ORIGINAL ARTICLE
Tumor Necrosis Factor-Alpha Gene Polymorphism Associated With Development of Hepatitis B Virus–associated Hepatocellular Carcinoma Young-Joo Jin, MD, PhD,* Danbi Lee, MD, PhD,w Young-Hwa Chung, MD, PhD,w Jeong A. Kim, MS,z Sung Eun Kim, MD, PhD,y Yoon-Seon Lee, MD, PhD,w Eun-Soon Shin, PhD,z Soo Hyung Ryu, MD, PhD,8 Myoung Kuk Jang, MD, PhD,z Jong-Eun Lee, PhD,z and Neung Hwa Park, MD, PhD#
Goal and Background: Host genetic diversity may play roles in development of HCC. This study was conducted to validate the effects of tumor necrosis factor-alpha (TNF-a) gene polymorphism on development of hepatocellular carcinoma (HCC) in patients chronically infected with hepatitis B virus (HBV). Study: The study cohort comprised 224 patients with HBV-associated HCC and 206 with HBV-associated liver cirrhosis (LC). Using chromosomal DNA, TNF-a promoter gene polymorphisms were determined at 3 common single-nucleotide polymorphism (SNP) sites (TNF-a-1031 T > C, TNF-a-857 C > T, and TNF-a308 G > A) using a single base extension method. The genotype distributions were compared between the 2 groups. All the HBVassociated LC patients were followed up regularly every 6 to 12 months for surveillance of HCC development. Results: In the cross-sectional analysis, the frequency of TNF-a-857 T allele was much higher in patients with HCC compared with those with LC (42% vs. 31%, P < 0.01). Of 206 HBV-associated LC patients, 12 (5.8%) developed HCC during the median followup period of 36 months. The cumulative occurrence rates of HCC were significantly higher in patients with TNF-a-857 T allele than those withTNF-a-857 C/C genotype (1-, 3-, and 5-y rates: 2.9%, 12.8%, and 20.7% vs. 0%, 3.1%, and 5.3%, respectively; P = 0.013). However, the other genetic polymorphisms of TNF-a promoter gene did not affect the development of HCC. In multivariate analysis, TNF-a-857 T allele was a significant predictor of HCC development (hazard ratio 6.29, P = 0.01).
Received for publication March 5, 2014; accepted September 11, 2014. From the *Department of Internal Medicine, Inha University Hospital, Inha University School of Medicine, Incheon; wDepartment of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center; zDNAlink Ltd; 8Department of Internal Medicine, University of Inje College of Medicine, Seoul Paik Hospital; zDepartment of Internal Medicine, Hallym University College of Medicine, Kangdong Sacred Heart Hospital, Seoul; yDepartment of Internal Medicine, Hallym University College of Medicine, Pyeongchon Sacred Heart Hospital, Pyeongchon; and #Department of Internal Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, Korea. Y.-J.J. and D.L. contributed equally. A part of this work was presented at the 63rd annual meeting of the American Association for the Study of the Liver Diseases (AASLD) in 2012 (The Liver Meeting 2012). Supported by a grant from Asan Institute for Life Science, Asan Medical Center, Republic of Korea (No. AILS10-498). The authors declare that they have nothing to disclose. Reprints: Young-Hwa Chung, MD, PhD, Department of Gastroenterology and Hepatology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro-43-gil, Songpa-gu, Seoul 138-736, South Korea (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
Conclusion: Our data suggest that TNF-a-857 T allele is closely associated with development of HCC in HBV-associated LC patients. Key Words: hepatocellular carcinoma, liver cirrhosis, tumor necrosis factor-alpha, genetic polymorphism, hepatitis B virus
(J Clin Gastroenterol 2015;49:e76–e81)
H
epatocellular carcinoma (HCC) is a worldwide health problem because of its critical effect on cancer-related death.1,2 Recently, its incidence has been increasing even in the Unites States and Europe.3,4 Epidemiological studies have established that liver cirrhosis (LC) is a major risk factor for HCC occurrence, and the incidence of HCC in cirrhotic patients has been approximately 3% to 9% per year.5–7 However, HCC develops with different incidences in various patient groups,6 even in LC patients associated with hepatitis B virus (HBV), suggesting that interindividual difference, such as host genetic diversity, may play a role in the development of HCC. Several mechanisms have been proposed to explain the progression from cirrhosis to HCC in patients with chronic hepatitis B (CHB), including the insertional mutagenesis of HBV genomes, and inflammation or regenerative hyperplasia initiated by an immune response to HBV infection.8–10 However, it is well known that HCC does not always occur in every patient with HBV-associated LC. Recently, studies have reported that chronic stimulation of aberrant cells with inflammatory mediators, such as tumor necrosis factor-alpha (TNF-a), may promote carcinogenesis of HCC by inducing compensatory proliferation and regeneration of aberrant cells.11–13 These reports also suggested the association of TNFa promoter gene polymorphism with development of HCC. These relationships are supported by another report using a TNF-a receptor type 1 knockout mice model.14 However, the studies investigating the associations between TNF-a promoter gene polymorphism and HCC development report conflicting results. Moreover, they seem to be inconclusive in CHB patients because of the enrollment of subjects with various etiologies of liver disease, and also because of lack of longitudinal analysis in patients with HBV-associated LC. Thus, in the current study, we intended to evaluate the difference of TNF-a promoter gene polymorphism (TNF-a308 G/A, TNF-a-857C/T, and TNF-a-1031 T/C SNP site), which has been suggested to be associated with HCC
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development in Korean population in the previous studies, between patients with HBV-associated HCC and those with HBV-associated LC using a cross-sectional analysis. In addition, using a longitudinal analysis, we aimed to validate the association of TNF-a gene polymorphism on the development of HCC in patients with HBV-associated LC.
MATERIALS AND METHODS Study Subject During the period between the year 2001 and 2005, 254 patients were diagnosed as HBV-associated HCC and 220 patients as HBV-associated LC at the liver clinic of Asan Medical Center in Seoul, Korea. Blood samples of all the subjects were obtained at the time of initial diagnosis. Of the 254 HBV-associated HCC patients, 30 patients were excluded because of simultaneous positivity for anti-hepatitis C virus (HCV) antibody (n = 22) and heavy alcoholism (n = 8). Of the 220 HBV-associated LC patients, 14 patients were also excluded because of simultaneous positivity for anti-HCV antibody (n = 8) and heavy alcoholism (n = 6). Therefore, 224 patients with HBV-associated HCC (HBV-HCC group) and 206 patients with HBV-associated LC (HBV-LC group) were analyzed in the cross-sectional study. In addition, longitudinally, the occurrence rates of HCC were calculated according to the polymorphism of TNF-a promoter gene using 206 subjects with HBV-associated LC. HCC was diagnosed according to the diagnostic guidelines issued by the 2005 American Association for the Study of Liver Diseases (AASLD).15 LC was diagnosed on the basis of clinical evidence of portal hypertension16 or by ultrasonography.17 LC patients, who showed no clinical evidence of HCC initially, were followed up regularly with serum a-fetoprotein (AFP) and ultrasonography or computed tomography every 6 to12 months for surveillance of HCC development. The study was approved by the institutional review board at Asan Medical Center, Seoul, Korea. Demographic and laboratory data were obtained using electronic medical records. Age, sex, aspartate aminotransferase, alanine aminotransferase (ALT), Child-Turcotte-Pugh (CTP) classification, anti-hepatitis B surface (HBs) antibody, anti-HCV antibody, hepatitis B e antigen (HBeAg), anti-HBe antibody, HBV-DNA titer, AFP levels, and Model for End-stage Liver Disease (MELD) score were analyzed. Serologic markers, including HBs antigen (HBsAg), anti-HBs, HBeAg, anti-HBe, and anti-HCV, were determined by enzyme immunoassays (Abbott Laboratories, Chicago, IL). Serum HBV-DNA titer was measured by a hybrid capture assay (Digene hybrid capture II assay, Digene Diagnostics, Gaithersburg, MD). The HBV genotypes were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the surface gene of HBV. The MELD score was calculated by the following formulation: MELD score = (0.957 ln(serum Cr) + 0.378 ln(serum bilirubin) + 1.120ln(INR) + 0.643)10 (if hemodialysis, value for creatinine is automatically set to 4.0).
Genotyping of TNF-a Promoter Gene (TNF-a-308, TNF-a-857, and TNF-a-1031) Blood samples were obtained at the time of initial diagnosis of HCC or LC. Chromosomal DNA was extracted from 5 mL of peripheral blood using the QuickGene DNA whole blood kit (Fuji Life Science, Tokyo, Japan), and stored at 701C until used. Genotyping was performed using a single base primer extension assay, using the ABI PRISM Copyright
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TNF-a Gene Polymorphism and HBV-HCC
SNaPShot Multiplex kit (ABI, Foster City, CA). Briefly, the chromosomal DNA flanking the SNP of interest was amplified by PCR reaction with forward and reverse primer pairs and standard PCR reagents in a 10 mL reaction volume, containing 10 ng of DNA, 0.5 pM of each oligonucleotide primer, 1 mL of 10 PCR buffer, 250 mM dNTP, and 0.25 U i-StarTaq DNA Polymerase(5 U/mL) (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea). The PCR reactions were performed as follows: 10 minutes at 951C for 1 cycle, and 35 cycles on denaturation at 951C for 30 seconds, annealing at 551C for 1 minute, extension at 721C for 1 minute, followed by 1 cycle of 721C for 10 minutes. After amplification, the PCR products were treated with 1 U each of shrimp alkaline phosphatase (Roche) and exonuclease I (USB Corporation) at 371C for 75 minutes and 721C for 15 minutes to purify the amplified products. One microliter of the purified amplification products was added to a SNaPshot Multiplex Ready reaction mixture containing 0.15 pmols of genotyping primer for the primer extension reaction. The primer extension reaction was performed for 25 cycles of 961C for 10 seconds, 501C for 5 seconds, and 601C for 30 seconds. The reaction products were treated with 1 U of shrimp alkaline phosphatase at 371C for 1 hour and 721C for 15 minutes for removal of excess fluorescent dye terminators. One microliter of the final reaction samples containing the extension products was added to 9 mL of Hi-Di formamide (ABI). The mixture was incubated at 951C for 5 minutes, followed by 5 minutes on ice, and then analyzed by electrophoresis in an ABI Prism 3730xl DNA analyzer. Analysis was performed using Genemapper software (version 4.0; Applied Biosystems). The genotype distributions were then compared between the 2 groups.
Statistical Analysis In the cross-sectional study, the frequencies of TNF-a promoter gene genotypes were compared between the two groups (HBV-associated HCC group vs. HBV-associated LC) using the w2 test. Odds ratios (OR) and 95% confidence intervals were calculated for comparison of genotypic frequency between the groups. In the longitudinal study,
TABLE 1. Baseline Characteristics of the Subjects
Variables Age (y)w Sex (male) [n (%)] ALT (IU/L)w CTP class, A or B/C [n (%)] MELD scorew HBeAg, presence HBV-DNA (log copies/mL) w HBV genotype C2 [n (%)] AFP (ng/mL)w
HBV-HCC (n = 224)
HBV-LC (n = 206)
52 (28-67) 55 (21-68) 170 (75.9) 143 (69.4) 32 (9-364) 35.5 (6-680) 211/13 (95.1/4.9) 122/84 (59.2/ 40.8) 7 (6-32) 19 (8-46) 74 (33.0) 84 (40.8) 4.0 (1.0-8.71) 4.2 (1.0-8.98) 224 (100)
P* 0.19 0.16 0.17 < 0.01 < 0.01 0.11 0.01
206 (100)
63.7 (10-1.7 106) 10.1 (1-2103)
1.00 0.06
*P-value for difference between the LC and HCC groups. wMedian (range). AFP indicates a-fetoprotein; ALT, alanine aminotransferase; CTP, Child-Turcotte-Pugh; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MELD, Model for Endstage Liver Disease.
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TABLE 2. Polymorphisms of Tumor Necrosis Factor-a Promoter Gene
Loci
Genotype
TNF-a-308
AG or AA GG CT or TT CC CT or TT CC
TNF-a-857 TNF-a-1031
HBV-HCC (N = 224) [n (%)] 29 195 94 130 209 15
(12.9) (87.1) (42.0) (58.0) (93.3) (6.7)
HBV-LC (N = 206) [n (%)] 24 182 63 143 192 14
(11.7) (88.3) (30.6) (69.4) (93.2) (6.8)
OR
95% CI
P*
1.13
0.63-2.01
0.68
1.64
1.10-2.44
0.02
0.98
0.46-2.09
0.97
*P-values using logistic regression model. A indicates adenine; C, cytosine; CI, confidence interval; G, guanine; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; OR, odds ratio; T, thymine; TNF, tumor necrosis factor.
cumulative rates of HCC development in HBV-associated LC patients were analyzed using Kaplan-Meier survival curves, and their differences were compared using the logrank test. Multivariate analysis of factors associated with occurrence for HCC was performed using a Cox regression hazard model. A P-value of T and T > T) was significantly higher in patients with HCC compared with those with LC [42% vs. 31%, OR 1.64, 95% confidence interval (1.10-2.44), P = 0.02]. However, no significant difference in the frequency of TNF-a 308 A allele was observed between the 2 groups (HBV-HCC vs. HBV-LC; 13% vs. 12%, OR 1.13, P = 0.68). In addition, no significant difference was observed between the 2 groups in the frequency of TNF-a 1031 T allele (HBV-HCC vs. HBV-LC; 93% vs. 93%, OR 0.98, P = 0.97). Clinical characteristics between patients with TNF-a-857 T allele and those with TNF-a-857 CC genotype were not significantly different.
Development of HCC in Relation to TNF-a Promoter Gene Polymorphism in HBV-associated LC Patients
FIGURE 1. Cumulative occurrence rates of HCC in HBV-LC patients. The 1-, 3-, 5-, and 7-year cumulative occurrence rates of HCC in HBV-associated LC patients were 0.9%, 5.5%, 9.1%, and 18.4%, respectively. HBV indicates hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis.
A total of 206 patients with HBV-associated LC were followed up for a median period of 36 months (range, 1 to 122 mo). During the follow-up period, HCC developed in 12 patients (5.8%). The 1-, 3-, 5-, and 7-year cumulative occurrence rates of HCC in HBV-associated LC patients were 0.9%, 5.5%, 9.1%, and 18.4%, respectively (Fig. 1). The 1-, 3-, 5-, and 7-year cumulative occurrence rates of HCC in patients with TNF-a857 TT/CT genotype were significantly higher than those in patients with TNF-a857 CC genotype (2.9%, 12.8%, 20.7%, and 42.2% vs. 0%, 3.1%, 5.3%, and 11.4%, respectively; P = 0.013) (Fig. 2A). However, the cumulative occurrence rates of HCC in patients with the TNF-a1031 TT/CT genotype were not different from those in patients with the TNF-a1031 CC genotype (P = 0.279) (Fig. 2B). Also, the cumulative occurrence rates of HCC in patients with the TNF-a308 GG genotype were not different from those in patients with the TNF-a308 GA/AA genotype (P = 0.367) (Fig. 2C). Clinical characteristics were not statistically different from each other between patients associated with HCC (n = 12) and those without HCC
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TNF-a Gene Polymorphism and HBV-HCC
FIGURE 2. Cumulative occurrence rates of HCC in relation to TNF-a promoter gene polymorphisms in HBV-LC patients. A, The cumulative occurrence rates of HCC in patients with the TNF-a857 TT/CT genotype were significantly higher than those with the TNFa857 CC genotype (P = 0.013). This significant association was not observed in other genetic polymorphisms [(TNF-a 1031CC vs. TNF-a 1031TT/CT, P = 0.279) (B) and (TNF-a 308GG vs. TNF-a 308AA/GA, P = 0.367) (C)]. C indicates cytosine; HCC, hepatocellular carcinoma; TNF, tumor necrosis factor; T, thymine.
(n = 194), except CTP class (A or B vs. C, P < 0.05) and MELD score (P < 0.01) (Table 3).
Multivariate Analysis for Predictors of HCC Development in HBV-associated LC Patients In univariate analysis, age [hazard ratio (HR) 1.04, P = 0.18], MELD score (HR 0.92, P = 0.18), and TNF-a-857 gene polymorphism (TT/CT vs. CC, HR 3.88, P = 0.02) were candidates for multivariate analysis (P < 0.2). In multivariate analysis, only TNF-a-857 T allele was an independent risk factor for development of HCC in patients with HBV-associated LC (HR 6.29, P = 0.01) (Table 4). Copyright
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DISCUSSION In the current study, the authors are suggesting a significant host genetic marker for susceptibility to development of HCC in patients with HBV-associated LC. In the crosssectional analysis, the data revealed that the frequency of TNF-a-857 T allele (genotype TNF-a-857 C > T and T > T) was significantly higher in patients associated with HBV-HCC compared with the frequency in patients with HBV-LC (42% vs. 31%). In addition, by the cohort-observational analysis, it was validated that HCC develops much more frequently in HBV-associated LC patients with TNF-a-857 T allele than in those with the TNF-a-857 CC genotype, and thus TNF-a-857 T allele was a significant predictor of HCC development in HBV-associated LC patients.
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TABLE 3. Clinical Characteristics of Hepatitis B Virus–associated Liver Cirrhosis Patients in Accordance With Development of Hepatocellular Carcinoma
Variables
HCC (n = 12)
Age (y)w Sex (male) [n (%)] ALT (IU/L)w CTP class, A or B/C [n (%)] MELD scorew HBeAg, presence HBV-DNA (log copies/mL)w AFP (ng/mL)w Follow-up duration (mo)w
56 7 41 11/1 12 5 5.9 20.5 46
No HCC (n = 194)
(34-60) (58.3) (6-106) (91.7/8.3) (9-21) (41.7) (1.0-8.1) (3-218) (10-87)
55 (21-68) 124 (63.9) 35 (6-680) 111/83 (57.2/42.8) 19 (8-46) 103 (53.1) 4.0 (1.0-9.4) 8.5 (1-2000) 28 (1-122)
P* 0.93 0.76 0.52 < 0.05 < 0.01 0.56 0.34 0.13 0.06
*P-value for difference between the LC and HCC groups. wMedian (range). AFP indicates a-fetoprotein; CTP, Child-Turcotte-Pugh; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MELD, Model for End-stage Liver Disease.
Recently, it has been suggested that polymorphisms of cytokine genes may have critical impacts on the outcome of patients with chronic HBV infection.18,19 TNF-a is a pleiotropic cytokine that may exert inflammatory and cytotoxic effects, and can be a potential target for treatment of solid tumors.20 TNF-a gene polymorphism has recently been emphasized to be associated with the progression of LC or the development of HCC.21 In relation to HCC development, it has been suggested that the TNF-a gene, which is located within the major histocompatibility complex class III region, may play a role by producing oxygen-derived free radicals or other reactive oxygen species.22,23 Several studies also have attempted to identify the specific TNF-a gene polymorphisms associated with the development of HCC.11–13 However, there has been no consistent result on this point so far. In the current study, the authors demonstrated that a specific TNF-a promoter gene polymorphism (TNF-a-857 T allele) was closely associated with HCC occurrence in the cross-sectional analysis of a relatively large number of subjects. In addition, the long-term cohort-observational study also validated that HCC developed much more frequently in patients with HBVassociated LC who had TNF-a-857 T allele. To improve the outcomes of patients with HCC, it is crucial to detect HCC earlier in patients with high risk for the development of HCC.15 Until now, a variety of factors,
such as age, sex, association with cirrhosis, serum ALT level, serum HBV-DNA level, or genomic changes of HBV, have been suggested as predictive factors for HCC development in patients chronically infected with HBV.24,25 Of these factors, in addition to the replicative activity or genomic changes of HBV, the association with cirrhosis also has been known to be a dominant risk factor for HCC development.26 Therefore, it is very important to monitor patients with HBV-associated LC for the early detection of HCC. Furthermore, the patients with higher genetic susceptibility to development of HCC should be followed up more closely in clinical practice. The current study indicates that it is necessary to monitor HBV-associated LC patients with the TNF-a857 T allele much more closely for the early detection of HCC. Thus, the results of our study suggest that TNF-a promoter gene polymorphism may be useful to provide HBV-associated LC patients with a tailored management, especially in the surveillance of HCC. In terms of TNF-a gene polymorphisms associated with HCC, a previous meta-analysis reported that TNF-a308G/A polymorphisms may be associated with the development of HCC, while TNF-a-857C/T and -1031T/C polymorphisms were not associated with the occurrence of HCC.27 However, the results of the current study showed a significant association of TNF-a-857C/T polymorphism
TABLE 4. Multivariate Analysis for Predictors of Hepatocellular Carcinoma Development in Patients With Hepatitis B Virus–associated Liver Cirrhosis
Univariate Analysis Variables Age (y) Sex (male) ALT (IU/L) HBeAg, presence HBV-DNA (log copies/mL) CTP class A or B vs. C MELD score TNF-a-857 TT/CT vs. CC TNF-a-1031 TT/CT vs. CC TNF-a-308 GG vs. GA/AA
Multivariate Analysis
HR
95% CI
P
HR
95% CI
P
1.04 0.50 0.99 0.95 1.07 0.31 0.92 3.88 23.28 1.99
0.96-1.12 0.16-1.59 0.99-1.01 0.30-3.01 0.84-1.37 0.04-2.43 0.82-1.04 1.23-12.20 0.01-1.5105 0.43-9.11
0.18 0.24 0.60 0.93 0.58 0.27 0.18 0.02 0.48 0.38
1.07 — — — — — 0.89 6.29 — —
0.99-1.15 — — — — — 0.78-1.01 1.62-24.43 — —
0.11 — — — — — 0.08 0.01 — —
A indicates adenine; ALT, alanine aminotransferase; C, cytosine; CI, confidence interval; CTP, Child-Turcotte-Pugh; G, guanine; HBV, hepatitis B virus; HR, hazard ratio; MELD, Model for End-stage Liver Disease; T, thymine; TNF, tumor necrosis factor.
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with the development of HCC in HBV-associated LC patients, which is supported by the results of a previous Korean study.12 Such differences in the results may arise due to the different characteristics of the subjects,11–13,27 and these may limit the application of the present results to other population. Thus, potential confounding factors such as the association of LC and the genotype of HBV can be minimized in the current study, although the clinical diagnosis of cirrhosis may cause some bias in the present study. Notably, in our multivariate analysis, other viral factors such as serum HBV-DNA titer or the presence of HBeAg were not confounders, indicating that TNF-a-857-T allele has an independent effect on HCC occurrence in HBVassociated LC patients. Unfortunately, in the current study, the functional role of TNF-a promoter gene polymorphism was not evaluated because the serum TNF-a level could not be measured because of lack of available stored serum. However, a previous Korean study had already demonstrated that the serum TNF-a levels were significantly higher in HCC patients with the TNF-a-857-TT/CT genotype, compared with the serum TNF-a levels in patients with TNF-a-857-CC genotype.12 Therefore, it is suggested that the presence of TNF-a-857-T allele may enhance the expression of serum TNF-a in patients with HBV-associated LC patients and thus ultimately increased the risk for the development of HCC. In conclusion, using a cross-sectional and longitudinal analysis, the current study demonstrated the close association of the TNF-a-857 T allele with development of HCC in patients with LC chronically infected with HBV. It suggests that TNF-a-857T allele may be useful as a biomarker for the identification of susceptibility to HCC in these patients. REFERENCES 1. Bosch FX, Ribes J, Diaz M, et al. Primary liver cancer: worldwide incidence and trends. Gastroenterology. 2004; 127(suppl 1):S5–S16. 2. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol. 2001;2:533–543. 3. El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med. 1999;340: 745–750. 4. Taylor-Robinson SD, Foster GR, Arora S, et al. Increase in primary liver cancer in the UK, 1979-94. Lancet. 1997;350: 1142–1143. 5. Velazquez RF, Rodriquez M, Navascue´s CA, et al. Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology. 2003;37:520–527. 6. Fattovich G, Stroffolini T, Zagni I, et al. Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology. 2004;127(suppl 1):S35–S50. 7. Ikeda K, Saitoh S, Koida I, et al. A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis. Hepatology. 1993;18:47–53. 8. Grisham JW. Interspecies comparison of liver carcinogenesis: implications for cancer risk assessment. Carcinogenesis. 1997; 18:59–81.
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Tumor Necrosis Factor-Alpha Gene Polymorphism Associated With Development of Hepatitis B Virus–associated Hepatocellular Carcinoma Young-Joo Jin, MD, PhD,* Danbi Lee, MD, PhD,w Young-Hwa Chung, MD, PhD,w Jeong A. Kim, MS,z Sung Eun Kim, MD, PhD,y Yoon-Seon Lee, MD, PhD,w Eun-Soon Shin, PhD,z Soo Hyung Ryu, MD, PhD,8 Myoung Kuk Jang, MD, PhD,z Jong-Eun Lee, PhD,z and Neung Hwa Park, MD, PhD#
Goal and Background: Host genetic diversity may play roles in development of HCC. This study was conducted to validate the effects of tumor necrosis factor-alpha (TNF-a) gene polymorphism on development of hepatocellular carcinoma (HCC) in patients chronically infected with hepatitis B virus (HBV). Study: The study cohort comprised 224 patients with HBV-associated HCC and 206 with HBV-associated liver cirrhosis (LC). Using chromosomal DNA, TNF-a promoter gene polymorphisms were determined at 3 common single-nucleotide polymorphism (SNP) sites (TNF-a-1031 T > C, TNF-a-857 C > T, and TNF-a308 G > A) using a single base extension method. The genotype distributions were compared between the 2 groups. All the HBVassociated LC patients were followed up regularly every 6 to 12 months for surveillance of HCC development. Results: In the cross-sectional analysis, the frequency of TNF-a-857 T allele was much higher in patients with HCC compared with those with LC (42% vs. 31%, P < 0.01). Of 206 HBV-associated LC patients, 12 (5.8%) developed HCC during the median followup period of 36 months. The cumulative occurrence rates of HCC were significantly higher in patients with TNF-a-857 T allele than those withTNF-a-857 C/C genotype (1-, 3-, and 5-y rates: 2.9%, 12.8%, and 20.7% vs. 0%, 3.1%, and 5.3%, respectively; P = 0.013). However, the other genetic polymorphisms of TNF-a promoter gene did not affect the development of HCC. In multivariate analysis, TNF-a-857 T allele was a significant predictor of HCC development (hazard ratio 6.29, P = 0.01).
Received for publication March 5, 2014; accepted September 11, 2014. From the *Department of Internal Medicine, Inha University Hospital, Inha University School of Medicine, Incheon; wDepartment of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center; zDNAlink Ltd; 8Department of Internal Medicine, University of Inje College of Medicine, Seoul Paik Hospital; zDepartment of Internal Medicine, Hallym University College of Medicine, Kangdong Sacred Heart Hospital, Seoul; yDepartment of Internal Medicine, Hallym University College of Medicine, Pyeongchon Sacred Heart Hospital, Pyeongchon; and #Department of Internal Medicine, University of Ulsan College of Medicine, Ulsan University Hospital, Ulsan, Korea. Y.-J.J. and D.L. contributed equally. A part of this work was presented at the 63rd annual meeting of the American Association for the Study of the Liver Diseases (AASLD) in 2012 (The Liver Meeting 2012). Supported by a grant from Asan Institute for Life Science, Asan Medical Center, Republic of Korea (No. AILS10-498). The authors declare that they have nothing to disclose. Reprints: Young-Hwa Chung, MD, PhD, Department of Gastroenterology and Hepatology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro-43-gil, Songpa-gu, Seoul 138-736, South Korea (e-mail: [email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
Conclusion: Our data suggest that TNF-a-857 T allele is closely associated with development of HCC in HBV-associated LC patients. Key Words: hepatocellular carcinoma, liver cirrhosis, tumor necrosis factor-alpha, genetic polymorphism, hepatitis B virus
(J Clin Gastroenterol 2015;49:e76–e81)
H
epatocellular carcinoma (HCC) is a worldwide health problem because of its critical effect on cancer-related death.1,2 Recently, its incidence has been increasing even in the Unites States and Europe.3,4 Epidemiological studies have established that liver cirrhosis (LC) is a major risk factor for HCC occurrence, and the incidence of HCC in cirrhotic patients has been approximately 3% to 9% per year.5–7 However, HCC develops with different incidences in various patient groups,6 even in LC patients associated with hepatitis B virus (HBV), suggesting that interindividual difference, such as host genetic diversity, may play a role in the development of HCC. Several mechanisms have been proposed to explain the progression from cirrhosis to HCC in patients with chronic hepatitis B (CHB), including the insertional mutagenesis of HBV genomes, and inflammation or regenerative hyperplasia initiated by an immune response to HBV infection.8–10 However, it is well known that HCC does not always occur in every patient with HBV-associated LC. Recently, studies have reported that chronic stimulation of aberrant cells with inflammatory mediators, such as tumor necrosis factor-alpha (TNF-a), may promote carcinogenesis of HCC by inducing compensatory proliferation and regeneration of aberrant cells.11–13 These reports also suggested the association of TNFa promoter gene polymorphism with development of HCC. These relationships are supported by another report using a TNF-a receptor type 1 knockout mice model.14 However, the studies investigating the associations between TNF-a promoter gene polymorphism and HCC development report conflicting results. Moreover, they seem to be inconclusive in CHB patients because of the enrollment of subjects with various etiologies of liver disease, and also because of lack of longitudinal analysis in patients with HBV-associated LC. Thus, in the current study, we intended to evaluate the difference of TNF-a promoter gene polymorphism (TNF-a308 G/A, TNF-a-857C/T, and TNF-a-1031 T/C SNP site), which has been suggested to be associated with HCC
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development in Korean population in the previous studies, between patients with HBV-associated HCC and those with HBV-associated LC using a cross-sectional analysis. In addition, using a longitudinal analysis, we aimed to validate the association of TNF-a gene polymorphism on the development of HCC in patients with HBV-associated LC.
MATERIALS AND METHODS Study Subject During the period between the year 2001 and 2005, 254 patients were diagnosed as HBV-associated HCC and 220 patients as HBV-associated LC at the liver clinic of Asan Medical Center in Seoul, Korea. Blood samples of all the subjects were obtained at the time of initial diagnosis. Of the 254 HBV-associated HCC patients, 30 patients were excluded because of simultaneous positivity for anti-hepatitis C virus (HCV) antibody (n = 22) and heavy alcoholism (n = 8). Of the 220 HBV-associated LC patients, 14 patients were also excluded because of simultaneous positivity for anti-HCV antibody (n = 8) and heavy alcoholism (n = 6). Therefore, 224 patients with HBV-associated HCC (HBV-HCC group) and 206 patients with HBV-associated LC (HBV-LC group) were analyzed in the cross-sectional study. In addition, longitudinally, the occurrence rates of HCC were calculated according to the polymorphism of TNF-a promoter gene using 206 subjects with HBV-associated LC. HCC was diagnosed according to the diagnostic guidelines issued by the 2005 American Association for the Study of Liver Diseases (AASLD).15 LC was diagnosed on the basis of clinical evidence of portal hypertension16 or by ultrasonography.17 LC patients, who showed no clinical evidence of HCC initially, were followed up regularly with serum a-fetoprotein (AFP) and ultrasonography or computed tomography every 6 to12 months for surveillance of HCC development. The study was approved by the institutional review board at Asan Medical Center, Seoul, Korea. Demographic and laboratory data were obtained using electronic medical records. Age, sex, aspartate aminotransferase, alanine aminotransferase (ALT), Child-Turcotte-Pugh (CTP) classification, anti-hepatitis B surface (HBs) antibody, anti-HCV antibody, hepatitis B e antigen (HBeAg), anti-HBe antibody, HBV-DNA titer, AFP levels, and Model for End-stage Liver Disease (MELD) score were analyzed. Serologic markers, including HBs antigen (HBsAg), anti-HBs, HBeAg, anti-HBe, and anti-HCV, were determined by enzyme immunoassays (Abbott Laboratories, Chicago, IL). Serum HBV-DNA titer was measured by a hybrid capture assay (Digene hybrid capture II assay, Digene Diagnostics, Gaithersburg, MD). The HBV genotypes were analyzed by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of the surface gene of HBV. The MELD score was calculated by the following formulation: MELD score = (0.957 ln(serum Cr) + 0.378 ln(serum bilirubin) + 1.120ln(INR) + 0.643)10 (if hemodialysis, value for creatinine is automatically set to 4.0).
Genotyping of TNF-a Promoter Gene (TNF-a-308, TNF-a-857, and TNF-a-1031) Blood samples were obtained at the time of initial diagnosis of HCC or LC. Chromosomal DNA was extracted from 5 mL of peripheral blood using the QuickGene DNA whole blood kit (Fuji Life Science, Tokyo, Japan), and stored at 701C until used. Genotyping was performed using a single base primer extension assay, using the ABI PRISM Copyright
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TNF-a Gene Polymorphism and HBV-HCC
SNaPShot Multiplex kit (ABI, Foster City, CA). Briefly, the chromosomal DNA flanking the SNP of interest was amplified by PCR reaction with forward and reverse primer pairs and standard PCR reagents in a 10 mL reaction volume, containing 10 ng of DNA, 0.5 pM of each oligonucleotide primer, 1 mL of 10 PCR buffer, 250 mM dNTP, and 0.25 U i-StarTaq DNA Polymerase(5 U/mL) (iNtRON Biotechnology, Sungnam, Kyungki-Do, Korea). The PCR reactions were performed as follows: 10 minutes at 951C for 1 cycle, and 35 cycles on denaturation at 951C for 30 seconds, annealing at 551C for 1 minute, extension at 721C for 1 minute, followed by 1 cycle of 721C for 10 minutes. After amplification, the PCR products were treated with 1 U each of shrimp alkaline phosphatase (Roche) and exonuclease I (USB Corporation) at 371C for 75 minutes and 721C for 15 minutes to purify the amplified products. One microliter of the purified amplification products was added to a SNaPshot Multiplex Ready reaction mixture containing 0.15 pmols of genotyping primer for the primer extension reaction. The primer extension reaction was performed for 25 cycles of 961C for 10 seconds, 501C for 5 seconds, and 601C for 30 seconds. The reaction products were treated with 1 U of shrimp alkaline phosphatase at 371C for 1 hour and 721C for 15 minutes for removal of excess fluorescent dye terminators. One microliter of the final reaction samples containing the extension products was added to 9 mL of Hi-Di formamide (ABI). The mixture was incubated at 951C for 5 minutes, followed by 5 minutes on ice, and then analyzed by electrophoresis in an ABI Prism 3730xl DNA analyzer. Analysis was performed using Genemapper software (version 4.0; Applied Biosystems). The genotype distributions were then compared between the 2 groups.
Statistical Analysis In the cross-sectional study, the frequencies of TNF-a promoter gene genotypes were compared between the two groups (HBV-associated HCC group vs. HBV-associated LC) using the w2 test. Odds ratios (OR) and 95% confidence intervals were calculated for comparison of genotypic frequency between the groups. In the longitudinal study,
TABLE 1. Baseline Characteristics of the Subjects
Variables Age (y)w Sex (male) [n (%)] ALT (IU/L)w CTP class, A or B/C [n (%)] MELD scorew HBeAg, presence HBV-DNA (log copies/mL) w HBV genotype C2 [n (%)] AFP (ng/mL)w
HBV-HCC (n = 224)
HBV-LC (n = 206)
52 (28-67) 55 (21-68) 170 (75.9) 143 (69.4) 32 (9-364) 35.5 (6-680) 211/13 (95.1/4.9) 122/84 (59.2/ 40.8) 7 (6-32) 19 (8-46) 74 (33.0) 84 (40.8) 4.0 (1.0-8.71) 4.2 (1.0-8.98) 224 (100)
P* 0.19 0.16 0.17 < 0.01 < 0.01 0.11 0.01
206 (100)
63.7 (10-1.7 106) 10.1 (1-2103)
1.00 0.06
*P-value for difference between the LC and HCC groups. wMedian (range). AFP indicates a-fetoprotein; ALT, alanine aminotransferase; CTP, Child-Turcotte-Pugh; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MELD, Model for Endstage Liver Disease.
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TABLE 2. Polymorphisms of Tumor Necrosis Factor-a Promoter Gene
Loci
Genotype
TNF-a-308
AG or AA GG CT or TT CC CT or TT CC
TNF-a-857 TNF-a-1031
HBV-HCC (N = 224) [n (%)] 29 195 94 130 209 15
(12.9) (87.1) (42.0) (58.0) (93.3) (6.7)
HBV-LC (N = 206) [n (%)] 24 182 63 143 192 14
(11.7) (88.3) (30.6) (69.4) (93.2) (6.8)
OR
95% CI
P*
1.13
0.63-2.01
0.68
1.64
1.10-2.44
0.02
0.98
0.46-2.09
0.97
*P-values using logistic regression model. A indicates adenine; C, cytosine; CI, confidence interval; G, guanine; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; OR, odds ratio; T, thymine; TNF, tumor necrosis factor.
cumulative rates of HCC development in HBV-associated LC patients were analyzed using Kaplan-Meier survival curves, and their differences were compared using the logrank test. Multivariate analysis of factors associated with occurrence for HCC was performed using a Cox regression hazard model. A P-value of T and T > T) was significantly higher in patients with HCC compared with those with LC [42% vs. 31%, OR 1.64, 95% confidence interval (1.10-2.44), P = 0.02]. However, no significant difference in the frequency of TNF-a 308 A allele was observed between the 2 groups (HBV-HCC vs. HBV-LC; 13% vs. 12%, OR 1.13, P = 0.68). In addition, no significant difference was observed between the 2 groups in the frequency of TNF-a 1031 T allele (HBV-HCC vs. HBV-LC; 93% vs. 93%, OR 0.98, P = 0.97). Clinical characteristics between patients with TNF-a-857 T allele and those with TNF-a-857 CC genotype were not significantly different.
Development of HCC in Relation to TNF-a Promoter Gene Polymorphism in HBV-associated LC Patients
FIGURE 1. Cumulative occurrence rates of HCC in HBV-LC patients. The 1-, 3-, 5-, and 7-year cumulative occurrence rates of HCC in HBV-associated LC patients were 0.9%, 5.5%, 9.1%, and 18.4%, respectively. HBV indicates hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis.
A total of 206 patients with HBV-associated LC were followed up for a median period of 36 months (range, 1 to 122 mo). During the follow-up period, HCC developed in 12 patients (5.8%). The 1-, 3-, 5-, and 7-year cumulative occurrence rates of HCC in HBV-associated LC patients were 0.9%, 5.5%, 9.1%, and 18.4%, respectively (Fig. 1). The 1-, 3-, 5-, and 7-year cumulative occurrence rates of HCC in patients with TNF-a857 TT/CT genotype were significantly higher than those in patients with TNF-a857 CC genotype (2.9%, 12.8%, 20.7%, and 42.2% vs. 0%, 3.1%, 5.3%, and 11.4%, respectively; P = 0.013) (Fig. 2A). However, the cumulative occurrence rates of HCC in patients with the TNF-a1031 TT/CT genotype were not different from those in patients with the TNF-a1031 CC genotype (P = 0.279) (Fig. 2B). Also, the cumulative occurrence rates of HCC in patients with the TNF-a308 GG genotype were not different from those in patients with the TNF-a308 GA/AA genotype (P = 0.367) (Fig. 2C). Clinical characteristics were not statistically different from each other between patients associated with HCC (n = 12) and those without HCC
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TNF-a Gene Polymorphism and HBV-HCC
FIGURE 2. Cumulative occurrence rates of HCC in relation to TNF-a promoter gene polymorphisms in HBV-LC patients. A, The cumulative occurrence rates of HCC in patients with the TNF-a857 TT/CT genotype were significantly higher than those with the TNFa857 CC genotype (P = 0.013). This significant association was not observed in other genetic polymorphisms [(TNF-a 1031CC vs. TNF-a 1031TT/CT, P = 0.279) (B) and (TNF-a 308GG vs. TNF-a 308AA/GA, P = 0.367) (C)]. C indicates cytosine; HCC, hepatocellular carcinoma; TNF, tumor necrosis factor; T, thymine.
(n = 194), except CTP class (A or B vs. C, P < 0.05) and MELD score (P < 0.01) (Table 3).
Multivariate Analysis for Predictors of HCC Development in HBV-associated LC Patients In univariate analysis, age [hazard ratio (HR) 1.04, P = 0.18], MELD score (HR 0.92, P = 0.18), and TNF-a-857 gene polymorphism (TT/CT vs. CC, HR 3.88, P = 0.02) were candidates for multivariate analysis (P < 0.2). In multivariate analysis, only TNF-a-857 T allele was an independent risk factor for development of HCC in patients with HBV-associated LC (HR 6.29, P = 0.01) (Table 4). Copyright
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DISCUSSION In the current study, the authors are suggesting a significant host genetic marker for susceptibility to development of HCC in patients with HBV-associated LC. In the crosssectional analysis, the data revealed that the frequency of TNF-a-857 T allele (genotype TNF-a-857 C > T and T > T) was significantly higher in patients associated with HBV-HCC compared with the frequency in patients with HBV-LC (42% vs. 31%). In addition, by the cohort-observational analysis, it was validated that HCC develops much more frequently in HBV-associated LC patients with TNF-a-857 T allele than in those with the TNF-a-857 CC genotype, and thus TNF-a-857 T allele was a significant predictor of HCC development in HBV-associated LC patients.
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TABLE 3. Clinical Characteristics of Hepatitis B Virus–associated Liver Cirrhosis Patients in Accordance With Development of Hepatocellular Carcinoma
Variables
HCC (n = 12)
Age (y)w Sex (male) [n (%)] ALT (IU/L)w CTP class, A or B/C [n (%)] MELD scorew HBeAg, presence HBV-DNA (log copies/mL)w AFP (ng/mL)w Follow-up duration (mo)w
56 7 41 11/1 12 5 5.9 20.5 46
No HCC (n = 194)
(34-60) (58.3) (6-106) (91.7/8.3) (9-21) (41.7) (1.0-8.1) (3-218) (10-87)
55 (21-68) 124 (63.9) 35 (6-680) 111/83 (57.2/42.8) 19 (8-46) 103 (53.1) 4.0 (1.0-9.4) 8.5 (1-2000) 28 (1-122)
P* 0.93 0.76 0.52 < 0.05 < 0.01 0.56 0.34 0.13 0.06
*P-value for difference between the LC and HCC groups. wMedian (range). AFP indicates a-fetoprotein; CTP, Child-Turcotte-Pugh; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis; MELD, Model for End-stage Liver Disease.
Recently, it has been suggested that polymorphisms of cytokine genes may have critical impacts on the outcome of patients with chronic HBV infection.18,19 TNF-a is a pleiotropic cytokine that may exert inflammatory and cytotoxic effects, and can be a potential target for treatment of solid tumors.20 TNF-a gene polymorphism has recently been emphasized to be associated with the progression of LC or the development of HCC.21 In relation to HCC development, it has been suggested that the TNF-a gene, which is located within the major histocompatibility complex class III region, may play a role by producing oxygen-derived free radicals or other reactive oxygen species.22,23 Several studies also have attempted to identify the specific TNF-a gene polymorphisms associated with the development of HCC.11–13 However, there has been no consistent result on this point so far. In the current study, the authors demonstrated that a specific TNF-a promoter gene polymorphism (TNF-a-857 T allele) was closely associated with HCC occurrence in the cross-sectional analysis of a relatively large number of subjects. In addition, the long-term cohort-observational study also validated that HCC developed much more frequently in patients with HBVassociated LC who had TNF-a-857 T allele. To improve the outcomes of patients with HCC, it is crucial to detect HCC earlier in patients with high risk for the development of HCC.15 Until now, a variety of factors,
such as age, sex, association with cirrhosis, serum ALT level, serum HBV-DNA level, or genomic changes of HBV, have been suggested as predictive factors for HCC development in patients chronically infected with HBV.24,25 Of these factors, in addition to the replicative activity or genomic changes of HBV, the association with cirrhosis also has been known to be a dominant risk factor for HCC development.26 Therefore, it is very important to monitor patients with HBV-associated LC for the early detection of HCC. Furthermore, the patients with higher genetic susceptibility to development of HCC should be followed up more closely in clinical practice. The current study indicates that it is necessary to monitor HBV-associated LC patients with the TNF-a857 T allele much more closely for the early detection of HCC. Thus, the results of our study suggest that TNF-a promoter gene polymorphism may be useful to provide HBV-associated LC patients with a tailored management, especially in the surveillance of HCC. In terms of TNF-a gene polymorphisms associated with HCC, a previous meta-analysis reported that TNF-a308G/A polymorphisms may be associated with the development of HCC, while TNF-a-857C/T and -1031T/C polymorphisms were not associated with the occurrence of HCC.27 However, the results of the current study showed a significant association of TNF-a-857C/T polymorphism
TABLE 4. Multivariate Analysis for Predictors of Hepatocellular Carcinoma Development in Patients With Hepatitis B Virus–associated Liver Cirrhosis
Univariate Analysis Variables Age (y) Sex (male) ALT (IU/L) HBeAg, presence HBV-DNA (log copies/mL) CTP class A or B vs. C MELD score TNF-a-857 TT/CT vs. CC TNF-a-1031 TT/CT vs. CC TNF-a-308 GG vs. GA/AA
Multivariate Analysis
HR
95% CI
P
HR
95% CI
P
1.04 0.50 0.99 0.95 1.07 0.31 0.92 3.88 23.28 1.99
0.96-1.12 0.16-1.59 0.99-1.01 0.30-3.01 0.84-1.37 0.04-2.43 0.82-1.04 1.23-12.20 0.01-1.5105 0.43-9.11
0.18 0.24 0.60 0.93 0.58 0.27 0.18 0.02 0.48 0.38
1.07 — — — — — 0.89 6.29 — —
0.99-1.15 — — — — — 0.78-1.01 1.62-24.43 — —
0.11 — — — — — 0.08 0.01 — —
A indicates adenine; ALT, alanine aminotransferase; C, cytosine; CI, confidence interval; CTP, Child-Turcotte-Pugh; G, guanine; HBV, hepatitis B virus; HR, hazard ratio; MELD, Model for End-stage Liver Disease; T, thymine; TNF, tumor necrosis factor.
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with the development of HCC in HBV-associated LC patients, which is supported by the results of a previous Korean study.12 Such differences in the results may arise due to the different characteristics of the subjects,11–13,27 and these may limit the application of the present results to other population. Thus, potential confounding factors such as the association of LC and the genotype of HBV can be minimized in the current study, although the clinical diagnosis of cirrhosis may cause some bias in the present study. Notably, in our multivariate analysis, other viral factors such as serum HBV-DNA titer or the presence of HBeAg were not confounders, indicating that TNF-a-857-T allele has an independent effect on HCC occurrence in HBVassociated LC patients. Unfortunately, in the current study, the functional role of TNF-a promoter gene polymorphism was not evaluated because the serum TNF-a level could not be measured because of lack of available stored serum. However, a previous Korean study had already demonstrated that the serum TNF-a levels were significantly higher in HCC patients with the TNF-a-857-TT/CT genotype, compared with the serum TNF-a levels in patients with TNF-a-857-CC genotype.12 Therefore, it is suggested that the presence of TNF-a-857-T allele may enhance the expression of serum TNF-a in patients with HBV-associated LC patients and thus ultimately increased the risk for the development of HCC. In conclusion, using a cross-sectional and longitudinal analysis, the current study demonstrated the close association of the TNF-a-857 T allele with development of HCC in patients with LC chronically infected with HBV. It suggests that TNF-a-857T allele may be useful as a biomarker for the identification of susceptibility to HCC in these patients. REFERENCES 1. Bosch FX, Ribes J, Diaz M, et al. Primary liver cancer: worldwide incidence and trends. Gastroenterology. 2004; 127(suppl 1):S5–S16. 2. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol. 2001;2:533–543. 3. El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med. 1999;340: 745–750. 4. Taylor-Robinson SD, Foster GR, Arora S, et al. Increase in primary liver cancer in the UK, 1979-94. Lancet. 1997;350: 1142–1143. 5. Velazquez RF, Rodriquez M, Navascue´s CA, et al. Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology. 2003;37:520–527. 6. Fattovich G, Stroffolini T, Zagni I, et al. Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology. 2004;127(suppl 1):S35–S50. 7. Ikeda K, Saitoh S, Koida I, et al. A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis. Hepatology. 1993;18:47–53. 8. Grisham JW. Interspecies comparison of liver carcinogenesis: implications for cancer risk assessment. Carcinogenesis. 1997; 18:59–81.
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