IJC International Journal of Cancer
Genetic variants in the IL1A gene region contribute to intestinal-type gastric carcinoma susceptibility in European populations ~es1, Xavier Mun ~oz2,3, Catalina Bonet4, Nadia Garcıa2,4, Adoracio n Vencesla 2,4, Fa tima Carneiro1,5,6, Cecılia Dura 5,7 5,7 5,7 8 rbara Peleteiro , Nuno Lunet , Henrique Barros , Bjo €rn Lindkvist , Marie-Christine Boutron-Ruault9,10,11, Ba 12,13,14 15 H. Bas Bueno-de-Mesquita , Cosmeri Rizzato , Antonia Trichopoulou16,17, Elisabete Weiderpass18,19,20,21, 22 Allessio Naccarati , Ruth C. Travis23, Anne Tjïnneland24, Aurelio Barricarte Gurrea25,26, Mattias Johansson27, Elio Riboli14, lez2, Gabriel Capella 3, Jose Carlos Machado1,5 and Nu ria Sala2,4 Ceu Figueiredo1,5, Carlos Alberto Gonza 1
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal Molecular Epidemiology Group, Translational Research Laboratory, Catalan Institute of Oncology (IDIBELL), Barcelona, Spain 3 Hereditary Cancer Program, Translational Research Laboratory, Catalan Institute of Oncology (ICO-IDIBELL), Barcelona, Spain 4 Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (IDIBELL), Barcelona, Spain 5 Faculty of Medicine of the University of Porto, Porto, Portugal 6 Department of Pathology, Hospital of S. Jo~ ao, Porto, Portugal 7 Institute of Public Health of the University of Porto (ISPUP), Porto, Portugal 8 Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 9 Institut National de la Sante et de la Recherche Medicale (INSERM), Centre for Research in Epidemiology and Population Health (CESP), U1018, Nutrition, Hormones and Women’s Health Team, Villejuif, France 10 Universite Paris-Sud, UMRS 1018, Villejuif, France 11 Institut Gustave Roussy (IGR), Villejuif, France 12 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands 13 Department of Gastroenterology and Hepatology, University Medical Centre, Utrecht, The Netherlands 14 School of Public Health, Imperial College London, St Mary’s Campus, Imperial College, London, United Kingdom 15 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: Fundacio ‘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 Generale de l’Education Nationale, Institut National de la Sante et de la Recherche Medicale (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 Andalucıa, 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: [email protected] or to Cecılia 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: [email protected]
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
Cancer Genetics
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IL1A genetic variants in gastric carcinoma
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Hellenic Health Foundation, Athens, Greece Department of Hygiene, Epidemiology and Medical Statistics, WHO Collaborating Center for Food and Nutrition Policies, University of Athens Medical School, Athens, Greece 18 Department of Community Medicine, Faculty of Health Sciences, University of Tromsï, Tromsï, Norway 19 Department of Research, Cancer Registry of Norway, Oslo, Norway 20 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 21 Samfundet Folkh€alsan, Helsinki, Finland 22 HuGeF (Human Genetics Foundation), Molecular and Genetic Epidemiology Unit, Torino, Italy 23 Cancer Epidemiology Unit, University of Oxford, Oxford, United Kingdom 24 Diet, Genes and Environment, Danish Cancer Society Research Center, Copenhagen, Denmark 25 Navarre Public Health Institute, Pamplona, Spain 26 blica-CIBERESP), Madrid, Spain Consortium for Biomedical Research in Epidemiology and Public Health (CIBER Epidemiologıa y Salud Pu 27 International Agency for Research on Cancer (IARC-WHO), Lyon, France
Cancer Genetics
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The most studied genetic susceptibility factors involved in gastric carcinoma (GC) risk are polymorphisms in the inflammationlinked 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 disequilibrium (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.
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.
Gastric carcinoma (GC), although declining in incidence in the last decades, is still the fourth most common malignancy and the second leading cause of cancer-related death worldwide.1 GC is an aggressive disease often diagnosed at an advanced stage. Surgery is the only curative option but despite improvements in surgical and chemotherapy treatment approaches, GC remains a global public health problem with a 5-year overall survival rate of less than 25%.2,3 Gastric carcinomas 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 defined glandular 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 observed for the intestinal type and non cardia localisation, in Caucasian populations. The most recognised etiological risk factor for non cardia GC development is the infection by Helico-
bacter pylori,5 a stomach-colonising bacterium that chronically infects up to 50% of the world’s human population.6 The major consequence of infection is chronic inflammation of the gastric mucosa, which can progress through chronic atrophic gastritis, intestinal metaplasia and dysplasia towards GC.7 This course of events is, however, rare leading to a conspicuous difference between the number of infected individuals and those that proceed to develop GC (1–3%).8 The reasons for this difference are poorly understood but it is considered that a combination of bacterial virulence factors,9,10 dietary and lifestyle habits11–14 and host genetic factors,15–17 greatly influence the risk of GC. Amongst the genetic susceptibility factors involved in GC risk, the most studied are polymorphisms in the inflammation-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
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
with increased mucosal levels of IL1b,18 have been shown to be significantly associated with risk of GC,15,19 particularly in combination with H. pylori virulence factors.16,20 These association results have found important support in studies showing that increased expression of IL1b does favour development of GC in an in vivo animal model.21 Recent studies have shown similar associations both with GC22,23 and gastric precancerous lesions,24 but others have, however, not confirmed them.25–27 In the follow-up of these studies, several metaanalyses confirmed the lack of reproducibility of the association of IL1 polymorphisms and GC among different populations.28–30 These results can be explained, in part, by poor study design, e.g. small number of samples thus low statistical power, disease phenotype heterogeneity and population admixture, but also by the linkage disequilibrium (LD) degree in the IL1 genomic region. The IL1B and IL1RN genes are located on chromosome 2q14 clustered with other genes of the same family,31 with which they share strong LD. Despite the evidence pointing to the IL1 region, neither the molecular basis for the associations nor the definite causal variant, have been elucidated so far. Therefore, if there are indeed in this region one or more susceptibility loci for GC, these can be identified by LD mapping. Focusing on this hypothesis, we investigated the association of haplotype tag single nucleotide polymorphisms (SNPs) in the IL1 genomic region with GC, histological subtypes and tumour localisation in two case-control studies including different European populations. In this study, we report a novel association between the rs17042407 SNP, 16Kb upstream of the IL1A gene, as well as a number of high frequency haplotypes in the IL1A region, and risk for GC of the intestinal type observed in both studies and in the combined analysis.
Methods Study populations
The discovery stage included a Portuguese group (Table 1) of 358 incident cases of gastric adenocarcinoma admitted to surgery at Hospital of S. Jo~ao and the Portuguese Institute of Oncology, Porto, Portugal, and 1,485 healthy controls, as previously described.32,33 Briefly, cancer patients were eligible to participate if there was no previous cancer diagnosis, except for skin nonmelanoma, and no sub-total gastrectomy for benign conditions. Cancer was diagnosed according to the routine procedures and patients submitted to gastrectomy had the tumours classified as intestinal, diffuse, or unclassifiable according to Lauren; a single experienced pathologist reviewed all pathology reports, and slides were reassessed by three pathologists whenever routine information was considered insufficient or inconsistent. The source of controls was a representative sample of the non-institutionalised adult population of Porto, Portugal, assembled as part of an health and nutrition survey. Participants were recruited by random digit dialing, using households as the sampling frame, followed by simple random sampling to select one eligible person among permanent residents. C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
The replication stage included a cohort-nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) study,34 comprising 365 GC cases and 1,284 controls (EPIC-EurGast study). The EPIC study includes 521,448 individuals recruited between 1992 and 2000 in 23 centres in 10 European countries. In the EPIC-EurGast study cases were subjects whose blood has been collected and were diagnosed with incident gastric adenocarcinoma (defined by code C16 of the International Classification of Diseases, 10th Revision) during the follow-up. An independent panel of pathologists confirmed and validated the diagnosis, tumour localisation and morphology.35 For each case, up to four control subjects were randomly selected among study members alive and free of cancer at the time of diagnosis, matched by centre, gender, age (62.5 years) and date of blood collection (645 days). All individuals provided informed consent and the study has been approved by ethical committees at the Hospital of S. Jo~ao, the Portuguese Institute of Oncology (Porto), the International Agency for Research on Cancer (Lyon) and in each of the EPIC recruitment centres. Sample sets characteristics are summarised in Table 1. The initial number of samples was larger both for the Porto (474 cases and 1,557 controls) and for the EPICEurGast (373 cases and 1,332 controls) studies. Following data filtering and quality control, 9.3% of the samples in the Porto study and 3.3% in the EPIC-EurGast study were excluded because their DNA was not amplified or had a genotyping call rate lower than 80%. SNP selection criteria
Genetic variation regarding the IL1 gene cluster (Fig. 1) in Caucasians was retrieved from HapMap (phase II CEU population, release #24 November 2008) and a list of SNPs was compiled from between 30 Kb downstream of IL1A and IL1RN genes (Fig. 1). Haplotype blocks were defined according to Gabriel et al.36 using the default parameters in Haploview v4.0.37 Haplotype tagging SNPs (tagSNPs) were selected using the Tagger algorithm implemented in Haploview and followed strict criteria relatively to the degree of LD between pairwise tagging SNPs (r2 0.8 and D0 5 1), the minor allele frequency (MAF) of the selected SNPs in Caucasians (0.01) and the haplotype frequency (0.01). A set of SNPs with functional potential in genes of the IL1 cluster that were not genotyped in HapMap, or that were not proxies of the selected tagSNPs, were also included. Following these criteria, 96 SNPs in a region spanning 420 Kb were selected for the Portuguese sample genotyping. The 96 tagSNPs were distributed along nine LD blocks (one block in the IL1A region, two in the IL1B region, four in the IL1F section and two in IL1RN region, Fig. 1). The SNPs selected for the replication stage using the EPIC-EurGast sample included a subset (43) of the 96 SNPs initially selected for the discovery analysis. These 43 SNPs were the ones included in the blocks which contained one or
Cancer Genetics
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~es et al. Dura
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IL1A genetic variants in gastric carcinoma
Table 1. Main characteristics of the Porto and EPIC-EurGast studies Cohort characteristics Porto
Controls
Cases
n 5 1,485
%
n 5 358
%
Men
576
38.8
203
56.7
Women
812
54.7
134
37.4
Unspecified
97
6.5
21
5.9
Gender
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
Cardia
36
10.0
Non cardia
244
68.2
78
21.8
Localisation
Unspecified
Cancer Genetics
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
Men
759
59.1
214
58.6
Women
525
40.9
151
41.4
Gender
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
Cardia
107
29.3
Non cardia
181
49.6
Unspecified
77
21.1
Localisation
SD: standard deviation.
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
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.
more statistically significant associations (p value
Genetic variants in the IL1A gene region contribute to intestinal-type gastric carcinoma susceptibility in European populations ~es1, Xavier Mun ~oz2,3, Catalina Bonet4, Nadia Garcıa2,4, Adoracio n Vencesla 2,4, Fa tima Carneiro1,5,6, Cecılia Dura 5,7 5,7 5,7 8 rbara Peleteiro , Nuno Lunet , Henrique Barros , Bjo €rn Lindkvist , Marie-Christine Boutron-Ruault9,10,11, Ba 12,13,14 15 H. Bas Bueno-de-Mesquita , Cosmeri Rizzato , Antonia Trichopoulou16,17, Elisabete Weiderpass18,19,20,21, 22 Allessio Naccarati , Ruth C. Travis23, Anne Tjïnneland24, Aurelio Barricarte Gurrea25,26, Mattias Johansson27, Elio Riboli14, lez2, Gabriel Capella 3, Jose Carlos Machado1,5 and Nu ria Sala2,4 Ceu Figueiredo1,5, Carlos Alberto Gonza 1
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal Molecular Epidemiology Group, Translational Research Laboratory, Catalan Institute of Oncology (IDIBELL), Barcelona, Spain 3 Hereditary Cancer Program, Translational Research Laboratory, Catalan Institute of Oncology (ICO-IDIBELL), Barcelona, Spain 4 Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (IDIBELL), Barcelona, Spain 5 Faculty of Medicine of the University of Porto, Porto, Portugal 6 Department of Pathology, Hospital of S. Jo~ ao, Porto, Portugal 7 Institute of Public Health of the University of Porto (ISPUP), Porto, Portugal 8 Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 9 Institut National de la Sante et de la Recherche Medicale (INSERM), Centre for Research in Epidemiology and Population Health (CESP), U1018, Nutrition, Hormones and Women’s Health Team, Villejuif, France 10 Universite Paris-Sud, UMRS 1018, Villejuif, France 11 Institut Gustave Roussy (IGR), Villejuif, France 12 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands 13 Department of Gastroenterology and Hepatology, University Medical Centre, Utrecht, The Netherlands 14 School of Public Health, Imperial College London, St Mary’s Campus, Imperial College, London, United Kingdom 15 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: Fundacio ‘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 Generale de l’Education Nationale, Institut National de la Sante et de la Recherche Medicale (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 Andalucıa, 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: [email protected] or to Cecılia 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: [email protected]
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
Cancer Genetics
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IL1A genetic variants in gastric carcinoma
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Hellenic Health Foundation, Athens, Greece Department of Hygiene, Epidemiology and Medical Statistics, WHO Collaborating Center for Food and Nutrition Policies, University of Athens Medical School, Athens, Greece 18 Department of Community Medicine, Faculty of Health Sciences, University of Tromsï, Tromsï, Norway 19 Department of Research, Cancer Registry of Norway, Oslo, Norway 20 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden 21 Samfundet Folkh€alsan, Helsinki, Finland 22 HuGeF (Human Genetics Foundation), Molecular and Genetic Epidemiology Unit, Torino, Italy 23 Cancer Epidemiology Unit, University of Oxford, Oxford, United Kingdom 24 Diet, Genes and Environment, Danish Cancer Society Research Center, Copenhagen, Denmark 25 Navarre Public Health Institute, Pamplona, Spain 26 blica-CIBERESP), Madrid, Spain Consortium for Biomedical Research in Epidemiology and Public Health (CIBER Epidemiologıa y Salud Pu 27 International Agency for Research on Cancer (IARC-WHO), Lyon, France
Cancer Genetics
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The most studied genetic susceptibility factors involved in gastric carcinoma (GC) risk are polymorphisms in the inflammationlinked 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 disequilibrium (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.
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.
Gastric carcinoma (GC), although declining in incidence in the last decades, is still the fourth most common malignancy and the second leading cause of cancer-related death worldwide.1 GC is an aggressive disease often diagnosed at an advanced stage. Surgery is the only curative option but despite improvements in surgical and chemotherapy treatment approaches, GC remains a global public health problem with a 5-year overall survival rate of less than 25%.2,3 Gastric carcinomas 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 defined glandular 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 observed for the intestinal type and non cardia localisation, in Caucasian populations. The most recognised etiological risk factor for non cardia GC development is the infection by Helico-
bacter pylori,5 a stomach-colonising bacterium that chronically infects up to 50% of the world’s human population.6 The major consequence of infection is chronic inflammation of the gastric mucosa, which can progress through chronic atrophic gastritis, intestinal metaplasia and dysplasia towards GC.7 This course of events is, however, rare leading to a conspicuous difference between the number of infected individuals and those that proceed to develop GC (1–3%).8 The reasons for this difference are poorly understood but it is considered that a combination of bacterial virulence factors,9,10 dietary and lifestyle habits11–14 and host genetic factors,15–17 greatly influence the risk of GC. Amongst the genetic susceptibility factors involved in GC risk, the most studied are polymorphisms in the inflammation-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
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
with increased mucosal levels of IL1b,18 have been shown to be significantly associated with risk of GC,15,19 particularly in combination with H. pylori virulence factors.16,20 These association results have found important support in studies showing that increased expression of IL1b does favour development of GC in an in vivo animal model.21 Recent studies have shown similar associations both with GC22,23 and gastric precancerous lesions,24 but others have, however, not confirmed them.25–27 In the follow-up of these studies, several metaanalyses confirmed the lack of reproducibility of the association of IL1 polymorphisms and GC among different populations.28–30 These results can be explained, in part, by poor study design, e.g. small number of samples thus low statistical power, disease phenotype heterogeneity and population admixture, but also by the linkage disequilibrium (LD) degree in the IL1 genomic region. The IL1B and IL1RN genes are located on chromosome 2q14 clustered with other genes of the same family,31 with which they share strong LD. Despite the evidence pointing to the IL1 region, neither the molecular basis for the associations nor the definite causal variant, have been elucidated so far. Therefore, if there are indeed in this region one or more susceptibility loci for GC, these can be identified by LD mapping. Focusing on this hypothesis, we investigated the association of haplotype tag single nucleotide polymorphisms (SNPs) in the IL1 genomic region with GC, histological subtypes and tumour localisation in two case-control studies including different European populations. In this study, we report a novel association between the rs17042407 SNP, 16Kb upstream of the IL1A gene, as well as a number of high frequency haplotypes in the IL1A region, and risk for GC of the intestinal type observed in both studies and in the combined analysis.
Methods Study populations
The discovery stage included a Portuguese group (Table 1) of 358 incident cases of gastric adenocarcinoma admitted to surgery at Hospital of S. Jo~ao and the Portuguese Institute of Oncology, Porto, Portugal, and 1,485 healthy controls, as previously described.32,33 Briefly, cancer patients were eligible to participate if there was no previous cancer diagnosis, except for skin nonmelanoma, and no sub-total gastrectomy for benign conditions. Cancer was diagnosed according to the routine procedures and patients submitted to gastrectomy had the tumours classified as intestinal, diffuse, or unclassifiable according to Lauren; a single experienced pathologist reviewed all pathology reports, and slides were reassessed by three pathologists whenever routine information was considered insufficient or inconsistent. The source of controls was a representative sample of the non-institutionalised adult population of Porto, Portugal, assembled as part of an health and nutrition survey. Participants were recruited by random digit dialing, using households as the sampling frame, followed by simple random sampling to select one eligible person among permanent residents. C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
The replication stage included a cohort-nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC) study,34 comprising 365 GC cases and 1,284 controls (EPIC-EurGast study). The EPIC study includes 521,448 individuals recruited between 1992 and 2000 in 23 centres in 10 European countries. In the EPIC-EurGast study cases were subjects whose blood has been collected and were diagnosed with incident gastric adenocarcinoma (defined by code C16 of the International Classification of Diseases, 10th Revision) during the follow-up. An independent panel of pathologists confirmed and validated the diagnosis, tumour localisation and morphology.35 For each case, up to four control subjects were randomly selected among study members alive and free of cancer at the time of diagnosis, matched by centre, gender, age (62.5 years) and date of blood collection (645 days). All individuals provided informed consent and the study has been approved by ethical committees at the Hospital of S. Jo~ao, the Portuguese Institute of Oncology (Porto), the International Agency for Research on Cancer (Lyon) and in each of the EPIC recruitment centres. Sample sets characteristics are summarised in Table 1. The initial number of samples was larger both for the Porto (474 cases and 1,557 controls) and for the EPICEurGast (373 cases and 1,332 controls) studies. Following data filtering and quality control, 9.3% of the samples in the Porto study and 3.3% in the EPIC-EurGast study were excluded because their DNA was not amplified or had a genotyping call rate lower than 80%. SNP selection criteria
Genetic variation regarding the IL1 gene cluster (Fig. 1) in Caucasians was retrieved from HapMap (phase II CEU population, release #24 November 2008) and a list of SNPs was compiled from between 30 Kb downstream of IL1A and IL1RN genes (Fig. 1). Haplotype blocks were defined according to Gabriel et al.36 using the default parameters in Haploview v4.0.37 Haplotype tagging SNPs (tagSNPs) were selected using the Tagger algorithm implemented in Haploview and followed strict criteria relatively to the degree of LD between pairwise tagging SNPs (r2 0.8 and D0 5 1), the minor allele frequency (MAF) of the selected SNPs in Caucasians (0.01) and the haplotype frequency (0.01). A set of SNPs with functional potential in genes of the IL1 cluster that were not genotyped in HapMap, or that were not proxies of the selected tagSNPs, were also included. Following these criteria, 96 SNPs in a region spanning 420 Kb were selected for the Portuguese sample genotyping. The 96 tagSNPs were distributed along nine LD blocks (one block in the IL1A region, two in the IL1B region, four in the IL1F section and two in IL1RN region, Fig. 1). The SNPs selected for the replication stage using the EPIC-EurGast sample included a subset (43) of the 96 SNPs initially selected for the discovery analysis. These 43 SNPs were the ones included in the blocks which contained one or
Cancer Genetics
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IL1A genetic variants in gastric carcinoma
Table 1. Main characteristics of the Porto and EPIC-EurGast studies Cohort characteristics Porto
Controls
Cases
n 5 1,485
%
n 5 358
%
Men
576
38.8
203
56.7
Women
812
54.7
134
37.4
Unspecified
97
6.5
21
5.9
Gender
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
Cardia
36
10.0
Non cardia
244
68.2
78
21.8
Localisation
Unspecified
Cancer Genetics
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
Men
759
59.1
214
58.6
Women
525
40.9
151
41.4
Gender
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
Cardia
107
29.3
Non cardia
181
49.6
Unspecified
77
21.1
Localisation
SD: standard deviation.
C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V
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.
more statistically significant associations (p value
