HEPATOBILIARY MALIGNANCIES
Prediagnostic Circulating Vitamin D Levels and Risk of Hepatocellular Carcinoma in European Populations: A Nested Case-Control Study Veronika Fedirko,1 Talita Duarte-Salles,2 Christina Bamia,3 Antonia Trichopoulou,3,4 Krasimira Aleksandrova,5 Dimitrios Trichopoulos,4,6,7 Elisabeth Trepo,8 Anne Tjønneland,9 Anja Olsen,9 Kim Overvad,10 Marie-Christine Boutron-Ruault,11,12 Franc¸oise Clavel-Chapelon,11,12 Marina Kvaskoff,11,12 Tilman K€uhn,13 Annie Lukanova,13 Heiner Boeing,5 Brian Buijsse,5 Eleni Klinaki,4 Chrysanthi Tsimakidi,4 Alessio Naccarati,14 Giovanna Tagliabue,15 Salvatore Panico,16 Rosario Tumino,17 Domenico Palli,18 H. Bas Bueno-de-Mesquita,19,20,21 Peter D. Siersema,20 Petra H. Peters,22 Eiliv Lund,23 Magritt Brustad,23 Karina Standahl Olsen,23 Elisabete Weiderpass,23,24,25,26 Raul Zamora-Ros,27 Marıa-Jose Sanchez,28,29,30 Eva Ardanaz,29,31 Pilar Amiano,32 Carmen Navarro,29,33,34 J. Ramon Quiros,35 Ma˚rten Werner,36,37 Malin Sund,38 Bj€orn Lindkvist,39 Johan Malm,40 Ruth C. Travis,41 Kay-Tee Khaw,42 Magdalena Stepien,2 Augustin Scalbert,2 Isabelle Romieu,2 Pagona Lagiou,3,6 Elio Riboli,21 and Mazda Jenab2 The association between vitamin D status and hepatocellular carcinoma (HCC) has not been well investigated, despite experimental evidence supporting an important role of vitamin D in liver pathophysiology. Our objective was to investigate the association between prediagnostic circulating 25-hydroxyvitamin D [25(OH)D] serum levels and the risk of HCC in a prospective, nested case-control study among 520,000 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Each case (n 5 138) diagnosed between 1992 and 2010 was matched to one control by age, sex, study center, date and time of blood collection, and fasting status. Serum baseline levels of 25(OH)D were measured by liquid chromatography/tandem mass spectrometry. Multivariable incident rate ratios (IRRs) of HCC associated with continuous (per 10 nmol/L) or categorical levels (tertiles or a priori-defined categories) of prediagnostic 25(OH)D were calculated using conditional logistic regression. Higher 25(OH)D levels were associated with a 49% reduction in the risk of HCC (highest versus lowest tertile: multivariable IRR 5 0.51, 95% confidence interval [CI], 0.26 to 0.99; Ptrend 5 0.04; per 10 nmol/L increase: IRR 5 0.80, 95% CI, 0.68-0.94). The finding did not vary substantially by time from enrolment to diagnosis, and did not change after adjustment for biomarkers of preexisting liver damage, nor chronic infection with hepatitis B or C viruses. The findings were not modified by body size or smoking status. Conclusion: In this prospective study on western European populations, serum levels of 25(OH)D were inversely associated with the risk of HCC. Given the rising incidence of this cancer in low-risk developed countries and the strong public health interest surrounding the potentially cancer-protective roles of vitamin D, additional studies in different populations are required. (HEPATOLOGY 2014;60:1222-1230)
See Editorial on Page 1130
H
epatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and the third most common cause of cancer-related
deaths worldwide.1 It is highly malignant, usually diagnosed at late stages, and often has very poor prognosis with limited treatment options. The major known risk factors for HCC are chronic infection with hepatitis B or C viruses (HBV/HCV), food contamination with aflatoxins, alcohol abuse accompanied by liver
Abbreviations: ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; EPIC, European Prospective Investigation into Cancer and Nutrition; IRR, incident rate ratio; NAFLD, nonalcoholic fatty liver disease. 1222
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cirrhosis, tobacco smoking, and obesity.2,3 However, little is known about the role of other risk factors including diet, particularly in regions where HBV/ HCV infections and exposure to aflatoxins are less prevalent. Vitamin D has been a focus of attention in cancer prevention during the last decade due to its antineoplastic effects including direct effects on cell proliferation, differentiation, programmed cell death, modulation of growth factor signaling, androgen/estrogen receptor pathways, inflammation, oxidative stress and immune responses, and inhibition of invasion, metastasis, and angiogenesis.4-6 Vitamin D may have a direct effect on the liver since it is the main synthesis
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site for vitamin D-binding protein and 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D reflecting vitamin D status.5 It was shown to inhibit liver fibrosis,7-9 affect expression of liver detoxifying enzymes,10 repress peroxisome proliferator activated receptor alpha (PPARa) signaling,11 and regulate bile acid synthesis.12-14 Also, vitamin D receptor (VDR), a mediator of vitamin D’s genomic actions, is expressed in nonparenchymal liver cells such as hepatic stellate cells,8,15 which are the main producers of extracellular matrix and central players in liver fibrosis,16 which can lead to cirrhosis and HCC. Furthermore, low vitamin D levels have also been shown to be associated with increased hepatic fibrosis
From the 1Department of Epidemiology, Rollins School of Public Health, Winship Cancer Institute, Emory University, Atlanta GA, USA; 2International Agency for Research on Cancer (IARC-WHO), Lyon, France; 3WHO Collaborating Center for Food and Nutrition Policies, Department of Hygiene, Epidemiology, Medical Statistics, University of Athens Medical School, Athens, Greece; 4Hellenic Health Foundation, Athens, Greece; 5Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany; 6Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA; 7Bureau of Epidemiologic Research, Academy of Athens, Athens, Greece; 8Centre de Bioloqie Republique, Lyon, France; 9Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark; 10Department of Public Health, Aarhus University, Aarhus, Denmark; 11Inserm, Centre for Research in Epidemiology and Population Health (CESP), Nutrition, Hormones and Women’s Health Team, Villejuif, France; 12Universit e Paris Sud, UMRS 1018, Institut Gustave Roussy, Villejuif, France; 13German Cancer Research Center (DKFZ), Heidelberg, Germany; 14HuGeF, Human Genetics Foundation, Torino, Molecular and Genetic Epidemiology Unit, Torino, Italy; 15Lombardy Cancer Registry and Environmental Epidemiology Unit Fondazione IRCCS Istituto Nazionale dei Tumori, Italy; 16Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy; 17Cancer Registry and Histopathology Unit, “ Civile M.P. Arezzo” Hospital, Ragusa, Italy; 18Molecular and Nutritional Epidemiology Unit, Cancer Research and Prevention Institute, ISPO, Florence, Italy; 19National Institute for Public Health and the Environment (RIVM), Bithoven, The Netherlands; 20Department of Gastroenterology and Hepatology, University Medical Centre, Utrecht, The Netherlands; 21School of Public Health, Imperial College London, London, UK; 22Julius Centre for Health Sciences and Primary Care, University Medical Centre, Utrecht, The Netherlands; 23Department of Community Medicine, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway; 24Department of Research, Cancer Registry of Norway, Oslo, Norway; 25Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; 26Samfundet Folkh€ alsan, Helsinki, Finland; 27Unit of Nutrition, Environment and Cancer, Catalan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain; 28Andalusian School of Public Health-Granada, Spain; 29CIBER Epidemiologıa y Salud P ublica-CIBERESP, Spain; 30Instituto de Investigaci on Biosanitaria de Granada, Granada, Spain; 31Navarre Public Health Institute, Pamplona, Spain; 32Public Health Division of Guipuzkoa, Basque Regional Health Department, San Sebastian, Spain; 33Department of Epidemiology, Murcia Regional Health Council, Murcia, Spain; 34Department of Health and Social Sciences, Universidad de Murcia, Murcia, Spain; 35Public Health Directorate, Asturias, Spain; 36Department of Public Health and Clinical Medicine, Umea˚ University, Sweden; 37Department of Public Health and Clinical Medicine, Umea˚ University, Umea˚, Sweden; 38Department of Surgical and Perioperative Sciences/Surgery, Umea˚ University, Umea˚, Sweden; 39Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 40Faculty of Medicine, Laboratory Medicine, Lund University, Malm€ o, Sweden; 41Cancer Epidemiology Unit, University of Oxford, Oxford, UK; 42Clinical Gerontology Unit, University of Cambridge School of Clinical Medicine, Cambridge, UK. Received November 28, 2013; accepted February 10, 2014. This work was supported by the French National Cancer Institute (L’Institut National du Cancer; INCA) (grant number 2009-139; PI: 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 G e n e rale de l’Education Nationale; and Institut National de la Sante et de la Recherche M e dicale (INSERM) (France); Deutsche Krebshilfe, Deutsches Krebsforschungszentrum (DKFZ); and Federal Ministry of Education and Research (Germany); Hellenic Health Foundation (Greece); Italian Association for Research on Cancer (AIRC); National Research Council; and AIRE-ONLUS Ragusa, AVIS Ragusa, Sicilian Government (Italy); Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy (Florence, 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); and Statistics Netherlands (the Netherlands); European Research Council (ERC) (grant number ERC-2009AdG 232997), Nordforsk, Nordic Center of Excellence Programme on Food, Nutrition and Health, European Research Council, Norwegian Cancer Society and Norwegian Research Council (Norway); Health Research Fund (FIS); Regional Governments of Andalucıa, Asturias, Basque Country, Murcia (No. 6236) and Navarra; and ISCIII RETIC (RD06/0020) (Spain); Swedish Cancer Society; Swedish Scientific Council; and Regional Government of Ska˚ne and V€ asterbotten (Sweden); Cancer Research UK; Medical Research Council; Stroke Association; British Heart Foundation; Department of Health; Food Standards Agency; and Wellcome Trust (UK). The funding sources had no influence on the design of the study; the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the article for publication. Address reprint requests to: Veronika Fedirko, Ph.D., Rollins School of Public Health, Winship Cancer Institute, Emory University, Atlanta, GA. E-mail: [email protected]; fax: 404-727-8737; or Mazda Jenab, Ph.D., International Agency for Research on Cancer (IARC-WHO), Lyon, France. E-mail: [email protected]. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27079 Potential conflict of interest: Nothing to report.
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in patients with chronic liver disease,17-19 type 2 diabetes,20,21 and nonalcoholic fatty liver disease (NAFLD),22,23 all of which could lead to HCC development. Multiple epidemiologic studies indicate that vitamin D deficiency may be associated with increased risks of some common cancers, particularly colorectal cancer, and that higher vitamin D concentrations are associated with better prognosis and improved outcomes.24-26 In contrast, pooled analyses to assess potential associations between vitamin D status and the seven rarer cancers (not including HCC) found no evidence of a protective effect.27 Existing data on HCC are scarce, with only one epidemiologic study to date showing a nonstatistically significant inverse association with HCC risk, and a strong inverse association with chronic liver disease mortality.28 The study was nested within the Linxian Nutrition Intervention Trial cohorts which were conducted in China among poorly nourished and micronutrientdepleted individuals residing in a region of elevated HCC incidence due to high prevalence of HBV/HCV infections and aflatoxin exposures. Information is still lacking on Western populations with different dietary and lifestyle patterns and a lower prevalence of HBV/ HCV infections. In consideration of these points, we investigated whether a prediagnostic circulating vitamin D concentration is associated with risk of HCC within the European Prospective Investigation into Cancer and Nutrition (EPIC) study, a large cohort based on geographically diverse European populations.
Materials and Methods Study Design. EPIC is a multicenter prospective cohort study designed to investigate the association between diet, lifestyle, and environmental factors and the incidence of various types of cancer and other chronic diseases. The rationale, study design, and methods of recruitment are described in detail elsewhere,29 including baseline assessment of lifestyle,30,31 anthropometrics,32 and diet.33 The study subjects were recruited from the general population, except for Utrecht and Florence (women attending breast cancer screening), the Oxford “Health conscious” subcohort (vegetarians), and subsamples of the Italian and Spanish cohorts (members of blood donor associations). Diet and lifestyle data were collected from 520,000 men and women aged 20-85 years enrolled between 1992 and 2000 in 23 centers throughout 10 European countries (Denmark, France, Germany, Greece, Italy, Norway, Spain, Sweden, the Netherlands, the United Kingdom).29 At recruitment, blood samples were col-
HEPATOLOGY, October 2014
lected from most participants and are stored at the International Agency for Research on Cancer (IARC, Lyon, France) in 2196 C liquid nitrogen for all countries except Denmark (2150 C, nitrogen vapor) and Sweden (280 C, freezers). Approval for this study was obtained from the IARC ethical review board (Lyon, France) as well as from relevant ethical review boards of participating EPIC centers. Follow-up for Cancer Incidence. Cancer incidence was determined through record linkage with population-based regional cancer registries (Denmark, Italy, the Netherlands, Norway, Spain, Sweden, and the United Kingdom; complete up to December 2008) or by way of a combination of methods, including the use of health insurance records, contacts with cancer and pathology registries, and active follow-up through study subjects and their next-of-kin (France, Germany, Greece; complete up to June 2010). Nested Case-Control Study. HCC was defined as first incident tumor in the liver (C22.0 as per the 10th Revision of the International Statistical Classification of Diseases, Injury and Causes of Death [ICD-10]). For each identified case, the histology, the methods used to diagnose the cancer, and a-fetoprotein levels were reviewed to exclude metastatic cases or other types of liver cancers. During the period between recruitment and 2010 (this date was 2006 for the centers in Malm€o, Sweden, and Denmark for administrative reasons and lack of biosample availability), a total of 204 HCC cases were identified. Sixty-six cases had no available serum samples for vitamin D analyses and so could not be included; however, they did not differ by lifestyle and demographic characteristics from cases with available serum sample. Thus, a final series of 138 HCC with available serum sample were identified and matched to one control by incidence density sampling from all cohort members alive and free of cancer (except nonmelanoma skin cancer) by age at blood collection (61 year), sex, study center, date (62 months) and time of the day at blood collection (63 hours), fasting status at blood collection (6 hours); among women, additionally by menopausal status (pre-/ peri-/postmenopausal), and hormone replacement therapy use at time of blood collection (yes/no). Laboratory Assays of HBV/HCV Status and Biomarker of Liver Injury. For a total of 100 of the HCC cases (those diagnosed before 2006) and their matched controls, existing data were available for HBV and HCV seropositivity (ARCHITECT HBsAg and anti-HCV chemiluminescent microparticle immunoassays; Abbott Diagnostics, France), and biomarkers of
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hepatic injury (alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyltransferase [GGT], liver-specific alkaline phosphatase [AP]; ARCHITECT c Systems; Abbott Diagnostics). For a total of 137 HCC cases and their matched controls, existing biomarker data were also available for measures of amino acids (Biocrates AbsoluteIDQ p150 Kit from Biocrates Life Science, Innsbruck, Austria) run on a QTRAP mass spectrometer (IARC, Lyon, France). These were used to compile the Fischer score (the molar ratio of branched-chain amino acids [leucine1valine1isoleucine] to aromatic amino acids [(phenylalanine1tyrosine1histidine1tryptophan) or (phenylalanine1tyrosine)]), an indicator of hepatic functional reserve and severity of liver dysfunction.34,35 25(OH)D Assessment. For the present study, serum 25(OH)D2 and 25(OH)D3 were measured on all cases and their matched controls using a liquid chromatography/tandem mass spectrometry (LC/MS/ MS) method as previously described36 (Heartland Assays, Ames, IA). The average intraassay coefficient of variation for serum 25(OH)D3 was 5%. Only the analyses for serum 25(OH)D3, the primary exposure variable of interest, are presented because only three participants (1.1%) had detectable 25(OH)D2 concentrations above the limit of assay sensitivity of 3.9 nmol/L. Accordingly, we included 25(OH)D2 measurements only in sensitivity analyses. Matched casecontrol pairs were handled identically and assayed in the same batch in a blinded fashion along with quality control standard. Statistical Analyses. Two conditional logistic models were applied to calculate the incidence rate ratio (IRR) as estimated by odds ratio (OR)37 with 95% confidence interval (CI) and test for trend: 1) with matching factors only, and 2) with adjustment for smoking status (never, former, current, unknown), body mass index (BMI; kg/m2), and baseline intakes of coffee (g/d) and alcohol (g/d). Other a priori-defined potential confounders examined, but not included in the model since their inclusion did not change the effect estimates substantially, were waist-to-hip ratio, level of education, total nonalcohol energy intake, dietary fiber, fish intake, red and processed meat intake, physical activity level (inactive, moderately inactive, moderately active, active, and missing), self-reported diabetes status at baseline (yes, no, unknown), and lifetime alcohol intake pattern (never, former light, former heavy, light, never heavy, periodically heavy, always heavy drinkers, unknown). 25(OH)D concentration was included in models as either continuous (per 10 nmol/L; 1 nmol/L 5 0.40064 ng/mL) or in tertiles with cutpoints based on the
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distribution of the control subjects. As an additional analysis, circulating 25(OH)D concentrations were also divided into three categories with predefined cutpoints38: 64 U/L, GGT women >36 U/L, AP >150 U/L, albumin 20.5 lmol/L); and 4) Fischer score, an indicator of hepatic functional reserve and severity of liver dysfunction. The season or month of blood collection may affect 25(OH)D levels. In order to assess this possibility in relation to HCC, two approaches were used: 1) adjustment for season/month of blood collection; 2) standardization of 25(OH)D levels by adding the overall mean of 25(OH)D for all subjects to the residuals derived from i) a simple regression model fitted to 25(OH)D concentration by season/
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Table 1. Baseline Characteristics of Incident Hepatocellular Carcinoma (HCC) Cases and Matched Control Subjects Within the European Prospective Investigation Into Cancer and Nutrition (EPIC) Study From 1992 to 2010* Characteristic
Case Subjects (N 5 138)
Matched Control Subjects (N 5 138)y
Men (%) 69.6 69.6 Age at blood collection 59.9 (7.3) 59.9 (7.3) (y), mean (SD) Follow-up from blood 6.0 (3.4) — collection (y), mean (SD) Smoking status (%) Never smoker 29.7 43.5 Former smoker 30.4 36.2 Current smoker 39.1 19.6 BMI (kg/m2), mean (SD) 28.5 (4.7) 27.3 (4.2) Physical activity (%) Inactive 7.3 10.9 Moderately inactive 36.2 31.2 Moderately active 46.4 48.6 Active 10.1 9.4 No. with diabetes (%) 13.0 7.3 Daily intake at baseline, mean (SD) Energy (kcal) 2,263.7 (1,011.3) 2,268.6 (631.9) Alcohol (g) 21.4 (33.6) 14.9 (19.2) Dietary calcium (mg) 1,063.4 (518.4) 1,041.6 (398.6) Dietary vitamin D (lg) 4.1 (3.2) 3.9 (2.4) Fish and shellfish (g) 30.8 (25.3) 34.5 (40.5) Red and processed meat (g) 82.7 (53.2) 88.7 (57.4) Fruits and vegetables (g) 414.1 (271.9) 464.9 (286.8) Coffee (g) 313.2 (358.0) 380.2 (424.5) HBV or HCV positive (%)‡ 35.6 3.0 Baseline liver damage score (%)§ 0 30.0 83.2 1-2 31.0 16.8 3 39.0 0 Baseline 25-OH-vitamin D3 (nmol/L), 41.7 (16.9-82.3) 49.9 (24.8-90.9) geometric mean (5th-95th percentile) Missing values were not excluded from percentage calculations; therefore, the sum of percent across subgroups may not add up to 100%. *From the following recruitment centers, number of HCC cases: Denmark (N 5 21), Germany (N 5 30), Greece (N 5 16), Italy (N 5 28), Spain (N 5 11), Sweden (N 5 13), the Netherlands (N 5 4), United Kingdom (N 5 15). No eligible case patients were identified in the cohorts of France and Norway, which include women only. No case patients from Umea, Sweden were included because serum samples were not collected for these participants. † Control subjects had to be alive as of the time of diagnosis of the corresponding case patients and were matched with case patients for study center, sex, age at the time of blood collection (612 months), date of blood collection (62 months), and time of day of blood collection (63 hours). Women were further matched by menopausal status (pre-, post-, or perimenopausal) and use of exogenous hormones (oral contraceptives for premenopausal women and hormone replacement therapy for postmenopausal women) at time of blood collection (yes or no). ‡ Available for 100 cases and 100 controls. § Ranges from 0 to 6; the score was grouped in categories as 0, 1-2, 3 abnormal liver function tests (ALT>55 U/L, AST>34 U/L, GGT men >64 U/L, GGT women > 36 U/L, AP > 150 U/L, albumin < 35 g/L, total bilirubin > 20.5 lmol/L; based on the values provided by the laboratory). Available for 101 cases and 101 controls.
month of blood collection,42 ii) a regression of 25(OH)D concentration on the periodic function 2sin(2pX/12) 2 cos(2pX/12), where X is the month
of blood collection43; and iii) a nonparametric local regression (Proc LOESS; SAS Institute, Cary, NC) with 25(OH)D as the dependent variable and day of the year of blood donation as the independent variable.44 Since the results were similar for different approaches to seasonal variation adjustment, adjustment by LOESS residuals was used in all final models. Potential biologically plausible effect modifying variables (sex, age at diagnosis [55 U/L, AST>34 U/L, GGT men >64 U/L, GGT women > 36 U/L, AP > 150 U/L, albumin < 35 g/L, total bilirubin > 1.2 mg/dL. Available for 100 cases and 100 controls. ‡ Additionally adjusted for lifetime alcohol intake pattern (never, former light, former heavy, light, never heavy, periodically heavy, and always heavy drinkers, unknown) and sex-specific physical activity level (inactive, moderately inactive, moderately active, active, and unknown). § Calculated as the molar ratio of branched-chain aminoacids (leucine, valine, isoleucine) to aromatic aminoacids (phenylalanine, tyrosine, histidine and tryptophan), an indicator of hepatic functional reserve and the severity of liver dysfunction.
HBV/HCV infections and aflatoxin exposures.28 The participants of these trials received a multiple vitamin/ mineral supplement (14 vitamins and 12 minerals), including 800 IU vitamin D in the Dysplasia Trial only, or matching placebo. The Linxian studies and the EPIC cohort populations are very different with respect to diet, lifestyle, environmental exposures, and HCC risk factor profiles. For example, the levels of circulating vitamin D were more than twice lower in the Linxian studies than in our cohort (Linxian 5 20.1 nmol/L versus EPIC 5 49.9 nmol/L among controls), and had a very narrow range of values (interquartile range 5 13.7 to 30.0 nmol/L). In addition, more than 59% of controls in the Linxian studies were either HBV or HCV seropositive and 36% were current smokers, whereas in the EPIC the corresponding numbers were 3% and 20%. In the Linxian studies, higher serum 25(OH)D levels were associated with significantly lower risk of chronic liver disease death. However, in contrast to our study, the association with liver cancer incidence was not statistically significant, likely due to the narrow range of 25(OH)D and differences in the study populations.
Key advantages of the present study are its prospective design, which allows the estimation of vitamin D status before cancer development, inclusion of only first primary HCCs, and measurement of 25(OH)D using the state-of-the-art, gold standard LC/MS/MS method rather than older enzyme-linked immunosorbent assay (ELISA) methods. This study also incorporates biomarkers of HBV/HCV infection and liver function into the analysis allowing the consideration of the 25(OH)D-HCC risk association in the hepatitis free subpopulation, and for adjustment by potential liver injury. In populations with a relatively low prevalence of HBV/HCV infection such as western Europeans, it is important to take into account a preexisting chronic liver dysfunction (e.g., from NAFLD, NASH, excessive alcohol consumption) since it may adversely influence circulating 25(OH)D levels, and may be independently associated with HCC risk. Overall, HCC research in humans has been hampered by the comparative rarity of the tumor, the cost of ascertaining the HBV/HCV status in large populations, the inaccurate diagnosis and reporting of primary tumors since the liver is a major site for cancer metastases, and by metabolic changes
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that may occur before cancer diagnosis. Therefore, this prospective study with long and near complete followup, detailed information on dietary/lifestyle factors, prediagnostic measures of circulating vitamin D levels and other biomarkers contributes considerably to our understanding of the role of vitamin D in the etiology of this important cancer, whose incidence is rising rapidly. There are some limitations as well. First, vitamin D concentration was measured once and only at baseline on average 6 years before HCC diagnosis; however, a previous study found that a single measurement of serum 25(OH)D has reasonable validity over a 5-year period.45 Multiple measurements of 25(OH)D would result in a better estimate of vitamin D status and could reduce the degree of nondifferential measurement error. Furthermore, vitamin D status might be susceptible to confounding since high vitamin D concentration in general might reflect a healthier lifestyle. In our models, we adjusted for other determinants of healthy lifestyle; however, the presence of possible residual confounding may not be ruled out. Another potential limitation is reverse causality, as several, but not all, studies have found lower circulating vitamin D levels among persons with chronic liver disease.46 However, we have not observed a statistically significant difference in vitamin D levels by HBV/HCV status, arguing against reverse causality as an explanation for our results. In addition, the results of the analyses adjusted for preexisting liver damage and after exclusion of the first 6 years after blood collection supported an inverse association between 25(OH)D and HCC, but were not statistically significant, likely due to smaller sample sizes. Although our study was the first to incorporate the liver function tests into the analysis of HCC in the context of a prospective cohort, we were not able to match cases to controls on liver dysfunction or use other indicators such as the Model for Endstage Liver Disease (MELD) score to adjust for the severity of chronic liver disease. Another limitation of our study is that for administrative reasons and lack of biosample availability, we had to exclude 66 HCC cases; however, they did not differ by lifestyle and demographic characteristics from cases with measured 25(OH)D. Finally, the sample size was relatively small, and no data were available on incidence of type 2 diabetes, prevalence and incidence of cirrhosis and other chronic liver diseases, sun exposure, use of sunscreen and vitamin D supplement, and on exposure to aflatoxins, although it is uncommon in western Europe.47 In summary, the findings from this prospective study support the hypothesis that higher 25(OH)D
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levels are associated with lower risk of HCC. Further research is needed to confirm or refute these findings in other populations and understand the underlying mechanisms of vitamin D’s actions. Acknowledgment: The authors thank Dr. Ronald L. Horst, PhD (Heartland Assays, LLC, Ames, Iowa) for conducting the measurements of 25-(OH)-vitamin D, and C. Biessy and B. Hemon for assistance in database preparation. Reagents for the hepatitis infection determinations were kindly provided to Labo Republique by Abbott Diagnostics Division, Lyon, France.
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16. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-218. 17. Lim LY, Chalasani N. Vitamin D deficiency in patients with chronic liver disease and cirrhosis. Curr Gastroenterol Rep 2012;14:67-73. 18. Terrier B, Carrat F, Geri G, Pol S, Piroth L, Halfon P, et al. Low 25OH vitamin D serum levels correlate with severe fibrosis in HIV-HCV co-infected patients with chronic hepatitis. J Hepatol 2011;55:756-761. 19. Petta S, Camma C, Scazzone C, Tripodo C, Di Marco V, Bono A, et al. Low vitamin D serum level is related to severe fibrosis and low responsiveness to interferon-based therapy in genotype 1 chronic hepatitis C. HEPATOLOGY 2010;51:1158-1167. 20. Pittas AG, Lau J, Hu FB, Dawson-Hughes B. The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis. J Clin Endocrinol Metab 2007;92:2017-2029. 21. Takiishi T, Gysemans C, Bouillon R, Mathieu C. Vitamin D and diabetes. Endocrinol Metab Clin North Am 2010;39:419-446. 22. Barchetta I, Angelico F, Del Ben M, Baroni MG, Pozzilli P, Morini S, et al. Strong association between non alcoholic fatty liver disease (NAFLD) and low 25(OH) vitamin D levels in an adult population with normal serum liver enzymes. BMC Med 2011;9:85. 23. Liangpunsakul S, Chalasani N. Serum vitamin D concentrations and unexplained elevation in ALT among US adults. Dig Dis Sci 2011;56: 2124-2129. 24. Krishnan AV, Feldman D. Mechanisms of the anti-cancer and antiinflammatory actions of vitamin D. Annu Rev Pharmacol Toxicol 2011;51:311-336. 25. Fedirko V, Riboli E, Tjonneland A, Ferrari P, Olsen A, Bueno-deMesquita HB, et al. Prediagnostic 25-hydroxyvitamin D, VDR and CASR polymorphisms, and survival in patients with colorectal cancer in western European ppulations. Cancer Epidemiol Biomarkers Prev 2012;21:582-593. 26. Jenab M, Bueno-de-Mesquita HB, Ferrari P, van Duijnhoven FJ, Norat T, Pischon T, et al. Association between pre-diagnostic circulating vitamin D concentration and risk of colorectal cancer in European populations:a nested case-control study. BMJ 2010;340:b5500. 27. Helzlsouer KJ. Overview of the cohort consortium vitamin D pooling project of rarer cancers. Am J Epidemiol 2010;172:4-9. 28. Wang JB, Abnet CC, Chen W, Dawsey SM, Fan JH, Yin LY, et al. Association between serum 25(OH) vitamin D, incident liver cancer and chronic liver disease mortality in the Linxian Nutrition Intervention Trials: a nested case-control study. Br J Cancer 2013;109:1997-2004. 29. Riboli E, Hunt KJ, Slimani N, Ferrari P, Norat T, Fahey M, et al. European Prospective Investigation into Cancer and Nutrition (EPIC): study populations and data collection. Public Health Nutr 2002;5:1113-1124. 30. Haftenberger M, Schuit AJ, Tormo MJ, Boeing H, Wareham N, Bueno-de-Mesquita HB, et al. Physical activity of subjects aged 50-64 years involved in the European Prospective Investigation into Cancer and Nutrition (EPIC). Public Health Nutr 2002;5:1163-1176. 31. Sieri S, Agudo A, Kesse E, Klipstein-Grobusch K, San-Jose B, Welch AA, et al. Patterns of alcohol consumption in 10 European countries
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participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) project. Public Health Nutr 2002;5:1287-1296. Haftenberger M, Lahmann PH, Panico S, Gonzalez CA, Seidell JC, Boeing H, et al. Overweight, obesity and fat distribution in 50- to 64year-old participants in the European Prospective Investigation into Cancer and Nutrition (EPIC). Public Health Nutr 2002;5:1147-1162. Margetts BM, Pietinen P. European Prospective Investigation into Cancer and Nutrition: validity studies on dietary assessment methods. Int J Epidemiol 1997;26:S1-S5. Fischer JE, Funovics JM, Aguirre A, James JH, Keane JM, Wesdorp RI, et al. The role of plasma amino acids in hepatic encephalopathy. Surgery 1975;78:276-290. Morgan MY, Milsom JP, Sherlock S. Plasma ratio of valine, leucine and isoleucine to phenylalanine and tyrosine in liver disease. Gut 1978; 19:1068-1073. Saenger AK, Laha TJ, Bremner DE, Sadrzadeh SM. Quantification of serum 25-hydroxyvitamin D(2) and D(3) using HPLC-tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency. Am J Clin Pathol 2006;125:914-920. Knol MJ, Vandenbroucke JP, Scott P, Egger M. What do case-control studies estimate? Survey of methods and assumptions in published casecontrol research. Am J Epidemiol 2008;168:1073-1081. Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011;96:1911-1930. Durrleman S, Simon R. Flexible regression models with cubic splines. Stat Med 1989;8:551-561. Govindarajulu US, Spiegelman D, Thurston SW, Ganguli B, Eisen EA. Comparing smoothing techniques in Cox models for exposure-response relationships. Stat Med 2007;26:3735-3752. http://www.hsph.harvard.edu/donna-spiegelman/software/lgtphcurv9/ Accessed September 1, 2013. Munger KL, Levin LI, Hollis BW, Howard NS, Ascherio A. Serum 25hydroxyvitamin D levels and risk of multiple sclerosis. JAMA 2006; 296:2832-2838. Bliss CI. Periodic regression in biology and climatology. New Haven: Connecticut Agricultural Experiment Station, 1958. Borkowf CB, Albert PS, Abnet CC. Using LOWESS to remove systematic trends over time in predictor variables prior to logistic regression with quantile categories. Stat Med 2003;22:1477-1493. Hofmann JN, Yu K, Horst RL, Hayes RB, Purdue MP. Long-term variation in serum 25-hydroxyvitamin D concentration among participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Cancer Epidemiol Biomarkers Prev 2010;19:927-931. Stokes CS, Volmer DA, Grunhage F, Lammert F. Vitamin D in chronic liver disease. Liver Int 2013;33:338-352. Liu Y, Wu F. Global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment. Environ Health Perspect 2010;118:818-824.
Prediagnostic Circulating Vitamin D Levels and Risk of Hepatocellular Carcinoma in European Populations: A Nested Case-Control Study Veronika Fedirko,1 Talita Duarte-Salles,2 Christina Bamia,3 Antonia Trichopoulou,3,4 Krasimira Aleksandrova,5 Dimitrios Trichopoulos,4,6,7 Elisabeth Trepo,8 Anne Tjønneland,9 Anja Olsen,9 Kim Overvad,10 Marie-Christine Boutron-Ruault,11,12 Franc¸oise Clavel-Chapelon,11,12 Marina Kvaskoff,11,12 Tilman K€uhn,13 Annie Lukanova,13 Heiner Boeing,5 Brian Buijsse,5 Eleni Klinaki,4 Chrysanthi Tsimakidi,4 Alessio Naccarati,14 Giovanna Tagliabue,15 Salvatore Panico,16 Rosario Tumino,17 Domenico Palli,18 H. Bas Bueno-de-Mesquita,19,20,21 Peter D. Siersema,20 Petra H. Peters,22 Eiliv Lund,23 Magritt Brustad,23 Karina Standahl Olsen,23 Elisabete Weiderpass,23,24,25,26 Raul Zamora-Ros,27 Marıa-Jose Sanchez,28,29,30 Eva Ardanaz,29,31 Pilar Amiano,32 Carmen Navarro,29,33,34 J. Ramon Quiros,35 Ma˚rten Werner,36,37 Malin Sund,38 Bj€orn Lindkvist,39 Johan Malm,40 Ruth C. Travis,41 Kay-Tee Khaw,42 Magdalena Stepien,2 Augustin Scalbert,2 Isabelle Romieu,2 Pagona Lagiou,3,6 Elio Riboli,21 and Mazda Jenab2 The association between vitamin D status and hepatocellular carcinoma (HCC) has not been well investigated, despite experimental evidence supporting an important role of vitamin D in liver pathophysiology. Our objective was to investigate the association between prediagnostic circulating 25-hydroxyvitamin D [25(OH)D] serum levels and the risk of HCC in a prospective, nested case-control study among 520,000 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Each case (n 5 138) diagnosed between 1992 and 2010 was matched to one control by age, sex, study center, date and time of blood collection, and fasting status. Serum baseline levels of 25(OH)D were measured by liquid chromatography/tandem mass spectrometry. Multivariable incident rate ratios (IRRs) of HCC associated with continuous (per 10 nmol/L) or categorical levels (tertiles or a priori-defined categories) of prediagnostic 25(OH)D were calculated using conditional logistic regression. Higher 25(OH)D levels were associated with a 49% reduction in the risk of HCC (highest versus lowest tertile: multivariable IRR 5 0.51, 95% confidence interval [CI], 0.26 to 0.99; Ptrend 5 0.04; per 10 nmol/L increase: IRR 5 0.80, 95% CI, 0.68-0.94). The finding did not vary substantially by time from enrolment to diagnosis, and did not change after adjustment for biomarkers of preexisting liver damage, nor chronic infection with hepatitis B or C viruses. The findings were not modified by body size or smoking status. Conclusion: In this prospective study on western European populations, serum levels of 25(OH)D were inversely associated with the risk of HCC. Given the rising incidence of this cancer in low-risk developed countries and the strong public health interest surrounding the potentially cancer-protective roles of vitamin D, additional studies in different populations are required. (HEPATOLOGY 2014;60:1222-1230)
See Editorial on Page 1130
H
epatocellular carcinoma (HCC) is the sixth most commonly diagnosed cancer and the third most common cause of cancer-related
deaths worldwide.1 It is highly malignant, usually diagnosed at late stages, and often has very poor prognosis with limited treatment options. The major known risk factors for HCC are chronic infection with hepatitis B or C viruses (HBV/HCV), food contamination with aflatoxins, alcohol abuse accompanied by liver
Abbreviations: ALT, alanine aminotransferase; AP, alkaline phosphatase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; EPIC, European Prospective Investigation into Cancer and Nutrition; IRR, incident rate ratio; NAFLD, nonalcoholic fatty liver disease. 1222
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cirrhosis, tobacco smoking, and obesity.2,3 However, little is known about the role of other risk factors including diet, particularly in regions where HBV/ HCV infections and exposure to aflatoxins are less prevalent. Vitamin D has been a focus of attention in cancer prevention during the last decade due to its antineoplastic effects including direct effects on cell proliferation, differentiation, programmed cell death, modulation of growth factor signaling, androgen/estrogen receptor pathways, inflammation, oxidative stress and immune responses, and inhibition of invasion, metastasis, and angiogenesis.4-6 Vitamin D may have a direct effect on the liver since it is the main synthesis
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site for vitamin D-binding protein and 25-hydroxyvitamin D [25(OH)D], the major circulating form of vitamin D reflecting vitamin D status.5 It was shown to inhibit liver fibrosis,7-9 affect expression of liver detoxifying enzymes,10 repress peroxisome proliferator activated receptor alpha (PPARa) signaling,11 and regulate bile acid synthesis.12-14 Also, vitamin D receptor (VDR), a mediator of vitamin D’s genomic actions, is expressed in nonparenchymal liver cells such as hepatic stellate cells,8,15 which are the main producers of extracellular matrix and central players in liver fibrosis,16 which can lead to cirrhosis and HCC. Furthermore, low vitamin D levels have also been shown to be associated with increased hepatic fibrosis
From the 1Department of Epidemiology, Rollins School of Public Health, Winship Cancer Institute, Emory University, Atlanta GA, USA; 2International Agency for Research on Cancer (IARC-WHO), Lyon, France; 3WHO Collaborating Center for Food and Nutrition Policies, Department of Hygiene, Epidemiology, Medical Statistics, University of Athens Medical School, Athens, Greece; 4Hellenic Health Foundation, Athens, Greece; 5Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany; 6Department of Epidemiology, Harvard School of Public Health, Boston, MA, USA; 7Bureau of Epidemiologic Research, Academy of Athens, Athens, Greece; 8Centre de Bioloqie Republique, Lyon, France; 9Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark; 10Department of Public Health, Aarhus University, Aarhus, Denmark; 11Inserm, Centre for Research in Epidemiology and Population Health (CESP), Nutrition, Hormones and Women’s Health Team, Villejuif, France; 12Universit e Paris Sud, UMRS 1018, Institut Gustave Roussy, Villejuif, France; 13German Cancer Research Center (DKFZ), Heidelberg, Germany; 14HuGeF, Human Genetics Foundation, Torino, Molecular and Genetic Epidemiology Unit, Torino, Italy; 15Lombardy Cancer Registry and Environmental Epidemiology Unit Fondazione IRCCS Istituto Nazionale dei Tumori, Italy; 16Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy; 17Cancer Registry and Histopathology Unit, “ Civile M.P. Arezzo” Hospital, Ragusa, Italy; 18Molecular and Nutritional Epidemiology Unit, Cancer Research and Prevention Institute, ISPO, Florence, Italy; 19National Institute for Public Health and the Environment (RIVM), Bithoven, The Netherlands; 20Department of Gastroenterology and Hepatology, University Medical Centre, Utrecht, The Netherlands; 21School of Public Health, Imperial College London, London, UK; 22Julius Centre for Health Sciences and Primary Care, University Medical Centre, Utrecht, The Netherlands; 23Department of Community Medicine, Faculty of Health Sciences, University of Tromsø, Tromsø, Norway; 24Department of Research, Cancer Registry of Norway, Oslo, Norway; 25Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; 26Samfundet Folkh€ alsan, Helsinki, Finland; 27Unit of Nutrition, Environment and Cancer, Catalan Institute of Oncology (ICO), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain; 28Andalusian School of Public Health-Granada, Spain; 29CIBER Epidemiologıa y Salud P ublica-CIBERESP, Spain; 30Instituto de Investigaci on Biosanitaria de Granada, Granada, Spain; 31Navarre Public Health Institute, Pamplona, Spain; 32Public Health Division of Guipuzkoa, Basque Regional Health Department, San Sebastian, Spain; 33Department of Epidemiology, Murcia Regional Health Council, Murcia, Spain; 34Department of Health and Social Sciences, Universidad de Murcia, Murcia, Spain; 35Public Health Directorate, Asturias, Spain; 36Department of Public Health and Clinical Medicine, Umea˚ University, Sweden; 37Department of Public Health and Clinical Medicine, Umea˚ University, Umea˚, Sweden; 38Department of Surgical and Perioperative Sciences/Surgery, Umea˚ University, Umea˚, Sweden; 39Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 40Faculty of Medicine, Laboratory Medicine, Lund University, Malm€ o, Sweden; 41Cancer Epidemiology Unit, University of Oxford, Oxford, UK; 42Clinical Gerontology Unit, University of Cambridge School of Clinical Medicine, Cambridge, UK. Received November 28, 2013; accepted February 10, 2014. This work was supported by the French National Cancer Institute (L’Institut National du Cancer; INCA) (grant number 2009-139; PI: 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 G e n e rale de l’Education Nationale; and Institut National de la Sante et de la Recherche M e dicale (INSERM) (France); Deutsche Krebshilfe, Deutsches Krebsforschungszentrum (DKFZ); and Federal Ministry of Education and Research (Germany); Hellenic Health Foundation (Greece); Italian Association for Research on Cancer (AIRC); National Research Council; and AIRE-ONLUS Ragusa, AVIS Ragusa, Sicilian Government (Italy); Associazione Italiana per la Ricerca sul Cancro-AIRC-Italy (Florence, 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); and Statistics Netherlands (the Netherlands); European Research Council (ERC) (grant number ERC-2009AdG 232997), Nordforsk, Nordic Center of Excellence Programme on Food, Nutrition and Health, European Research Council, Norwegian Cancer Society and Norwegian Research Council (Norway); Health Research Fund (FIS); Regional Governments of Andalucıa, Asturias, Basque Country, Murcia (No. 6236) and Navarra; and ISCIII RETIC (RD06/0020) (Spain); Swedish Cancer Society; Swedish Scientific Council; and Regional Government of Ska˚ne and V€ asterbotten (Sweden); Cancer Research UK; Medical Research Council; Stroke Association; British Heart Foundation; Department of Health; Food Standards Agency; and Wellcome Trust (UK). The funding sources had no influence on the design of the study; the collection, analysis, and interpretation of data; the writing of the report; or the decision to submit the article for publication. Address reprint requests to: Veronika Fedirko, Ph.D., Rollins School of Public Health, Winship Cancer Institute, Emory University, Atlanta, GA. E-mail: [email protected]; fax: 404-727-8737; or Mazda Jenab, Ph.D., International Agency for Research on Cancer (IARC-WHO), Lyon, France. E-mail: [email protected]. C 2014 by the American Association for the Study of Liver Diseases. Copyright V View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.27079 Potential conflict of interest: Nothing to report.
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in patients with chronic liver disease,17-19 type 2 diabetes,20,21 and nonalcoholic fatty liver disease (NAFLD),22,23 all of which could lead to HCC development. Multiple epidemiologic studies indicate that vitamin D deficiency may be associated with increased risks of some common cancers, particularly colorectal cancer, and that higher vitamin D concentrations are associated with better prognosis and improved outcomes.24-26 In contrast, pooled analyses to assess potential associations between vitamin D status and the seven rarer cancers (not including HCC) found no evidence of a protective effect.27 Existing data on HCC are scarce, with only one epidemiologic study to date showing a nonstatistically significant inverse association with HCC risk, and a strong inverse association with chronic liver disease mortality.28 The study was nested within the Linxian Nutrition Intervention Trial cohorts which were conducted in China among poorly nourished and micronutrientdepleted individuals residing in a region of elevated HCC incidence due to high prevalence of HBV/HCV infections and aflatoxin exposures. Information is still lacking on Western populations with different dietary and lifestyle patterns and a lower prevalence of HBV/ HCV infections. In consideration of these points, we investigated whether a prediagnostic circulating vitamin D concentration is associated with risk of HCC within the European Prospective Investigation into Cancer and Nutrition (EPIC) study, a large cohort based on geographically diverse European populations.
Materials and Methods Study Design. EPIC is a multicenter prospective cohort study designed to investigate the association between diet, lifestyle, and environmental factors and the incidence of various types of cancer and other chronic diseases. The rationale, study design, and methods of recruitment are described in detail elsewhere,29 including baseline assessment of lifestyle,30,31 anthropometrics,32 and diet.33 The study subjects were recruited from the general population, except for Utrecht and Florence (women attending breast cancer screening), the Oxford “Health conscious” subcohort (vegetarians), and subsamples of the Italian and Spanish cohorts (members of blood donor associations). Diet and lifestyle data were collected from 520,000 men and women aged 20-85 years enrolled between 1992 and 2000 in 23 centers throughout 10 European countries (Denmark, France, Germany, Greece, Italy, Norway, Spain, Sweden, the Netherlands, the United Kingdom).29 At recruitment, blood samples were col-
HEPATOLOGY, October 2014
lected from most participants and are stored at the International Agency for Research on Cancer (IARC, Lyon, France) in 2196 C liquid nitrogen for all countries except Denmark (2150 C, nitrogen vapor) and Sweden (280 C, freezers). Approval for this study was obtained from the IARC ethical review board (Lyon, France) as well as from relevant ethical review boards of participating EPIC centers. Follow-up for Cancer Incidence. Cancer incidence was determined through record linkage with population-based regional cancer registries (Denmark, Italy, the Netherlands, Norway, Spain, Sweden, and the United Kingdom; complete up to December 2008) or by way of a combination of methods, including the use of health insurance records, contacts with cancer and pathology registries, and active follow-up through study subjects and their next-of-kin (France, Germany, Greece; complete up to June 2010). Nested Case-Control Study. HCC was defined as first incident tumor in the liver (C22.0 as per the 10th Revision of the International Statistical Classification of Diseases, Injury and Causes of Death [ICD-10]). For each identified case, the histology, the methods used to diagnose the cancer, and a-fetoprotein levels were reviewed to exclude metastatic cases or other types of liver cancers. During the period between recruitment and 2010 (this date was 2006 for the centers in Malm€o, Sweden, and Denmark for administrative reasons and lack of biosample availability), a total of 204 HCC cases were identified. Sixty-six cases had no available serum samples for vitamin D analyses and so could not be included; however, they did not differ by lifestyle and demographic characteristics from cases with available serum sample. Thus, a final series of 138 HCC with available serum sample were identified and matched to one control by incidence density sampling from all cohort members alive and free of cancer (except nonmelanoma skin cancer) by age at blood collection (61 year), sex, study center, date (62 months) and time of the day at blood collection (63 hours), fasting status at blood collection (6 hours); among women, additionally by menopausal status (pre-/ peri-/postmenopausal), and hormone replacement therapy use at time of blood collection (yes/no). Laboratory Assays of HBV/HCV Status and Biomarker of Liver Injury. For a total of 100 of the HCC cases (those diagnosed before 2006) and their matched controls, existing data were available for HBV and HCV seropositivity (ARCHITECT HBsAg and anti-HCV chemiluminescent microparticle immunoassays; Abbott Diagnostics, France), and biomarkers of
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hepatic injury (alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyltransferase [GGT], liver-specific alkaline phosphatase [AP]; ARCHITECT c Systems; Abbott Diagnostics). For a total of 137 HCC cases and their matched controls, existing biomarker data were also available for measures of amino acids (Biocrates AbsoluteIDQ p150 Kit from Biocrates Life Science, Innsbruck, Austria) run on a QTRAP mass spectrometer (IARC, Lyon, France). These were used to compile the Fischer score (the molar ratio of branched-chain amino acids [leucine1valine1isoleucine] to aromatic amino acids [(phenylalanine1tyrosine1histidine1tryptophan) or (phenylalanine1tyrosine)]), an indicator of hepatic functional reserve and severity of liver dysfunction.34,35 25(OH)D Assessment. For the present study, serum 25(OH)D2 and 25(OH)D3 were measured on all cases and their matched controls using a liquid chromatography/tandem mass spectrometry (LC/MS/ MS) method as previously described36 (Heartland Assays, Ames, IA). The average intraassay coefficient of variation for serum 25(OH)D3 was 5%. Only the analyses for serum 25(OH)D3, the primary exposure variable of interest, are presented because only three participants (1.1%) had detectable 25(OH)D2 concentrations above the limit of assay sensitivity of 3.9 nmol/L. Accordingly, we included 25(OH)D2 measurements only in sensitivity analyses. Matched casecontrol pairs were handled identically and assayed in the same batch in a blinded fashion along with quality control standard. Statistical Analyses. Two conditional logistic models were applied to calculate the incidence rate ratio (IRR) as estimated by odds ratio (OR)37 with 95% confidence interval (CI) and test for trend: 1) with matching factors only, and 2) with adjustment for smoking status (never, former, current, unknown), body mass index (BMI; kg/m2), and baseline intakes of coffee (g/d) and alcohol (g/d). Other a priori-defined potential confounders examined, but not included in the model since their inclusion did not change the effect estimates substantially, were waist-to-hip ratio, level of education, total nonalcohol energy intake, dietary fiber, fish intake, red and processed meat intake, physical activity level (inactive, moderately inactive, moderately active, active, and missing), self-reported diabetes status at baseline (yes, no, unknown), and lifetime alcohol intake pattern (never, former light, former heavy, light, never heavy, periodically heavy, always heavy drinkers, unknown). 25(OH)D concentration was included in models as either continuous (per 10 nmol/L; 1 nmol/L 5 0.40064 ng/mL) or in tertiles with cutpoints based on the
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distribution of the control subjects. As an additional analysis, circulating 25(OH)D concentrations were also divided into three categories with predefined cutpoints38: 64 U/L, GGT women >36 U/L, AP >150 U/L, albumin 20.5 lmol/L); and 4) Fischer score, an indicator of hepatic functional reserve and severity of liver dysfunction. The season or month of blood collection may affect 25(OH)D levels. In order to assess this possibility in relation to HCC, two approaches were used: 1) adjustment for season/month of blood collection; 2) standardization of 25(OH)D levels by adding the overall mean of 25(OH)D for all subjects to the residuals derived from i) a simple regression model fitted to 25(OH)D concentration by season/
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Table 1. Baseline Characteristics of Incident Hepatocellular Carcinoma (HCC) Cases and Matched Control Subjects Within the European Prospective Investigation Into Cancer and Nutrition (EPIC) Study From 1992 to 2010* Characteristic
Case Subjects (N 5 138)
Matched Control Subjects (N 5 138)y
Men (%) 69.6 69.6 Age at blood collection 59.9 (7.3) 59.9 (7.3) (y), mean (SD) Follow-up from blood 6.0 (3.4) — collection (y), mean (SD) Smoking status (%) Never smoker 29.7 43.5 Former smoker 30.4 36.2 Current smoker 39.1 19.6 BMI (kg/m2), mean (SD) 28.5 (4.7) 27.3 (4.2) Physical activity (%) Inactive 7.3 10.9 Moderately inactive 36.2 31.2 Moderately active 46.4 48.6 Active 10.1 9.4 No. with diabetes (%) 13.0 7.3 Daily intake at baseline, mean (SD) Energy (kcal) 2,263.7 (1,011.3) 2,268.6 (631.9) Alcohol (g) 21.4 (33.6) 14.9 (19.2) Dietary calcium (mg) 1,063.4 (518.4) 1,041.6 (398.6) Dietary vitamin D (lg) 4.1 (3.2) 3.9 (2.4) Fish and shellfish (g) 30.8 (25.3) 34.5 (40.5) Red and processed meat (g) 82.7 (53.2) 88.7 (57.4) Fruits and vegetables (g) 414.1 (271.9) 464.9 (286.8) Coffee (g) 313.2 (358.0) 380.2 (424.5) HBV or HCV positive (%)‡ 35.6 3.0 Baseline liver damage score (%)§ 0 30.0 83.2 1-2 31.0 16.8 3 39.0 0 Baseline 25-OH-vitamin D3 (nmol/L), 41.7 (16.9-82.3) 49.9 (24.8-90.9) geometric mean (5th-95th percentile) Missing values were not excluded from percentage calculations; therefore, the sum of percent across subgroups may not add up to 100%. *From the following recruitment centers, number of HCC cases: Denmark (N 5 21), Germany (N 5 30), Greece (N 5 16), Italy (N 5 28), Spain (N 5 11), Sweden (N 5 13), the Netherlands (N 5 4), United Kingdom (N 5 15). No eligible case patients were identified in the cohorts of France and Norway, which include women only. No case patients from Umea, Sweden were included because serum samples were not collected for these participants. † Control subjects had to be alive as of the time of diagnosis of the corresponding case patients and were matched with case patients for study center, sex, age at the time of blood collection (612 months), date of blood collection (62 months), and time of day of blood collection (63 hours). Women were further matched by menopausal status (pre-, post-, or perimenopausal) and use of exogenous hormones (oral contraceptives for premenopausal women and hormone replacement therapy for postmenopausal women) at time of blood collection (yes or no). ‡ Available for 100 cases and 100 controls. § Ranges from 0 to 6; the score was grouped in categories as 0, 1-2, 3 abnormal liver function tests (ALT>55 U/L, AST>34 U/L, GGT men >64 U/L, GGT women > 36 U/L, AP > 150 U/L, albumin < 35 g/L, total bilirubin > 20.5 lmol/L; based on the values provided by the laboratory). Available for 101 cases and 101 controls.
month of blood collection,42 ii) a regression of 25(OH)D concentration on the periodic function 2sin(2pX/12) 2 cos(2pX/12), where X is the month
of blood collection43; and iii) a nonparametric local regression (Proc LOESS; SAS Institute, Cary, NC) with 25(OH)D as the dependent variable and day of the year of blood donation as the independent variable.44 Since the results were similar for different approaches to seasonal variation adjustment, adjustment by LOESS residuals was used in all final models. Potential biologically plausible effect modifying variables (sex, age at diagnosis [55 U/L, AST>34 U/L, GGT men >64 U/L, GGT women > 36 U/L, AP > 150 U/L, albumin < 35 g/L, total bilirubin > 1.2 mg/dL. Available for 100 cases and 100 controls. ‡ Additionally adjusted for lifetime alcohol intake pattern (never, former light, former heavy, light, never heavy, periodically heavy, and always heavy drinkers, unknown) and sex-specific physical activity level (inactive, moderately inactive, moderately active, active, and unknown). § Calculated as the molar ratio of branched-chain aminoacids (leucine, valine, isoleucine) to aromatic aminoacids (phenylalanine, tyrosine, histidine and tryptophan), an indicator of hepatic functional reserve and the severity of liver dysfunction.
HBV/HCV infections and aflatoxin exposures.28 The participants of these trials received a multiple vitamin/ mineral supplement (14 vitamins and 12 minerals), including 800 IU vitamin D in the Dysplasia Trial only, or matching placebo. The Linxian studies and the EPIC cohort populations are very different with respect to diet, lifestyle, environmental exposures, and HCC risk factor profiles. For example, the levels of circulating vitamin D were more than twice lower in the Linxian studies than in our cohort (Linxian 5 20.1 nmol/L versus EPIC 5 49.9 nmol/L among controls), and had a very narrow range of values (interquartile range 5 13.7 to 30.0 nmol/L). In addition, more than 59% of controls in the Linxian studies were either HBV or HCV seropositive and 36% were current smokers, whereas in the EPIC the corresponding numbers were 3% and 20%. In the Linxian studies, higher serum 25(OH)D levels were associated with significantly lower risk of chronic liver disease death. However, in contrast to our study, the association with liver cancer incidence was not statistically significant, likely due to the narrow range of 25(OH)D and differences in the study populations.
Key advantages of the present study are its prospective design, which allows the estimation of vitamin D status before cancer development, inclusion of only first primary HCCs, and measurement of 25(OH)D using the state-of-the-art, gold standard LC/MS/MS method rather than older enzyme-linked immunosorbent assay (ELISA) methods. This study also incorporates biomarkers of HBV/HCV infection and liver function into the analysis allowing the consideration of the 25(OH)D-HCC risk association in the hepatitis free subpopulation, and for adjustment by potential liver injury. In populations with a relatively low prevalence of HBV/HCV infection such as western Europeans, it is important to take into account a preexisting chronic liver dysfunction (e.g., from NAFLD, NASH, excessive alcohol consumption) since it may adversely influence circulating 25(OH)D levels, and may be independently associated with HCC risk. Overall, HCC research in humans has been hampered by the comparative rarity of the tumor, the cost of ascertaining the HBV/HCV status in large populations, the inaccurate diagnosis and reporting of primary tumors since the liver is a major site for cancer metastases, and by metabolic changes
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that may occur before cancer diagnosis. Therefore, this prospective study with long and near complete followup, detailed information on dietary/lifestyle factors, prediagnostic measures of circulating vitamin D levels and other biomarkers contributes considerably to our understanding of the role of vitamin D in the etiology of this important cancer, whose incidence is rising rapidly. There are some limitations as well. First, vitamin D concentration was measured once and only at baseline on average 6 years before HCC diagnosis; however, a previous study found that a single measurement of serum 25(OH)D has reasonable validity over a 5-year period.45 Multiple measurements of 25(OH)D would result in a better estimate of vitamin D status and could reduce the degree of nondifferential measurement error. Furthermore, vitamin D status might be susceptible to confounding since high vitamin D concentration in general might reflect a healthier lifestyle. In our models, we adjusted for other determinants of healthy lifestyle; however, the presence of possible residual confounding may not be ruled out. Another potential limitation is reverse causality, as several, but not all, studies have found lower circulating vitamin D levels among persons with chronic liver disease.46 However, we have not observed a statistically significant difference in vitamin D levels by HBV/HCV status, arguing against reverse causality as an explanation for our results. In addition, the results of the analyses adjusted for preexisting liver damage and after exclusion of the first 6 years after blood collection supported an inverse association between 25(OH)D and HCC, but were not statistically significant, likely due to smaller sample sizes. Although our study was the first to incorporate the liver function tests into the analysis of HCC in the context of a prospective cohort, we were not able to match cases to controls on liver dysfunction or use other indicators such as the Model for Endstage Liver Disease (MELD) score to adjust for the severity of chronic liver disease. Another limitation of our study is that for administrative reasons and lack of biosample availability, we had to exclude 66 HCC cases; however, they did not differ by lifestyle and demographic characteristics from cases with measured 25(OH)D. Finally, the sample size was relatively small, and no data were available on incidence of type 2 diabetes, prevalence and incidence of cirrhosis and other chronic liver diseases, sun exposure, use of sunscreen and vitamin D supplement, and on exposure to aflatoxins, although it is uncommon in western Europe.47 In summary, the findings from this prospective study support the hypothesis that higher 25(OH)D
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levels are associated with lower risk of HCC. Further research is needed to confirm or refute these findings in other populations and understand the underlying mechanisms of vitamin D’s actions. Acknowledgment: The authors thank Dr. Ronald L. Horst, PhD (Heartland Assays, LLC, Ames, Iowa) for conducting the measurements of 25-(OH)-vitamin D, and C. Biessy and B. Hemon for assistance in database preparation. Reagents for the hepatitis infection determinations were kindly provided to Labo Republique by Abbott Diagnostics Division, Lyon, France.
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