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Hepatic steatosis in HIV/HCV co-infected patients: Correlates, efficacy and outcomes of anti-HCV therapy: A paired liver biopsy study
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Journal of Hepatology May 2008
Maribel Rodriguez-Torres1, Sugantha Govindarajan2, Ricard Sola3, Nathan Clumeck4, Eduardo Lissen5, Mario Pessoa6, Peter Buggisch7, Janice Main8, Jean DePamphilis9, Douglas T. Dieterich10
1 Fundacion de Investigacion de Diego, Ave. De Diego # 359, Suite 302, Santurce, PR 09909, USA
2 Rancho Los Amigos Medical Center, Downey, CA, USA
3 Universitat Autonoma de Barcelona, Barcelona, Spain
4 CHU Saint-Pierre, Brussels, Belgium
5 Virgen del Rocio, University Hospital, Seville, Spain
6 Instituto de Infectologia Emilio Ribas, Sao Paulo, Brazil
7 Universitatsklinik, Eppendorf, Hamburg, Germany
8 St. Mary's Hospital, London, UK
9 Roche Nutley, NJ, USA
10 Mount Sinai School of Medicine, New York, NY, USA
ABSTRACT
Background/Aims: Hepatic steatosis is caused by the complex interaction of host and viral factors, such as metabolic syndrome (MS), alcoholism and HCV genotype, and in HIV-HCV co-infected patients, antiretroviral therapy may also play a role. A large population of patients from the AIDS Pegasys Ribavirin International Co-infection Trial (APRICOT) had paired liver biopsies interpreted and graded for steatosis along with lipid measurements and anthropometric data.
Methods: We analyzed these patients to determine the prevalence of steatosis, baseline factors associated with steatosis, effect of steatosis in HCV therapy efficacy and the impact of anti-HCV treatment on steatosis.
Results: A total of 65/283 (23%) patients with paired biopsies were positive for steatosis.
Patients with steatosis were significantly more likely to have HCV genotype 3, bridging fibrosis/cirrhosis, higher HCV RNA levels, increased triglycerides and lower cholesterol levels. The only different body measurement was neck circumference which was greater in patients with steatosis and significantly decreased from baseline during the study. Hip circumference was predictive of steatosis at baseline.
Conclusions: Factors associated to the metabolic syndrome are important in co-infected patients. Treatment outcome affected steatosis in that viral eradication reduced steatosis in genotype 3 patients, but altogether steatosis did not affect efficacy of treatment in any genotype.
".....We have confirmed that nucleoside analogs appear to be important in the aetiology of steatosis in co-infected patients infected with HCV non-3 genotypes. An association between nucleoside analogs and hepatic steatosis has been reported previously [15], [17], [37], as has an association between nucleoside analogs and MS [16]. However, this is the first time that nucleoside analogs have been implicated to steatosis in co-infected patients with HCV non-3 genotypes.....
....Multiple regression analysis demonstrated that hip circumference is significantly associated with steatosis. This association of a specific anthropometric change with steatosis is a novel finding, and adds to existing evidence that body weight, BMI or non-specific lipodeposition/lipoatrophy are important prognostic factors for steatosis [13], [15]. Further research is required to confirm a strong association between MS and steatosis in HIV-HCV co-infected patients...."
Introduction
Hepatic steatosis is a frequent histological occurrence in patients infected with hepatitis C virus (HCV) [1] and has been associated with accelerated progression of fibrosis [1], [2].
In patients infected with HCV, steatosis may be caused by metabolic factors, host factors such as alcoholism or age, or by viral factors, such as infection with HCV genotype 3 [3], [4]. Recent investigations suggest two forms of steatosis in HCV monoinfection: one is by direct effect of the HCV genotype 3 virus through interactions between hepatocytes and the HCV core protein [4]; the second is related to metabolic risk factors for insulin resistance (IR), such as obesity, type 2 diabetes and hyperlipidemia [5]. In the case of steatosis in non-alcoholic fatty liver disease, metabolic syndrome (MS) is especially implicated [6], [7], [8]. MS is a cluster of phenomena associated to atherosclerotic cardiovascular disease, with clinical diagnosis based on findings of abdominal obesity (waist-to-hip ratio and/or BMI), dyslipidemia, hypertension, and (IR) or glucose intolerance [9].
HCV infection is seen in around 30% of patients with HIV infection. The progression of fibrosis is more rapid in patients with HIV/HCV co-infection [10] and several studies have identified factors that contribute to hepatic steatosis [11], [12], [13], [14], [15], and characteristics associated to MS, as older age, lipodystrophy and BMI [16].
In addition, antiretroviral therapy (ART) has been implicated in metabolic disturbances in HIV-infected patients [16], [17], [18] and is generally accepted that ART is associated with dyslipidemia [19], [20], [21]. However, there is conflicting evidence of a role for ART influencing steatosis in co-infected patients [13], [15], [22].
The treatment of choice for chronic hepatitis C in patients with HIV/HCV co-infection is pegylated interferon (Peg-IFN) plus ribavirin (RBV) [23], [24]. This is based on the results of the AIDS Pegasys Ribavirin International Co-infection Trial (APRICOT), a large, randomized, international study conducted in patients with HIV/HCV co-infection [25]. Significantly higher sustained virological response (SVR) rates were obtained with Peg-IFN alfa-2a plus RBV treatment, than with Peg-IFN alfa-2a/Placebo or IFN alfa-2a plus RBV (40% vs. 20% and 12%, respectively; p<0.001) [25], [26].
As yet, no study has reported the effect of treatment for chronic hepatitis C on steatosis in co-infected patients, although in HCV monoinfection there have been conflicting reports [27], [28], [29].
A large subset of centrally interpreted paired liver biopsies from APRICOT, graded for steatosis and multiple lipid and anthropometric measurements, was analyzed to assess the effect of steatosis on the efficacy of anti-HCV therapy and to examine the effect of treatment on steatosis. Secondary endpoints of the study were to determine the prevalence of steatosis and to identify baseline characteristics associated to steatosis and the effect of anti-HCV therapy on body measurements.
Discussion
In this analysis of hepatic steatosis in a large population of HIV/HCV co-infected persons, we found the prevalence of hepatic steatosis to be 23%, lower than reported in other studies, 40-70% [15], [22], [34]. There are possible explanations for this result. First, we did not have information for the total ITT APRICOT population as baseline biopsies were not collected nor centrally interpreted except for this cohort. So, prevalence of steatosis in the overall population could have been different. Second, APRICOT comprised patients from all continents and many countries with diverse genetic/constitutional/diet differences, whereas most reported prevalence studies are from single countries/region or even cities. Third,in view of the association of ART to steatosis, the population of APRICOT who started anti-HCV treatment in 2000, as a whole was less ART "experienced", than more recent studied populations. Finally, assessment of steatosis relies on the quality of liver biopsy and the pathological interpretation. In order to study our end points, we were careful to include only paired and centrally reviewed biopsies. The number of subjects in this cohort (283) was robust enough to demonstrate important conclusions from this study.
This study demonstrates that HCV genotype 3 is significantly associated with steatosis in patients with HIV/HCV co-infection. Although this association has been shown in HCV monoinfection [35], previous studies in co-infected patients have failed to show this correlation possibly because other studies recruited only 1-11% of patients with HCV genotype 3 [13], [14], [15] compared with 27% of patients with HCV genotype 3 recruited to APRICOT [25].
While studies in HCV monoinfection have hypothesized that steatosis is caused by either the direct action of HCV genotype 3 on hepatic lipid homeostasis or secondary to metabolic factors (alcohol use or MS) [4], causes of steatosis in co-infection may be more complex. Here, we have shown that as well as being more likely to have HCV genotype 3, patients with steatosis were more prone to have histological diagnosis of bridging fibrosis/cirrhosis, higher levels of HCV RNA, and random elevated blood glucose. Our results expand upon those previously published by Lapadula et al., where co-infected patients infected with HCV genotype 3 had lower risk of hypercholesterolemia and hypertriglyceridemia [36].
We have confirmed that nucleoside analogs appear to be important in the aetiology of steatosis in co-infected patients infected with HCV non-3 genotypes. An association between nucleoside analogs and hepatic steatosis has been reported previously [15], [17], [37], as has an association between nucleoside analogs and MS [16]. However, this is the first time that nucleoside analogs have been implicated to steatosis in co-infected patients with HCV non-3 genotypes.
MS has been implicated as the main cause of steatosis in HCV monoinfection for patients with non-3 genotypes and the prevalence of MS has been reported around 17 and 45%, similar to the 24% found in this study [16], [38], [39].
Lipoatrophy/Lipodeposition has been shown to be associated with MS in HIV/HCV co-infected patients [16]. Although this study was not powered to examine body measurements differences, in our study most anthropometric measurements were not different, and did not alter over time in patients with and without steatosis, with the exception of neck circumference. We have no explanation for these findings and further prospective studies are needed to examine the affect of IFN therapy in body measurements.
Multiple regression analysis demonstrated that hip circumference is significantly associated with steatosis. This association of a specific anthropometric change with steatosis is a novel finding, and adds to existing evidence that body weight, BMI or non-specific lipodeposition/lipoatrophy are important prognostic factors for steatosis [13], [15]. Further research is required to confirm a strong association between MS and steatosis in HIV-HCV co-infected patients.
In patients with HCV monoinfection, a decrease in steatosis has been observed in those achieving SVR, especially in patients infected with genotype 3 [4], [28]. In our analysis, co-infected patients achieving SVR significantly reduced steatosis in patients infected with HCV genotype 3, but not in those infected with other HCV genotypes. There are two important conclusions from these findings. First, this again suggests that at least two separate mechanisms may contribute to the overall incidence of steatosis in patients with co-infection, with factors other than genotype implicated in patients with HCV non-3 genotype. A recent study in HIV/HCV co-infection, confirmed that IR was associated to lower SVR, as has been reported in HCV monoinfected patients [40], [41], [42]. Patients who achieved an SVR also had decrease in IR, suggesting a role for HCV in the hyperinsulinemic status of co-infected patients and also providing a possible mechanism for improved steatosis in genotype 3 patients with SVR [40].
Second, in non-3 genotype patients, we have shown that HCV clearance does not improve hepatic steatosis, with the possibility of further progression of hepatic fibrosis. This finding has important clinical relevance in co-infected patients as severe liver function impairment may still occur after HCV clearance.
We established that the probability of achieving SVR was not affected by steatosis at baseline in patients with co-infection. This is consistent with findings in HCV monoinfected patients [27], and indicates that steatosis should not be used as baseline predictor of response to anti-HCV therapy.
Results
The intent-to-treat (ITT) population of the APRICOT study comprised 860 patients. Paired biopsies from 283 of 860 patients (33%) in the ITT population were submitted for central reading and assessed for steatosis and constitute the study cohort for this study.
Steatosis at baseline
At baseline, 65 of the 283 patients (23%) had steatosis. The baseline characteristics of these patients (Table 1) were similar to the overall population of the APRICOT study [25]. Patients with steatosis were more likely to have HCV genotype 3 infection (p<0.0001), bridging fibrosis/cirrhosis (p=0.0311), higher HCV RNA level (p=0.0132), higher ALT quotient (p=0.0067), increased serum triglyceride (p=0.0132), lower total cholesterol (p=0.0004) HDL cholesterol (p<0.0001) and LDL cholesterol (p=0.0008) and random elevated blood glucose (p=0.0097) levels than patients without steatosis.
There were no significant differences in rates of history of diabetes, hyperglycemia, hypercholesterolemia or hypertriglyceridemia among patients with and without steatosis. A total of 277 patients were evaluable for MS at baseline, and 261 at final assessment. A larger proportion of patients with baseline steatosis had MS than those without steatosis (38.5% vs. 19.8%; p=0.0026).
Baseline factors associated with steatosis
Model 1 - all patients (without HDL and anthropometric data)
Data from the total study cohort were included in this analysis (n=282). The final model of independent factors associated with steatosis included HCV genotype (genotype 3 vs. non-3; p<0.0001), cirrhosis vs. no cirrhosis; p=0.0356, serum triglycerides (per 1mmol/L increase; p=0.0025), serum glucose (per 1mmol/L increase; p=0.0489) and serum cholesterol (per 1mmol/L increase; p=0.0176) (Fig. 1a).
Model 2 - included HDL and anthropometric data
A total of 258 patients were included in this analysis, with 25 patients from the cohort with at least one missing value excluded from the model. Genotype (genotype 3 vs. non-3; p=<0.0001) HDL (per 0.1mmol/L decrease; p=0.0003), hip circumference (per cm increase; p=0.0083), inflammation score (<10 vs. >10; p=0.0161), ALT ratio (per 1mmol/L increase; p=0.0272), serum glucose (per 1mmol/L increase; p=0.0282) and HCV RNA (per Log10 increase; p=0.0411) were all independently associated with steatosis at baseline (Fig. 1b).
Model 3 - by genotype
Because of the magnitude of the effect exerted by HCV genotype in the overall model and the established association to steatosis, separate models were developed for HCV genotype 3 and non-3 (Fig. 2). Baseline factors significantly associated with hepatic steatosis in patients infected with HCV genotype 3 (n=84) were; HCV RNA level (per Log10 increase; p=0.0307), systolic blood pressure (per 10mmHg increase; p=0.0270), decreasing serum cholesterol level (per 1mmol/L change; p=0.0022) and necroinflammatory grade (L10 vs. <10; p=0.0109).
Baseline factors significantly associated with hepatic steatosis in patients with non-3 genotypes (n=198) were; treatment with zidovudine (yes vs. no; p=0.0015), treatment with didanosine, stavudine or zalcitabine (yes vs. no; p=0.0402), increasing necroinflammatory grade (L10 vs. <10; p=0.002), triglyceride level (per 1mmol/L increase; p<0.0001) and BMI (per 1U increase; p<0.003).
Effect of steatosis on treatment efficacy
The presence of steatosis at baseline did not affect the probability of achieving an SVR in any of the three treatment groups (Fig. 3). For this reason, data from the three treatment groups were combined for the purposes of examining the influence of a SVR on steatosis within particular genotypes.
Effect of treatment outcome on steatosis
Among patients who achieved SVR, there was a significant (p=0.0003) reduction in the prevalence of steatosis between baseline and the end of follow-up in patients infected with HCV genotype 3, but not in those infected with other HCV genotypes (Fig. 4). Among patients who did not achieve SVR, there was no significant difference in the prevalence of steatosis between baseline and the end of follow-up for any HCV genotype.
Body composition measurements
Hip circumference increase (cm) was independently associated to steatosis at baseline in logistic regression analysis (Fig. 1b). The only measurement different at baseline among patients with and without steatosis was neck circumference. (38.6, 37.3cm p=0.0054). Mean hip circumference was higher in patients with steatosis, (95.2cm compared to 93.4cm), but did not reach statistical significance. We found a significant decrease in neck circumference from baseline to final assessment in patients with steatosis. (-0.2 and -1.3cm p=0.0287).
Also decrease in neck circumference was significantly different (-1.3cm for patients with improvement in steatosis vs. -0.4cm) in patients with no improvement; p=0.0272.
Methods
Patient characteristics
The complete inclusion and exclusion criteria and primary results of APRICOT study have been published elsewhere [25]. The study was conducted at 95 centers in 19 countries between June 2000 and September 2003. The study was conducted conforming to ethical guidelines of the Declaration of Helsinki, after Institutional Review Board approval at each institution. Informed Consent Document was explained and signed by every patient enrolled in the study.
Briefly, patients were HCV treatment-naive adults with confirmed HIV and HCV infection, elevated serum alanine aminotransferase (ALT) activity and compensated liver disease. Patients were also required to be either receiving stable ART (no changes expected during the first 8weeks of study treatment) or no ART.
Study design
As described elsewhere [25], eligible patients were randomized to 48weeks of once-weekly treatment with subcutaneous Peg-IFN alfa-2a 180μg plus either oral RBV 400mg twice daily or placebo, or subcutaneous interferon alfa-2a three times weekly at a dose of 3 million international units plus RBV 400mg twice daily [25].
The primary efficacy endpoint in APRICOT was sustained virological response (SVR), defined as undetectable serum HCV RNA (<50IU/mL) by qualitative polymerase chain reaction assay (COBAS AMPLICOR_ HCV Test, v2.0), at the end of follow-up (week 72) [25].
Histological assessments and outcome measures
Baseline liver biopsies for the APRICOT study population were not centrally interpreted. The post-treatment liver biopsy was voluntary, and only patients that agreed to have a second biopsy had paired biopsies with central interpretation.
Histological assessments were based on liver biopsies obtained at baseline (K15 months before enrollment) and at the end of follow-up. For the second biopsy, the range of assessment time was from 360days (week 56) to 806days (week 115) after baseline. The lower end of assessment time was for patients that had early discontinuation and had liver biopsy performed 6months after end of study treatment but before week 60. The mean value of time for the second biopsy is week 72 (day 509) and standard deviation is 47days. All pre- and post-treatment paired biopsies were analyzed by a central pathologist blinded to host and viral factors, treatment and treatment outcome. Pre- and post-treatment liver biopsies were scored using the Ishak-modified Histological Activity Index (HAI) scoring system [30]. Steatosis was categorized by the proportion of hepatocytes with fatty changes per low power field: K5%, >5-33%, 34-66%, >66% [31]. For the purposes of this analysis, patients with >5% of hepatocytes with fatty changes per lower power field were considered to have hepatic steatosis. Improvement in steatosis was defined as a change to <5% in the second biopsy.
The patients with paired liver biopsies analyzed by the central pathologist, comprise the study cohort for this study.
Factors known to be associated with steatosis, including lipid levels, hyperglycemia and body composition (lipoatrophy and lipodeposition) were analyzed. The effect of steatosis on SVR rates and the effect of HCV treatment on steatosis were also examined. The number of patients with MS was assessed using the criteria of the American Heart Association [9], where MS is identified by the presence of three or more of the following components: elevated waist circumference (men - equal to or greater than 102cm, women - equal to or greater than 88cm); elevated triglycerides (equal to or greater than 150mg/dL); reduced HDL cholesterol (men - less than 40mg/dL, women - less than 50mg/dL); elevated blood pressure (equal to or greater than 130/85mmHg); elevated fasting glucose (equal to or greater than 100mg/dL).
Laboratory and metabolic assessments
Hematology, chemistry and fasting lipid assessments were performed at baseline and at multiple time points. Body composition measurements including neck, arm, waist, hip, mid-thigh, and chest measurements were also performed at baseline and at multiple time points.
Statistical analysis
Associations between baseline characteristics and steatosis (univariate logistic regression) were analyzed by Wald Chi-square test. Baseline factors studied included sex, age, body mass index (BMI), weight, HCV genotype, serum HCV RNA level, ALT quotient (ALT/upper limit of normal/ULN), histological diagnosis and serum levels of cholesterol, triglycerides and glucose (non-fasting measurements). The use of ART and hyperglycemia, hypercholesterolemia, hypertriglyceridemia, and hypertension at baseline were also included in the assessment.
t-Tests were used to analyze body measurements changes from baseline to end of treatment between patients with and without steatosis. When the assumption of equal variances in both groups was not reasonable, the Satterthwaite method was used to estimate the variance for the t-statistic and the degree of freedom of the corresponding t-distribution [32].
Body measurements were compared for patients with SVR and those who failed to achieve SVR, and also between patients with or without improvement in steatosis after end of treatment. Stepwise multiple logistic regression analyses were performed to explore factors associated with steatosis at baseline across all treatment groups. In the stepwise model building process, a variable was added to the model if the adjusted Chi-square statistic was significant at the 0.1 level and a variable was deleted from the model if the Wald Chi-square statistic was not significant at the 0.05 level. Baseline factors considered were age, sex, BMI, body weight, HCV genotype, serum HCV RNA level, ALT quotient, bridging fibrosis/cirrhosis vs. no bridging fibrosis/cirrhosis, fibrosis stage and necroinflammatory grade, serum levels of cholesterol, triglyceride and glucose, diagnosis of hypertriglyceridemia, hypercholesterolemia, hyperglycemia, diabetes, hypertension, and/or elevated blood pressure, and ART (any ART; treatment with nucleoside analogs; treatment with didanosine, stavudine or zalcitabine; treatment with zidovudine) and CD4+ cell count. Due to the fact that HDL and anthropometric data were missing in several patients, models with and without these factors were analyzed. Changes in the prevalence of steatosis between baseline and end of follow-up were analyzed by McNemar test [33].
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