hepatocellular carcinoma ( hcc ) is a primary malignancy of the liver ; it is a major threat to human health and has poor prognosis . hcc is the fifth and seventh most frequent cancer in men and women worldwide , respectively , and is the third and second leading cause of cancer related deaths worldwide and in taiwan , respectively 1 , 2 . major risk factors for hcc include infections of hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) , exposure to substances ( e.g. , alphatoxins ) that are toxic to the liver , and alcohol and tobacco consumption . moreover , immune system - mediated chronic inflammation of the liver can lead to hcc 3 - 5 . in addition , various inflammatory cytokines , such as interleukins ( il)-1 , -18 , -6 3 - 5 , and -17 3 , 6 , 7 , participate in chronic hepatic inflammation , leading to tumorigenesis . 8 reported that the serum level of il-18 is a useful biological marker of tumor invasiveness and an independent prognostic factor for survival among patients with hcc . shiraki et al . 9 reported that the serum level of il-18 increased in patients with hcv - related stage iv hcc compared with patients with earlier - stage hcc . although genetic predisposition is one of the factors critical for hcc progression , few studies have focused on il-18 single nucleotide polymorphisms ( snps ) in patients with hcc . moreover , research on the combined effect of il-18 snps and hbv infection on the risk and clinicopathologic development of hcc remain scant . il-18 , an 18-kda cytokine , originally known as interferon- ( ifn-)-inducing factor , shares structural and functional properties with il-1 . this cytokine is mainly produced by activated macrophages and kupffer cells and can promote ifn- production 10 , 11 . il-18 is located on chromosome 11q23 . 1 and contains six exons spanning over 20.8 kb and five introns 12 , 13 . several snps in the promoter region and two polymorphisms in the 5-nontranslated region of il-18 have been identified 12 , 14 - 16 . among these , two functional polymorphisms in the promoter region at loci -607a / c and -137g / c are the most studied . the structure of il-18 promoter suggests that transcription factor pu.1 might be a critical regulator of its activity 17 . serum levels of il-18 may influence the risk of coronary artery disease and asthma and is a crucial event in oral carcinoma cells for oncogenesis 10 , 18 . 17 revealed the influence of serum levels of il-18 on the risk of diseases and reported that variations in il-18 may influence il-18 synthesis . 19 reported that improper synthesis of il-18 contributes to cancer pathogenesis and may influence the clinical outcome in patients . in addition , snp is the most common type of dna sequence variation , influencing the occurrence and progression of gene - related hepatocarcinogenesis . our previous studies reported that snps in cd44 , fibroblast growth factor receptor 4 , intercellular adhesion molecule-1 , metallothionein-1 , reversion - inducing - cysteine - rich protein with kazal motifs , and ccr2 genes may predict the risk of hcc 20 - 25 . however , few studies have focused on il-18 snps in patients with hcc who are hbv positive . therefore , the aim of this study was to identify il-18 polymorphisms specifically in patients showing hbv - related susceptibility and clinicopathological status of hcc . in this study , we recruited 342 patients with hcc at the chung shan medical university hospital , taiwan . a diagnosis of hcc was made according to the criteria specified in the national guidelines for hcc detection . all 559 control subjects were recruited at the same hospital and these control groups had neither self - reported history of cancer of any sites . personal information and characteristics collected from the study subjects using interviewer - administered questionnaires contained questions involving demographic characteristics . the blood samples which obtained from the controls and hcc patients were stored in edta tubes , centrifuged immediately and stored at -80c . the institutional review board of chung shan medical university hospital approved this study ( csmuh no : cs15099 ) , and informed written consent was obtained from each participant . a total of two snps in il-18 were selected from the international hapmap project data for this study . we included -607a / c ( rs1946518 ) and il-18 -137g / c ( rs187238 ) in the promoter region which were selected in this study since these 2 snps were found to modify the binding affinities 17 . genomic dna was extracted from edta anti - coagulated venous blood using a qiaamp dna blood mini kit based on the manufacturer 's protocol as described in detail previously 26 . allelic discrimination of -607a / c ( rs1946518 ) and -137g / c ( rs187238 ) polymorphisms of the il-18 gene was assessed with the abi stepone real - time pcr system ( applied biosystems , foster city , ca , usa ) and analyzed using sds vers . 3.0 software ( applied biosystems ) , with the taqman assay 18 . the distributions of demographic characteristics and genotype frequencies for different genotypes between the study participants and controls were analyzed using the chi - square test for certain categories of variables . student 's t - test was used to evaluate the differences in the laboratory findings between the 2 groups . the odds ratios ( ors ) and their 95% confidence intervals ( cis ) of the association between the genotype frequencies and hcc were estimated using multiple logistic regression models by controlling for covariates . in this study , we recruited 342 patients with hcc at the chung shan medical university hospital , taiwan . a diagnosis of hcc was made according to the criteria specified in the national guidelines for hcc detection . all 559 control subjects were recruited at the same hospital and these control groups had neither self - reported history of cancer of any sites . personal information and characteristics collected from the study subjects using interviewer - administered questionnaires contained questions involving demographic characteristics . the blood samples which obtained from the controls and hcc patients were stored in edta tubes , centrifuged immediately and stored at -80c . the institutional review board of chung shan medical university hospital approved this study ( csmuh no : cs15099 ) , and informed written consent was obtained from each participant . a total of two snps in il-18 were selected from the international hapmap project data for this study . we included -607a / c ( rs1946518 ) and il-18 -137g / c ( rs187238 ) in the promoter region which were selected in this study since these 2 snps were found to modify the binding affinities 17 . genomic dna was extracted from edta anti - coagulated venous blood using a qiaamp dna blood mini kit based on the manufacturer 's protocol as described in detail previously 26 . allelic discrimination of -607a / c ( rs1946518 ) and -137g / c ( rs187238 ) polymorphisms of the il-18 gene was assessed with the abi stepone real - time pcr system ( applied biosystems , foster city , ca , usa ) and analyzed using sds vers . 3.0 software ( applied biosystems ) , with the taqman assay 18 . the distributions of demographic characteristics and genotype frequencies for different genotypes between the study participants and controls were analyzed using the chi - square test for certain categories of variables . student 's t - test was used to evaluate the differences in the laboratory findings between the 2 groups . the odds ratios ( ors ) and their 95% confidence intervals ( cis ) of the association between the genotype frequencies and hcc were estimated using multiple logistic regression models by controlling for covariates . demographic characteristics and clinical parameters of the 559 healthy controls and 342 patients with hcc are summarized in table 1 . the mean age ( sd ) in the control and patient groups was 51.86  14.71 and 62.96  11.67 years , respectively . the genotypic distributions and associations between patients with hcc and healthy controls carrying the il-18 polymorphisms , -607a / c ( rs1946518 ) and -137g / c ( rs187238 ) , are listed in table 2 . in our recruited control group , the frequencies of il-18 -607a / c ( 2 value : 0.080 , p=0.777 ) and -137g / c ( 2 value : 0.803 , p=0.370 ) were in hardy - weinberg equilibrium , respectively . after adjusting for the covariates of age , sex , and alcohol consumption , the il-18 -137g / c ( rs187238 ) polymorphism genotypes , gc+cc and gg , in the healthy controls and patients with hcc differed significantly ( adjusted odds ratio [ aor ] = 1.987 ; 95 % confidence interval [ ci ] = 1.301 - 3.032 ; . table 3 shows the distribution frequency of the il-18 -137 polymorphism among the 342 patients with hcc along with the clinical status . the patients with hcc were evaluated to understand the influence of il-18 -137 polymorphism genotypes , gc+cc and gg , on the clinical tnm stage , primary tumor size , lymph node involvement , distant metastasis , vascular invasion , child - pugh grade , presence of an hbv or hcv infection , and liver cirrhosis . no significant differences were observed in the influence of the aforementioned il-18 -137 polymorphism genotypes on the clinical tnm stage and clinical variables , except for hbv infection ( gc+cc vs gg ; aor = 1.668 ; 95 % ci = 1.001 - 2.786 ; p < 0.05 ) . in addition , we explored the potential association between the il-18 -137 polymorphism genotypes , gc+cc and gg , in the patients with hcc who were hbv positive ( table 4 ) . among the 142 patients with hcc who were hbv positive , significant vascular invasion was observed in those carrying il-18 -137 polymorphism genotype gc+cc compared with those carrying gg genotype ( aor = 2.825 ; 95% ci = 1.168 - 6.833 ; p = 0.018 ) . as shown in table 5 , the level of -fetoprotein differed significantly ( p < 0.05 ) in the patients with hcc under the influence of il-18 -137g / c polymorphism genotypes gg and gc+cc . the other clinicopathological statuses for hcc , including alt , ast , and ast / alt ratio , showed no significant differences . compared with healthy people , the serum level of il-18 in patients with hcc is controversial . 27 reported that il-18 levels in patients with hcc were significantly higher than those in healthy controls . however , bao et al . 12 showed that the level of serum il-18 were significantly lower in patients with hcc than in healthy people . in addition , they reported that the il-18 -137g / c polymorphism was significantly correlated with the risk of hcc ; however , il-18 snps were not associated with the serum concentration of il-18 . thus , we suggested that serum levels of il-18 may be distinct in various diseases and cancer stages . in addition , we revealed that the genetic of patients profile is one of the key factors for the serum level of il-18 to regulate the development of hcc . in our study , the presence of il-18 -607a / c ( rs1946518 ) and -137g / c ( rs187238 ) polymorphisms was analyzed in 559 healthy controls and 342 patients with hcc . the number of patients with hcc carrying the il-18 -137g / c ( rs187238 ) polymorphism genotypes gc+cc and gg differed significantly ( p < 0.001 ) . our results confirmed that patients carrying the il-18 -137g / c polymorphism genotype gc+cc have a high risk of hcc , similar to the results obtained by bao et al 12 , reporting a significant association between the il-18 -137g / c polymorphism and the risk of hcc , where a high frequency of the g allele was associated with an increased risk of hcc . no association between the il-18 -137 polymorphism genotypes gc+cc and gg and the aforementioned clinical parameters except hbv infection was observed in our study . we found that among the patients with hcc who were hbv positive , the risk of hcc was higher in those who were carrying the il-18 -137 polymorphism genotype gc+cc ( 39/76 = 51.3% ) than those who were carrying the gg genotype ( 103/266 = 38.7% ) with an aor of 1.668 ( p < 0.05 ) . this result is similar to the results of a study performed by bouzgarrou et al . 28 , reporting that a polymorphism in c allele at position -607 ( cc + c / a ) was associated with an increased risk of cirrhosis and hcc in patients who were hbv positive . because hbv infection can lead to severe liver diseases , such as chronic hepatitis , cirrhosis , and hcc 29 , 30 , the natural course of hbv infection is probably associated with host immune factors , and il-18 is crucial in immune defense . 31 showed that the -148c , + 8925 g , and + 13925c alleles of il-18 are associated with the development of hcc , and the -148g > c snp was functionally essential in determining the disease outcome . however , the association between il-18 snp and hbv - related hcc has been studies seldom . 32 reported that polymorphisms in the il-18 promoter region at positions -607 and -137 combining with hbv infection can be associated with various outcomes , including spontaneous recovery , chronic hepatitis , liver cirrhosis , and hcc . therefore , as reported by thio et al . 33 , the genetic background of an individual might influence the clinical outcome of hbv infection . interestingly , in a meta - analysis , yang et al . suggested that il-18 - 137g / c polymorphism , but not -607c / a polymorphism , was associated with chronic hepatitis c virus infections 34 . coordinating innate and adaptive humoral and cell - mediated immunities , various cytokines , such as il-18 , and associations with snps may reveal some clues in some spontaneous clearance , while others develop cirrhosis and liver cancer ; these results can provide new clues to explain the mechanism of hbv infection 35 , 36 . an advanced statistics on the 142 patients with hcc who were hbv positive revealed that the gc genotype and c allele of the il-18 -137 snp were associated with a significantly increased risk of vascular invasion compared with the gg genotype and g allele . 37 reported that the polymorphisms at -607 and -137 in the il-18 promoter region may affect the development and progression of hbv - related liver diseases ; we obtained similar results with an aor of 2.825 ( p = 0.018 ) . 31 confirmed that the -148c , + 8925 g , and + 13925c alleles of il-18 in patients with hbv infection are associated with the presence of hcc , and the 148g > c snp is functionally crucial in determining the disease outcome . 38 reported that il-18 and il-18r polymorphisms may contribute to the development and lymph node metastasis of papillary thyroid carcinoma . we revealed that the il-18 -137c polymorphism can be a crucial factor for the risk of hbv - related hcc outcome of vascular invasion . moreover , il-18 and il-18r polymorphisms in various cancers have different influence on the outcomes and specific characteristic phenomena . 39 reported no significant difference among patients with hcc and no correlation between cytokines and other evaluated variables , such as hcv infection , rna , -fetoprotein , genotype , and demographic characteristics of patients with hcc . our results suggest that the clinicopathological statuses for hcc , including alt , ast , and ast / alt ratio , showed no significant differences except the -fetoprotein level . the level of -fetoprotein was significantly associated with the presence of the il-18 -137g / c polymorphism genotypes gg and gc+cc in the patients with hcc ( p < 0.05 ) . therefore , the il-18 -137g / c polymorphism might be crucial for the pathogenesis of hcc , and the gc+cc genotype in the il-18 -137 snp combining with -fetoprotein levels can be utilized as markers for an early diagnosis of hcc . in conclusion , our results confirmed that the il-18 -137g / c ( rs187238 ) snp can be a factor that increases the risk of hcc . moreover , the association of the gc genotype of the il-18 -137 snp with hbv - related hcc can increase the risk of vascular invasion . in addition , the -fetoprotein level combined with the il-18 -137 snp can be utilized as a marker for the early diagnosis of hcc .
we investigated the association between interleukin-18 ( il-18 ) polymorphisms and the susceptibility and clinicopathological state of hepatocellular carcinoma ( hcc ) . in total , 901 participants , including 559 healthy controls and 342 patients with hcc , were recruited . 
 the allelic discrimination of -607a / c ( rs1946518 ) and -137g / c ( rs187238 ) polymorphisms of il-18 was assessed through real - time polymerase chain reaction by performing the taqman assay . 
 the il-18 -137g / c polymorphism but not the -607a / c polymorphism showed a significant association with the risk of hcc . 
 participants carrying the il-18 -137 polymorphism with heterozygous g / c and homozygous cc genotypes showed a 1.987-fold increase ( 95% ci = 1.301 - 3.032 ; p = 0.001 ) in the risk of hcc compared with those homozygous for wild - type g / g . 
 the 342 patients with hcc carrying the il-18 -137g / c polymorphism were positive for hepatitis b virus ( hbv ) infection with an adjusted odds ratio of 1.668 . moreover , the 142 hbv positive patients with hcc and the il-18 -137 polymorphism were positive for at least one c genotype and showed significant vascular invasion ( p = 0.018 ) . 
 furthermore , the level of -fetoprotein was high in the patients carrying the il-18 -137 polymorphism with gc+cc alleles ( p = 0.011 ) . in conclusion 
 , the il-18 -137g / c polymorphism with a gc+cc genotype could be a factor that increases the risk of hcc . 
 furthermore , the correlation between the il-18 -137g / c polymorphism and hcc - related hbv infection is a risk factor for vascular invasion and has a synergistic effect that can further enhance hcc prognosis .