Serum Endotoxins and Flagellin and Risk of Colorectal Cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC) Cohort

This article is featured in Highlights of This Issue, p. 227

Colorectal cancer is one of the most commonly diagnosed cancers and a leading cause of death worldwide (1). It has been postulated that dietary and metabolic factors, such as energy excess and obesity, can cause breakdown of the colonic epithelial barrier function, allowing the interaction of innate immune system with bacterial products, such as lipopolysaccharide (LPS), also known as endotoxin (2). The human gastrointestinal (GI) tract is colonized by a complex community of approximately 10¹⁴ commensal bacteria, representing approximately 1,000 species (3). Colonic microbiota are being increasingly recognized as important contributors to GI health and likely also to colorectal cancer development (4).

LPS is an integral part of the outer membrane of gram-negative bacterial cell wall and also has a major role in both acute and chronic inflammation (5). A related bacterial product is flagellin, the primary structural component of flagella and a dominant target of humoral immunity in response to infection (6). Emerging evidence suggests that an overabundance of bacterial LPS from the gut microbiota may trigger chronic inflammation and increased production of proinflammatory cytokines and increased reactive oxygen species (2, 7). These proinflammatory cytokines can activate the nuclear factor κβ (NF-κβ) pathway, which has been implicated in cell proliferation and DNA damage leading to carcinogenesis (8). Chronic inflammation has been associated with increased risk of colorectal cancer by several studies (9). Thus, hypothetically, long-term exposure to the localized inflammatory responses resulting from LPS exposure may promote colorectal cancer development.

Direct in-vivo measurement of LPS and flagellin levels is challenging, in part because their appearance in blood and organs is sporadic and partly because their presence is quite transient. Hence, a few recent studies have measured levels of immunoglobulins against LPS and flagellin, whose levels can persist for months following exposure to these products, in an attempt to broadly assess systemic exposure to these gut microbial products and probe their potential associations with various disease states (10, 11). In a recent study by Ziegler and colleagues (10), flagellin- and LPS-specific serum immunoglobulin levels (IgM, IgA, and IgG) were markedly increased in patients with short bowel syndrome (SBS) compared with healthy controls. In another study, IgA and IgG antibodies specific for flagellin monomers were shown to be a target of the elevated adaptive immune response associated with Crohn disease, a chronic inflammatory disease of the GI tract (12). Another line of evidence has emerged from a recent animal study that explored the intricate relationship between intestinal barrier function, microbial environment, and inflammation in colorectal cancer by demonstrating that an inflammatory microenvironment promotes colorectal cancer progression in mice (13). The study highlighted that defective intestinal barrier function at tumour sites facilitates invasion of microbial products, triggering inflammation and subsequent tumor growth.

Although the role of microbiota in development of colorectal carcinogenesis has been explored in basic science and animal studies (13, 14), there is currently no direct epidemiologic evidence for the role of endotoxemia and gut barrier dysfunction in colorectal cancer etiology. In the present study, we aimed to examine the association between serum LPS- and flagellin-specific immunoglobulin levels (IgA and IgG) and risk of colorectal cancer development within a nested case–control study in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort.

We used a case–control design nested within the EPIC cohort, a large prospective cohort study with over 520,000 subjects enrolled from 23 centers in 10 Western European countries (Denmark, France, Greece, Germany, Italy, the Netherlands, Norway, Spain, Sweden, and United Kingdom). Details of the design and methods of the EPIC study, including information on dietary assessment methods, blood collection protocols, and follow-up procedures, have been previously described (15). Briefly, individuals who were eligible for the study were selected from the general population of a specific geographical area, town, or province. Exceptions included the French subcohort, which is based on members of the health insurance system or state-school employees, and the Utrecht (Netherlands) subcohort, which is based on women who underwent screening for breast cancer. Between 1992 and 1998, standardized lifestyle and personal history questionnaires, anthropometric data, and blood samples were collected from most participants at recruitment. Diet over the previous 12 months was assessed at recruitment by validated country-specific questionnaires designed to ensure high compliance and improved measures of local dietary habits (16).

In each of the study centers, fasting or nonfasting blood samples were drawn from participants who provided a blood sample and stored at 5°C to 10°C, protected from light, and transported to local laboratories for processing and aliquoting as previously described (15, 16). In all countries, except Denmark and Sweden, blood was separated in the local EPIC centeres and stored at the International Agency for Research on Cancer (Lyon, France; −196°C, nitrogen vapor). In Denmark, blood samples were stored locally at −150°C under nitrogen vapor. In Sweden, samples were stored in −80°C freezers.

Vital status follow-up (98.4% complete) is collected by record linkage with regional and/or national mortality registries in all countries except Germany and Greece, and the Italian center of Naples, where data are collected actively. Incident cancer cases were determined through record linkage with regional cancer registries (Denmark, other Italian centers, the Netherlands, Norway, Spain, Sweden, and United Kingdom; completed up to June 2003) or via a combination of methods, including linkage with health insurance records, contacts with cancer and pathology registries, and active follow-up through study subjects or their next-of-kin (France, Germany, and Greece; completed up to June 2002). Follow-up began at the date of enrollment and ended at the date of colorectal cancer diagnosis.

Eligible colorectal cancer cases were first incident, histologically confirmed cases diagnosed within the EPIC study population. Colon cancers were defined as tumors in the cecum, appendix, ascending colon, hepatic flexure, transverse colon, splenic flexure, and descending and sigmoid (C18.0–C18.7, according to the 10th Revision of the International Statistical Classification of Diseases, Injury, and Cause of Death), as well as tumors that were overlapping or unspecified (C18.8 and C18.9). Rectal cancers were defined as tumors occurring at the rectosigmoid junction (C19) or rectum (C20). Subjects with anal canal tumors were excluded from the study. Colorectal cancer is defined as a combination of the colon and rectal cancer cases. After exclusions of 23 subjects with missing laboratory measurements of LPS or flagellin and 49 subjects with incomplete matching, a total of 1,065 incident colorectal cancer cases (colon, n = 667; rectal, n = 398) with available biomarker measurements were included in the study.

For each identified cancer case, one control was matched by incidence density sampling by age (within 2.5 years), gender, administrative center, time of the day at blood collection, and fasting status at the time of blood collection (less than 3 hours, 3–6 hours, and more than 6 hours). Women were additionally matched on menopausal status (premenopausal, perimenopausal, postmenopausal, or surgically menopausal). Premenopausal women were further matched on phase of the menstrual cycle at blood collection, and postmenopausal women were matched on current use of hormone replacement therapy. Controls were defined as free of cancer, except nonmelanoma skin cancer, at the time of diagnosis of the case.

Serum anti–LPS- and anti–flagellin-specific IgA and IgG levels were quantitated by ELISA at Georgia State University as previously described (10, 11). Briefly, microtiter plates (DYNEX) were coated overnight with purified laboratory-made flagellin (100 ng/well) or purified E.coli LPS (2 μg/well; from E. coli 0128: B12, Sigma; catolog No. 2887) in 9.6 pH bicarbonate buffer. Serum samples from cases and controls diluted at a ratio of 1:200 were applied to wells coated with flagellin or LPS. After incubation and washing, the wells were incubated either with IgG coupled to horseradish peroxidase (GE; catalog No. 375112) or, in the case of IgA-specific antibodies, with peroxidase-labeled IgA (KPL; catolog No. 14-10-01). Quantitation of total immunoglobulins was performed using the colorimetric peroxidase substrate tetramethylbenzidine (TMZ), and optical density (OD) was read at 450 nm and 540 nm (the difference was taken to compensate for optical interference from the plate), with an ELISA plate reader. Data are reported as OD corrected by subtracting background (determined by readings in blank samples) and are normalized to each plate's control sample, which was prepared in bulk, aliquoted, frozen, and thawed daily as used. Only adjusted ODs were used in the analysis. Standardization was performed using preparations of known concentrations of IgA and IgG. Because previously performed assays for these biomarkers in replicates had a very low intraassay coefficient of variation (<5%; ref. 17), our samples were analyzed in singleton to minimize biosample volume requirement, cost, and time. Interassay coefficients of variation were between 3.8% and 6.8%. For all analyses, cases and matched controls were run in the same batch, and the case–control status of the samples was blinded to laboratory technicians.

In the present study, secondary use was made of relevant biomarker measures that had been conducted previously on the same series of subjects (18–20). Briefly, measurements of glycated hemoglobin (HbA1c) were done on erythrocyte hemolysate using the high-performance liquid chromatography method (Bio-Rad Variant II instrument; Bio Lad Laboratories) with intrabatch coefficient of variations of 2.5% (18). High-sensitivity C-reactive protein (hs-CRP) concentrations were measured using a high-sensitivity assay (Beckman-Coulter) on a Synchron LX-20 Pro autoanalyzer (Beckman-Coulter). The interassay coefficients of variation were 6.0% to 6.5% at various concentrations of hs-CRP (19).

The distributions of selected characteristics between colon and rectal cases and the matched controls were compared. Normality of each biomarker was checked by visual inspection, and all were deemed to be approximately normal. Each individual biomarker, as well as anti-flagellin (flagellin IgA+flagellin IgG), anti-LPS (LPS IgA+LPS IgG), and anti-flagellin+LPS exposure (flagellin IgA + flagellin IgG + LPS IgA + LPS IgG) levels were categorized into quartiles based on the distribution among the controls with the lowest quartile as the reference category.

Conditional logistic regression was used to estimate the ORs and 95% confidence intervals (CI) of colorectal cancer, and by anatomical subsite of cancers of colon and rectum in relation to levels of each circulating biomarkers. Risk estimates were computed from both univariate analyses adjusted for the matching factors (matching-adjusted) and multivariable analyses, with additional adjustments for established confounding variables (fully-adjusted), including smoking status (status/duration/intensity of smoking), body mass index (BMI, kg/m²), waist circumference (cm), education level, total alcohol consumption (g/d), physical activity (sex-specific combined total physical activity index), total energy intake (kcal/day), and total daily intakes of fiber (g/day), fruits and vegetables (g/day), and red/processed meats (g/day; refs. 21–26). For all models, collinearity was assessed, and tests for linear trend were performed using a score variable with values from 1 to 4 included in the model, consistent with the quartile grouping.

We evaluated interactions by several factors relevant for colorectal cancer risk that may be also related to anti–LPS- and anti–flagellin-IgA and -IgG concentrations, total bacterial load, and/or to colonic barrier function (27). Sex and tumor location (colon, rectum) were proposed a priori as potential interactions so results are presented stratified by these factors, as well as combined. Other variables (i.e., hs-CRP, waist circumference, BMI, dietary fat, and alcohol intake) were studied for hypothesis generation analyses. Continuous analyses were conducted using a cross-product term of each biomarker and potential interaction term in the model, followed by a likelihood ratio test. Discrete analyses were also undertaken for hs-CRP, waist circumference, BMI, dietary fat, and alcohol intake by including an interaction term formed by the product of the total anti-flagellin+LPS tertile (cutoff points: <5.58, 5.58 to <7.19, ≥7.19) and the sex-specific dichotomized high and low categories of the potential interaction. As with continuous analysis, a likelihood ratio test was used to assess statistical significance.

As a sensitivity analysis, we repeated the main multivariable-adjusted models after excluding cases that occurred in the first 2 years of follow-up and their matched controls to avoid possible reverse causality, as well as after exclusion of countries with lowest (Denmark) and highest (Greece) anti–LPS- and anti-flagellin exposure levels.

Conditional logistic–restricted cubic spline models were used to explore possible deviation from linear relationships between each biomarker and colorectal cancer, with four knots specific at the median of each quartile of biomarker levels (28).

A two-tailed P value of <0.05 was considered to be statistically significant. All statistical analyses were performed with SAS version 9.3 (SAS Institute) statistical software package.

Selected baseline characteristics of the colon and rectal cases and their matched controls are compared in Table 1. Colon and rectal cancer cases were on average 58.8 years and 58.1 years old, respectively. Both colon and rectal cancer cases were more likely to be current smokers, inactive, had higher education, higher total daily energy, and consume less fruit and vegetable than their matched controls. For colon cancer, female cases had lower median concentrations of anti–flagellin-IgA (1.05 vs. 1.17), anti–LPS-IgA (1.56 vs. 1.71), and anti–LPS-IgG (1.36 vs. 1.44) than their matched controls, whereas the concentrations of each of these serologic biomarkers were higher in cases than controls in men (anti–flagellin-IgA: 1.33 vs. 1.30; anti–LPS-IgA: 1.83 vs. 1.68; anti–LPS-IgG: 1.45 vs. 1.37). For rectal cancer, concentrations of each of the serologic biomarkers were higher in cases than controls in women, except anti–LPS-IgA, where cases had lower concentrations than controls (1.38 vs. 1.48). On the other hand, concentrations of all the serologic biomarkers were slightly lower in male rectal cancer cases than controls, except anti–flagellin-IgA, where cases had higher concentrations than controls (1.29 vs. 1.17).

All models found no association between colorectal cancer and biomarkers of either anti-LPS or anti-flagellin (Supplementary Table S1). However, when analyses were stratified by sex, a significant interaction of the colorectal cancer-LPS/flagellin risk association (total anti-flagellin+LPS, P_interaction < 0.05) was observed. Among men, there was a significant, positive association between colorectal cancer risk and levels of total anti-flagellin+LPS exposure (Table 2) with a fully-adjusted OR of 1.66 (95% CI, 1.10–2.51) comparing the higest versus lowest quartiles, and a significant test for trend (P_trend 0.049). In contrast, among women, there were inverse associations with colorectal cancer risk. Anti–flagellin-IgA was negatively associated with risk of colorectal cancer (fully-adjusted OR, 0.65 comparing highest vs. lowest quartiles; 95% CI, 0.44–0.96; P_trend = 0.02). In addition, there was a trend of significant inverse association between anti–LPS-IgA and risk of colorectal cancer with P_trend of 0.02. Unlike among men, the levels of total anti-flagellin+LPS exposure were also negatively related to colorectal cancer risk, though the association did not reach significance (fully-adjusted OR, 0.70 comparing highest vs. lowest quartiles; 95% CI, 0.47–1.02; P_trend = 0.18).

In stratified analyses by anatomical subsites (P_{heterogeneity} = 0.64), colon cancer risk in men continued to be significantly positively associated with total anti-flagellin+LPS concentrations (fully-adjusted OR, 1.80 comparing highest vs. lowest quartiles; 95% CI, 1.04–3.10; P_trend < 0.049; Table 3), as well as with total anti-LPS (fully-adjusted OR, 1.97; 95% CI, 1.15–3.39; P_trend = 0.01). However, among women, higher concentrations of several biomarkers remained associated with reduced risk of colon cancer with fully-adjusted ORs of 0.59 (95% CI, 0.37–0.93), 0.57 (95% CI, 0.35–0.91), and 0.62 (95% CI, 0.39–0.98), comparing those with highest quartiles of anti–flagellin-IgA, anti–LPS-IgG, and total anti-LPS to reference, respectively (Table 3).

No significant association was observed between risk of rectal cancer and any of the measures for either men or women (Table 4).

The analysis of the interaction between total anti-LPS+flagellin level and inflammation (hs-CRP), body size (waist circumference and BMI), dietary fat intake, and alcohol consumption showed that, among men, the positive association between colorectal cancer risk and total anti-LPS+flagellin level was stronger at higher levels of hs-CRP (OR, 2.35 comparing highest hs-CRP and highest tertile of total anti-LPS+flagellin vs. lowest hs-CRP and lowest tertile of total anti-LPS+flagellin; 95% CI, 1.45–3.81; P_interaction = 0.002), waist circumference (OR, 1.97; 95% CI, 1.24–3.13; P_interaction = 0.01), BMI (OR, 1.77; 95% CI, 1.13–2.78; P_interaction = 0.03), and alcohol (OR, 1.71; 95% CI, 1.09–2.69; P_interaction = 0.02). No interaction was observed in any of these factors among women (P_interaction > 0.05 for all; Supplementary Table S2).

After excluding cases that occurred during the first 2 years of follow-up and their matched controls to avoid possible reverse causality, the findings did not change substantially for any of the serologic biomarkers in both colon and rectal cancers for either sex (Supplementary Table S3). Similar results were observed after excluding participants in the countries with lowest (Denmark) and highest (Greece) anti–LPS- and anti-flagellin biomarker exposure levels (data not shown). Spline models showed that the associations between anti-flagellin and anti-LPS biomarkers and risk of colon or rectal cancers were linear (data not shown).

In this nested case–control study, we investigated the associations of serologic bacterial markers of anti–LPS- and anti–flagellin-IgA and IgG with colorectal cancer risk. No significant associations were observed with colorectal cancer risk, but subgroup analyses by sex revealed a positive association in men for anti-LPS and anti-flagellin markers combined, whereas in women, the associations were inverse.

One key mechanism whereby microbiota may influence colorectal cancer development is through intestinal barrier dysfunction (29). There is an emerging recognition of the ability of the GI tract to regulate the trafficking of macromolecules between the environment and the host through a barrier mechanism (30). A growing body of evidence supports a link between increased intestinal permeability and several GI disorders such as inflammatory bowel disease (IBD) (31), which is a known risk factor for colorectal cancer. It has been suggested that some dietary/lifestyle exposures (e.g., total fat intake, body weight) and physiologic factors (e.g., inflammation) may exacerbate intestinal permeability, leading to increased exposure of the colonic epithelium to endotoxins and greater leakage of endotoxins into the systemic circulation (32, 33).

The impact of bacteria on the development of colorectal cancer has been mostly studied from the perspective of inflammatory responses. It has become clear that the microbiota has a major influence on immune responses, and chronic inflammation is a well-established risk factor for colorectal cancer (34). LPS has been suggested to be involved in colorectal cancer development through their roles in stimulating the immune system by binding cell-surface Toll-like receptor (TLR)-4, the predominant receptor for LPS, and activating transcription factors, such as NF-κB, resulting in an increased production of proinflammatory cytokines, such as TNFα, IL1, and IL6 (35). Flagellin is recognized by both TLR-5 and the NLRC4 inflammasome, which elicits immune signals by activation of NF-κB and caspase-1, respectively, and hence promotes systemic inflammation by production of multiple inflammatory cytokines (36, 37).

Despite a growing body of evidence from in vitro and in vivo studies on the role of the microbiome in the development of colorectal cancer, limited epidemiologic studies have thus far been available to show associations between bacterial endotoxin exposure and colorectal adenomas or colorectal cancer. Two recent studies have observed a positive relationship between endotoxin and colorectal adenomas (38, 39) with the strongest associations observed for dysplastic lesions (39). Our results showing a positive association of serum LPS and flagellin biomarkers and colorectal cancer in men are in line with the results of these studies on the role of bacteria exposure in colorectal cancer carcinogenesis. However, these studies did not report sex-stratified findings so do not permit compassion with our findings in women.

We observed in hypothesis-generating analyses that the positive associations between anti-LPS and anti-flagellin levels and risk of colorectal cancer in men were stronger in higher levels of hs-CRP, waist circumference, BMI, and alcohol intake, results which, if replicated, suggest that these factors may play a role in exacerbating the colorectal cancer–promotive effects of LPS and flagellin. Also worthy of examination is the possibility that body size, inflammation, and alcohol intake may influence intestinal permeability and so leads to increased exposure to bacterial products.

Based on the observations from the above-mentioned studies, an inverse association between anti-LPS and anti-flagellin levels and colorectal cancer risk that we observed among women was unexpected. However, other studies have previously demonstrated inverse associations between environmental endotoxin exposures and the risk of lung and other cancers in occupational settings. Protective effects of environmental/occupational endotoxin exposure on lung cancer have been consistently demonstrated in studies of cotton textile due to raw cotton fiber or dust being contaminated with bacterial endotoxin (40–42) and farming industries (43). Differences between men and women have also been observed among cotton plant workers where there was an increased risk of colon and liver cancers in men while women had lower risk of rectal/anal and liver cancers (44). However, these previous studies were based on occupational cohorts with high endotoxin exposures, whereas the endotoxin measures of our study subjects are likely to be derived largely from the colonic bacteria rather than the environment. Therefore, careful interpretation is required when comparing our findings with those of the previous studies looking at specific subject groups.

Two mechanisms may be involved in the differences between men and women that we observed in the associations between endotoxin and risk of colorectal cancer. First, complex interactions between the innate and adaptive immune systems are important underlying mechanisms of associations between endotoxin and carcinogenesis (45). The differences between men and women are observed and could therefore result from well-established sex-based differences in the immune systems that result in women having a more vigorous immune response, both cellular and humoral, than men (46–48). Second, it is possible that the composition of microbiota could differ in men and women as sex differences have been observed in the composition of skin microbiota (49). It is therefore possible that different organisms might have different associations with colon carcinogenesis and so account for the differences between men and women we observed. Such possibilities have yet to be studied in detail.

Lastly, it is possible that our gender-specific observations are due to chance, despite the relatively large size of the present study. Therefore, replication of these findings and deeper exploration of the sex-specific bacterial exposure and colorectal cancer hypothesis is required.

The present study has several strengths. Our study is the largest prospective cohort so far to investigate bacterial exposures and colorectal cancer risk. Therefore, we had a large enough sample size to be able to stratify by anatomical subsites of colorectal cancer and by gender. To our knowledge, no previous studies on bacterial exposure and colorectal cancer risk have had a sufficiently large enough sample size to conduct stratified analyses.

We also have several limitations in the study. First, because the gut is colonized by complex bacterial communities, elevated anti-LPS or anti-flagellin levels alone may not be sufficient to promote inflammation and tumor progression (38). Another limitation is that we measured the anti-LPS and anti-flagellin concentrations in serum, not in the colonic mucosa, which could be more relevant for colorectal carcinoma formation. Indeed, the assay we applied measures serum immunoreactivity to common bacterial flagellin monomers, which have highly conserved regions common to many flagellins in the microbiota. Although differences in such flagellin immunoreactivity have been thought to reflect differences in gut permeability, they may also arise from differences in microbiota composition and/or gene expression. Thus, better clarification of the source and biologic properties of these compounds is a task for future research. Moreover, biosamples were available only from the time of recruitment into the cohort, and thus we only had a single-blood measure taken at one point in time.

In summary, we found no overall association between bacterial exposure levels, measured by anti–LPS- and anti-flagellin-IgA and IgG, and risk of colorectal cancer. However, in subgroup analysis by sex, we found some biomarker levels to be positively associated with colorectal cancer risk among men, whereas they were inversely associated with colorectal cancer risk among women. Further studies are warranted to elucidate the underlying mechanisms of bacterial exposure and colorectal cancer by sex as well as the sex-specific role of inflammation and immune response on colorectal cancer risk.

No potential conflicts of interest were disclosed.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.

Conception and design: G. McKeown-Eyssen, A. Tjønneland, K. Overvad, V. Krogh, R. Tumino, H.B. Bueno-de-Mesquita, P.H. Peeters, E. Weiderpass, M.-J. Sánchez, A. Barricarte, M. Dorronsoro, K.-T. Khaw, H. Freisling, W.R. Bruce, M. Jenab

Development of methodology: G. McKeown-Eyssen, R. Tumino, H.B. Bueno-de-Mesquita, E. Weiderpass, A. Barricarte, M. Dorronsoro, M. Jenab

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.Q. Tran, A.T. Gewirtz, K. Overvad, M.-C. Boutron-Ruault, T. Kühn, R. Kaaks, H. Boeing, A. Trichopoulou, E. Vasilopoulou, D. Palli, V. Krogh, A. Mattiello, R. Tumino, A. Naccarati, H.B. Bueno-de-Mesquita, P.H. Peeters, E. Weiderpass, J.R. Quirós, N. Sala, M.-J. Sánchez, A. Barricarte, M. Dorronsoro, M. Werner, N.J. Wareham, K.-T. Khaw, K.E. Bradbury, M. Jenab

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.Y. Kong, H.Q. Tran, A.T. Gewirtz, G. McKeown-Eyssen, V. Fedirko, A. Tjønneland, D. Palli, E. Weiderpass, M. Werner, N.J. Wareham, H. Freisling, P. Ferrari, M.J. Gunter, M. Jenab

Writing, review, and/or revision of the manuscript: S.Y. Kong, G. McKeown-Eyssen, V. Fedirko, I. Romieu, A. Tjønneland, A. Olsen, K. Overvad, M.-C. Boutron-Ruault, N. Bastide, A. Affret, T. Kühn, R. Kaaks, H. Boeing, K. Aleksandrova, A. Trichopoulou, M. Kritikou, E. Vasilopoulou, D. Palli, V. Krogh, A. Mattiello, A. Naccarati, H.B. Bueno-de-Mesquita, P.H. Peeters, E. Weiderpass, J.R. Quirós, N. Sala, M.-J. Sánchez, J.M. Huerta Castaño, A. Barricarte, M. Dorronsoro, M. Werner, N.J. Wareham, K.-T. Khaw, K.E. Bradbury, H. Freisling, F. Stavropoulou, P. Ferrari, M.J. Gunter, A.J. Cross, E. Riboli, W.R. Bruce, M. Jenab

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.Y. Kong, T. Kühn, H. Boeing, M. Kritikou, R. Tumino, E. Weiderpass, J.R. Quirós, K.-T. Khaw, K.E. Bradbury, M. Jenab

Study supervision: M. Kritikou, R. Tumino, H.B. Bueno-de-Mesquita, P.H. Peeters, E. Weiderpass, M. Dorronsoro, M. Jenab

Other (co-author on the grant which supported the research): G. McKeown-Eyssen

This work was funded by Wereld Kanker Onderzoek Fonds as part of the World Cancer Research Fund (WCRF) International Regular Grant Programme (grant number 2010–251; pricipal investigator: M. Jenab). The coordination of EPIC is financially supported by the European Commission (DG-SANCO) and the International Agency for Research on Cancer. The national cohorts are supported by Danish Cancer Society (Denmark); Ligue Contre le Cancer, Institut Gustave Roussy, Mutuelle Générale de l'Education Nationale, Institut National de la Santé et de la Recherche Médicale (INSERM, France); Deutsche Krebshilfe, Deutsches Krebsforschungszentrum and Federal Ministry of Education and Research (Germany); the Hellenic Health Foundation (Greece); Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy and National Research Council (Italy); Dutch Ministry of Public Health, Welfare, and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (the Netherlands); Nordic Centre of Excellence programme on Food, Nutrition, and Health (Norway); Health Research Fund (FIS), PI13/00061 to Granada, Regional Governments of Andalucía, Asturias, Basque Country, Murcia (no. 6236) and Navarra, ISCIII RETIC (RD06/0020; Spain); Swedish Cancer Society, Swedish Scientific Council and County Councils of Skåne and Västerbotten (Sweden); Cancer Research UK (14136 to EPIC-Norfolk; C570/A16491 to EPIC-Oxford; K.E. Bradbury and K.-T. Khaw), Medical Research Council (1000143 to EPIC-Norfolk, United Kingdom).

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