. tobacco smoking, alcohol drinking), personal medical history, family history of cancer and, for women, menstrual and reproductive factors. Volume 23 | No. 6 | June 2012 Odds ratios (ORs) for pancreatic cancer and their corresponding 95% confidence intervals were estimated using conditional multiple logistic regression models [38] conditioned on gender, age (quinquennia, categorically), and study center. Quintiles based on the distribution of controls were computed both directly on the flavonoids and on the residuals of the regression of flavonoids on energy [39]. Since both analyses yielded similar results, only findings from the first approach are presented. All the regression models included terms for year of interview (continuously), years of education (<7, 7–11, ‡12, categorically), history of diabetes, tobacco smoking (never, ex, current smokers of <15 and of ‡15 cigarettes per day, categorically), alcohol drinking (never and current drinkers of <0.5 and ‡0.5 drink per day, categorically), number of drinks per day (continuously) and non-alcohol energy intake (quintiles, categorically). Ex-drinkers were included in the category of drinkers. Additional models included terms for intakes of vitamin C, vitamin E, potassium and folate (continuously). Besides quintiles, flavonoids were entered in the model as continuous variables, with the measurement unit set at one standard deviation of the distribution of controls. Tests for trend were based on the likelihood ratio test between models with and without a linear term for each class of flavonoids and proanthocyanidins. Given the high correlation between some classes of proanthocyanidins, we combined monomers with dimers, as well as polymers with three or doi:10.1093/annonc/mdr475 | 1489 original articles Annals of Oncology more mers. We further investigated foods rich in proanthocyanidins with three or more mers and their combination. results Table 1 shows the distribution of 326 cases of pancreatic cancer and 652 controls according to gender, age and other selected covariates. By design, cases and controls had similar distribution by gender, age and study center. Cases were more frequently smokers than controls, and the proportion of subjects reporting a history of diabetes was higher in cases than in controls. Table 2 shows the distribution of cases according to quintiles of flavonoids among controls. The mean daily intake among controls was 63.4 mg for flavanols, 36.2 mg for flavanones, 24.7 mg for flavonols, 18.6 mg for anthocyanidins, 0.5 mg for flavones, 103.5 mg for monomer and dimer proanthocyanidins combined and 261.7 mg for proanthocyanidins with three or more mers. Table 3 gives the ORs of pancreatic cancer according to quintiles of intake and continuous ORs for the studied compounds. Inverse associations were evident for proanthocyanidins with three or more mers: the ORs were similar in all classes of polymers with three or more mers, as well as in their combination (OR for the highest versus the lowest quintile of intake, 0.41; 95% CI 0.24–0.69; P for trend = 0.001). Weak inverse associations were also found for Table 1. Distribution of 326 patients with pancreatic cancer and 652 control patients according to gender, age, education and other selected variables (Italy, 1991–2008) Characteristic Cases No. (%) Gender Men 174 Women 152 Age (years) <50 32 50–59 89 60–69 122 ‡70 83 Center Milan 151 Aviano/Pordenone 175 Education (years)a <7 166 7–12 86 ‡12 72 Smoking statusa Never smokers 137 Ex-smokers 88 Current smokers (cigarettes per day) <15 35 ‡15 63 History of diabetes No 269 Yes 57 a Controls No.(%) (53.4) (46.6) 348 (53.4) 304 (46.6) (9.8) (27.3) (37.4) (25.5) 64 178 244 166 (46.3) (53.7) 302 (46.3) 350 (53.7) (51.2) (26.5) (22.2) 350 (53.9) 192 (29.5) 108 (16.6) (42.4) (27.2) 328 (50.5) 195 (30.0) (10.8) (19.5) 59 (9.1) 67 (10.3) (82.5) (17.5) (9.8) (27.3) (37.4) (25.5) 615 (94.3) 37 (5.7) The sum does not add up to the total because of some missing values. 1490 | Rossi et al. flavanones (P for trend = 0.04) and flavonols (P for trend = 0.06). No noticeable associations emerged for other flavonoids including flavanols, anthocyanidins, flavones and monomer and dimer proanthocyanidins. After adjustment for fruit and vegetables, the ORs for flavonoids and proanthocyanidins did not substantially change. After adjustment for vitamin C intake, the OR for proanthocyanidins with three or more mers for the highest versus the lowest quintile of intake changed from Table 2. Distribution of 326 patients with pancreatic cancer according to quintiles of flavonoid intakes among controls (Italy, 1991–2008) Mean (SD)a Quintiles of flavonoid intakeb 1 2 3 4 5 Flavanols (mg) Upper cut-off point Cases Flavanones (mg) Upper cut-off point Cases Flavonols (mg) Upper cut-off point Cases Anthocyanidins (mg) Upper cut-off point Cases Flavones (mg) Upper cut-off point Cases Proanthocyanidins <3 mers (mg) Upper cut-off point Cases Monomers (mg) Upper cut-off point Cases Dimers (mg) Upper cut-off point Cases Proanthocyanidins ‡3 mers (mg) Upper cut-off point Cases Trimers (mg) Upper cut-off point Cases 4–6 mers (mg) Upper cut-off point Cases 7–10 mers (mg) Upper cut-off point Cases >10 mers (mg) Upper cut-off point Cases 63.4 (57.8) 23.8 80 38.9 56 56.6 57 97.7 — 70 63 9.6 67 24.6 78 34.7 65 61.0 — 57 59 13.9 78 18.0 59 23.9 64 31.1 — 48 77 4.5 64 11.0 61 18.6 60 31.1 — 59 82 0.3 67 0.4 58 0.5 64 0.7 — 67 70 45.7 66 73.7 103.4 149.2 — 66 59 56 79 19.1 66 32.1 64 44.6 59 65.4 — 56 81 26.1 70 42.2 65 58.8 58 82.9 — 54 79 36.2 (32.7) 24.7 (18.8) 18.6 (16.8) 0.5 (0.3) 103.5 (71.5) 45.2 (32.6) 58.3 (39.3) 261.7 (129.9) 158.1 216.3 270.1 366.9 — 89 64 67 54 52 20.4 (11.1) 11.1 76 16.1 73 20.9 66 28.7 — 62 49 40.0 83 55.9 67 70.6 62 96.8 — 62 52 32.3 84 45.3 74 56.7 61 77.6 — 57 50 72.3 90 97.1 120.2 160.4 — 58 62 59 57 69.1 (35.5) 55.5 (28.7) 116.7 (56.5) a Mean intake and SD among controls. Computed among controls. Number of controls across quintiles could slightly vary between 129 and 131 according to the distributions of each class of flavonoids and proanthocyanidins. SD, standard deviation. b Volume 23 | No. 6 | June 2012 original articles Annals of Oncology 0.41 to 0.57 (95% CI 0.33–0.98), whereas after adjusting for folate intake, the OR changed to 0.50 (95% CI 0.29–0.84). For all other flavonoids, the associations with pancreatic cancer risk disappeared after adjusting for vitamin C or folate. Table 4 shows the OR of pancreatic cancer according to the number of portions per day of selected foods rich in proanthocyanidins with three or more mers. The combination of apples, pears, pulses, peaches, apricots and prunes was inversely associated with the risk of pancreatic cancer: the OR for three or more portions versus less than half portion per day was 0.34 (95% CI 0.17–0.66) and the continuous OR per one portion per day was 0.75 (95% CI 0.64–0.88). We found similar ORs for combined consumption of apples and pears only. discussion We found that proanthocyanidins with three or more mers were inversely related to the risk of pancreatic cancer, even after adjusting for fruit and vegetable consumption or for vitamin C or folate intake. We then examined the major food sources of Table 3. Odds ratios (ORs)a for pancreatic cancer and corresponding 95% confidence intervals (CIs) according to quintiles of flavonoid intakes (Italy, 1991–2008) Quintiles of flavonoid intakeb 1d 2 3 Flavanols Flavanones Flavonols Anthocyanidins Flavones Proanthocyanidins <3 mers Monomers Dimers Proanthocyanidins ‡3 mers Trimers 4–6 mers 7–10 mers >10 mers 1 1 1 1 1 1 1 1 1 1 1 1 1 0.74 1.31 0.68 0.91 0.74 1.03 1.08 0.87 0.56 0.82 0.64 0.69 0.50 (0.46–1.20) (0.83–2.09) (0.42–1.08) (0.55–1.49) (0.45–1.20) (0.64–1.67) (0.62–1.65) (0.54–1.42) (0.36–0.89) (0.52–1.30) (0.41–1.02) (0.44–1.08) (0.31–0.80) 0.65 0.95 0.72 0.75 0.93 0.77 0.73 0.71 0.63 0.72 0.61 0.58 0.57 4 (0.39–1.08) (0.59–1.52) (0.44–1.19) (0.44–1.27) (0.57–1.51) (0.45–1.30) (0.43–1.24) (0.42–1.19) (0.40–1.01) (0.45–1.16) (0.38–0.98) (0.36–0.94) (0.36–0.92) 0.85 0.89 0.41 0.72 0.81 0.67 0.69 0.59 0.49 0.62 0.59 0.56 0.53 5 (0.52–1.40) (0.54–1.47) (0.24–0.70) (0.40–1.30) (0.49–1.35) (0.38–1.17) (0.39–1.22) (0.34–1.03) (0.30–0.81) (0.38–1.01) (0.36–0.96) (0.34–0.92) (0.32–0.85) 0.63 0.68 0.69 0.83 0.88 0.72 0.82 0.68 0.41 0.46 0.41 0.41 0.48 (0.38–1.03) (0.41–1.14) (0.42–1.13) (0.43–1.60) (0.53–1.46) (0.39–1.34) (0.45–1.51) (0.36–1.26) (0.24–0.69) (0.27–0.77) (0.25–0.71) (0.24–0.69) (0.29–0.81) v2 trend ordinal (P-value) OR continuousc 0.17 0.04 0.06 0.38 0.83 0.16 0.28 0.10 0.001 0.002 0.002 <0.001 0.01 0.98 0.85 0.90 1.10 0.97 0.92 0.96 0.90 0.76 0.77 0.76 0.75 0.77 (0.84–1.14) (0.72–1.00) (0.76–1.07) (0.89–1.35) (0.83–1.13) (0.75–1.13) (0.78–1.17) (0.73–1.11) (0.64–0.91) (0.65–0.92) (0.63–0.90) (0.63–0.90) (0.65–0.92) a Estimates from conditional logistic regression models conditioned on gender, age and center of study, and adjusted for year of interview, education, history of diabetes, tobacco smoking, alcohol drinking and non-alcohol energy intake. b Computed among controls. c Estimated for an increment of intake equal to 1 standard deviation (computed among controls). d Reference category. Table 4. Odds ratios (ORs)a for pancreatic cancer and corresponding 95% confidence intervals (CIs) according to number of portions of selected foods rich in proanthocyanidins with three or more mers, and their combination (Italy, 1991–2008) No of portions per day of proanthocyanidins rich foods <0.5b 0.5 to <1 ‡1 to <2 ‡2 to <3 Apples and pears No cases : No controls 121 : 187 OR (95% CI) 1 Pulses No cases : No controls 276 : 496 OR (95% CI) 1 Peaches, apricots and prunes No cases : No controls 231 : 453 OR (95% CI) 1 Combination of foodsc No cases : No controls 40 : 46 OR (95% CI)d 1 3+ v2 trend ordinal (P-value) OR continuous per one portion per day <0.001 0.73 (0.60–0.90) 60 : 91 1.03 (0.66–1.62) 101 : 225 0.67 (0.46–0.97) 34 : 115 0.47 (0.29–0.78) 10 : 34 0.35 (0.15–0.82) 52 : 128 0.73 (0.49–1.09) 4 : 28 0.32 (0.11–0.98) — — — — 0.016 0.44 (0.22–0.88) 74 : 133 1.05 (0.73–1.52) 21 : 66c 0.75 (0.42–1.31) — — — — 0.4 0.74 (0.48–1.15) 60 : 92 0.69 (0.38–1.25) 136 : 277 0.49 (0.29–0.83) 61 : 144 0.44 (0.24–0.79) 29 : 93 0.34 (0.17–0.66) 0.001 0.75 (0.64–0.88) a Estimates from conditional logistic regression models conditioned on gender, age and center of study, and adjusted for year of interview, education, history of diabetes, tobacco smoking, alcohol drinking, and non-alcohol energy intake. b Reference category. c Apples, pears, peaches, apricots, plums, and pulses. d One case and four controls with more than two portions of peaches, apricots or plums were included in this category. Volume 23 | No. 6 | June 2012 doi:10.1093/annonc/mdr475 | 1491 original articles these compounds and found that eating one more portion of apples, pears, pulses, peaches, apricots or prunes every day reduced the risk of pancreatic cancer by 25%. There was also a suggestion of an inverse association of pancreatic cancer risk with flavanone and flavonol intakes, which, however, disappeared after adjustment for other dietary micronutrients. To our knowledge, no previous studies investigated proanthocyanidins and flavanones in relation to pancreatic cancer risk. In the four cohort studies that examined the relation between flavonoids and pancreatic cancer risk [27–30], only flavanols, flavones and flavonols have been studied. Of these, flavonols, in particular kaempferol, were inversely associated with pancreatic cancer risk in two large cohort studies [27, 28] but only in selected subgroups of their populations, i.e. among male smokers in the placebo branch including 79 cases in the ATBC Study [27] and among current smokers including 80 cases in the multiethnic cohort [28]. The hazard ratios in these studies were not adjusted for other dietary factors [20]. In our study, the weak inverse association between flavonols and pancreatic cancer risk did not persist after adjusting for vegetables or folate intake. Flavanols, the monomeric form of proanthocyanidins with added sugar, have been inversely related to pancreatic cancer risk in the placebo branch of the ATBC Study [27] and among postmenopausal women in the Iowa Women’s Health Study, but in our study, the inverse association was rather weak and statistically nonsignificant. We also found no association between flavones and pancreatic cancer, in line with the results of previous studies [40, 41]. Our results with respect to proanthocyanidins are supported by several in vitro and in vivo studies reporting that these compounds have greater antioxidant properties and superior radical scavenging capacity compared with the monomeric flavonoids [42–44]. The inverse relation we found between proanthocyanidins with three or more mers and pancreatic cancer risk points to apples, the major source of these flavonoids, as a food with potential beneficial properties against pancreatic cancer. Few epidemiological studies have investigated this neoplasm in relation to consumption of apples and their results have been inconsistent [27, 45, 46], although apple procyanidins have been suggested to have a strong anticarcinogenic effect on pancreatic cancer cell [24, 47]. We also identified a combination of foods inversely associated with pancreatic cancer that was different from that suggested by Nothlings et al. [28]. Using data from the multiethnic cohort, they found a flavonol-based food pattern, consisting mainly of tea, fruit, cabbage and wine, that was inversely associated to the risk of pancreatic cancer in that population; they could not, however, replicate this association in the EPIC study [48]. Besides apples and pears, we found that pulses were strongly inversely associated with pancreatic cancer risk, in agreement with other studies [46, 49]. Further studies are needed to evaluate the role of flavonoids, especially proanthocyanidins and their food sources, on pancreatic cancer risk. In our studies, the OR estimates were generally more evident for women compared with men. This could be due to the fact that women drink less wine than men. Wine is rich in both alcohol, which has been reported to increase pancreatic cancer risk [32], and in flavonoids, some of which are likely to reduce this risk. Thus, when wine is an important contributor to 1492 | Rossi et al. Annals of Oncology proanthocyanidin intake, the favorable effect of these compounds with respect to pancreatic cancer risk could be reduced. For this reason, we did not include wine in the proanthocyanidin-rich food combination that we suggested as ‘protective’ against pancreatic cancer risk. Cases and controls in our study came from comparable catchment areas, were interviewed by uniformly trained interviewers in their hospital settings and were unaware of any particular dietary hypothesis related to pancreatic cancer, thereby reducing the likelihood of potential selection and recall bias [50]. The FFQ was satisfactorily reliable [33] and valid [34]. Participation among eligible cases and controls was virtually complete, and we excluded from the control series patients hospitalized for diseases likely to be related to tobacco smoking, alcohol consumption or long-term dietary modifications. Strengths of this study are also the relatively high number of pancreatic cancer cases, the reliable food composition databases [33, 34] and the ability to control for energy and micronutrients intakes. Limitations of the study are generic concerns about hospital-based case–control studies, and questions concerning the adaptability of USA flavonoid food composition data to the Italian diet. Though total flavonoid intake may have not been accurately estimated in our study, however, this should not have affected the comparisons between cases and controls. The same case–control study has generated results on other pancreatic risk factors that were in line with those reported from most pancreatic investigations, a fact that provides some assurance that major biases did not operate during the implementation of the study [32]. In conclusion, we found compelling evidence that proanthocyanidins with three or more mers and foods rich in them are inversely associated with pancreatic cancer risk. These compounds deserve to be further investigated as possibly contributing against the risk of developing this serious malignancy for which few therapeutic options are currently available. acknowledgements The authors thank Ivana Garimoldi for editorial assistance. funding This work was supported by the Italian Association for Cancer Research (n.10068), Milan, Italy. The work in this paper was partially supported by Assomela. disclosure The authors declare no conflicts of interest. references 1. Manach C, Scalbert A, Morand C et al. 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