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Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection†
Version of Record online: 29 MAY 2003
Copyright © 2003 American Cancer Society
Volume 97, Issue 12, pages 3036–3043, 15 June 2003
How to Cite
Ohata, K., Hamasaki, K., Toriyama, K., Matsumoto, K., Saeki, A., Yanagi, K., Abiru, S., Nakagawa, Y., Shigeno, M., Miyazoe, S., Ichikawa, T., Ishikawa, H., Nakao, K. and Eguchi, K. (2003), Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Cancer, 97: 3036–3043. doi: 10.1002/cncr.11427
See editorial on pages 2948–50, this issue.
- Issue online: 29 MAY 2003
- Version of Record online: 29 MAY 2003
- Manuscript Accepted: 17 FEB 2003
- Manuscript Revised: 5 JAN 2003
- Manuscript Received: 2 DEC 2002
- core protein;
- hepatic steatosis;
- hepatitis C virus;
- hepatocellular carcinoma
Hepatic steatosis is one of the histopathologic features of chronic hepatitis C. It was reported recently that the expression of hepatitis C virus (HCV) core protein in transgenic mice induced hepatocellular carcinoma (HCC) in association with steatosis. The objective of this study was to determine the relation between hepatic steatosis and hepatocarcinogenesis in patients with chronic HCV infection.
The authors studied 161 patients with chronic HCV infection who were diagnosed at Nagasaki University Hospital, Nagasaki, Japan, between January 1980 and December 1999. Age, gender, body mass index (BMI), habitual drinking, diabetes mellitus, serum alanine aminotransferase (ALT) level, HCV serotype, serum level of HCV core protein, interferon (IFN) treatment, hepatic fibrosis inflammation, and hepatic steatosis were studied with regard to their significance in the development of HCC using univariate and multivariate analyses.
The cumulative incidence rates of HCC were 24%, 51%, and 63% at 5 years, 10 years, and 15 years, respectively. Multivariate analysis identified hepatic steatosis, together with aging, cirrhosis, and no IFN treatment, as independent and significant risk factors for HCC (P = 0.0135, P = 0.0390, P = 0.0068, and P = 0.0142, respectively). In addition, hepatic steatosis was correlated with BMI, serum ALT levels, and triglyceride levels.
The findings of the current study indicate that hepatic steatosis is a risk factor for HCC in patients with chronic HCV infection. Patients with chronic HCV and hepatic steatosis should be monitored carefully for HCC. Cancer 2003;97:3036–43. © 2003 American Cancer Society.
Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The incidence of HCC has increased substantially in Japan during the past several decades1, 2 and also has increased slightly in the United Kingdom, France,3, 4 and the U.S.5 These increases in the incidence of HCC have been attributed to the increased numbers of patients with hepatitis C virus (HCV)-associated liver cirrhosis. Several risk factors for the development of HCV-associated HCC have been reported, such as aging, gender, total alcohol intake,6, 7 cirrhosis,8 irregular regeneration of hepatocytes,9, 10 and HCV genotype.11, 12 Recent studies have indicated that interferon (IFN) treatment can reduce the incidence of HCC.13, 14 The identification of additional variables associated with changes in the risk of HCC would be particularly important with regard to optimizing preventive medical programs.
Hepatic steatosis is one of the histopathologic features of chronic hepatitis C.15–18 According to previous reports, the prevalence of hepatic steatosis ranges from 31 to 72%.15–20 The pathogenesis of hepatic steatosis in patients with chronic HCV infection has been postulated recently. Both in vitro studies and in vivo studies have shown that HCV core protein expression either in cell cultures or in transgenic mice led to the development of hepatic steatosis, contributing to carcinogenesis.21–23 In addition, hyperplasia of hepatocytes has been described in ob/ob mice with fatty liver disease, suggestive of malignant changes secondary to hepatic steatosis.24 These observations suggest that hepatic steatosis may be involved in the development of HCC.
The objective of this study was to determine whether hepatic steatosis is an independent risk factor in the development of HCC in patients with chronic HCV. To this end, univariate and multivariate analyses were performed to identify independent risk factors, including hepatic steatosis, for HCC in 161 patients with chronic HCV infection.
MATERIALS AND METHODS
Liver biopsies were obtained from 560 patients who had no detectable HCC at the Nagasaki University Hospital, Nagasaki, Japan, between January 1980 and December 1999. Patients with bleeding tendencies or clinically evident cirrhosis associated with ascites or hepatic encephalopathy were excluded from the biopsy procedure. Of these 560 patients, 218 patients who were diagnosed with chronic hepatitis or cirrhosis had confirmed negative results for hepatitis B surface antigen (HBsAg) but positive results for the antibody to HCV (anti-HCV). Of these 218 patients, 161 patients who were followed at the Nagasaki University Hospital for > 6 months were enrolled in this study. Patients who had other causes of liver disease, such as primary biliary cirrhosis or autoimmune hepatitis, were excluded from this study. The body mass index (BMI) was calculated as the patient's weight (kilograms) divided by height (meters) squared (kg/m2). At the time patients underwent liver biopsy, information regarding alcohol consumption was obtained through an interview by physicians. Habitual drinking was defined as an average daily consumption of an amount equivalent to 80 g per day of pure ethanol over a period of > 5 years. Diabetes mellitus was diagnosed based on fasting serum glucose levels that exceeded 7.8 mmol/L (140 mg/dL), abnormal results for a 75-g oral glucose tolerant test, or the need for insulin or an oral antihyperglycemic drug to control glucose levels. Informed consent was obtained from each patient at the time they underwent liver biopsy.
A serum sample was obtained from each patient at the time of liver biopsy and stored at − 40 °C for later analysis of viral markers. Anti-HCV was determined using a second-generatoin or third-generation enzyme-linked immunosorbent assay (Ortho Diagnostics Systems, Tokyo, Japan). HCV serotype was determined using the genotyping enzyme-linked immunosorbent assay (International Reagents Corporation, Tokyo, Japan) reported by Tanaka et al.25 In this assay, HCV serotypes 1 and 2 correspond to genotypes 1 and 2 of Simmonds classification, respectively. Serum HCV core protein level was measured by enzyme immunoassay (Ortho Clinical Diagnostics, Tokyo, Japan) for quantitative evaluation of HCV viremia.26
Follow-Up of Patients
Clinical evaluation and biochemical tests were performed every 1–3 months. Patients received ultrasonography or computed tomography studies of the liver at least every 3–6 months. The diagnosis of HCC was based on the histopathologic findings in tumor tissue or on the characteristic appearance on ultrasonography, computed tomography, and hepatic arteriography. The endpoint used in the current study was the appearance of HCC, and the reference date used was December 31, 2000. The numbers of patients who were followed without showing any appearance of HCC for 5 years, 10 years, and 15 years after liver biopsy were 90 patients, 29 patients, and 16 patients, respectively. Overall, 70 patients were followed until the endpoint of this study. The average observation period was 76.5 months (6.4 years).
Seventy-one patients received IFN treatment during the follow-up period. IFN therapy was initiated within 1 year after liver biopsy, and each patient was followed for at least 48 weeks after the completion of IFN therapy. A sustained response (SR) was defined as negative results for serum HCV RNA according to polymerase chain reaction analysis and normal alanine aminotransferase (ALT) levels for > 24 weeks after completion of IFN therapy. A nonresponse (NR) was defined as any other response.
Histopathologic Examination of the Liver
Liver biopsy specimens were fixed in 10% formalin, embedded in paraffin, cut to thickness of 4 μm, and stained with hematoxylin-eosin and Azan. All liver tissue specimens were evaluated by one pathologist (K. T.) who was unaware of the clinical condition of the patient. Liver histology was evaluated according to the degree of fibrosis, necroinflammatory activity, and steatosis. The extent of fibrosis (staging) and the degree of necroinflammatory activity (grading) were classified according to Desmet et al.27 as follows: F1 (periportal expansion), F2 (portoportal septa), and F3 (portocentral linkage or bridging fibrosis) were categorized as noncirrhosis; and F4 (cirrhosis) was categorized as cirrhosis. In terms of necroinflammatory activity, A1 represented mild activity and A2 or A3 represented severe activity. The severity of steatosis was graded as Grade 0 (absent), Grade 1 (1–10% of hepatocytes affected), Grade 2 (11–30% of hepatocytes affected), or Grade 3 (> 30% of hepatocytes affected).
Data were expressed as the mean ± standard deviation for continuous variables and as counts for categorical variables. Continuous variables and categorical variables were compared using the Student t test and the chi-square test, respectively. Cumulative incidence curves were determined with the Kaplan–Meier method, and the differences between groups were assessed with the log-rank test. Univariate and multivariate analyses of the risk ratios for the occurrence of HCC were conducted by using Cox proportional hazards regression analysis. The factors examined included age, gender, BMI, habitual drinking, diabetes mellitus, serum ALT level, HCV serotype, serum HCV core protein level, IFN treatment during follow-up, histopathologic staging (noncirrhosis or cirrhosis), histopathologic grading (mild activity or severe activity), and hepatic steatosis (absence or presence). In addition, factors that were correlated with hepatic steatosis were analyzed. All P values were two-tailed, and P values < 0.05 were considered significant. Statistical analysis was performed by using Stat View software (version 5.0; SAS Institute Inc., Cary, NC).
Table 1 shows the clinical features of patients at study entry. The study included 106 males and 55 females with a mean age of 53 years (range, 19–78 years). The mean BMI was 22.7 kg/m2, and 31 patients had a BMI > 25 kg/m2 (19%). This study included 11 patients (7%) who were habitual drinkers and 26 patients (16%) with diabetes mellitus. Serotype was available for 126 patients; 106 patients had serotype 1, 17 patients had serotype 2, and serotype 3 was not determined. The mean level of serum HCV core protein in 143 patients was 1623.9 fmol/L (range, 0–20000 fmol/L). During follow-up, 71 of 161 patients received IFN treatment: Twenty of those 71 patients (28%) had an SR, and 51 patients (72%) had NR. None of the patients with an SR and eight patients with NR developed HCC. Of the 161 patients examined, only 0 patients, 5 patients, and 6 patients had serum total bilirubin levels > 2.0 mg/dL, serum albumin levels < 3.5g/dL, and prothrombin time < 80%, respectively.
|Characteristic||No of patients (%)|
|Median age (yrs; mean ± SD)||53 ± 12|
|BMI (kg/m2, mean ± SD)||22.7 ± 0.24|
|No. of patients with BMI > 25 kg/m2||31 (19)|
|Habitual drinking||11 (7)|
|Diabetes mellitus||26 (16)|
|ALT (IU/L; mean ± SD)||103.4 ± 94.8|
|γ-GTP (IU/L; mean ± SD)||78.3 ± 74.9|
|Triglyceride (mg/dL; mean ± SD)||102.3 ± 53.0|
|Cholesterol (mg/dL; mean ± SD)||162.9 ± 34.5|
|HCV core protein(fmol/L mean ± SD)||1623.9 ± 2668.2|
Examples of histopathologic findings are shown in Table 2. According to the histopathologic staging of the extent of fibrosis, 45 patients (28%) had F1 fibrosis, 28 patients (17%) had F2 fibrosis, 25 patients (16%) had F3 fibrosis, and 63 patients (39%) had F4 fibrosis. According to the histopathologic grading of necroinflammatory activity, 79 patients (49%) had A1 activity, 79 patients (49%) had A2 activity, and 3 patients (2%) had A3 activity. Hepatic steatosis was present in 91 of 161 patients (56%). The distribution of steatosis showed that 71 patients (43%) had Grade 0 steatosis, 79 patients (49%) had Grade 1 steatosis, 8 patients (6%) had Grade 2 steatosis, and 3 patients (2%) had Grade 3 steatosis.
|Characteristic||No. of patients (%)|
|Distribution of stage of fibrosis|
|Distribution of grade of inflammation|
|Distribution of grade of steatosis|
|0 (absent)||71 (44)|
|1 (1–10% of hepatocytes affected)||79 (49)|
|2 (11–30% of hepatocytes affected)||8 (5)|
|3 (> 30% of hepatocytes affected)||3 (2)|
Analysis of Clinicopathologic Factors that Influence the Development of HCC
Figure 1 shows that the cumulative incidence rates of HCC were 24% 5 years after biopsy, 51% 10 years after biopsy, and 63% 15 years after patients underwent liver biopsy. Recently, Hashem and El-Serag28 reported that the incidence of HCC in patients with chronic HCV tended to be greater in Japan compared with other countries. In a large retrospective cohort study from Japan, the annual incidence of HCC was 0.5% in patients with HCV who had low-grade fibrosis and 7.9% in patients who had severe fibrosis.13 Those results are in agreement with the current findings drawn from a cohort in which > 50% of patients had severe fibrosis (F3 or F4).
To determine the factors that may influence the development of HCC, a Cox proportional hazards regression analysis was performed. Based on univariate analysis, the following four factors influenced the incidence of HCC significantly: patient age at the time of liver biopsy (relative risk, 5.52; 95% confidence interval [95% CI], 2.51–12.04 [P < 0.0001]), IFN treatment (relative risk: 0.18; 95% CI, 0.09–0.38 [P < 0.0001]), histopathologic staging (cirrhosis: relative risk, 2.65; 95%CI, 1.62–4.34 [P = 0.0001]), and hepatic steatosis (steatosis present: relative risk, 2.56; 95% CI, 1.49–4.41 [P = 0.0007]) (Table 3). Furthermore, a multivariate analysis of determinants of HCC was applied using a Cox regression model. Age at the time of liver biopsy (relative risk, 2.96; 95% CI, 1.06–8.26 [P = 0.0390]), IFN treatment (relative risk, 0.30; 95% CI, 0.12–0.79 [P = 0.0142]), cirrhosis (relative risk, 3.21; 95% CI, 1.38–7.48 [P = 0.0068]), and hepatic steatosis (relative risk, 2.81; 95% CI, 1.24–6.37 [P = 0.0135]) were identified as statistically independent risk factors (Table 4). Figure 2 shows the cumulative incidence of HCC based on the prevalence of hepatic steatosis. The cumulative incidence rate of HCC in 90 patients who had steatosis was significantly greater compared with the rate in 71 patients who did not have steatosis (P = 0.0012).
|Characteristic||Risk ratio||95% CI||P value|
|Age (compared with ≤ 50 yrs)||5.52||2.51–12.04||0.0001a|
|Gender (compared with female)||1.30||0.76–2.23||0.3435|
|Diabetes mellitus (compared with absent)||1.46||0.82–2.60||0.2003|
|BMI (compared with < 24 kg/m2)||1.04||0.63–1.73||0.8690|
|Habitual drinking (compared with no habitual drinking)||1.22||0.51–2.88||0.6581|
|ALT (compared with < 90 IU/L)||1.56||0.96–2.53||0.0725|
|HCV serotype (compared with serotype 2)||1.02||0.40–2.62||0.9620|
|HCV core (compared with 500 fmol/L)||0.85||0.51–1.42||0.5365|
|IFN (compared with no treatment)||0.18||0.09–0.38||0.0001a|
|Cirrhosis (compared with no cirrhosis)||2.65||1.62–4.34||0.0001a|
|Grading (compared with mild)||1.37||0.85–2.23||0.1995|
|Steatosis (compared with absent)||2.56||1.49–4.41||0.0007a|
|Characteristic||Risk ratio||95% CI||P value|
|Age (compared with ≤ 50 yrs)||2.96||1.06–8.26||0.0390a|
|Gender (compared with female)||1.45||0.67–3.13||0.3484|
|Diabetes mellitus (compared with absent)||1.58||0.62–3.99||0.3351|
|BMI (compared with < 24 kg/m2)||1.67||0.80–3.46||0.1700|
|Habitual drinking (compared with no habitual drinking)||1.56||0.38–6.33||0.5375|
|ALT (compared with < 90 IU/L)||1.17||0.59–2.33||0.6596|
|HCV serotype (compared with serotype 2)||1.15||0.38–3.48||0.8073|
|HCV core (compared with < 500 fmol/L)||1.24||0.60–2.56||0.5698|
|IFN (compared with no treatment)||0.30||0.12–0.79||0.0142a|
|Cirrhosis (compared with no cirrhosis)||3.21||1.38–7.48||0.0068a|
|Grading (compared with mild)||1.01||0.44–2.29||0.9887|
|Steatosis (compared with absent)||2.81||1.24–6.37||0.0135a|
Analysis of Factors Associated with Hepatic Steatosis
The characteristics and laboratory features of patients with and without steatosis are summarized in Table 5. The Student t test or the chi-square test was performed to evaluate the various factors that affected the presence of hepatic steatosis. The results showed significant associations between the presence of steatosis and BMI (P = 0.0067), ALT levels (P = 0.026), and triglyceride levels (P = 0.0044) (Table 5). There was no correlation between BMI and the grade of steatosis (Table 6). Diabetes and habitual drinking were not correlated with the presence of steatosis.
|No. of patients||71||90||—|
|Median age (yrs; mean ± SD)||51 ± 13.8||55 ± 10.1||0.0540|
|BMI (kg/m2; mean ± SD)||21.9 ± 2.83||23.3 ± 3.14||0.0067a|
|Habitual drinking (%)||5 (7)||6 (7)||0.9056|
|Diabetes mellitus (%)||11 (16)||15 (17)||0.8200|
|AST (IU/L; mean ± SD)||77.6 ± 78.5||97.9 ± 89.4||0.1500|
|ALT (IU/L; mean ± SD)||84.8 ± 72.2||118.2 ± 107.7||0.0260a|
|γ-GTP (IU/L; mean ± SD)||71.3 ± 85.4||83.9 ± 65.6||0.3000|
|Triglyceride (mg/dL; mean ± SD)||88.4 ± 30.9||113.2 ± 63.5||0.0044a|
|Cholesterol (mg/dL; mean ± SD)||161.8 ± 35.1||163.7 ± 34.2||0.7300|
|HCV core (fmol/L; mean ± SD)||2154.1 ± 3990.0||1376.5 ± 1942.2||0.1309|
|Staging (no cirrhosis:cirrhosis)||48:50||23:40||0.1198|
|BMI (kg/m2)||Grade of hepatic steatosisa||Total|
|Below normal (BMI < 18.5)||10||4||1||0||15|
|Normal (BMI 18.5–25.0)||51||58||3||1||113|
|Overweight (BMI 25.0–30.0)||10||15||4||—||29|
|Obese (BMI ≥ 30.0)||—||2||—||2||4|
Several histopathologic features characterize chronic hepatitis C and distinguish it from other forms of hepatitis, including bile duct damage, lymphoid follicles, and steatosis.15–18 In a comparative study, steatosis was observed in 72% of patients who had chronic hepatitis C, compared with 19% of patients who had autoimmune hepatitis.16 In another study, fatty changes were observed at a significantly greater rate in patients who had chronic hepatitis C compared with patients who had chronic hepatitis B.17 The prevalence of steatosis observed in the current study was compatible with previous reports, although most patients with steatosis had the low-grade variety.
In the current study, we analyzed the factors that affected the incidence of HCC in patients with chronic HCV infection who were diagnosed by liver biopsy. Univariate and multivariate analyses identified hepatic steatosis, together with aging, cirrhosis, and no IFN treatment, as significant independent risk factors for HCC. Recently, it was shown that the expression of HCV core protein in transgenic mice induced hepatic steatosis and HCC without inflammation.23, 29 Lerat et al.30 provided additional evidence in support of the direct causative roles of both the structural and nonstructural proteins of HCV in steatosis and carcinogenesis. In addition, several investigators have revealed that hepatic steatosis, including steatosis induced by HCV core protein, predisposes to lipid peroxidation and excess free-radical activity with the potential risk of genomic mutations.31 In addition, Marreto et al.32 reported that nonalcoholic fatty liver disease may be a common underlying liver disease in patients with HCC in the U.S. Those studies support our results showing that hepatic steatosis plays a role in hepatocarcinogenesis in patients with chronic HCV.
Hepatic steatosis is seen frequently in obese patients and in patients with diabetes mellitus.33–35 Several lines of evidence indicate that diabetes mellitus is a risk factor for HCC in patients with cirrhosis,36 although the exact correlation remains to be determined. Nair et al.37 also showed that obesity is an independent risk factor for HCC in patients with alcoholic liver disease and cryptogenic cirrhosis, but not in patients with viral hepatitis, based on an analysis of explanted liver specimens in patients with advanced cirrhosis. We did not find direct effects of diabetes or obesity on hepatocarcinogenesis. This may be attributable to the fact that our study did not include patients who had advanced cirrhosis at baseline and/or that the studied population included relatively small numbers of obese patients and patients with diabetes.
Hepatic steatosis has been cited as a characteristic feature of chronic HCV, although it remains uncertain whether it is related directly to the virus or is secondary to host factors. Several investigators have reported that an increase in BMI or visceral obesity is related to steatosis in patients with chronic hepatitis C.38, 39 In concordance with these observations, we found a significant correlation between hepatic steatosis and BMI, although the prevalence of a BMI > 25 kg/m2 was low in our patients. In addition, higher serum levels of ALT and triglycerides were observed in patients who had steatosis compared with patients who were without steatosis. These results suggest that weight reduction in patients with chronic hepatitis C may be associated with a reduction in steatosis and abnormal liver enzymes. However, it is unclear whether weight reduction leads to a favorable outcome in patients with chronic HCV, because BMI had no significant effect on the development of HCC in our study. The lack of such association also may suggest that additional factors are associated with the presence of steatosis, although other host factors (such as age, gender, habitual drinking, diabetes, and cholesterol levels) were not statistically significant. Polymorphism of one or more host factors may provide an intriguing and likely mechanism. Several such factors have been described in various experimental models of steatosis and steatohepatitis, including tumor necrosis factor40 and peroxisome proliferator-activated receptor α.41
Hourigan et al.38 showed a significant association between steatosis and hepatic fibrosis, suggesting that steatosis is an important cofactor in accelerating the development of hepatic fibrosis and inflammatory activity. In the current study, however, no relation was found between these factors. This discrepancy may be attributable to differences in the background of patients studied, including a lower prevalence of steatosis > Grade 2 in our study compared with previous reports. Alternatively, our results may suggest that steatosis contributes to hepatocarcinogenesis independent of necroinflammatory reaction, as suggested in experimental models.23
Recent studies have reported that hepatic steatosis is more likely to be associated with genotype 3 than with other HCV genotypes.42, 43 We could not find an effect for HCV type on hepatic steatosis, because the majority of patients studied were infected with serotype 1, which corresponds to genotype 1. Other studies also demonstrated that viral load was associated with hepatic steatosis in patients infected with HCV genotype 3 but not with genotype 1.42, 43 In agreement with those observations, our results showed no significant difference in serum HCV core protein levels between patients with and without hepatic steatosis, most of whom were infected with serotype 1. Fujie et al.44 showed that the intrahepatic concentration of HCV, but not the serum concentration, was associated with the development of steatosis in the liver of patients with chronic HCV. Therefore, it is possible that HCV itself or the core protein may play a role in the pathogenesis of steatosis in patients with chronic HCV, although information on intrahepatic HCV RNA was not available in the current study.
The results of the current study demonstrate that hepatic steatosis is an independent risk factor for HCC in patients with chronic HCV infection, although the factors responsible for steatosis could not be identified clearly. Therefore, our data emphasize the need for the careful monitoring of patients with chronic HCV and hepatic steatosis for the development of HCC. Because of the small number of patients studied, the current findings need to be confirmed in a larger population of patients with chronic HCV infection.
The authors thank Kokichi Arisawa, M.D., for his advice regarding statistical analysis. They also thank Dr. F. G. Issa for the careful reading and editing of the article.
- 13Interferon therapy reduces the risk for hepatocellular carcinoma: national surveillance program of cirrhotic and noncirrhotic patients with chronic hepatitis C in Japan. IHIT Study Group. Inhibition of hepatocarcinogenesis by interferon therapy. Ann Intern Med. 1999; 131: 174–181., , , et al.
- 35Association between nonalcoholic fatty liver, markers of obesity, and serum leptin level in young adults. Am J Gastroenterol. 2002; 97: 1796–1801., , , et al.Direct Link: