Hepatic steatosis is associated with increased frequency of hepatocellular carcinoma in patients with hepatitis C-related cirrhosis

Authors


Abstract

BACKGROUND.

Chronic hepatitis C can result in fatty changes in the liver. Previous studies have suggested that hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with hepatitis C virus (HCV) infection. The authors sought to determine whether hepatic steatosis is associated with hepatocellular carcinoma (HCC) in a cohort of patients with hepatitis C-related cirrhosis.

METHODS.

The authors retrospectively identified 94 consecutive patients with hepatitis C cirrhosis who underwent liver transplantation from 1992 to 2005 and had pathology available for review. Of these, 32 had evidence of HCC, and 62 had no HCC on explant histology. All explant specimens were graded again for steatosis by a single, blinded pathologist. Steatosis, age, sex, body mass index, HCV RNA, HCV genotype, Model for End-Stage Liver Disease (MELD) score, chronic alcohol use, and diabetes were examined in univariate and multivariate analyses for association with HCC.

RESULTS.

In total, 69% of patients in the HCC group and 50% of patients in the control group had evidence of steatosis (1+) on histology. Odds ratios for the development of HCC for each grade of steatosis compared with grade 0 were as follows: grade 1 (1.61 [0.6–4.3]), grade 2 (3.68 [1.1–12.8]), and grade 3 or 4 (8.02 [0.6–108.3]) (P = .03 for the trend). In univariate analysis, there was a significant association between increasing steatosis grade (P = .03), older age (56 years vs 49 years; P < .02), higher aspartate aminotransferase (122.5 U/L vs 91.5 U/L; P = .005), higher alanine aminotransferase (95.8 U/L vs 57.2 U/L; P = .002), higher alpha-fetoprotein (113.5 ng/mL vs 17.8 ng/mL; P < .001), lower median HCV RNA (239,000 IU/mL vs 496,500 IU/mL; P = .02), higher biologic MELD score (21.8 vs 20.3; P = .03), and risk of HCC. In multivariate analysis, age (P = .02), AFP (P = .007), and steatosis (P = .045) were significantly associated with HCC.

CONCLUSIONS.

In patients with HCV-related cirrhosis, the presence of hepatic steatosis is independently associated with the development of hepatocellular carcinoma. These findings suggest that steatosis poses an additional risk for HCC and that increased vigilance should be practiced in surveillance of persons with both HCV and steatosis. Cancer 2007. © 2007 American Cancer Society.

Hepatocellular carcinoma (HCC) is a life-threatening complication of hepatic cirrhosis. Over the last 2 decades, there has been a substantial increase in incidence of HCC in the US, which is likely due to the high prevalence and long duration of hepatitis C virus (HCV) among the general population as well as higher prevalence of obesity in society.1, 2 Known risk factors in the development of hepatocellular carcinoma include age, sex, HCV, hepatitis B virus (HBV), diabetes, and non-Caucasian ethnicity.3, 4, 5 After adjustment for alcohol use, recent data also suggest an increased risk of HCC in obese patients compared with the general population.6, 7, 8

Fatty changes that occur in the liver are termed steatosis and are associated with ethanol use, obesity, HCV infection, and numerous medications. When pathologic examination reveals steatosis with or without associated inflammation in the absence of alcohol use, this is termed nonalcoholic fatty liver disease (NAFLD). There have been several small case series linking obesity, nonalcoholic fatty liver disease (NAFLD), and HCC, even in the absence of cirrhosis.9, 10, 11 In addition, nearly half of patients who are diagnosed with cryptogenic cirrhosis and HCC will have NAFLD at the time of biopsy, indicating that there are probably more cases of NAFLD leading to HCC than previously thought.12, 13 Although the mechanism of carcinogenesis is unknown, animal models imply that obesity and fatty liver disease lead to hepatic hyperplasia and decreased apoptosis.14 As such, NAFLD may provide a prototypical example of how steatohepatitis can progress to carcinoma.

Studies have also shown an association of chronic hepatitis C virus infection and hepatic steatosis.15, 16, 17 Factors that are thought to promote steatosis include: body mass index (BMI), alcohol use, and genotype 3 hepatitis C.16, 18 Although it is well established that patients with HCV infection are at increased risk for the development of HCC, it is unclear what role steatosis plays in the development of HCC. Steatosis promotes lipid peroxidation and reactive oxidative species, and may be a common mediator of HCC in hepatitis C, NAFLD, and alcoholic liver disease.

Recently, hepatic steatosis was described in a prospective observational study as an independent risk factor for HCC in patients with hepatitis C virus infection, although an additional study failed to confirm these findings.19, 20 Steatosis has not been examined as a predictor of HCC in patients with chronic hepatitis C cirrhosis, or in a US population with a higher prevalence of diabetes, obesity, and alcohol use. We designed a retrospective case-control study to determine if steatosis was associated with HCC in patients with HCV-related cirrhosis who underwent liver transplantation.

MATERIALS AND METHODS

Patient Population

The protocol was approved by the institutional review board at the Massachusetts General Hospital. We retrospectively identified as subjects all patients who underwent liver transplantation at our institution between October 1992 and September 2005. Inclusion criteria included patients older than 18 years, hepatitis C antibody-positive serology, and cirrhosis on pathology of the explanted liver. Exclusion criteria included age younger than 18 years, fulminant hepatic failure, and patients with other known causes of liver disease, such as evidence of current hepatitis B infection, HFE-related hemochromatosis, primary biliary cirrhosis, and primary sclerosing cholangitis. All subjects who met eligibility criteria and had their liver transplantation histology available for review were assigned on the basis of their original explanted liver pathology report to either a group with hepatocellular carcinoma or a control group.

Data Collection

Data were collected on age at transplant, race, sex, HCV genotype, presence of HCC based on pathologic examination of the transplanted liver, and stage of fibrosis. Additional data collected by chart review included alcohol use and presence of diabetes before transplantation. Patients were deemed to have chronic alcohol use if they were identified by a physician in their medical records as having alcohol dependence or alcohol-induced liver disease. Diabetes was defined as a fasting glucose >126 mg/dL, or a patient taking insulin or oral hypoglycemic agents. Serum aspartate trasaminase, alanine aminotransferase, alpha-fetoprotein, hepatitis C virus RNA, fasting cholesterol, and fasting triglycerides at the most recent time point before transplantation were recorded. Height and weight at the time of listing and at transplantation were obtained through a United Network for Organ Sharing (UNOS) query. BMI was calculated by using the following formula, weight in kilograms ÷ height in meters squared. Serum total bilirubin, international normalized ratio (for anticoagulant monitoring), and creatinine were collected to calculate a biologic Model for End-Stage Liver Disease (MELD) score by using the MELD score calculator from the UNOS website (www.unos.org). Patients' HCC status was not used in our MELD score calculation.

Pathologic Examination

All liver specimens were reanalyzed by a single, blinded pathologist (A.K.B.). H & E stained, paraffin-embedded sections used for routine diagnostic purposes were evaluated for steatosis. One representative section from each case was selected to determine the grade of steatosis represented by a percentage of hepatocytes containing fat droplets. Tissues used for diagnosis were obtained at the time resected specimens were received. Resected specimens were assessed to determine the grade of steatosis represented by a percentage of hepatocytes containing fat droplets. The following grading system was used, grade 0 = absent, grade 1 = 1% to 5% of hepatocytes affected, grade 2 = 6–32% of hepatocytes affected, grade 3 = 33% to 66 % of hepatocytes affected, and grade 4 = >66% of hepatocytes affected.21

Statistical Analysis

Statistical analysis was performed by use of SAS software (Cary, NC). P values between the 2 groups were compared by using Fisher exact test for binary variables, Wilcoxon rank-sum test for continuous variables, and logistic regression for multivariate analyses. Chi-squared test of the trend was used to test the association between steatosis and HCC. Odds ratios and 95% confidence intervals were calculated for each grade of steatosis as compared with grade 0 (no fat present).

RESULTS

Baseline Characteristics

In total, 94 consecutive patients with hepatitis C who underwent liver transplantation, met eligibility criteria, and had pathology slides from their liver explants available for review were included in the study. The majority of patients were men (78%), white (80%), and infected with genotype 1 (57%). Genotype 3 HCV comprised 13% of the population. Forty-three percent of patients were identified as having a past history of chronic alcohol use, and 17% had diabetes mellitus. Four patients had undergone antiviral therapy for HCV before liver transplantation. Of these, 2 experienced a sustained virologic response (1 in each group) to treatment, and 2 were nonresponders (1 in each group).

Thirty-two patients had histological evidence of hepatocellular carcinoma. The control group participants (n = 62) all had a history of hepatitis C cirrhosis, but they did not have evidence of HCC on pathology. All patients in the study had cirrhosis (Ishak score 5 or 6).22 Compared with the control group, the HCC group had a slightly lower percentage of subjects with chronic alcohol use (31% vs 48%; P = .13), diabetes (9% vs 21%; P = .25), and white race (76% vs 85%; P = .26). The HCC group was significantly older than the control group (56 years vs 49 years; P < .002). The 2 groups were roughly equal in their distribution of HCV genotypes as well as sex (Table 1).

Table 1. Baseline Characteristics
 HCCNo HCCP
  1. HCV indicates hepatitis C virus; DM, diabetes mellitus.

No.3262NA
Alcohol dependence (%)10 (31)30 (48).13
Sex (%)  1.0
 Men25 (78)48 (77)
 Women7 (22)14 (23)
Race (%)  .26
 Hispanic3 (9)6 (10)
 Black2 (6)2 (3)
 Asian2 (6)0 (0)
 White23 (72)52 (84)
 Other2 (6)2 (3)
HCV Genotype (%)  .28
 119 (59)35 (56)
 22 (6)4 (6)
 36 (19)6 (10)
 41 (3)1 (2)
 Unknown4 (13)16 (26)
Age, y56± 849± 8<.002
DM  .25
 No (%)29 (91)49 (79)
 Yes (%)3 (9)13 (21)

Univariate Analysis

In univariate analysis, steatosis trend (P = .03), older age (56 years vs 49 years; P < .02), higher AST (122.5 U/L vs 91.5 U/L; P = .005), higher ALT (95.8 U/L vs 57.2 U/L; P = .002), lower median HCV RNA (239,000 IU/mL vs 496,500 IU/mL; P = .02), higher biologic MELD score (21.8 vs 20.3; P = .03), and higher AFP (113.5 ng/mL vs 17.8 ng/mL; P < .001) were associated with HCC. There was no statistically significant difference in cholesterol and triglyceride values between the 2 groups. Calculated BMI at listing (26.3 kg/m2 vs 28.1 kg/m2; P = .10) and at transplant (25.4 kg/m2 vs 27.2 kg/m2; P = .09) was lower in the HCC group although not statistically significant (Table 2). We also evaluated BMI >25 compared with BMI ≤25 and did not find a significant difference between the 2 groups (P = .29). In addition, we examined diabetes, alcohol use, and lipid-lowering medication, which are also known to influence steatosis, and we found no correlation with HCC.

Table 2. Laboratory Values
 HCCNo HCCP
  • Values± standard deviation.

  • HCC indicates hepatocellular carcinoma; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index; HCV, hepatitis C virus; MELD, model for end-stage liver disease; AFP, α-fetoprotein.

  • *

    Median (25th, 75th percentile).

Triglycerides, mg/dL108.9± 36.8123.3± 109.6.13
Cholesterol, mg/dL121.0± 46.4116.6± 53.6.71
AST, U/L122.5± 58.391.5± 55.0.005
ALT, U/L95.8± 71.757.2± 34.3.002
BMI at listing26.3± 4.528.1± 4.7.10
BMI at transplant25.4± 4.027.2± 4.8.09
HCV RNA, IU/mL*239,000 (17,242, 492,292)496,500 (132,100, 500,000).02
MELD score21.8± 8.620.3± 8.0.03
AFP, ng/mL113.5± 20017.8± 41<.001

Steatosis

In total, 69% of patients in the HCC group and 50% of patients in the control group had evidence of steatosis (1+) on histology. There was a linear relation between the grade of steatosis and its association with HCC. The odds ratio for HCC with each incremental grade of steatosis compared with grade 0 was as follows: grade 1 (1.61 [0.6–4.3]), grade 2 (3.68 [1.2–12.9]), and grade 3 or 4 (8.02 [0.6–108.3]) (Fig. 1). In the statistical analysis, grades 3 and 4 were grouped together because there was only 1 patient with grade 4 steatosis. There was a significant association between the trend in steatosis grade and risk of HCC (P = .03) (Table 3). We also evaluated differences in steatosis with age, BMI, and history of alcohol dependence. Age was not associated with the presence of steatosis (P = .49) nor was it associated with steatosis when steatosis grade was examined as a continuous variable (P = .79). Additionally, BMI (P = .19), and alcohol use (P = .67) were not associated with steatosis.

Figure 1.

Odds ratio of hepatocellular carcinoma for each grade of steatosis is compared with grade 0.

Table 3. Steatosis Grade in the HCC and non-HCC Groups
Steatosis grade0123/4Total with any steatosisTrend
  1. HCC indicates hepatocellular carcinoma; CI, confidence interval.

  2. Odds ratios (OR) for HCC for each grade of steatosis compared with grade 0. P values are reported for each indicated grade compared with grade 0 and for the trend of steatosis.

HCC No. (%)10 (31)12 (37)8 (25)2 (6)22 (69) 
No HCC No. (%)31 (50)23 (37)7 (11)1 (2)31 (50) 
OR (95% CI)1.61 (0.6–4.3)3.68 (1.1–12.8)8.02 (0.6–108.3)2.1 (0.9–5.0) 
P0.450.040.110.080.03

Multivariate Analysis

We conducted multivariate analysis by using logistic regression modeling to determine which variables were independently associated with HCC. Variables included in the model were those that were significant in univariate analysis, excluding HCV RNA. HCV RNA was excluded because the limited number of patients with HCV RNA values decreased the power of the multivariate analysis. Odds ratios for the variables in the multivariate analysis are shown in Table 4. Of these variables, AFP (P = .007), age (P = .02), and steatosis (P = .045) were each independently associated with HCC (Table 4).

Table 4. Multivariate Analysis
 Odds ratio (95% CI)P
  1. Odds ratios with 95% confidence intervals and P values for multivariate analysis.

  2. CI indicates confidence interval; AFP, α-fetoprotein; AST, aspartate transaminase; ALT, alanine aminotransferase; MELD, model for end-stage liver disease.

AFP1.11 (1.00, 1.03).007
AST1.00 (0.92, 1.18).76
ALT1.02 (1.0, 1.05).10
Age1.10 (1.01, 1.20).03
MELD1.07 (0.98, 1.18).13
Steatosis6.39 (1.04, 39.35).045

DISCUSSION

In our series of 94 patients with hepatitis C cirrhosis, hepatic steatosis was associated with hepatocellular carcinoma in patients with hepatitis C cirrhosis. In total, 53 (56%) patients in the analysis had evidence of steatosis on histology, which is consistent with previous studies.17, 20, 23 Nineteen percent of patients had grade 2 steatosis or higher (>6% of hepatocytes affected), which is lower than most previous studies that have examined patients with HCV. However, most of the earlier studies included predominantly noncirrhotic specimens, which are likely to harbor higher degrees of steatosis compared with cirrhotic specimens.16, 17, 24 Prior data suggest that genotype 3 is associated with higher rates of steatosis. Although our genotype 3 patients did not show this correlation, the small number of patients with this genotype precludes our ability to make a meaningful comparison.25, 26 As with previous studies, the HCC group was older. Contrary to previous observational investigations, BMI did not correlate with either steatosis or hepatocellular carcinoma. Several previous studies demonstrate a relation between steatosis and BMI. There are 2 possible explanations why our study did not confirm this relation. Fatty changes might have been masked in our study because of the reliance on cirrhotic liver specimens. Indeed, prior analysis has shown that steatosis is associated with increasing fibrosis but then decreases upon development of cirrhosis.27 Thus, we were less likely to observe the strict relation between BMI and steatosis. Second, our subjects had advanced cirrhosis and were awaiting transplantation, in contrast to previous studies. Patients without HCC were more likely to have decompensated liver disease than those with HCC, because the latter group is given increased priority for transplant as a direct result of their cancer diagnosis. By extension, the control group was more likely to have ascites, which could have increased the recorded BMI.

Interestingly, lower HCV RNA levels appeared to be associated with HCC. We speculate that this finding could reflect the observation that HCV replication is diminished in HCC relative to nonmalignant surrounding tissue.28 Virologic responders to treatment are known to have a lower incidence of HCC than nonresponders. However, HCV RNA, when present, despite level, does not correlate with disease activity or progression to HCC. Because this study included only 2 patients with response to previous antiviral therapy, the observed HCV RNA levels do not merely reflect a response to therapy.

There are few studies that have addressed the association between hepatic steatosis, HCV, and hepatocellular carcinoma. One prospective biopsy-based study concluded that hepatic steatosis is a risk factor for hepatocellular carcinoma.19 On the other hand, a second retrospective case-control study of liver biopsies did not identify an association between steatosis and HCC.20

Our study confirms the association between steatosis grade and HCC. Because our study involved the evaluation of explanted livers, it offers 2 clear advantages in interpretation of these data. Patients with cirrhosis are known to be at increased risk for development of HCC. We minimized the contribution of differences in fibrosis stage between the 2 groups, because all of our subjects had cirrhosis on pathology. In addition, there is substantially less likelihood of experiencing sampling error in the explant specimens compared with biopsies because of the extensive area covered by explants.

How can the discordant findings be reconciled between our study and that of Kumar et al.? It is possible that steatosis could have been undetected in the Kumar study as a result of biopsy-based sampling error in a relatively small cohort compared with our larger explant-based study. It is also possible that in the time that elapsed between biopsies in the Kumar study and the development of HCC, steatosis could have been a late event, functioning as a “second hit” for carcinogenesis superimposed on chronic HCV.

There are several possible limitations to our study. The retrospective nature of our study design, where we examined steatosis grade after diagnosis of HCC, may make it more difficult to generalize our findings to a clinical setting, where biopsies would be expected to occur at an interval of months to years before diagnosis of HCC. Further prospective study of patients with steatosis for subsequent HCC risk is warranted. Furthermore, fatty changes observed in the liver at the time of transplantation may actually underestimate the magnitude of steatosis that precedes development of hepatocellular carcinoma. This is supported by the observation by others that steatosis increases in parallel with increasing fibrosis but appears to drop once cirrhosis has developed.29–31 This apparent reduction of steatosis in cirrhosis may be related to reduced access of triglycerides to hepatocytes as a result of portosystemic shunting or loss of sinusoidal fenestrations.29, 32, 33 These observations may account for the relatively limited number of subjects with high-grade (2+) steatosis in this study of cirrhotic liver patients. Notwithstanding this apparent limitation, we were still able to observe a significant relation between steatosis and HCC.

The mechanism of hepatocarcinogenesis that links hepatitis C to HCC remains ill defined; however, several previous investigations have proposed that steatosis may play a role in the development of HCC. Prior data suggest that HCV is associated with steatosis in a large portion of cases and that steatosis is associated with worsening fibrosis.15, 16, 19, 24, 34–36 In addition, steatosis induces chronic hepatic inflammation, reactive oxygen species, and DNA damage in animal models.37–39 Moreover, in HCV transgenic mice that develop HCC, carcinogenesis is preceded by development of steatosis40, 41 Additional evidence implicating steatosis as a carcinogen comes from data demonstrating that nonalcoholic steatohepatitis (NASH) is a risk factor for HCC. Although this was a cross-sectional analysis, the finding of increasing HCC frequency with higher grades of steatosis argues for a dose-response relation between steatosis and malignant transformation. Larger longitudinal studies of patients with HCC and steatosis will help to confirm this hypothesis.

In conclusion, steatosis is independently associated with hepatocellular carcinoma in patients with hepatitis C cirrhosis and may, therefore, be a useful marker in clinical practice for identifying patients at high risk for HCC. It is likely that viral factors are acting in concert with steatosis to abet malignant transformation. As such, steatosis should be used with other variables to develop a relative-risk profile for each patient to identify those who require more intensive surveillance. Further studies focusing on identification of steatosis to identify persons at particular risk, as well as interventions aimed at reducing steatosis, will be important steps in modifying the risk of HCC among HCV-infected patients.

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