This article has Open Peer Review reports available.
Inflammatory gene variants and the risk of biliary tract cancers and stones: a population-based study in China
© Castro et al.; licensee BioMed Central Ltd. 2012
Received: 3 May 2012
Accepted: 2 October 2012
Published: 11 October 2012
Genetic variants in inflammation-related genes have been associated with biliary stones and biliary tract cancers in previous studies.
To follow-up on these findings, we examined 35 single nucleotide polymorphism (SNPs) in 5 genes related to inflammation (IL8, NFKBIL, RNASEL, TNF, and VEGFA) in 456 participants with incident biliary tract cancer cases (262 gallbladder, 141 extrahepatic bile duct, 53 ampulla of Vater), 982 participants with biliary stones, and 860 healthy controls in a population–based case–control study in Shanghai, China.
Suggestive associations were observed for SNPs in VEGFA with biliary stones, IL8 with gallbladder and ampulla of Vater cancers, and RNASEL with ampulla of Vater cancer (false discovery rate≤0.2).
These findings provide additional support for the role of inflammation in biliary stones and biliary tract cancer risk and need further validation.
Biliary tract cancers, which include cancers of the gallbladder, extrahepatic bile duct, and ampulla of Vater, are rare, yet highly fatal malignancies . Elevated incidence rates have been reported in Native Americans and Hispanic immigrants in the United States, certain populations in Asia (including China, Korea, Japan and India), and in some parts of Eastern Europe and South America . Previous clinical and population-based studies have linked various inflammatory factors and mechanisms with the development of biliary tract cancers [3–6]. For example, gallstones, the predominant risk factor for biliary tract cancers, are thought to cause repeated irritation of the biliary tact mucosa, leading to chronic inflammation and eventual malignant changes .
Variants in genes involved in inflammatory pathways have been linked to biliary tract cancer and biliary stones. For example, studies in India, a high-risk population for gallbladder cancer, have shown that polymorphisms in IL1, TNF alpha, and CCR5 are associated with gallbladder cancer. Data from our population-based study of biliary tract cancers in Shanghai suggested that variants in PTGS2, IL8, IL8RB, RNASEL, NOS2 and VEGF were associated with biliary tract cancer and/or stones . To follow-up on these initial findings, in this analysis we examined an additional 28 SNPs in four of the candidate genes we previously identified (IL8, RNASEL, TNF, and VEGFA) in our population-based study in Shanghai. We also evaluated five SNPs in NFKBIL, a novel gene in the major histocompatibility complex (MHC) class I region that was not evaluated previously.
The study protocol was approved by the Institutional Review Boards of the USA National Cancer Institute and Shanghai Cancer Institute. All participants provided written informed consent. The methods of the Shanghai Biliary Tract Cancer Study have been reported . Briefly, cases were residents of urban Shanghai between the ages of 40 and 74. They were diagnosed with biliary tract cancer (ICD-9 156) between 1997 and 2000, and identified at 42 collaborating hospitals. Biliary stone cases without a history of cancer were ascertained from the same hospitals and matched to index cancer cases on hospital, gender, and age (within 5 years). Biliary tract cancer and biliary stone cases were confirmed by an expert panel review of clinical and pathology records. Population controls were healthy subjects without a history of cancer selected from the Shanghai Resident Registry and frequency-matched to cancer cases in a 1-to-1 ratio by gender and age (5-yr intervals). Biliary stone status among population controls was assessed by abdominal ultrasound or self-reported history. Cases were interviewed within three weeks of diagnosis. The interview response rate was over 95% for cases and 82% for controls.
Gene and SNP selection and genotyping
Selected inflammatory genes and their association with biliary stones and cancer in the Shanghai population
Gene symbol (name)
Examined in the current study
Previously reported variants1
High linkage disequilibrium
IL8 (interleukin 8)
rs12506479, rs10805066 rs2227543, rs7657356
rs4073, rs2227307, rs2227306
rs2227307, rs4073, rs2227543 and rs2227306 (r2>0.8)
NFKBIL (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1)
Encodes divergent member of I-kappa-B family proteins
rs2230365, rs2239707, rs2857605, rs928815, rs13215091
RNASEL (ribonuclease L)
Encodes an interferon-inducible ribonuclease
rs11807829, rs474939, rs533259, rs682585, rs627839, rs627928, rs635261, rs672527, rs579006
rs11807829 and rs11072 (r2=0.98), rs635261 and rs627839 (r2=0.8), rs579006 with rs486907 (r2=0.95)
TNF (tumor necrosis factor)
Inflammatory cytokine that promotes hyperlipidemia by increasing hepatic triglyceride production and decreasing clearance
rs2857708, rs769177, rs769178
rs1800750, rs1800629, rs361525, rs673, rs1799724, rs1800630, rs1800610, rs1799964
rs769178 and rs1799724 (r2=0.95)
VEGFA (vascular endothelial growth factor A)
Vascular permeability, angiogenesis, vasculogenesis, cell growth, cell migration, apoptosis
rs25648, rs3025000, rs3025033, rs3025035, rs833052, rs866236, rs9367173, rs9394963, rs998584, rs10434, rs6905288, rs6899540, rs4714696, rs833070
(rs3025033 and rs3025039 (r2=0.93)
The final analysis included subjects who completed the interview and for whom we had DNA samples and genotyping results. A total of 456 biliary tract cancer cases (262 gallbladder, 141 extrahepatic bile duct and 53 ampulla of Vater), 982 biliary stone cases (252 bile duct, 730 gallstone), and 860 controls, were included. Hardy-Weinberg equilibrium of allele frequencies for all SNPs was tested among controls using the chi-square test. Unconditional logistic regression was conducted to estimate odds ratios (OR) and 95% confidence intervals (CI) for the associations between SNPs (using additive and co-dominant genetic models) and biliary stone and cancer risks. Biliary stone cases were compared with healthy controls without stones; gallbladder cancer cases were compared with population controls without a history of cholecystectomy; and bile duct and ampulla of Vater cancer cases were compared with all population controls. Statistical interactions between SNPs and biliary stones were examined using the likelihood ratio test in a logistic regression model. Risk estimates were adjusted for age (categorical) and sex, and further evaluated for other potential confounding factors. To account for multiple comparisons, we used the Benjamini-Hochberg method to control for the false discovery rate (FDR) , considering a FDR≤0.2 being noteworthy, as per previous studies [15, 16]. We also calculated a gene-based summary p-value using the minP test  for each of the five genes using the SNPs examined in this analysis and the SNPs from our previous study .
Odds Ratios (ORs) and 95% Confidence Intervals (CIs) for the association between inflammatory variants and biliary stones, gallbladder and ampulla of Vater cancers in the Shanghai population
OR2 (95% CI)
VEGFA 9921bp 3' of STP A>G
VEGFA 6507bp 3' of STP G>A
Ampulla of Vater Cancer
Following-up on findings suggesting an association between inflammation-related genes and biliary stones and cancer , in this analysis we expanded the gene coverage of four previously identified genes and examined another gene not previously studied in the Shanghai Biliary Tract Cancer Study. We observed suggestive associations for SNPs in VEGFA with biliary stones, RNASEL with ampulla of Vater cancer, and IL8 with gallbladder and ampulla of Vater cancers after correcting for multiple comparisons (FDR≤0.2).
Genetic susceptibility to biliary stones was linked to two SNPs in VEGFA. These two SNPs, rs9367173 and rs6905288, are located downstream of VEGFA and are neither in linkage disequilibrium (LD) with each other, nor in LD with the other VEGFA SNPs we examined. VEGFA is a signal protein that is fundamental in vascular permeability and angiogenesis . Thus, the role of VEGFA in gallstones susceptibility could be attributed to the process of blood vascularization during acute and/or chronic inflammation; however, the functional effects of these variants are unknown. To our knowledge, this study is the first to report an association between VEGFA and biliary stones. In our previous study, VEGFA was not associated with biliary stones, but rather VEGFA rs3025039 was associated with gallbladder cancer. This variant is not in LD with the two VEGFA SNPs we identified in this analysis.
We also found an increased risk of ampulla of Vater cancer with RNASEL rs672527, which is located in an intronic region of the gene. RNASEL is a key component of the innate immune system and participates in the process of apoptosis , but the functional effect of rs672527 is unknown. Other polymorphisms in the RNASEL gene have been associated with an increased risk of such cancers as prostate, head and neck, uterine cervix and breast [20, 21]; however, this is the first report to our knowledge of an association of rs672527 with cancer. We have previously shown that another RNASEL SNP (rs486907) was associated with biliary stones. These two SNPs are not in LD.
IL8 rs10805066, which was linked with increased risks of ampulla of Vater and gallbladder cancers, is located outside the promoter region of the gene. No functional effects of the variant have been reported. IL8 chemokine has been recognized as a potent mitogenic/angiogenic and inflammatory factor , which could support its participation in biliary tract cancer development. In our previous study, other variants in IL8 (rs4073, rs2227307 and rs27306, all in LD) were associated with bile duct stones . These three SNPs are not in LD with rs10805066.
The associations between RNASEL and IL8 variants with biliary tract cancers were independent of biliary stones. In our previous study, some, but not all genetic variants interacted significantly with biliary stone status to effect biliary cancer risk (Hsing, 2008). It has been suggested that most of the inflammatory processes of biliary tract cancer are linked to biliary stones; however, not all biliary cancer cases have prior stones, and other inflammatory conditions such as cholecystitis, history of gastric or duodenal ulcers have been reported to play a role . The cascade of inflammatory events in relation to the genes detected in this or previous studies, with or without the presence of biliary stones is unclear. In general, in both humans and mouse models, inflammatory processes, such as edema of the gallbladder, increased organ wall thickness, inflammatory infiltrates (the presence of inflammatory cells), and increases in transforming growth factor (TGF)-β production ) lead to chronic inflammation [25, 26], which may eventually lead to carcinogenesis.
Although we extended the analysis of common genetic variants to approximately 80% coverage for VEGFA (chr6: 43827369–43870265), 90% for RNASEL (chr1: 180805238–180826133) and 75% for IL8 (chr4: 74831698–74808648), none of the overall effects for each of the tested genes resulted in significant associations at the 0.05 level with either biliary stones or cancer. Our findings from this analysis, together with our findings from our previous study conducted in the same population do not strongly support an association between a particular gene and a particular biliary disease. It may be possible that each of the current and previously examined SNPs have independent effects on stones or biliary tract susceptibility, which is supported by the absence of LD (D’ or r2) between the SNPs. In addition, these variants may influence expression of different genes, which it is supported by the findings of recent follow-up of genome-wide association studies and the rare candidate genes [27, 28]; however, we did not find evidence of regulatory consequences for the associated variants.
Strengths of this study include the population-based design, the high case ascertainment and response rates, and the detailed review of pathology and clinical data to confirm the diagnosis of cancer cases. The use of ultrasound among controls also minimized misclassification of gallstones. Although, this study is the largest population-based study of biliary tract cancer to date, we have limited statistical power to detect a modest association, in particular for cancer of the ampulla of Vater.
In conclusion, after evaluating a new set of SNPs in a priori selected inflammatory genes, our results provide additional data to support that genetic susceptibility related to inflammatory mechanisms play a role in biliary tract cancer and stones. Further validation of these findings in other populations with full coverage of the genetic variability of each of the genes would help clarify the role of these genes in biliary tract carcinogenesis.
We thank the collaborating surgeons and pathologists in Shanghai for assistance in patient recruitment and pathology review; Chia–Rong Cheng, Lu Sun, and Kai Wu of the Shanghai Cancer Institute for coordinating data and specimen collection; and Shelley Niwa and Ann Truelove of Westat for support with study and data management. The study was funded by the Intramural Research Program of the National Institute of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics, USA.
- Randi G, Malvezzi M, Levi F, Ferlay J, Negri E, Franceschi S, La Vecchia C: Epidemiology of biliary tract cancers: an update. Ann Oncol. 2009, 20 (1): 146-159.View ArticlePubMedGoogle Scholar
- Hsing A, Rashid A, Devesa S, Fraumeni JJ: Biliary Tract Cancer. Cancer Epidemiology and Prevention. Edited by: Jr DSJF. 2006, New York: Oxford University Press, Inc, 787-800. Third EditionView ArticleGoogle Scholar
- Hsing AW, Gao YT, Han TQ, Rashid A, Sakoda LC, Wang BS, Shen MC, Zhang BH, Niwa S, Chen J, et al: Gallstones and the risk of biliary tract cancer: a population-based study in China. Br J Cancer. 2007, 97 (11): 1577-1582. 10.1038/sj.bjc.6604047.View ArticlePubMedPubMed CentralGoogle Scholar
- Hsing AW, Gao YT, McGlynn KA, Niwa S, Zhang M, Han TQ, Wang BS, Chen J, Sakoda LC, Shen MC, et al: Biliary tract cancer and stones in relation to chronic liver conditions: A population-based study in Shanghai, China. Int J Cancer. 2007, 120 (9): 1981-1985. 10.1002/ijc.22375.View ArticlePubMedGoogle Scholar
- Hsing AW, Zhang M, Rashid A, McGlynn KA, Wang BS, Niwa S, Ortiz-Conde BA, Goedert JJ, Fraumeni JF, O'Brien TR, et al: Hepatitis B and C virus infection and the risk of biliary tract cancer: a population-based study in China. Int J Cancer. 2008, 122 (8): 1849-1853.View ArticlePubMedGoogle Scholar
- Liu E, Sakoda LC, Gao YT, Rashid A, Shen MC, Wang BS, Deng J, Han TQ, Zhang BH, Fraumeni JF, et al: Aspirin use and risk of biliary tract cancer: a population-based study in Shanghai, China. Cancer Epidemiol Biomarkers Prev. 2005, 14 (5): 1315-1318. 10.1158/1055-9965.EPI-05-0032.View ArticlePubMedGoogle Scholar
- Vishnoi M, Pandey SN, Choudhuri G, Mittal B: IL-1 gene polymorphisms and genetic susceptibility of gallbladder cancer in a north Indian population. Cancer Genet Cytogenet. 2008, 186 (2): 63-68. 10.1016/j.cancergencyto.2008.05.004.View ArticlePubMedGoogle Scholar
- Vishnoi M, Pandey SN, Choudhury G, Kumar A, Modi DR, Mittal B: Do TNFA −308 G/A and IL6–174 G/C gene polymorphisms modulate risk of gallbladder cancer in the north Indian population?. Asian Pac J Cancer Prev. 2007, 8 (4): 567-572.PubMedGoogle Scholar
- Srivastava A, Pandey SN, Choudhuri G, Mittal B: CCR5 Delta32 polymorphism: associated with gallbladder cancer susceptibility. Scand J Immunol. 2008, 67 (5): 516-522. 10.1111/j.1365-3083.2008.02097.x.View ArticlePubMedGoogle Scholar
- Sakoda LC, Gao YT, Chen BE, Chen J, Rosenberg PS, Rashid A, Deng J, Shen MC, Wang BS, Han TQ, et al: Prostaglandin-endoperoxide synthase 2 (PTGS2) gene polymorphisms and risk of biliary tract cancer and gallstones: a population-based study in Shanghai, China. Carcinogenesis. 2006, 27 (6): 1251-1256. 10.1093/carcin/bgi314.View ArticlePubMedGoogle Scholar
- Hsing AW, Sakoda LC, Rashid A, Andreotti G, Chen J, Wang BS, Shen MC, Chen BE, Rosenberg PS, Zhang M, et al: Variants in inflammation genes and the risk of biliary tract cancers and stones: a population-based study in China. Cancer Res. 2008, 68 (15): 6442-6452. 10.1158/0008-5472.CAN-08-0444.View ArticlePubMedPubMed CentralGoogle Scholar
- Ghosh S, May MJ, Kopp EB: NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol. 1998, 16: 225-260. 10.1146/annurev.immunol.16.1.225.View ArticlePubMedGoogle Scholar
- Grivennikov SI, Greten FR, Karin M: Immunity, inflammation, and cancer. Cell. 2010, 140 (6): 883-899. 10.1016/j.cell.2010.01.025.View ArticlePubMedPubMed CentralGoogle Scholar
- Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I: Controlling the false discovery rate in behavior genetics research. Behav Brain Res. 2001, 125 (1–2): 279-284.View ArticlePubMedGoogle Scholar
- Han S, Lee KM, Park SK, Lee JE, Ahn HS, Shin HY, Kang HJ, Koo HH, Seo JJ, Choi JE, et al: Genome-wide association study of childhood acute lymphoblastic leukemia in Korea. Leuk Res. 2010, 34 (10): 1271-1274. 10.1016/j.leukres.2010.02.001.View ArticlePubMedGoogle Scholar
- Barry KH, Koutros S, Andreotti G, Sandler DP, Burdette LA, Yeager M, Beane Freeman LE, Lubin JH, Ma X, Zheng T, et al: Genetic variation in nucleotide excision repair pathway genes, pesticide exposure and prostate cancer risk. Carcinogenesis. 2011, 33 (2): 331-337.View ArticlePubMedPubMed CentralGoogle Scholar
- Westfall PH, Young SS: Resampling-Based Multiple Testing: Examples and Methods for p-Value Adjustment. 1993, New York: John Wiley & SonsGoogle Scholar
- Kerbel RS: Tumor angiogenesis. N Engl J Med. 2008, 358 (19): 2039-2049. 10.1056/NEJMra0706596.View ArticlePubMedPubMed CentralGoogle Scholar
- Castelli J, Wood KA, Youle RJ: The 2-5A system in viral infection and apoptosis. Biomed Pharmacother. 1998, 52 (9): 386-390. 10.1016/S0753-3322(99)80006-7.View ArticlePubMedGoogle Scholar
- Wiklund F, Jonsson BA, Brookes AJ, Stromqvist L, Adolfsson J, Emanuelsson M, Adami HO, Augustsson-Balter K, Gronberg H: Genetic analysis of the RNASEL gene in hereditary, familial, and sporadic prostate cancer. Clin Cancer Res. 2004, 10 (21): 7150-7156. 10.1158/1078-0432.CCR-04-0982.View ArticlePubMedGoogle Scholar
- Madsen BE, Ramos EM, Boulard M, Duda K, Overgaard J, Nordsmark M, Wiuf C, Hansen LL: Germline mutation in RNASEL predicts increased risk of head and neck, uterine cervix and breast cancer. PLoS One. 2008, 3 (6): e2492-10.1371/journal.pone.0002492.View ArticlePubMedPubMed CentralGoogle Scholar
- Xie K: Interleukin-8 and human cancer biology. Cytokine Growth Factor Rev. 2001, 12 (4): 375-391. 10.1016/S1359-6101(01)00016-8.View ArticlePubMedGoogle Scholar
- Andreotti G, Liu E, Gao YT, Safaeian M, Rashid A, Shen MC, Wang BS, Deng J, Han TQ, Zhang BH, et al: Medical history and the risk of biliary tract cancers in Shanghai, China: implications for a role of inflammation. Cancer Causes Control. 2011, 22 (9): 1289-1296. 10.1007/s10552-011-9802-z.View ArticlePubMedGoogle Scholar
- Maurer KJ, Carey MC, Fox JG: Roles of infection, inflammation, and the immune system in cholesterol gallstone formation. Gastroenterology. 2009, 136 (2): 425-440. 10.1053/j.gastro.2008.12.031.View ArticlePubMedGoogle Scholar
- Tazuma S, Kajiyama G: Carcinogenesis of malignant lesions of the gall bladder. The impact of chronic inflammation and gallstones. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie. 2001, 386 (3): 224-229. 10.1007/s004230100220.View ArticlePubMedGoogle Scholar
- Spirli C, Nathanson MH, Fiorotto R, Duner E, Denson LA, Sanz JM, Di Virgilio F, Okolicsanyi L, Casagrande F, Strazzabosco M: Proinflammatory cytokines inhibit secretion in rat bile duct epithelium. Gastroenterology. 2001, 121 (1): 156-169. 10.1053/gast.2001.25516.View ArticlePubMedGoogle Scholar
- Rotival M, Zeller T, Wild PS, Maouche S, Szymczak S, Schillert A, Castagne R, Deiseroth A, Proust C, Brocheton J, et al: Integrating genome-wide genetic variations and monocyte expression data reveals trans-regulated gene modules in humans. PLoS Genet. 2011, 7 (12): e1002367-10.1371/journal.pgen.1002367.View ArticlePubMedPubMed CentralGoogle Scholar
- Freedman ML, Monteiro AN, Gayther SA, Coetzee GA, Risch A, Plass C, Casey G, De Biasi M, Carlson C, Duggan D, et al: Principles for the post-GWAS functional characterization of cancer risk loci. Nat Genet. 2011, 43 (6): 513-518. 10.1038/ng.840.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/468/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.