Genetic variants in the IL1A gene region contribute tointestinal-type gastric carcinoma susceptibility in Europeanpopulations

Cec�ılia Dur~aes1, Xavier Mu~noz2,3, Catalina Bonet4, Nadia Garc�ıa2,4, Adoraci�on Vencesl�a2,4, F�atima Carneiro1,5,6,

B�arbara Peleteiro5,7, Nuno Lunet5,7, Henrique Barros5,7, Bj€orn Lindkvist8, Marie-Christine Boutron-Ruault9,10,11,

H. Bas Bueno-de-Mesquita12,13,14, Cosmeri Rizzato15, Antonia Trichopoulou16,17, Elisabete Weiderpass18,19,20,21,

Allessio Naccarati22, Ruth C. Travis23, Anne Tj�nneland24, Aurelio Barricarte Gurrea25,26, Mattias Johansson27, Elio Riboli14,

C�eu Figueiredo1,5, Carlos Alberto Gonz�alez2, Gabriel Capell�a3, Jos�e Carlos Machado1,5 and N�uria Sala2,4

1 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal2 Molecular Epidemiology Group, Translational Research Laboratory, Catalan Institute of Oncology (IDIBELL), Barcelona, Spain3 Hereditary Cancer Program, Translational Research Laboratory, Catalan Institute of Oncology (ICO-IDIBELL), Barcelona, Spain4 Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (IDIBELL), Barcelona, Spain5 Faculty of Medicine of the University of Porto, Porto, Portugal6 Department of Pathology, Hospital of S. Jo~ao, Porto, Portugal7 Institute of Public Health of the University of Porto (ISPUP), Porto, Portugal8 Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden9 Institut National de la Sant�e et de la Recherche M�edicale (INSERM), Centre for Research in Epidemiology and Population Health (CESP), U1018, Nutrition,

Hormones and Women’s Health Team, Villejuif, France10 Universit�e Paris-Sud, UMRS 1018, Villejuif, France11 Institut Gustave Roussy (IGR), Villejuif, France12 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands13 Department of Gastroenterology and Hepatology, University Medical Centre, Utrecht, The Netherlands14 School of Public Health, Imperial College London, St Mary’s Campus, Imperial College, London, United Kingdom15 German Cancer Research Center (DKFZ), Heideflberg, Germany

Key words: gastric carcinoma, genetic susceptibility, IL1 gene cluster, IL1A, haplotypes, intestinal-type gastric carcinoma

Additional Supporting Information may be found in the online version of this article.

Grant sponsor: Health Research Fund of the Spanish Ministry of Health (ISCIII); Grant numbers: PI081420, PI070130 and PI1201187;

Grant sponsor: Integrated Actions of the Spanish Ministry of Science and Innovation (MICINN); Grant number: PT2009-0177; Grant

sponsor: Council of Rectors of Portuguese Universities (CRUP); Grant number: Luso-Espanholas E-83/10; Grant sponsor: Fundaci�o

‘LaCaixa’, Grant number: BM06–130-0; Grant sponsor: Spanish Ministry of Health Network RTICCC; Grant number: ISCIII RD06/0020/

0091; Grant sponsor: Generalitat de Catalunya (AGAUR); Grant number: 2009SGR939; Grant sponsor: Portuguese Foundation of Science

and Technology (FCT); Grant number: ERA-NET Pathogenomics (HELDIVPAT) ERA-PTG/0001/2010; FCT, POCTI/SAU-ESP/61685/2004;

FCT POCI/SAU-ESP/56126/2004; FCT, PTDC/SAU-ESA/71517/2006; Grant sponsors: The European Commission (DG-SANCO) and the

International Agency for Research on Cancer (coordination of EPIC), Danish Cancer Society (Denmark), Ligue Contre le Cancer, Institut

Gustave Roussy, Mutuelle G�en�erale de l’Education Nationale, Institut National de la Sant�e et de la Recherche M�edicale (INSERM) (France),

Deutsche Krebshilfe e.V., German Cancer Research Center and Federal Ministry of Education and Research (Germany); Hellenic Health

Foundation (Greece); Italian Association for Research on Cancer (AIRC) and National Research Council (Italy); Dutch Ministry of Public

Health, Welfare and Sports (VWS), Netherlands Cancer Registry (NKR), LK Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg

Onderzoek Nederland), World Cancer Research Fund (WCRF), Statistics Netherlands (The Netherlands); Grant number: ERC-2009-AdG

232997; Grant sponsor: Nordforsk, Nordic Centre of Excellence programme on Food, Nutrition and Health. (Norway); Health Research

Fund (FIS), Regional Governments of Andaluc�ıa, Asturias, Basque Country, Murcia; Grant number: 6236; Grant sponsor: Navarra, ISCIII

RTICC; Grant numbers: RD06/0020/0091 and RD12/0036/0018 (Spain); Grant sponsors: Swedish Cancer Society, Swedish Scientific

Council and Regional Government of Skåne and V€asterbotten (Sweden); Cancer Research UK, Medical Research Council (United Kingdom);

Grant sponsor: FCT; Grant number: SFRH/BPD/62974/2009; Grant sponsor: Instituto de Salud Carlos III of the National Health System;

Grant number: exp.CA06/0200; Grant sponsor: MICINN; Grant number: JCI 2010-0836

DOI: 10.1002/ijc.28776

History: Received 21 Oct 2013; Accepted 16 Jan 2014; Online 12 Feb 2014

Correspondence to: N�uria Sala, Translational Research Laboratory and Unit of Nutrition, Environment and Cancer, Catalan Institute of

Oncology (IDIBELL), Gran Via de l’Hospitalet 199–203, 08907-Hospitalet de Llobregat, Barcelona, Spain, Tel.: 134932607464, Fax:

134932607466, E-mail: nsala@iconcologia.net or to Cec�ılia Dur~aes, Institute of Molecular Pathology and Immunology of the University of Porto

(IPATIMUP), Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal; Tel: 1351225570700; Fax: +351225570799; E-mail: cduraes@ipatimup.pt

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International Journal of Cancer

IJC

16 Hellenic Health Foundation, Athens, Greece17 Department of Hygiene, Epidemiology and Medical Statistics, WHO Collaborating Center for Food and Nutrition Policies, University of Athens Medical

School, Athens, Greece18 Department of Community Medicine, Faculty of Health Sciences, University of Troms�, Troms�, Norway19 Department of Research, Cancer Registry of Norway, Oslo, Norway20 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden21 Samfundet Folkh€alsan, Helsinki, Finland22 HuGeF (Human Genetics Foundation), Molecular and Genetic Epidemiology Unit, Torino, Italy23 Cancer Epidemiology Unit, University of Oxford, Oxford, United Kingdom24 Diet, Genes and Environment, Danish Cancer Society Research Center, Copenhagen, Denmark25 Navarre Public Health Institute, Pamplona, Spain26 Consortium for Biomedical Research in Epidemiology and Public Health (CIBER Epidemiolog�ıa y Salud P�ublica-CIBERESP), Madrid, Spain27 International Agency for Research on Cancer (IARC-WHO), Lyon, France

The most studied genetic susceptibility factors involved in gastric carcinoma (GC) risk are polymorphisms in the inflammation-

linked genes interleukin 1 (IL1) B and IL1RN. Despite the evidence pointing to the IL1 region, definite functional variants

reproducible across populations of different genetic background have not been discovered so far. A high density linkage dise-

quilibrium (LD) map of the IL1 gene cluster was established using HapMap to identify haplotype tagSNPs. Eighty-seven SNPs

were genotyped in a Portuguese case-control study (358 cases, 1,485 controls) for the discovery analysis. A replication study,

including a subset of those tagSNPs (43), was performed in an independent analysis (EPIC-EurGast) containing individuals

from 10 European countries (365 cases, 1284 controls). Single SNP and haplotype block associations were determined for GC

overall and anatomopathological subtypes. The most robust association was observed for SNP rs17042407, 16Kb upstream of

the IL1A gene. Although several other SNP associations were observed, only the inverse association of rs17042407 allele C

with GC of the intestinal type was observed in both studies, retaining significance after multiple testing correction

(p 5 0.0042) in the combined analysis. The haplotype analysis of the IL1A LD block in the combined dataset revealed the

association between a common haplotype carrying the rs17042407 variant and GC, particularly of the intestinal type (p 5 3.1

3 1025) and non cardia localisation (p 5 4.6 3 1023). These results confirm the association of IL1 gene variants with GC and

reveal a novel SNP and haplotypes in the IL1A region associated with intestinal type GC in European populations.

Gastric carcinoma (GC), although declining in incidence inthe last decades, is still the fourth most common malignancyand the second leading cause of cancer-related death world-wide.1 GC is an aggressive disease often diagnosed at anadvanced stage. Surgery is the only curative option butdespite improvements in surgical and chemotherapy treat-ment approaches, GC remains a global public health problemwith a 5-year overall survival rate of less than 25%.2,3 Gastriccarcinomas are localised in the proximal part of the stomach(cardia) or in the distal region (non cardia). Histologically,the main variants are the intestinal type, with clearly definedglandular structures and the undifferentiated diffuse type.4

Although non cardia is the most common localisation of GC,a decrease in GC risk has been more consistently observedfor the intestinal type and non cardia localisation, in Cauca-sian populations. The most recognised etiological risk factorfor non cardia GC development is the infection by Helico-

bacter pylori,5 a stomach-colonising bacterium that chroni-cally infects up to 50% of the world’s human population.6

The major consequence of infection is chronic inflammationof the gastric mucosa, which can progress through chronicatrophic gastritis, intestinal metaplasia and dysplasia towardsGC.7 This course of events is, however, rare leading to a con-spicuous difference between the number of infected individu-als and those that proceed to develop GC (1–3%).8 Thereasons for this difference are poorly understood but it isconsidered that a combination of bacterial virulence fac-tors,9,10 dietary and lifestyle habits11–14 and host genetic fac-tors,15–17 greatly influence the risk of GC.

Amongst the genetic susceptibility factors involved in GCrisk, the most studied are polymorphisms in theinflammation-linked genes interleukin 1 (IL1) B and IL1RN.A decade ago, the IL1B2511 (rs16944) T and IL1RN VNTR(rs2234663) 2 repeats alleles, which are putatively associated

What’s new?

Genetic susceptibility to gastric cancer lurks in the region of the interleukin 1 gene cluster, but no one yet knows just how

genetic variation contributes to risk. These authors searched for other variants within this genetic neighborhood by assessing

linkage disequilibrium. There they found several small nucleotide polymorphisms (SNPs), mainly in the IL1A gene region, that

associated with gastric carcinoma, particularly the intestinal subtype. By identifying these SNPs, they hope to shed more

light on how the disease develops or help identify people who are at risk.

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with increased mucosal levels of IL1b,18 have been shown tobe significantly associated with risk of GC,15,19 particularly incombination with H. pylori virulence factors.16,20 These associ-ation results have found important support in studies showingthat increased expression of IL1b does favour development ofGC in an in vivo animal model.21 Recent studies have shownsimilar associations both with GC22,23 and gastric precancer-ous lesions,24 but others have, however, not confirmedthem.25–27 In the follow-up of these studies, several meta-analyses confirmed the lack of reproducibility of the associa-tion of IL1 polymorphisms and GC among different popula-tions.28–30 These results can be explained, in part, by poorstudy design, e.g. small number of samples thus low statisti-cal power, disease phenotype heterogeneity and populationadmixture, but also by the linkage disequilibrium (LD)degree in the IL1 genomic region. The IL1B and IL1RNgenes are located on chromosome 2q14 clustered with othergenes of the same family,31 with which they share strong LD.Despite the evidence pointing to the IL1 region, neither themolecular basis for the associations nor the definite causalvariant, have been elucidated so far. Therefore, if there areindeed in this region one or more susceptibility loci for GC,these can be identified by LD mapping.

Focusing on this hypothesis, we investigated the associa-tion of haplotype tag single nucleotide polymorphisms(SNPs) in the IL1 genomic region with GC, histological sub-types and tumour localisation in two case-control studiesincluding different European populations. In this study, wereport a novel association between the rs17042407 SNP,16Kb upstream of the IL1A gene, as well as a number ofhigh frequency haplotypes in the IL1A region, and risk forGC of the intestinal type observed in both studies and in thecombined analysis.

MethodsStudy populations

The discovery stage included a Portuguese group (Table 1) of358 incident cases of gastric adenocarcinoma admitted to sur-gery at Hospital of S. Jo~ao and the Portuguese Institute ofOncology, Porto, Portugal, and 1,485 healthy controls, as previ-ously described.32,33 Briefly, cancer patients were eligible to par-ticipate if there was no previous cancer diagnosis, except forskin nonmelanoma, and no sub-total gastrectomy for benignconditions. Cancer was diagnosed according to the routine pro-cedures and patients submitted to gastrectomy had the tumoursclassified as intestinal, diffuse, or unclassifiable according toLaur�en; a single experienced pathologist reviewed all pathologyreports, and slides were reassessed by three pathologists when-ever routine information was considered insufficient or inconsis-tent. The source of controls was a representative sample of thenon-institutionalised adult population of Porto, Portugal,assembled as part of an health and nutrition survey. Participantswere recruited by random digit dialing, using households as thesampling frame, followed by simple random sampling to selectone eligible person among permanent residents.

The replication stage included a cohort-nested case-con-trol study within the European Prospective Investigation intoCancer and Nutrition (EPIC) study,34 comprising 365 GCcases and 1,284 controls (EPIC-EurGast study). The EPICstudy includes 521,448 individuals recruited between 1992and 2000 in 23 centres in 10 European countries. In theEPIC-EurGast study cases were subjects whose blood hasbeen collected and were diagnosed with incident gastric ade-nocarcinoma (defined by code C16 of the International Clas-sification of Diseases, 10th Revision) during the follow-up.An independent panel of pathologists confirmed and vali-dated the diagnosis, tumour localisation and morphology.35

For each case, up to four control subjects were randomlyselected among study members alive and free of cancer at thetime of diagnosis, matched by centre, gender, age (62.5years) and date of blood collection (645 days).

All individuals provided informed consent and the studyhas been approved by ethical committees at the Hospital ofS. Jo~ao, the Portuguese Institute of Oncology (Porto), theInternational Agency for Research on Cancer (Lyon) and ineach of the EPIC recruitment centres. Sample sets character-istics are summarised in Table 1.

The initial number of samples was larger both for thePorto (474 cases and 1,557 controls) and for the EPIC-EurGast (373 cases and 1,332 controls) studies. Followingdata filtering and quality control, 9.3% of the samples in thePorto study and 3.3% in the EPIC-EurGast study wereexcluded because their DNA was not amplified or had a gen-otyping call rate lower than 80%.

SNP selection criteria

Genetic variation regarding the IL1 gene cluster (Fig. 1) inCaucasians was retrieved from HapMap (phase II CEU popu-lation, release #24 November 2008) and a list of SNPs wascompiled from between 30 Kb downstream of IL1A andIL1RN genes (Fig. 1). Haplotype blocks were defined accord-ing to Gabriel et al.36 using the default parameters in Haplo-view v4.0.37 Haplotype tagging SNPs (tagSNPs) were selectedusing the Tagger algorithm implemented in Haploview andfollowed strict criteria relatively to the degree of LD betweenpairwise tagging SNPs (r2� 0.8 and D05 1), the minor allelefrequency (MAF) of the selected SNPs in Caucasians (�0.01)and the haplotype frequency (�0.01). A set of SNPs withfunctional potential in genes of the IL1 cluster that were notgenotyped in HapMap, or that were not proxies of theselected tagSNPs, were also included. Following these criteria,96 SNPs in a region spanning 420 Kb were selected for thePortuguese sample genotyping. The 96 tagSNPs were distrib-uted along nine LD blocks (one block in the IL1A region,two in the IL1B region, four in the IL1F section and two inIL1RN region, Fig. 1).

The SNPs selected for the replication stage using theEPIC-EurGast sample included a subset (43) of the 96 SNPsinitially selected for the discovery analysis. These 43 SNPswere the ones included in the blocks which contained one or

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Table 1. Main characteristics of the Porto and EPIC-EurGast studies

Cohort characteristics Controls Cases

Porto n 5 1,485 % n 5 358 %

Gender

Men 576 38.8 203 56.7

Women 812 54.7 134 37.4

Unspecified 97 6.5 21 5.9

Age (yrs)

Median (SD) 55 (615) 63 (613)

25–75 Percentiles 42–66 53–71

Histological type

Intestinal 157 43.9

Diffuse 70 19.5

Unspecified 131 36.6

Localisation

Cardia 36 10.0

Non cardia 244 68.2

Unspecified 78 21.8

EPIC-EurGast n 5 1,284 n 5 365

France 3 0.2 2 0.6

Italy 206 16.1 56 15.3

Spain 134 10.4 41 11.2

United Kingdom 135 10.5 41 11.2

The Netherlands 99 7.7 26 7.1

Greece 88 6.9 24 6.6

Germany 186 14.5 48 13.2

Sweden 220 17.1 64 17.5

Denmark 205 16.0 61 16.7

Norway 8 0.6 2 0.6

Gender

Men 759 59.1 214 58.6

Women 525 40.9 151 41.4

Age (yrs)

Median (SD) 60 (68) 60 (68)

25–75 Percentiles 54–64 54–64

Histological type

Intestinal 126 34.5

Diffuse 128 35.1

Unspecified 111 30.4

Localisation

Cardia 107 29.3

Non cardia 181 49.6

Unspecified 77 21.1

SD: standard deviation.

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more statistically significant associations (p value <0.05 orFDR value <0.2) with GC and/or its subtypes in the discov-ery stage (IL1A, IL1B and IL1RN blocks).

Sample preparation, genotyping and quality control

Genomic DNA from subjects of the Portuguese study was iso-lated from whole blood cells using standard proteinase K diges-tion with phenol/chloroform extraction and the FlexiGeneDNA Kit (Qiagen, Germany). DNA from subjects of the EPIC-EurGast study was extracted from a 0.5 ml aliquot of buffycoat, following previously reported methods.38 DNA was quan-tified with the PicoGreen dsDNA quantitation assay (Molecu-lar Probes, The Netherlands). DNA (0.75–1.0 lg) at about 50ng/ll was inserted into 96-well plates for genotyping at theBarcelona laboratory of the Spanish National GenotypingCentre (CEGEN). Genotyping of the Portuguese SNPs panelwas performed using the Illumina Veracode Technology (Illu-mina, San Diego, CA) according to the manufacturer’s proto-cols. As previously described,38 genotyping of the EPIC-EurGast samples was performed using the Illumina BeadSta-tion Platform and GoldenGate Technology (Illumina, SanDiego, CA) according to the manufacturer’s protocols. In addi-tion to the internal genotyping controls included by CEGEN,5% of the samples were genotyped in duplicate to assess the

genotyping error rate.38 Concordance of their genotypes was100% in the Porto and EPIC-EurGast studies. Samples inwhich more than 20% of the SNPs failed genotyping wereexcluded from the analysis (Porto study: cases5 116, con-trols5 72; EPIC-EurGast study: cases5 8, controls5 48).SNPs were excluded if they failed in more than 20% of thesamples, were monomorphic or deviated from Hardy-Weinberg equilibrium among controls (Fisher’s exact testp< 0.0001). In the Portuguese and EPIC-EurGast sample sets,10 and 6 SNPs, respectively, were excluded because they couldnot be amplified. Subsequently, one SNP in the Portuguesestudy (rs16944 in the IL1B block) and three in the EPIC study(rs3783512 in the IL1A block, rs16944 in the IL1B block andrs7578034 in IL1F block) were genotyped using TaqMan Pre-Designed SNP Genotyping Assays (Applied Biosystems, Carls-bad, USA). The total number of SNPs successfully genotypedand available for analysis was, respectively for the Portugueseand EPIC-EurGast studies, 87 and 40 (35 common to both),and these were the numbers used for multiple testingcorrections.

Statistical analyses

Compliance of alleles at individual loci with the Hardy-Weinbergequilibrium was measured at the level of the control population

Figure 1. IL1 gene cluster on chromosome 2 and linkage disequilibrium (LD) map according to Haploview analysis of genotype data

obtained for the Portuguese study (87 SNPs). Each gene is represented by an arrow-shaped rectangular grey block indicating the direction

of transcription (position of genes on chromosome 2 are adapted from the International HapMap Project). SNPs order from left to right is

the same as from top to bottom in the online Supporting Information Table 1. Vertical solid lines represent the limits of each haplotype

block (nine haplotype blocks). LD map key: D’ (0�D’�1) is a measure of LD between two genetic markers, higher values representing

strong LD; LOD is the logarithm of the odds for LD between two genetics markers. Can

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using a v2 test (p< 0.0001). Associations between SNP genotypesand groups defined by status (cases versus controls) and anatomo-pathological characteristics were assessed by unconditional logisticregression (level of significance set to p< 0.05) with the SNPassocpackage implemented in R (http://www.creal.cat/jrgonzalez/soft-ware.htm). The models included gender and age for the Portu-guese study and gender, age and country for the EPIC-EurGaststudy and combined analysis. The log-additive model of inheri-tance (per allele) was considered. Odds ratios (OR) with respec-tive confidence intervals (95% CI) were calculated for one copy ofthe allele with minor frequency. Additional genetic models suchas codominant, dominant and recessive were explored in subse-quent analyses.

The adjustment for multiple testing was performed by thefalse discovery rate (FDR) method.39 Differential SNP effectson GC subgroups (intestinal versus diffuse and cardia versusnon cardia) were assessed by a heterogeneity test based on alikelihood ratio test. Haplotype frequencies were inferred usingthe haplo.stats package implemented in R. Haplotype associa-tion with GC, with and without anatomopathological charac-teristics subgrouping, was assessed for those with a minimumhaplotype frequency (MHF) above 0.01 and using as referencethe most frequent haplotype in the analysed population. Allpower calculations were performed using Quanto 1.2.4.

ResultsIL1 gene cluster genotype frequencies and association

with GC overall

The genotype frequencies of the 87 SNPs in the Porto studyand the 40 SNPs in the EPIC-EurGast study were in Hardy-Weinberg equilibrium (Tables 1 and 2 of the SupportingInformation). All genotyping results are summarised inTables (1–3) of the Supporting Information. The SNPs fre-quency analysis in controls shows that there is strong correla-tion between the Porto study and HapMap CEU populationallele frequencies (r2 5 0.90), EPIC-EurGast study and Hap-Map (r2 5 0.97), and Porto and EPIC-EurGast studies(r2 5 0.98). Such observation demonstrates the effectivenessof using the CEU HapMap database to select tagSNPs for theassociation study with the Portuguese and EPIC-EurGaststudies.

The discovery analysis performed in the Portuguese studyand the replication analysis performed in the EPIC-EurGaststudy revealed several associations with GC overall (p< 0.05,Table 2 and Supporting Information in Tables 4–6). In thecombined analysis four of these associations were maintainedas inversely associated with GC (Table 2), one in the IL1Ablock (rs17042407: OR5 0.85, CI5 0.74–0.97, p5 0.0171),two in the IL1RN block 1 (rs13409371: OR5 0.85,

Table 2. Association of SNPs in the IL1A haplotype block with gastric carcinoma overall in the Porto study, EPIC-EurGast study and combinedanalysis

Porto, n 5 358 EPIC, n 5 365 Combined analysis, n 5 723

Locus/SNP Alleles OR 95% CI p OR 95% CI p OR 95% CI p

IL1A

rs7592947 A/G 1.21 0.91–1.62 0.20

rs2048874 C/T 0.98 0.75–1.27 0.86

rs4848300 T/C 1.15 0.95–1.39 0.16 1.03 0.86–1.24 0.73 1.09 0.96–1.25 0.19

rs17561 G/T 1.15 0.95–1.40 0.15 1.01 0.85–1.22 0.88 1.08 0.95–1.24 0.23

rs3783512 G/A 1.12 0.93–1.34 0.23

rs6722023 C/T 0.73 0.29–1.81 na

rs17042407 T/C 0.81 0.66–0.99 0.0347 0.87 0.72–1.05 0.15 0.85 0.74–0.97 0.0171

rs4849122 A/G 1.02 0.69–1.51 0.93 0.81 0.56–1.18 0.26 0.91 0.69–1.19 0.47

rs4849124 G/A 1.03 0.86–1.24 0.75 0.97 0.81–1.16 0.74 1.01 0.89–1.15 0.87

IL1B

rs16944 C/T 1.25 1.03–1.52 0.0283 1.00 0.83–1.21 0.97 1.14 0.99–1.31 0.06

IL1RN

rs13409371 G/A 0.90 0.76–1.06 0.21 0.83 0.70–0.98 0.0293 0.85 0.76–0.97 0.0113

rs10188292 A/T 0.88 0.74–1.05 0.15 0.82 0.69–0.98 0.0236 0.84 0.75–0.95 0.0062

rs17669228 C/T 0.90 0.73–1.12 0.34 0.80 0.65–0.99 0.0386 0.87 0.75–1.02 0.08

rs17042917 G/A 1.15 0.85–1.57 0.36 1.32 1.01–1.72 0.0476 1.18 0.96–1.45 0.12

rs4251961 T/C 0.88 0.74–1.05 0.17 0.79 0.67–0.94 0.0070 0.85 0.75–0.96 0.0073

rs2637988 A/G 1.19 1.01–1.41 0.0380

Also indicated are the SNPs in the IL1B and IL1RN haplotype blocks with p < 0.05 in at least one of the studies. ORs and 95% CIs were calculatedconsidering a log-additive model, adjusted for gender, age and country.na: not applicable (the frequency of the homozygous minor allele is zero).Bold font indicates to highlight nominally significant results.C

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31

.08

–1

.89

0.0

14

60

.98

0.7

3–

1.3

30

.91

0.0

34

1

rs6

72

20

23

C/T

0.8

10

.24

–2

.80

na

na

na

na

0.1

3

rs1

70

42

40

7T/

C0

.61

0.4

5–

0.8

30

.00

09

1.2

30

.85

–1

.79

0.2

70

.00

07

0.7

20

.52

–1

.00

0.0

45

40

.91

0.6

7–

1.2

30

.52

0.4

20

.66

0.5

3–

0.8

20

.00

01

1.0

10

.80

–1

.28

0.9

40

.00

71

rs4

84

91

22

A/G

1.4

20

.87

–2

.31

0.1

70

.98

0.4

2–

2.2

90

.97

0.4

40

.77

0.4

3–

1.3

90

.38

0.9

90

.57

–1

.74

0.9

80

.23

1.0

90

.75

–1

.58

0.6

60

.99

0.6

2–

1.5

70

.97

0.7

7

rs4

84

91

24

G/A

1.1

10

.86

–1

.44

0.4

41

.04

0.7

2–

1.5

10

.82

0.9

30

.90

0.6

7–

1.2

10

.49

0.8

40

.63

–1

.12

0.2

30

.82

1.0

00

.83

–1

.22

0.9

60

.91

0.7

2–

1.1

40

.41

0.8

9

IL1

B

rs1

69

44

C/T

1.4

51

.11

–1

.89

0.0

06

51

.23

0.8

6–

1.7

80

.26

0.2

81

.10

0.8

1–

1.4

80

.56

0.8

30

.61

–1

.13

0.2

40

.22

1.2

81

.05

–1

.56

0.0

14

40

.99

0.7

8–

1.2

50

.91

0.1

1

IL1

RN

rs3

15

92

1C

/T1

.16

0.8

3–

1.6

10

.38

1.2

10

.76

–1

.93

0.4

30

.34

1.4

01

.02

–1

.93

0.0

41

90

.82

0.5

7–

1.1

90

.28

0.0

18

11

.26

1.0

0–

1.5

80

.05

0.9

20

.69

–1

.23

0.5

80

.30

rs4

25

19

61

T/C

0.9

20

.72

–1

.18

0.5

00

.96

0.6

7–

1.3

70

.81

0.6

90

.72

0.5

5–

0.9

50

.01

71

0.8

50

.65

–1

.11

0.2

30

.43

0.8

30

.69

–0

.99

0.0

40

30

.89

0.7

2–

1.1

10

.30

0.8

6

rs2

63

79

88

A/G

1.1

80

.91

–1

.54

0.2

01

.32

1.0

1–

1.7

20

.04

02

0.4

8

Als

oin

dic

ate

da

reth

eS

NP

sin

the

IL1

Ba

nd

IL1

RN

ha

plo

typ

eb

lock

sw

ith

p<

0.0

5in

at

lea

sto

ne

of

the

stu

die

s.O

Rs

an

d9

5%

CIs

we

reca

lcu

late

dco

nsi

de

rin

ga

log

-ad

dit

ive

mo

de

l,a

dju

ste

dfo

rg

en

de

r,a

ge

an

dco

un

try.

1p

valu

efo

rh

ete

rog

en

eit

y;n

a:

no

ta

pp

lica

ble

(th

efr

eq

ue

ncy

of

som

eg

en

oty

pe

sis

zero

);a

sso

cia

tio

ns

wit

hp<

0.0

5a

reh

igh

lig

hte

din

bo

ld.

Bo

ldfo

nt

ind

ica

tes

toh

igh

lig

ht

no

min

all

ysi

gn

ifica

nt

resu

lts.

Can

cerGenetics

Dur~aes et al. 7

Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC

CI5 0.76–0.97, p5 0.0113; rs10188292: OR5 0.84,CI5 0.75–0.95, p5 0.0062) and one in the IL1RN block 2(rs4251961: OR5 0.85, CI5 0.75–0.96, p5 0.0073). None ofthe associations observed for GC overall were retained afterFDR multiple test corrections. The previous reported riskassociation of SNP rs16944 (IL1B2511 C/T) with GC wasonly observed in the Porto study (OR5 1.25, CI5 1.03–1.52,p5 0.0283), however, it was not retained after correction formultiple testing (p5 0.99, data not shown). These results aresummarised in Table 2. Analysis by other inheritance models(data not shown) did not improve the results with the excep-tion of IL1B rs16944 under the dominant model in the Portostudy (OR5 1.43, CI5 1.09–1.88, p5 0.0103) and in thecombined analysis (OR5 1.24, CI5 1.03–1.50, p5 0.0220),and IL1A rs17042407 under the dominant model in theEPIC-EurGast study (OR5 0.79, CI5 0.62–1.00, p5 0.0480)and in the combined analysis (OR5 0.80, CI5 0.68–0.93,p5 0.0127). However, as for the log-additive model, theseassociations did not retain significance after FDR multipletest corrections.

IL1 gene cluster association with GC histological and

anatomical types

Several associations were observed when the SNPs were ana-lysed according to tumour histological subtype and localisa-tion, both in the Porto and EPIC-EurGast studies, and in thecombined analysis, particularly with the intestinal subtypeand non cardia localisation (Tables 3 and 4; SupportingInformation in Tables 4–6). The more robust associationswere observed in the IL1A block, with the C allele ofrs17042407 being inversely associated with GC of the intesti-nal type in the Porto, EPIC-EurGast and combined analysis.However, only the association of rs17042407 with GC of theintestinal type in the combined analysis (OR5 0.66,CI5 0.53-0.82, p5 0.00012) retained statistical significanceafter FDR multiple testing correction (p5 0.0042, data notshown). Independence of this association was verified afteradjustment for the other SNPs in the IL1A block (p< 0.0007,online Supporting Information Table 7). Analysis by otherinheritance models (data not shown) did not substantiallymodify the results with the exception of the associationbetween IL1B rs16944 and non cardia GC in the mergedstudy, which reached significance under the dominant model(OR5 1.27, CI5 1.01-1.60, p5 0.0398). However, as for thelog-additive model, these associations did not retain signifi-cance after FDR multiple test corrections.

The heterogeneity analysis of SNP effects according toGC subgrouping (anatomical and histological subtypes)revealed only significant risk differences in the histologicalsubgroup for rs17042407 (IL1A block) in the Porto and com-bined analysis, and for rs3783512 (IL1A block) and rs315921(IL1RN block 2) in the EPIC-EurGast study (Table 3). Signif-icant risk differences according to tumour localisation sub-grouping were only observed for rs7596461 (IL1B block 1)(Table 4).

IL1 haplotypes association with GC, histological and

anatomical types

The haplotype analysis of all the blocks specified prior togenotyping, both in the Porto and EPIC-EurGast studiesrevealed several associations with GC overall, histologicalsubtypes and tumour localisation (Supporting Information inTables 8–10). Most of these associations present low rele-vance because, either the haplotype frequency is very low(<5%) or the p is not very robust (0.05> p> 0.01). Further-more, they do not provide any additional information thanthat already provided by single SNP analyses. Nevertheless,in the IL1RN block 2 there is an haplotype with a frequencyof 9% that is associated with GC overall, intestinal subtypeand non cardia localisation in the EPIC and combined analy-sis, which carries the A (rs17042917) and T (rs4251961) riskalleles for GC and its intestinal subtype in the single SNPanalyses. The most robust haplotype associations wereobserved for the IL1A block in both studies (Table 5), how-ever, the interpretation of haplotype associations is bestsuited for the combined analysis, where only SNPs in com-mon have been analysed.

The Porto study revealed two common haplotypes(ACTGACTAG and ACCTACTAA) which are statisticallysignificant risk factors for GC overall (OR5 1.29, CI5 1.01-1.64, p5 0.0388 for ACTGACTAG; and OR5 1.28,CI5 1.00–1.64, p5 0.0525 for ACCTACTAA); the associa-tion with the intestinal type is more robust with ORs (CI; pvalue) of, respectively, 1.48 (1.03–2.12; 0.0323) and 1.67(1.15–2.42; 0.0067). There are another three haplotypes asso-ciated with GC of the intestinal type, but their frequency isbelow 5%. The reference haplotype contains, as opposed tothe others, the minor allele C of rs17042407 (IL1A block,underlined in the haplotype sequence in the text above).The results are reported in Table 5; the order ofhaplotype SNPs is presented according to their localisation inthe chromosome 2 DNA forward strand and is indicated inTable 5.

The EPIC-EurGast study contains a common haplotype(AATGGACAG) inversely associated with GC of the intesti-nal type. The OR (CI; p) is 0.59 (0.40–0.86; 0.0057), aninverse association which can be explained not only by a dif-ferent SNP content but also, and most likely, by the presenceof the “protective” C allele of SNP rs17042407. This isexplained by the fact that in this haplotype context, in con-trast with that observed in the Porto study, the referencehaplotype carries the major T allele of rs17042407. Threeother haplotypes, also carrying the T allele of rs17042407,were identified to be associated with GC or any of its sub-types in the EPIC-EurGast study. These haplotypes are pres-ent, however, at low frequencies (Table 5).

The combined analysis, containing only five SNPs, alsorevealed a common haplotype (A ATGA ACAG, at a fre-quency of 27.6% in the controls and 23.4% in the cases) that isassociated with GC overall, the intestinal subtype and non car-dia localisation (Table 5). The OR (CI; p-value) is, respectively,

Can

cerGenetics

8 IL1A genetic variants in gastric carcinoma

Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC

Tab

le4

.A

sso

cia

tio

no

fS

NP

sin

the

IL1

Ah

ap

loty

pe

blo

ckw

ith

ga

stri

cca

rcin

om

aa

na

tom

ica

lty

pe

sin

the

Po

rto

stu

dy,

EP

IC-E

urG

ast

stu

dy

an

dco

mb

ine

da

na

lysi

s

Po

rto

EP

ICC

om

bin

ed

an

aly

sis

Ca

rdia

,n

53

6N

on

card

ia,

n5

24

4C

ard

ia,

n5

10

7N

on

card

ia,

n5

18

1C

ard

ia,

n5

14

3N

on

card

ia,

n5

42

5

Locu

s/S

NP

3O

R9

5%

CI

pO

R9

5%

CI

pp

-he

t1O

R9

5%

CI

pO

R9

5%

CI

pp

-het

1O

R9

5%

CI

pO

R9

5%

CI

pp

-het

1

IL1

A

rs7

59

29

47

A/G

1.1

80

.72

–1

.94

0.5

11

.33

0.9

0–

1.9

70

.16

0.8

5

rs2

04

88

74

C/T

0.8

90

.41

–1

.93

0.7

70

.96

0.7

0–

1.3

10

.79

0.8

6

rs4

84

83

00

T/C

1.3

10

.76

–2

.24

0.3

41

.22

0.9

8–

1.5

20

.08

0.9

70

.88

0.6

4–

1.2

10

.41

1.1

50

.91

–1

.47

0.2

50

.07

0.9

40

.72

–1

.24

0.6

81

.18

1.0

0–

1.3

80

.05

0.1

5

rs1

75

61

G/T

1.2

50

.72

–2

.17

0.4

31

.22

0.9

8–

1.5

30

.08

0.8

80

.82

0.6

0–

1.1

40

.24

1.1

60

.91

–1

.47

0.2

40

.03

31

0.8

90

.68

–1

.18

0.4

21

.18

1.0

0–

1.3

90

.04

60

0.0

8

rs3

78

35

12

G/A

1.0

70

.78

–1

.47

0.6

61

.16

0.9

1–

1.4

90

.23

0.8

5

rs6

72

20

23

C/T

na

na

na

0.6

90

.23

–2

.06

na

0.4

7

rs1

70

42

40

7T/

C0

.73

0.4

1–

1.3

00

.27

0.8

30

.66

–1

.04

0.1

10

.67

1.0

60

.77

–1

.47

0.7

10

.79

0.6

1–

1.0

40

.08

0.1

40

.98

0.7

4–

1.2

90

.87

0.8

10

.68

–0

.97

0.0

17

20

.38

rs4

84

91

22

A/G

0.8

10

.24

–2

.75

0.7

31

.23

0.7

9–

1.9

00

.36

0.4

10

.62

0.3

0–

1.2

80

.17

0.8

00

.48

–1

.33

0.3

80

.37

0.6

70

.36

–1

.24

0.1

81

.00

0.7

2–

1.3

90

.98

0.2

7

rs4

84

91

24

G/A

1.2

20

.73

–2

.05

0.4

41

.02

0.8

3–

1.2

70

.82

0.6

01

.01

0.7

4–

1.3

70

.96

0.9

90

.78

–1

.27

0.9

60

.77

1.0

40

.80

–1

.36

0.7

51

.01

0.8

6–

1.1

80

.94

0.8

7

IL1

B

rs7

59

64

61

C/T

1.4

20

.65

–3

.11

0.4

00

.72

0.4

8–

1.0

70

.09

0.2

30

.88

0.5

3–

1.4

70

.63

0.6

30

.39

–1

.00

0.0

38

50

.10

1.0

10

.66

–1

.55

0.9

70

.68

0.5

0-0

.91

0.0

07

90

.02

77

rs1

69

44

C/T

1.4

50

.86

–2

.44

0.1

71

.29

1.0

3–

1.6

10

.02

83

0.4

91

.02

0.7

3–

1.4

20

.92

0.9

00

.69

–1

.18

0.4

50

.46

1.1

50

.87

–1

.52

0.3

21

.11

0.9

4–

1.3

10

.21

0.2

4

IL1

RN

rs1

34

09

37

1G

/A1

.21

0.7

5–

1.9

50

.43

0.8

10

.66

–0

.99

0.0

42

50

.05

0.7

60

.56

–1

.02

0.0

60

.86

0.6

8–

1.0

80

.18

0.8

20

.86

0.6

7–

1.1

10

.24

0.8

40

.72

–0

.98

0.0

22

40

.30

rs1

01

88

29

2A

/T1

.17

0.7

3–

1.8

70

.52

0.8

20

.67

–1

.01

0.0

60

.08

0.7

40

.55

–1

.00

0.0

46

60

.87

0.6

9–

1.0

90

.23

0.6

50

.85

0.6

6–

1.0

80

.18

0.8

50

.73

–0

.99

0.0

32

20

.47

rs1

18

86

75

4C

/G0

.67

0.2

3–

1.9

30

.43

1.0

30

.72

–1

.48

0.8

70

.47

1.5

21

.03

–2

.23

0.0

39

11

.17

0.8

3–

1.6

40

.38

0.4

21

.34

0.9

4–

1.9

10

.12

1.1

00

.86

––

1.4

00

.47

0.6

9

rs1

76

69

22

8C

/T1

.41

0.8

3–

2.4

00

.21

0.8

70

.68

–1

.13

0.2

90

.04

40

0.6

70

.45

–0

.98

0.0

32

10

.87

0.6

6–

1.1

60

.34

0.6

50

.85

0.6

2–

1.1

50

.29

0.8

80

.73

–1

.06

0.1

70

.36

rs1

70

42

91

7G

/A0

.55

0.1

7–

1.8

10

.29

1.0

90

.76

–1

.56

0.6

30

.32

1.3

40

.87

–2

.08

0.2

01

.46

1.0

3–

2.0

90

.04

14

0.7

51

.13

0.7

6–

1.7

00

.55

1.2

30

.96

–1

.58

0.1

10

.40

rs4

25

19

61

T/C

1.1

60

.71

–1

.89

0.5

60

.85

0.6

9–

1.0

50

.12

0.1

50

.74

0.5

5–

1.0

00

.04

79

0.8

20

.65

–1

.03

0.0

80

.96

0.8

40

.65

–1

.08

0.1

70

.85

0.7

3–

0.9

90

.03

25

0.3

5

Als

oin

dic

ate

da

reth

eS

NP

sin

the

IL1

Ba

nd

IL1

RN

ha

plo

typ

eb

lock

sw

ith

p<

0.0

5in

at

lea

sto

ne

of

the

stu

die

s.O

Rs

an

d9

5%

CIs

we

reca

lcu

late

dco

nsi

de

rin

ga

log

-ad

dit

ive

mo

de

l,a

dju

ste

dfo

rg

en

de

r,a

ge

an

dco

un

try.

1p

valu

efo

rh

ete

rog

en

eit

y;n

a:

no

ta

pp

lica

ble

(th

efr

eq

ue

ncy

of

som

eg

en

oty

pe

sis

zero

);a

sso

cia

tio

ns

wit

hp<

0.0

5a

reh

igh

lig

hte

din

bo

ld.

Bo

ldfo

nt

ind

ica

tes

toh

igh

lig

ht

no

min

all

ysi

gn

ifica

nt

resu

lts.

Can

cerGenetics

Dur~aes et al. 9

Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC

Tab

le5

.IL

1A

blo

ckh

ap

loty

pe

sa

nd

ass

oci

ati

on

wit

hg

ast

ric

carc

ino

ma

ove

rall

,h

isto

log

ica

la

nd

an

ato

mic

al

sub

typ

es

inth

eP

ort

ost

ud

y,E

PIC

-Eu

rGa

stst

ud

ya

nd

com

bin

ed

an

aly

sis

Ga

stri

cca

nce

r,n

53

58

Inte

stin

al,

n5

15

7D

iffu

se,

n5

70

Ca

rdia

,n

53

6N

on

card

ia,

n5

24

4

Co

ho

rtH

ap

loty

pe

s1Fr

eq

ue

ncy

con

tro

ls/c

ase

sO

R9

5%

CI

pO

R9

5%

CI

pO

R9

5%

CI

pO

R9

5%

CI

pO

R9

5%

CI

p

Po

rto

AC

TGA

CC

AG

0.2

85

/0.2

35

1.0

01

.00

1.0

01

.00

1.0

0

AC

TGA

CTA

G0

.25

3/0

.28

71

.29

1.0

1–

1.6

40

.03

88

1.4

81

.03

–2

.12

0.0

32

30

.82

0.5

1–

1.3

30

.42

1.3

20

.66

–2

.61

0.4

31

.24

0.9

4–

1.6

50

.13

AC

CTA

CTA

A0

.22

1/0

.24

31

.28

1.0

0–

1.6

40

.05

25

1.6

71

.15

–2

.42

0.0

06

71

.02

0.6

5–

1.6

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90

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00

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0.0

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4–

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40

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3–

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.87

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9

AC

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7/0

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20

.98

0.5

2–

1.8

40

.95

0.9

80

.37

–2

.60

0.9

70

.84

0.2

5–

2.8

10

.77

1.4

70

.34

–6

.33

0.6

10

.54

0.2

1–

1.3

70

.19

AC

TGA

CTG

G0

.01

9/0

.01

70

.97

0.4

8–

1.9

80

.94

1.1

90

.42

–3

.36

0.7

41

.20

0.3

5–

4.1

60

.77

0.8

10

.10

–6

.78

0.8

51

.17

0.5

2–

2.6

20

.71

EP

ICAA

TGAA

TAG

0.2

69

/0.2

97

1.0

01

.00

1.0

01

.00

1.0

0

AA

TGGA

CA

G0

.26

2/0

.23

40

.81

0.6

4–

1.0

20

.08

0.5

90

.40

–0

.86

0.0

05

70

.98

0.6

7–

1.4

20

.91

0.9

30

.63

–1

.38

0.7

20

.76

0.5

5–

1.0

40

.09

AA

CTG

ATA

A0

.15

3/0

.15

30

.90

0.6

8–

1.1

70

.43

0.8

70

.58

–1

.31

0.5

10

.74

0.4

6–

1.1

90

.22

0.9

10

.57

–1

.44

0.6

80

.90

0.6

3–

1.3

00

.59

GA

CTG

ATA

A0

.08

6/0

.10

11

.07

0.7

8–

1.4

70

.69

0.6

10

.35

–1

.09

0.1

01

.42

0.8

8–

2.3

20

.15

0.9

90

.58

–1

.72

0.9

81

.13

0.7

4–

1.7

20

.57

AA

TGGA

TAA

0.0

73

/0.0

55

0.6

80

.46

–1

.00

0.0

50

.52

0.2

7–

0.9

90

.04

64

0.7

40

.39

–1

.40

0.3

50

.87

0.4

7–

1.6

10

.66

0.5

60

.31

–0

.99

0.0

45

4

AA

TGGA

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0.0

53

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65

1.1

20

.77

–1

.64

0.5

60

.89

0.4

9–

1.6

10

.70

2.0

11

.17

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.46

0.0

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10

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0.3

7–

1.5

90

.48

1.2

00

.72

–1

.98

0.4

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CTG

ATG

G0

.04

4/0

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70

.57

0.3

3–

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60

.03

35

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50

.94

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1.9

80

.86

0.2

00

.05

–0

.86

0.0

30

70

.68

0.3

5–

1.3

30

.26

AA

CTG

ATA

G0

.02

2/0

.02

81

.10

0.6

2–

1.9

60

.74

1.0

90

.47

–2

.56

0.8

41

.43

0.6

3–

3.2

40

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30

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61

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70

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0.0

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2.4

90

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0.0

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08

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0.1

80

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0.2

5–

2.3

60

.63

0.7

00

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.78

0.6

10

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0.0

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2.5

90

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da

na

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76

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40

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90

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50

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50

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0.7

3–

1.2

90

.85

0.9

70

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.35

0.8

51

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0.8

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1.2

30

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40

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Can

cerGenetics

10 IL1A genetic variants in gastric carcinoma

Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC

0.78 (0.67-0.92; 0.0036), 0.58 (0.45-0.75; 3.1 3 1025) and 0.75(0.61–0.91; 0.0046). This haplotype is the only one containingthe allele C of rs17042407. As already observed with the EPIC-EurGast samples, two other haplotypes, both at lower frequen-cies and containing the T allele of rs17042407 were alsoinversely associated with GC or any of its subtypes. One ofthem (A ATGA ATAA, 9.5% in controls) was associated withGC overall, non cardia localisation and intestinal subtype; theother (A ACTA ATGG, 3.5% in controls) was inversely asso-ciated with the cardia localisation (Table 5).

DiscussionThe IL1 gene cluster on chromosome 2q has been implicatedin susceptibility to a high number of neoplastic, autoimmuneand chronic inflammatory disorders,40–42 with special empha-sis on polymorphisms in the IL1B and IL1RN genes. Whereasnumerous studies have reported significant associations link-ing the IL1 region to GC, no causal genetic variants havebeen identified so far as being reproducibly associated withGC. In this investigation, haplotype tagSNPs covering about400Kb of the IL1 region were genotyped in a Portuguesepopulation of GC cases and controls for the discovery analy-sis. A second study, including a subset of those tagSNPs, wasperformed in samples from the EPIC cohort.34

The most robust single SNP association was observed forthe rs17042407 (T/C) in the IL1A LD block, located 16Kbupstream of the IL1A gene. Several other SNP associationswere observed but only the association of rs17042407 withGC of the intestinal type was confirmed in both studies,retaining significance after FDR multiple testing correction inthe combined analysis with all populations. Although therewas an a priori hypothesis about the potential relationship ofthe disease with the genes analysed, and each test could beconsidered independent, we opted for a multiple test correc-tion as to reinforce the results obtained. As a consequence,the association of rs16944 (IL1B2511 C/T), observed both inthe Porto population and in the combined analysis, was notretained. This is in agreement with previous results from thefirst phase of the EPIC-EurGast study in which an associationwith SNP rs1143627, in complete LD with rs16944, was notobserved.22

The protective effect of the rs17042407 C allele on GCwas observed for the intestinal but not the diffuse histologicalsubtype, and in the non cardia localisation. However, associa-tion with the non cardia localisation was only observed inthe combined analysis and the heterogeneity test did notreveal a significantly different effect of this variant on thetwo GC localisations. More studies with larger populationsare required to clarify whether this is due to an insufficientpower of the study to detect a significant association with theless common cardia localisation or to a real differential effectof the variant allele. Power calculations indicated that, in thecombined study, and with MAFs �20% (as is the case ofrs17042407 and more than half of the SNPs analysed) wehad more than 80% power to detect a significant association

of OR �1.25 (�0.75) with GC, OR �1.30 (�0.70) with noncardia localisation, OR�1.35 (�0.65) with the intestinal type,OR�1.45 (�0.55) with the diffuse type and OR�1.50(�0.50) with the cardia localisation.

The haplotype analysis of the IL1A LD block also confirmeda strong inverse association between a common haplotype car-rying the minor C allele of rs17042407 and GC, particularly ofthe intestinal subtype. Nevertheless, haplotype results are diffi-cult to interpret due to the fact that, while the C allele ofrs17042407 is mainly observed in a single common haplotypeof approximately 26% frequency, the major T allele is detectedin several different haplotypes of frequencies ranging from 32to less than 5%. Depending on the number and characteristicsof the SNPs analysed, the commonest, and hence the referencehaplotype, may be the one carrying the T allele of rs17042407or the one carrying the C allele. When this is the case, as it hap-pens in the Porto population study, the most common haplo-types carrying the T allele become risk haplotypes. However, ifthe reference haplotype carries the T allele, as it occurs in theEPIC-EurGast study and in the combined analyses, then thehaplotype carrying the C allele acts as protective against GCand its intestinal type.

Additional haplotypes associated with GC overall and itssubtypes were observed, particularly in the IL1RN blocks, buttheir frequency was either low (<5%) or the p not veryrobust (0.05> p> 0.01). Since in this study we did not ana-lyse the IL1RN 86bp VNTR variant (rs2234663), we cannotrule out the possibility of these haplotype associations reflect-ing the previously described association between this IL1RNVNTR and GC.

Recent studies have reported associations of polymorphismsin IL1A and adjacent regions with risk of glaucoma(rs1800587),43 ovarian cancer (rs4848300, rs17561),44 breastcancer (rs2856836, rs17561),40,45 nasopharyngeal carcinoma(rs3783553),46 and obesity (rs1800587, rs17561).47 SNPsrs17651 and rs4848300 are located in the same haplotype blockas rs17042407 and are highly correlated, both in the aforemen-tioned ovarian cancer report (r2 5 0.99)44 and in our study(r2 5 0.99 for Porto, EPIC-EurGast and combined analysis).Furthermore, although their LD with rs17042407 is low whenmeasured by r2 (r2 5 0.16 in Porto and 0.16 in EPIC-EurGast),it is high when measured by D’ (D’� 0.98 in both studies) andboth SNPs have been found associated with intestinal GC inthe Porto population, although not in the EPIC-EurGast studynor in the combined cohort. Moreover, in our study, SNPrs17042407 is independent of rs17651 and rs4848300 as it isthe only one maintaining an association after adjusting forthose SNPs, while rs17651 and rs4848300 lose the associationwhen adjusted for rs17042407. The report of SNP rs2856836association with breast cancer in a Korean population40 alsoincludes rs17042407 in the haplotype analysis, where the onlysignificant haplotype, like in our study, contains the risk alleleT. Although all these studies include Caucasian populationswith similar rs17651 and rs4848300 MAFs, the geographicalregions analysed are different.

Can

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Dur~aes et al. 11

Int. J. Cancer: 00, 00–00 (2014) VC 2014 UICC

There is no data on the effect of rs17042407 on expressionor function of any IL1 gene, something that can be due to itsintergenic location or that it has not captured the attention ofany study. Nevertheless, this SNP is worth pursuing in subse-quent investigations as indicated by the number of variantswith which it is in perfect LD and that have functional interest(explored in HaploReg v2 but not indicated; http://www.broadinstitute.org/mammals/haploreg/haploreg.php). Othervariation in the IL1A region has been recently investigated,suggesting its involvement in cancer pathways. An insertion/deletion in the 3’ UTR of IL1A (rs3783553) has been reportedto be associated with hepatocellular carcinoma in a Chinesepopulation.48 This study shows that the TTCA insertion alleledisrupts a binding site for miR-122 and miR-378, therebyincreasing the transcription of IL1a in vitro and in vivo. Thesame variant is also associated with nasopharyngeal carcinomaand higher levels of serum IL1a in patients.46 We have notevaluated this variant in our study (or any other variantlocated in the same indel cluster), however, the Haploviewanalysis of the 1000 Genomes49 data of the IL1 region indi-cates that it is not in LD with rs17042407 (data not reported).In the context of IL1a activity, Sakamoto et al.50 reported thatIL1a induced the proliferation of GC cells and showed that ithas a role in gastric carcinogenesis. Therefore, functional var-iants in the IL1A gene could affect GC by altering the IL1a

pathway.The present study was designed to search for variants in

LD with published IL1B and IL1RN variants associated withGC, and a novel, independent association in the IL1A regionwas identified. It is here reported that the IL1A region con-tains single variants and haplotypes associated with risk forGC, particularly of the intestinal and non cardia subtypes.Whether these variants are also associated with GC prognosisor response to treatment is something worth to be exploredin future studies. Increasing our understanding of host genet-

ics in cancer development may allow particularly susceptibleindividuals to be targeted for screening or treatment toreduce risk of future malignant transformation.

AcknowledgmentsTo Dr Mireia Vilardell for the helpful comments and assistance with somestatistical analyses. To Dr Magda Montfort and Dr Sebasti�an Mor�an fromthe Spanish National Genotyping Center (CEGEN, Barcelona node). To theEPIC-EurGast study pathology panel members Drs Hendrik Blaker, ClausFenger, Laszlo Igali, Gabriella Nesi and Roger Stenling for their contribu-tion to the collection and review of paraffin tumour blocks, slides andpathology reports. To Ana Moleirinho for the assistance with the 1,000Genomes data analysis.

Cec�ılia Dur~aes is supported by a posdoctoral grant from FCT, ref.SFRH/BPD/62974/2009. Xavier Mu~noz was partially supported by Institutode Salud Carlos III of the National Health System (exp.CA06/0200). Ador-aci�on Vencesl�a is supported by ‘Juan de la Cierva’ posdoctoral subprogramfrom MICINN, ref. JCI 2010-0836. IPATIMUP is an Associate Laboratoryof the Portuguese Ministry of Science, Technology and Higher Educationand is partially supported by the Portuguese Foundation for Science andTechnology.

Specific author contributionsC.D. contributed to project design including SNP selection,performed experiments, statistical analysis and drafted themanuscript; X.M. contributed to project design includingSNP selection and performed statistical analysis; C.B. per-formed statistical analysis and interpretation of data; N.G.and A.V. contributed to experimental analyses and datainterpretation; G.C., J.C.M. and N.S contributed to projectdesign, results discussion, supervision of the work and criticalrevision of the manuscript for important intellectual content;N.L. and C.A.G. contributed as principal investigators of thePorto and EPIC-EurGast studies, respectively. The remainingauthors contributed with sample collection, patient diagnosis,tumour characterisation and database setup; all authors havereviewed and approved the final draft submitted.

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