Background
Biliary tract cancers, which include cancers of the gallbladder, extrahepatic bile duct, and ampulla of Vater, are rare, yet highly fatal malignancies [
1]. 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 [
2]. 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 [
2].
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[
7],
TNF alpha[
8], and
CCR5[
9] are associated with gallbladder cancer. Data from our population-based study of biliary tract cancers in Shanghai suggested that variants in
PTGS2[
10],
IL8,
IL8RB,
RNASEL,
NOS2 and
VEGF were associated with biliary tract cancer and/or stones [
11]. 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.
Results
The distribution of selected characteristics among cases and controls used in the current analysis (Additional file
1: Table S1) is similar to the distributions reported previously (11). Of the 35 SNPs examined, statistically significant (p<0.05) associations were seen for
VEGFA rs9367173 and rs6905288 with biliary stones,
IL8 rs10805066 with gallbladder and ampulla of Vater cancers, and
RNASEL rs672527 with ampulla of Vater cancer (Table
2). Individual SNP-associations are shown in Additional file
2: Table S2. Adjustment for biliary stones did not change the effect of these SNPs on cancer, except for
IL8 rs10805066 and gallbladder cancer, which was no longer statistically significant (data not shown). Stratifying by biliary stone status did not identify additional associations. The associations noted above were robust to multiple comparisons at the FDR p≤0.2 level, with FDR p-values ranging between 0.09 and 0.2. Including SNPs from this analysis and our previous study [
11] in the minP test did not result in additional significant gene-based associations (Additional file
3: Table S3).
Table 2
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
Biliary stones
| | | | | | |
VEGFA 9921bp 3' of STP A>G | rs9367173 | | | | | |
GG | | 539 (82.9) | 837 (86.8) | 1.0 | | |
AG | | 102 (15.7) | 126 (13.1) | 0.8 (0.60-1.07) | | |
AA | | 9 (1.4) | 6 (0.6) | 0.4 (0.14-1.15) | | |
trend
3
| | | | |
0.03
|
0.2
|
AG+AA | | 111 (17.1) | 132 (13.7) | 0.77 (0.58-1.01) | | |
VEGFA 6507bp 3' of STP G>A | rs6905288 | | | | | |
AA | | 357 (55.3) | 499 (51.8) | 1.0 | | |
AG | | 249 (38.5) | 386 (40.0) | 1.14 (0.92-1.41) | | |
GG | | 40 (6.2) | 81 (8.4) | 1.5 (1.00-2.25) | | |
trend
3
| | | | |
0.04
|
0.2
|
AG+GG | | 289 (44.7) | 467 (48.4) | 1.19 (0.97-1.46) | | |
Gallbladder cancer
|
IL8 -13985C>G | rs10805066 | | | | | |
CC | | 647 (81.1) | 187 (73.0) | 1.0 | | |
CG | | 140 (17.5) | 68 (26.6) | 1.67 (1.20-2.34) | | |
GG | | 11 (1.4) | 1 (0.4) | | | |
trend
3
| | | | |
0.03
|
0.2
|
CG+GG | | 151 (18.9) | 69 (27.0) | 1.57 (1.13-2.18) | | |
Ampulla of Vater Cancer
|
RNASEL IVS5+170G>A | rs672527 | | | | | |
GG | | 712 (83.6) | 39 (73.6) | 1.0 | | |
AG | | 133 (15.6) | 11 (20.8) | 1.54 (0.77-3.10) | | |
AA | | 7 (0.8) | 3 (5.7) | 8.40 (2.06-34.14) | | |
trend
3
| | | | |
0.01
|
0.09
|
AG+AA | | 140 (16.4) | 14 (26.4) | 1.88 (0.99-3.57) | | |
IL8 -13985C>G | rs10805066 | | | | | |
CC | | 685 (80.4) | 37 (69.8) | 1.0 | | |
CG | | 156 (18.3) | 13 (24.5) | 1.59 (0.82-3.07) | | |
GG | | 11 (1.3) | 3 (5.7) | 5.6 (1.47-21.42) | | |
trend
3
| | | | |
0.02
|
0.1
|
CG+GG | | 167 (19.6) | 16 (30.2) | 1.83 (0.99-3.39) | | |
Discussion
Following-up on findings suggesting an association between inflammation-related genes and biliary stones and cancer [
11], 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 [
18]. 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 [
19], 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 [
22], 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 [
11]. 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 [
23]. 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 [
24]) 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 r
2) 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.
Competing interests
The author declare that they have no competing interests.
Authors’ contributions
Design, coordination, and execution of the study (A.W.H, G.A. and L.W.C.). Design and conduct of the Population-based case–control study (A.W.H., Y.T.G., A.R., M.C.S., B.S.W., T.Q.H., and B.H.Z.). Contractor (S.N.). Genotyping (S.C.). Data analyses (F.A.C., G.A., H.Z. and K.Y.). First draft of manuscript (F.A.C., G.A., J.K. and A.W.H.). All authors read and approved the final manuscript.