Potential conflict of interest: Nothing to report.
Hepatic steatosis is common in patients with chronic hepatitis C (CHC) and is reported to be a risk factor for progression of fibrosis. The aims of this study were to evaluate the interactions between hepatic steatosis and fibrosis in a well-defined cohort of patients with CHC. The computerized pathology database at the National Institutes of Health Clinical Center was searched for patients with CHC who had undergone liver biopsy between 1980 and 2003. Biopsies were scored for necroinflammation using a modified histology activity index, fibrosis using the Ishak system, and steatosis as either none (<5% of cells), mild (5%–25%), or moderate-to-severe (>25%). Four hundred ninety-four patients were identified. The mean age was 44 ± 9.8 years; 60% were male, 80% Caucasian, and 65% were infected with genotype 1. Steatosis was mild in 31% and moderate to severe in 9% of patients. In univariate analysis, steatosis was associated with increased age, body weight, body mass index (BMI), alanine aminotransferase (ALT) levels, histological necroinflammatory activity, and fibrosis. However, in multivariate analysis, steatosis was associated only with increased age, BMI, and ALT levels and not with fibrosis. One hundred thirty-six patients had 2 liver biopsies separated by 0.5 to 17 years. Worsening of fibrosis occurred in 40% of patients and correlated independently with increasing age, periportal necroinflammation, and ALT elevations but not with steatosis. In conclusion, in this cohort of patients with CHC, steatosis was associated with older age, higher BMI, and higher serum ALT levels but not with the presence of or subsequent progression of fibrosis. (HEPATOLOGY 2006;43:780–787.)
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Chronic hepatitis C is a major cause of cirrhosis, end-stage liver disease, and hepatocellular carcinoma in the United States and worldwide.1 The natural history of the disease is not well understood because of the protracted and silent course of infection, but several studies have identified the progression of fibrosis as the best indicator of long-term outcome.2 Factors that have been recognized to be associated with worsening of fibrosis include age at time of infection, male sex, duration of infection, alcohol consumption, degree of necroinflammation on liver biopsy, and serum alanine aminotransferase levels (ALT).3–5 However, even in the absence of these factors, progression of fibrosis may occur, suggesting a role of other unidentified co-factors.
Hepatic steatosis is a common finding on radiologic imaging and liver biopsy, present in 10% to 24% of the general population.6, 7 Risk factors for fatty liver include overweight and obesity, type 2 diabetes, and dyslipidemia.8 Steatosis is also a frequent histopathological finding in patients with chronic hepatitis C virus (HCV) infection, occurring in 30% to 70% of cases, which suggests that HCV may have a direct causative role in steatosis.9–11In vitro studies using HCV-transfected cell lines and in vivo studies using HCV transgenic mice suggest that the HCV core protein induces steatosis and inhibits lipoprotein synthesis and secretion of triglyceride-rich very-low-density lipoproteins through an interaction with microsomal triglyceride transfer protein.12, 13 Furthermore, in persons with chronic HCV infection, hepatic steatosis has been associated with infection with genotype 3 virus.14 Moreover, resolution of HCV infection after antiviral therapy is associated with a decrease in hepatic steatosis, particularly in patients with genotype 3.15
Although hepatic steatosis has been considered a benign finding, recent studies have suggested that both steatosis and the more severe lesion of non-alcoholic steatohepatitis may be associated with progression of fibrosis in patients with hepatitis C.16–18 However, many of the studies were cross-sectional and involved selected populations with unknown duration of steatosis. Furthermore, progression of fibrosis was estimated based on a single liver biopsy together with the clinical history of exposure and assumed that progression of fibrosis was linear over time. Three studies that analyzed progression of fibrosis from paired liver biopsies in untreated patients suggested that worsening of fibrosis correlated with either worsening of steatosis or simply the presence of steatosis on liver biopsy in patients with19, 20 and without genotype 3 infection.21 However, in two studies genotype 3 infections were overrepresented, which may have been a source of bias, and the other study examined progression of fibrosis in patients with mild hepatitis C initially. Thus, the role of hepatic steatosis in the development of fibrosis in patients with chronic hepatitis C is still unclear. The aims of this study were to assess factors associated with hepatic steatosis in a large cohort of untreated patients with chronic hepatitis C and to determine whether hepatic steatosis was associated with worsening hepatic fibrosis in a paired liver biopsy study.
ALT, alanine aminotransferase; HCV, hepatitis C virus; AST, aspartate aminotransferase; BMI, body mass index; HAI, histology activity index.
Patients and Methods
The computerized pathology database of the Department of Pathology at the Clinical Center of the National Institutes of Health was searched for all patients who had the diagnosis of chronic hepatitis C and who had undergone liver biopsy between 1980 and 2003. Chronic hepatitis C was defined in patients accessed before 1992 on the basis of a positive test for anti-HCV together with raised aminotransferase levels for more than 6 months and in subsequent patients on the basis of detection of HCV RNA in serum. Patients who were hepatitis B surface antigen or anti–HIV-1 positive, who had other known causes of liver disease, who had a known malignancy, decompensated liver disease, or who received treatment before initial and follow-up liver biopsy were excluded. Furthermore, individuals with heavy alcohol consumption defined as ≥60 g/d for men and ≥40 g/d for women, and those with concurrent corticosteroid therapy were excluded. A subset of patients from the cohort had two liver biopsies performed separated by 6 months or more and without receiving therapy between the two biopsies. This group consisted of 59 blood donors enrolled in a study of the natural history of hepatitis C,22 52 untreated control patients who were participating in randomized trials evaluating interferon alfa or ribavirin,23, 24 and 30 patients who were referred for evaluation and possible therapy who elected not to undergo treatment.4 A proportion of these patients were the subject of a previous report on fibrosis; however, steatosis was not specifically analyzed.4 All patients were participants in clinical research protocols that had been approved by the NIDDK Institutional Review Board and gave written informed consent for liver biopsy and evaluation.
Patients' charts were reviewed, and the following clinical and laboratory information was recorded: age, age at biopsy, gender, weight, height, alcohol consumption, presence of diabetes, route of transmission, suspected date of exposure, and laboratory results at time of initial liver biopsy, including serum alkaline phosphatase, ALT, aspartate aminotransferase (AST), direct and total bilirubin, cholesterol and triglycerides, and viral genotype. Duration of infection was estimated from the time of suspected exposure in cases where this information was available. Body mass index (BMI) was calculated by dividing a patient's weight in kilograms (kg) by height in meters (m) squared. Alcohol consumption was estimated based on retrospective review of patients' self-reported use of alcohol and by the Michigan Alcohol Screening Test(MAST).
All liver biopsies were read under code by a hepatopathologist without knowledge of the patient's clinical information. A liver biopsy was considered adequate if it contained more than 10 portal tracts. Liver biopsies were scored using a modification of the histology activity index (HAI), which grades necrosis and inflammation on a scale of 0 to 18 (periportal inflammation and necrosis, 0–10; lobular inflammation and necrosis, 0–4; and portal inflammation, 0–4).23, 25 Fibrosis was scored using the Ishak fibrosis scale of 0 to 6 (0 = no fibrosis, 1–2 = portal fibrosis, 3–4 = bridging fibrosis, and 5–6 = cirrhosis).26 Hepatic steatosis was graded based on the proportion of hepatocytes with fat on a 6-point scale as none, <5%, 5% to 25%, 26% to 50%, 51% to 75%, and 76% to 100%. After an initial analysis, steatosis scores were grouped as none (<5%), mild (5% to <25%), and moderate-to-severe (>25%) (Grouping those with <5% steatosis as mild did not affect variables identified with steatosis in either the univariate or multivariate analysis, data not shown). Worsening of fibrosis was defined as an increase by at least 1 point in the fibrosis score and improvement as a decrease of at least one point. Similarly, worsening of steatosis was defined as an increase and improvement as a decrease in steatosis scores by at least 1 grade.
Analysis of variance (ANOVA) controlling for degree of fibrosis was used to analyze factors that were associated with steatosis. Multivariate regression analysis using backward selection was used to identify factors independently associated with hepatic steatosis and progression of fibrosis. An alpha less than 0.05 was considered significant.
Search through the computerized database indicated 709 patients with chronic hepatitis C, of whom 494 met the criteria for inclusion in this analysis. Reasons for exclusion included previous treatment with antiviral agents (n = 65), anti-HIV positivity (n = 52), ongoing heavy alcohol intake (n = 31), underlying malignancy (n = 37), immunosuppression (n = 17), presence of another liver disease (n = 10), decompensated cirrhosis (n = 2), and age younger than 18 years (n = 1). The clinical, demographic, laboratory, and histological characteristics of the cohort by degree of hepatic steatosis are shown in Table 1.
Table 1. Clinical Factors Associated With Hepatic Steatosis
None (<5%) (n = 298)
Mild (5–25%) (n = 154)
Moderate–severe (>25%) (n = 42)
Abbreviations: BMI, body mass index; Alkaline Phos, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Protime, prothrombin time; HAI, histology activity index; y, years.
Sixty percent of patients had minimal or no hepatic steatosis, 31% had mild steatosis, and 9% had moderate-to-severe steatosis. Analysis of variance was used to identify clinical factors associated with hepatic steatosis. Age at biopsy, male sex, body weight, BMI, genotype 3, serum alkaline phosphatase, serum ALT and AST, total protein, serum iron, triglycerides, periportal and lobular inflammation/necrosis, and Ishak fibrosis scores were all found to be associated with hepatic steatosis on univariate analysis. Importantly, diabetes mellitus and alcohol consumption were not associated with hepatic steatosis in this cohort, perhaps because the number of patients with diabetes (n = 17) was small, and most patients did not consume alcohol. In multivariate analysis, age at biopsy, serum ALT level, and BMI were the only factors that were independently associated with steatosis. Genotype 3 was no longer significantly associated with presence of steatosis.
We found a complex association between prevalence of steatosis and age. The prevalence of steatosis increased from 36% in those younger than 40 years of age to 50% in those older than 60 years (Fig. 1A), although this was mostly attributable to an increase in mild steatosis. Paradoxically, the severity of steatosis decreased with increasing age. An increase in the prevalence and severity of steatosis also was observed with increasing ALT and increasing BMI levels. Steatosis was present in 27% of persons with normal ALT values (97% of the steatosis was mild) compared with 46% to 47% (89% to 91% of which was mild) in patients with ALT levels greater than 3 times the upper limit of normal (Fig. 1B). The most striking association was found between steatosis and BMI categories (Fig. 1C) with a stepwise increase in prevalence and severity of steatosis with increasing BMI. The prevalence of steatosis was 23% in patients with a normal or low BMI, 41% in those who were overweight, and 64% in those who were obese, an almost three-fold increase in the rate of steatosis. Additionally, the proportion of patients with moderate to severe steatosis increased from 2% in those with BMI < 25 to 17% among those with a BMI > 30 kg/m2. The association of steatosis with ALT levels was independent of its association with BMI levels, as shown in Fig. 2. Thus, within each BMI category (<25 or normal; 25–30 or overweight; >30, obese), the frequency of steatosis was higher among patients with higher ALT levels, although the effect was most striking among persons with a normal BMI.
Strikingly, most patients in this cohort had at least one of the three factors that correlated independently with steatosis. Thus, only 6 patients were younger than 40 years of age and had a normal BMI and normal ALT levels. None of these 6 had steatosis on liver biopsy.
Factors Associated With Progression of Fibrosis: Paired Liver Biopsy Cohort.
Paired liver biopsies were available from 136 subjects. The clinical, laboratory, and histological characteristics of this sub-group were not significantly different from the remainder of the cohort with single liver biopsies (data not shown). The mean duration between liver biopsies was 3.6 years (range, 0.5–17 years). For the sake of analysis, patients were categorized into two groups: the 54 patients (40%) who showed progression in fibrosis, defined as at least a 1-point worsening in fibrosis score (range = +1 to +4) and the 82 patients (60%) who showed no change or apparent improvement in fibrosis score, (range = 0 to −2). On univariate analysis, only serum ALT, AST, and total protein levels were associated with progression of fibrosis (Table 2). In multivariate regression analysis with backward selection, older age, higher serum ALT, more severe periportal inflammation/necrosis, and lower initial fibrosis score were independently associated with progression of fibrosis. Steatosis grade did not correlate with progression of fibrosis in either univariate or multivariate analysis. Even after exclusion of patients with genotype 3 (n = 4) and those with less than 2 years' duration between liver biopsies (n = 62), steatosis grade was not found to be significantly associated with fibrosis progression between liver biopsies. Three of the four parameters previously identified, older age, lower Ishak score at initial biopsy, and higher AST (AST replaced ALT) remained in the model.
Table 2. Factors Associated With Progression in Fibrosis in Paired Liver Biopsy Cohort
Progression (n = 54)
No progression (n = 82)
Abbreviations: MAST, Michigan Alcohol Screening Test; BMI, body mass index; Alkaline phos, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Protime, prothrombin time; HAI, histology activity index; y, years.
Factors Associated With Worsening of Steatosis: Paired Liver Biopsy Cohort.
At the second liver biopsy, the prevalence of steatosis was 45%. Fifty-five percent of patients had minimal or no steatosis, whereas steatosis was graded as mild in 34% and moderate to severe in 11%. Worsening of steatosis occurred in a minority (13%) of patients, whereas 20% had improvement and 67% no change between liver biopsies. Half of patients with worsening of steatosis had no steatosis on their index liver biopsy. No demographic, clinical, biochemical, or histological feature was associated with worsening of steatosis (data not shown). Notably, neither change in BMI nor duration between initial and follow-up liver biopsies correlated with worsening steatosis. The mean BMI was 27.2 kg/m2 at the time of the initial biopsy, and 27.9 kg/m2 at the time of the second biopsy, an average difference of only 0.7 kg/m2. The numbers of subjects with diabetes or who drank alcohol between biopsies were too few to reliably assess the role of diabetes or alcohol intake with change in steatosis.
Steatosis and Progression of Fibrosis: Paired Liver Biopsy Cohort.
To further assess the contribution of steatosis to progression of fibrosis, we examined the association between baseline steatosis (none, mild, or moderate–severe) and change in fibrosis score. Baseline steatosis score was not associated with change in fibrosis score, P = .58. Furthermore, there was no correlation between the change in steatosis scores and change in fibrosis scores between paired liver biopsies (r2 = 0.0007, P = NS).
In this retrospective analysis from a large, well-characterized cohort of untreated patients with chronic hepatitis C, the prevalence of steatosis was 40% on index liver biopsy. Several factors were found to correlate with steatosis on univariate analysis, including fibrosis, but only age, ALT, and BMI were identified as being independently associated on multivariate analysis. To examine the role of steatosis on the progression of fibrosis, multivariate linear regression analysis was applied to identify factors that predicted progression of fibrosis from paired liver biopsies separated an average of 3.7 years. Age at biopsy, serum ALT levels, and degree of periportal inflammation and necrosis but not steatosis correlated with progression of fibrosis.
This study confirmed the previously reported high prevalence of steatosis in chronic hepatitis C.16, 17, 21, 27 However, the results obtained from analysis of clinical correlates of steatosis suggested a multifactorial origin for steatosis in this disease. Many clinical features were identified as associated with steatosis on univariate analysis of the cross-sectional sample of patients; however, multivariate analysis suggested that the presence and severity of hepatic steatosis were attributable to three major factors: age, disease severity (as reflected best by ALT), and metabolic factors (as reflected best by BMI).17
In this cohort, steatosis on liver biopsy in chronic hepatitis C appeared to reflect the severity of the liver disease. Hepatic necrosis and inflammation may stimulate metabolic pathways involved in fat deposition in the liver. The pro-inflammatory cytokine milieu such as tumor necrosis factor alpha or oxidative stress associated with HCV infection may promote hepatic steatosis.28 This possibility would explain why steatosis correlated with elevations in liver-associated enzymes (alkaline phosphatase, ALT, AST) as well as liver biopsy features of disease activity and stage (HAI inflammatory scores, periportal, lobular, and fibrosis scores). Thus, steatosis was more common in patients with active and more advanced disease. This possibility would also explain why steatosis is often associated with fibrosis in cross-sectional studies and might correlate with progression of fibrosis in longitudinal studies when initial disease severity was not controlled for adequately.16–20, 29 Thus, these findings might indicate that steatosis is the result rather than the cause of disease severity and disease progression.
The strongest associations identified in this study were between hepatic steatosis and metabolic factors. In univariate analysis, steatosis was significantly associated with body weight, BMI, and serum triglyceride levels. In multivariate analysis, BMI was most strongly associated with presence and degree of steatosis. BMI is a fairly reliable marker for obesity and is a surrogate marker for the dysmetabolic syndrome. The pathogenesis of steatosis in obesity is uncertain, but insulin resistance probably plays a major role. Insulin resistance is associated with an increase in adipocyte lipolysis and elevated circulating free fatty acids as well as inhibition of hepatic lipid oxidation and export, all of which serve to promote triglyceride deposition in the liver.30, 31
We observed an inverse association between age and degree of steatosis: milder steatosis is associated with older age. This discrepancy may be due to type II error because of the small number of patients older than 60 years of age relative to the younger age groups.
In contrast to previous studies, the current study did not identify an association between steatosis and diabetes, alcohol, or genotype 3. None of these factors was present in high enough numbers to adequately evaluate them, however. Thus, only 23 patients had genotype 3, of whom 65% had steatosis (P = .034), a rate similar to that described in other studies.19, 21 Notably, the severity of steatosis was mild (<25%) in most (87%) genotype 3 patients. In multivariable analysis, however, the significant association of genotype 3 and steatosis was lost. Similarly, this cohort had a low prevalence of diabetes (<3%), and 50% of patients consumed no alcohol, whereas two thirds drank less than two alcoholic drinks per week.
An important aim of the study was to evaluate whether steatosis was associated with progression of fibrosis. Previous studies have shown that the severity of steatosis on index liver biopsy was an independent predictor of fibrosis stage and progression of fibrosis. Many of these studies were conducted on small, selected populations, and a major shortcoming was that they were mostly cross-sectional, where neither the duration of steatosis nor duration of infection was well documented. One of the strengths of the current study was the large number of persons with paired biopsies, which allowed a more accurate assessment of changes in both steatosis and fibrosis over time. In the cross-sectional analysis, a linear relationship was found between the severity of steatosis and the stage of fibrosis. However, in the paired biopsy analysis, no association was found between baseline steatosis and change in fibrosis score. Furthermore, no association was seen between the change in steatosis grade and change in fibrosis stage between liver biopsies. Together these results argue against a role of steatosis in progression of fibrosis. Instead, factors previously associated with progression of fibrosis such as older age, higher ALT level, and more severe necroinflammatory changes were identified.
The reasons for the differences in results of this cohort compared with previous studies are not entirely clear. Features that might explain the discrepant results include the nature of the population studied (American vs. European patients, referral patterns, varying rates of obesity); differences in systems for scoring steatosis, fibrosis, and disease activity; the retrospective nature of the study and its inherent biases; and the types of factors included in the analyses. In all analyses, sampling errors in detection and variabilities in scoring systems of steatosis, inflammation/necrosis, and fibrosis are important variables that may explain discrepant results. Furthermore, in performing multivariate analysis, the factors that are entered into regression models can greatly affect the outcome. In this study, steatosis at baseline correlated with disease activity (HAI scores and ALT levels) as well as fibrosis. Thus, in previous studies, steatosis might have appeared to correlate with progressive disease, because it was confounded by disease activity and preexisting fibrosis. Conversely, in this analysis, the assumption was made that steatosis was the result rather than the cause of more severe disease activity because of the strong association of markers of disease activity (ALT levels and periportal inflammation/necrosis on liver biopsy) with disease progression (as reflected by worsening fibrosis).
In summary, steatosis was present in 40% of untreated patients with chronic HCV infection but was usually mild. Steatosis was associated with features of the metabolic syndrome (BMI, advancing age) and more severe disease activity (higher ALT, HAI, and fibrosis scores). Using paired liver biopsies to assess the relationship between steatosis and fibrosis, worsening of steatosis was uncommon, occurring in only 13% of patients, and did not correlate with progression in liver fibrosis. Instead, progression of fibrosis was associated with older age and more active liver injury as evidenced by higher ALT levels and periportal inflammation and necrosis on the initial liver biopsy. This study serves to highlight the differences between data derived from cross-sectional and longitudinal analyses. The role of steatosis in outcome of chronic HCV infection deserves further investigation, and the current findings need to be validated in different patient populations and assessed in separate cohorts of patients with genotype 3, diabetes mellitus, and moderate to heavy alcohol abuse.