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The global burden of hepatocellular carcinoma (HCC; primary liver cancer) is increasing. HCC is often unaccompanied by clear symptomatology, causing patients to be unaware of their disease. Moreover, effective treatment for those with advanced disease is lacking. As such, effective surveillance and early detection of HCC are essential. However, current screening and surveillance guidelines are not being fully implemented. Some at-risk populations fall outside of the guidelines, and patients who are screened are often not diagnosed at an early enough stage for treatment to be effective. From March 17 to 19, 2015, the Hepatitis B Foundation sponsored a workshop to identify gaps and limitations in current approaches to the detection and treatment of HCC and to define research priorities and opportunities for advocacy. In this Commentary, we summarize areas for further research and action that were discussed throughout the workshop to improve the recognition of liver disease generally, improve the recognition of liver cancer risk, and improve the recognition that screening for HCC makes a life-saving difference. Participants agreed that primary prevention of HCC relies on prevention and treatment of viral hepatitis and other underlying etiologies. Earlier diagnosis (secondary prevention) needs to be substantially improved. Areas for attention include increasing practitioner awareness, better definition of at-risk populations, and improved performance of screening approaches (ultrasound, biomarkers for detection, risk stratification, targeted therapies). The heterogeneous nature of HCC makes it unlikely that a single therapeutic agent will be universally effective. Medical management will benefit from the development of new, targeted treatment approaches., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Dichlorodiphenyltrichloroethane (p,p'-DDT), an organochlorine pesticide known to have deleterious health effects in humans, has been linked to hepatocellular carcinoma (HCC) in rodents. A recent study has reported that p,p'-DDT and its most persistent metabolite, dichlorodiphenyldichloroethylene (p,p'-DDE), may also be associated with HCC in humans. To examine whether there is an association between p,p'-DDT and/or p,p'-DDE in a population at high-risk of developing HCC, a nested case-control study was conducted within the 83,794 person Haimen City Cohort in China. Sera and questionnaire data were collected from all participants between 1992 and 1993. This study included 473 persons who developed HCC and 492 who did not, frequency matched on sex, age and area of residence. p,p'-DDT and p,p'-DDE levels were determined by mass spectrometry. Hepatitis B viral infection status (based on hepatitis B virus surface antigen; HBsAg) was also determined. p,p'-DDT and/or p,p'-DDE serum levels were significantly associated with sex, area of residence, occupation, alcohol consumption and cigarette smoking. Adjusting for age, sex, area of residence, HBsAg, family history of HCC, history of acute hepatitis, smoking, alcohol, occupation (farmer vs. other) and levels of p,p'-DDT or p,p'-DDE, odds ratios (OR) and 95% confidence intervals (CI) were calculated via unconditional logistic regression. Overall, the highest quintile of p,p'-DDT was associated with an increased risk of HCC, OR = 2.96 95% CI; 1.19-7.40. There were no statistically significant associations with p,p'-DDE. Overall, these results suggest that recent exposure to p,p'-DDT may increase risk of HCC., (Copyright © 2012 UICC.)

Fumonisin B1 (FB1), a mycotoxin that contaminates corn in certain climates, has been demonstrated to cause hepatocellular cancer (HCC) in animal models. Whether a relationship between FB1 and HCC exists in humans is not known. To examine the hypothesis, we conducted case-control studies nested within two large cohorts in China; the Haimen City Cohort and the General Population Study of the Nutritional Intervention Trials cohort in Linxian. In the Haimen City Cohort, nail FB1 levels were determined in 271 HCC cases and 280 controls. In the General Population Nutritional Intervention Trial, nail FB1 levels were determined in 72 HCC cases and 147 controls. In each population, odds ratios and 95% confidence intervals (95%CI) from logistic regression models estimated the association between measurable FB1 and HCC, adjusting for hepatitis B virus infection and other factors. A meta-analysis that included both populations was also conducted. The analysis revealed no statistically significant association between FB1 and HCC in either Haimen City (OR=1.10, 95%CI=0.64-1.89) or in Linxian (OR=1.47, 95%CI=0.70-3.07). Similarly, the pooled meta-analysis showed no statistically significant association between FB1 exposure and HCC (OR=1.22, 95%CI=0.79-1.89). These findings, although somewhat preliminary, do not support an associated between FB1 and HCC., (Published by Elsevier Ltd.)

Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.

Background: Studies in experimental animals suggest that low folate levels may play a role in liver damage and hepatocarcinogenesis. To examine this association in humans, folate levels in blood and risk for subsequent liver damage and hepatocellular carcinoma (HCC) were assessed in a population at high risk of liver cancer in China., Methods: Four hundred fifteen hepatitis B surface antigen-positive participants of the Haimen City Cohort were prospectively followed between 1998 and 2002. Serum and RBC folate levels were determined at baseline. Alanine aminotransferase (ALT) and hepatitis B virus DNA levels were measured semiannually. Logistic regression modeling was used to examine the presence of hepatitis B virus DNA and HCC, whereas linear regression with a log-link function was used to examine ALT levels., Results: There was a statistically significant inverse association between serum folate level and ALT level. ALT levels decreased with each quartile increase in serum folate (adjusted odds ratio, 0.86; 95% confidence interval, 0.76-0.97 for the highest compared with the lowest quartile; Ptrend = 0.002). After exclusion of three persons with prevalent HCC, 20 (4.9%) of the 412 study participants developed HCC during follow-up, with a median time between enrollment and HCC diagnosis of 2.66 years (interquartile range, 1.8-4.1). When comparing persons in the lowest quartile RBC folate to persons in all other quartiles, the analysis found that higher RBC folate levels were associated with reduced risk of hepatocarcinogenesis (odds ratio, 0.33, 95% confidence interval, 0.13-0.86; P(trend) = 0.02)., Conclusions: This study suggests that increased folate levels in humans may be inversely associated with the development of liver damage and HCC.

Background and Aims: In a prospective cohort study with 11 yr of follow-up, we assessed the relationship between past hepatitis B virus (HBV) viral load and mortality. Surviving cohort members were evaluated for current liver disease., Methods: We measured HBV viral load by real-time polymerase chain reaction on stored samples from cohort entry (1992-1993) in 2,763 hepatitis B surface antigen (HBsAg)-positive adults. Major end points were death from hepatocellular carcinoma (HCC) or chronic liver disease (CLD). There were 447 deaths. In the 1,683 survivors, we assessed severity of liver disease on a return visit in 2003. Viral load was divided into three categories: undetected (<1.6 x 10(3) copies/mL); low titer (<10(5) copies/mL); and high titer (> or =10(5) copies/mL)., Results: For HCC, there was a significant increase in mortality across viral load categories (p(trend) < 0.001). Compared to the HBV undetected category, the relative risk (RR) for HCC mortality in the low viral load group was 1.7 (95% confidence interval [CI] 0.5-5.7) and 11.2 (3.6-35.0) in the high viral load group. For CLD mortality, the RRs were 1.5 (0.2-12.1) and 15.2 (2.1-109.8), respectively (p(trend) < 0.001). The RR associated with high viral load did not change with increased follow-up time. In surviving cohort members evaluated for liver disease in 2003, there was also a significant association of viral load with disease severity., Conclusion: In this prospective study, viral load is associated with increased mortality from HCC and CLD in HBV-infected subjects. Viral load may be a useful prognostic tool in HBV infection, and interventions aimed at its reduction should be explored.

Changes in N-linked glycosylation are known to occur during the development of cancer. For example, increased branching of oligosaccharides has been associated with metastasis and has been correlated to tumor progression in human cancers of the breast, colon and melanomas. Increases in core fucosylation have also been associated with the development of hepatocellular carcinoma (HCC). Chronic infection with the hepatitis B virus is associated with more than 55% of all cases of hepatocellular carcinoma. We show here that increased levels of core fucosylation can be observed via glycan analysis of total serum and are associated with the development of HCC. In a blinded study, the serum glycoproteins derived from people diagnosed with HBV induced liver cancer were found to possess a dramatically higher level of fucosylation. This change occurs on both immunoglobulin molecules and on other serum glycoproteins. Targeted glycoproteomic analysis was used to identify those glycoproteins that are hyperfucosylated in cancer. In total, 19 proteins were found to be hyperfucosylated in cancer. The potential of these proteins as biomarkers of cancer is discussed.

Selenium (Se) is an essential trace mineral with known anticarcinogenic properties in humans. However, few studies have examined the association between Se nutrient status and risk of liver cancer. We conducted a nested case-control study comparing the Se content in toenail clippings of 166 individuals (154 men, 12 women) with hepatocellular carcinoma (HCC) to 394 healthy controls (360 men, 34 women) in Haimen City, China, where HCC is a leading cause of mortality. Toenail Se concentration was measured by inductively coupled plasma-optical emission spectroscopy. Median toenail Se was lower for HCC cases than controls (p = 0.03). Adjusted odds ratios and 95% confidence intervals for HCC mortality by increasing quartile of toenail Se were 1.00 (reference), 0.58 (0.32-1.03), 0.83 (0.48-1.42) and 0.50 (0.28-0.90), with a marginally significant trend in risk observed (p for trend = 0.06). This inverse association appeared stronger among those who did not consume alcohol and among women. Future studies are needed to examine the interrelationship between Se, viral hepatitis infection and HCC in order to better understand the etiologic mechanisms involved and evaluate the true chemopreventive potential of Se compounds for liver diseases., (Copyright 2005 Wiley-Liss, Inc.)

The role of quantitative viral load in development of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers was evaluated using real-time PCR (TaqMan PCR), a highly sensitive method for quantitative detection of HBV DNA. Serum samples collected at study entry from HCC cases and matched controls were chosen separately from ongoing prospective cohort studies in Senegal, West Africa, and Haimen City, China. For 14 HCC cases and 28 controls from Senegal, the relative risk (RR, 95% CI) of HCC was 15.6 (2.0-124.3) for those positive by the TaqMan PCR assay. Average length of follow-up (study entry to death from HCC) among cases was 2.8 (+/-1.6) years. The paired median difference between cases and controls was 3.8 x 10(4) virions/ml, with cases higher (P = 0.09). In order to clarify the relationship with lower-titer viremia, we selected 55 cases and 55 matched controls from the Chinese cohort all negative for serum HBV DNA by conventional dot blot hybridization. In this group, the RR associated with HBV DNA positivity by TaqMan PCR was 3.1 (1.1-9.2), with an average duration of follow-up of 3.3 (+/-2.1) years. The median difference in quantitative viremia between cases and controls was 6.0 x 10(4) virions/ml, with cases higher (P < 0.0001). Increased risk appeared to be confined to subjects with viral loads >2.3 x 10(4) virions/ml. In conclusion, HBV viremia, except perhaps at extremely low levels, is associated with increased risk for HCC in prospective studies of chronic carriers in two disparate populations., (Copyright 2004 Wiley-Liss, Inc.)

Chronic hepatitis B virus (HBV) carriers with high-titer viremia (>10(5) virions/ml) are at increased risk for hepatocellular carcinoma (HCC). The aim of this study was to determine the relationship between clearance of high-titer viremia and subsequent risk of HCC. The study population was a prospective cohort of 114 adults from Haimen City, China, all HBV DNA(+) at study entry and followed for 797.8 person-years in total. During follow-up, 54 (47.4%) subjects spontaneously cleared high-titer viremia at least once. Of these, 27 were considered to have undergone stable seroconversion, 16 were considered unstable (12 reversions to HBV DNA positivity and 4 multiple clearances), and 11 did not have sufficient follow-up to determine stability. Of the 114 persons, 26 (22.8%) died during follow-up, 21 (18.4%) from HCC. Using Cox proportional hazards models, the RR of HCC death associated with seroconversion was 2.8 (95% CI = 1.1-7.4), controlling for age, sex, family HCC history, history of acute hepatitis, alcohol use and cigarette smoking. In conclusion, fluctuations of high-titer viremia may indicate increased hepatocellular damage and at least short-term increases in HCC risk. Long-term longitudinal studies are needed to clarify this relationship and its potential usefulness as a prognostic marker in chronic HBV infection.

The genetic basis of disease susceptibility can be studied by several means, including research on animal models and epidemiological investigations in humans. The two methods are infrequently used simultaneously, but their joint use may overcome the disadvantages of either method alone. We used both approaches in an attempt to understand the genetic basis of aflatoxin B(1) (AFB(1))-related susceptibility to hepatocellular carcinoma (HCC). Ingestion of AFB(1) is a major risk factor for HCC in many areas of the world where HCC is common. Whether humans vary in their ability to detoxify the active intermediate metabolite of AFB(1), AFB(1)-exo-8,9-epoxide, is not certain but may explain why all exposed individuals do not develop HCC. To determine whether human variability in detoxification may exist, in a study of 231 HCC cases and 256 controls, we genotyped eleven loci in two families of AFB(1) detoxification genes; the glutathione S-transferases (GSTs) and the epoxide hydrolases (EPHX). After adjustment for multiple comparisons, only one polymorphism in the epoxide hydrolase family 2 locus remained significantly associated with HCC (odds ratio = 2.06, 95% confidence interval = 1.13-3.12). To determine whether additional susceptibility loci exist, we developed a mouse model system to examine AFB(1)-induced HCC. Susceptibility of 7-day-old mice from two common inbred strains (C57BL/6J, DBA/2J) was assessed. DBA/2J animals were 3-fold more sensitive to AFB(1)-induced HCC and significantly more sensitive to AFB(1) acute toxicity than were C57BL/6J animals. Analysis of the xenobiotic metabolizing genes in the two strains revealed single nucleotide polymorphisms in three genes, Gsta4, Gstt1, and Ephx1. Although the GSTT1 and EPHX1 loci did not appear to be related to HCC in the total population of the human study, a polymorphism in GSTA4 was significantly related to risk in the male subset. The mouse model also demonstrated that absent or compromised p53 was not necessary for the development of carcinogenesis. These results indicate that the comparison of results from human studies and the AFB(1)-susceptible mouse model may provide new insights into hepatocarcinogenesis.

Many of the emerging technologies for the global evaluation of gene expression, at both the RNA and protein level, are being applied to the problem of finding biomarkers for human disease progression. These analyses can be made difficult, however, by variation between samples that arises from both technical and nondisease related physiological or genetic causes. In an effort to identify serum polypeptides whose presence or absence correlates with the clinical status of patients at high risk for hepatocellular carcinoma (HCC), we have developed a strategy that helps to focus the analysis on meaningful changes in protein levels above the background of variation. For the current study we divided the patient population into four clinically defined diagnostic groups that represent a generally increasing risk for HCC. Chronic infection with hepatitis B virus (HBV) is a major risk factor for HCC and our groups included patients with no indication of liver disease (healthy), those with inactive chronic HBV, those with active chronic HBV, and patients with a diagnosis of HCC and history of chronic HBV infection. Serum polypeptides from these patients were first analyzed in two-dimensional gels by combining the serum from patients in each of the four groups to generate composite gel profiles. Analysis of these composite gels allowed us to identify two relatively abundant features that were reduced in the HCC group as compared to the healthy group. Tryptic fragment mass fingerprinting identified the features as a carboxy terminal fragment of complement C3 and an isoform of apolipoprotein A1. These two features were examined by two-dimensional gel electrophoresis of serum from each individual in the four groups in order to verify that the inter-group differences seen in composite gels reported changes in abundance for most members of the group, rather than extreme changes for a small fraction of the group. These preliminary studies suggest that a proteomic methodology can be used for the identification of serum biomarkers for HCC and other liver disease.

In an 8-year follow-up of a prospective cohort study in Haimen City, China, we sought to identify hepatocellular carcinoma (HCC) risk factors in addition to hepatitis B virus (HBV) infection. Two cohorts of adults between ages 25 and 64 years at study entry were followed from 1992-1993 to 2000. The male cohort included 58,545 men, 15.0% of whom were HBV carriers. The female cohort included 25,340 women, 10.7% of whom were HBV carriers. 434,718 person-years of follow-up were accumulated, and 1092 deaths from HCC occurred. The relationship of potential risk factors measured at study entry to HCC mortality was analyzed using Cox proportional hazards models. For males, HCC mortality was significantly associated with HBV infection [relative risk (RR) 18.8; 95% confidence interval (CI), 15.7-22.5], history of acute hepatitis (RR, 2.3; 95% CI, 2.0-2.7), family history of HCC (RR, 2.3; 95% CI, 1.9-2.7), and occupation as a peasant (RR, 1.5; 95% CI, 1.3-1.8). For females, HCC mortality was significantly associated with HBV infection (RR, 33.5; 95% CI, 17.1-65.5) and acute hepatitis history (RR, 4.7; 95% CI, 3.0-7.5). HCC risk was not significantly associated with alcohol consumption, water source, or staple foods in either sex. There was no association with smoking in males, but there was a positive association for females. Environmental and genetic risk factors besides HBV infection play a significant role in HCC mortality in this extremely high-risk population. Gender differences in HCC mortality and known risk factors are substantial and warrant further study. Identification of risk factors amenable to intervention should be a high priority in the prevention of HCC.

Individuals chronically infected with hepatitis B or C virus (HBV, HCV) are at high risk for the development of hepatocellular carcinoma (HCC), with disease progression occurring relentlessly over many years. The diagnosis of HCC usually occurs at late stages in the disease when there are few effective treatment options and the prognosis for patients with HCC is very poor. The long latency period, together with clearly identified at risk populations, provide opportunities for earlier detection that will allow more timely and effective treatment of this devastating cancer. We are using a proteomic approach to test the hypothesis that changes in the amount of certain serum polypeptides, or changes in their post-translational modifications, can be used to predict the onset of HCC. Advances in the standardization of two dimensional gel electrophoresis (2DE) coupled with computerized image analysis now permit the reproducible resolution of thousands of polypeptides per run. Serum polypeptides from individuals at different stages in the disease continuum are being resolved by 2DE to identify those that change with disease progression. Polypeptides found by this method can be further characterized by mass spectrometry. In addition, the potential for changes in the glycan structure of certain polypeptides to serve as a marker for disease progression can be explored. The proteomic approach is expected to liberate us from the need to "cherry pick" or guess the best biomarkers and let the data tell us which are the best indicators of disease. Information may also be gleaned about the pathobiology of the disease process.

The risk of hepatocellular carcinoma (HCC) varies significantly among hepatitis B virus (HBV) carriers from different geographic regions. We compared serological markers of HBV infection in adult male carriers from Haimen City, China and Senegal, West Africa, where the prevalence of chronic infection is similar. HCC mortality among HBV carriers is much higher in Haimen City than it is in Senegal (age-standardized rate, 878 versus 68 per l0(5) person-years). A dramatic difference was observed when HBV DNA levels in serum were assessed among carriers by Southern blot. In the Senegalese group (n = 289), 14.5% were HBV DNA positive by Southern blot in their 20s, and this percentage declined in each subsequent decade of age to 3.3, 2.9, and 0% thereafter. In the Chinese group (n = 285), a higher prevalence of HBV DNA positivity and a less consistent reduction were seen; 29.4% were positive in their 20s, and 30.2, 23.6, and 20.6%, respectively, were positive in each subsequent decade of age. Among 102 male Asian-American HBV carriers, the prevalence of HBV DNA positivity was intermediate between the Chinese and Senegalese populations (36.8, 10.7, 3.0, and 4.6% in each subsequent decade of age). Viral titers were similar among those who were HBV DNA positive in all three populations [median value, 10(7) virions/ml (range, 10(6)-10(9) virions/ml)]. The presence of HBV DNA in serum was positively associated with serum glutathione S-transferase, a marker of liver damage. These findings suggest that the more prolonged maintenance of productive virus infection in the Chinese carriers compared with the Senegalese carriers may explain their higher risk of HCC. This profound difference in the natural history of chronic infection may be due to earlier age of infection in China or to as yet unknown environmental or genetic factors.

Hepatitis B virus (HBV) and aflatoxin B1 represent the main risk factors for the development of hepatocellular carcinoma (HCC) in areas endemic for liver cancer. The glutathione S-transferases (GSTs) are a family of Phase II detoxification enzymes that catalyze the conjugation of a wide variety of endogenous and exogenous toxins, including aflatoxin B1, with glutathione. This study characterizes the GST isoenzyme composition (alpha, mu, and pi) of both HBV-infected normal hepatic tissues and HCCs. Analysis of matched pairs of hepatic tissue (normal and tumor) from 32 HCC patients indicated that total GST activity was significantly higher in normal tissues than in tumor tissues, although the percentage of samples expressing GST alpha and pi was equivalent. GST mu was detected by Western blot in the normal tissue from 87.5% of the subjects possessing the GST M1 gene but only 28.6% of the corresponding tumor tissues. The GST activity of normal tissue from GST M1 null patients was significantly decreased as compared to that of subjects possessing the GST M1 gene (264.6 and 422.2 nmol/min/mg, respectively; P = 0.005). GST pi appeared to be overexpressed in the normal tissue of GST M1 null patients, a potential compensatory effect. Patients positive for HBV DNA had significantly lower GST activity than those who were HBV negative (302.1 versus 450.0 nmol/min/mg, respectively; P = 0.02). These results suggest that cellular protection within the human liver is compromised by HBV infection and further decreased during hepatocellular tumorigenesis.

To identify environmental, viral, and genetic factors that may influence the risk of developing hepatocellular carcinoma (HCC), large prospective studies are being conducted in Haimen City, China and Senegal, and a case-control study of genetic variation in the detoxification of aflatoxin-B1 was carried out in Shanghai, China. Analysis of 78 HCCs that have occurred among 51,020 men enrolled in a large prospective study in Haimen City, China showed a strong association of HCC with chronic hepatitis B virus (HBV) infection. There were also significant associations of HCC risk with occupation (farming), history of a clinical episode of hepatitis in adulthood, and a family history of HCC. Study of 52 HCC cases and 116 controls for genetic polymorphisms and HCC risk showed a significant association with epoxide hydrolase (EPHX) mutant alleles (1/2, 2/2) and a borderline association with homozygous deletion of the glutathione-S-transferase mu (GSTM1) gene. There was a multiplicative interaction of these polymorphisms with chronic HBV infection such that HBsAg-positive persons who were GSTM1 null and were EPHX 1/2 or 2/2 had 135 times the risk of HCC as HBsAg-negative persons with the wild type genotypes for GSTM1 and EPHX. The risk of HCC is not uniform among persons with chronic HBV or HCV infections. Studies of genetic, viral, and environmental interactions may permit identification of those individuals at highest risk within groups at increased risk of HCC. Prevention strategies could then be targeted at those individuals.

To identify specific environmental, viral, and genetic risk factors for hepatocellular carcinoma (HCC) and the interaction of such factors, we are conducting a prospective study in a high-incidence area of China. Questionnaires were completed and biosamples collected by 60,984 men ages 30-64 years, at study entry. Within 2.5 years, 183 deaths from HCC had occurred. Each HCC case was matched with 5 controls and compared for items on the questionnaire. In addition to chronic hepatitis B virus (HBV) infection, the significant risk factors were: occupation (peasant), corn consumption (in the 1970s), family history of HCC, and history of an episode of acute hepatitis as an adult. HBV, consumption of aflatoxins, a genetic factor, and possibly a second hepatitis virus infection contribute to the risk of HCC.