Assessing the role of insulin-like growth factors and binding proteins in prostate cancer using Mendelian randomization : Genetic variants as instruments for circulating levels

Assessing the role of insulin-like growth factors and binding proteins in prostate cancer using Mendelian randomization: Genetic variants as instruments for circulating levels Carolina Bonilla 1,2 , Sarah J. Lewis 1,2 , Mari-Anne Rowlands 1 , Tom R. Gaunt 1,2 , George Davey Smith 1,2 , David Gunnell 1 , Tom Palmer 3 , Jenny L. Donovan 1 , Freddie C. Hamdy 4 , David E. Neal 4,5 , Rosalind Eeles 6,7 , Doug Easton 8 , Zsofia Kote-Jarai 6 , Ali Amin Al Olama 8 , Sara Benlloch 8 , Kenneth Muir 9,10 , Graham G. Giles 11,12 , Fredrik Wiklund 13 , Henrik Gr€ onberg 13 , Christopher A. Haiman 14 , Johanna Schleutker 15,16 , Børge G. Nordestgaard 17 , Ruth C. Travis 18 , Nora Pashayan 19,20 , Kay-Tee Khaw 21 , Janet L. Stanford 22,23 , William J. Blot 24 , Stephen Thibodeau 25 , Christiane Maier 26,27 , Adam S. Kibel 28,29 , Cezary Cybulski 30 , Lisa Cannon-Albright 31 , Hermann Brenner 32,33,34 , Jong Park 35 , Radka Kaneva 36 , Jyotsna Batra 37 , Manuel R. Teixeira 38,39 , Hardev Pandha 40 , the PRACTICAL consortium, Mark Lathrop 41,42 , Richard M. Martin 1,2,43 * and Jeff M. P. Holly 43,44 * 1 School of Social and Community Medicine, University of Bristol, Bristol, United Kingdom 2 MRC/University of Bristol Integrative Epidemiology Unit, University of Bristol, Bristol, United Kingdom 3 Department of Mathematics and Statistics, Lancaster University, Lancaster, United Kingdom 4 Nuffield Department of Surgery, University of Oxford, Oxford, United Kingdom 5 Surgical Oncology (Uro-Oncology: S4), University of Cambridge, Box 279, Addenbrooke’s Hospital, Hills Road, Cambridge, United Kingdom Key words: insulin-like growth factors, insulin-like growth factor-binding proteins, prostate cancer, Mendelian randomization, single nucleotide polymorphisms, IGFBP3, ProtecT, PRACTICAL, ALSPAC, UKHLS Abbreviations: BPH: benign prostatic hyperplasia; CV: coefficient of variation; GWAS: genome-wide association study; IBD: identity by descent; ICC: intra-class correlation; IGF: insulin-like growth factor; IGFBP: insulin-like growth factor-binding proteins; IV: instrumental variable; LD: Linkage disequilibrium; MR: Mendelian randomization; PSA: prostate specific antigen; QC: quality control; RIA: radioimmunoassay; SD: standard deviation; SNP: single nucleotide polymorphism; UKHLS: UK Household Longitudinal Study. Additional Supporting Information may be found in the online version of this article. For information on how to submit an application for gaining access to EPIC data and/or biospecimens, please follow the instructions at http://epic.iarc.fr/access/index.php, http://www.metadac.ac.uk/data-access-through-metadac/. R.M.M., S.J.L., G.D.S., D.G. and J.M.P.H. developed the hypotheses and secured funding. C.B., M-AR, T.P. and T.R.G. undertook statistical analyses. C.B., M.-A.R., S.J.L. and R.M.M. wrote the first draft of the paper. M.L. organized the genome-wide genotyping of controls from the ProtecT study. J.L.D., F.C.H. and D.E.N. are PIs of the ProtecT (Prostate testing for cancer and Treatment) study. D.G., J.L.D., F.C.H. and D.E.N. are NIHR Senior Investigators. All authors critically commented on and approved the final submitted version of the paper. Disclosure: TRG: In addition to funding from the UK Medical Research Council the MRC Integrative Epidemiology Unit receives funding from a number of commercial organizations, including Pfizer, Sanofi-Aventis, Astra Zeneca and Merck Sharp & Dohme. *R.M.M. and J.M.P.H contributed equally to this work This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Grant sponsor: World Cancer Research Fund; Grant number: 2011/419; Grant sponsor: Cancer Research UK; Grant number: C18281/ A19169; Grant sponsor: MRC and the University of Bristol; Grant number: G0600705, MC_UU_12013/19 (to Integrative Epidemiology Unit (IEU)); Grant sponsor: Cancer Research UK; Grant number: C18281/A19169 (to Integrative Cancer Epidemiology Programme); Grant sponsor: National Institute for Health Research (NIHR) (to Bristol Biomedical Research Unit in Nutrition, which is a partnership between University Hospitals Bristol NHS Foundation Trust and the University of Bristol); Grant sponsor: National Institute for Health Research (NIHR) Health Technology Assessment (HTA) Programme (to ProtecT study); Grant number: HTA 96/20/99; ISRCTN20141297; Grant sponsor: European Community’s Seventh Framework Programme (grant agreement n8223175); Grant number: HEALTH-F2-2009–223175) (COGS) (to PRACTICAL and the iCOGS infrastructure); Grant sponsor: Cancer Research UK; Grant numbers: C1287/A10118, C1287/A 10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692, C8197/A16565; Grant sponsor: National Institutes of Health; Grant number: CA128978; Grant sponsor: Post-Cancer GWAS initiative; Grant numbers: 1U19 CA148537, 1U19 CA148065 and 1U19 CA148112 - the GAME-ON initiative; Grant sponsor: Department of Defense; Grant number: W81XWH-10–1-0341; Grant sponsors: Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer, Komen Foundation for the Cure, the Breast Cancer Research Foundation and the Ovarian Cancer Research Fund DOI: 10.1002/ijc.30206 History: Received 24 Nov 2015; Accepted 7 Apr 2016; Online 26 May 2016 Correspondence to: Richard Martin, University of Bristol, School of Social and Community Medicine, Canynge Hall, 39 Whatley Road, Bristol BS8 2PS, United Kingdom, Tel: 144 117 928 7321, Fax: 144 117 928 7236, E-mail: [email protected] Cancer Epidemiology Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC International Journal of Cancer IJC 6 The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey, SM2 5NG, United Kingdom 7 Royal Marsden NHS Foundation Trust, Fulham and Sutton, London and Surrey, United Kingdom 8 Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, University of Cambridge, Strangeways Research Laboratory, Worts Causeway, Cambridge, United Kingdom 9 University of Warwick, Coventry, United Kingdom 10 Institute of Population Health, University of Manchester, Manchester, M13 9PL, United Kingdom 11 The Cancer Council Victoria, 615 St. Kilda Road, Melbourne, Victoria, 3004, Australia 12 Centre for Epidemiology and Biostatistics, Melbourne School of Population and Global Health, the University of Melbourne, Victoria, 3010, Australia 13 Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden 14 Department of Preventive Medicine, Keck School of Medicine, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, California 15 Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland 16 Institute of Biomedical Technology/BioMediTech, University of Tampere and FimLab Laboratories, Tampere, Finland 17 Department of Clinical Biochemistry, Herlev Hospital, Copenhagen University Hospital, Herlev Ringvej 75, Herlev, DK, 2730, Denmark 18 Cancer Epidemiology Unit, Nuffield Department of Population Health, University of Oxford, Oxford, United Kingdom 19 Centre for Cancer Genetic Epidemiology, Department of Oncology, University of Cambridge, Strangeways Research Laboratory, Worts Causeway, Cambridge, United Kingdom 20 Department of Applied Health Research, University College London, 1-19 Torrington Place, London, WC1E 7HB, United Kingdom 21 Forvie Site, Cambridge Institute of Public Health, University of Cambridge, Robinson Way, Cambridge, CB2 0SR, United Kingdom 22 Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 23 Department of Epidemiology, School of Public Health, University of Washington, Seattle, Washington 24 International Epidemiology Institute, 1455 Research Blvd, Suite 550, Rockville, Maryland 25 Mayo Clinic, Rochester, Minnesota 26 Department of Urology, University Hospital Ulm, Germany 27 Institute of Human Genetics, University Hospital Ulm, Germany 28 Brigham and Women’s Hospital/Dana-Farber Cancer Institute, 45 Francis Street-ASB II-3, Boston, Massachussets 29 Washington University, St Louis, Missouri 30 International Hereditary Cancer Center, Department of Genetics and Pathology, Pomeranian Medical University, Szczecin, Poland 31 Division of Genetic Epidemiology, Department of Medicine, University of Utah School of Medicine, Salt Lake City, Utah 32 Division of Clinical Epidemiology and Aging Research, German Cancer Research Center (DKFZ), Heidelberg, Germany 33 Division of Preventive Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany 34 German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 35 Division of Cancer Prevention and Control, H. Lee Moffitt Cancer Center, 12902 Magnolia Dr, Tampa, Florida 36 Molecular Medicine Center and Department of Medical Chemistry and Biochemistry, Medical University - Sofia, 2 Zdrave St, Sofia, 1431, Bulgaria 37 Australian Prostate Cancer Research Centre-Qld, Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, Australia 38 Department of Genetics, Portuguese Oncology Institute, Porto, Portugal 39 Biomedical Sciences Institute (ICBAS), Porto University, Porto, Portugal 40 The University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom 41 Commissariat  a L’Energie Atomique, Center National De G enotypage, Evry, France 42 McGill University-G enome Qu ebec Innovation Centre, Montreal, Canada 43 NIHR Bristol Biomedical Research Unit in Nutrition, Bristol, United Kingdom 44 IGFs and Metabolic Endocrinology Group, School of Clinical Sciences North Bristol, University of Bristol, Bristol, United Kingdom Circulating insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are associated with prostate cancer. Using genetic variants as instruments for IGF peptides, we investigated whether these associations are likely to be causal. We identified from the literature 56 single nucleotide polymorphisms (SNPs) in the IGF axis previously associated with biomarker levels (8 from a genome-wide association study [GWAS] and 48 in reported candidate genes). In ~700 men without prostate cancer and two replication cohorts (N~900 and ~9,000), we examined the properties of these SNPS as instrumental variables (IVs) for IGF-I, IGF-II, IGFBP-2 and IGFBP-3. Those confirmed as strong IVs were tested for association with prostate cancer risk, low (<7) vs. high (7) Gleason grade, localised vs. advanced stage, and mortality, in 22,936 controls and 22,992 cases. IV analysis was used in an attempt to estimate the causal effect of circulating IGF peptides on prostate cancer. Published SNPs in the IGFBP1/IGFBP3 gene region, particularly rs11977526, were strong instruments for IGF-II and IGFBP-3, less so for IGF-I. Rs11977526 was associated with high (vs. low) Gleason grade (OR per IGF-II/IGFBP-3 levelraising allele 1.05; 95% CI: 1.00, 1.10). Using rs11977526 as an IV we estimated the causal effect of a one SD increase in IGF-II (~265 ng/mL) on risk of high vs. low grade disease as 1.14 (95% CI: 1.00, 1.31). Because of the potential for pleiotropy of the genetic instruments, these findings can only causally implicate the IGF pathway in general, not any one specific biomarker. Cancer Epidemiology Bonilla et al. 1521 Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Prostate cancer is the most common male cancer in industrialised countries, yet there are no established, potentially modifiable risk factors for prevention. 1 The nutritionally regulated IGFs, and their modulating binding proteins (IGFBPs) play a key role in somatic growth, and activate carcinogenic intracellular signalling networks. 1 Meta-analyses of epidemiological studies generally observe positive associations of circulating IGF-I with prostate cancer, 2–4 but substantial differences exist between studies. 5,6 Such diverse evidence indicates that causation remains to be established. Alternative explanations for the observed association of IGF-axis peptides with prostate cancer include: reverse causality, because tumours may promote an endocrine response 7 ; confounding by dietary, 8 nutritional 9 and lifestyle 10 factors; measurement error, 11 as single serum measurements may inadequately reflect long-term exposure; or detection bias, 11 occurring, for example, if IGF-I causes symptomatic benign prostatic hyperplasia (BPH) that results in the serendipitous finding of latent cancer on diagnostic biopsy. Mendelian randomization (MR) 12 seeks to establish causality by using genetic variants as proxies for the exposure of interest. Since alleles randomly assort at gamete formation and segregate randomly at conception to generate genotypes, associations between genotypes and outcome are not generally confounded by behavioural or environmental factors and cannot be explained by reverse causation. Genetic variation may also be a better measure of exposure over a lifetime than a single serum measurement, as those with genotypes causing high (or low) IGF levels will have been, in effect, randomly allocated to high (or low) IGF levels from birth. To determine causality, MR relies on an association between genetic variant (also known as instrument) and exposure so that the greater the correlation between the two, and thus the more variation in the exposure phenotype explained by the genotype, the more reliable the causal inference. Additionally, the instrument is expected to influence the outcome only via the exposure (i.e., absence of horizontal pleiotropy 13 ) and to be independent from confounders of the relationship between exposure and outcome. We used an MR approach in an attempt to assess the causal role of the IGF axis in prostate cancer. First, we validated genetic variants previously associated with IGF levels in the literature to confirm reported associations of the SNPs (especially SNPs selected from candidate gene studies), and to assess the potential for pleiotropic effects of the genetic variants on more than one IGF protein. Second, we performed a large case–control study based on an international prostate cancer consortium of >22,000 case/control pairs using the validated polymorphisms. Material and Methods Study populations ProtecT (Prostate testing for cancer and Treatment) study. The association of genetic variants with IGF levels was evaluated in the control arm of a case–control study nested within ProtecT, a UK multicentre study to identify localised prostate cancer and evaluate its management in a randomly allocated controlled trial. 5 All men without evidence of prostate cancer were eligible for selection as controls; that is, men with a prostate specific antigen (PSA) test <3 ng/mL, or men with a raised PSA (3 ng/mL) combined with at least one negative diagnostic biopsy. Of the 2,766 controls who underwent measures of IGFs in ProtecT 5 , 700 men also had genome-wide genotype data available (mean age 6SD: 62.1 65.0 years). Blood samples for IGF measurement in ProtecT were drawn at the time of the PSA test, frozen at 2808C within 36 hr, then transferred on dry ice for assay. 4 Total IGF-I, IGF-II and IGFBP-3 levels were measured by in-house radioimmunoassay (RIA) and circulating IGFBP-2 was measured using a one-step sandwich ELISA (DSL-10–7100; Diagnostic Systems Laboratories). The intra-class correlations (ICC) for within-assay variability for IGF-I, IGF-II, IGFBP-2 and IGFBP-3 were 0.86, 0.91, 0.95 and 0.88; the ICCs for between-assay variability were 0.66, 0.84, 0.81 and 0.71, respectively. Genome-wide genotyping of participants was carried out at the Centre National de G enotypage (CNG, Evry, France), using the Illumina Human660W-Quad_v1_A array (Illumina Inc., San Diego, CA). The quality control (QC) process performed before imputation excluded individuals on the basis of the following: sex mismatches, minimal (<0.325) or excessive (>0.345) heterozygosity, disproportionate levels of individual missingness (>3%), cryptic relatedness measured as a proportion of identity by descent (IBD >0.1), and insufficient sample replication (IBD <0.8). All individuals with non-European ancestry, and SNPs with a minor allele frequency (MAF) below 1%, a call rate of <95% or out of Hardy-Weinberg equilibrium (p<5310 27 ) were removed. Autosomal genotypic data were imputed using Markov Chain Haplotyping software (MACH v.1.0.16) 14 and phased What’s new? Circulating insulin-like growth factors (IGF) and their binding proteins have been associated with prostate cancer risk in observational epidemiological studies but it is not clear whether there is a causal relationship with disease. To address this question, the authors used Mendelian randomization, a method that uses genetic variants as proxies for measured exposures. Their results implicate the IGF pathway in general in prostate cancer development but specific biomarkers remain to be determined. Cancer Epidemiology 1522 Mendelian randomization study of the IGF axis and prostate cancer Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC haplotype data from European (CEU) individuals (HapMap release 22, Phase II NCBI B36, dbSNP 126) based on 514,432 autosomal SNPs. After imputation, all SNPs with indication of poor imputation quality (r 2 hat <0.3) were eliminated. The working dataset consisted of 2,927 individuals (1,136 cases, 1,791 controls) of European ancestry. Trent Multicenter Research Ethics Committee (MREC) approved both the ProtecT study (MREC/01/4/025), and the associated ProMPT study which collected biological material (MREC/01/4/061). Written informed consent was obtained from all men. ALSPAC (Avon Longitudinal Study of Parents and Children). We used ALSPAC to replicate ProtecT findings. ALSPAC is a population-based prospective cohort study of children and their parents. The study is described in detail elsewhere 15–17 (http://www.bristol.ac.uk/alspac/). Measurement of circulating IGF-I, IGF-II and IGFBP-3 in plasma or serum was carried out as in ProtecT. IGFBP-2 was not measured. The intraand inter-assay coefficients of variation (CV) were 7.0 and 14.3% for IGFI, 7.9 and 18.6% for IGF-II, and 6.1 and 8.7% for IGFBP-3. 18 Genome-wide association study (GWAS) data for the children were generated by Sample Logistics and Genotyping Facilities at the Wellcome Trust Sanger Institute (Cambridge, UK) and the Laboratory Corporation of America (Burlington, NC, USA) with support from 23andMe (Mountain View, CA, USA) using the Illumina HumanHap550 quad chip. The mothers were genotyped at CNG using the Illumina Human660W quad array. All individuals of non-European ancestry, ambiguous sex, extreme heterozygosity, cryptic relatedness (IBD >0.1 in children, >0.125 in mothers), high missingness (>3% in children, >5% in mothers) and insufficient sample replication (IBD <0.8) were removed. SNPs with genotyping rate <95%, MAF <1%, or out of HardyWeinberg equilibrium (p<5310 27 in children, p<13 10 26 in mothers) were excluded. Genotypic data was subsequently phased with ShapeIT v2.r644, 19 and imputed using IMPUTE v2.2.2 20 and phased haplotype data from the 1000 Genomes reference panel (phase 1, version 3), based on 465,740 SNPs. The cleaned dataset consisted of 8,237 children and 8,196 mothers. Up to 400 pregnant women (mean 6SD age at delivery: 28.7 65.4 years) and 450 children at different ages (mean 6SD age: 61.8 60.8 months, 54% male; 7.5 60.2 years, 54% male; 8.2 60.1 years, 56% male), as well as 500 umbilical cord samples had genotypes and IGF measures for analysis. Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees (http://www.bristol.ac.uk/alspac/researchers/data-access/ethics/lrec-approvals/#d.en.164120). Written informed consent was obtained from all participants in the study. Understanding Society: the UK Household Longitudinal Study (UKHLS). SNPs validated in ProtecT were also examined in the UKHLS study, which is a stratified clustered random sample of households, representative of the UK population (https://www.understandingsociety.ac.uk/). Serum IGF-I levels were measured using an electrochemiluminescent immunoassay on an IDS ISYS analyser. The interand intra-assay C vs. were <14%. No measurements of IGF-II, IGFBP-2 or IGFBP-3 were available. In total, 10,480 samples were genotyped on the Illumina HumanCoreExome chip (v1.0) at the Wellcome Trust Sanger Institute. Data QC was performed at the sample-level using the following filters: call rate <98%, autosomal heterozygosity outliers (>3 SD), gender mismatches, duplicates as established by IBD analysis (PI_HAT >0.9), ethnic outliers. Variants with a Hardy-Weinberg equilibrium pvalues <10 24 ,a call rate below 98% and poor genotype clustering values (< 0.4) were removed, as well as mitochondrial polymorphisms, leaving 518,542 variants. Imputation was performed at the UCL Genetics Institute using Minimac version 5–29-12, 21 MaCH 14 for phasing, and the 1000 Genomes Project, March 2012, version 3, NCBI build GRCh37/hg19 as a reference sample. The final sample consisted of 9,944 individuals. As UKHLS is a household study we additionally eliminated individuals who were related (>5%), thus the working sample included 9,237 participants (mean 6SD age: 54.1 616.1 years, 44% male). UKHLS is designed and conducted in accordance with the ESRC Research Ethics Framework and the ISER Code of Ethics. The University of Essex Ethics Committee approved waves 1–5 of UKHLS. Approval from the National Research Ethics Service was obtained for the collection of biosocial data by trained nurses in waves 2 and 3 of the main survey (Oxfordshire A REC, Reference: 10/H0604/2). PRACTICAL Consortium (PRostate cancer AssoCiation group to Investigate Cancer-Associated aLterations in the genome). We investigated associations of published IGF-related genetic variants, evaluated as instruments in ProtecT and replicated in ALSPAC and/or UKHLS, with prostate cancer risk, progression and mortality in men from 25 studies contributing to the international PRACTICAL consortium 22 (http://practical.ccge.medschl.cam.ac.uk). Seventeen studies were from Europe, six from North America and two from Australia, and comprised population samples of predominantly European ancestry 22 (Table 1). Data on cancer stage, grade and method of diagnosis were collected by each study using a variety of methods. We categorised cancers as localised (T1 or T2 on TNM staging, or if not available, “localised” on SEER staging) or advanced (T3 or T4, or “regional” or “distant” on SEER staging). Genotyping of PRACTICAL samples was carried out using an Illumina Custom Infinium genotyping array (iCOGS), designed for the Collaborative Oncological GeneEnvironment Study (COGS) (http://www.cogseu.org/) and Cancer Epidemiology Bonilla et al. 1523 Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC consisting of 211,155 SNPs. 22 This array was devised to evaluate associations of genetic variants with breast, ovarian and prostate cancer (85,278 were specifically chosen for their potential relevance to prostate cancer). A total of 201,598 SNPs passed QC for the European ancestry samples. 22 Imputation of 17 million SNPs/indels using the 1000 Genomes Project (version 3, March 2012 release) as a reference panel was performed with the program IMPUTE v.2. 20 Polymorphisms with quality information scores of (r 2 )>0.3 and MAF >0.5 were taken forward for analysis. 23 Overall there were 22,992 prostate cancer cases and 22,936 controls with genotype data available. All studies have the relevant Institutional Review Board approval in each country in accordance with the Declaration of Helsinki. Identification of genetic variants associated with IGF levels in the literature We selected single nucleotide polymorphisms (SNPs) associated with circulating IGF levels from the National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) catalog of genome-wide association studies (GWAS) (https://www.ebi.ac.uk/gwas/) and by conducting a PubMed literature search. All SNPs chosen were associated Table 1. Clinical characteristics of prostate cancer cases in 25 PRACTICAL studies Study Country N controls N cases Mean age at diagnosis (years) Mean PSA at diagnosis (ng/mL) European ethnicity (%) 1 Family history of prostate cancer (%) 1,2 High Gleason score (7, %) 1 Advanced stage (%) 1,3 Screendetected cancer (%) 1 CAPS Sweden 664 1153 66.1 79.6 100 17.4 49.9 2 30.3 0.0 CPCS1 Denmark 2756 848 69.5 48.0 99.6 8.2 2 71.2 2 n/a 0.0 CPCS2 Denmark 1001 265 64.9 36.0 99.4 14.7 2 52.2 2 n/a 0.0 EPIC Europe 1079 722 64.9 0.2 100 n/a 27.9 2 4.0 2 0.0 EPIC-Norfolk UK 911 481 72.1 n/a 99.9 2.5 39.4 2 n/a n/a ESTHER Germany 318 313 65.5 58.7 100 8.9 2 48.0 27.6 61.9 2 FHCRC USA 729 761 59.7 16.1 99.9 21.7 41.7 20.2 N/a IPO-Porto Portugal 66 183 59.3 8.3 100 20.0 2 84.2 64.5 82.8 2 MAYO USA 488 767 65.2 15.5 100 29.1 55.3 2 45.5 73.7 2 MCCS 3 Australia 1169 1650 58.5 18.8 98.8 23.5 2 53.4 14.5 N/a MEC USA 829 819 69.5 n/a 100 13.0 n/a 12.5 N/a MOFFITT USA 96 404 65.0 7.3 97.5 22.3 43.4 3.6 0.0 2 PCMUS Bulgaria 140 151 69.3 32.5 100 5.3 59.6 46.7 21.2 Poland Poland 359 438 67.7 40.2 100 10.6 32.8 2 37.1 2 0.0 2 PPF-UNIS UK 187 244 68.9 32.1 99.8 25.3 45.2 2 28.8 2 N/a ProMPT UK 2 166 66.3 33.0 100 34.6 74.3 2 34.7 0.0 2 ProtecT UK 1458 1545 62.7 9.6 99.7 8.0 2 29.9 11.4 100.0 QLD Australia 85 139 61.4 7.4 99.1 4 37.8 83.6 0.0 2 N/a SEARCH UK 1231 1354 63.1 53.2 100 16.3 56.9 2 18.0 2 36.7 2 STHM1 Sweden 2224 2006 66.2 n/a 100 20.2 45.5 2 14.4 2 N/a TAMPERE Finland 2413 2754 68.2 69.2 100 n/a 43.8 2 21.4 46.8 UKGPCS UK 4132 3838 63.6 88.0 99.8 22.4 2 50.5 2 36.4 2 28.0 2 ULM Germany 354 603 63.8 19.1 100 44.9 51.3 2 40.5 N/a UTAH USA 245 440 62.6 n/a 100 51.4 n/a 17.2 2 N/a WUGS USA 0 948 60.8 6.1 95.8 42.6 2 59.3 24.2 N/a N545,928 men. Information in the table is given for the subset of individuals whose ethnicity was “European” (except for the study’s European ethnicity percentage). 1 Percent of cases with data available. Family history of prostate cancer in a first degree relative. T3 or T4 on TNM staging, or if not available, “regional” or “distant” on SEER staging. 2 Information missing for >10% of patients. 3 MCCS includes Risk Factors for Prostate Cancer Study (RFPCS) and The Early Onset Prostate Cancer Study (EOPCS). 4 Information missing for >10% of individuals. n/a not available. Cancer Epidemiology 1524 Mendelian randomization study of the IGF axis and prostate cancer Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC with IGF concentration at the significance thresholds established by each study (p<5310 27 in the discovery GWAS; usually p<0.05 in candidate gene studies). Validation of genetic variants as instruments of IGF levels The properties of the SNPs as instrumental variables (IV) were assessed in ProtecT controls by examination of: (i) F statistics (with values lower than 10 taken as evidence of a weak instrument 24 ) and R 2 values (the proportion of variation in IGF levels explained by the genetic variant) from the linear regression of each biomarker on the SNP; (ii) associations of the genetic variants with potential confounding factors and other variables (age, PSA at recruitment, body mass index (BMI), height, leg-length, BPH and diabetes); and (iii) possible pleiotropic effects of the variants on more than one IGF peptide. 25 The validated genetic instruments were tested for replication in ALSPAC mothers and children, and UKHLS participants. Statistical analysis All SNPs were examined for deviation from Hardy-Weinberg equilibrium using the hwsnp function in the statistical package Stata. Linear and logistic regression were used as appropriate to investigate the effect of SNPs on IGF-I, IGF-II, IGFBP-2, IGFBP-3, PSA and potential confounders. For the validated SNPs we ran meta-analyses across all PRACTICAL studies to evaluate between-study heterogeneity in the association with prostate cancer risk, Gleason grade (low: <7vs. high: 7) and stage (localised vs. advanced). We computed pooled ORs assuming a fixed-effects model when there was no evidence of heterogeneity (p>0.05), otherwise we used a random-effects model. Logistic regression with robust standard errors, to account for within-study clustering, was performed to test for associations of all polymorphisms across the IGFBP1/IGFBP3 region and SNPs in other chromosomal regions with the above prostate cancer outcomes. Linkage disequilibrium (LD) between pairs of variants in the IGFBP-1/IGFBP-3 gene region was calculated with the program LDlink using data for the GBR population (English and Scottish) in Phase 3 of the 1,000 Genomes Project. 26 r 2 values obtained with LDlink were then used to create an LD plot of the region with the R package LDheatmap (http:// www.R-project.org). Functional consequences of genetic polymorphisms were predicted using SNPnexus (http://www.snpnexus.org/). Survival analysis. Amongst men with prostate cancer, we estimated associations of the validated SNPs with long-term (15-year) survival, examining all-cause and prostate cancerspecific mortality using Cox proportional hazards regression with date at diagnosis as the start date and date at death or final follow-up time-point as the exit date, with robust standard errors to account for within-study clustering. Instrumental variable (IV) analysis. To estimate the causal effect of IGF levels on prostate cancer, we used validated SNPs as the instruments in a two-sample ratio estimator IV analysis 27,28 (Fig. 1). The ratio represents the causal log odds ratio of a one unit increase in circulating IGF on the risk of prostate cancer. IV analysis was conducted for the SNPs showing the strongest association with prostate cancer, which were also associated with circulating IGFs in ProtecT, ALSPAC or UKHLS, and the estimates are given per standard deviation (SD) increase in IGF levels. Adjustments. Principal components reflecting each population’s genetic structure were included as covariates in the regression models to account for confounding by population stratification. Additional adjustments for age at diagnosis, age at blood sample collection, gestational age and sex were made when appropriate. Unless otherwise specified, all analyses were carried out in Stata version 13 (StataCorp LP, 2013, College Station, TX). Figure 1. Directed acyclic graph (DAG) showing the instrumental variable (IV) assumptions underpinning a Mendelian randomization analysis of circulating IGF levels with prostate cancer. IV models use associations A and B to estimate the causal effect of IGF on prostate cancer C(C5B/A). The instrument is assumed not to have a direct effect on the outcome, hence the dashed line is to illustrate that association B is required for IV estimation. The effect of genotype on the outcome should be mediated only through the intermediate phenotype (no pleiotropy). The numerator of the two sample IV estimator is the log odds ratio from a logistic regression of the outcome (Y) on the instrument (Z) in the PRACTICAL population and the denominator is the beta coefficient from a linear regression of the exposure (X) on the instrument (Z) in the ProtecT or UKHLS population or obtained from the literature. Cancer Epidemiology Bonilla et al. 1525 Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Table 2. Association of published SNPs with IGF biomarkers in ProtecT controls ProtecT: effect on published biomarkers ProtecT: effect on other biomarkers SNP Effect allele/ non-effect allele 1 Published associations Mean difference in IGF levels (ng/mL) per effect allele 95% CI pvalue Other associations Mean difference in IGF levels (ng/mL) per effect allele 95% CI pvalue F R 2 (%) rs3770473 G/T IGF-I 1.06 (28.77,10.89) 0.83 IGFBP-3 243.89 (2225.24,137.47) 0.64 rs300982 G/A IGFBP-3 2139.80 (2420.66, 141.05) 0.33 rs4234798 T/G IGFBP-3 249.51 (2165.48,66.45) 0.40 rs7703713 A/G IGF-I 21.32 (28.14,5.49) 0.70 IGFBP-2 20.07 (20.14, 20.001) 0.04 2.5 0.34 rs2153960 A/G IGF-I 3.67 (23.16,10.49) 0.29 IGFBP-2 0.07 (0.002,0.14) 0.04 3.6 0.50 rs998075 G/A IGF-I 1.78 (24.14,7.71) 0.56 rs998074 C/T IGF-I 1.78 (24.14,7.71) 0.56 rs7780564 C/A IGF-I 4.35 (21.46,10.15) 0.14 rs10228265 A/G IGFBP-3 211.25 (2126.51,104.00) 0.85 IGF-II 27.31 (21.71,56.33) 0.07 3.8 0.52 rs1908751 T/C IGF-I 20.40 (26.98,6.18) 0.91 rs2270628 C/T IGFBP-3 3.35 (2129.87,136.56) 0.96 IGF-II 34.97 (1.40,68.54) 0.04 4.9 0.68 rs6670 T/A IGF-I 25.62 (214.58,3.35) 0.22 rs3110697 G/A IGFBP-3 234.10 (2144.90,76.69) 0.55 IGF-II 55.26 (27.60,82.92) 9.64x10 25 14.3 1.94 rs9282734 G/T IGFBP-3 360.75 (2574.69,1296.20) 0.45 rs2471551 G/C IGFBP-3 7.96 (2128.43,144.34) 0.91 IGF-I 9.03 (21.65,16.42) 0.02 5.6 0.76 IGF-II 244.24 (278.55, 29.93) 0.01 6.0 0.82 rs2132572 C/T IGFBP-3 252.69 (2180.87,75.48) 0.42 IGF-II 35.09 (2.79,67.38) 0.03 4.3 0.59 IGF-I 24.32 (211.30,2.65) 0.22 rs2132571 C/T IGFBP-3 62.68 (253.82,179.19) 0.29 IGF-II 55.35 (26.15,84.55) 2.14x10 24 11.6 1.58 IGF-I 6.79 (0.45,13.13) 0.04 4.0 0.54 rs924140 T/C IGFBP-3 13.33 (297.43,124.10) 0.81 IGF-II 76.49 (49.08,103.89) 5.92x10 28 26.0 3.47 rs1496499 G/T IGF-I 2 3.12 (22.48,8.72) 0.27 IGF-II 77.18 (49.81,104.55) 4.35x10 28 26.3 3.52 rs11977526 A/G IGFBP-3 83.98 (231.18,199.14) 0.15 IGF-II 94.78 (66.48,123.09) 9.53x10 211 37.8 4.98 IGF-I 2 3.07 (22.77,8.91) 0.30 rs700752 G/C IGF-I 9.22 (3.19,15.24) 0.003 7.7 1.05 IGFBP-3 219.21 (108.61,329.81) 1.09x10 24 13.6 1.86 rs1245541 G/A IGF-I 20.79 (26.93,5.34) 0.80 rs217727 A/G IGF2 14.65 (220.09,49.39) 0.41 IGFBP-3 135.16 (22.00,272.33) 0.053 2.0 0.28 rs6214 T/C IGF-I 2.64 (23.51,8.79) 0.40 Cancer Epidemiology 1526 Mendelian randomization study of the IGF axis and prostate cancer Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Table 2. Association of published SNPs with IGF biomarkers in ProtecT controls (Continued) ProtecT: effect on published biomarkers ProtecT: effect on other biomarkers SNP Effect allele/ non-effect allele 1 Published associations Mean difference in IGF levels (ng/mL) per effect allele 95% CI pvalue Other associations Mean difference in IGF levels (ng/mL) per effect allele 95% CI pvalue F R 2 (%) rs1520220 G/C IGF-I 6.37 (21.88,14.61) 0.13 rs5742694 A/C IGF-I 25.59 (212.74,1.56) 0.13 rs978458 T/C IGF-I 5.22 (21.79,12.23) 0.14 rs5742678 C/G IGF-I 5.22 (21.79,12.23) 0.14 rs972936 C/T IGF-I 25.22 (212.23,1.79) 0.14 rs2288378 T/C IGF-I 5.60 (21.55,12.74) 0.12 rs7136446 C/T IGF-I 3.81 (22.19,9.81) 0.21 rs10735380 G/A IGF-I 6.13 (20.71,12.96) 0.08 3.4 0.47 rs2195239 G/C IGF-I 5.89 (21.35,13.13) 0.11 rs12821878 G/A IGF-I 6.93 (20.18,14.05) 0.06 3.2 0.43 rs5742615 T/G IGF-I 3.99 (228.62,36.60) 0.81 rs2162679 T/C IGFBP-3 238.78 (2201.52,123.96) 0.64 rs5742612 G/A IGF-I 28.36 (225.99,9.26) 0.35 IGFBP-3 281.73 (2409.99,246.53) 0.63 rs35767 A/G IGF-I 1.27 (27.47,10.01) 0.78 IGFBP-3 38.78 (2123.96,201.52) 0.64 rs35766 C/T IGF-I 3.58 (24.85,12.02) 0.41 rs35765 T/G IGF-I 6.45 (23.14,16.04) 0.19 rs7965399 C/T IGF-I 24.86 (220.59,10.86) 0.54 rs11111285 G/A IGF-I 24.96 (220.73,10.80) 0.54 IGFBP-2 0.003 (20.15,0.16) 0.97 rs855211 A/G IGF-I 2.50 (25.75,10.75) 0.55 rs10778177 C/T IGF-I 21.22 (29.80,7.36) 0.78 rs855203 C/A IGF-I 2.52 (27.95,12.99) 0.64 rs1457596 A/G IGF-I 2.94 (27.83,13.71) 0.59 rs7964748 A/G IGF-I 1.25 (26.44,8.94) 0.75 rs907806 G/A IGFBP-3 2112.72 (2285.09,59.66) 0.20 rs213656 T/G IGF-I 4.32 (21.66,10.30) 0.16 IGFBP-2 20.06 (20.12,0.00) 0.05 4.1 0.56 rs3751830 C/T IGF-I 3.23 (22.80,9.26) 0.29 IGFBP-2 20.05 (20.11,0.01) 0.09 3.3 0.44 rs197056 A/G IGF-I 6.70 (0.61,12.78) 0.03 IGFBP-2 20.06 (20.12,0.00) 0.06 3.6 0.50 Cancer Epidemiology Bonilla et al. 1527 Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC Results We identified 56 SNPs that were associated with circulating IGF peptides in GWAS (n58) or candidate gene studies (n548) (Supporting Information Table 1). Most of these SNPs were located in the IGF1 and IGFBP1/IGFBP3 gene regions on chromosomes 12q23.2 and 7p12.3, respectively, and showed associations with IGF-I and IGFBP-3 levels. We could only find one candidate gene study that had examined the relationship of blood IGF-II with genetic polymorphisms, 29 and one that had similarly considered IGFBP-2 concentrations. 30 Validation of the association of published SNPs with IGF levels in ProtecT controls IGF-I, IGF-II and IGFBP-3 blood concentrations were approximately normally distributed, as opposed to IGFBP-2, which was natural log-transformed for analysis. Mean (6 SD) levels are given in Supporting Information Table 2. All SNPs, with the exception of rs3770473 (p<0.0001), conformed with Hardy-Weinberg equilibrium. Six SNPs in the IGFBP1/IGFBP3 gene region were strongly associated with circulating IGFs (F-statistic >10), 31 individually explaining 2 – 5% of variation in biomarker concentration (Table 2). The genetic variant showing the strongest association, and thus ranking as the best instrument, was rs11977526 (F 538, R 2 55%), the lead SNP in a GWAS of IGF-I and IGFBP-3 levels. 32 Five out of the six SNPs (including rs11977526) were not associated with the IGF biomarker reported in the literature but with IGF-II instead. Only one SNP (rs700752) was consistent with published reports, showing associations with both IGF-I and IGFBP-3 (although it qualified as a strong instrument only for IGFBP-3) (Table 2). Three of the most robustly associated variants (rs11977526, rs1496499, rs700752) had been identified in a GWAS including over 10,000 participants, 32 and the remaining three (rs3110697, rs2132571, rs924140) were in strong LD with the first two (Supporting Information Fig. 1). Other SNPs identified in the same GWAS, but located in different chromosomal regions, were either not associated with the serum concentration of any biomarker (rs4234798, rs7780564 and rs1245541), marginally associated with a biomarker other than the one reported in the GWAS (rs2153960 with IGFBP-2 instead of IGF-I), or showed an association with the GWAS-reported biomarker (IGFBP-3) but did not satisfy the requirements of a strong instrument (rs1065656) (Table 2). The validated SNPs were not correlated with potential confounders or PSA, after applying a Bonferroni correction for multiple testing (pvalues >0.001) (Supporting Information Table 3). Replication in ALSPAC Mean (6SD) levels of IGF-I, IGF-II and IGFBP-3 for mothers and children are shown in Supporting Information Table Table 2. Association of published SNPs with IGF biomarkers in ProtecT controls (Continued) ProtecT: effect on published biomarkers ProtecT: effect on other biomarkers SNP Effect allele/ non-effect allele 1 Published associations Mean difference in IGF levels (ng/mL) per effect allele 95% CI pvalue Other associations Mean difference in IGF levels (ng/mL) per effect allele 95% CI pvalue F R 2 (%) rs174643 G/A IGF-I 4.24 (21.64,10.11) 0.16 IGFBP-2 20.05 (20.11,0.01) 0.09 3.3 0.45 rs1178436 C/T IGFBP-3 188.47 (45.27,331.67) 0.01 7.1 0.98 rs1065656 G/C IGFBP-3 146.47 (27.31,265.63) 0.02 5.3 0.73 rs17559 A/G IGFBP-3 100.90 (274.95,276.75) 0.26 rs11865665 G/A IGFBP-3 164.35 (240.99,369.68) 0.12 1 The effect allele is expected to increase the levels of biomarkers reported in the literature. 2 IGF-I adjusted for IGFBP-3. Circulating IGFBP-2 was natural log transformed. The regression models were adjusted for age and 10 principal components. IGF-I N5727, IGF-II N5718, IGFBP-2 N5724, IGFBP-3 N5712. In bold, SNPs uncovered in a GWAS of IGF-I and IGFBP-3 levels. Cancer Epidemiology 1528 Mendelian randomization study of the IGF axis and prostate cancer Int. J. Cancer: 139, 1520–1533 (2016) V C2016 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC