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Clin Liver Dis 12 (2008) xiiixiv

Preface

K. Rajender Reddy, MD David E. Kaplan, MD, MSc Guest Editors

Despite major advances in the understanding of viral pathogenesis and signicant evolution in antiviral therapies, chronic infection with the hepatitis C virus (HCV) remains a highly prevalent and burdensome disease worldwide. It aects approximately 3% of individuals worldwide and is the cause for approximately 7,500 annual deaths in the United States alone. While the current treatments oer reasonable response rates, there remain enormous challenges that include therapy-related adverse events, the rising population of non-responders, and special populations such as those with cirrhosisdparticularly decompensated liver disease. Novel HCV-specic antiviral agents loom on the horizon, bringing with them the hope of major improvements in cure rates and dramatic alterations of the natural history of HCV infection. Challenges undoubtedly are to come up in the search for treatment strategies that are better tolerated, have higher response rates with shortened duration of therapy, while we maintain a high genetic barrier to prevent the phenotypic expression of viral resistance. Therefore, in this issue of the Clinics in Liver Disease, we focus on topics related to optimization of currently available therapies, evolution of noninvasive disease-staging techniques, and management of important conditions associated with or complicating chronic HCV infection while providing an update on emerging antiviral therapies and a snapshot of advances in the understanding of viral pathogenesis. Along with discussion of tailored

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antivirals in the article by Bernd Kronenberger, Christoph Welsch, Nicole Forestier, and Stefan Zeuzem, we present two articles discussing optimization of interferon-based approaches by Mitchell L. Shiman and Thomas Berg. We examine the evolving state of non-invasive staging modalities in the article by David S. Kotlyar, Wojciech Blonski, and Vinod K. Rustgi. Three translational articles focus on important hostvirus interactions that contribute to innate and adaptive immune responses in the setting of chronic viral infection of hepatocytes that are likely to impact the ecacy of novel antiviral therapies and antiviral vaccine approaches in the articles by Gyongyi Szabo and Angela Dolganiuc; Zania Stamataki, Joe Grove, Peter Balfe, and Jane A McKeating; and Chloe L. Thio. We conclude with ve articles focusing on conditions frequently and concomitantly aecting HCV patients (HIV co-infection, fatty liver disease), dicult-to-manage complications (hepatocellular carcinoma, extrahepatic manifestations of HCV), and issues relating to liver transplantation as reviewed by Vincent Lo Re III, Jay R. Kostman, and Valerianna K. Amorosa; Onpan Cheung and Arun J. Sanyal; Anna Linda Zignego and Antonio Crax` ; Wojciech Blonski and K. Rajender Reddy; and Elizabeth C. Verna and Robert S. Brown. We hope that these articles prove informative and provocative in their choice of both clinical and translational topics critically assessing the current state of the art and providing a glimpse of the future of hepatitis C management. K. Rajender Reddy, MD Department of Hepatology Hospital of the University of Pennsylvania 3 Dulles 3400 Spruce Street Philadelphia, PA 19104 E-mail address: rajender.reddy@uphs.upenn.edu David E. Kaplan, MD, MSc Division of Gastroenterology University of Pennsylvania 600 CRB 415 Curie Boulevard Philadelphia, PA 19104 Research Section Philadelphia Veterans Administration Medical Center Research A402A 3900 Woodland Avenue Philadelphia, PA 19104 E-mail address: dakaplan@mail.med.upenn.edu

Clin Liver Dis 12 (2008) 487505

Optimizing the Current Therapy for Chronic Hepatitis C Virus: Peginterferon and Ribavirin Dosing and the Utility of Growth Factors
Mitchell L. Shiman, MD
Hepatology Section, Virginia Commonwealth University Medical Center, Box 980341, Richmond, VA 23298, USA

The primary goal when treating patients who have chronic hepatitis C virus (HCV) is to achieve a sustained virologic response (SVR). For patients to achieve this goal, however, three independent milestones must be sequentially achieved (Table 1). The rst step is that the patient must achieve a virologic response and become HCV RNA undetectable during treatment. It is fairly obvious but rarely stated that patients who do not achieve a virologic response cannot possibly achieve an SVR. The second step is that the patient must maintain this response by remaining HCV RNA undetectable throughout the duration of therapy. Patients who break through and become HCV RNA-positive during treatment also cannot achieve an SVR. The nal step is that the patient must not relapse after treatment has been competed; thus: SVR (virologic response) (breakthrough plus relapse). The current treatment for chronic HCV infection is the combination of peginterferon and ribavirin [1,2]. These medications are eective for the treatment of chronic HCV. Approximately 80% of patients who have HCV genotype 1 and virtually all patents who have genotype 2 or 3 have a rapid and profound reduction in HCV RNA within the rst 3 months after initiating treatment [14]. Unfortunately, not all patients with this early virologic response (EVR) pattern achieve an SVR [1,4]. There are three major reasons for this. Some patients do not become HCV RNA undetectable even though they achieved an EVR and had a 2-log reduction in HCV RNA within the rst 12 weeks of treatment. In a previous review, this was referred to as a partial early virologic response [5]. Because these patients do not

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Table 1 Milestones for achieving a sustained virologic response Virologic response Preventing breakthrough Preventing relapse Patients must become HCV RNA undetectable within 24 weeks after initiating treatment Patients must remain HCV RNA undetectable throughout the duration of treatment Patients must remain HCV RNA undetectable after treatment is discontinued

become HCV RNA undetectable, they cannot achieve an SVR. Peginterferon and ribavirin are associated with adverse events, which, at times, may be so severe that dosing with one or both of these medications has to be adjusted, interrupted, or prematurely discontinued. These actions may reduce the SVR by preventing an EVR from being achieved [6] or by enhancing breakthrough and relapse [79]. Finally, some patients have a slow response to treatment and only become HCV RNA undetectable late during the course of treatment. These slow to respond patients have an extremely low SVR because they experience a high rate of relapse [1012]. This article focuses on how altering the doses of peginterferon and ribavirin can aect the three milestones leading to SVR, namely, virologic response, breakthrough, and relapse. This discussion rst reviews the various virologic patterns and denitions of response, nonresponse, breakthrough, and relapse. The discussion will then address the roles that peginterferon and ribavirin play in achieving virologic response, how adjusting the doses of peginterferon or ribavirin could adversely aect these response patterns, and when it would be appropriate to use hematologic growth factors in the treatment of chronic HCV. Patterns of virologic response The term early virologic response refers to a 2-log decline in HCV RNA from the pretreatment baseline or being HCV RNA undetectable within 12 weeks after the initiation of treatment [1,13,14]. Approximately 80% of patients who have genotype 1 and virtually all patients who have genotypes 2 and 3 achieve an EVR [1,4,6,10,13,14]. Patients without an EVR rarely if ever achieve an SVR [1,6,10,13,14]. These patients are referred to as having a null response [5]. At the Second National Institutes of Health Consensus Development Conference on the Management of HCV, it was recommended that all patients who have genotype 1 undergo HCV RNA testing at baseline and at week 12 [13,14]. Patients without an EVR (a null response) should stop treatment because they cannot achieve an SVR. It was further recommended that all genotype 1infected patients with an EVR continue treatment for 48 weeks and that all patients who have genotypes 2 and 3 simply be treated for 24 weeks [1,13,14]. Unfortunately, not all patients

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with an EVR become HCV RNA undetectable, and continuing treatment in these patients cannot lead to an SVR. It is therefore apparent that these recommendations need to be modied. Fig. 1 illustrates various virologic response patterns in patients with an EVR. It is apparent that an EVR constitutes a wide spectrum of virologic responses that include patients who become HCV RNA undetectable within 4 weeks to as late as 24 weeks after initiating treatment. Overall, 80% of patients who have genotype 1 achieve an EVR, but only 65% actually become HCV RNA undetectable [1,10]. The remaining 15% of patients have a partial virologic response (Fig. 2). Approximately 10% of patients with a partial virologic response eventually become HCV RNA undetectable if treatment is continued past week 24 [10]. These patients rarely, if ever, achieve an SVR when treated for only 48 weeks. It is critically important that the partial virologic response pattern be recognized and treatment be discontinued in these patients at week 24 unless an alternative strategy is implemented (see section on management of partial responders). Dening the time when the patient rst becomes HCV RNA undetectable is critically important in the management of HCV treatment because this is directly related to the likelihood of an SVR [10]. Patients who become HCV RNA undetectable within 4 weeks of initiating treatment are referred to as having a rapid virologic response (RVR). As illustrated in Fig. 2, approximately 15% of patients who have genotype 1 and 66% of patients who have genotypes 2 and 3 achieve an RVR [4,10]. These patients are exquisitely sensitive to treatment; they have an SVR rate of 90% regardless of their genotype and the type of therapy they receive [4,10]. In a retrospective analysis of a large clinical trial database, patients who had genotype 1 and an RVR had an SVR of 90% whether they were treated with peginterferon
8

Log HCV RNA (IU/mL)

7 6 5 4 3 2 1 0 -6 0 6

Peginterferon/Ribavirin

2-log decline

Partial response
Limit of detection

SVR 12 18 24 30 36 42 48 54 60 66 72 78

WEEKS
Fig. 1. Patterns of early virologic response. All patients in this example had an EVR. The time at which these patients became HCV RNA undetectable varied from week 4 to week 24, however. Patients who become HCV RNA undetectable within 4 weeks of initiating treatment are referred to as having a rapid virologic response (RVR). Those who do not become HCV RNA undetectable until week 24 of treatment are referred to as slow to respond. Patients with a partial virologic response have a 2-log decline in HCV RNA from the pretreatment baseline but do not become HCV RNA undetectable.

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Early Virologic Response = 80%

% of Patients

80 60 40 20 0 4 12 24 Partial Non Response Response


Genotype 1 Genotypes 2 and 3

Week Became HCV RNA (-)


Fig. 2. The percentage of patients who have HCV genotype 1 or genotypes 2 and 3 and become HCV RNA undetectable at various time points during treatment. Patients who have genotype 1 were treated with peginterferon alfa-2a at a dosage of 180 mg/wk and ribavirin at a dosage of 1000 to 1200 mg/d. Patients who have genotypes 2 and 3 were treated with peginterferon alfa-2a at a dosage of 180 mg/wk and ribavirin at a dosage of 800 mg/d. (Data from Shiman ML, Suter F, Bacon BR, et al. Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3. N Engl J Med 2007;357:12434; and Davis GL, Wong JB, McHutchison JG, et al. Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology 2003;38:64552.)

and full-dose ribavirin, a lower dose of ribavirin, standard interferon and ribavirin, or even peginterferon monotherapy [10,15]. Shortening the duration of therapy from 48 to only 24 weeks also seemed to be eective in achieving high rates of SVR in genotype 1infected patients with an RVR [15]. These observations have signicant implications for dosing peginterferon and ribavirin. Because patients with an RVR are so responsive to treatment, the primary response to adverse events in these patients should be to reduce the doe of peginterferon or ribavirin and not to add growth factors or other adjuvant therapies. The later during treatment a patient becomes HCV RNA undetectable, the lower is the rate of SVR [10]. Patients who have genotype 1 who become HCV RNA undetectable between treatment weeks 4 and 12 have an SVR of approximately 66%, but those who become HCV RNA undetectable between weeks 12 and 24 have an SVR of only 45%. The low SVR in slow responders occurs because of a higher rate of relapse. Genotype 1infected patients who become HCV RNA undetectable between weeks 12 and 24 have been referred to as slow to respond, and recent studies have demonstrated that prolonging the duration of treatment in these patients from 48 to 72 weeks can significantly reduce the relapse rate [11,12]. This would obviously enhance the SVR. Patients who have genotypes 2 and 3 who do not become HCV RNA undetectable after week 4 have an SVR of only 49% [4], and a recent retrospective analysis has suggested that prolonging treatment in these patients could also reduce relapse [16]. Adjusting the duration of therapy in response to on-treatment virologic response is discussed in a later chapter in this issue of Clinics in Liver Disease (see the article by Berg elsewhere in this issue).

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Two additional patterns of virologic response are important to recognize: breakthrough and relapse (Fig. 3). Patients with breakthrough initially become HCV RNA undetectable in serum during treatment but then have reappearance of HCV RNA in serum despite ongoing treatment. Patients with relapse become and remain HCV RNA undetectable in serum throughout treatment but then have reappearance of HCV RNA in serum after treatment is discontinued. Common reasons for breakthrough and relapse include the premature termination or temporary interruption of peginterferon or ribavirin [8].

Eect of interferon dosing on virologic response It has long been recognized that the ability of interferon to reduce HCV RNA in serum is a dose-dependent process. Studies conducted at the authors center longer than a decade ago demonstrated that increasing the dosage of standard interferon from 3 to 5 to 10 mU and then to 20 mU three times weekly every 3 months in those patients who remained HCV RNApositive led to a stepwise increase in the overall percentage of patients becoming HCV RNA undetectable [17]. Up to 80% of patients treated with this approach eventually became HCV RNA undetectable. Increasing the dosage of peginterferon alfa-2a from 45 to 180 mg/wk or peginterferon alfa-2b from 0.5 to 1.5 mg/kg/wk also led to a stepwise increase in virologic response [18,19]. High daily dosages of consensus interferon (915 mg/d) and higher dosages of peginterferon, up to a peginterferon alfa-2a dose of 360 mg/wk and a peginterferon alfa-2b dose of 3.0 mg/kg/wk, respectively, have been shown to lead to a virologic response in approximately 15% to 20% of patients who previously failed to become HCV RNA undetectable after treatment with standard doses of interferon or peginterferon [2023]. The major limitation to using higher doses of interferon or peginterferon is the adverse events of these agents, which increase with increasing dose and

HCV RNA (IU/mL)

7 6 5 4 3 2 1 0 -6 0 6

Peginterferon/Ribavirin

Relapse

Null response

Breakthrough

2-log decline

Limit of detection

12 18 24 30 36 42 48 54 60 66 72 78

WEEKS
Fig. 3. The patterns of null response, breakthrough, and relapse.

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cause patients to discontinue treatment [2023]. The current recommended starting doses for the two peginterferons were derived by balancing response and discontinuation rates to achieve the highest overall rates of SVR. Because most patients respond to the current starting dosages of peginterferon (180 mg/wk for peginterferon alfa-2a and 1.5 mg/kg/wk for peginterferon alfa-2b), it is not appropriate to initiate treatment with higher doses of these medications. In contrast, it is quite rationale to consider escalating the dose of peginterferon or switching to high doses of daily interferon in patients with a suboptimal response who could tolerate such a treatment strategy. Increasing the interferon dose to overcome nonresponse As depicted in Figs. 2 and 3, approximately 20% of patients who have genotype 1 have a null response during treatment and fail to achieve an EVR [1,2,10]. Such patients are likely resistant to the antiviral eects of interferon. In vitro studies have demonstrated that interferon binds to its membrane receptor with the same anity in nonresponders [24], but the downstream immunologic response is downregulated [25]. As a result, increasing the dose of interferon or peginterferon is unlikely to lead to a virologic response in patients with a null response. Approximately 15% of patients who have genotype 1 and up to 7% of patients who have genotypes 2 and 3 have a partial response to treatment; they achieve an EVR but never become HCV RNA undetectable [1,4,10]. These patients are sensitive to the antiviral and immunologic eects of interferon and ribavirin, and two previous studies have suggested that most nonresponders who responded to higher doses of interferon or peginterferon during retreatment are those with a previous partial virologic response [26,27]. In both of these studies, most patients, if not the only patients, who achieved an SVR during retreatment with a higher dose of interferon or peginterferon were those who had a decline in HCV RNA to less than 100,000 copies/mL (approximately 50,000 IU/mL) during the initial course of treatment. It is therefore reasonable to consider escalating the interferon dose in patients with a partial virologic response as soon as this response pattern is recognized. This typically occurs between treatment weeks 12 and 24 (Fig. 4). If patients do not have a further decline in HCV RNA and become virus RNA undetectable with 3 additional months of high-dose interferon therapy, it is highly unlikely that a virologic response is going to occur and treatment thereafter should be discontinued. Patients with a prior partial virologic response can also be retreated with higher doses of interferon. If such patients do not achieve an EVR or become HCV RNA undetectable within 12 to 24 weeks of treatment, however, this approach should also be abandoned. In either situation, a higher dose of peginterferon or high doses of daily consensus interferon can be used.

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HCV RNA (IU/mL)

7 6 5 4 3 2 1 0 -6 0

Peginterferon/Ribavirin Intensify interferon dose


2-log decline

Limit of detection

SVR 6 12 18 24 30 36 42 48 54 60 66 72 78

WEEKS
Fig. 4. The impact of intensifying the interferon or peginterferon dose in a patient with a partial virologic response. If the patient does not respond and becomes HCV RNA undetectable within 3 months of dose intensication, treatment should be discontinued.

Eect of ribavirin dosing on virologic response Ribavirin is an essential ingredient in the treatment of chronic HCV and aects all three of the milestones required to achieve an SVR (see Table 1). Ribavirin is a weak antiviral agent and does not cause a reduction in serum HCV RNA when used alone [28]. When combined with interferon, however, it more than doubles the virologic response rate (from 24% to 50%) compared with that observed with standard interferon alone [29]. Adding ribavirin to peginterferon also enhances virologic response [1]. Because peginterferon is more eective than standard interferon, however, this represents only a 10% increase (from 59% to 69%) compared with that observed with peginterferon monotherapy. Ribavirin enhances virologic response by accelerating the decline in HCV RNA over that observed with interferon alone [30]. Although the combination of peginterferon and ribavirin does not increase the percentage of patients who achieve an RVR, it more than doubles (from 18% to 38%) the percentage of patients who become HCV RNA undetectable by treatment week 12 [10]. As noted previously, becoming HCV RNA undetectable sooner during treatment reduces relapse. The starting dose of ribavirin may also play a role in enhancing virologic response. In several randomized controlled clinical studies a 3% to 9% higher virologic response rate was consistently observed when the starting ribavirin dosage was increased by 200 to 400 mg/d [3,31,32]. It remains unclear from these studies, however, if this dierence is statistically or clinically signicant. Ribavirin also helps to maintain the response and enhances the SVR by preventing breakthrough and reducing relapse. Unfortunately, the adverse events associated with ribavirin prevent higher doses of this medication from being used and, in some patients, from maintaining adequate ribavirin exposure. Managing the adverse events of ribavirin has been the subject of considerable debate.

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Impact of adverse events and dose reduction The treatment of chronic HCV with peginterferon and ribavirin is associated with numerous adverse events [1,2]. The most common of these can be classied as systemic u-like symptoms, psychiatric manifestations, autoimmune reactions, and hematologic toxicities. The management of these adverse events has been discussed previously [33]. Although adverse events can be successfully managed in many cases, approximately 20% to 40% of patients require that the dose of peginterferon or ribavirin be reduced or temporarily interrupted. In 5% of patients, adverse events are so severe that treatment must be discontinued [1,2]. Higher doses of peginterferon and ribavirin have been associated with a greater incidence of adverse events [1823,31,32,34]. The need to alter the dosing of peginterferon or ribavirin in response to adverse events may have a negative impact on all three milestones leading to an SVR. Two studies have clearly demonstrated that when the dose of peginterferon is reduced to less than 80% within the rst 12 to 20 weeks of treatment, the ability to achieve an EVR and become HCV RNA undetectable is signicantly impaired [6,7]. This is consistent with the observations that achieving a virologic response highly depends on the dose of interferon [35] or peginterferon dose [1823] and depends far less on the dose of ribavirin [3,31,32]. Because achieving a virologic response is the rst essential milestone leading to an SVR (see Table 1), any reduction in this response has a negative impact on the SVR. Prematurely discontinuing ribavirin leads to breakthrough and a higher relapse rate, even if the peginterferon dose remains unaltered. In a recent study, patients who were HCV RNA undetectable at treatment week 24 were randomly assigned to stop ribavirin and continue peginterferon alone or to remain on both drugs [8]. Within 6 weeks of stopping ribavirin, breakthrough began to occur and this increased stepwise over time. At treatment week 48, 24 weeks after stopping ribavirin, breakthrough had developed in 12% of patients. In contrast, breakthrough occurred in only 3% of patients randomized to continue peginterferon and ribavirin, and each of these patients prematurely discontinued ribavirin or both drugs in response to adverse events. It is therefore apparent that the primary, if not the only, reason for breakthrough is an interruption or permanent premature discontinuation of ribavirin. Relapse was also signicantly greater in patients who prematurely stopped ribavirin: 42% compared with only 29% in patients who remained on peginterferon and ribavirin. The only subgroup of patients who did not develop breakthrough or have an increased relapse rate after prematurely stopping ribavirin were those with an RVR. This strongly suggests that the primary response to adverse events in patients with an RVR should be dose reduction rather than growth factor support. As opposed to interrupting or prematurely stopping ribavirin, which enhances breakthrough and relapse, recent data suggest that merely reducing

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the dose of ribavirin in response to adverse events does not signicantly aect these milestones, and therefore has little impact on the SVR. The rst report to evaluate the impact of dose reduction on the SVR demonstrated that only those patients whose total cumulative peginterferon or ribavirin dose declined to less than 80% of expected after treatment week 12 had a signicant decline in SVR [36]. So let us consider the following question: how much of a reduction in ribavirin dose was required to decrease to less than 80% by week 12 in this study? The answered is depicted in Fig. 5. In this study, the starting dosage of ribavirin was 800 mg/d [36]. The total cumulative expected ribavirin dosage was therefore: 800 mg/d 7 d/wk 48 wk of therapy 268,800 mg. Fig. 5A illustrates the impact of reducing the ribavirin dosage from 800 to 600 mg/d, as was done in this study [36], at various time points during the 48 weeks of treatment. If the ribavirin dose reduction occurred at treatment week 24, the patient still received 88% of the total
% MAXIMAL RIBAVIRIN DOSE

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WEEKS

% MAXIMAL RIBAVIRIN DOSE

100 90 80 70 60

12

18

24

30

36

42

48

WEEKS
Fig. 5. (A) Impact of dose reduction on the total cumulative dose of ribavirin. The starting dosage of ribavirin was 800 mg/d, and this was subsequently reduced to 600 mg/d at various time points during treatment. It was assumed that no doses of ribavirin were missed. (B) Impact of dose interruption on the total cumulative dose of ribavirin. The starting dosage of ribavirin was 800 mg/d. Dosing was interrupted for 3, 7, or 14 days starting at week 6 and then restarted at 600 mg/d for the remaining 48 weeks of treatment. Calculations in both gures were based on the description of ribavirin dosing from the study by McHutchison and colleagues. (Data from McHutchison JG, Manns M, Patel K, et al. Adherence to combination therapy enhances sustained response in genotype-1-infected patients with chronic hepatitis C. Gastroenterology 2002;123:10619.)

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cumulative expected ribavirin dose. Moving the time at which this dose reduction occurred to treatment week 12 or 6 failed to lower the cumulative ribavirin dose to less than 80% within the rst 24 weeks of treatment. Even if dose reduction occurred at treatment week 4, the patient still received 79% of the cumulative ribavirin dose by week 24. It is therefore apparent that patients could not have received less than 80% of their ribavirin dose in this study through dose reduction alone. The only way this could have happened was for patients to have missed doses. The impact of interrupting ribavirin dosing is illustrated in Fig. 5B. In this gure, ribavirin dosing was interrupted at the start of treatment week 6 for variable periods before the dosage was reduced from 800 to 600 mg/d. Missing just 3 days of ribavirin reduced cumulative ribavirin exposure at week 12 to 81%, and missing 7 and 14 days reduced this to only 77% and 71%, respectively. A more recent analysis has evaluated the impact of stepwise ribavirin dosing, from greater than 97% to less than 60% of the total expected cumulative dose, on virologic response and SVR [9]. It is important to note that this study included only those patients who remained on full-dose peginterferon for 48 weeks, and therefore evaluated the impact of reducing only the ribavirin dose. No signicant impact of ribavirin dose on virologic response was observed. A signicant decline in SVR (from 57% to 67% to only 34%) did occur but only when the total cumulative ribavirin dose declined to less than 60%. As illustrated in Fig. 5, this can only occur by interrupting or prematurely discontinuing ribavirin dosing. Because no signicant decline in virologic response was observed in this analysis, the decline in SVR must have been secondary to breakthrough and relapse. The impact of dose reducing peginterferon or ribavirin independent of each other was examined in patients with prior nonresponse to interferon (with or without ribavirin) undergoing retreatment with peginterferon and ribavirin in the Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) clinical trial [7]. This study included more than 900 patients who had genotype 1, making it the largest and most comprehensive analysis of dose reduction performed to date. Reducing the total cumulative dose of peginterferon to less than 80% led to a decline in virologic response and SVR. In contrast, reducing the dose of ribavirin had no impact on virologic response or SVR as long as patients remained on full-dose peginterferon and ribavirin dosing was not interrupted or prematurely terminated. It is therefore apparent that missed doses of ribavirin rather than dose reduction is what has an adverse impact on SVR and that this occurs by increasing breakthrough and relapse. Role of epoetin alfa in the treatment of chronic hepatitis C virus One of the most common adverse events in patients receiving peginterferon and ribavirin for treatment of chronic HCV is anemia [1,2]. This is secondary to a dose-dependent hemolysis induced by ribavirin- and

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interferon-induced bone marrow suppression, which inhibits a compensatory reticulocytosis [37]. The mean decline in hemoglobin observed with peginterferon and ribavirin in large registration trials is 2.5 to 3 g/dL [1,2]. Twenty percent of persons have a decline in hemoglobin of 4 g/dL or more, however [38]. Anemia signicantly lowers the quality of life and exacerbates the fatigue experienced by patients receiving HCV therapy [39]. In general, it is recommended that the ribavirin dose be reduced when the hemoglobin decreases by more than 4 g/dL from the pretreatment baseline or to less than 10 g/mL and that it be discontinued if the hemoglobin decreases to less than 8.5 g/dL [1,2]. The role of epoetin alfa for managing anemia in patients receiving peginterferon and ribavirin is controversial. Several years ago, an open-label trial demonstrated that epoetin alfa could reverse the amenia that developed during treatment with peginterferon and ribavirin [40]. This observation led to a double-blind placebo-controlled trial demonstrating that epoetin alfa could reduce anemia, allow patients to remain on a higher mean dose of ribavirin [41], and improve quality of life during HCV treatment [42]. A recent cost analysis suggested that using a hematologic growth factor in lieu of dose reduction would be cost-eective [43]. Unfortunately, this analysis was based on the premise that using epoetin alfa would actually enhance the SVR, without any data to support this claim. Despite these shortcomings, these reports were fueled with the belief that reducing the ribavirin dose would have a negative impact on SVR and led to the widespread use of epoetin alfa and darbepoetin alfa in patients who developed anemia while being treated with peginterferon and ribavirin [39]. The only randomized, prospective, controlled trial evaluating epoetin alfa in patients receiving peginterferon and weight-based ribavirin for chronic HCV failed to demonstrate that this approach could enhance the SVR, even though this agent was administered at the onset of therapy before any patients developed anemia or required dose reduction [32]. There are several reasons why patients who received epoetin alfa in this study failed to demonstrate any improvement in SVR. First, ribavirin was reduced sequentially in 200-mg steps with the goal being not to interrupt or prematurely discontinue dosing. Second, using epoetin-alfa did reduce but did not completely eliminate the need to dose reduce ribavirin. Finally, reducing the dose of ribavirin did not adversely aect the ability of all patients to achieve an SVR, especially those with an RVR. Another study conducted in patients who had HCV and were coinfected with HIV has also demonstrated that the routine use of epoetin alfa did not enhance the SVR during treatment with peginterferon and ribavirin [44]. In this study, no relation between ribavirin dose and anemia was apparent, and the commonly used protease inhibitor zidovudine rather than ribavirin was found to be the major cause of anemia. Although widely used and generally safe, hematologic growth factors do cause adverse events, including hypertension, headache, arthralgias, paresthesias, and injection site erythema [4547]. More recently, an increased

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rate of thromboembolic events has been observed in patients receiving these agents, and this has led the US Food and Drug Administration to administer a warning regarding the overuse of these agents. Antibody-mediated pure red blood cell aplasia has also been reported in an HCV-infected patient receiving epoetin alfa during treatment with peginterferon and ribavirin [48]. It is therefore apparent that dose reduction rather than epoetin alfa should be the rst strategy in a patient receiving peginterferon and ribavirin for HCV treatment, especially if this patient has achieved an RVR or if anemia has developed after the patient has become HCV RNA undetectable. As opposed to reducing ribavirin to 600 mg as recommended in previous studies [1,2,36], the authors group reduces ribavirin by 200-mg steps. This approach maximizes total cumulative ribavirin exposure, as illustrated in Fig. 6. In fact, the medical and nursing sta at the authors institution frequently perform a one-step (200 mg) ribavirin reduction in a patient who has developed severe fatigue even if the hemoglobin has not declined to less than 10 mg/dL. This small reduction in the ribavirin dose does not have a signicant impact on the total cumulative ribavirin exposure (see Fig. 6) and, in many cases, yields signicant symptomatic improvement and allows the patient to be more enthusiastic about continuing therapy. The goal of dose reduction is to prevent the hemoglobin from decreasing so much that the patient must interrupt ribavirin dosing, which clearly increases the risks of breakthrough and relapse [7,8]. These observations do not mean that epoetin alfa should be abandoned as an adjuvant therapy in the treatment of chronic HCV. There is no doubt that some patients develop anemia so rapid and severe in its onset that they cannot possibly remain on ribavirin, and therefore cannot be successfully treated for chronic HCV without the use of epoetin alfa. Profound anemia occurs in approximately 5% to 10% of patients within the rst 12 weeks of initiating HCV treatment [1,2,38], and most of these patients may need to stop or interrupt ribavirin dosing. Many physicians faced with this clinical situation initiate epoetin alfa and then restart ribavirin 1 to 2 weeks later,
% MAXIMAL RIBAVIRIN DOSE
110 100 90 80 70 60 50 40 0 6 12 18 24 30 36 42 48 54
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WEEKS
Fig. 6. The impact of dose reduction by 200-mg steps on the total cumulative dose of ribavirin received during 48 weeks of treatment. The starting dosage of ribavirin in this example was 1200 mg/d.

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after the hemoglobin has increased to greater than 10 g/dL. Unfortunately, this is not an eective strategy, because these patients have a high rate of breakthrough and relapse. As a result, the strategy of the author and his group is simply to discontinue therapy, allow the anemia to resolve, obtain approval to use epoetin alfa, and then restart HCV treatment along with growth factor support from the onset. Severe anemia is more common during HCV treatment in patients who have advanced cirrhosis or chronic renal insuciency and in liver transplant recipients [39]. Most of these patients cannot possibly remain on ribavirin and be successfully treated without the use of epoetin alfa. Role of granulocyte stimulation factor in the treatment of chronic hepatitis C virus Peginterferon-induced bone marrow suppression may also lead to severe neutropenia [1,2]. This is most commonly observed when treating patients who have cirrhosis or African Americans who have essential neutropenia [49]. Although it is recommended that the dose of peginterferon be reduced when the absolute neutrophil count decreases to 750 cells/mL, many physicians experienced in the care of patients who have cirrhosis and chronic HCV allow the absolute neutrophil count to decline to 500 cells/mL. A previous study in patients who had cirrhosis has demonstrated that the risk of infection is not signicantly increased in patients receiving peginterferon [50]. A controlled trial has demonstrated that granulocyte colony-stimulating factor signicantly increases the neutrophil count in these patients [51]. Most patients who have chronic HCV, even those who have cirrhosis, do not develop signicant neutropenia and are not at increased risk for severe infections during treatment with peginterferon. The exceptions are patients who have decompensated cirrhosis, particularly those who have ascites [52]. Utilizing serum hepatitis C virus RNA to optimize current dosing Successfully treating chronic HCV requires that the milestones that lead to SVR be balanced against the adverse event prole experienced by patients receiving peginterferon and ribavirin. This can only be accomplished if the specic virologic response pattern achieved during treatment is recognized. It is therefore essential that HCV RNA be monitored at regular intervals during treatment until the patient becomes HCV RNA undetectable or a virologic nonresponse pattern is dened, as illustrated in Figs. 1 and 3. This is best accomplished by monitoring HCV RNA at monthly intervals. At the authors institution, the medical and nursing sta treating patients with chronic HCV measure HCV RNA at baseline and every month until the patient has become HCV RNA undetectable or a non-response virologic pattern is identied and the treatment is stopped. This allows them to balance adverse events, virologic response, and dose modication as described in Table 2.

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Table 2 Balancing adverse events, virologic response, and dose modication Timing of adverse event Strategy Weeks 14 Weeks 412 Discontinue treatment, allow adverse event to recover, develop strategy to prevent adverse event from recurring, and initiate retreatment If patient achieved an RVR: modify dose and continue treatment If no RVR: consider discontinuing treatment, allow adverse event to recover, develop strategy to prevent adverse event from recurring, and consider retreatment If HCV RNA undetectable: modify dose in small steps (see text and Fig. 6); recheck HCV RNA 1 month after dose modication to ensure breakthrough has not occurred If HCV RNA-positive: modify dose in small steps as stated previously; continue monitoring HCV RNA at monthly intervals to determine if a virologic response has occurred; if not, discontinue treatment, allow adverse event to resolve, develop strategy to prevent adverse event from recurring, and consider retreatment Modify dose in small steps (see text and Fig. 6)

Weeks 1224

After week 24

The patient with minimal or infrequent side eects during treatment is easy to treat. Virologic response is monitored at monthly intervals, and if the patient becomes HCV RNA undetectable, treatment is continued for the appropriate duration of therapy. This is based on genotype and the time at which the patient becomes HCV RNA undetectable during treatment (see the article by Berg elsewhere in this issue). If the patient does not have a decline in HCV RNA by more than 2 log units, treatment should be discontinued by week 12 or as soon as the null response pattern is recognized. In patients with a partial response, the dose of peginterferon could be escalated for an additional 3 months. Alternatively, high-dose daily consensus interferon could be used [23]. The goal of this maneuver is to render patients HCV RNA undetectable. Thus, if the patient fails to become HCV RNA undetectable after 3 additional months of dose intensication, treatment should be discontinued. The patient who is most dicult to treat develops adverse events so severe that the patient must temporarily interrupt or discontinue treatment within the rst few weeks to months after treatment is initiated. Dose modication so early into treatment frequently leads to a null response. If the patient does achieve an EVR or becomes HCV RNA undetectable, however, the interruption in treatment signicantly increases the risk of breakthrough or relapse [79]. It is therefore preferable for such patients simply to discontinue treatment, allow the adverse eects to resolve, and then reinitiate peginterferon and ribavirin with the knowledge and tools required to lessen the severity or prevent these adverse events from recurring. If the adverse event in question is severe anemia, the use of epoetin alfa during retreatment is justied. Possibly the only exception to this recommendation would be in

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the patient who has already achieved an RVR. In this case, simply completing treatment even with lower doses of peginterferon and ribavirin has a high likelihood of yielding an SVR [10]. It is important to realize that these treatment decisions cannot be made unless HCV RNA is monitored frequently and the virologic response pattern is clearly dened. Most patients receiving HCV treatment develop adverse events of moderate severity, which progressively worsen and require that peginterferon or ribavirin be dose modied at some point after the patient has become HCV RNA undetectable. The primary reason for dose modication in these patients is progressive fatigue, myalgias, arthralgias, insomnia, and depression. The management of these adverse events has been discussed previously [33]. At the authors institution, it has been found that reducing the dose of ribavirin by 200 mg or the dosage of peginterferon by 20% (from 180 to 135 mg for peginterferon alfa-2a or from 1.5 to 1.2 mg/kg for peginterferon alfa-2b) can signicantly improve adverse events and enable many of these patients to complete treatment. Several studies [9,35,36] and the analysis in Figs. 5 and 6 clearly suggest that such a strategy is unlikely to have a negative impact on the SVR. Nevertheless, whenever the dose of peginterferon or ribavirin is modied, HCV RNA should be assessed 1 month later so that breakthrough can be identied. If breakthrough does occur, treatment should be discontinued, because continuing treatment in a patient with breakthrough cannot lead to an SVR. Before treatment is abandoned, however, the author advises that repeat HCV RNA testing be performed to conrm that the previous result was not a false-positive value. False-positive and false-negative values do occur during HCV RNA testing [53]. Discordant virologic results cannot be recognized unless HCV RNA testing is performed at regular intervals during treatment [5]. Pushing patients to remain on full-dose therapy when they are experiencing adverse events forces them to seek relief by skipping doses or prematurely stopping treatment on their own. This clearly enhances the likelihood of breakthrough and relapse. Summary In summary, the ability to achieve an SVR depends on the patient achieving the three milestones of treatment: virologic response, maintaining the virologic response, and not relapsing. Achieving virologic response is primarily a function of the peginterferon dose, and some patients may require that the peginterferon dose be escalated to become HCV RNA undetectable. Ribavirin aects all three of the milestones that lead to an SVR. The major impact of ribavirin is to prevent breakthrough and reduce relapse, however. Interrupting or prematurely discontinuing ribavirin therapy increases the likelihood of breakthrough and relapse, and this reduces the SVR. In contrast, reducing the dose of ribavirin, especially after the patient has become HCV RNA undetectable, does not seem to aect virologic response, relapse, or the SVR. Ideally, dose reduction should be performed in 200-mg steps,

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because this maximizes total cumulative ribavirin exposure and often alleviates the adverse event. A randomized controlled trial has demonstrated that using epoetin alfa in lieu of this stepwise dose reduction strategy does not affect the SVR. Modifying the doses of peginterferon and ribavirin during treatment can only be accomplished when the virologic response pattern is well dened. This demands that HCV RNA be monitored at regular intervals, preferably monthly, until the patient has become HCV RNA undetectable or a nonresponse pattern has been recognized and treatment is discontinued. Acknowledgments The author acknowledges the support and insight of the medical and nursing sta at his institution (Sarah Hubbard, PA, Charlotte Hofmann, RN, April Long, NP, Jennifer Salvatori, RN, Denice Shelton, RN, Paula Smith, RN and Kimberly Williams, RN) who have contributed to many of the observations and concepts detailed in this article and have helped to improve the treatment of chronic HCV for our patients. Dr. Shiman is a consultant for Roche Laboratories, Roche Molecular Systems, Valeant Pharmaceuticals, and Wyeth Pharmaceuticals. He receives research support from Schering-Plough, Roche Laboratories, Valeant Pharmaceuticals, Vertex Pharmaceuticals, and Human Genome Sciences and is a speaker for Roche Laboratories and Valeant Pharmaceuticals. References
[1] Fried MW, Shiman ML, Reddy KR, et al. Combination of peginterferon alfa-2a (40 kd) plus ribavirin in patients with chronic hepatitis C virus infection. N Engl J Med 2002;347: 97582. [2] Manns MP, McHutchinson JG, Gordon SC, et al. Peginterferon-alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 2001;358:95865. [3] Hadziyannis SJ, Sette H Jr, Morgan TR, et al. Peginterferon-alfa 2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;140:34655. [4] Shiman ML, Suter F, Bacon BR, et al. Peginterferon alfa-2a and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3. N Engl J Med 2007;357:12434. [5] Sethi A, Shiman ML. Approach to the management of patients with chronic hepatitis C who failed to achieve sustained virologic response. Clin Liver Dis 2005;9:45371. [6] Davis GL, Wong JB, McHutchison JG, et al. Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology 2003; 38:64552. [7] Shiman ML, Ghany MG, Morgan TR, et al. Impact of reducing peginterferon alfa-2a and ribavirin dose during retreatment in patients with chronic hepatitis C. Gastroenterology 2007;132:10312. [8] Bronowicki JP, Ouzan D, Asselah T, et al. Eect of ribavirin in genotype 1 patients with hepatitis C responding to pegylated interferon alfa-2a plus ribavirin. Gastroenterology 2006; 131:10408.

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[9] Reddy KR, Shiman ML, Morgan TR, et al. Impact of ribavirin dose reductions in hepatitis C virus genotype 1 patients completing peginterferon alfa-2a/ribavirin treatment. Clin Gastroenterol Hepatol 2006;5:1249. [10] Ferenci P, Fried MW, Shiman ML, et al. Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alfa-2a (40 KD)/ribavirin. J Hepatol 2005;43:45371. [11] Berg T, von Wagner M, Nasser S, et al. Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon alfa-2a plus ribavirin. Gastroenterology 2006;130:108697. [12] Sanchez-Tapias JM, Diago M, Escartin P, et al. Peginterferon alfa-2a plus ribavirin for 48 versus 72 weeks in patients with detectable hepatitis C virus RNA at week 4 of treatment. Gastroenterology 2006;131:45160. [13] Lindsay KL. Introduction to therapy of hepatitis C. Hepatology 2002;36(Suppl 1):S11420. [14] Davis GL. Monitoring of viral levels during therapy of hepatitis C. Hepatology 2002; 36(Suppl 1):S14551. [15] Jensen DM, Morgan TR, Marcellin P, et al. Early identication of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology 2006; 43:95460. [16] Willems B, Hadziyannis SJ, Morgan TR, et al. Should treatment with peginterferon plus ribavirin be intensied in patients with HCV genotype 2/3 without a rapid virological response? J Hepatol 2007;46:S6. [17] Shiman ML, Hofmann CM, Luketic VA, et al. Treatment of chronic hepatitis C with escalating doses of interferon-a-2b increases both biochemical and virologic response. Hepatology 1995;22:152A. [18] Lindsay KL, Trepo C, Heintges T, et al. A randomized, double-blind trial comparing peginterferon alfa-2b to interferon alfa-2b as initial treatment for chronic hepatitis C. Hepatology 2001;34:395403. [19] Reddy KR, Wright TL, Pockros PJ, et al. Ecacy and safety of pegylated (40-KD) interferon a-2a compared with interferon a-2a in non-cirrhotic patients with chronic hepatitis C. Hepatology 2001;33:4338. [20] Gross J, Johnson S, Kwo P, et al. Double-dose peginterferon alfa-2b with weight-based ribavirin improves response for interferon/ribavirin non-responders with hepatitis C: nal results of RENEW. Hepatology 2005;42(Suppl 1):219A. [21] Marcellcin P, Jensen D. Retreatment with Pegasysin patients not responding to prior peginterferon alfa-2b/ribavirin combination therapy. Ecacy analysis of the 12 week induction period of the repeat trial. Hepatology 2005;42(Suppl 1):749A. [22] Heathcote EJ, Keee EB, Lee SS, et al. Re-treatment of chronic hepatitis C with consensus interferon. Hepatology 1998;27:113643. [23] Bacon B, Regev A, Ghalib R, et al. Use of daily interferon alfacon-1 plus ribavirin in patients infected with hepatitis C who are non-responders to previous pegylated interferon plus ribavirin therapy: 24-week data from the Direct Trial. Hepatology 2006;44(Suppl 1):698A. [24] Kimball P, Verbeke S, Shiman ML. Comparison of 125I-alpha interferon binding to peripheral blood cells from African Americans and Caucasians with hepatitis C. J Viral Hepat 2003;10:3549. [25] Rosen HR, Weston SJ, Im K, et al. Selective decrease in hepatitis C virus-specic immunity among African Americans and outcome of antiviral therapy. Hepatology 2007; 46:3508. [26] Shiman ML, Hofmann CM, Gabbay J, et al. Treatment of chronic hepatitis C in patients who failed interferon monotherapy: eects of higher doses of interferon and ribavirin combination therapy. Am J Gastroenterol 2000;95:292835. [27] Jacobson IM, Gonzalez SA, Ahmed F, et al. A randomized trial of pegylated interferon alpha-2b plus ribavirin in the retreatment of chronic hepatitis C. Am J Gastroenterol 2005;100:245362.

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[28] Bodenheimer HC Jr, Lindsay KL, Davis GL, et al. Tolerance and ecacy of oral ribavirin treatment of chronic hepatitis C: a multicenter trial. Hepatology 1997;26:4737. [29] McHutchinson JG, Gordon SC, Schi ER, et al. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998;339:148592. [30] Herrmann E, Lee JH, Marinos G, et al. Eect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon. Hepatology 2003;37:13518. [31] Jacobson IM, Brown R, Freilich B, et al. Weight-based ribavirin dosing increases sustained viral response in patients with chronic hepatitis C: nal results of the WIN-R study, a US community based trial. Hepatology 2005;42(Suppl 1):749A. [32] Shiman ML, Salvatore J, Hubbard S, et al. Treatment of chronic hepatitis C virus genotype 1 with peginterferon, ribavirin, and epoetin alpha. Hepatology 2007;46:3719. [33] Shiman ML. Side eects of medical therapy for chronic hepatitis C. Ann Hepatol 2004;3: 510. [34] Snoeck E, Wade JR, Du F, et al. Predicting sustained virological response and anaemia in chronic hepatitis C patients treated with peginterferon alfa-2a (40 KD) plus ribavirin. Br J Clin Pharmacol 2006;62:699709. [35] Fried MW, Shiman ML, Sterling RK, et al. A multicenter, randomized trial of daily highdose interferon-alpha 2b for the treatment of chronic hepatitis C: pretreatment stratication by viral burden and genotype. Am J Gastroenterol 2000;95:32259. [36] McHutchison JG, Manns M, Patel K, et al. Adherence to combination therapy enhances sustained response in genotype-1-infected patients with chronic hepatitis C. Gastroenterology 2002;123:10619. [37] De Franceschi L, Fattovich G, Turrini F, et al. Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage. Hepatology 2000;31:9971004. [38] Maddrey WC. Safety of combination interferon alfa-2b/ribavirin therapy in chronic hepatitis C-relapsed and treatment-naive patients. Semin Liver Dis 1999;19(Suppl 1):6775. [39] McHutchison JG, Manns MP, Brown RS Jr, et al. Strategies for managing anemia in hepatitis C patients undergoing antiviral therapy. Am J Gastroenterol 2007;102:8809. [40] Dieterich DT, Wasserman R, Brau N, et al. Once-weekly epoetin alfa improves anemia and facilitates maintenance of ribavirin dosing in hepatitis C virus-infected patients receiving ribavirin plus interferon alfa. Am J Gastroenterol 2003;98:24919. [41] Afdhal NH, Dieterich DT, Pockros PJ, et al. Epoetin alfa maintains ribavirin dose in HCV-infected patients: a prospective, double-blind, randomized controlled study. Gastroenterology 2004;126:130211. [42] Pockros PJ, Shiman ML, Schi ER, et al. Epoetin alfa improves quality of life in anemic HCV-infected patients receiving combination therapy. Hepatology 2004;40:14508. [43] Del Rio RA, Post AB, Singer ME. Cost-eectiveness of hematologic growth factors for anemia occurring during hepatitis C combination therapy. Hepatology 2006;44:1598606. [44] Alvarez D, Dieterich DT, Brau N, et al. Zidovudine use but not weight-based ribavirin dosing impacts anaemia during HCV treatment in HIV-infected persons. J Viral Hepat 2006;13:6839. [45] Epogen. In: Physicians desk reference. 60th edition. 2006. p. 237484. [46] Darbepoetin. In: Physicians desk reference. 60th edition. 2006. p. 57680. [47] Ross SD, Allen IE, Henry DH, et al. Clinical benets and risks associated with epoetin and darbepoetin in patients with chemotherapy induced anemia: a systematic review of the literature. Clin Ther 2006;28(6):80131. [48] Stravitz RT, Chung H, Sterling RK, et al. Antibody-mediated pure red cell aplasia due to epoetin alfa during antiviral therapy of chronic hepatitis C. Am J Gastroenterol 2005;100: 14159. [49] Reed WW, Diehl LF. Leukopenia, neutropenia and reduced hemoglobin levels in healthy American blacks. Ann Intern Med 1991;151:5015.

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[50] Heathcote EJ, Shiman ML, Cookesly WG, et al. Peginterferon alfa-2a in patients with chronic hepatitis C and cirrhosis. N Engl J Med 2000;343:167380. [51] Carreno V, Martin J, Pardo M, et al. Randomized controlled trial of recombinant human granulocyte macrophage stimulating factor for the treatment of chronic hepatitis C. Cytokine 2000;12:16570. [52] Crippin JS, McCashland T, Terrault N, et al. A pilot study of the tolerability and ecacy of antiviral therapy in hepatitis C virus-infected patients awaiting liver transplantation. Liver Transpl 2002;8:3505. [53] Shiman ML, Ferreira-Gonzalez A, Reddy KR, et al. Comparison of three commercially available assays for HCV RNA using the international unit standard: implications for management of patients with chronic hepatitis C virus infection in clinical practice. Am J Gastroenterol 2003;98:115966.

Clin Liver Dis 12 (2008) 507528

Tailored Treatment for Hepatitis C


Thomas Berg, MD*
Medical Department Hepatology and Gastroenterology, Charite, Campus Virchow-Klinikum, Universitatsmedizin Berlin, Berlin, Germany

Hepatitis C virus (HCV) infection is a serious global health threat. Despite considerable reduction of the incidence of new infections, the prevalence of HCV is predicted to remain constant in the near future [1]. Furthermore, the current combined interferon (IFN)-based therapy is eective in only a fraction of the patients and is also plagued with adverse eects. There is therefore a need to develop new therapies. Early clinical trials have just begun on some promising HCV-targeted compounds, such as the substrate specic NS3-4A protease inhibitors or the NS5B polymerase inhibitors that have been tested alone or in combination with pegylated interferon alfa (peg-IFNa) with or without ribavirin. For the time being, however, the currently recommended therapy for chronic hepatitis C remains the combination of peg-IFNa and ribavirin for 24 or 48 weeks. Several ongoing studies have recently been put forth to optimize therapeutic strategies by tailoring the duration of treatment and also by evaluating the optimal dose of peg-IFNa and ribavirin. Also explored were dierent baseline factors, which were shown to improve the prediction of the outcome, especially when correlated with viral kinetics. This review therefore focuses on the analysis of current concepts and strategies in favor of a more individualized treatment regimen in patients who have HCV infection.

T. Berg is supported by the German Competence Network for Viral Hepatitis (Hep-Net), which is funded by the German Ministry of Education and Research (BMBF, 01 KI 0437), and by the European Union-Vigilance Network of Excellence Combating Viral Resistance (VIRGIL, LSHM-CT-2004-503359). , Campus * Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Charite Virchow-Klinikum, Universita tsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail address: thomas.berg@charite.de 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.011 liver.theclinics.com

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Principles of tailored treatment in hepatitis C virus type 1 Treatment response in patients who have chronic HCV infection is quite heterogeneous and depends on host factors (ie, age, gender, alanine aminotransferase [ALT] levels, stage of brosis, insulin resistance) and viral factors, such as serum concentration of HCV RNA at the time of initiation of antiviral therapy, including HCV genotypes [27]. Thus, virologic factors and host factors must be considered if one tries to get sound information on the individual likelihood of response. If one is aware of the inuence of viral and host factors with respect to the possible consequences of therapeutic response, it indeed seems rather illogical to treat all patients with the same xed therapeutic regimen. Viral kinetic studies have further expanded our knowledge of the mechanisms of IFNa action and have shown that the likelihood of achieving a sustained virologic response (SVR) clearly depends on the speed of the response [810]. It is well established that the rate of SVR is inversely correlated with the time on treatment that is necessary to clear HCV RNA from serum. Individual prognosis with respect to the long-term treatment outcome can therefore be best predicted by early viral kinetics, because all baseline factors nally alter the speed of response (Fig. 1). Indeed, if one includes viral kinetic parameters in multivariate models to predict SVR, most of the baseline factors lose their predictive power. The rational background of any individualized therapy is based on the concept that rapid responders need less therapy as compared with those patients who are slow responders.

Virologic factors HCV genotype level of replication mutational pattern (ISDR) (HCV genetic mutations)

Host factors age gender stage of fibrosis insulin resistance/steatosis cholesterol level ALT levels GGT levels genetic polymorphisms

Early viral kinetics

Summarizing the individual likelihood of response

Fig. 1. Individual prognosis of a patient with respect to treatment outcome is best reected by viral kinetics, because all known host and viral baseline predictors nally aect this type of response.

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Table 1 summarizes the available data on long-term treatment outcome seen in HCV type 1 infection treated for 48 weeks with standard combination therapy according to the early virologic response pattern. In a previous article, Shiman (see the article by Shiman elsewhere in this issue) discussed how to identify the optimal doses in an individual-based strategy to inuence the ecacy of the response. Perhaps even more important to solve the problem is to try to inuence the therapeutic response by modifying treatment duration, thereby dierentiating between slow and rapid virologic responders. Still, there remains the intriguing question as to what the critical criteria are to be sure that relapses do not occur even when we can now anticipate (provided that sensitive tests have been applied) that the HCV RNA has become undetectable on treatment. Thus, a clear denition of the duration of the negative HCV RNA interval required to prevent any relapse is still lacking and is of the utmost importance (Fig. 2A).

Importance of the sensitivity of the hepatitis C virus RNA test Undetectable hepatitis C viremia on treatment, even when evaluated by standard qualitative HCV RNA tests (detection limit of 50 IU/mL) does not, per se, indicate that the virus was completely eliminated from the serum. There is now emerging evidence that by applying more sensitive assays (eg, transcription-mediated amplication [TMA] or real-time polymerase chain reaction [PCR] assays with a detection limit !10 IU/mL), a signicant proportion of patients who were shown to have undetectable HCV RNA levels by standard assay are still viremic (Figs. 3 and 4) [11,12]. This implies that many patients considered so far to have had a relapse might, in fact, have been nonresponders with a minimal replication level (minimal residual viremia) (see Fig. 2B). Quite importantly, the detection of minimal residual HCV RNA levels at week 12 must be taken as a serious predictor for a possible later relapse event. Morishima and colleagues [12] provided evidence that all patients in whom a minimal residual viremia could be detected at week 12 and still conrmed at week 20 had a relapse. The application of extremely sensitive HCV RNA tests to design individualized therapeutic strategies is therefore indispensable.

Experiences with shortening treatment duration in hepatitis C virus type 1 infection Treatment guidelines recommend that patients infected with HCV genotype 1 should be treated for 48 weeks with the combination of peg-IFN plus ribavirin [13,14]. The recommendation of this xed treatment duration is based on the outcome of a large randomized international trial published in 2004 by Hadziyannis and colleagues [6], in which patients were

510 Table 1 Overview of the predicted long-term outcome in hepatitis C virus type 1infected patients treated for 48 weeks with pegylated interferon alfa plus ribavirin combination therapy according to early virologic response patterns Frequency of response Virologic response pattern (approximate % of patients) Rapid virologic response Early virologic response 20% 40% Predicted long-term outcome Denitions HCV RNA !50 IU/mL at week 4 HCV RNA O50 IU/mL at week 4 but !50 IU/mL at week 12 HCV RNA rst !50 IU/mL at week 24 !2-log10 HCV RNA decline at week 12 or O50 IU/mL at week 24 SVR O90% 70%80% Relapse !10% !15% Possible consequences to improve outcome Overtreatment (shorten treatment duration) None, adequate therapy
BERG

Slow virologic response Nonresponse

20% 20%

!30% !5%

O70% d

Undertreatment (extending treatment duration) Treatment intensication (dosage and duration)

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A7
6

Peg-IFNa + Ribavirin

HCV RNA (log10 IU/mL)

5 4 3 2 Detection limit

Individually different on-treatment HCV-RNA negative interval

Relapse

SVR
1 0 Time

B7
6

Peg-IFNa + Ribavirin

HCV RNA (log10 IU/mL)

5 4 3 2 Detection limit (standard assay) 1 Detection limit (real-time PCR or TMA)

Relapse

SVR
0 Time

Fig. 2. Current hypotheses for the reasons why patients with an end-of-treatment virologic response may experience a relapse. (A) HCV RNA-negative interval is too short in patients with a slow virologic response to eradicate the infection. (B) Patients were, in fact, nonresponders because of minimal residual viremia that remained undetectable with assays currently available.

randomized to treatment for 24 or 48 weeks with peg-IFNa-2a plus ribavirin at a dosage of 800 or 1000/1200 mg/d. SVR rates ranged from 29% to 52% across the four treatment groups, and the highest SVR rates were achieved with a 48-week treatment regimen plus ribavirin at a dosage of 1000/1200 mg/d.

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Percentage of Apmlicor-negative samples with detectable HCV RNA by TMA

100

80

60 37 40 27 20 20 6

0 Week 12 Week 20 Week 24 Week 48

Time of evaluation on treatment


Fig. 3. Percentage of HCV RNA-negative samples by the Amplicor assay (Roche Diagnostics; detection limit !50 IU/mL) in which minimal residual viremia could be detected by the TMA assay; Siemens (Deereld, IL); detection limit !5.3 IU/mL) at various time points during antiviral combination therapy. (Data from Morishima C, Morgan TR, Everhart JE, et al. HALT-C Trial Group. HCV RNA detection by TMA during the hepatitis C antiviral longterm treatment against cirrhosis (HALT-C) trial. Hepatology 2006;44:3607.)

Extension of treatment duration up to 48 weeks increased the SVR rate, especially in patients who had high baseline viremia (R800,000 IU/mL) from 26% to 47%, whereas increasing treatment duration from 24 to 48 weeks in patients who had low viremia (!800,000 IU/mL) yielded only a moderate eect on SVR rates (52% versus 65%). These data indicate that a subgroup of patients dened by pretreatment HCV RNA levels of
7 6 5 4 3 2 Detection limit < 50 IU/mL 1 0 12 HCV RNA decline > 2 log10 TaqMan-PCR positive (> 10 IU/mL)

Peg-IFNa + Ribavirin

+
HCV RNA negativeby standard assay

24

36

48

Weeks
Fig. 4. Example of a patients who remained minimally viremic throughout the whole treatment course when evaluated by a real-time polymerase chain reaction (PCR) test, although attaining a virologic response (HCV RNA !50 IU/mL) at treatment week 24.

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less than 800,000 IU/mL can achieve long-term response rates up to 52% even after a treatment period of 24 weeks. Based on these ndings, Zeuzem and colleagues [15] performed a study to examine whether patients who have HCV genotype 1 and low baseline HCV RNA levels (dened as !600,000 IU/mL) may require just 24 weeks of treatment with peg-IFNa plus ribavirin. Two hundred thirty-ve treatment-naive patients infected with HCV genotype 1 were treated for 24 weeks with pegIFNa-2b at a dosage of 1.5 mg/kg/wk and weight-based ribavirin (8001400 mg/d). A comparable group of patients who had type 1 infection and low baseline viremia who had been treated for 48 weeks within the Manns trial [3] served as a historical control. The end-of-treatment virologic response rates were comparable between both studies (ie, 80% versus 74%). SVR rates were signicantly dierent (50% versus 71%), however, because of higher relapse rates observed in patients treated for only 24 weeks (37% versus 4%). Thus, baseline factors alone, like HCV RNA levels, do not really help to get sound information concerning tailoring of treatment duration. These investigators then re-evaluated their ndings and found that a subgroup of patients expressing a rapid virologic response (RVR) already at week 4 (dened as HCV RNA levels !29 IU/mL by real-time PCR) had an 89% chance to achieve an SVR even after only 24 weeks of combination therapy, which was comparable to the SVR rates observed in patients with an RVR treated for 48 weeks (85% in historical control group with RVRs). Jensen and colleagues [16] aimed to explore the prognostic factors for an RVR (HCV RNA !50 IU/mL at week 4) and an SVR in type 1infected patients treated in the study by Hadziyannis and colleagues [6] for 24 weeks with peg-IFNa-2a at a dose of 180 mg plus ribavirin at a dosage of 800 or 1000 to 1200 mg/d by stepwise multiple logistic regression analysis. Their ndings conrmed that RVR was the single best predictor of SVR. Thus, 89% with an RVR versus 19% without an RVR had an SVR (Table 2) when treated for only 24 weeks. A multiple regression model demonstrates that HCV RNA level was the only independent predictor of RVR. These ndings could also be conrmed in a prospective study by Ferenci and colleagues [17], in which type 1infected patients and a small proportion of type 4infected patients with an RVR (!50 IU/mL at week 4) were treated for only 24 weeks with peg-IFNa-2a plus weight-based ribavirin. Overall SVR rates were 87%. Patients with a high viral load at baseline (dened as O600,000 IU/mL) achieved SVR rates lower as compared with those with a low viral load of less than 600,000 IU/mL (74% versus 93%). Stage of brosis also inuenced SVR rates in patients dened as super-responders (SVR rates were 90% with stage F0F2 versus 79% with stage F3F4). Also of interest are these investigators observations that frequency of relapse rates could be determined according to the assays applied to determine HCV RNA. In the case of a negative real-time PCR HCV RNA at week 4, the relapse rate was 10% as compared with relapse rates of 30% in patients still positive with the sensitive assay at week 4.

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Table 2 Virologic response rates in patients with and without rapid virologic response at week 4 in relation to treatment duration and ribavirin dosage Treatment arms duration of combination therapy with peg-IFNa-2a, 180 mg, and RBV dosage 24 weeks; RBV, 800 mg 24 weeks; RBV, 1000 1200 mg 48 weeks; RBV, 800 mg 24 weeks, 10001200 mg RBV End-of-treatment virologic response rates (HCV RNA !50 IU/mL) Patients with RVR 94% 97% 73% 93% Patients without RVR 63% 70% 56% 63%

SVR rates Patients with RVR 89% 88% 73% 91% Patients without RVR 16% 23% 35% 44%

RVR is dened by HCV RNA !50 IU/mL at week 4. Abbreviation: RBV, ribavirin. Data from Jensen DM, Morgan TR, Marcellin P, et al. Early identication of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology 2006;43:95460.

Between February 2001 and November 2003, the Dynamically Individualized Treatment of Hepatitis C Infection and Correlates of Viral/Host dynamics (DITTO-HCV) study group evaluated a dynamically individualized treatment schedule according to the early virologic response (compared with a standard-of-care peg-IFNa plus ribavirin combination therapy for 48 weeks) [18]. Patients categorized as having an RVR (denition: total log10 decline of HCV RNA during the rst 4 weeks R2 and a second phase decline R0.09 per day) were randomized to 24 or 48 weeks of treatment. SVR rates in HCV type 1infected patients with an RVR who were treated for only 24 weeks were substantial (65%) but were lower than in those treated for 48 weeks (83%). No signicant dierence, however, was observed when patients with a low viral load (ie, !800,000 IU/mL) were considered, in whom SVR rates were nearly identical regardless of whether they were treated for 24 or 48 weeks (82% versus 83%) [18]. Would it be possible to shorten treatment duration further? In a prospective, multicenter, randomized controlled study from Germany, HCV type 1infected patients received an individualized treatment duration from 18 to 48 weeks tailored according to early viral kinetics. Patients with a low viral load (!800,000 IU/mL and RVR dened as !5.3 IU/mL by means of TMA assay) achieved an SVR of 95% after the 18-week treatment duration, and these ndings were similar to the comparable control group treated for 48 weeks (94% SVR rate) [19]. Denition of a new hepatitis C virus RNA cuto to predict sustained virologic response The determination of viral load has turned out to be of such importance to predict SVR that a more rened denition of the cuto level indicating

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low versus high viral load is desired so as to tailor treatment duration more accurately. In the past, patients have been classied as having a high or low viral load using a cuto of 2 106 copies/mL (corresponding to 600,000800,000 IU/mL, depending on the assay). This was dened in the era of conventional IFNs, however. Several retrospective studies have now been undertaken with the aim of determining the most eective cuto to dierentiate between high and low viral loads based on the probability of an SVR with peg-IFNa plus ribavirin therapy [2022]. Using the logistic regression model taking into account discrete (eg, gender, race, cirrhosis status) and continuous (eg, age, weight, baseline viral load, pretreatment ALT quotient) variables, HCV RNA turned out to be a strong independent predictor of SVR, but this eect was not linear, indicating that at greater than a cuto of 5.6 log10 IU/mL, the contribution of viral load in predicting an SVR was minimal. Thus, a baseline HCV RNA level of approximately 400,000 IU/mL was found to be optimal for use as a cuto for the probability of achieving an SVR in patients who had type 1 HCV treated for 48 weeks. Less than the limit of 400,000 IU/mL, there is a nearly linear correlation between the amount of HCV RNA and the chance to establish an SVR, whereas HCV RNA levels greater than 400,000 IU/mL showed no signicant eect on the SVR rates [20]. The reason for the nding that a viral load greater than 400,000 IU/mL had no signicant eect on SVR is far from clear. One might speculate, however, that at greater than this level, the chance to achieve complete HCV RNA elimination from serum is reduced and many patients have minimal residual hepatitis C viremia. Nevertheless, it still remains to be seen whether the now established cuto based on the standard PCR test holds true when the real-time PCR assays are used to measure HCV RNA levels.

Experiences with extending treatment duration in hepatitis C virus type 1 Extension of treatment with peg-IFNa plus ribavirin from 24 to 48 weeks has signicantly increased the SVR rate in patients who have HCV genotype 1. Thus, the standard duration of therapy for patients who have chronic HCV is 48 weeks. The ability to achieve an SVR depends on the time at which HCV RNA becomes undetectable, however, and patients who have a slow virologic response have an increased risk for relapse after treatment has been discontinued, even when HCV RNA was undetectable at the end of the 48-week treatment period [8]. Obviously, the HCV RNA-negative interval was too short to clear HCV from the host tissue permanently. Extension of treatment duration may be one possibility for improving SVR rates by reducing relapse rates in these patients [23]. This concept is, however, confronted with some limitations, considering the troublesome side eects associated with this combination therapy, which, as a consequence, may be responsible for an increasing number of dropouts when treatment duration

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is extended. In this situation, the intention-to-treat analysis may not be optimal to demonstrate the potential benet of extending treatment duration, because the higher withdrawal rates (when extending treatment duration) and higher relapse rates (when shortening treatment duration) may neutralize each other. In two randomized and controlled multicenter studies from Germany and Spain, researchers analyzed whether the extension of treatment duration beyond 48 weeks may increase the SVR rate in patients who have HCV type 1 [24,25]. In the German study, patients were randomized to one of the two treatment groups with active treatment for 48 weeks (group A, n 230) or 72 weeks (group B, n 225) [24]. All patients received peg-IFNa-2a at a dosage of 180 mg/wk plus ribavirin at a dosage of 800 mg/d. After the end of treatment, patients from both groups were followed up for a further 24 weeks. The frequency of side eects was similar in both groups, but the on-treatment discontinuation rate between groups varied greatly (24% in group A as compared with 41% in group B). The intention-to treat analysis revealed no major dierences in SVR rates between the two groups (53% in group A and 54% in group B). From a per-protocol analysis, however, by including only patients who received at least 80% of the peg-IFNa and ribavirin dose and who completed the planned (4 weeks) treatment and follow-up period, a 10% dierence in SVR rate in favor of group B became evident (68% in group A versus 78% in group B). This indicates that a subgroup of patients may prot from the 72-week regimen. It could be shown that patients who still were HCV RNA-positive at week 12 had signicantly higher SVR rates when treated for 72 weeks instead of 48 weeks (29% versus 17%). Relapse rates were signicantly higher in slow virologic responders, dened as becoming HCV RNA undetectable for the rst time at week 24 when treated for only 48 weeks instead of 72 weeks (relapse rate of 64% versus 40% in groups A and B). In contrast, patients with an early virologic response in whom HCV RNA levels were less than 50 IU/mL at weeks 4 and 12 had excellent SVR rates, ranging between 76% and 84% at the end of the followup period independent of the treatment period. In the study from Spain, a total of 510 HCV-infected patients (also including genotypes 2 and 3) were treated with peg-IFNa-2a at a rate of 180 mg/wk plus ribavirin at a dose of 800 mg [25]. Only patients who had detectable HCV RNA at week 4 were randomized to complete 48 (group A, n 165) or 72 (group B, n 161) weeks of treatment, however. The end-of treatment response rate was similar in both groups (61%). The SVR rates were signicantly higher in the 72-week group as compared with the 48-week group (45% versus 32%), however, and it also became evident that the 72- weektreated patients demonstrated signicantly lower relapse rates (13% versus 48% in the 48-week group). Analyzing only patients with HCV genotype 1, SVR rates were 28% and 44% in group A (n 149) and group B (n 142), respectively.

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Also, in two other studies by Perlman and colleagues [26] and Ferenci and colleagues [27] analyzing patients who had HCV genotype 1 with a slow virologic response, it became clear that extending the treatment duration with peg-IFNa and weight-based ribavirin from 48 weeks to 72 weeks signicantly improved SVR rates by reducing relapse rates. In Fig. 5, the expected SVR rates are presented in those patients who had a slow virologic response and had been treated for 48 or 72 weeks.

Predicting relapse according to the hepatitis C virus RNA-negative interval during treatment? Drusano and Preston [28] recently presented a mathematic model to predict whether patients may achieve an SVR or experience relapse. It was concluded that HCV type 1infected patients require continuous absence of detectable of HCV RNA in serum for 36 weeks to attain 90% probabilities of an SVR (ie, relapse rate of 10%). These data conrm the previously mentioned ndings indicating that patients with an early virologic response at week 12 hardly experienced relapse independent of treatment duration (ie, 48 or 72 weeks), with an HCV RNA-negative interval on treatment of 36 to 60 weeks. Late responders who rst became HCV RNA-negative at week 24 and were treated for 72 weeks (ie, who had undetectable HCV RNA levels during the last 48 weeks of the total 72-week treatment period) still had relapse rates of approximately 30% to 40% [24], however, a nding that clearly contradicts the proposed model by Drusano and Preston [28]. Obviously, the HCV RNA-negative phase required to prevent a relapse

100 80

SVR Rate %

58 60 40 20 0 Sanchez-Tapias et al. Berg et al. Pearlman et al. 28 44 26 18 38

Fig. 5. Summary of three studies evaluating 72-week treatment duration in HCV type 1 infection. Shown are the observed SVR rates in patients who had a slow virologic response determined either at week 4 (HCV RNA > 50 IU/mL at week 4; Sanchez-Tapies et al [25]) or at week 12 (HCV RNA is 50 IU/mL or greater, and week 24 HCV RNA is less than 50 IU/mL; Berg et al [24] and Pearlman et al [26]) and had been treated for either 48 or 72 weeks. Black bars indicate 48 weeks. White bars indicate 72 weeks. In the studies by Sanchez-Tapias et al and Berg et al patients were treated with 180 mg Peg-IFNa-2a/wk plus 800 mg RBV/d whereas in the study by Pearlman et al patients received Peg-IFNa-2b 1.5 mg/kg/wk plus 8001400 mg RBV/d.

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must be calculated in a more exponential way and seems to depend on how early a patient becomes HCV RNA-negative during treatment (Fig. 6).

Summary of the current concepts of tailoring treatment duration in patients who have hepatitis C virus type 1 Patients who have an RVR and low baseline viremia may be potential candidates for shortening treatment duration from 48 to 24 weeks. Around 10% to 15% of all HCV type 1infected patients reach these criteria. The optimal cuto to dene low viremia (400,000, 600,000, or 800,000 IU/mL) in this setting is still unclear. As long as HCV RNA is undetectable by sensitive real-time PCR assays or TMA tests at week 4, however, levels of less than 600,000 IU/mL seem to be appropriate (more conservative physicians may even choose 400,000 IU/mL). Because hepatitis C viremia may uctuate in the natural course of the disease, however, it is desirable to have at least two measurements of HCV RNA before starting therapy to conrm low-level replication over time. To exclude to the best possible level the presence of minimal residual viremia at week 4, which clearly increases the likelihood of experiencing a relapse, the most sensitive HCV RNA assays should be used when shortening treatment duration is considered. Furthermore,

Treatment period HCV RNA negative interval 20 weeks detection limit BL W4 W 24 Treatment period HCV RNA negative interval 36 weeks detection limit BL W 12 Treatment period W 48 SVR 70-80% Relapse rate ~ 20% SVR > 90% (if < 800.000 IU/mL) Relapse rate < 10%

HCV RNA negative interval 48 weeks detection limit BL W 24 W 72

SVR 40-60% Relapse ~ 30%

Fig. 6. Overview of the importance of the time to HCV RNA-negative status and the HCV RNA-negative interval with respect to the prediction of the SVR and relapse in HCV type 1 infection. Although the HCV RNA-negative interval was increased in patients with a slow virologic response, the relapse rates were still higher as compared with patients with an RVR (data for the 72-week treatment duration were obtained from studies using only ribavirin at a dose of 800 mg). BL, baseline; W, week.

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patients who have advanced brosis or established cirrhosis, or those who need a marked dose reduction within the rst 24 weeks of treatment, should at present be excluded from abbreviated treatment regimens because of insucient data for those cohorts. In contrast, there is now emerging evidence that patients who have a slow virologic response not reaching an undetectable HCV RNA level before week 24 who are exposed to a 48-week standard treatment period run a high risk for relapse (reaching up to 70%). Extension of the treatment period up to 72 weeks can help to reduce relapse rates in these patients. In this context, it should be pointed out that the thus far generally accepted stopping rule for patients with a less than 2-log10 decline of HCV RNA within the initial 12 weeks of therapy should be reconsidered, because the high negative predictive value of this stopping rule of 98% to 100% has so far only been established for the 48-week treatment duration. Therefore, this cuto may not be relevant when patients are treated for 72 weeks [24].

Principles of tailored treatment in hepatitis C virus type 2 and 3 infection Treatment guidelines recommend that patients infected with HCV genotype 2 or 3 infection can be treated for 24 weeks with the combination of peg-IFNa plus ribavirin at a dosage of 800 mg/d [13,14]. This recommendation is based on the outcome of a large randomized international trial in which patients were randomized to treatment for 24 or 48 weeks with pegIFNa-2a plus ribavirin at a dosage of 800 mg/d or 1000/1200 mg/d [6]. SVR rates ranged from 79% to 84% across the four treatment groups in this trial, with no signicant dierences between any combination of treatment duration and ribavirin dose. In Fig. 7, the SVR rates and end-of-treatment response rates of the four groups are compared. Treatment duration up to 24 weeks was associated with higher end-of-treatment response rates than observed in patients treated for 48 weeks, but SVR rates were nearly identical. As a consequence, relapse rates were twice as high in patients only treated for 24 weeks instead of 48 weeks (Fig. 8). More patients randomized to the 48-week group withdrew from treatment than in the abbreviated treatment group. Thus, the higher relapse rates in the 24-week treatment groups and the higher dropout rates in the 48-week groups tend to cancel each other out and create the appearance that the outcomes are similar with the two strategies. Based on these ndings, it can again be deduced that the optimal therapeutic regimen still remains unsettled and that there are certainly also patients who have HCV genotypes 2 and 3 who do not achieve an SVR after 24 weeks of treatment with the standard regimen. From previous studies, we know that various factors, such as high viral load, age (O55 years), male gender, and advanced brosis, are associated with the risk for experiencing relapse after xed standard combination therapy given for

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100

94% 84%

90% 81% 82% 79%

Virological response (% HCV RNA <50 IU/mL)

85%

80%

80 60 40 20 0 n= 96 96 144 144 99

99

153

153

RBV 800 mg/day

RBV 1000/1200 mg/day

RBV 800 mg/day

RBV 1000/1200 mg/day

24 weeks

48 weeks

Fig. 7. End-of-treatment (EOT) response (black bars) and SVR rates (white bars) in HCV type 2 and type 3infected patients treated for 24 or 48 weeks with low (800 mg/d) or weight-based (10001200 mg/d) ribavirin (RBV). Although EOT response rates were higher in the 24-week groups, SVR rates were nearly identical. Peg-IFNa-2a at a dosage of 180 mg/wk plus RBV. (Data from Hadziyannis S, Sette H Jr, Morgan TR, et al. Peginterferon-alfa-2a plus ribavirin combination therapy in chronic hepatitis C. A randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;40:34655.)

24 weeks [29]. For instance, relapse rates were nearly three times higher in type 3infected patients with a high viral load (O600,000 IU/mL) as compared with those with low viremia (23% versus 8%) after a 24-week course of peg-IFNa-2b at a dose of 1.5 mg plus weight-based ribavirin (8001400 mg). Also in type 2 and 3 infection, viral kinetics (ie, time to HCV RNA negative status) are of increasing importance to optimize antiviral treatment individually. Compared with type 1 infection, viral decline is more rapid in type 2 and 3 infection [30]. Because most patients achieve an RVR at treatment week 4, this explains, analogous to the situation in type 1 infection, why most patients are suciently treated with a 24-week treatment course. In Table 3, it is outlined how viral kinetics inuence the expected SVR rates in patients who have genotypes 2 and 3.

Experiences with shortening treatment duration in hepatitis C virus type 2 and 3 infection The nding that maximal SVR rates could be obtained with 24 weeks of treatment has spurred researchers to investigate even shorter durations of treatment for patients infected with HCV genotype 2 or 3. To date, ve trials (four national investigator-initiated studies [3134] and one large international F. Homann-La Roche Ltd. (Basel, Switzerland)sponsored study,

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A 25
20

Relapse (%)

15 10% 10 6% 5 0 RBV 800 mg/day RBV 1000/1200 mg/day RBV 800 mg/day RBV 1000/1200 mg/day 4% 10%

24 weeks

48 weeks

B 25
20

Relapse (%)

15 10

13%

13%

5% 5 0 RBV 800 mg/day RBV 1000/1200 mg/day RBV 800 mg/day RBV 1000/1200 mg/day 3%

24 weeks

48 weeks

Fig. 8. (A) Relapse rates in HCV type 2 and type 3infected patients treated for 24 or 48 weeks with low (800 mg/d) or weight-based (10001200 mg/d) ribavirin (RBV). Peg-IFNa-2a at a dosage of 180 mg/wk plus RBV. (B) Relapse rates in the subgroup of patients with high baseline HCV RNA levels (O800,000 IU/mL). (Data from Hadziyannis S, Sette H Jr, Morgan TR, et al. Peginterferon-alfa-2a plus ribavirin combination therapy in chronic hepatitis C. A randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;40:34655.)

the ACCELERATE study [35]) have examined abbreviated 12- to 16-week treatment regimens for genotypes 2 and 3. The various trials dier greatly in study design, treatment regimen (especially ribavirin dosage), and patient characteristics with respect to the percentage of patients who have type 2 being included; thus, great care is necessary when examining the results and especially when attempting to compare results of studies or generalize the ndings to a broad population (Table 4). In three trials, treatment duration was determined on the basis of RVR status at week 4 [3133]. In contrast, two studies randomized patients at baseline to abbreviated treatment or the standard 24-week duration [34,35].

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Table 3 Overview of the predicted long-term outcome in hepatitis C virus type 2 and type 3infected patients treated for 24 weeks with pegylated interferon alfa plus ribavirin combination therapy according to early virologic response patterns Virologic response pattern RVR Frequency of response (approximate) Denitions O65% Predicted long-term outcome SVR

Possible Relapse consequences Overtreatment (shorter duration possible) Undertreatment (extending treatment duration) Treatment intensication (dosage and duration)

HCV RNA !50 R85% IU/mL at week 4

!10%

No RVR

30%

HCV RNA O50 w50% w50% IU/mL at week 4 (36%77%)

No early !5% virologic response Data from Refs. [3135].

HCV RNA O50 IU/mL at week 12

w5%

Table 5 summarizes the principal ndings of these four investigatorinitiated studies, which included approximately 700 patients. Interestingly, patients with an RVR indeed achieved SVR rates between 82% and 94% even when treated for 12, 14, or 16 weeks. Dierences from the 24-week treatment period were marginal and statistically not signicant, but overall relapse rates were higher. In contrast, patients without an RVR had significantly lower SVR rates even when treated for 24 weeks (36%77%). The largest trial to examine the merits of abbreviated therapy in patients who have HCV genotype 2 or 3 infection was the ACCELERATE study, which randomized 1469 patients at baseline to 16 weeks or 24 weeks of treatment with peg-IFNa-2a at a dosage of 180 mg/wk plus ribavirin at a dosage of 800 mg/d [35]. In the intention-to-treat analysis, SVR rates were 70% versus 62% for 24 weeks versus 16 weeks, respectively (P!.001). These ndings were obtained despite a higher rate of withdrawal in patients randomized to 24 weeks compared with 16 weeks of treatment. The ndings demonstrate conclusively that it is not appropriate to recommend abbreviated treatment as a general strategy for patients who have genotype 2 or 3 infection. Nevertheless, closer inspection of data conrm that RVR is a powerful predictor of outcomes in patients who have HCV genotype 2 or 3 and also sheds light on the appropriateness of assigning patients to abbreviated treatment based on their RVR status. Two thirds (67%) of all treated patients achieved an RVR. The SVR rates in these patients being treated for 24 weeks were higher (90%) than in patients treated only for 16 weeks (82%). In patients without an RVR, the SVR rates were considerably

Table 4 Basic information concerning studies that have investigated abbreviated treatment for hepatitis C virus genotype 2 or 3 infection Stratication/ randomization according to week 4 response No Denition of RVR, serum HCV RNA level, IU/mL
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Study

No. patients treated

% HCV genotype 2 50%

Treatment duration, weeks 16 versus 24

Treatment regimen Peg-IFNa-2a, 180 mg/wk RBV Peg-IFNa-2a, 180 mg/wk RBV Peg-IFNa-2a, 180 mg/wk RBV Peg-IFNa-2b, 1.0 mg/kg/wk RBV Peg-IFNa-2b, 1.5 mg/kg/wk RBV

RBV dosage, mg/d 800

Randomized trials Shiman et al 1463 [35] Yu et al [34] 150

!50

100%

16 versus 24

No

1000/1200

!50

von Wagner et al [33] Mangia et al [32] Nonrandomized Dalgard et al [31]

153

27%

16 versus 24

Yes

8001200

!600

283

75%

12 versus 24

Yes

1000/1200

!50

122

24%

14 versus 24

Yes

8001400

!50

Abbreviation: RBV, ribavirin.

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Table 5 Presentation of the virologic response rates in studies evaluating abbreviated treatment duration in hepatitis C virus type 2 and 3 infection according to the rapid virologic response pattern Study Mangia et al [32] Treatment group 12 weeks in RVR pts (n 133) 24 weeks regardless of RVR status (n 70) 24 weeks, no RVR (n 80) 16 weeks in RVR pts (n 71) 24 weeks in RVR pts (n 71) 24 weeks, no RVR (n 11) 14 weeks in RVR pts (n 95) 24 weeks, no RVR (n 27) 16 weeks (n 50) 24 weeks (n 100) 16 weeks in RVR pts 24 weeks in RVR pts 16 weeks, no RVR 24 weeks, no RVR 16 weeks (n 733) 24 weeks (n 732) 16 weeks in RVR pts (n 489) 24 weeks in RVR pts (n 470) 16 weeks, no RVR (n 220) 24 weeks, no RVR (n 247) End-of-treatment response 95% 79% 68% 94% 85% 72% n.a. n.a. 100% 98% n.a. n.a. n.a. n.a. 89% 82% n.a. n.a. n.a. n.a. SVR rate 85% 76% 64% 82% 80% 36% 90% 56% 94% 95% 100% 98% 57% 77% 62% 70% 79% 85% 26% 45% Relapse rate 10% 4% 6% 13% 5% n.a. 10% 26% 6% 3% d n.a. 43% 9% 31% 18% n.a. n.a. n.a. n.a.

Von Wagner et al [33]

Dalgard et al [31]

Yu et al [34]

Shimann et al [35]

Abbreviations: n.a., not available; pts, patients.

lower (49% in patients treated for 24 weeks versus 27% in patients treated for 16 weeks). When baseline HCV RNA levels are superimposed on an RVR, it is apparent that these two factors exert a powerful inuence on SVR rates. In patients with low HCV RNA levels, dened as 400,000 IU/mL or less, the SVR rates were 95% in those treated for 24 weeks and 90% in those treated for 16 weeks [35]. In contrast, SVR rates were 88% in patients with a baseline HCV RNA level greater than 800,000 IU/mL who were treated for 24 weeks and 78% in patients treated for 16 weeks.

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100 76 67 60 65 67

Percentage of patients

80

40 26 20 n= 21 34 30 37 19 29 24 13 4 0 23 27 SVR Relapse

Fig. 9. SVR and relapse rates in HCV type 2 and type 3infected patients without an RVR treated for 24 or 48 weeks with low (800 mg/d) or weight-based (10001200 mg/d) ribavirin. Black bars indicate 24 weeks, ribavirin (RBV) at a dosage of 800 mg/d. Dark gray bars indicate 24 weeks, RBV at a dosage of 1000/1200 mg/d. Light gray bars indicate 48 weeks, RBV at a dosage of 800 mg/d. White bars indicate 48 weeks, RBV at a dosage of 1000/1200 mg/d. (Data from Hadziyannis S, Sette H Jr, Morgan TR, et al. Peginterferon-alfa-2a plus ribavirin combination therapy in chronic hepatitis C. A randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;40:34655.)

Extending treatment duration in those who are slow to respond There are data from randomized prospective trials that show prolonging the duration of treatment increases SVR rates in genotype 1infected patients who are slow to respond [2427]. This raises the question of whether intensication of the therapeutic regimen could increase SVR rates in genotype 2 or 3infected patients who do not achieve an RVR. There are no data from prospective trials available on this topic. This question has been addressed through a retrospective analysis of data [36] from the trial by Hadziyannis and colleagues [6]. In patients who did not have RVRs, end-of-treatment response rates ranged from 65% to 76% across the four treatment groups (Fig. 9). Consistent with results observed in genotype 1-infected patients, relapse rates decreased in inverse proportion to the intensity of treatment and were lowest in those treated for 48 weeks with peg-IFNa2a plus ribavirin at a dosage of 1000/1200 mg/d (4%) and highest in those treated for 24 weeks with ribavirin at a dosage of 800 mg/d (26%) [36]. Although these results must be conrmed in a prospective study, they suggest that intensication of therapy may be eective in increasing SVR rates in genotype 2 or 3infected patients who do not achieve an RVR at week 4.

Summary: tailored treatment for hepatitis C virus type 2 and 3 It is no longer appropriate to generalize that patients who have HCV genotype 1 and 4 are dicult to cure because of viral kinetic evidence to the

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contrary. Conversely, it is no longer appropriate to think of all patients who have HCV genotype 2 or 3 as easy to cure. Although genotype is an important driver of response and is useful in designing the initial treatment plan, it is clear that once treatment is initiated, RVR is the most important and powerful predictor of SVR. In genotype 2 or 3infected patients, response-guided therapy using measurement of the virologic response after 4 weeks of combination therapy allows the treatment regimen to be tailored to the individual. At present, available data from ve studies, including a total of more than 2000 patients, demonstrate that genotype 2 or 3infected patients with an RVR can achieve high SVR rates with abbreviated therapy. There is a trade-o between slightly higher relapse rates in patients treated with abbreviated regimens and slightly higher withdrawal rates in those treated for the standard duration. The highest SVR rates and lowest relapse rates are obtained in patients with an RVR who have low baseline HCV RNA levels (%400,000 IU/mL) even when treated for only 16 weeks, and such therapy may be a reasonable option for these patients. In contrast, patients without an RVR run a high risk for relapse even when treated for 24 weeks. From retrospective analysis, however, there is evidence that prolongation of treatment duration can reduce relapse rates. References
[1] Brown RS, Gaglio JP. Scope of worldwide hepatitis C problem. Liver Transpl 2003;9(11): S103. [2] Zeuzem S. Heterogeneous virologic response rates to interferon-based therapy in patients with chronic hepatitis C: who responds less well? Ann Intern Med 2004;140(5):37081. [3] Manns MP, McHutchison JG, Gordon SC, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomized trial. Lancet 2001;358(9286):95865. [4] Fried MW, Shiman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347(13):97582. [5] Zeuzem S, Feinmann SV, Rasenack J, et al. Peginterferon alfa-2a in patients with chronic hepatitis C. N Engl J Med 2000;343(23):166672. [6] Hadziyannis S, Sette H Jr, Morgan TR, et al. Peginterferon-alfa-2a plus ribavirin combination therapy in chronic hepatitis C. A randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004;40(5):34655. [7] Berg T, Sarrazin C, Herrmann E, et al. Prediction of treatment outcome in patients with chronic hepatitis C: signicance of baseline parameters and viral dynamics during therapy. Hepatology 2003;37(3):6009. [8] Ferenci P, Fried MW, Shiman ML, et al. Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alfa-2a (40 KD)/ribavirin. J Hepatol 2005;43(3):42533. [9] Zeuzem S, Lee JH, Franke A, et al. Quantication of the initial decline of serum hepatitis C virus RNA and response to interferon alfa. Hepatology 1998;27:114956. [10] Davis GL, Wong JB, McHutchison J, et al. Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology 2003;38(3): 64552.

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[11] Sarrazin C, Teuber G, Kokka R, et al. Detection of residual hepatitis C virus RNA by transcription-mediated amplication in patients with complete virologic response according to polymerase chain reaction-based assays. Hepatology 2000;32(4 Pt 1):81823. [12] Morishima C, Morgan TR, Everhart JE, et al, HALT-C Trial Group. HCV RNA detection by TMA during the Hepatitis C antiviral long-term treatment against cirrhosis (HALT-C) trial. Hepatology 2006;44(2):3607. [13] Dienstag JL, McHutchison JG. American Gastroenterological Association medical position statement on the management of hepatitis C. Gastroenterology 2006;130(1):22530. [14] Strader DB, Wright T, Thomas DL, et al. Diagnosis, management, and treatment of hepatitis C. Hepatology 2004;39(4):114771. [15] Zeuzem S, Buti M, Ferenci P, et al. Ecacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol 2006;44(1):97103. [16] Jensen DM, Morgan TR, Marcellin P, et al. Early identication of HCV genotype 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hepatology 2006; 43(5):95460. [17] Ferenci P, Bergholz U, Laferl H, et al. 24 Week treatment regimen with peginterferon-alpha2a (40 KD) (Pegasys) plus ribavirin (Copegus) in HCV genotype 1 or 4 super-responders. J Hepatol 2006;44(Suppl 2):S6. [18] Zeuzem S, Pawlotsky JM, Lukasiewicz E, et al. International, multicenter, randomized, controlled study comparing dynamically individualized versus standard treatment in patients with chronic hepatitis C. J Hepatol 2005;43(2):2507. [19] Berg T, Weich V, Teuber G, et al. Evaluation of the ecacy of an 18 week short treatment duration in HCV type 1 infected patients based upon early viral kinetics: an approach to recognise super-responders. Hepatology 2006;44(Suppl 1):319A. [20] Zeuzem S, Fried MW, Reddy KR, et al. Improving the clinical relevance of pre-treatment viral load as a predictor of sustained virological response (SVR) in patients infected with hepatitis C genotype 1 treated with peginterferon alfa-2A (40 KD) (Pegasys) plus ribavirin (Copegus). Hepatology 2006;44(Suppl 1):267A. [21] Zehnter E, Mauss S, John C, et al. Better prediction of SVR in patients with HCV genotype 1 (G1) with peginterferon alfa-2a (Pegasys) plus ribavirin: improving dierentiation between low (lvl) and high baseline viral load (hvl). Hepatology 2006;44(Suppl 1):328A. [22] Berg T, von Wagner M, Hinrichsen H, et al. Denition of a pre-treatment viral load cut-o for an optimized prediction of treatment outcome in patients with genotype 1 infection receiving either 48 or 72 weeks of peginterferon alfa-2a plus ribavirin. Hepatology 2006; 44(Suppl 1):321A. [23] Buti M, Valdez A, Sanchez-Avila F, et al. Extending combination therapy with peginterferon alfa-2b plus ribavirin for genotype 1 chronic hepatitis C late responders: a report of 9 cases. Hepatology 2003;37(5):12267. [24] Berg T, von Wagner M, Nasser S, et al. Extended treatment duration for hepatitis C virus type 1: comparing 48 versus 72 weeks of peginterferon-alfa-2a plus ribavirin. Gastroenterology 2006;130(4):108697. [25] Sanchez-Tapias JM, Diago M, Escartin P, et al, TeraViC-4 Study Group. Peginterferonalfa2a plus ribavirin for 48 versus 72 weeks in patients with detectable hepatitis C virus RNA at week 4 of treatment. Gastroenterology 2006;131(2):45160. [26] Pearlman BL, Ehleben C, Saifee S. Treatment extension to 72 weeks of peginterferon and ribavirin in hepatitis c genotype 1-infected slow responders. Hepatology 2007;46(6): 168894. [27] Ferenci P, Laferl H, Scherzer T-M, et al. Customizing treatment with peginterferon alfa2a (40 KD) (Pegasys) plus ribavirin (Copegus) in patients with HCV genotype 1 or 4 infection, interim results of a prospective randomized trial. Hepatology 2006;44(Suppl 1): 336A.

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[28] Drusano GL, Preston SL. A 48-week duration of therapy with pegylated interferon-alpha 2b plus ribavirin may be too short to maximize long-term response among patients infected with genotype-1 hepatitis C virus. J Infect Dis 2004;189:96470. [29] Zeuzem S, Hultcrantz R, Bourliere M, et al. Peginterferon alfa-2b plus ribavirin for treatment of chronic hepatitis C in previously untreated patients infected with HCV genotype 2 or 3. J Hepatol 2004;40(6):9939. [30] Neumann AU, Lam NP, Dahari H, et al. Dierences in viral dynamics between genotypes 1 and 2 of hepatitis C virus. J Infect Dis 2000;182:2835. [31] Dalgard O, Bjoro K, Hellum KB, et al. Treatment with pegylated interferon and ribavirin in HCV infection with genotype 2 or 3 for 14 weeks: a pilot study. Hepatology 2004;40(6): 12605. [32] Mangia A, Santoro R, Minerva N, et al. Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N Engl J Med 2005;352(25):260917. [33] von Wagner M, Huber M, Berg T, et al. Randomized multicenter study comparing 16 vs. 24 weeks of combination therapy with peginterferon alfa-2a plus ribavirin in patients chronically infected with HCV genotype 2 or 3. Gastroenterology 2005;129(2):5227. [34] Yu ML, Dai CY, Huang JF, et al. A randomised study of peginterferon and ribavirin for 16 versus 24 weeks in patients with genotype 2 chronic hepatitis C. Gut 2007;56(4):5539. [35] Shiman ML, Suter F, Bacon B, et al. Peginterferon alfa-2A and ribavirin for 16 or 24 weeks in HCV genotype 2 or 3. N Engl J Med 2007;357(2):12434. [36] Willems B, Hadziyannis SJ, Morgan TR, et al. Should treatment with peginterferon plus ribavirin be intensied in patients with HCV genotype 2/3 without a rapid virologic response? J Hepatol 2007;46(Suppl 1):S6, [abstract no. 8].

Clin Liver Dis 12 (2008) 529555

Novel Hepatitis C Drugs in Current Trials


Bernd Kronenberger, MD, Christoph Welsch, MD, Nicole Forestier, MD, Stefan Zeuzem, MD*
Zentrum der Inneren Medizin, Medizinische Klinik 1, Klinikum der Johann Wolfgang Goethe-Universitat, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany

Almost half of the patients who have chronic hepatitis C cannot be cured with the current standard treatment consisting of pegylated interferon-alfa and ribavirin. For those patients who have chronic hepatitis C and did not respond to interferon-alfabased antiviral therapy, there is currently no approved treatment option available. Dicult-to-cure patient populations include patients infected with hepatitis C virus (HCV) genotype 1, HIV/HCV-coinfected patients, patients who have advanced liver cirrhosis, and patients who have recurrent hepatitis C after liver transplantation. Recent progress in structure determination of HCV proteins and development of a subgenomic replicon system [1] and, more recently, of a cell culture infectious HCV clone [2,3] enables the development of a specically targeted antiviral therapy for hepatitis C (STAT-C). Many HCV-specic compounds are under investigation in preclinical and clinical trials. It is anticipated that HCV-specic inhibitors can improve treatment opportunities for patients who have chronic hepatitis C, especially in patients who are dicult to cure. Therapeutic approaches for specically targeted antiviral therapy for hepatitis C virus The HCV genome is a positive-sense 9.6-kb RNA molecule, comprising 50 and 30 untranslated regions (UTRs) anking a single open reading frame encoding for a polyprotein of approximately 3100 amino acids. Translation of the HCV polyprotein is initiated by an internal ribosome entry site (IRES)

* Corresponding author. E-mail address: zeuzem@em.uni-frankfurt.de (S. Zeuzem). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.001 liver.theclinics.com

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located in the 50 UTR of the HCV genome (Fig. 1). The HCV polyprotein is co- and posttranslationally processed by host- and virally encoded proteases into structural (core, envelope E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (Fig. 2). The functions of the HCV proteins and therapeutic approaches for specically targeted antiviral therapy are listed in Table 1. The core protein binds and packages the viral RNA genome and forms the viral nucleocapsid [4,5]. In addition, the core protein interacts with the envelope protein E1. In infected cells, the core protein is found on membranes of the endoplasmic reticulum, in membranous webs, and on the surface of lipid droplets. The core protein interacts with various cellular proteins. The association with lipid droplets may have a role during viral replication or virion morphogenesis. The deletion of the C-terminal hydrophobic region of the core protein causes translocation to the nucleus. Because of the various interactions with cellular proteins and the consecutive eects on signal transduction pathways and transcription, the core protein has been implicated in hepatocarcinogenesis and the alteration of apoptosis. Blocking of the core protein could alter viral morphogenesis, alter viral replication, and reverse liver steatosis. To date, small-molecule inhibitors for the core protein are not available. The envelope proteins E1 and E2 are essential for host cell entry and are potential targets of antiviral therapy. Blocking of HCV entry could be achieved by blocking the envelope proteins or by blocking cellular receptors of HCV, including the tetraspanin CD81, the scavenger receptor class B type

(+)RNA 2. release and uncoating 3. IRES mediated translation 4. polyprotein processing 6. replication (+)RNA (-)RNA

1. binding and internalization 7. assembly and release

5. membraneous web formation

Endoplasmic reticulum

Fig. 1. Life cycle of HCV. IRES-mediated translation, polyprotein processing, membraneous web formation, and replication are illustrated as separate steps; however, they might occur in a tightly coupled fashion. Note that IRES-mediated translation and polyprotein processing occur at the endoplasmic reticulum.

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NS5B

E1

E2

p7 NS2

NS3

NS4A NS4B

NS5A

HCV polyprotein co- and posttranslational polyprotein processing NS2/3 Cysteine Protease NS3/4A Serine Protease

C Core

E1

E2

p7

NS2

NS3

NS4A NS4B Mebr. web

NS5A

NS5B RNA-dependent RNA-Polymerase

Envelope Glycoproteins

Cystein Serine Helicase Protease Protease

Ion Channel

Serine Protease co-factor

IFN-resistance?

structural proteins

non-structural proteins

Fig. 2. Polyprotein processing occurs co- and posttranslationally. The structural proteins and the p7 protein are cleaved by the endoplasmic reticulum signal peptidase. Mebr, membranous.

I (SR-BI), heparan sulfate (HS), dentritic cell and liver and lymph node-specic ICAM-3 grabbing non-integrin (DC-SIGN/L-SIGN), the low-density lipoprotein (LDL) receptor, and claudin-1 [68]. Testing of entry inhibitors is still at the preclinical stage. P7 is a small hydrophobic structural protein [9,10] that homo-oligomerizes into a circular hexamer and is believed to form an ion channel in its center [11]. The role of p7 in HCV replication is still unclear. In chimpanzees, p7 has been shown to be important for infectivity. An antiviral compound that has been shown in one study to block the p7 ion channel in vitro is amantadine [12]; however, the results on antiviral activity of amantadine in patients who have chronic hepatitis C are conicting. A recent study of the antiviral eect of amantadine on cell culture infectious HCV particles showed that across a spectrum of HCV isolates and genotypes, amantadine aected neither RNA replication nor the release or infectivity of HCV particles [13]. Furthermore, the same study showed that the p7 ion channel activity was not aected by amantadine. Overall, the recent results demonstrate that amantadine is not an HCV-selective antiviral. The NS proteins include enzymes necessary for protein maturation (NS2/ 3 cysteine protease and NS3/4A serine protease) and viral replication (NS3 helicase/nucleoside triphosphatase and NS5B RNA-dependent RNA-polymerase). The NS2/3 protease mediates a single cleavage at the NS2/NS3 junction and releases the mature NS3 protease. NS4A enhances the proteolytic activity of the NS3 serine protease domain (Fig. 3). The NS3/4A protease cleaves at four downstream sites in the polyprotein to generate the N-termini of the NS4A, NS4B, NS5A, and NS5B proteins. The structure

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Table 1 Hepatitis C virus structural and nonstructural proteins and potential targets for specically targeted antiviral therapy for hepatitis C virus HCV protein 50 -UTR Function Contains the IRES necessary for initiation of translation of the HCV polyprotein Therapeutic approach Inhibition of IRES blocks initiation of translation; inhibitors are antisense oligonucleotides, ribozymes, and small molecules (eg, VGX-410C, siRNA) in vitro Inhibition might inuence viral replication and viral morphogenesis and inhibit the development of liver steatosis Blocking of envelope proteins or corresponding receptors may inhibit HCV entry and reduce de novo infection of susceptible cells Blocking may reduce infectivity Inhibition blocks HCV polyprotein processing Inhibition blocks polyprotein procession and viral replication; furthermore, blocking may restore the innate antiviral response; inhibitors (telaprevir, boceprevir) are in phase 2 clinical trials

Core

E1/E2

Nucleocapsid, viral morphogenesis, RNA-binding and replication, association with lipid droplets Envelope, attachment, entry, interferon-alfa resistance (?)

p7 NS2

NS3

NS4A

NS4B

NS5A

NS5B

Contains an ion channel potentially involved in viral infection Forms with NS3, a dimeric cysteine protease, for cleavage of the NS2/3 junction  NS2/3 cysteine protease  NS3/4A serine protease  Part of the viral RNA complex  RNA helicase  Nucleotide triphosphatase  Inhibition of the innate antiviral response  Cofactor of the NS3/4A serine protease  Enhances proteolytic activity of the NS3 protease  Membrane protein localized at the endoplasmatic reticulum  Forms a membranous web potentially harboring the HCV replication complex  Potential eect on interferon resistance  Involved in RNA replication  Modulation of cellular signaling pathways (apoptosis, cell growth) RNA-dependent RNA-polymerase

Inhibitor in phase 1 trial; inhibition may overcome interferon resistance and reduce HCV replication

Inhibition blocks HCV replication; inhibitors (nucleoside/nonnucleoside) in phase 2 clinical trials

30 -UTR

 RNA replication  Stimulation of IRES-dependent translation  Potential 30 -50 end interaction necessary for a switch between translation and RNA replication

Abbreviation: siRNA, small interfering RNA.

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Fig. 3. Structure of the HCV NS3/4A serine protease. The protease subdomains are marked in light and dark gray with residues of the catalytic triad in the crevice between the subdomains. An inhibitor (ball-and-stick model) is covalently bound to the active site catalytic triad. The NS4A cofactor (ball-model) is bound to NS3.

of both proteases has been determined. Therefore, NS2/3 and NS3/4A are promising targets for the development of specic inhibitors. The NS3/4A serine protease has also been shown to cleave and inactivate the host proteins (Toll-like receptor domain-containing adapter inducing IFNb [Trif] and Cardif). Both proteins have important roles in the interferon response mediated by Toll-like receptor 3 (TLR3) and retinoic-acid inducible gene I (RIG-I), respectively [14,15]. Inactivation of both proteins blocks the double-stranded (ds) RNAdependent innate immune response and indicates that the NS3/4A serine protease may have an important role in evasion of the innate immune response. Furthermore, it has been shown that NS3 is not only a protease but an integral part of the viral RNA replication complex and functions as an RNA helicase and a nucleotide triphosphatase (NTPase). Because of the multiple functions, it can be assumed that inhibitors against NS3 are highly ecient in blocking HCV replication. Several antiviral drugs directed against the NS3/4A protease have been developed and are listed in Table 2. NS4B is a membrane protein with four transmembrane regions localized at the endoplasmatic reticulum membrane. The function of NS4B is not well understood. NS4B is assumed to form a membranous web that is necessary for the HCV replication complex [16]. The functions of NS4B need to be understood better before it can be considered a promising target of antiviral therapy. NS5A is a pleiotropic protein with key roles in viral RNA replication and modulation of the physiology of the host cell. NS5A is mostly known because of its potential eect on interferon-alfa signaling. Furthermore, NS5A has been shown to aect cell growth of target cells and apoptosis. The crystal structure of domain I of NS5A has recently been solved [17], and specic inhibitors are currently being developed.

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Table 2 Emerging therapies against hepatitis C virus Name Long-acting interferons Albinterferon Pegamax Producer Human Genome Sciences/Novartis Maxygen/Roche Study phase Phase 3 Preclinical/phase 1 (stopped) Phase 2 Phase 2

Locteron (BLX-883) Biolex Interferon-omega Intarcia Therapeutics Oral interferon Belerofon Nautilus Biotech STAT-C NS3/4 serine protease inhibitors Ciluprevir (BILN 2061) Boehringer Ingelheim Telaprevir (VX-950) Vertex Boceprevir (SCH 503034) Schering-Plough ITMN-191 InterMune GS9132/ACH-806 Gilead Sciences/Achillion NS5B RNA-dependent RNA-polymerase inhibitors Nucleoside analogues Valopicitabine (NM283) Idenix/Novartis R1626 (prodrug of R1479) Roche R1656/R7128 Pharmasset/Roche XTL-2125 XTL-Biopharmaceuticals MK-608 Merck Nonnucleoside polymerase inhibitors HCV-796 ViroPharma/Wyeth BILB 1941 Boehringer Ingelheim A-837093 Abbott GS-9190 Gilead NS5A inhibitors A-831 Arrow Therapeutics/AstraZeneca A-689 Arrow Therapeutics/AstraZeneca Cyclophilin inhibitors DEBIO-25 Debiopharm NIM811 Novartis

Stopped Phase 2 Phase 2 Phase 1 Stopped

Stopped Phase 2 Phase 1 Stopped Preclinical Stopped Stopped Preclinical Phase 1 Phase 1 Preclinical Phase 1 in HCV/HIVcoinfected patients Preclinical

The NS5B RNA-dependent RNA-polymerase is another promising target for the development of HCV-specic compounds. NS5B reveals the typical polymerase structure, a classic right-hand shape of thumb, palm, and nger domains encircling the active site (Fig. 4) [18]. To date, many inhibitors against the NS5B RNA-dependent RNA-polymerase have been developed, including nucleoside analog polymerase inhibitors and nonnucleoside polymerase inhibitors (see Table 2). The IRES located in the 50 -UTR is required for the initiation of HCV polyprotein translation. The IRES has been targeted by the antisense oligonucleotide inhibitor ISIS 14803 and the small-molecule organic drug mifepristone (VGX 410C). The development of ISIS 14803 was stopped

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Fig. 4. Structure of the HCV NS5B polymerase. Ligands are given as ball-and-stick models. The active site is located in the palm domain. Regions important for inhibitor binding are indicated for nucleoside and nonnucleoside compounds.

because of weak and highly variable antiviral activity and substantial increases of aminotransferases in a phase 1 trial in patients who had chronic hepatitis C [19]. Mifepristone is currently being investigated in a phase 2 trial in patients who have chronic hepatitis C, and data are pending [20]. The 30 -UTR is necessary for RNA replication and amplication of IRESdependent protein translation and also seems to be a promising antiviral target. To date, most new antiviral drugs for HCV focus on the NS3/4A serine protease and on the NS5B RNA-dependent RNA-polymerase. The results of clinical trials are presented in the following sections. Current therapies for hepatitis C virus Pegylated interferon-alfa and ribavirin are the current standard of care for treatment of patients who have chronic hepatitis C. Great eorts have been made for the individualization of antiviral therapy to reduce adverse events and to improve sustained virologic response rates. HCV genotype and early virologic response during treatment are important factors for individualization of antiviral therapy. An extended treatment duration of 72 weeks has been shown to reduce relapse rates signicantly in patients who have chronic HCV genotype 1 infection and a slow virologic response compared with the standard duration of 48 weeks [21,22]. Conversely, HCV genotype 1infected patients with a low baseline HCV RNA concentration who become HCV RNA-negative at week 4 may be treated for 24 weeks without compromising sustained virologic response rates [23].

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In patients who have HCV genotype 2 or 3 infection, who have a better response to interferon-alfa than patients infected with HCV genotype 1, the standard treatment duration is 24 weeks. Several trials investigated whether a shorter treatment duration is possible in patients who have genotype 2 or 3 infection without compromising the sustained virologic response. Smaller trials showed that a shorter treatment duration of 12 to 16 weeks is equally eective as the standard treatment duration in those patients infected with HCV genotype 2 or 3 who achieve a rapid virologic response after 4 weeks of therapy [24,25]. The large ACCELERATE trial comparing 16 versus 24 weeks of treatment in patients who had HCV genotype 2 or 3 infection showed that a shorter treatment duration of 16 weeks results in reduced sustained virologic response rates compared with the standard treatment duration, however [26]. In the ACCELERATE trial, a shorter course of therapy over 16 weeks has been shown to be as eective as a 24-week course in those patients who have genotype 2 or 3 infection, have a baseline viral load of 400,000 IU/mL or less, and achieve an early virologic response at week 4 [26]. In patients who have genotype (2 and) 3 infection without a rapid virologic response (!50 IU/mL) at week 4, a longer treatment duration may be necessary to optimize sustained virologic response rates [27]. The ribavirin dose may also inuence the rate of sustained virologic response. The Weight-based dosing of pegINterferon alfa-2b and Ribavirin (WIN-R) trial investigated pegylated interferon alfa-2b plus weight-based ribavirin (8001400 mg) or at-dose ribavirin (800 mg) in patients who had chronic hepatitis C [28]. Sustained virologic response but not end-of-treatment rates were signicantly higher in patients treated with weight-based ribavirin than in patients treated with at-dose ribavirin (44.2% versus 40.5%). Sustained virologic response rates by intention-to-treat analysis were 34.0% and 28.9%, respectively, in genotype 1infected patients. In genotype 2 or 3infected patients, sustained virologic response rates were not signicantly dierent (61.8% and 59.5%, respectively), regardless of treatment duration [28]. From the current point of view, further individualization of standard therapy seems unlikely to improve the convenience and outcome of antiviral therapy markedly. New interferons have been developed, however, and are currently being investigated in clinical trials.

Further development of interferon therapy Albinterferon Albinterferon is a novel long-acting form of interferon-alfa that results from the genetic fusion of interferon-alfa with human albumin. Albinterferon has a longer half-life than pegylated interferon-alfa. A recent phase 2b clinical trial investigated antiviral ecacy and tolerability of albinterferon in combination with ribavirin in patients who had chronic hepatitis C genotype 1 infection and were naive to

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interferon-alfabased treatment regimens [29]. In this trial, the virologic response rates 12 weeks after treatment were 58% and 55% in patients treated with albinterferon, 900 mg and 1200 mg every 2 weeks, plus ribavirin versus 58% in patients treated with pegylated interferon-alfa-2a, 180 mg once weekly, plus ribavirin. The health-related quality-of-life scores were more favorable in the albinterferon group than in the pegylated interferon-alfa2a group. Overall, these interim results indicate that albinterferon plus ribavirin may oer ecacy comparable to pegylated interferon-alfa-2a plus ribavirin with half of the injections and the potential for less impairment of quality of life. Albinterferon is currently being investigated in two randomized, openlabel, active controlled, multicenter, noninferiority phase 3 trials to evaluate the ecacy, safety, and impact on health-related quality of life of albinterferon in combination with ribavirin versus pegylated interferonalfa-2a in combination with ribavirin. The ACHIEVE 1 trial is conducted in patients infected with HCV genotype 1 randomized into three treatment groups, including two groups with subcutaneously administered albinterferon once every 2 weeks (900 or 1200 mg), and a control group with pegylated interferon-alfa-2a administered once every week at a dose of 180 mg. All patients receive oral ribavirin (10001200 mg) concomitantly and are treated for 48 weeks. The ACHIEVE 2/3 trial is being conducted in patients infected with HCV genotype 3. The ACHIEVE 2/3 trial has the same design as the ACHIEVE 1 trial with a shorter treatment duration of 24 weeks and a lower ribavirin dose of 800 mg. The primary ecacy end point in both trials is a sustained virologic response, dened as an undetectable HCV RNA level (!10 IU/mL) at weeks 72 and 48, respectively. Higher doses of albinterferon administered every 4 weeks, in combination with ribavirin, are to be explored in separate trials, which are expected to begin in 2007. Interferon-omega Interferon-omega is a type 1 interferon that shares 70% homology of the amino-acid sequence to interferon-alfa and is derived from Chinese hamster ovary cells. Interferon-omega, which is fully glycosylated, has been broadly studied in phase 1 and 2 clinical studies. These studies have demonstrated that interferon-omega is safe and has potent antiHCV activity. Preliminary results from a phase 2 study comparing interferon-omega (25 mg/d) with the combination of interferon-omega and ribavirin (10001200 mg/d) have shown virologic response rates 12 weeks after the end of treatment of 6% and 36%, respectively [30]. It is planned to administer interferon-omega by an implantable osmotic minipump, requiring changes every 3 months, which delivers consistent drug levels through the device outlet.

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Locteron (BLX-883) Locteron is a recombinant interferon-alfa that is released by a special biodegradable polymeric drug delivery system. The polymeric drug delivery system consists of polyester or polyether copolymers that are degraded by hydrolysis and oxidation and enables linear release of compounds. Locteron is designed to be administered every 2 weeks. A phase 1 clinical study investigating the safety, pharmacokinetics, and pharmacodynamics of locteron in healthy volunteers was completed [31]. Results of this phase 1 clinical trial showed that a single dose of locteron was safe and well tolerated. In particular, groups receiving locteron reported fewer, less severe, and shorter lasting u-like symptoms than those subjects receiving pegylated interferon-alfa-2b. Maxy-alpha (R7025/RO5014583, pegamax) Maxy-alpha (R7025) is an interferon-alfa variant that has been created through a directed molecular evolution gene shuing technology to have stronger antiviral activity against the hepatitis C virus and to be more eective in stimulating immune responses than standard interferon-alfa. A pegylated form of maxy-alpha has been developed using the same pegylation technology as for pegylated interferon-alfa-2a. Preclinical data comparing maxy-alpha with pegylated interferon-alfa-2a show that maxy-alpha has increased antiviral activity in the HeLa encephalomyocarditis virus assay and stronger immune stimulatory activity on T helper 1 (Th1) cytokine induction and dendritic cell maturation than pegylated interferon-alfa-2a [32]. A double-blind, dose-escalation, controlled phase 1 study of a single subcutaneous administration of maxy-alpha has been initiated in healthy volunteers [32], and data are pending. Oral interferon (belerofon) Belerofon is a variant of human interferon-alfa with a single amino-acid replacement that has been designed to lower the susceptibility of interferonalfa to proteolytic degradation and to make it longer lasting in serum [33]. In animal models, appropriate oral doses have shown that belerofon can be absorbed from the intestine into the bloodstream and reaches blood levels comparable to those obtained by subcutaneously injected interferon-alfa [33]. Oral belerofon is formulated as enteric-coated tablets containing the lyophilized belerofon protein. Phase 1 results on the safety, tolerability, and pharmacokinetics of oral belerofon are not yet available. Further development of ribavirin Taribavirin (previously known as viramidine) is a prodrug of ribavirin with a distinct pharmacologic prole. Taribavirin is preferentially taken

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up by the liver and is converted to ribavirin by hepatic adenosine deaminase. In contrast to ribavirin, taribavirin is poorly taken up by red blood cells. The ecacy and safety of taribavirin versus ribavirin combined with pegylated interferon in patients who have chronic hepatitis C was investigated in a phase 2 open-label study [34]. Patients were stratied according to HCV genotype and randomized to receive pegylated interferon-alfa-2a at a dosage of 180 mg/wk plus taribavirin at a dosage of 800, 1200 or 1600 mg/d or ribavirin at a dosage of 1000 or 1200 mg/d. Patients infected with HCV genotype 1, 4, 5, or 6; mixed genotypes; or an indeterminate genotype were treated for 48 weeks, and those infected with HCV genotype 2 or 3 were treated for 24 weeks. All patients were followed for 24 weeks after the end of treatment to determine the rate of sustained virologic response. The rates of sustained virologic response were 23%, 37%, and 29% for the dierent taribavirin dose groups, respectively, and 44% for pegylated interferon alfa-2a plus ribavirin. Fewer patients on any dose of taribavirin had severe anemia (hemoglobin !10 g/dL) than on ribavirin (4% versus 27%). Two large-scale randomized phase 3 clinical trials comparing the safety and ecacy of taribavirin plus pegylated interferon-alfa-2b (VISER-1) and pegylated interferon-alfa-2a (VISER-2) versus pegylated interferonalfa-2b/2a plus ribavirin, respectively, were recently completed. In both trials, patients were stratied according to genotype, baseline viral load, and weight. The VISER-1 trial showed a signicantly lower rate of anemia among patients treated with taribavirin compared with ribavirin; however, the overall rate of sustained virologic response in the VISER-1 trial was lower in taribavirin-treated patients versus ribavirin-treated patients (38% versus 52%) [35]. Similar results were obtained in the VISER-2 study (40% versus 55% for taribavirin-treated versus ribavirin-treated patients) [36]. Future studies may be warranted to examine higher weight-based doses of taribavirin in combination with pegylated interferon [34].

Specically targeted antiviral therapy for hepatitis C virus Protease inhibitors Ciluprevir (BILN 2061) Ciluprevir is the rst potent and specic inhibitor of the NS3/4A serine protease [37] that was tested within a randomized, multiple-dose, doubleblind, placebo-controlled pilot study in patients who had chronic hepatitis C [38]. In this study, the oral administration of ciluprevir (25500 mg twice per day) to patients who had chronic hepatitis C genotype 1 infection for 2 days resulted in viral RNA reductions of 2- to 3-log10 copies/mL in most of the patients. The study provided proof of concept that HCV NS3/4A protease inhibitors are a therapeutic option for patients who have chronic hepatitis C.

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The HCV genotype is currently the most important baseline predictive factor for virologic response to interferon-alfabased treatment. The antiviral ecacy of ciluprevir was also investigated in patients who had chronic HCV genotype 2 or 3 infection [39]. The antiviral ecacy of ciluprevir was less pronounced and more variable in HCV genotype 2 or genotype 3infected patients compared with HCV genotype 1infected patients, showing that treatment response to protease inhibitors may also depend on HCV genotype [38,39]. Safety and tolerability are important issues in the development of new antiviral drugs. In rhesus monkeys treated with high doses of ciluprevir for 4 weeks, histologic signs of cardiotoxicity were observed. Because of this cardiotoxicity, the clinical development of ciluprevir was stopped. The development of resistance against ciluprevir was studied in HCV genotype 1b replicon cells [40]. Several mutations in the NS3 protease were identied that were associated with resistance against ciluprevir (Table 3). Modeling studies indicate that all mutations are located in close proximity to the inhibitor binding site [40]. Telaprevir (VX-950) Telaprevir is a peptidomimetic inhibitor of the NS3/4A serine protease that has been developed for the specic treatment of hepatitis C. Compared with ciluprevir, telaprevir exhibits a longer half-life of the bound enzyme inhibitor complex [41]. The rst placebo-controlled double-blind phase 1 study with telaprevir was started in June 2004. In this study, 34 patients who had chronic genotype 1 infection were randomized to receive placebo or telaprevir at a dosage of 450 mg or 750 mg every 8 hours or 1250 mg every 12 hours for 14 days [42]. Most of the included patients in this study (27 of 34 patients) had failed prior interferon-based treatment. During treatment with telaprevir, all
Table 3 Mutations in the protease conferring resistance to various protease inhibitors BILN 2061 In vitro R155Q A156V/T D168V/A/Y Telaprevir A156S/V/T Boceprevir T54A A156S/T V170A ITMN-191 V23A Q41R S138T D168A/V/E D168V/A156S/V S489L No data

In vivo

No data

V36M/A T54A R155K/T 36/155 A156S/V/T 36/156

T54A

Data from Refs. [43,45,76].

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patients showed a decline in viral load of 2 log10 or greater. Twenty-six of 28 patients had a decline in viral load of 3 log10 or greater. In the 750-mg dose group, the median reduction of HCV RNA was 4.4 log10 after 14 days. In the 450-mg dose group and the 1250-mg dose group, the maximum eect was seen between days 3 and 7 of dosing, followed by an increase of HCV RNA [42]. The increase of HCV RNA between days 7 and 14 indicates the selection of variants with reduced sensitivity to telaprevir [42,43]. Several mutations conferring resistance to telaprevir were detected in the replicon system and during monotherapy with telaprevir [43]. The development of resistant variants was investigated in patients during telaprevir monotherapy [43]. The known in vitro and in vivo mutations conferring resistance to telaprevir are listed in Table 3. Mutations that confer low-level resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36155, 36156) to telaprevir were detected and correlated with telaprevir exposure and virologic response. Viral tness generally refers to the relative replication competence of a virus under dened circumstances. Sarrazin and colleagues [43] generated an algorithm to calculate viral tness using the HCV RNA data and sequence data at the end of treatment and at follow-up. Changes in the frequency of mutations after the end of dosing showed an inverse relation between in vivo viral tness and resistance. In the absence of telaprevir selective pressure, most resistant variants were replaced by wild-type virus within 3 to 7 months. The rapid development of resistance during telaprevir monotherapy indicates that combination therapy with pegylated interferon-alfa or other direct antiviral drugs seems to be necessary to avoid the development of resistance [43]. Subsequently, telaprevir was investigated in combination with pegylated interferon-alfa-2a [44]. Treatment-naive patients who had chronic hepatitis C genotype 1 infection were randomized for treatment with pegylated interferon-alfa-2a plus placebo (n 4), telaprevir monotherapy (n 8), or a combination of telaprevir and pegylated interferon-alfa-2a (n 8) for 2 weeks. The median changes in HCV RNA level from baseline to day 15 were 1.09 log10 IU/mL, 3.99 log10 IU/mL, and 5.49 log10 IU/mL in the pegylated interferon alfa-2a plus placebo, the telaprevir monotherapy, and the telaprevir plus pegylated interferon alfa-2a combination groups, respectively. The results of this study demonstrated at least additive antiviral eects of telaprevir in combination with pegylated interferon-alfa-2a. The treatment was well tolerated, all patients completed dosing, and no serious adverse events were reported. A detailed kinetic analysis of variants in patients treated with telaprevir alone or with telaprevir plus pegylated interferon-alfa-2a for 14 days was performed [45]. This analysis indicates that the initial antiviral response to telaprevir is attributable to a sharp reduction in wild-type virus, which uncovers preexisting telaprevir-resistant variants. The combination of telaprevir and pegylated interferon-alfa-2a inhibited wild-type and resistant

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variants, indicating that telaprevir-resistant variants are sensitive to pegylated interferon-alfa-2a. Combination therapy with pegylated interferon-alfa-2a plus ribavirin was oered to the patients after the initial 14-day dosing period [44,45]. Nineteen of the 20 patients began standard therapy after completing the dosing period (1 patient of the previous telaprevir monotherapy group declined treatment). Twelve weeks after starting standard therapy, HCV RNA was undetectable in 5 patients of the previous telaprevir group and in all 8 patients of the previous telaprevir plus pegylated interferon-alfa-2a group. At 24 weeks, HCV RNA was undetectable in all patients who started standard therapy in the previous telaprevir monotherapy group (n 7) and the previous telaprevir plus pegylated interferon-alfa-2a group (n 8) [44,45]. In consecutive studies, telaprevir is being combined with pegylated interferon-alfa-2a and ribavirin [46,47] and is currently being further investigated in this combination in two phase 2 studies (PROVE-1 and -2). In the PROVE-1 trial (conducted in the United States), treatment-naive patients infected with HCV genotype 1 (n 260) have been randomized into four groups: 1. Twelve weeks of therapy with telaprevir (750 mg every 8 hours) in combination with pegylated interferon-alfa-2a and ribavirin (total duration of 12 weeks, n 20) 2. Twelve weeks of therapy with telaprevir (750 mg every 8 hours) in combination with pegylated interferon-alfa-2a and ribavirin, followed by pegylated interferon-alfa-2a and ribavirin alone for 12 weeks (total duration of 24 weeks, n 80) 3. Telaprevir (750 mg every 8 hours) in combination with pegylated interferon-alfa-2a and ribavirin for 12 weeks, followed by pegylated interferon-alfa-2a and ribavirin alone for 36 weeks (total duration of 48 weeks, n 80) 4. Control arm with pegylated interferon-alfa-2a and ribavirin for 48 weeks (total duration of 48 weeks, n 80) Only patients in the 12- and 24-week treatment arms who achieve a rapid viral response (HCV RNA level !10 IU/mL) by the end of week 4 and who maintain this status through to week 10 or 20, respectively, are planned to stop all treatment at the 12- or 24-week time point (according to the study arms) and are to be followed after treatment to evaluate whether they achieve a sustained virologic response. Patients in these treatment arms without a rapid virologic response are required to continue on pegylated interferon-alfa-2a and ribavirin for a total of 48 weeks, however. The PROVE-2 study is conducted in Europe (n 320, n 80 for each treatment arm) and has a similar study design as the PROVE-1 study. In the PROVE-2 trial, the 12-week treatment arm with telaprevir plus pegylated interferon-alfa-2a plus ribavirin followed by 36 weeks of treatment with pegylated interferon-alfa-2a plus ribavirin is replaced by 12 weeks of

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treatment with telaprevir plus pegylated interferon-alfa-2a only. Another important dierence is that patients within the PROVE-2 trial can stop treatment according to study arm assignment independent of a rapid virologic response criterion. The interim analysis of the PROVE-1 trial showed that a rapid virologic response after 4 weeks of treatment (HCV RNA level !10 IU/mL) was achieved in 79% of patients in the triple-therapy arm, including telaprevir, and in 11% of patients in the standard combination arm. The virologic response rate after 12 weeks of treatment (HCV RNA level !10 IU/mL) was 70% in the triple-therapy arm and 39% in the standard combination arm. Nine patients who were treated with pegylated interferon-alfa-2a, ribavirin, plus telaprevir in study arm A and achieved a rapid virologic response at week 4 (!10 IU/mL) discontinued triple therapy after 12 weeks. Twenty weeks after discontinuation of triple therapy, six of nine patients had undetectable HCV RNA in serum. This is the rst result indicating that specically targeted antiviral therapy against HCV may shorten the required treatment duration and improve the sustained virologic response rate. In the PROVE-1 trial, the total incidence of adverse events in patients treated with telaprevir, interferon-alfa-2a, and ribavirin was similar to that of the control group. Discontinuation attributable to adverse events was more frequent in the telaprevir arm compared with the control arm (9% versus 3%), however. Gastrointestinal events, rashes (severe in several cases), and anemia were more common in the triple-therapy arms, which included telaprevir, than in the standard double-combination treatment arm. The ecacy of telaprevir in patients infected with HCV genotype 1 who have not achieved a sustained virologic response with a previous interferonbased treatment is to be investigated in the PROVE-3 trial (n 400 with 60% nonresponders and 40% relapsers to previous interferon-based treatment). The PROVE-3 trial is a double-blind randomized phase 2 study investigating telaprevir or placebo with peginterferon-alfa-2a, with or without ribavirin. In the PROVE-3 trial, patients are to be randomized into four groups [48]: 1. Twelve weeks of therapy with telaprevir, pegylated interferon-alfa-2a and ribavirin, followed by 12 weeks of treatment with placebo, pegylated interferon-alfa-2a, and ribavirin (total duration of 24 weeks) 2. Twenty-four weeks of therapy with telaprevir and pegylated interferonalfa-2a (total duration 24 weeks) 3. Twenty-four weeks of therapy with telaprevir plus pegylated interferonalfa-2a and ribavirin, followed by 24 weeks of treatment with pegylated interferon-alfa-2a and ribavirin (total duration of 48 weeks) 4. Control arm with placebo plus pegylated interferon-alfa-2a and ribavirin for 24 weeks, followed by 24 weeks of treatment with pegylated interferon-alfa-2a and ribavirin only (total duration of 48 weeks)

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All patients who do not achieve an early virologic response at week 4 discontinue treatment. Patients in the control group 4 have the option to receive telaprevir through a rollover study after week 24. Final data are awaited in 2008. Boceprevir (SCH 503034) Boceprevir binds reversibly to the NS3 protease active site and has potent activity in the replicon system alone [49] and in combination with interferonalfa-2b [49]. In a phase 1 open-label combination study, boceprevir was evaluated in combination with pegylated interferon-alfa-2b versus either agent alone in a crossover design in adult patients who have HCV genotype 1 and were previous nonresponders to pegylated interferon-alfa-2bbased therapy [50]. Patients were randomized to receive in random sequence (1) boceprevir (200 mg or 400 mg every 8 hours) as a monotherapy for 7 days, (2) pegylated interferon-alfa-2b (1.5 mg/kg/wk) as monotherapy for 14 days, and (3) boceprevir plus pegylated interferon-alfa-2b combination therapy for 14 days in a three-period crossover design with a 3-week washout between treatments. Mean maximum log10 changes in HCV RNA were 2.45 0.22 and 2.88 0.22 for pegylated interferon-alfa-2b plus boceprevir at a rate of 200 mg or 400 mg, respectively, compared with 1.08 0.22 and 1.61 0.21 for boceprevir at a rate of 200 mg and 400 mg, respectively, and 1.08 0.22 and 1.26 0.20 for pegylated interferon-alfa-2b alone in the groups with boceprevir administered at 200 mg and 400 mg, respectively [50]. Several mutations conferring resistance to boceprevir were observed in the replicon system, but only a single mutation has to date been described in one patient by direct sequencing (see Table 3). Based on the results of the phase 1 clinical program and extensive preclinical safety and pharmacology studies, a large randomized phase 2 dose-nding study has been initiated [51]. This study was designed to evaluate the safety and ecacy of boceprevir in combination with pegylated interferon-alfa-2b, with and without added ribavirin, for 24 or 48 weeks in patients infected with chronic HCV genotype 1 who were nonresponders to previous pegylated interferon and ribavirin combination therapy. The primary objective of this study was to determine the safe and eective dose range of boceprevir in combination with pegylated interferon-alfa-2b in this patient population. A secondary objective was to explore whether ribavirin provides an additional benet when combined with boceprevir plus pegylated interferon-alfa-2b. The study was completed in 2007, and the results are pending [51]. The safety and ecacy of boceprevir in treatment-naive patients who have chronic HCV genotype 1 infection is currently being investigated in a phase 2, randomized, 5-arm, comparative, open-label safety and ecacy study. The study compares treatment with pegylated interferon-alfa-2b plus ribavirin with treatment with boceprevir plus pegylated interferonalfa-2b and ribavirin.

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Optimizing pharmacokinetics: ritonavir boosting Ritonavir is a protease inhibitor approved for treatment of HIV with potent inhibitory eects against cytochrome P450 3A (CYP3A). Codosing with ritonavir leads to pharmacokinetic enhancement (boosting) of peptidomimetic HIV protease inhibitors, which are metabolized by CYP3A. The interaction of the investigational HCV protease inhibitors telaprevir and boceprevir with ritonavir was studied in vitro and in vivo [52]. In rat and human liver microsomes, the metabolism of telaprevir and boceprevir was strongly inhibited by ritonavir. On codosing of telaprevir or boceprevir with ritonavir in rats, plasma exposure of the HCV protease inhibitors was increased by greater than 15-fold and plasma concentrations 8 hours after dosing were increased by greater than 50-fold. ITMN-191 ITMN-191 is another inhibitor of the NS3/4A protease with potent antiviral activity in vitro [53]. Several mutations associated with resistance to ITMN-191 were found in the replicon system (see Table 3). A phase 1a trial in healthy volunteers was completed in May 2007. The phase 1b study in patients who have chronic hepatitis C is designed to assess the eect on viral kinetics, viral resistance, pharmacokinetics, safety, and tolerability of multiple ascending doses of ITMN-191 given as monotherapy for 14 days. Twice per day and three times per day dosage regimens are to be studied. [54]. NS4A inhibitors ACH-806, ACH-1095. ACH-806 is an antagonist of the NS4A protein, which is a cofactor of the NS3 protease. Thus, ACH-806 is a protease inhibitor with a distinct mechanism of action compared with the previously described NS3/4A protease inhibitors. ACH-806 prevents the formation of the replicase complex after viral protein processing, a necessary step in viral replication that occurs before copying the viral RNA genome. This unique mechanism may contribute to a lack of cross-resistance between ACH-806 and other HCV NS3/4A protease inhibitors in vitro. Data from a phase 1b trial indicated that ACH-806 has antiviral activity against HCV, validating the novel anti-HCV mechanism. Based on elevations of serum creatinine, which were reversible after completion of dosing, however, further development of ACH-806 was stopped [55]. ACH-1095 is one of a series of next-generation compounds with potent antiviral potency in the replicon system [56]. Results from clinical trials on this compound are not yet available. Polymerase inhibitors Two classes of polymerase inhibitors, nucleoside and nonnucleoside polymerase inhibitors, have been developed. Nucleoside analog polymerase inhibitors are converted to triphosphates by cellular kinases and

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incorporated into the elongating RNA strand as chain terminators. Generally, they show similar ecacy against all HCV genotypes. Nucleoside analog polymerase inhibitors Valopicitabine (NM283) Valopicitabine is an orally bioavailable prodrug of a nucleoside analogue inhibiting the HCV NS5B RNA-dependent RNA-polymerase [57]. Valopicitabine inhibits the viral polymerase directly and is incorporated into growing strands of viral RNA, with subsequent termination of RNA chain extension. Human polymerases do not seem to be aected by valopicitabine. Valopicitabine was investigated in patients who had chronic hepatitis C alone and in combination with pegylated interferon. Patients infected with HCV genotype 1 and prior nonresponse to interferon-based antiviral treatment showed a mean reduction of 0.15 to 1.21 log10 IU/mL after 14 days of treatment with dierent doses of valopicitabine ranging from 50 to 800 mg/d [58]. Combination therapy was generally well tolerated; however, higher doses of valopicitabine were associated with gastrointestinal side eects that were severe in some patients [59]. Therefore, the maximum dose of valopicitabine was reduced from 800 to 400 mg/d by protocol amendment during the progress of the phase 2 trials. An interim analysis showed that the combination of pegylated interferon-alfa-2a plus valopicitabine in treatment-naive patients who had chronic hepatitis C genotype 1 infection was associated with a mean decline of HCV RNA of 3.90 to 4.56 log10 IU/mL and 3.75 to 4.41 log10 IU/mL after 24 and 36 weeks of treatment, respectively [59]. Valopicitabine in combination with pegylated interferon-alfa-2a was also investigated in patients with prior nonresponse to interferon-alfabased treatment [60]. The interim analysis of this study after 24 weeks of treatment showed a signicantly stronger decline of HCV RNA in the combination of valopicitabine (800 mg) and pegylated interferon-alfa-2a versus pegylated interferon-alfa-2a and ribavirin treatment (3.32 versus 2.31 log10 IU/mL) [60]. After 40 weeks of treatment, the maximum dose of valopicitabine also had to be reduced to 400 mg/d by protocol amendment [61]. After 48 weeks of treatment, the decline of HCV RNA in the previous high-dose valopicitabine plus pegylated interferon-alfa-2a arms was still 0.8 log10 IU greater than in the pegylated interferon-alfa-2a plus ribavirin combination arm; however, the difference was not signicant [61]. Based on the overall risk-benet prole observed in clinical testing, the development program of valopicitabine for the treatment of hepatitis C has been placed on clinical hold. Resistance to valopicitabine was investigated using the replicon system. Replicon variants bearing an S282T mutation in the viral polymerase showed resistance to the active metabolite of valopicitabine [62,63]. Replicon variants bearing this mutation showed reduced tness compared with the wild-type replicon.

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Nonsynergistic interactions of antiviral drug combinations could be a problem for future therapies. It was found that ribavirin antagonizes the in vitro anti-HCV activity of 20 -C-methylcytidine, the active metabolite of valopicitabine. These ndings may have implications in future clinical studies with specic nucleoside analog polymerase inhibitors [64]. R1479 and R1626 The nucleoside analogue R1479 (40 -azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture [65]. R1626 is a prodrug of R1479 [66]. A multiple dose ascending phase 1 study was designed to evaluate the safety, tolerability, pharmacokinetics, and antiviral activity of R1626 in previously untreated patients infected with chronic hepatitis C genotype 1 [66]. In this study, patients were randomized for treatment with dierent doses of R1626 ranging from 500 to 4500 mg of R1626 or placebo twice daily. Patients were treated for 14 days and followed up for another 14 days. Mean viral load reductions of 1.2, 2.6, and 3.7 log10 IU/mL were observed with R1626 at doses of 1500, 3000, and 4500 mg, respectively. A phase 2 trial evaluates safety and ecacy of R1626 in combination with peginterferon-alfa-2a and ribavirin. Development of resistance to R1479 was investigated in the replicon system. Resistance was associated with the presence of amino-acid substitutions S96T and S96T/N142T in the NS5B polymerase [67]. PSI-6130 and R7128 R7128 is another nucleoside type polymerase inhibitor that has been developed for the treatment of chronic hepatitis C [68]. R7128 is a prodrug of PSI-6130, an oral cytidine nucleoside analogue. In preclinical studies, no toxicity was observed in various human cells, including liver cells, bone marrow cells, and white blood cells. When compared in laboratory studies with several other compounds in development for the treatment of HCV, PSI6130 was found to be more active at low concentrations or less toxic. In combination with interferon, PSI-6130 was active and additive to the activity of interferon alone in these preclinical assays. Results from phase 1 studies on R7128 are pending. Nonnucleoside polymerase inhibitors The mechanisms of action of nonnucleoside polymerase inhibitors are dierent from those of nucleoside polymerase inhibitors. Therefore, crossresistance between these two classes is unlikely to occur. Several structurally distinct nonnucleoside inhibitors of the HCV RNAdependent RNApolymerase NS5B have been reported to date, including benzimidazole, benzothiadiazine, and disubstituted phenylalanine or thiophene or dihydropyranone derivatives. They target dierent sites within the thumb domain of

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the polymerase. Nonnucleoside inhibitors based on the structure of a benzimidazole or an indole core bind to an exposed guanosine triphosphate (GTP) site on the protein surface, whereas inhibitors with a phenylalanine, thiophene, or dihydropyranone scaold bind 1.01.5 nm from that external GTP site in the region of a hydrophobic cleft at the base of the thumb domain (see Fig. 4). Dierent resistance proles attributable to distinct target sites can be expected for the class of nonnucleoside inhibitors. As with protease inhibitors, however, a single mutation may already confer resistance to nonnucleoside polymerase inhibitors. In contrast to nucleoside polymerase inhibitors, a restricted spectrum of activity of nonnucleoside polymerase inhibitors against dierent HCV genotypes and subtypes has been described. In addition to classic nonnucleoside analog inhibitors, several pyrophosphate mimics have been described that interact with catalytic metal ions in the active site of the enzyme. HCV-796 HCV-796 is a nonnucleoside polymerase inhibitor of the NS5B RNAdependent RNA-polymerase that has demonstrated potent antiviral activity in vitro and in patients who have chronic hepatitis C. Monotherapy showed a maximum antiviral eect after 4 days of treatment with a mean reduction of HCV RNA of 1.4 log10 IU/mL. Viral load started to increase thereafter, however, indicating that resistance might be an issue. The emergence of a C316Y amino-acid substitution in NS5B in isolates of patients treated with HCV-796 was associated with the development of resistance to HCV-796. The combination of HCV-796 and pegylated interferon-alfa-2b was investigated in treatment-naive patients who had chronic hepatitis C. The combination of HCV-796 and pegylated interferon-alfa-2b produced a mean viral reduction of 3.3 to 3.5 log10 IU/mL after 14 days of treatment compared with 1.6 log10 IU/mL with pegylated interferon-alfa-2b alone [69]. The antiviral activity of HCV-796 diered by HCV genotype. Mean reductions at day 14 for patients infected with HCV genotype 1 ranged from 2.6 to 3.2 log10 IU/mL in the combination groups versus l.2 log10 IU/mL for pegylated interferon-alfa-2b alone. For HCV genotype non1-infected patients, the respective reductions of HCV RNA were 3.5 to 4.8 log10 IU/mL versus 2.6 log10 IU/mL. Common adverse events in all groups were those typically associated with interferons, including headache, chills, rash, and myalgia [69]. In a consecutive phase 2 study evaluating HCV-796 in combination with pegylated interferon and ribavirin, clinically signicant elevations of liver enzymes were observed in approximately 8% of patients receiving HCV796, including two patients who experienced serious adverse events leading to withdrawal from active therapy with HCV-796, pegylated interferon, and ribavirin [70]. Because of this potential safety issue, HCV-796 treatment was discontinued in the phase 2 program.

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GS-9190 GS-9190 is another nonnucleoside polymerase inhibitor with potent antiviral activity in the replicon system [71]. The antiviral activity of GS-9190 is higher in HCV genotype 1 replicons compared with HCV genotype 2 replicons. GS-9190 is currently being investigated in phase 1 clinical trials. BILB 1941 BILB 1941 is an orally bioavailable reversible nonnucleoside inhibitor of the RNA-dependent RNA-polymerase of the hepatitis C virus. The compound exhibits potent and specic inhibition of the HCV RNAdependent RNA-polymerase in enzymatic- and cell-based assays. In a phase 1 trial, BILB 1941 was given as monotherapy in a liquid formulation for 5 days and demonstrated signicant antiviral activity in patients infected with HCV genotype 1 [72]. Increased virologic response was limited by gastrointestinal intolerance that precluded testing at higher doses. The contribution to the gastrointestinal side eects by BILB 1941 versus the constituents of the liquid formulation remains uncertain. NS5A antagonists A-831 and A-689 A-831 targets the NS5A protein and has shown potent activity in the replicon assay. The drug has an excellent therapeutic index and good pharmacokinetic properties. A-831 is currently being investigated in a phase 1 trial, and results are pending. A-689 is another NS5A inhibitor currently in preclinical development. A-689 has a dierent chemical structure from A-831 and binds to the NS5A target at a dierent site [73]. Cyclophilin inhibitors DEBIO-25, NIM811 Cyclophilins are ubiquitous proteins in human cells that are involved in protein folding. Moreover, cyclophilins participate in HCV replication. It was shown that cyclophilin B binds to the HCV NS5B polymerase and stimulates its RNA-binding activity. Cyclophilin inhibitors show strong antiviral activity in vitro and in vivo. Cyclophilin inhibitors may not only be eective against HCV but against HIV. The cyclophilin inhibitor DEBIO-025 showed a strong dual antiviral activity against HCV and HIV in a phase 1 trial with HCV/HIV-coinfected patients [74]. In this study, the mean maximal decrease in HIV-1 viral load was 1.0 log10 IU/mL. A pronounced eect on HCV RNA was found, with a mean maximal decrease of 3.6 log10 IU/ mL. All patients except one showed an HCV RNA reduction of more than 2 log10 IU/mL, with dierences depending on genotype. DEBIO-025 was well tolerated. Laboratory abnormalities observed were hyperbilirubinemia and low platelet count. Clinical data for another cyclophilin inhibitor, NIM811, are currently not available.

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Alpha-glucosidase I inhibitor Celgosivir (MX-3253) Celgosivir (MX-3253) is a new class of antiviral in clinical development for treatment of patients who have chronic hepatitis C. The active metabolite of celgosivir is castanospermine, which is a potent inhibitor of the alphaglucosidase I that is a host enzyme required for viral assembly and release. Celgosivir is potentially synergistic with other mechanistically diverse antiHCV drugs. A phase 2, multicenter, double-blind, controlled study on the ecacy of celgosivir in patients who had chronic hepatitis genotype 1 infection with prior nonresponse to interferon-based antiviral treatment was performed [75]. In this study, virologic nonresponders to previous interferon-alfabased antiviral treatment showed a 1.2-log10 decline of HCV RNA after treatment with celgosivir, pegylated interferon-alfa-2b, and ribavirin compared with a 0.4-log10 decline after treatment with placebo plus pegylated interferon-alfa-2b and ribavirin (one-sided P!.05).

Summary Research in the past years has focused on individualization of interferonalfabased treatment regimens for patients who have chronic hepatitis C. New long-acting interferons may improve convenience of application and potentially improve the adverse event prole. Further modication of interferon therapy is unlikely to improve sustained virologic response rates markedly. Specic targeted antiviral therapy for HCV is a new perspective in the treatment of chronic hepatitis C especially for patient populations that are dicult to treat. The results from recent clinical trials indicate that several compounds have potent antiviral activity; however, not all were considered as safe and well tolerated. A central question remains whether the new antiviral compounds not only increase the virologic response during treatment but increase the rate of sustained virologic response after treatment. A rst interim analysis on sustained virologic response after treatment with telaprevir in combination with pegylated interferon-alfa and ribavirin has recently been presented, and the results are promising. The results suggest that combination therapy with telaprevir with pegylated interferon-alfa plus ribavirin may not only increase the rate of sustained virologic response compared with standard therapy but may allow a reduction of treatment duration. The viral RNA-polymerase of HCV has a high error rate. As a result, different HCV variants are continuously produced during replication. HCV does not integrate into host DNA. Therefore, every infected cell has the potential of producing multiple drug-resistant mutants over time. The emergence of resistance might possibly limit the success of HCV-specic antiviral compounds, and is therefore a highly clinically relevant issue.

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Selection of drug-resistant HCV strains may occur when viral replication continues while drugs are taken. Not all viral strains have the same ability to replicate. The inherent ability of a virus to replicate is termed viral tness. The tness of variants present in a patient may lead to dierences in viral response. The tness of viral variants was investigated in patients who had chronic hepatitis C after stopping monotherapy with telaprevir [43]. In this study, high-level resistant strains were replaced more rapidly than low-level resistant strains by wild-type virus. The results indicate that telaprevir resistance is inversely correlated with viral tness to replicate [43]. Innate and adaptive immune responses play an important role in the control of viral diseases. In most patients infected with the hepatitis C virus, the innate and adaptive immune responses are too weak for complete elimination of HCV-infected cells, which is the prerequisite for a cure from HCV. The consequence of an impaired immune response is the persistence of infected cells and the development of chronic hepatitis C. Several mechanisms have been identied by which the hepatitis C virus attenuates innate and adaptive immune responses. It can be anticipated that inhibition of HCV replication by new HCV-specic antiviral compounds leads to a reconstitution of the innate and adaptive immune response against HCV during antiviral therapy. A potential reconstitution of the immune response against HCV could enhance the infected cell loss during treatment, and thereby improve the antiviral eciency of HCV-specic compounds [50]. Primary or secondary resistance to HCV specic inhibitors is potentially a serious problem. Combination of (noncross-resistant) antiviral compounds inhibiting dierent viral and cellular mechanisms may reduce the problem of resistance. Therefore, future research should not only focus on the development of new compounds but on optimal drug combinations. A recent study on cross-resistance of telaprevir and interferon-alfa indicates that telaprevir-resistant strains are sensitive to interferon-alfa [45]. This study supports the concept that combination therapy may reduce the development and selection of resistant strains. The development of agents in different classes may allow construction of antiviral combinations that enhance the eectiveness of antiviral treatment, reduce the duration of treatment, and, eventually, may even avoid the use of interferon-alfa. References
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Clin Liver Dis 12 (2008) 557571

Noninvasive Monitoring of Hepatitis C Fibrosis Progression


David S. Kotlyar, BSa, Wojciech Blonski, MD, PhDb,c, Vinod K. Rustgi, MD, FACPd,*
University of Pennsylvania School of Medicine, 3 Ravdin/3400 Spruce Street, Philadelphia, PA 19104, USA b Hospital of the University of Pennsylvania, 3 Dulles, 3400 Spruce Street, Philadelphia, PA 19104, USA c Department of Gastroenterology and Hepatology, Wroclaw Medical University, Borowska 213 Street, 50-556 Wroclaw, Poland d Metropolitan Liver Diseases/Gastroenterology Center, Georgetown University Medical Center, 8316 Arlington Boulevard, Suite 515, Fairfax, VA 22031, USA
a

Hepatitis C remains the most common cause of chronic liver disease in the United States. Monitoring of the progression of brosis is an important tool for the clinician in assessing the aggressiveness of infection and level of hepatic injury. Historically, liver biopsy has been used as the gold standard in determining the degree of brosis and the degree of hepatitic necroinammatory injury [1,2]. The degree of inammation and brosis gleaned from a liver biopsy also has been used as a vital tool in strategizing treatment for hepatitis C [3]. Biopsy is not only an invasive and possibly painful procedure; it also carries a small risk for morbidity and death [3]. Further, there is the issue of sampling error, dened as variable levels of brosis throughout the liver, with biopsy only examining a small (1/50,000) portion of the liver [4]. In one study, several patients had minimal brosis on biopsy but by means of transient elastography, a newly developed form of ultrasound, they were found to have severe brosis and severely elevated portal pressures, as conrmed by hepatic-portal venous gradient measurement [4]. Biopsy has been shown to have signicant inter- and intraobserver variability among pathologists, with an average 20% error rate in the staging of brosis [5]. One study with 124 patients noted that approximately 33% of
* Corresponding author. E-mail address: hepgi@aol.com (V.K. Rustgi). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.006 liver.theclinics.com

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samples had dierent brosis scores among dierent pathologists, although only 9.7% were deemed to be clinically signicant if grade 3 and grade 4 brosis were considered similar [6]. The amount of tissue procured aects the staging as well; in one study, staging was signicantly underscored when the sample was smaller [7]. Interpretation of a liver biopsy by expert pathologists with more than 10 years of experience in an academic center along with a minimum biopsy sample of 2 cm and 11 or more portal tracts may have much improved interobserver agreement and less sample error [8,9]. Although biopsy remains an invaluable tool in determining liver injury, there remains a need to supplement biopsy with noninvasive methods of assessing liver brosis. Importance and pathophysiology of brosis Fibrosis is usually secondary to hepatic injury and inammation. Often, although transaminase levels may be normal in a patient who had chronic hepatitis C infection, the patient may have signicant levels of brosis, which suggests that transaminase levels are a suboptimal surrogate marker for brosis progression [1]. Of those with moderate inammation, most develop cirrhosis within 20 years and those with bridging brosis or severe inammation usually develop cirrhosis within 10 years [1]. Fibrosis is a dynamic process; in the healthy individual, although there is no change in the structure of the extracellular matrix (ECM) on histology, there are simultaneous catabolic and metabolic processes that reach equilibrium with each other [10]. In the brotic state, there is excessive ECM production, which outstrips the catabolism of ECM elements [10]. It has been shown that a major player in brosis is the hepatic stellate cell (HSC, also known as Ito cells), which normally functions to store vitamin A and usually remains morphologically stable [11]. In liver injury, however, these cells become activated, wherein their morphology changes from spheroid cells to more elongated and spindle-shaped cells reminiscent of myobroblasts [11]. They have a reduction in the amount of vitamin A and begin to secrete dense forms of collagen, such as collagen I [11]. They also express matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), which alter the makeup of the ECM [11]. The HSCs also begin to proliferate, responding to increased levels of plateletderived growth factor (PDGF) and increase production of collagen I and other proteins, which increases brosis production because of cytokines, such as transforming growth factor-b (TGFb) [11]. Progressive scarring makes the entire liver more sti (as measured by the shear modulus), which is highly signicant for noninvasive testing [12]. The ECM may also aect brotic production of HSCs, making the pathophysiology of brosis a vicious cycle [12]. Interestingly, there is also phosphorylation of focal adhesion kinase (FAK) in HSCs, a key protein involved in the activation of cell movement [13]. There is also increased expression of

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nuclear factor (NF)-kB, which promotes HSC proliferation and antiapoptotic properties [13]. FAK activation secondary to ECM (and extracellular lysyl oxidase) has also been observed in hypoxic metastases of tumors; this parallels brosis as a model in which the ECM greatly inuences cellular behavior [14]. Fig. 1 is a schematic showing a model of the progression of brosis [12]. Transient elastography Within the past 4 years, transient elastography has emerged as a highly useful noninvasive form of ultrasound that can help to assess the level of brosis in a patient who has hepatitis C. Elastography is also known by the name FibroScan, as developed in France. The scan was developed on the principle that livers with increasing degrees of scarring have decreasing elasticity and that a shear wave propagating through stier material would progress faster than in one with more elastic material [15]. The transient nature of measuring the shear wave (w100 milliseconds) is ideal,

Fig. 1. Biology of injury, inammation, and brogenesis. In most diseases leading to brosis, injury and subsequent inammation are prominent. Injury to the liver, typically involving hepatocytes, leads to activation of immune cells and an inammatory response. Inammatory mediators are an important stimulus for stellate cell activation, and thus brogenesis. Importantly, once activated, the stellate cell activation arm becomes self-perpetuating through several autocrine systems (involving TGFb, PDGF, endothelin, and others). (From Rockey DC, Bissell DM. Noninvasive measures of liver brosis. Hepatology 2006;43(Suppl):S113; with permission.)

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because the livers position moves with breathing, and other reected elastic waves can be excluded from analysis by this method [15]. The elastography device is a vibrating probe with an ultrasound transducer on its end. The transducer emits a shear wave of low frequency at 50 Hz, and pulse-echo ultrasound acquisitions are used to measure the amount of time the wave takes to go through a set window of tissue [16]. The amount of tissue scanned is 1 cm by 4 cm [15], an area that is 100 times the size of a standard biopsy [16]. Stiness is measured by the shear modulus, which uses kilopascal (kPa) units [15]. Fig. 2 shows a schematic of how transient elastography acquires the data on the stiness of tissue [17]. In one of the initial studies of elastography with 106 patients, the mean receiver operating curves (ROC) for dierent METAVIR stages of brosis were 0.9 for stage F1 or greater, 0.88 for stage F2 or greater, 0.91 for stage F3 or greater, and 0.99 for stage F4 [15]. Intraobserver and interobserver variability were 3.3% and 3.3%, respectively, which is signicantly better than a liver biopsy [15]. A recent observation by Rigamonti and Fraquelli [18] describes such variability as less than 1% after a 1-month training period. The advantages of elastography include its relative low cost, noninvasive approach, rapid acquisition of information (within about 5 minutes), and ease of use [15]. The limitations seen in the rst study included one scan with an interobserver variability of 18%, which was then attributed to heterogenic distribution of brosis. Also, there was a measurement failure rate of 6%; this was likely attributable to patients who had ascites or who were obese, because the low frequency shear wave can be dampened by fat or ascites, and thus become not measurable [15]. Those with narrow intercostal spaces are also a challenge to assess with elastography [15]. A follow-up prospective study with 183 patients showed that elastography and combined elastography with a multiple serum marker test (FibroTest) had good ROC results [19]. The ROC area under the curve (AUC) for elastography showed 0.83, 0.90, 0.95 for METAVIR results on biopsy for stage F2 or greater, stage F3 or greater, and stage F4, respectively [19]. Combining elastography with the FibroTest showed an AUC of 0.88 for stage F2 or greater and 0.95 for stage F3 or F4 [19]. Diagnostic cuto values for staging were somewhat dierent than in the original study; these were 7.1 kPa, 9.5 kPa, and 12.5 kPa for stage F2 or greater, stage F3 or greater, and stage F4, respectively [19]. A third major follow-up trial by Ziol and colleagues [20] had 327 patients, of whom 251 had biopsy and elastography compared. This also showed good ROC results with an AUC of 0.81, 0.95, and 0.99 for stage F2 or greater, stage F3 or greater, and stage F4, respectively, when compared with biopsies that were at least 2.5 cm in length [20]. Cuto values for diagnosis were again similar in range but dierent than in other studies by Ziol using 8.8 kPa, 9.6 kPa, and 14.6 kPa for stage F2 or greater, stage F3 or greater, and stage F4, respectively [20].

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Fig. 2. Schematic of transient elastography (FibroScan). (Courtesy of Echosens SA, Paris, France; with permission.)

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Another trial with 711 patients showed good ROC results as well. The ROC AUC was 0.8, 0.9, and 0.96 for stage F2 or greater, stage F3 or greater, and stage F4, respectively [21]. The investigators used cuto values for various METAVIR stages based on a positive predictive value of at least 90%. Their cuto values were 7.2 kPa, 12.5 kPa, and 17.6 kPa for stage F2 or greater, stage F3 or greater, and stage F4, respectively [21]. Also of note, cuto values for complications of cirrhosis were calculated for a negative predictive value of greater than 90%. These were 27.5 kPa for esophageal varices, 37.5 kPa for Child-Pugh B or C score, 49.1 kPa for a past history of ascites, 53.7 kPa for hepatocellular carcinoma, and 62.7 kPa for esophageal bleeding [21]. Two trials from Japan also have validated the use of elastography. In one study of 237 patients who had chronic hepatitis C, elastography was found to be highly superior to biochemical markers in distinguishing stages of brosis [22]. Median stiness values were 4.1 kPa (range: 3.54.9 kPa) for 50 stage 0 patients, 6.3 kPa (range: 4.88.5 kPa) at stage 1, 8.8 (range: 6.812.0 kPa) at stage 2, 14.6 (range: 10.518.6 kPa) at stage 3, and 22.2 (range: 15.428.0) at stage 4 [22]. Patients who had attained a sustained virologic response had a stiness value of 3.8 kPa, 5.7 kPa, 6.8 kPa, and 6.1 kPa for stages 1, 2, 3, and 4, respectively [22]. The other Japanese study examined resections of liver in 30 patients and compared digital image analysis (DIA) of resections with elastography. The ROC AUC was 0.932 when DIA showed brotic areas of greater than10% in the liver, and it was 0.991 when DIA showed brotic areas of more than 20% of the liver [23]. One limitation of the study was that it did not compare DIA and METAVIR scores. Another analysis from Germany with 79 patients showed somewhat worse AUC values, but these became comparable when elastography was combined with the aspartate transaminaseto-platelet ratio (APRI) [24]. Instead of transient elastography, the investigators used real-time elastography, which is used in breast, thyroid, and prostate imaging and is a type of three-dimensional (3D) imaging [24]. Original AUC values were 0.75, 0.73, and 0.69, but combined AUC values were 0.93, 0.95, and 0.91 for METAVIR stage F2 or greater, stage F3 or greater, and stage F4, respectively [24]. Accuracy in the diagnosis of cirrhosis was also examined in a separate analysis of 775 patients [25]. The AUC was excellent at 0.95, with the highest sensitivity and specicity being at 9.4 kPa and 17.1 kPa, respectively, for the nding of cirrhosis [25]. A cuto value of 11.7 kPa had a sensitivity of 0.91 and specicity of 0.87 for nding cirrhosis [25]. Patients with an elastography value of less than 9.4 kPa had a greater than 95% chance that they did not have cirrhosis, with those having a value greater than 17.1 kPa having a 95% chance that they did indeed have cirrhosis [25]. Being male or having steatosis increased the risk for being among those having been misclassied (which occurred in 80 [10.3%] of 775 patients) [25].

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One trial quantied failure during elastographic measurement. There were 935 patients studied in this prospective trial, and on multivariate analysis of 888 patients, it was found that physicians with more experience, specically with more than 100 elastography examinations on patients, were more likely to be successful when measuring liver stiness [26]. Male patients who were obese were dicult to measure, having an odds ratio of 0.11 of a successful measurement, and obese women had an odds ratio of 0.16 of a successful measurement; older patients had a small decrease from baseline of 0.96, but it was statistically signicant [26]. AUC values were also calculated in the study and were 0.79, 0.89, and 0.91 for stage F2 or greater, stage F3 or greater, and stage F4, respectively [26].

Radiology Single photon emission computed tomography (SPECT) and in vivo phosphorus 31 (31P) magnetic resonance spectroscopy (MRS) have been proposed as valuable noninvasive methods of assessing severity of liver disease in patients who have chronic hepatitis C [27,28]. It was observed that the two SPECT parameters, minimum spleen pixel count and maximum right hepatic lobe pixel count, identied an accurate brosis score when compared with liver biopsy in 39 of 46 patients, yielding a positive and negative predictive value of 86% [28]. A study of 48 patients who had chronic hepatitis Crelated liver disease demonstrated that 31P MRS can dierentiate between patients who have mild, moderate, and cirrhotic liver disease [27]. It was observed on spectroscopy that a phosphomonoester (PME)/ phosphodiester (PDE) ratio of 0.2 or less yielded 76% sensitivity and 83% specicity of identifying mild hepatitis, a PME/PDE ratio of greater than 0.2 but less than 0.3 yielded 47% sensitivity and 87% specicity in identifying moderate hepatitis, and a PME/PDE ratio of 0.3 or greater yielded 82% sensitivity and 81% specicity in identifying liver cirrhosis [27]. It was proposed that 31P MRS might be useful in particular in evaluating the progression of liver disease in patients who have chronic hepatitis C [27]. The use of Doppler sonography in assessing the severity of liver disease in patients who have chronic hepatitis C remains controversial. A recent study of 65 patients who had liver disease related to hepatitis C demonstrated no signicant dierences in the Doppler indices with increasing severity of liver disease [29]. In light of these ndings, Doppler sonography cannot be recommended as a reliable noninvasive method evaluating the severity of liver disease related to chronic hepatitis C [29].

Direct serum markers of liver brosis It has been suggested that measurement of direct serum markers of brogenesis, such as procollagen type III N-terminal peptide (PIIINP) and direct

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serum marker of brolysis MMP-1 might be helpful in evaluating liver brosis in patients who have chronic hepatitis C (PIIINP/MMP-1 score) [30]. The brosis index (PIIINP/MMP score) was recently shown to be a useful marker in analyzing liver brosis in patients who have chronic hepatitis C during and after treatment with interferon-a and ribavirin because of its signicant correlation (r 0.68, P!.001) with the METAVIR brosis score [31]. A brosis index score less than 0.20 allowed for ruling out stage F3 to F4 and stage F2 to F4 brosis with respective negative predictive values of 96% and 88%, whereas a score greater than 0.50 yielded 89% and 100% positive predictive values for identication of stage F3 to F4 and stage F2 to F4 brosis, respectively [31]. An early and regular decrease in the brosis index was observed during treatment, with stable values in patients after treatment response, whereas patients who did not respond to treatment demonstrated no signicant changes in the brosis index [31]. It was proposed that the FIBROSpect II, a brosis panel including several ECM remodeling proteins, such as serum hyaluronic acid (HA), TIMP-1, and a2- macroglobulin may separate patients who have moderate to severe liver brosis from those who do not have liver brosis or have only mild liver brosis [32]. Among 696 patients who had chronic hepatitis C and moderate to severe liver brosis (METAVIR stage F2F4), a FIBROSpect II cuto point greater than 0.36 demonstrated a positive predictive value of 74.3%, negative predictive value of 75.8%, and accuracy of 75% [32]. Another study also suggested that this index might also be a good alternative to liver biopsy by excluding advanced liver brosis or cirrhosis (score O0.42) in patients who have chronic hepatitis C [33]. An analysis of 142 liver specimens from 136 patients who had chronic hepatitis C and a 38% prevalence for advanced liver brosis observed a positive predictive value of 63%, negative predictive value of 94%, and accuracy of 76% using a FIBROSpect cuto point greater than 0.42 [33]. It was suggested that patients with FIBROSpect II scores between 0.42 and 0.8 should be referred for liver biopsy, however, because of the decreased sensitivity and specicity in establishing the stage of liver brosis for this range of values [33]. These data were further supported by a recent prospective study that validated the diagnostic value of FIBROSpect II in identifying patients who have chronic hepatitis C who do not have liver brosis [34]. Conversely, this method has not been recommended to identify and dierentiate among intermediate stages of liver brosis [34]. Among 108 consecutive patients who had chronic hepatitis C with a 36.1% prevalence of advanced liver brosis, a FIBROSpect II cuto point of greater than 0.42 was characterized by a positive predictive value of 60.9%, negative predictive value of 82.3%, and accuracy of 73.1% [34]. It was also suggested that serum levels of insulin-like growth factor-I (IGF-I) could serve as noninvasive marker of liver brosis in patients who have chronic hepatitis C [35]. Patients who have chronic hepatitis C have abnormal synthesis of IGF-1, and its serum levels were consistent with the

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severity of liver brosis, demonstrating signicantly lower values in patients who had liver cirrhosis (METAVIR stage F4) when compared with other degrees of brosis (METAVIR stage F0F3) [35]. In addition, a serum brogenesis marker, YKL-40, was recently proposed as a noninvasive marker of identication of liver brosis in patients who have chronic hepatitis C [36]. YKL-40 was found to be superior to other brosis markers, such as HA or PIIINP, in separating severe brosis (stage F2F4) from mild brosis (stage F0F1) [36]. Among brosis markers, however, HA was the best predictor of dierentiating between liver cirrhosis (stage F4) and liver brosis (stage F0F3) in patients who have chronic hepatitis C [36]. After therapy with interferon, YKL-40 was the only marker that signicantly decreased in responders and nonresponders [36].

Biochemical markers of liver brosis Several noninvasive tests based on biochemical markers have been proposed as surrogates of invasive methods evaluating the severity of liver disease related to chronic hepatitis C. The analyzed tests included the FibroTest score (calculated with a patients age; gender; and levels of serum haptoglobin, a2-macroglobulin, apolipoprotein A1, g-glutamyl transpeptidase [GGTP], and bilirubin), APRI, Forns score (calculated based on platelet count, GGTP, cholesterol level, and age), and FIB-4 (combination of platelets, alanine aminotransferase [ALT], and aspartate aminotransferase [AST]) [3739]. The FibroTest score was found to yield a 100% negative predictive value for the absence of signicant liver brosis and cirrhosis and more than 90% positive predictive value for the presence of signicant liver brosis and cirrhosis [38]. It was observed that staging of liver brosis by the FibroTest had a 5-year prognostic value comparable with that done by liver biopsy in patients who have chronic hepatitis C [40]. Staging by the FibroTest was superior to liver biopsy in predicting complications and deaths related to chronic hepatitis C [40]. Moreover, it was suggested that the use of the FibroTest could reduce the number of liver biopsies by 32% in patients who have chronic hepatitis C and undergo hemodialysis and renal transplantation [41]. It was shown that the APRI can accurately identify the presence or absence of liver brosis or liver cirrhosis in 51% and 81% of treatment-naive patients who had chronic hepatitis C, respectively [39]. By determining appropriate cuto values for the APRI to achieve predictive values of 90%, Snyder and colleagues [42], were able to identify the severity of liver disease accurately in up to 59% of patients who had chronic hepatitis C when compared with their results of liver biopsies. Conversely, Lackner and colleagues [43] observed that the APRI identied signicant liver brosis only in 24% of patients who had chronic hepatitis C and excluded liver cirrhosis in 80% of patients.

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The Forns score yielded a 96% negative predictive value and 66% positive predictive value for identifying signicant liver brosis in patients who had chronic hepatitis C in one study [37]. The optimal proposed cuto points were less than 4.21 for absence of liver brosis and greater than 6.9 for presence of signicant liver brosis [37]. A recent study of 235 consecutive patients who had chronic hepatitis C observed that a combination of the FibroTest, APRI, and Forns score without liver biopsy identied 81.3% of patients who had liver brosis, whereas in the remaining 18.7% of patients, liver biopsy was required to identify liver brosis [44]. A prospective comparison of discordant results between the FibroTest and liver biopsy in 537 patients who had chronic hepatitis C demonstrated that failure of liver biopsy was more than sevenfold more common than failure of the FibroTest among patients with an identied reason for discordant results [45]. A recent comparison between FIB-4, liver biopsy, and the FibroTest showed that values less than 1.45 and more than 3.25 allow for accurate identication of patients who have moderate or severe liver brosis, respectively [46]. Results of FIB-4 less than 1.45 and greater than 3.25 agreed with those achieved by the FibroTest in 92.1% and 76% of patients, respectively, and with the results of liver biopsy in 72.8% [46]. Several other biochemical parameters have been proposed as indices detecting severity of liver disease in patients who have chronic hepatitis C. These include an AST/ALT ratio of 1 or greater, level of platelets less than 140,000 cells/mL, and globulin/albumin ratio greater than 1 [47]. The APRI and Forns score in 243 patients who had chronic hepatitis C and signicant liver brosis proved by liver biopsy showed at least a 90% negative predictive value in ruling out signicant liver brosis, whereas only a platelet count less than 140,000 cells/mL and a globulin/albumin ratio greater than 1 yielded an acceptably similar positive predictive value in identifying signicant liver brosis [47]. An analysis of these tests among 78 patients who had biopsy-conrmed hepatitis Crelated liver cirrhosis demonstrated that although all had excellent performance in excluding liver cirrhosis with a negative predictive value of at least 90%, all were characterized by a low 50% positive predictive value in identifying liver cirrhosis [47]. Recently, Metwally and colleagues [48] developed a noninvasive scoring system (range: 09) for detecting severe liver brosis in 173 patients who had chronic hepatitis C. This system incorporated the three most signicant independent predictors of severe liver brosis: platelet count, AST, and albumin [48]. That noninvasive scoring system was shown to yield a 95% negative predictive value for a cuto point of 2 and a 94% positive predictive value for a cuto point of 4 [48]. Another scoring system recently developed and internally validated by Alsatie and colleagues [49] included ve factors independently associated with advanced liver brosis in 286 patients who had chronic hepatitis C, such as diabetes mellitus, platelet count less than 150,000 cells/mL, AST of 65 IU/mL or greater, international normalized

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ratio (INR) of 1.1 or greater, and bilirubin of 0.85 mg/dL or greater. Based on the proposed scoring system, patients were subsequently divided into low-risk (score of 0), intermediate-risk (score of 13), and high-risk groups of advanced liver brosis (score R4) [49]. When the results of the scoring system were compared with the results of liver biopsies among analyzed patients, 9% of patients in the low-risk group, 34% of patients in the intermediate-risk group, and 92% of patients in the high-risk group had actual advanced liver brosis [49]. Noninvasive and simple indices were also suggested as good predictors of liver brosis in patients who underwent liver transplantation attributable to hepatitis Crelated liver cirrhosis [50,51]. Benlloch and colleagues [50] developed a brosis index based on four independent predictors of liver brosis, including prothrombin time, albumin/total protein ratio, AST, and time since liver transplantation. The proposed threshold of 0.2 for separating bridging brosis or cirrhosis from portal or no brosis yielded a positive predictive value of 49% and negative predictive value of 95% in validating this test, which included 96 patients [50]. These investigators proposed that their brosis index might be accurately used to identify patients who have posttransplant hepatitis C with a low risk for signicant liver brosis, thus allowing them to avoid liver biopsy [50]. A subsequent study by Schmilovitz-Weiss and colleagues [51] suggested that serum levels of globulin and immunoglobulin G (IgG) were the only predictors of extent of liver brosis, and thus could become noninvasive markers of liver brosis in patients who had recurrent hepatitis C after liver transplantation. Table 1 summarizes the predictive values for biochemical and serum markers. Summary Noninvasive approaches in the diagnosis and monitoring of brosis are still evolving. Although, historically, liver biopsy has remained the test of choice in determining the level of brosis, there remains a need for supplemental noninvasive options. Transient elastography is an inexpensive, rapid, and relatively accurate form of noninvasive monitoring, especially in severe brosis (METAVIR stage F3 or F4) [25,26]. It is a nascent technology, however, and there is no clear indication that elastography is better than biopsy for less severe brosis; most ROC values are between 0.80 and 0.90 with experienced pathologists and liver biopsy samples more than 2 cm in length [1921,26]. With improved resolution and longer term data, it may become a vital supplement. Radiologic tests, such as new forms of MRS, are intriguing but require further studies and technological development before implementation. Biochemical tests are quite good at ruling out advanced brosis but not for conrming brosis, especially intermediate forms [37,38,43]. Several studies have shown that combining serum markers with elastography improve results [19,24].

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Table 1 Noninvasive brosis and biochemical markers dierentiating between patients who have mild and severe brosis Positive predictive value 100% 74% 63% 61% 80% 79% 76% 67% O90% 76% Negative predictive value 96% 76% 94% 82% 79% 76% 77% 66% 100% 71%

Name of test Fibrosis index by Trocme FibroSpect II

Variables PIIINP, MMP-1 serum HA, TIMP-1, a2-macroglobulin d

Study Trocme et al [31] Patel et al [32] Christensen et al [33] Zaman et al [34] Saitou et al [36]

YKL-40 Hyaluronic acid PIIINP intravenous collagen FibroTest

APRI

Age, gender, haptoglobin, a2-macroglobulin, apolipoprotein A1, GGTP, bilirubin AST/PLT

Imbert-Bismut et al [38] Bourliere et al [44]

Forns score FIB-4 Fibrosis index by Benlloch (transplanted liver)

Age, PLT, GGTP, cholesterol Age, PLT, ALT, AST PT, albumin/total protein, AST, time since liver transplantation

Wai et al [39] (signicant brosis) (cirrhosis) Snyder et al [42] (retrospective) (prospective) Bourliere et al [44] Forns et al [37] Bourliere et al [44] Vallet-Pichard et al [46] Benlloch et al [50]

91% 65% 90% 92% 79% 66% 83% 82% 49%

90% 100% 88% 95% 90% 96% 79% 95% 95%

Direct markers of brosis are somewhat less useful. The exception, HA, has been shown to be a sensitive test at ruling out cirrhosis but is otherwise nonspecic [36]. The combined use of transient elastography and biochemical markers seems to be the most promising noninvasive technique; technological improvements and increased data may help to inform the clinician whether a liver biopsy is necessary in some patients, and this can facilitate guidance of treatment.

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[2] de Franchis R, DellEra A. Non-invasive diagnosis of cirrhosis and the natural history of its complications. Best Pract Res Clin Gastroenterol 2007;21:3. [3] Bravo AA, Sheth SG, Chopra S. Liver biopsy. N Engl J Med 2001;344:495. [4] Carrion JA, Navasa M, Bosch J, et al. Transient elastography for diagnosis of advanced brosis and portal hypertension in patients with hepatitis C recurrence after liver transplantation. Liver Transpl 2006;12:1791. [5] Afdhal NH. Diagnosing brosis in hepatitis C: is the pendulum swinging from biopsy to blood tests? Hepatology 2003;37:972. [6] Regev A, Berho M, Jeers LJ, et al. Sampling error and intraobserver variation in liver biopsy in patients with chronic HCV infection. Am J Gastroenterol 2002;97:2614. [7] Colloredo G, Guido M, Sonzogni A, et al. Impact of liver biopsy size on histological evaluation of chronic viral hepatitis: the smaller the sample, the milder the disease. J Hepatol 2003; 39:239. [8] Lefkowitch JH. Liver biopsy assessment in chronic hepatitis. Arch Med Res 2007;38:634. [9] Rousselet MC, Michalak S, Dupre F, et al. Sources of variability in histological scoring of chronic viral hepatitis. Hepatology 2005;41:257. [10] Albanis E, Friedman SL. Diagnosis of hepatic brosis in patients with chronic hepatitis C. Clin Liver Dis 2006;10:821. [11] Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic brogenesis. J Gastroenterol Hepatol 2007;22(Suppl 1):S79. [12] Rockey DC, Bissell DM. Noninvasive measures of liver brosis. Hepatology 2006;43:S113. [13] Friedman SL, Rockey DC, Bissell DM. Hepatic brosis 2006: report of the Third AASLD Single Topic Conference. Hepatology 2007;45:242. [14] Erler JT, Bennewith KL, Nicolau M, et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 2006;440:1222. [15] Sandrin L, Fourquet B, Hasquenoph JM, et al. Transient elastography: a new noninvasive method for assessment of hepatic brosis. Ultrasound Med Biol 2003;29:1705. [16] Nguyen-Khac E, Capron D. Noninvasive diagnosis of liver brosis by ultrasonic transient elastography (Fibroscan). Eur J Gastroenterol Hepatol 2006;18:1321. [17] Echosens SA. Available at: http://www.echosens.com/en/ex1.html. Accessed 2007. [18] Rigamonti C, Fraquelli M. Do not trivialize the Fibroscan examination, value its accuracy. J Hepatol 2007;46:1149. [19] Castera L, Vergniol J, Foucher J, et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of brosis in chronic hepatitis C. Gastroenterology 2005;128:343. [20] Ziol M, Handra-Luca A, Kettaneh A, et al. Noninvasive assessment of liver brosis by measurement of stiness in patients with chronic hepatitis C. Hepatology 2005;41:48. [21] Foucher J, Chanteloup E, Vergniol J, et al. Diagnosis of cirrhosis by transient elastography (FibroScan): a prospective study. Gut 2006;55:403. [22] Takeda T, Yasuda T, Nakayama Y, et al. Usefulness of noninvasive transient elastography for assessment of liver brosis stage in chronic hepatitis C. World J Gastroenterol 2006;12: 7768. [23] Kawamoto M, Mizuguchi T, Katsuramaki T, et al. Assessment of liver brosis by a noninvasive method of transient elastography and biochemical markers. World J Gastroenterol 2006;12:4325. [24] Friedrich-Rust M, Ong MF, Herrmann E, et al. Real-time elastography for noninvasive assessment of liver brosis in chronic viral hepatitis. AJR Am J Roentgenol 2007;188:758. [25] Ganne-Carrie N, Ziol M, de Ledinghen V, et al. Accuracy of liver stiness measurement for the diagnosis of cirrhosis in patients with chronic liver diseases. Hepatology 2006;44: 1511. [26] Kettaneh A, Marcellin P, Douvin C, et al. Features associated with success rate and performance of FibroScan measurements for the diagnosis of cirrhosis in HCV patients: a prospective study of 935 patients. J Hepatol 2007;46:628.

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Clin Liver Dis 12 (2008) 573585

Hepatitis C Infection and Nonalcoholic Fatty Liver Disease


Onpan Cheung, MDa, Arun J. Sanyal, MBBS, MDb,*
Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, Virginia Commonwealth University Medical Center, 1101 East Marshall Street, Sanger Hall B3-051, Richmond, VA 23298, USA b Division of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine, Virginia Commonwealth University Medical Center, MCV Box 980341, Richmond, VA 232980341, USA
a

Hepatitis C virus (HCV) is a major cause of chronic liver disease, aecting 170 million (3%) of the worlds population and approximately 2.7 million Americans; cirrhosis can occur in 20% of these patients [13]. There are six HCV genotypes; of these, type 1 is the most common in the United States and is also the most dicult genotype for which to achieve a sustained virologic response (SVR) to standard antiviral therapy. Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in North America and aects up to 30% of the general population [4]. Its clinical-histologic spectrum ranges from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis in 15% to 20% of subjects [5]. NASH is distinguished from NAFL by the presence of inammation and cytologic ballooning with or without Mallory hyaline or pericellular brosis in addition to steatosis [6]. NAFL is estimated to aect up to two thirds of obese adults and nearly 50% of obese children. Approximately 30% to 50% of adult Americans who are obese may have NASH [7,8]. Although NAFLD can occur in all ages and ethnic groups, those of Hispanic origin have the highest risk [4]. African Americans, conversely, have a low risk for developing NAFLD [9]. In addition to obesity, NAFLD is strongly linked to insulin resistance and is considered the hepatic manifestation of the metabolic syndrome [10,11]. Given that hepatitis C and NAFLD occur commonly in the general population, it is not surprising that these conditions often coexist in the same patient. Also, emerging data indicate that HCV itself may promote the
* Corresponding author. E-mail address: ajsanyal@hsc.vcu.edu (A.J. Sanyal). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.005 liver.theclinics.com

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development of hepatic steatosis. In this article, the authors review the mechanisms by which hepatic steatosis and steatohepatitis develop in subjects who have hepatitis C and the implications of concurrent hepatitis C and NAFLD for liver disease and antiviral therapy.

Mechanisms of hepatic steatosis Overview Hepatic steatosis develops when fat inux and synthesis exceed fat catabolism and export [12]. The predominant lipid that accumulates in hepatic steatosis is triglyceride [13,14], and this is derived by esterication of a glycerol moiety with free fatty acids (FFAs). FFA inux is derived from dietary fat intake or from peripheral lipolysis. FFA synthesis in the liver is also referred to as de novo lipogenesis (DNL) and is mediated by a series of enzymes whose activity is rate-limited by the action of acetyl coenzyme A (CoA) carboxylase on the conversion of acetyl-CoA to malonyl-CoA. Several steps in FFA synthesis are regulated by transcription factors, such as sterol regulatory element binding protein (SREBP). b-oxidation of FFAs for energy generation occurs in the mitochondria and is regulated by adenosine monophosphateactivated kinase (AMPK) and peroxisome proliferatoractivated receptor (PPAR-a). FFA can also be hydrolyzed by lipases and exported from the liver while bound to apolipoprotein (apo) B as very-low-density lipoprotein (VLDL). Factors that are known to be associated with increased circulating FFA are obesity and insulin resistance [15]. FFAs further interfere with insulin signaling in striated muscle and adipocytes by reducing their ability to take up glucose [16]. This excess glucose is potentially available for conversion to FFA in the liver by means of acetyl CoA, a downstream product of glucose metabolism. Simultaneously, there is an increase in DNL secondary to retained sensitivity to insulin for this pathway. Under conditions of FFA loading, there is a preferential use of FFA instead of glucose for mitochondrial ATP generation. Thus, intrahepatic insulin resistance attributable to FFA promotes mitochondrial fatty acid b-oxidation. There are, however, conicting reports of the status of mitochondrial fatty acid oxidation in NASH. The circulating beta-hydroxybutyrate (b-OH butyrate) levels, a product of mitochondrial fatty acid oxidation, are increased in NASH. At the same time, there are reports of decreased mitochondrial fatty acid oxidation based on exhalation of deuterated hydrogen after ingestion of labeled fatty acids. Hepatic steatosis, in turn, can exacerbate total body insulin resistance. Excess FFAs from circulation, increased DNL, and impaired fatty acid boxidation induce a vicious cycle of hepatic insulin resistance by means of failure of phosphorylation of insulin receptor that ultimately leads to failure of phosphorylation of insulin receptor substrate (IRS)-2. As a result,

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activation of glycogen synthase (GSK3) and hepatocyte nuclear factor (FOXO) is downregulated. Failure to phosphorylate FOXO results in its nuclear translocation, upregulation of phosphoenolpyruvate carboxykinase (PEPCK), and export of glucose by means of glucose transporter (GLUT) 2r [17]. PEPCK is the rate-limiting enzyme for gluconeogenesis, and its increased activity results in increased hepatic glucose output and worsening of hyperglycemia. Despite the strong link between insulin resistance and NAFLD, there is some increasing evidence that NASH may occur in the absence of overt insulin resistance [1820]. Although poorly understood, a potential mechanism may be related to an underlying intrinsic alteration in the regulation of fatty acid metabolism. Studies have suggested that hepatic steatosis in NAFLD can begin as overstorage of unmetabolized energy in hepatocytes in those who consume excess energy that exceeds the energy combustion capability of the PPAR-amediated b-oxidation [18,20,21]. Mechanisms of steatosis in hepatitis C virus genotype 3 Hepatic steatosis is a common nonspecic histologic feature found in greater than 50% of individuals infected with HCV [22]. Genotype 3a, in particular, has been shown to link to steatosis more strongly than with other genotypes [23]. Obesity is also a well-recognized risk factor for the development of steatosis and brosis in HCV-infected patients [24,25]. Genotype 3 is the only subtype that has been shown to correlate with a higher grade of steatosis independent of other host-related factors, such as the presence of NAFLD [26]. The severity of steatosis in these patients is directly related to the burden of the HCV RNA viral load, and resolution of steatosis is often observed with the loss of viremia after antiviral treatment [2729]. It has been postulated that HCV genotype 3 can cause steatosis also by interfering with triglyceride secretion. The exact mechanism of HCV-induced steatosis is not completely understood, although research studies have been slowly unraveling several potential contributors. In particular, HCV core protein has been studied in vitro and in transgenic mice. It seems that intracellular lipid accumulation occurs when the HCV core protein is strongly expressed [30] and that the core protein can be found on the surface of lipid droplets within the cytoplasm in cell cultures transfected with HCV, although it is absent in control cells [31]. In a recent report, HCV core protein and NS4b proteins derived from genotype 3a were found to be capable of inducing proteolytic cleavage of SREBP, a transcription factor that controls the enzymes responsible for fatty acid synthesis [32]. Mechanisms of steatosis in hepatitis C virus genotype 1 It is believed that steatosis in genotype 1 infection is attributable to metabolic perturbations caused by activation of proinammatory mechanisms and underlying obesity and insulin resistance. The degree of steatosis in

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this genotype is independent of HCV viral load, and the use of antiviral therapy alone does not lead to improvement in steatosis in these patients. Similar data have been obtained for genotype 4 infection, whereas few data are available regarding genotype 2 infection [33]. HCV virus, regardless of genotype, has been shown to cause insulin resistance through direct eects on viral proinammatory cytokines and suppressors of cytokine signaling [3437]. Using HCV core gene transgenic mice, a recent study has demonstrated the presence of impaired glucose tolerance and marked insulin resistance in those animals [38]. Furthermore, the same transgenic mice showed an increased level of tumor necrosis factor-a (TNFa) that has been previously shown, in vivo and in vitro, to induce insulin resistance by means of inhibition of tyrosine phosphorylation of IRS-1 and IRS-2 [39,40]. Additional studies, as shown in Table 1, have identied factors associated with hepatic steatosis in HCV. HCV entry into hepatocytes may be mediated by low-density lipoprotein (LDL) receptor, and that HCV core protein may interact with apoA2, which is a major component of high-density lipoproteins, and this interaction can lead to hepatocellular steatosis by inhibiting microsomal triglyceride transfer protein activity [41]. Another nonstructural protein, NS5A, has been found to interact with apoA1 and apoA2, and therefore can lead to altered cholesterol tracking [42,43]. Impact of concomitant presence of nonalcoholic fatty liver disease and hepatitis C virus on the liver Clinical and histologic features of hepatitis C virus and nonalcoholic fatty liver disease The concomitant presence of HCV and NAFLD is inferred from the presence of greater than 5% steatosis in the liver in subjects who do not consume clinically relevant (O20 g/d for women and O30 g/d for men) amounts of alcohol. The diagnosis of steatohepatitis is somewhat more challenging. Lobular inammation can be seen in NASH and HCV, and its
Table 1 Summary of studies regarding hepatic steatosis in hepatitis C virus Author, year Hourigan et al [75] Rubbia-Brandt et al [23] Adinol et al [45] Hickman et al [76] Sanyal et al [77] Younossi et al [49] Castera et al [27] Patton et al [29] Liu et al [78] Patients (n) 148 101 180 160 144 120 151 574 95 Characteristics associated with steatosis (P) BMI (0.0001), age (0.002) Genotype 3 (0.002) Genotype 3 (!0.01), BMI in genotype 1 (!0.001), visceral fat distribution (!0.001) Genotype 3 (0.0001) BMI (!0.001), weight (!0.003) BMI (0.03), genotype 3 (0.03) BMI O25 kg/m2 (0.02), genotype 3 (0.07) Genotype 3 (!0.03) Hyperglycemia (0.01), BMI O27 kg/m2 (!0.01)

Abbreviation: BMI, body mass index.

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presence cannot therefore be taken as a feature of NASH in such patients. The presence of cytologic ballooning along with steatosis serves as the minimal requirement to make the diagnosis of steatohepatitis in subjects who have HCV. Although Mallorys hyaline strengthens the case for steatohepatitis, it is not always present and its absence does not exclude steatohepatitis. Similarly, pericellular brosis is not always present in steatohepatitis, and its absence does not exclude the diagnosis. It is always important to remember that the nonalcoholic nature of the disease has to be assessed clinically. As shown in Table 2, steatosis secondary to metabolic reasons (genotype 1, NAFLD) or HCV virus (genotype 3) worsens the sequence of events leading to advanced brosis in patients who have HCV. Progression of HCV toward cirrhosis is also accelerated by several factors, such as male gender, age, alcohol use, HIV coinfection, HCV duration and genotype, the presence of diabetes mellitus, insulin resistance, and obesity [4447]. In patients infected with HCV, NASH has been reported to be more commonly seen in those who are obese, especially with central obesity, and in those with advanced age, hyperglycemia, hypertriglyceridemia, elevated transaminases, and HCV genotype 3 [48]. These patients also tend to have more advanced stages of brosis compared with those with superimposed steatosis alone or no steatosis at all [49]. Mechanisms As a pathophysiologic hallmark of NAFLD, insulin resistance is strongly linked to oxidative stress and inammatory cytokines [50,51]. As previously mentioned, patients who have NAFLD have high circulating levels of lipid peroxidation products that are proinammatory and probrotic. Interestingly, the same observation has also been reported in patients who have
Table 2 Factors associated with advanced brosis in hepatitis C virus Author, year Mihm et al [79] Hourigan et al [44] Adinol et al [45] Clouston et al [80] Hui et al [34] Poynard et al [81] Ratziu et al [82] Sanyal et al [77] Younossi et al [49] Rubbia-Brandt et al [26] Bugianesi et al [83] Patients (n) 85 148 180 80 260 1428 710 144 120 755 132 Characteristics associated with brosis (P) Hepatic steatosis (n/a) Hepatic steatosis (!0.03) Hepatic steatosis (!0.001), age (!0.001) Hepatic steatosis (0.001), age (0.003) HOMA-IR (!0.001) Hepatic steatosis (0.007) Hyperglycemia (!0.01), BMI (!0.01), steatosis (!0.01) BMI (!0.003), cytologic ballooning (!0.003), diabetes (!0.03) Superimposed NASH (!0.001) Hepatic steatosis (!0.001 in genotype 3) HOMA-IR (0.02 in genotype 3)

Abbreviations: BMI, body mass index; HOMA-IR, Insulin resistance was calculated using the homeostasis model assessment (HOMA) method as per the following equation: insulin resistance (HOMA-IR) fasting insulin (mU/mL) fasting glucose (mmol/L)/22.5; n/a, data not available.

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chronic HCV [5254]. In general, steatosis leads to increased lipid peroxidation in hepatocytes, which, in turn, activates hepatic stellate cells (HSCs) to produce extracellular matrix components and collagen deposition [5557]. The potential use of antioxidant therapy to halt hepatic brogenesis, reduce lipid peroxidation, and attenuate HSC activation has been investigated. Early studies have shown that the use of vitamin E in patients who have chronic HCV results in a greater chance of obtaining a complete virologic response to interferon (IFN) therapy and a signicantly greater reduction in viral load than in patients without vitamin E treatment [58]. There is also evidence that the use of vitamin E in chronic HCV signicantly reduces ultrasonographic scores for liver steatosis and improves liver enzyme levels, hyperinsulinemia, and liver brosis [59]. Although the exact mechanism of how antioxidant improves these parameters is poorly understood, further large-scale studies are needed to investigate the impact of oxidative stress on disease progression in chronic HCV. Impact of nonalcoholic fatty liver disease on response to antiviral therapy Clinical response to antiviral therapy in subjects who have concurrent hepatic steatosis Generally, the presence of hepatic steatosis reduces the likelihood of achieving an SVR to pegylated IFN and ribavirin combination therapy, especially if steatosis is greater than 33% [60]. Some studies have implicated insulin resistance and cytologic ballooning as additional markers for failure of antiviral therapy. Although steatosis is an important cofactor in the progression of brosis and necroinammation in HCV, the exact mechanism whereby steatosis may have a negative impact on the response to antiviral therapy remains poorly understood. The current concepts related to this are reviewed here. Mechanisms Hepatic steatosis is associated with obesity and insulin resistance. Obesity, insulin resistance, and HCV genotype have also been reported to aect antiviral response (Table 3). In addition to causing steatosis and brosis progression in HCV, insulin resistance has been shown to correlate with a poor response to antiviral therapy, particularly in HCV genotype 1 [61]. Obesity, per se, has been previously reported to be a risk factor for nonresponse to antiviral therapy independent of HCV genotype and the presence of cirrhosis. Obese patients have approximately an 80% lower chance of achieving an SVR to antiviral therapy compared with nonobese patients [62]. Although the actual mechanism whereby obesity aects the response to antiviral therapy is not completely understood, it is speculated that steatosis in obese patients who have HCV has increased accumulation of lipid droplets within hepatocytes, which can act as a functional barrier for the interaction between

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Table 3 Factors associated with poor response to antiviral therapy in hepatitis C virus Author, year Akuta et al [84] Bressler et al [62] Poynard et al [81] Sanyal et al [77] Patton et al [29] Harrison et al [60] Patients (n) 394 174 1428 144 574 231 Characteristics associated with nonresponse (P) Hepatic steatosis, genotype 1 (n/a) BMI O30 kg/m2 (0.01), cirrhosis (!0.01), genotype 1 (!0.01) BMI, hepatic steatosis (!0.001) Presence of NAFLD (p!0.01) Genotype 1 (0.02) Steatosis O33% (0.001)

Abbreviations: BMI, body mass index; n/a, data not available.

antiviral drugs and the hepatocytes containing the virus [63]. Additionally, depending on the size and structural property of some pegylated IFN, the drug may be preferentially absorbed through blood capillaries or the lymphatic circulation. Because obese people are known to have poor lymphatic circulation [64], this can potentially lead to suboptimal serum levels of pegylated IFN, thus reducing the likelihood of a successful antiviral response. Another potential mechanism whereby obesity may aect antiviral response is through modulation of the interferon signaling pathway. Normally, IFNa-activated cellular signaling is negatively regulated by inhibitory factors, such as the suppressor of cytokine signaling (SOCS) family (Fig. 1).

Fig. 1. Signaling pathway of interferon (IFN) resistance in HCV. Normally, when IFN binds to its receptor and leads to a conformational change, the associated janus kinase (JAK) is phosphorylated and becomes activated. Signal transducer and activator of transcription (STAT) then binds to the activated receptor complex at its docking site through the src homology 2 domains and is phosphorylated by JAK kinases as ATP is converted to adenosine diphosphate (ADP). The STAT molecule binds to another phosphorylated STAT to form a dimer, which translocates to the nucleus, binds to the response elements (RE), and induces the expression of IFN-stimulated genes (ISG) to produce an antiviral state. Suppressor of cytokine signaling (SOCS) inhibits the phosphorylation of STAT, thus impairing IFN sensitivity in treatment ecacy.

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As reported in a recent study, obese subjects who have HCV genotype 1 were found to have increased mRNA expression of SOCS-3 compared with controls [65]. This potential association between obesity and altered cytokinemediated regulation of interferon signaling may contribute to poor antiviral response in patients who have HCV with superimposed NAFLD or NASH. Future direction in managing patients who have hepatitis C virus with superimposed nonalcoholic fatty liver disease Statins and hepatitis C virus viral replication Dyslipidemia is common in NAFLD and HCV genotype 1. Cholesterol is an integral part of HCV replication, and it is believed that HCV virus enters hepatocytes by means of LDL receptors [66]. Hydroxy-3-methylglutarylCoenzyme A reductase (HMG-CoA reductase) is associated with upregulation of LDL receptors [67], and its inhibitor (statins) has been shown to be capable of inhibiting HCV viral replication in vitro [6870]. The same nding, however, has not been observed in vivo at a similar conventional dose of statins [71]. Ikeda and colleagues [68] have recently demonstrated in vitro that uvastatin, when compared with other statins, exhibits the strongest anti-HCV activity, with a much lower dose required to achieve a 50% reduction in RL activity (median inhibitory concentration [IC50]). The group also reported that the use of a statin with IFNa seems to enhance the antiviral eect of IFN well beyond the activity observed when the drug is used alone. The anti-HCV activity of statins results from the inhibition of geranylgeranylation of HCV cellular proteins rather than from direct inhibition of cholesterol biosynthesis. Geranylgeranyl is a cholesterol intermediate that has been shown in vitro to be crucial in HCV replication [69,70]. Statins may seem to be a potential therapeutic option for HCV infection; however, because of the lack of in vivo data supporting their antiviral ecacy and the fact that higher total cholesterol and LDL levels have been observed and reported to be associated with increased SVR rates after IFN-based therapy, it is suggested that lowering LDL levels may not necessarily be benecial for antiviral therapy [72,73]. Further study is therefore required to dene the interaction between statins and IFN response. Increase the ecacy of antiviral therapy for hepatitis C virusinfected patients who have concomitant nonalcoholic steatohepatitis Subjects infected with HCV genotypes 2 and 3 have a higher probability of achieving an SVR to combination antiviral therapy, regardless of the presence of hepatic steatosis, compared with those infected with genotype 1. In such subjects, it may not be necessary to treat the underlying NAFLD or NASH before anti-HCV therapy. Conversely, subjects who have genotype 1 infection are at greater risk for becoming virologic nonresponders. There is therefore a need to modify HCV treatment to increase the response rate.

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Although the presence of concomitant NAFLD increases the risk for antiHCV treatment failure, there are no data on the ecacy of treating NAFLD as a way to improve virologic response. Despite this lack of evidence, there is still considerable enthusiasm to treat NAFLD as a modiable risk factor to improve the response to antiviral therapy in subjects who have HCV. Although optimal therapy for NAFLD remains to be dened, the focus of treatment on the underlying risks (ie, obesity, insulin resistance) is a reasonable therapeutic goal on the basis of achieving improvement in cardiovascular risks. It is therefore reasonable to treat obesity and the metabolic syndrome when they are present. In the absence of any data on treating NASH to improve virologic response, antiviral therapy may not need to be withheld, but in addition, one should consider instituting a diet and lifestyle plan shortly before starting treatment. For those in whom a virologic response is most urgently required, such as those who have severe inammation or bridging brosis, it may be necessary to treat obesity aggressively with the goal of improving insulin resistance and steatosis. There are emerging data suggesting that thiazolidinediones (eg, pioglitazone) may be eective in improving steatohepatitis [74]. Finally, the use of inhibitors of cholesterol and lipid biosynthesis as adjuncts to HCV therapy, especially in patients who do not have dyslipidemia, merits further evaluation.

Summary Hepatitis C is a common chronic liver disease in the world. Disease progression and virologic response to antiviral therapy are greatly aected by several crucial host and viral factors, with two such important contributors being the presence of NAFLD and insulin resistance. Obesity and insulin resistance are signicant public health concerns in North America, and it has become increasingly evident that NAFLD is a frequently encountered challenge in managing patients who have HCV in terms of clinical and histologic manifestations in addition to treatment ecacy. The presence of concomitant HCV and NAFLD is associated with greater brosis and a low rate of SVR to antiviral therapy.

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[52] Konishi M, Iwasa M, Araki J, et al. Increased lipid peroxidation in patients with non-alcoholic fatty liver disease and chronic hepatitis C as measured by the plasma level of 8-isoprostane. J Gastroenterol Hepatol 2006;21:18215. [53] Par A, Roth E, Rumi G Jr, et al [Oxidative stress and antioxidant defense in alcoholic liver disease and chronic hepatitis C]. Orv Hetil 2000;141:16559. [54] Madan K, Bhardwaj P, Thareja S, et al. Oxidant stress and antioxidant status among patients with nonalcoholic fatty liver disease (NAFLD). J Clin Gastroenterol 2006;40:9305. [55] Britton RS, Bacon BR. Role of free radicals in liver diseases and hepatic brosis. Hepatogastroenterology 1994;41:3438. [56] Bedossa P, Houglum K, Trautwein C, et al. Stimulation of collagen alpha 1(I) gene expression is associated with lipid peroxidation in hepatocellular injury: a link to tissue brosis? Hepatology 1994;19:126271. [57] Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991;11:81128. [58] Look MP, Gerard A, Rao GS, et al. Interferon/antioxidant combination therapy for chronic hepatitis Ca controlled pilot trial. Antiviral Res 1999;43:11322. [59] Loguercio C, Federico A, Trappoliere M, et al. The Eect of a Silybin-Vitamin E-Phospholipid Complex on Nonalcoholic Fatty Liver Disease: A Pilot Study. Dig Dis Sci 2007;52(9): 238795. [60] Harrison SA, Brunt EM, Qazi RA, et al. Eect of signicant histologic steatosis or steatohepatitis on response to antiviral therapy in patients with chronic hepatitis C. Clin Gastroenterol Hepatol 2005;3:6049. [61] Romero-Gomez M, Del Mar Viloria M, Andrade RJ, et al. Insulin resistance impairs sustained response rate to peginterferon plus ribavirin in chronic hepatitis C patients. Gastroenterology 2005;128:63641. [62] Bressler BL, Guindi M, Tomlinson G, et al. High body mass index is an independent risk factor for nonresponse to antiviral treatment in chronic hepatitis C. Hepatology 2003;38: 63944. [63] Giannini E, Ceppa P, Testa R. Steatosis in chronic hepatitis C: can weight reduction improve therapeutic ecacy? J Hepatol 2001;35:4323. [64] Banerjee D, Williams EV, Ilott J, et al. Obesity predisposes to increased drainage following axillary node clearance: a prospective audit. Ann R Coll Surg Engl 2001;83:26871. [65] Walsh MJ, Jonsson JR, Richardson MM, et al. Non-response to antiviral therapy is associated with obesity and increased hepatic expression of suppressor of cytokine signalling 3 (SOCS-3) in patients with chronic hepatitis C, viral genotype 1. Gut 2006;55:52935. [66] Monazahian M, Bohme I, Bonk S, et al. Low density lipoprotein receptor as a candidate receptor for hepatitis C virus. J Med Virol 1999;57:2239. [67] Brown MS, Goldstein JL. Lowering plasma cholesterol by raising LDL receptors. N Engl J Med 1981;305:5157. [68] Ikeda M, Abe K, Yamada M, et al. Dierent anti-HCV proles of statins and their potential for combination therapy with interferon. Hepatology 2006;44:11725. [69] Ye J, Wang C, Sumpter R Jr, et al. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci U S A 2003;100: 1586570. [70] Kapadia SB, Chisari FV. Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids. Proc Natl Acad Sci U S A 2005;102:25616. [71] OLeary JG, Chan JL, McMahon CM, et al. Atorvastatin does not exhibit antiviral activity against HCV at conventional doses: a pilot clinical trial. Hepatology 2007;45:8958. [72] Gopal K, Johnson TC, Gopal S, et al. Correlation between beta-lipoprotein levels and outcome of hepatitis C treatment. Hepatology 2006;44:33540. [73] Hamamoto S, Uchida Y, Wada T, et al. Changes in serum lipid concentrations in patients with chronic hepatitis C virus positive hepatitis responsive or non-responsive to interferon therapy. J Gastroenterol Hepatol 2005;20:2048.

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[74] Belfort R, Harrison SA, Brown K, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006;355:2297307. [75] Hourigan LF, Macdonald GA, Purdie D, et al. Fibrosis in chronic hepatitis C correlates signicantly with body mass index and steatosis. Hepatology 1999;29:12159. [76] Hickman IJ, Powell EE, Prins JB, et al. In overweight patients with chronic hepatitis C, circulating insulin is associated with hepatic brosis: implications for therapy. J Hepatol 2003; 39:10428. [77] Sanyal AJ, Contos MJ, Sterling RK, et al. Nonalcoholic fatty liver disease in patients with hepatitis C is associated with features of the metabolic syndrome. Am J Gastroenterol 2003; 98:206471. [78] Liu CJ, Jeng YM, Chen PL, et al. Inuence of metabolic syndrome, viral genotype and antiviral therapy on superimposed fatty liver disease in chronic hepatitis C. Antivir Ther 2005; 10:40415. [79] Mihm S, Fayyazi A, Hartmann H, et al. Analysis of histopathological manifestations of chronic hepatitis C virus infection with respect to virus genotype. Hepatology 1997;25:7359. [80] Clouston AD, Jonsson JR, Purdie DM, et al. Steatosis and chronic hepatitis C: analysis of brosis and stellate cell activation. J Hepatol 2001;34:31420. [81] Poynard T, Ratziu V, McHutchison J, et al. Eect of treatment with peginterferon or interferon alfa-2b and ribavirin on steatosis in patients infected with hepatitis C. Hepatology 2003;38:7585. [82] Ratziu V, Munteanu M, Charlotte F, et al. Fibrogenic impact of high serum glucose in chronic hepatitis C. J Hepatol 2003;39:104955. [83] Bugianesi E, Marchesini G, Gentilcore E, et al. Fibrosis in genotype 3 chronic hepatitis C and nonalcoholic fatty liver disease: Role of insulin resistance and hepatic steatosis. Hepatology 2006;44:164855. [84] Akuta N, Suzuki F, Tsubota A, et al. Ecacy of interferon monotherapy to 394 consecutive naive cases infected with hepatitis C virus genotype 2a in Japan: therapy ecacy as consequence of tripartite interaction of viral, host and interferon treatment-related factors. J Hepatol 2002;37:8316.

Clin Liver Dis 12 (2008) 587609

Management Complexities of HIV/Hepatitis C Virus Coinfection in the Twenty-First Century


Vincent Lo Re III, MD, MSCEa,b,*, Jay R. Kostman, MDa, Valerianna K. Amorosa, MDa,c
a

Division of Infectious Diseases, Department of Medicine, 502 Robert Wood Johnson Pavilion, University of Pennsylvania School of Medicine, Philadelphia, PA 191046073, USA b Department of Biostatistics and Epidemiology and Center for Clinical Epidemiology and Biostatistics, 423 Guardian Drive, Philadelphia, PA 19104-6021, USA c Infectious Diseases Section, Room 809A, Philadelphia Veterans Aairs Medical Center, 3900 Woodland Avenue, Philadelphia, PA 19104, USA

Because of shared risk factors, approximately one third of patients who have HIV type 1 (HIV) are coinfected with chronic hepatitis C virus (HCV) infection [1,2]. HIV coinfection accelerates the course of HCV-associated liver disease, and compared with those infected with chronic HCV alone (ie, HCV monoinfected), there is more rapid progression of hepatic brosis in HIV/HCV-coinfected individuals [35]. Thus, because HIV has become a chronic illness as a result of the eectiveness of highly active antiretroviral therapy (HAART), HCV-related liver disease has emerged as a major cause of morbidity and mortality among HIV-infected patients in the developed world [68]. Because HIV/HCV coinfection is prevalent and increases the risk of HCV-associated liver disease, eective anti-HCV therapy is critical for the long-term survival of these patients. Among HIV-infected individuals, HCV therapy can lead to viral eradication [912]; may halt or regress hepatic brosis [13,14]; and has been shown to be cost-eective [15]. A variety of complexities, including overall reluctance by patients and providers to initiate HCV therapy, increased hepatotoxicity of antiretroviral therapy,
This article was supported by National Institutes of Health research grant K01 AI07000101A1 from the National Institute for Allergy and Infectious Diseases (to V. Lo Re). * Corresponding author. Division of Infectious Diseases, Department of Medicine, 502 Robert Wood Johnson Pavilion, University of Pennsylvania School of Medicine, Philadelphia, PA 191046073. E-mail address: vincent.lore@uphs.upenn.edu (V. Lo Re). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.009 liver.theclinics.com

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drug-drug interactions, and adverse eects of HCV therapy, have made management of chronic HCV infection a major challenge in the HIV-infected population, however. In this article, the authors review the (1) epidemiology of HCV among HIV-infected individuals, (2) eect of HIV on the natural history of chronic HCV, (3) impact of antiretroviral therapy on HCV coinfection, and (4) management of chronic HCV in the HIV-infected person. Epidemiology of hepatitis C virus infection in HIV HIV and HCV are transmitted eciently by percutaneous exposure to contaminated blood, through sexual intercourse, and from mother to infant. Because both viruses have similar routes of transmission, coinfection with HCV is common among HIV-infected individuals. In the United States, a cross-sectional analysis of two large HIV trials (n 1687 subjects) demonstrated that the overall prevalence of HCV coinfection was 16.1% (95% condence interval [CI]: 14.3%17.8%) [1]. Approximately 80% of these patients were infected with HCV genotype 1, and 75% had high HCV RNA levels (ie, O800,000 IU/mL) [1]. Similar HCV prevalence rates have been demonstrated among HIV-infected populations in France [16], Germany [17], Switzerland [18], and Greece [19]. In contrast, the prevalence of HCV infection in the general US population is 1.6% [20]. The prevalence of HCV infection varies with the mode of transmission of HIV. As such, chronic HCV has been reported in up to 90% of HIV-infected hemophiliacs [2123] and 90% of HIV-positive injection drug users [2,2427]. Transmission of HIV and HCV through blood products has been reduced markedly in the United States by screening of blood donations for both viruses, however [28]. In contrast, the incidence of HCV infection among HIV-infected homosexual men has increased recently, and unprotected anal intercourse, traumatic sexual practices, and concomitant sexually transmitted diseases have been the main risk factors for HCV acquisition [29]. The risk of perinatal transmission of HCV is increased for infants born to HIV/HCV-coinfected mothers. A recent meta-analysis of 10 studies demonstrated that the risk for HCV vertical transmission among HIV/ HCV-coinfected mothers was 2.82 (95% CI: 1.84.5) compared with HCVmonoinfected mothers [30]. The cumulative incidence of HCV infection is 17.1% in infants born to HIV/HCV-coinfected mothers compared with 5.4% for those born to mothers with only HCV infection [3133]. Higher HCV RNA levels are associated with increased perinatal transmission [32]. Eect of HIV on the natural history of hepatitis C virus HIV infection adversely aects every aspect of the natural history of chronic HCV. Although 14% to 45% of HCV-monoinfected individuals spontaneously clear HCV after acute infection, HCV clearance occurs in

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only 5% of HIV/HCV-coinfected persons, and less often in those with lower CD4 cell counts [3436]. HIV/HCV coinfection is associated with higher HCV RNA levels compared with HCV-monoinfected patients [5,3740], and HCV RNA levels increase as the CD4 cell count decreases, suggesting that HIV-induced immune deciency allows for increased HCV replication [5,38]. In addition, liver brosis progression is accelerated in HIV/HCV-coinfected patients, with a more rapid progression to cirrhosis compared with HCV-monoinfected individuals [3,5]. HIV/HCV-coinfected persons are at higher risk for advanced hepatic brosis and cirrhosis compared with HCV-monoinfected individuals [3,5,37,4144]. Among 67 HIV/HCV-coinfected patients undergoing paired liver biopsies separated by a median of 2.8 years, Sulkowski and colleagues [45] demonstrated that 28% of patients had an increase of at least two modied Ishak stages of hepatic brosis. Among those with mild brosis on initial biopsy, 26% had a two-stage progression on follow-up biopsy. HIV/HCV coinfection increases the risk of hepatocellular carcinoma compared with HIV-monoinfected patients (adjusted hazard ratio [HR] 5.35, 95% CI: 2.3412.20) [46] but not compared with HCV-monoinfected patients (adjusted HR 0.84, 95% CI: 0.551.27) [47]. This cancer presents sooner after cirrhosis develops [48] and more commonly as inltrating and multifocal lesions [49] in HIV/HCV-coinfected patients compared with those with chronic HCV alone. The eect of HIV on HCV-related end-stage liver disease has been examined exclusively in patients who have hemophilia. These studies show that the cumulative incidence of hepatic failure is 11% to 35% over a mean follow-up of 10 to 24 years from initial factor concentrate exposure, corresponding to a yearly incidence of 1.5% per year [2123,5052]. The results also indicate a 3- to 21-fold increase in the risk of end-stage liver disease in HIV/HCV-coinfected patients compared with HCV-monoinfected patients. HIV coinfection decreased the median survival time of patients who had HCV-associated end-stage liver disease compared with HCV-monoinfected patients (16 versus 48 months; P!.001) in a Spanish cohort [53]. The risk of death in HIV-infected patients is substantially higher in HIV/HCV-coinfected patients compared with those with chronic HCV alone (relative risk [RR] 2.26, 95% CI: 1.513.38) [53]. Predictors of death among HIV/ HCV-coinfected patients were Child-Pugh score (HR 1.20, 95% CI: 1.081.37), CD4 count less than 100 cells/mm3 (HR 2.48, 95% CI: 1.524.06), and hepatic encephalopathy at the time of decompensation (HR 2.45, 95% CI: 1.414.27) [54]. With the increased longevity of HIV-infected patients as a result of potent antiretroviral therapy and the prophylaxis of traditional opportunistic pathogens, HCV-related liver disease has emerged as a major cause of morbidity and mortality in this population [6,7,5558]. Results from the Data

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Collection on Adverse Events of AntiHIV Drugs (D:A:D) Study, a collaborative network of 11 HIV-infected cohorts from North America, Europe, and Australia, demonstrate that HCV-related liver disease is now the second leading cause of death in the HIV-infected population [8]. Among 23,441 HIV-infected patients enrolled in the study between December 1999 and February 2004 and followed for a median of 3.5 years, 1246 deaths occurred and 181 (14.5%) were liver related. Among these liver-related deaths, 66% were attributable to chronic HCV, 17% were attributable to chronic hepatitis B, and 7% were attributable to hepatitis B and HCV. In addition, recent data from the Strategies for Management of Antiretroviral Therapy (SMART) study demonstrate that mortality rates among HIV/viral hepatitis-coinfected individuals are nearly four times those of HIV-monoinfected persons [59]. In summary, these data clearly show that chronic HCV is more severe in HIV/HCV-coinfected patients. A greater proportion of HIV/HCV-coinfected patients develop chronic HCV, and HIV/HCV-coinfected patients are at substantially higher risk for liver-related complications, such as cirrhosis, end-stage liver disease, and hepatocellular carcinoma compared with HCV-monoinfected patients. In addition, HCV-associated liver disease is now the major cause of morbidity and mortality among HIV-infected patients in the developed world.

Impact of antiretroviral therapy on hepatitis C virus infection Eect of antiretroviral therapy on the natural history of hepatitis C virus Recent cohort studies have shown that use of HAART, particularly achieving HIV suppression, is associated with better hepatic outcomes in HIV/HCV-coinfected patients. Mehta and colleagues [60] reported that detectable HIV RNA (adjusted odds ratio [OR] 3.4, 95% CI: 1.67.1) and shorter cumulative HAART exposure (adjusted OR per year of exposure 0.8, 95% CI: 0.700.96) were associated with necroinammatory activity among coinfected persons. Brau and colleagues [61] found that HIV/HCVcoinfected patients with HIV RNA suppression had a slower brosis progression rate than those with detectable HIV RNA (0.122 versus 0.151 Ishak brosis units per year; P .013) but similar to that of HCV-monoinfected patients (0.122 versus 0.128 Ishak brosis units per year; P .52). Similarly, Verma and colleagues [62] reported that HIV/HCV-coinfected persons on HAART had comparable liver histology ndings to those of HCV-monoinfected persons. In a study of 162 HIV/HCV-coinfected individuals who underwent liver biopsy, absence of protease inhibitor therapy as part of the antiretroviral regimen was an independent predictor of progression to cirrhosis (RR 4.74, 95% CI: 1.3416.67) [63]. Use of HAART has also been shown to be associated with reduced liverrelated mortality among HIV/HCV-coinfected individuals. A German

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cohort study that followed coinfected patients over a 12-year period showed that HAART was an independent predictor of liver-related survival (OR 0.106, 95% CI: 0.0200.564) [64]. Among HIV/HCV-coinfected patients with preexisting hepatic failure, HAART was associated with improved survival (HR 0.5, 95% CI: 0.300.90) and 40% of HIV/HCV-coinfected patients receiving HAART were alive after 3 years compared with 18% not on HAART [54]. Finally, the recent D:A:D study [8] showed that there was little change in the liver-related death rate with increasing cumulative duration of HAART use. Taken together, the available data suggest that HAART favorably aects the course of HCV in HIV-infected patients, decreases the rate of death attributable to liver disease, and should not be withheld from HIV/HCVcoinfected persons because of fears regarding toxicity. Hepatotoxicity of antiretroviral therapy in hepatitis C virus coinfection Although the use of HAART has had enormous benets for HIV/HCVcoinfected patients, liver toxicity associated with these medications remains a concern for physicians who treat this population. Four main mechanisms of antiretroviral-related hepatotoxicity have been described in HIV-infected individuals: (1) mitochondrial toxicity, (2) hypersensitivity reactions involving the liver, (3) direct drug toxicity, and (4) immune reconstitution after HAART initiation in the presence of hepatitis coinfection [65]. More than one mechanism may occur simultaneously. Among studies of HIV-infected persons, the AIDS Clinical Trial Group (ACTG) scale of liver toxicity typically has been used to categorize the severity of liver injury [66]. Severe hepatotoxicity, the primary outcome in most hepatotoxicity studies in the HIV-positive population, has been dened as a grade 3 (5.110 times the upper limit of normal) or grade 4 (O10 times upper limit of normal) change in aspartate aminotransferase (AST) or alanine aminotransferase levels during HAART treatment or a greater than 3.5-fold increase in these levels higher than baseline if aminotransferases are elevated at HAART initiation [67]. Using these criteria, studies have shown that HCV coinfection increases the risk of severe hepatotoxicity in HIV-infected patients treated with HAART [6775]. Discontinuation of HAART is more frequent and occurs earlier among HIV/HCV-coinfected patients than for those with HIV alone [76]. Moreover, the risk of severe hepatotoxicity with HAART is increased for HIV/HCV-coinfected patients with advanced (METAVIR stage 3 or 4) brosis (RR 2.75, 95% CI: 1.086.97) [77]. Anxiety related to hepatotoxicity should not, as it often does, dissuade or delay patients and physicians from initiating a therapy that may attenuate HCV-related liver disease and reduce liver-related mortality, however. Given the increased risk of hepatotoxicity in the HIV/HCV-infected population, eradication of HCV with antiviral therapy might improve tolerance

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to HAART. In a recent study among 132 HIV/HCV-coinfected patients treated with interferon-based therapy in Spain, the yearly incidence of severe hepatotoxicity was greater in patients who did not achieve a sustained virologic response (SVR; dened as an undetectable HCV RNA level at the end of treatment and 24 weeks later) than in those who did (12.9% versus 3.1%; P!.001) and in patients who had advanced liver brosis than in those who did not have it (14.4% versus 7.6%; P .003) [78]. Failure to achieve SVR (OR 6.13, 95% CI: 1.8337.45) and use of didanosine or stavudine as part of the antiretroviral regimen (OR 3.59, 95% CI: 1.2310.42) were independent predictors of hepatotoxicity after interferon therapy. These data demonstrate that achieving SVR after antiHCV treatment can reduce the risk of hepatotoxicity during antiretroviral therapy, which should further encourage the treatment of chronic HCV in HIV. Certain antiretroviral medications or classes may be more likely to produce elevated aminotransferases or lead to clinically apparent hepatotoxicity (Table 1). The use of the nucleoside analogues stavudine, didanosine, or their combination can lead to a higher rate of hepatic steatosis in the coinfected population [79]. The nonnucleoside reverse transcriptase inhibitor nevirapine has been shown to increase the risk of severe hepatotoxicity in HIV/HCV-coinfected patients, and elevations in aminotransferase levels may develop 4 to 6 months after initiation of the medication [67,80,81]. Nevirapine use has also been associated with more advanced hepatic brosis (adjusted OR 2.56, 95% CI: 1.026.58) in a cross-sectional study of 152 HIV/HCV-coinfected patients who underwent a liver biopsy [82]. In contrast, most HIV/HCV-coinfected patients who initiate a protease inhibitorcontaining regimen do not experience treatment-limiting hepatotoxicity. Although liver enzyme elevations may develop with any protease inhibitor, lopinavir-ritonavir, fosamprenavir-ritonavir, and nelnavir seem to be less hepatotoxic [83] and tipranavir-ritonavir seems to be the most hepatotoxic [84]. An unconjugated hyperbilirubinemia can occur during atazanavir and indinavir therapy but does not reect liver damage and is related to the inhibition of the uridine diphosphate glucuronosyl transferase enzyme [85,86]. Dual protease inhibitor therapy does not increase the rate of hepatotoxicity [87]. Data on the hepatotoxicity of fusion inhibitors, integrase inhibitors, and chemokine receptor antagonists in HIV/HCV-coinfected patients are lacking.

Integrating hepatitis C virus care in HIV practice Multidisciplinary approach HIV/HCV-coinfected patients should be referred to a hepatologist or infectious diseases physician with expertise in HIV/HCV coinfection to provide more information about diagnosis, natural history, and therapeutic options. Clinical management should subsequently be multidisciplinary,

Table 1 Specic antiretroviral concerns in persons coinfected with HIV and hepatitis C virus Drug Abacavir Comment  May decrease virologic response to HCV therapy, especially when serum ribavirin levels are low, possibly by competing with ribavirin for phosphorylation at an intracellular level [139,140]  Can have increased intracellular levels when administered with ribavirin [132]  May be associated with hepatic steatosis in coinfected persons [79]  Increased risk of lactic acidosis and pancreatitis when administered with ribavirin [133136]  Increased risk of hepatic decompensation during HCV therapy in coinfected patients who have cirrhosis [137,138]  Increased rate of severe hepatotoxicity in HIV/HCV-coinfected persons [67,80,81]  May be associated with hepatic brosis in coinfected individuals [82]  May be associated with hepatic steatosis in HIV/HCV-coinfected persons [79]  Can potentiate ribavirin-induced anemia, possibly through suppression of hematopoiesis [131] Recommendation  Ensure adequate weight-based ribavirin dosing  Emphasize ribavirin adherence
MANAGEMENT COMPLEXITIES OF HIV/HEPATITIS C VIRUS COINFECTION

Didanosine

 Concomitant use with ribavirin contraindicated

Nevirapine

 Consider alternate antiretroviral agent in coinfected persons

Stavudine Zidovudine

 Consider avoiding use of stavudine, if possible  Avoid concomitant use with ribavirin  Monitor hemoglobin levels closely if no other antiretroviral options

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with input from advanced practice nurses, psychiatrists, pharmacists, dieticians, and addiction management experts [88]. Support and education are crucial to the management of chronic HCV in HIV-infected patients, and health care providers should provide additional educational materials and oer referral to support groups to those undergoing evaluation for established HCV infection. Patients should be counseled to prevent liver damage and HCV transmission. Screening for drug and alcohol abuse Heavy alcohol consumption, particularly in quantities greater than 50 g/d (approximately three drinks) can worsen the course and outcome of chronic HCV [89] and may compromise antiviral therapy by decreasing adherence or interfering with the antiviral action of interferon-based therapy [90]. Eorts to diagnose and treat alcohol abuse should be performed in all HIV/HCV-coinfected patients before beginning HCV therapy, and relapse into drug and alcohol use should be repeatedly assessed. Treatment for drug and alcohol abuse should be made available to all who want and need it. Safe levels of alcohol consumption in HIV/HCV-coinfected patients remain unclear, but even moderate levels of consumption may accelerate disease progression [91]. All HIV/HCV-coinfected patients should therefore be advised to abstain from alcohol [92,93]. Treatment of neuropsychiatric disorders Neuropsychiatric disorders, particularly depression, are common among HIV/HCV-coinfected patients [94] and are a frequent adverse eect of pegylated interferon therapy [911]. Identication and treatment of neuropsychiatric disorders should be pursued before and during HCV therapy [91]. Referral to a psychiatrist should also be considered. Immunization against hepatitis A and B HIV/HCV-coinfected individuals should be tested for prior exposure to hepatitis A virus infection (antihepatitis A immunoglobulin G [IgG] antibody) and previous or concurrent hepatitis B virus infection (hepatitis B surface antigen, antihepatitis B core IgG antibody, and antihepatitis B surface antibody). Acute infection with hepatitis A or B in those with underlying chronic HCV increases the risk for fulminant hepatitis and can result in high morbidity [95,96]. Despite evidence of decreased response in HIV-infected persons, hepatitis A and B vaccination should be performed in those who are seronegative for these viruses [97]. Liver brosis assessment Evaluation of liver histology with a liver biopsy is the best tool for assessing the likelihood of progression of hepatic injury and is a better predictor of

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subsequent clinical events than hepatic aminotransferase elevations, HCV genotype, or HCV RNA level [98,99]. In one study, 29% of HIV/HCVcoinfected patients with persistently normal alanine aminotransferase levels had advanced brosis on liver biopsy [100]. As such, many experts recommend a liver biopsy in HIV/HCV-coinfected patients to assess the extent of underlying liver damage. The liver biopsy can help to guide HCV treatment decisions, permits direct determination of the degree of necroinammation, and allows detection of other hepatic abnormalities (eg, steatosis, iron overload, concomitant infections) [101]. Evaluation of liver histology with a liver biopsy can also inform the need for hepatocellular carcinoma screening, which is recommended once cirrhosis is present. Despite the value of the liver biopsy, it possesses several limitations that have contributed to low acceptance by HIV/HCV-coinfected patients, particularly its invasive nature, occasional serious complications [101], sampling error attributable to the small size of the extracted tissue and inherent heterogeneity of hepatic brosis [102,103], and high cost [104]. As a result, noninvasive modalities to evaluate hepatic brosis have been increasingly examined in the HIV/HCV-coinfected population [105]. These noninvasive tools are currently divided into two major categories: (1) serum biochemical markers (eg, AST-to-platelet ratio index, serum testing for hyaluronic acid, albumin, and AST [SHASTA index], FIB-4, FibroTest [Biopredictive, Paris, France], HCV-FibroSure [LabCorp, Burlington, NC]) [106109] and (2) imaging techniques, primarily elastometry (FibroScan, Echosens, Paris, France) [110,111]. These tools have high predictive value in identifying advanced hepatic brosis and cirrhosis, but they have been imprecise in distinguishing among the intermediate stages of hepatic brosis [112]. In addition, serum brosis markers have generally been less reliable in coinfected patients because of the inammatory nature of HIV disease and coadministration of medications that may interfere with test results. Given the accelerated progression of HCV-related liver disease among HIV-infected patients, improvements in the ecacy of HCV therapy in the HIV-infected population, the high predictive value of the early virologic response to HCV therapy (at week 12 of treatment) to identify who does and who does not respond, and the acknowledged limitations of the liver biopsy, current HIV/HCV coinfection management guidelines no longer require that a liver biopsy be performed before initiation of HCV therapy [112]. Hepatitis C virus treatment Pegylated interferon plus ribavirin Given the accelerated progression to end-stage liver disease among HIV/ HCV-coinfected patients, treatment of chronic HCV should be considered in all coinfected patients [113]. Combination pegylated interferon plus ribavirin for 48 weeks represents the standard of care for treating chronic HCV in HIV-infected individuals, as it is for HCV-monoinfected persons. As with

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HCV-monoinfected patients, the primary goal of treatment is viral eradication (ie, SVR) [91,112]. A second potential benet of HCV therapy is a reduction in the risk for liver-related complications. Among a Spanish cohort of antiretroviral-treated HIV/HCV-coinfected patients, receipt of HCV therapy was associated with improved survival, and no episodes of hepatic decompensation were noted among subjects who achieved an SVR [114]. In an Italian retrospective cohort study, HIV/HCV-coinfected patients with cirrhosis who received a mean of 9 months of pegylated interferon plus ribavirin without virologic response were less likely to develop adverse liver-related outcomes (ie, ascites, jaundice, encephalopathy, variceal bleeding, hepatocellular carcinoma, death) compared with age- and Child-Pugh scorematched coinfected patients who had not received HCV therapy [115]. Three randomized controlled trials (AIDS Clinical Trials Group [ACTG] Study 5071, AIDS Pegasys Ribavirin International Coinfection Trial [APRICOT], and Agence Nationale de Recherches sur le Sida [ANRS] HC-02 RIBAVIC trial) were published in 2004 demonstrating that pegylated interferon plus ribavirin is the optimal therapy for chronic HCV among HIV-infected patients [911]. There were notable dierences in type of pegylated interferon use, dose regimen of ribavirin, and type of coinfected patients enrolled between the studies, and these are highlighted in Table 2. These dierences make it impossible to compare overall results between these clinical trials. In each study, the highest SVR rates were observed in the pegylated interferon plus ribavirin arms. SVR rates for HCV genotype 1infected persons in these arms were 14% for ACTG Study 5071, 17% for the RIBAVIC trial, and 29% for the APRICOT [911]. For subjects infected with HCV genotype 2 or 3, SVR rates were considerably higher: 44% (RIBAVIC trial), 62% (APRICOT), and 72% (ACTG Study 5071) [911], emphasizing the importance of HCV genotype as a predictor of SVR, as in studies of HCV-monoinfected patients. Similarly, low pretreatment HCV RNA level (%800,000 IU/mL) is also associated with SVR [9]. This was highlighted in the APRICOT, in which SVR rates were 61% among persons who had HCV genotype 1 and a baseline HCV RNA level of 800,000 IU/mL or less who were randomized to pegylated interferon plus ribavirin [9]. In contrast, the SVR rate was only 18% for those who had HCV genotype 1 and an HCV RNA level greater than 800,000 IU/mL who received pegylated interferon plus ribavirin. Additional predictors of SVR that have been reported include absence of prior history of injection drug use [10], detectable HIV RNA [10], age of 40 years or younger [11], baseline alanine aminotransferase level greater than three times the upper limit of normal [11], antiretroviral therapy with a nonnucleoside reverse transcriptase inhibitor or protease inhibitor [116], and non-black race [116]. The early virologic response, dened as a greater than 2-log IU/mL decrease in HCV RNA level after 12 weeks of therapy, has high predictive value in patients who have coinfection [911]. Thus, if a patient has not had an early virologic response,

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Table 2 Data from pegylated interferon plus ribavirin arms from four pivotal studies examining the treatment of chronic hepatitis C virus infection in HIV-infected individuals Characteristic Site Peginterferon formulation Ribavirin dosage HCV genotype 1 (%) Bridging brosis/cirrhosis (%) On antiretroviral therapy (%) CD4 count (cells/mm3) Undetectable HIV RNA (%) SVR rate, genotype 1 (%) SVR rate, genotype 2/3 (%) Withdrawal rate (%) ACTG 5071 (n 133) United States Peginterferon a-2a 6001000 mg/d 77 11 85 453 (median) 61 (!50 copies/mL) 14 73 12 APRICOT (n 868) United States, Europe, Australia Peginterferon a-2a 800 mg/d 61 12 84 520 (mean) 60 (!50 copies/mL) 29 62 25 RIBAVIC (n 412) France Peginterferon a-2b 800 mg/d 48 39 83 547 (median) 70 (!400 copies/mL) 17 44 39 PRESCO (n 389) Spain Peginterferon a-2a 10001200 mg/d 51 27 67 540 (mean) 72 (!50 copies/mL) 35 72 45

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the likelihood of SVR is negligible. Extending therapy in patients who do not have an early virologic response does not increase SVR rates [117]. The SVR rates reported in the pivotal HIV/HCV treatment trials are considerably lower than those reported for HCV-monoinfected patients (42%52% in HCV genotype 1 and 78%84% in HIV genotypes 2 and 3). Possible reasons for the poorer SVR rates among HIV/HCV-coinfected patients include (1) high HCV RNA levels in subjects with HIV coinfection, (2) qualitative defects in the cellular and innate immune response, (3) and lower doses of ribavirin or dose escalation administered in the treatment trials (because of concern for potential increased risk of hematologic toxicity in this population). Recently, the Spanish Pegasys Ribavirina Espan a Coin n (PRESCO) study examined whether administration of weight-based feccio ribavirin (1000 mg/d if body weight !75 kg and 1200 mg/d if body weight O75 kg) in combination with pegylated interferon, as is used in HCV-monoinfected patients, improves the SVR rate in HIV/HCV-coinfected patients [118,119]. Substantial improvements in virologic outcomes were reported, with SVR achieved in 72.4% of HCV genotype 2 or 3infected patients and 35% of genotype 1 or 4infected patients. Only 3% of patients stopped HCV therapy because of severe anemia. As such, recent HIV/HCV management guidelines now recommend weight-based ribavirin dosing in HIV-positive persons undergoing combination HCV therapy. When SVR is achieved, HCV therapy can halt or regress hepatic brosis in HIV/HCV-coinfected patients, even in the absence of SVR [13,14]. Data from the APRICOT show that 70% of patients who achieved SVR with pegylated interferon plus ribavirin had a two-stage improvement in Ishak brosis score on a repeat liver biopsy 24 weeks after the end of HCV treatment. Among those who did not achieve an SVR, 43% receiving pegylated interferon plus ribavirin also had this histologic response. Combination HCV therapy has also been shown to decrease progression of HCV-related brosis in HIV-infected individuals [14]. For coinfected patients who have not responded to HCV therapy or who have relapsed after treatment, clinical trials are currently examining whether the long-term administration of interferon can prevent liver brosis progression even in the absence of SVR [120]. Preliminary results from ACTG 5178, which randomized coinfected subjects who did not achieve early virologic response to pegylated interferon alfa-2a monotherapy or observation for 72 weeks, demonstrated no dierence in brosis progression on paired liver biopsies between the groups, although no signicant brosis progression was observed in either arm [121]. The lack of brotic progression in the observation arm precluded the ability to nd ecacy in the pegylated interferon maintenance arm. Thus, since there was no improvement in hepatic brosis among the coinfected non-responders who received pegylated interferon maintenance therapy, this treatment modality cannot be recommended in routine clinical practice. New agents with specic anti-HCV activity are being tested [122], and clinical trials examining the ecacy and safety of these drugs in

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coinfected patients should be prioritized without waiting for the nal results of phase III trials conducted in HCV-monoinfected patients. Adverse eects of hepatitis C virus therapy The toxicities and intolerabilities of HCV therapy tend to dominate HCV treatment in HIV-infected patients, but these do not lead to treatment discontinuation more frequently among HIV/HCV-coinfected patients (12%17% withdrawal rates in randomized trials Refs. [911]) compared with HCV-monoinfected patients (14%22% withdrawal rates in clinical trials Refs. [12,123]). Results from the APRICOT show that pegylated interferon reduces HIV RNA levels approximately 1.0 log copies among subjects with detectable HIV RNA [9]. In the same study, HCV therapy precipitated a decrease in absolute CD4 cell counts throughout the 48-week treatment phase of the study, which then returned to baseline values by 24 weeks after completing HCV therapy [9]. Despite the decrease in CD4 cell counts, the CD4 cell percentage is typically unchanged, and clinical progression to AIDS was not observed in any subject in the APRICOT during the study period. Cooper and colleagues [124] recently compared infection rates between HIV/HCV-coinfected and HCV-monoinfected patients receiving interferon-based therapy for chronic HCV. Rates of infection did not dier by HIV status, and HIV was not found to be an independent predictor of infection during HCV therapy. Leukopenia and thrombocytopenia are dose-related adverse eects of pegylated interferon. In particular, use of granulocyte colony-stimulating factor was allowed in two of the pivotal HIV/HCV coinfection treatment trials to improve leukopenia [9,10]. Anemia is also a common adverse eect during combination anti-HCV therapy [125]. It arises because of the suppression of erythropoiesis induced by interferon [126] and the reversible hemolysis induced by ribavirin [127,128]. Reduction of the ribavirin dose had been recommended if anemia developed during HCV therapy, but this is associated with reduced SVR rates [123]. Recombinant human erythropoietin can counteract the anemia associated with HCV therapy in HIV/HCV-coinfected subjects and helps to avoid ribavirin dose reduction [129], maximizing the eectiveness of antiviral therapy. The use of hematopoietic growth factors in HIV/HCV-coinfected patients has been associated with an improved clinical response to pegylated interferon plus ribavirin therapy [130]. A recent retrospective cohort study found that the incidence of signicant weight loss (dened as loss of at least 5% of baseline body weight) was substantially greater in dually treated HIV/HCV-coinfected subjects compared with treated HCV- or HIV-monoinfected subjects [131]. Among 192 patients (n 63 HIV/HCV-coinfected, n 64 HCV-monoinfected, n 65 HIV-monoinfected), signicant weight loss occurred in 48 (76%) HIV/ HCV-coinfected subjects versus 25 (39%) HCV-infected subjects (P!.001) and 2 (3%) HIV subjects (P!.001), yielding an adjusted HR of 2.76 (95% CI: 1.674.55) and 38.5 (95% CI: 8.5174.7), respectively.

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The degree of weight loss was also greater among the HIV/HCV-coinfected cohort compared with both monoinfected cohorts. Body weights for HIV/HCV-coinfected and HCV-monoinfected subjects were stable before initiation of HCV therapy, but both cohorts lost weight after HCV treatment initiation, with the rate of weight loss being greater for dually treated HIV/HCV-infected subjects. Receipt of more than two nucleoside reverse transcriptase inhibitors increased the risk of clinically signicant weight loss (adjusted HR 8.17, 95% CI: 2.3728.20), suggesting that mitochondrial toxicity might play some role in weight loss during dual HIV/HCV therapy. Drug-drug interactions during hepatitis C virus therapy An additional concern for dually treated HIV/HCV-coinfected patients is the potential for drug-drug interactions between ribavirin and the nucleoside reverse transcriptase inhibitors included in the antiretroviral regimen (see Table 1). A retrospective cohort study among 217 HIV/HCV-coinfected patients receiving pegylated interferon plus ribavirin found that zidovudine use was associated with a greater mean hemoglobin decline at 4 weeks of HCV therapy (3.1 versus 2.1 g/dL; P!.001) compared with nonusers [132]. By week 12 of HCV therapy, zidovudine use was more frequently associated with ribavirin dose reduction (52% versus 18%; P!.001) and erythropoietin use (49% versus 23%; P!.001) compared with those who did not receive the drug. Zidovudine likely exacerbates ribavirin-related anemia by inhibiting the hematopoietic response to ribavirin-induced hemolysis. Thus, it is advisable to avoid zidovudine use during HCV therapy, and providers should considering switching to an alternative nucleoside reverse transcriptase inhibitor before initiation of HCV treatment [112]. Because ribavirin increases the intracellular active metabolite of didanosine [133], concomitant use of both medications increases the likelihood of mitochondrial toxicity, which may lead to pancreatitis and symptomatic lactic acidosis [134137]. In the APRICOT and RIBAVIC trial, didanosine use increased the risk of hepatic decompensation when used in conjunction with ribavirin [138,139]. As a result, ribavirin should not be administered to persons taking didanosine as part of their HAART regimen. A recent retrospective substudy of the RIBAVIC trial reported that abacavir use increased the risk of early virologic failure to combination HCV therapy (OR 4.9, 95% CI: 1.516.1), possibly because of the intracellular competition between ribavirin and abacavir, both guanosine analogues, for activation through phosphorylation [140]. In a Spanish cohort that controlled for serum ribavirin levels, however, abacavir use was not associated with early virologic failure, suggesting that appropriate weight-based ribavirin dosing and adherence are important when abacavir is part of the antiretroviral regimen [141].

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Advanced liver disease The management of advanced liver disease in HIV/HCV-coinfected patients is complex. Patients who have cirrhosis should have regular monitoring for evidence of decompensation and hepatocellular carcinoma. Individuals who have decompensated liver disease are generally not candidates for HCV therapy because treatment increases the risk of life-threatening complications [139]. HIV therapy may improve hepatic outcomes and survival in coinfected patients who have liver failure [54]. Administration of these medications in this setting is challenging, however, because of alteration of hepatic metabolism and the risk of drug-induced liver injury. Of note, hepatic metabolism of nonnucleoside reverse transcriptase inhibitors, particularly efavirenz, is impaired in coinfected patients who have cirrhosis, but no similar eect is seen for protease inhibitors [142]. Orthotopic liver transplantation is an option for coinfected patients who have decompensated liver disease. HIV coinfection is a major determinant of poor outcomes and death in HCV-infected persons undergoing liver transplantation, however [143]. de Vera and colleagues [144] demonstrated substantially reduced 5-year survival after liver transplantation among HIV/HCV-coinfected patients compared with HCV-monoinfected patients (33% versus 72%; P .07). In addition, 22% of coinfected patients were unable to tolerate HAART after transplantation, which was a major determinant of death. Early treatment of HCV after transplantation may be a potential strategy to reduce these adverse outcomes, but more study is needed and ongoing in this area. Summary Because of shared routes of transmission, HCV coinfection is common among HIV-infected persons. Because of the eectiveness of antiretroviral therapy, chronic HCV has now emerged as a major cause of morbidity and mortality in this population. Because chronic HCV is highly prevalent among HIV-infected patients and has a rapid disease progression, antiviral therapy with pegylated interferon plus ribavirin is critical for the long-term survival of HIV/HCV-coinfected patients. Additional studies are needed to examine the natural history of chronic HCV in HIV/HCV-coinfected patients, identify the appropriate treatment candidates, and identify additional interventions that can improve response to antiviral therapy. References
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Clin Liver Dis 12 (2008) 611636

Extrahepatic Manifestations of Hepatitis C Virus Infection


Anna Linda Zignego, MD, PhDa,*, Antonio Crax` , MDb
Center for Systemic Manifestations of Hepatitis Viruses (MaSVE), Department of Internal Medicine, University of Florence, Viale GB Morgagni 85, 50134 Firenze, Italy b Gastroenterologia and Epatologia, Di.Bi.M.I.S., University of Palermo, Piazza Marina, 3490133 Palermo, Italy
a

Hepatitis C virus (HCV) is at the same time a hepatotropic and a lymphotropic virus and may cause hepatic and extrahepatic diseases. Extrahepatic manifestations linked to HCV range from disorders for which a signicant association with viral infection is supported by epidemiologic data and by biological plausibility, to anecdotal observations without clear proof of causality. B cell lymphoproliferative disorders (ie, mixed cryoglobulinemia and non-Hodgkins lymphoma) are the extrahepatic conditions most closely linked to HCV, having been investigated extensively, and represent a model for both pathogenetic and clinicotherapeutic deductions. An association between HCV infection and other morbid conditions, including dermatologic, nephrological, neurologic, endocrinologic, cardiocirculatory, and lung disorders also has been suggested. Overlap syndromes characterized by the presence in one patient of manifestations belonging to various pathologic conditionsdtypically of autoimmune/lymphoproliferative natured would suggest that chronic HCV infection is a distinct systemic disease with a varying spectrum of clinical manifestations. Interferon-based antiviral therapy is considered, when feasible, the mainstay of treatment for most HCV-linked extrahepatic diseases. Although its ecacy in curtailing a nonhepatic manifestation after obtaining viral clearance often is seen as a conrmation of the key pathogenetic role played by HCV, clinical symptoms and viral persistence also may be disjointed, the former persisting even beyond a sustained viral response to therapy. Because of this fact and the intrinsic potential inecacy of interferon, which in some cases may even exacerbate

* Corresponding author. E-mail address: a.zignego@dmi.unifi.it (A.L. Zignego). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.012 liver.theclinics.com

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extrahepatic conditions, an individualized tailoring of therapy is needed in these patients.

Classication of extrahepatic manifestations of hepatitis C virus Extrahepatic manifestations of HCV infection (EHMs-HCV) range from disorders for which a signicant association with HCV infection is supported clearly by multiple lines of evidence to anecdotal observations without clear proof of causality [1,2]. A tentative classication of EHMs-HCV is suggested in Box 1. According to such classication, extrahepatic manifestations of HCV infection are distinguished, taking into account the robustness of available scientic data. It is thus likely that the nosography of some EHMs-HCV may change over time. Group A includes EHMs-HCV characterized by a strong association proven by both epidemiologic and pathogenetic evidence. This category includes B-cell lymphoproliferative disorders (LPDs). Group B includes disorders for which a signicant association with HCV infection is supported by substantial epidemiologic data and groups C and D associations still require conrmation and/or a more detailed characterization as opposed to observations that are of similar pathologic nature but of dierent etiology, or idiopathic in nature, or only anecdotal (see Box 1).

Hepatitis C virus-related lymphoproliferative disorders Mixed cryoglobulinemia Mixed cryoglobulinemia (MC) is a systemic vasculitis caused by deposition of circulating immune complexes in the small vessels and characterized by multiple organ involvement, mainly skin, peripheral nerves, kidney, and salivary glands, and less frequently associated with widespread vasculitis and malignant lymphoma [36]. The strong association between HCV and MC has been conrmed repeatedly by serologic and molecular investigations [4,6,7]. Generally speaking, cryoglobulinemias are conditions characterized by the presence of serum immunoglobulins that become insoluble below 37 C and can dissolve by warming serum (cryoglobulins, CGs). According to Brouet and colleagues [8], CGs are classied on the basis of their immunoglobulin composition. In type I, they are composed of a pure monoclonal component and usually associated with an indolent B-cell lymphoma, and in types II and III mixed CGs, they are composed of a mixture of polyclonal IgG and monoclonal IgM or polyclonal IgG and polyclonal IgM, respectively. In MC, the IgM represents an autoantibody bearing rheumatoid factor (RF) activity. In type II MC (MC II), the IgM RF molecules most frequently display the WA cross-reactive idiotype [9]. MC II accounts for 50% to 60%, and type III (MC III) for the remaining 40% to 50% of MC.

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Box 1. Classication of extrahepatic manifestations of hepatitis C virus infection Association dened on the basis of high prevalence and pathogenesis Mixed cryoglobulinemia (complete or incomplete clinical syndrome) B-cell non-Hodgkins lymphoma Association dened on the basis of higher prevalences than in controls Monoclonal gammopathies Porphyria cutanea tarda Lichen planus Diabetes mellitus Associations to be conrmed/characterized Autoimmune thyroiditis Thyroid cancer Sicca syndrome Alveolitislung brosis Noncryoglobulinemic nephropathies Erectile dysfunctions Carotid Atherosclerosis Psychopathological disorders Anecdotal observations Psoriasis Peripheral/central neuropathies Chronic polyarthritis Rheumatoid arthritis Polyarthritis nodosa Bechets syndrome Myositis/dermatomyositis Fibromyalgia Chronic urticaria Chronic pruritus Kaposis pseudosarcoma Vitiligo Cardiomyopathies Mooren corneal ulcer Necrolytic acral erythema

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The prevalence of chronic HCV infection in patients who have CGs in their serum ranges from 19% to more than 50% according to various studies [10,11]. CGs, however, are generally present at low levels, and symptoms are generally absent or mild in chronically HCV-infected patients, whereas clinically overt MC syndrome (MCS) would be evident in 10% to 30% of MC subjects [1012]. Serum mixed CGs, high RF values, and reduced C4 values are the most frequent laboratory data. The most common symptoms of MCS are weakness, arthralgias, and purpura (Meltzer and Franklin triad). Raynauds phenomenondmicrocirculatory changes of the small vessels of the hands and feet, identied as color changes in response to colddperipheral neuropathy, sicca syndrome, renal involvement, lung disorders, fever, and cytopenias also may be observed [3]. In a recent study involving 231 Italian MC patients, peripheral neuropathy was observed in most cases, representing the most frequent clinical feature after the triad, followed by sicca syndrome, Raynaud phenomenon, and renal involvement [13]. MC-related peripheral neuropathy typically includes mixed neuropathies, which are more often sensitive and axonal. They can manifest themselves as symmetric distal neuropathies, multiple mononeuritis, or mononeuropathies. Involvement of the central nervous system is unusual and generally presents as transient dysarthria and hemiplegia. Pathologic ndings show axonal damage with epineural vasculitic inltrates and endoneural microangiopathy. A sicca syndrome (xerostomia and xerophthalmia) caused by involvement of salivary and lacrimal glands is recognized in a large proportion of MC patients [1418]. This syndrome close resembles primary Sjo grens syndrome; however it typically lacks antinuclear autoantibodies and antiepithelial neutrophil-activating peptide (ENA, SSA/Ro, SSB/La) [13]. The pathogenetic role of HCV infection in sicca syndrome and the characteristics distinguishing classic Sjo grens syndrome from those associated with HCV remain at issue [19]. It has been proposed that HCV infection is a criterion to exclude diagnosis of primary Sjo grens syndrome, especially if mixed cryoglobulins and hypocomplementemia are present, and anti-SSA/Ro antibodies are absent [20]. Signicant renal involvement is present in up to one third of patients who have MC, being observed in about 20% of patients upon clinical presentation of MC and in 35% to 60% of patients over long-term follow-up [13,21]. The most common features at diagnosis are one or more subclinical features of renal involvement including microscopic hematuria, proteinuria below the nephrotic range (!3 g/24 h), and with normal or only fairly reduced renal function (creatinine !1.5 mg%). Arterial hypertension is observed in up to 80% of cases [22]. In about 20% of patients, proteinuria is in the nephrotic range, and in them a nephrotic syndrome may represent the principal manifestation of MC. About 20% to 30% of cases present with an acute nephritic syndrome as their rst renal

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manifestation [4,23]. Presence of a signicant renal involvement is among the worst prognostic indices in patients who have MC, even when its course is variable [13,23]. In a study of 231 patients who had MC, glomerulonephritis with subsequent renal failure was the main complication (33%), leading to death during long-term follow-up [13]. The typical histologic pattern of renal damage observed in patients who have MC type I is membranoproliferative glomerulonephritis (MPGN) [13]. The presence of capillary thrombi, made up of precipitated cryoglobulins and deposits of IgM in capillary loops typically dierentiates the cryoglobulinemic form from idiopathic MPGN. In a minority of cases (generally type III MC), dierent pathologic ndings have been described (ie, focal and mesangioproliferative glomerulonephritis and membranous glomerulonephritis) [24,25]. The association between MC and severe liver damage has been discussed widely [1,2,9,2629]. Several studies have shown an epidemiologic association between MC and severe liver damage [10,30]. An association between MC and liver steatosis also has been suggested [31]. Overall, it seems that both MC and the stage of liver damage are more related to a long duration of infection. The occurrence of MC generally has an important impact on the quality of life and survival of patients who have MC. Survival analysis according to the Kaplan-Meier method shows a cumulative 10-year survival, calculated from time of diagnosis, to be signicantly lower in patients who have MC as compared with an age- and sex-matched general population. Lowest survival rates were observed in males and in subjects who had renal involvement, the main causes of death being nephropathy (33%), malignancies (23%), liver failure (13%), and diuse vasculitis (13%) [13]. Because of its variable presentation, no standardized criteria for the diagnosis and staging of MCS are available, even if classications have been proposed [32]. In the presence of purpura, mixed CGs, reduced C4 values, and organ involvement, the diagnosis of MCS is relatively easy. Quite frequently, however, it is suggested by abnormal laboratory data (RF test or mixed CGs or reduced C4 values) with or without mild symptoms like arthralgias or asthenia. Moreover, some subjects who have HCV may show clinically evident MCS, though incomplete from a serologic point of view, mainly with the temporary absence of circulating CGs. This may be explained by the diculty in a proper determination of the presence of CGs, because of their thermolability and the variability of the rate of CGs responsible for vasculitic damage [13,33]. Because some mixed CGs are present in low concentrations, the dierentiation between type II and type III CGs often requires a more sensitive method for immunochemical characterization such as electroimmunoxation or Western blot, than conventional immunoelectrophoresis [34]. Several data, including the presence of a clonal expansion of B-lymphocytes (BL) in peripheral blood or liver inltrates [3538], and the histopathological

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features of the bone marrow and liver lymphoid inltrates, conrm the lymphoproliferative nature of MC. Some studies have shown that: B-cell clonal expansion (in particular of RF B-cells) underlies MC. This condition is associated with Bcl2/JH rearrangement. MC II can evolve into a frank b-cell non-Hodgkins lymphoma (NHL) in approximately 8% to 10% of cases after a long period of time [13]. From a histopathological point of view, the presence of monoclonal lymphoproliferation of uncertain signicance (MLDUS) in subjects who have clinicolaboratory features of MC II is typical [33,3941]. MLDUS represents oligoclonal proliferations of small BL, preferentially located in the bone marrow and liver. In these organs, MLDUS is generally present with phenotypic and histologic aspects comparable to indolent B-cell lymphoma. Further immunemorphologic analysis reveals two dierent varieties: the rst and more frequent variety, with analogous features to B-cell chronic lymphatic leukemia (CLL)/small cell lymphoma and a second, less frequent, lymphoplasmacytic-like form. The prevalence of these histologic patterns has varied in dierent reports [40,4248]. Lymphoma HCV-associated lymphoid malignancies can be observed during the course of MC or as non-MC related idiopathic forms. About 8% to 10% of MC II evolves into a frank lymphoma [43,49], generally after long-lasting infection. According to recent data, MC patients had a 35 times higher risk of NHL than the general population [50]. An association between B-cell derived NHL and HCV infection initially was suggested by studies performed in populations from southern Europe and the southern United States, [46,5155], whereas some northern European and northern United States or Canadian surveys did not conrm a higher prevalence of chronic HCV infection in patients with lymphoma than in the general population [5661]. During the last decade, many studies performed at dierent latitudesdgenerally larger in size than initial onesd as well as meta-analysis, were able to conrm the existence of such association, even with a clear south/north gradient of prevalence. These dierences at least partly reect how HCV is diuse at dierent rates in the specic populations studied, but also suggest a likely contribution of environmental or genetic factors [62] to lymphomagenesis. Although virtually all types of lymphoid malignancy can be found in patients with HCV infection, the strongest association is with B-cell derived NHL [18,40,51,53,54,6365]. The use of dierent classications presented in the various studies, however, may confound the evaluation of the actual incidences of each histotype [48,6567]. Peripheral B-cell derived indolent NHL appears to be the most frequent HCV-associated lymphoma in many studies. According to the Revised European-American Lymphoma

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(REAL) classication/World Health Organization (WHO) Classication [68,69], the main HCV-related histotypes include B-cell chronic lymphocytic leukemia/small lymphocyte lymphoma, diuse large B-cell lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, and marginal zone lymphoma [40]. A recent European multicenter survey suggests a specic role of HCV infection in the pathogenesis of diuse large B-cell lymphoma [70]. Among marginal zone lymphomas, a special association with HCV infection was reported for the mucosa-associated lymphoid tissue (MALT) lymphoma [65,71,72] and the splenic forms. This is conrmed by reports of regression of marginal splenic lymphoma after successful HCV clearance because of antiviral therapy, in spite of previous ineective chemotherapy [7375]. In general, the observations of a hematological regression of some HCV-associated NHL and expanded B-cell clones following eective antiviral therapy represent a consistent argument in favor of the pathogenetic role played by HCV infection, even if a direct antineoplastic role of interferon cannot be excluded. A serum monoclonal gammopathy, more frequently type IgM/K, has been described among HCV-associated LPDs [76]. In most patients with HCV, however, MG was classied as MGUS (monoclonal gammopathies of uncertain signicance), whereas a few patients who have HCV with monoclonal gammopathy can be considered as aected by myeloma according to their clinicopathologic characteristics [69,77,78]. Pathogenesis of hepatitis C virus-related lymphoproliferative disorders It now is accepted widely that the pathogenesis of LPDs is a complex, multistep process. Several studies investigating the pathogenetic mechanisms involved in the evolution of HCV infection to B-cell LPDs are available, but in spite of interesting hypotheses, the exact mechanisms involved are not known. Originally, the observation of HCV lymphotropism at the beginning of the 1990s led to the hypothesis of a causal link between infection of lymphatic cells and autoimmunelymphoproliferative disorders [79]. Earlier, it was observed that both HCV-positive strand (genomic) and -negative strand (antigenomic replicative intermediates) could be detected in peripheral blood mononuclear cells (PBMC) taken from patients who had chronic HCV infection [80]. During the past decade, ex vivo and in vitro studies with techniques of increasing specicity and sensitivity have led to better characterization of HCV lymphotropism [8187]. Nonetheless, no clear proof of a direct link between HCV lymphotropism and LPD pathogenesis has been obtained, despite the demonstration of a more extensive involvement of the lymphatic system by HCV infection in patients who have B-cell LPDs than those who do not [5,88,89], and of an enhancing eect of B-cell infection by HCV in promoting the proliferation of lymphoid cells [90]. By contrast, several studies have supported an indirect role of HCV through activation of the hosts immune response [37,9194]. The

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importance of a sustained antigenic stimulation by viral epitopes and of the specic binding between the HCV E2 protein and the CD81 molecule has been stressed [95], supporting the key role played by the promotion of a strong polyclonal B-cell response to chronic viral infection that progressively would favor lymphomagenesis until nal malignant transformation. Contrasting data, showing the possibility that HCV may favor mutations of immunoglobulin genes and oncogenes by a hit and run mechanism, were obtained in cell lines and in cultured cells taken from patients with HCV [96]. This analysis originated from previous observations showing a signicant association between Bcl-2 rearrangement (14;18 translocation) and chronic HCV infection, especially in those subjects who developed type 2 MC [97 102], and MALT lymphoma [103]. In patients who had type II MC, the analysis of synchronous and metachronous blood samples showed the clonal expansion of B-cells harboring this chromosomal rearrangement [99]. In addition, it was possible to demonstrate an overexpression of the antiapoptotic Bcl-2 protein with a higher bcl-2/bax ratio in t (14;18)-positive B-cell samples [99], and a modication of t (14;18) B-cell clone detection following antiviral treatment [104]. Only treatments leading to sustained clearance of HCV RNA from serum led to the regression of clones, with consequent lack of t (14;18)-positive cells in PBMC at the end of treatment, whereas this eect was not observed in nonresponder patients [104]. An extensive follow-up of HCV-positive MC patients after sustained virological response to IFN-based treatments has revealed the possibility of viral persistence in the lymphatic compartment in the absence of serum HCV RNA or liver infection. Interestingly, isolated lymphatic infection was associated strictly with the persistence of both MC-syndrome and t (14;18)-bearing B-cell clones. These observations rearm, after more than a decade since the original suggestions, the strong likelihood of a role played by HCV lymphotropism in lymphomagenesis, but the exact mechanisms involved remain unclear. On the other hand, they strongly suggest that the pathogenesis of MC is not necessarily related to the intrahepatic challenge between HCV epitopes and lymphoid inltrates. Considering the complexity of LPDs and the heterogeneity of HCV-associated LPDs, it is conceivable that MC and other HCV-related LPDs may have dierent pathogenetic pathways. An accurate understanding of these dierent pathways and the frequency of their involvement in the pathogenetic mechanisms would be essential for the correct appraisal of both therapeutic and preventive measures [105,106]. An attempt to summarize current knowledge of the interrelations between HCV and LPDs is presented in Fig. 1. Treatment of hepatitis C virus-related mixed cryoglobulinemia and lymphoproliferative disorders Before the identication of its viral etiology, MC treatment included a variable combination of anti-inammatory and immunosuppressive

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+
HCV
HCV E2 /CD81 binding B-cell infection Likelihood of reversion after HCV eradication
HCV-induced mutagenesis

Sustained Bcell activation

t(14;18)/others? Bcl-2 overexpression B-cell apoptosis inhibition

Genetic and/ or environmental factors

Prolonged BL survival

MC

Additional genetic aberrations

Malignant NHL

Fig. 1. Pathogenesis of hepatitis C virus-related lymphoproliferative disorders: an evidencebased hypothesis.

strategies. Soon after linking chronic HCV infection to MC, several studies were carried out to assess the eect of interferon (Table 1). Interferon monotherapy initially was used, sometimes in association with corticosteroids (CS) [24,26,107116], and resulted generally in eective improvement in MCS, even if associated with a very high relapse rate after discontinuation of therapy (see Table 1). In later studies, the combination of interferon with ribavirin (RBV) oered better results than interferon monotherapy [117 120]. Further improvement in the sustained virological response (SVR) rate was obtained by the introduction of pegylated interferons (IFNs) [121123] (see Table 1). Additional controlled studies, however, are needed to gain denitive information. Interestingly, all available studies show that clinicoimmunologic and virologic responses generally are related [112,117,119,122124]. The persistence of isolated lymphatic infection after therapy was associated with persistence of MC syndrome stigmata [105]. Disappearance of BL monoclonal inltrate from bone marrow and BL expansion in peripheral blood following IFN therapy also has been shown. In particular, the antiviral response was proven to be signicantly associated with the absence of circulating B-cell clones bearing t (14;18) translocation [99,104,125]. The

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Table 1 Antiviral therapy in hepatitis C virus-related mixed cryoglobulinemia Author Ferri Ferri Marcellin Johnson Misiani Year 1993 1993 1993 1993 1994 Number of patients 15 26 2 4 27 Treatment Interferon 2 MIU/d (1 m)2 MIU three times weekly (5 m) (CS) Interferon 2 MIU /d (1 m)2 MIU three times weekly (5 m) (CS) Interferon 3 MIU three times weekly Interferon 110 MIU Interferon 1.5 MIU three times weekly (1 w)3 MIU three times weekly (23 w) Interferon 3 MIU three times weekly Interferon 3 MIU three times weekly (CS) Variable interferon Interferon 3 MIU three times weekly Interferon 3 MIU three times weekly Interferon 3 MIU three times weekly Interferon 6 MIU three times weekly Interferon 3 MIU three times weekly Interferon 35 MIU three times weekly Interferon 3 MIU/d (3 m)3 MIU three times weekly (R9 m) RBV Interferon 3 MIU three times weekly Interferon 3 MIU three times weekly RBV Interferon 3 MIU three times weekly RBV Treatment duration (months) 6 6 6 212 6 End of treatment 80% 100% 50% 75%a 60% 0 Response sustained

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Dammacco

1994

15 16 14 18 18 18 25 20 20 31 5 NR 18 8 NR 9 NR

12 12

53.3% 52.9% 0a 28% 28% 39% 52%b 60%c 65%b 62% 100% 55% 63% 78%

25% 33.3%

Johnson Mazzaro Mazzaro Casaril Cohen Akriviadis Casato Durand Calleja

1994 1994 1995 1996 1996 1997 1997 1998 1999

6 12 6 612 R12 1036 12 12 6

11% 22% 9%c 33%b

0% 28% 38%

Zuckerman

2000

Cacoub Mazzaro Alric Cacoub Saadoun

2002 2003 2004 2005 2006

14 27 NR or Rel 18 9 32 40

Variable interferon RBV ( variable CS) Interferon 3 MIU three times weekly RBV Interferon 3 MIU three times weekly or pegylated interferon RBV Pegylated interferon 1.5 mg/kg/w RBV Interferon 3 MIU three times weekly RBV Pegylated interferon RBV

656 12 R18 R10 R6 85%

71%

70% 88%
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67.5% 56.3%

Kidney function improvement. Cryoglobulins disappearance. c Both complete and partial mixed cryoglobulinemia syndrome response. Abbreviations: CS, corticosteroids; d, daily; MIU, millions of international units; m, months; NR, nonresponders; RBV, ribavirin; Rel, relapsers; w, week. Data from: Zignego AL, Giannini C, Ferri C. Hepatitis C virus-related lymphoproliferative disorders: an overview. World J Gastroenterol 2007;13:246778.
b

621

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reappearance of circulating translocated BL clones after virological relapse at the end of treatment, and their persistent detection in subjects with unmodied viral load after antiviral therapy, strongly indicates that clonal expansion of translocated cells depends on modications of viral replication induced by antiviral treatment [104,125]. More recently, long-term analysis of patients with HCVand MCS showing SVR after therapy indicated that occult lymphatic infection and persistence of MCS stigmata also were associated with persistent determination of expanded t (14;18) carrying B-cell clones [105]. These data suggest the critical role played by a complete viral eradication and its possible role in preventing the evolution of LPD. An algorithmic approach to the treatment of HCV-related MCS is outlined in Fig. 2. Overall, available data show that combination antiviral treatment with pegylated interferon and RBV should be considered as the rst option in subjects who have HCV-positive MCS. Interferon-based antiviral treatment in HCV patients who have MCS is usually more complex to handle than in patients without MC for several reasons, including: the absence of standardized treatment protocols, the frequent presence of contraindications, and the diculties in the accurate interpretation of results. Biochemical markers of MC response (cryocrit, RF, or complement values) may be related less strictly to virological response than alanine aminotransferase (ALT) levels. This may conrm the importance of a multistage pathogenetic

Assessment of MCS and of contraindications to IFN-based therapy


No contraindications and mild/moderate MCS

Contraindications to IFN and/or severe MCS

Mild-moderate purpura Weakness Arthralgias Mild neuropathy

Moderate-severe MPGN, skin vasculitis or Severe-rapidly progressive MPGN Severe sensomotor neuropathies Widespread vasculitis

PEG IFN plus ribavirin*


Rituximab LAC diet Low-medium doses CS +/- other sympto matic treatment PE + CS +/-CF
se ve re

SR

NR

NON RESPONDERS

RESPONDERS

Antiviral therapy ?

Fig. 2. An algorithmic approach to the treatment of hepatitis C virus-positive mixed cryoglobulinemia (MC). *Schedule as for non-MC patients. Abbreviations: CS, corticosteroids; CF, cyclophosphamide; NR, no MCs response; PE-plasma exchange; SR, sustained MCs response.

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mechanism in MCS, suggesting the need for precise monitoring of this category of patients and the denition of predictive markers. For patients in whom antiviral treatment is contraindicated or is not tolerated or in patients who are nonresponders, alternative therapeutic approaches should be used, CS, immunosuppressive drugs, Non-Steroidal Anti-Inammatory Drugs (NSAIDs), plasmapheresis (PE) and a hypoantigenic diet (low antigen content [LAC] diet) administration [41,126]. Treatment should be tailored to the individual patient according to the severity of clinical symptoms, also considering the possible additional factors involved (age, renal failure, comorbidities), and the time (weeks or months) required for symptom remission. Corticosteroids represent the most commonly used nonantiviral therapy for MCS and generally allow the control of most MC symptoms even at low doses. They may favor HCV replication, however, induce several adverse eects, and do not signicantly modify the natural history of the disease. Cytostaticimmunosuppressive drugs (ie, cyclophosphamide, chlorambucil, and azathioprine)may be used, especially during the acute phases of MCS, but they may cause severe adverse eects [127]. A special note must be made of new B-cell specic immunosuppressive therapy based on the use of chimeric antibodies (rituximab) against the CD20, a B-cell specic surface antigen [128,129]. Rituximab is eective in most patients who have MC, leading to marked improvement or resolution of the syndrome, especially of skin lesions, and to regression of the expanded B-cell clones [128,129]. This therapeutic approach appears to be very promising for managing patients who have MCS, but future controlled studies still are required to establish its ultimate role in treating HCV-related MCS. It should be stressed that rituximab, as an immunosuppressive agent, leads to an increase in HCV replication, but no signicant reactivation of HCV-related liver disease has been reported. Its use in combination with direct antiviral molecules [41,128131], when available, probably will lead to further improvement in management. Other therapeutic measures include plasma exchange (PE) and LAC diets. PE (ie, the apheretic removal of circulating immunocomplexes) specically is indicated in the presence of clinically signicant acute manifestations (cryoglobulinemic nephritis, severe sensorimotor neuropathies, cutaneous ulcers, and hyperviscosity syndrome). The LAC diets, generally prescribed at the initial stage of MCS, essentially act by reducing the antigen load to the reticulo endothelial system, thus allowing a more ecient removal of CGs. Recent studies, performed in specic subgroups of patients with HCVassociated NHLs, such as marginal zone lymphomas [73,132], support the rationale for the use of antiviral therapy also in the setting of malignant HCV-associated LPDs. In the study by Hermine and colleagues [73], a complete remission of Splenic Lymphoma with Villous Lymphocytes (SLVL) was observed in most HCV-positive patients but in none of HCV-negative cases following treatment with IFN. In addition, regression of clonal proliferation in response to antiviral treatment was shown to be

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associated clearly with virological response [99,104,125]. Seemingly only a proportion of HCV-associated lymphomas are cured with antiviral therapy. Also in responsive patients who had SLVL, the rearrangement of the monoclonal immunoglobulin genes persistently was detected in the blood even after a complete hematological response [75]. Such data suggest that the multistep lymphomagenetic cascade may have points of no-return, making the LPD progressively independent from HCV infection (see Fig. 1). Although antiviral therapy appears to be an attractive therapeutic tool for low-grade HCV-positive NHL, in intermediate and high-grade NHL, chemotherapy is most likely to be necessary while antiviral treatment possibly could represent a maintenance therapy [133]. Further studies are needed to standardize antiviral therapy better in HCV-associated NHL. The use of rituximab in HCV-associated NHL, in monotherapy or in combination with antiviral treatment and/or chemotherapy, appears very promising, particularly in the setting of low-grade NHL, where rituximab monotherapy has been proposed as rst-line treatment [134136]. In spite of the limited number of described cases, rituximab may be considered a safe and eective therapy for HCV-related indolent B-cell lymphoma.

Other extrahepatic disorders and overlap syndromes Many and dierent disorders have been linked to HCV infection. In most cases, because of possible methodological bias, mainly in patient selection in the various studies, it is dicult to verify whether the suggested association is coincidental or whether a real pathogenetic link exists. Several conditions are observed more frequently in the context of an MC and quite rarely as idiopathic forms. This is the case of skin, kidney, salivary glands, or lung disorders. In addition, a clinicoserological overlap between dierent EHMs-HCV often is observed [32,137,138]. In spite of the heterogeneity of the morbid conditions that have been linked to HCV, two issues are quite constant: the pathogenetic involvement of the hosts immune system and the symptom severity with consequent worsening of quality of life [139]. In addition, extrahepatic manifestations may be observed variably in the presence or absence of hepatic damage. On the whole, these observations support the view of HCV infection as a distinct systemic disease with widely variable clinical expressions. Dermatopathologic manifestations In addition to MC-related purpura, HCV infection also has been associated with several cutaneous disorders, including the sporadic variant of porphyria cutanea tarda (PCT) [1,140143], a metabolic disorder characterized by reduced hepatic activity of uroporphyrinogen decarboxylase, and with oral lichen planus (OLP) [144154]. A strong association between the

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sporadic form of PCT and HCV was suggested by the high prevalence (greater than 50%) of HCV markers in these patients, mainly in studies from southern Europe [1,140143]. In HCV-positive patients without PCT, however, no signicant alteration in porphyrin metabolism was shown [142,155,156]. Epidemiologic studies also proved the existence of a correlation between OLP and chronic HCV infection [144]. The prevalence of HCV infection in a large population of patients who had OLP was about 27% [145]. Data supporting this correlation essentially stem from studies performed in Japan [144] and southern Europe [145148], but these were not conrmed in other populations [149152]. It generally is agreed that in these cutaneous disorders, HCV infection plays an indirect role, probably acting as a triggering factor in genetically predisposed individuals. As a matter of fact, attempts to cure these disorders with antiviral therapy led to discordant, but generally negative results with the possible induction of a clinical manifestation in previously unaected patients or worsening of the disease [33]. Nephrological disorders A causative association between HCV and non-MC related forms of renal damage (membranoproliferative glomerulonephritis without simultaneous presence of cryoglobulins, or membranous nephropathy) has been suggested, but requires conrmation [33]. Neurologic disorders Neurologic complications have been associated with HCV infection mostly in the context of MC, but also in the absence of this condition [157,158]. Only cryoglobulinemic neuropathy, however, has been associated clearly with HCV infection. Disorders of the joints, bones and muscles HCV-related chronic polyarthritis can be observed in HCV-positive patients both with and without MC [13,159]. True rheumatoid arthritis (RA), in keeping with classic criteria, seems to be uncommon in subjects who have HCV. In patients who have HCV, tests are usually negative for antibodies to cyclic citrullinated peptides (anti-CCP), which may help to differentiate the two conditions (true RA and HCV-related RA-like form). By contrast, intermittent oligoarthritis, generally not erosive and involving the big and middle-sized joints, is observed frequently [13,32,159]. An association of HCV infection with osteosclerosis and a correlation between such manifestation and the imbalance in the osteoprotegerin/RANKL (Receptor Activator of NF-kB Ligand) system with a predominance of osteoprotegerin also were observed in some studies [160,161]. HCV infection also was suggested to be associated with myositis and dermatomyositis [162].

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Endocrinologic disorders Practically all known manifestations of a thyroid disorder have been described in patients who have HCV infection, but frequently with discordant data [163167]. Geographic, genetic, or environmental cofactors [168171] and dierent methodological approaches partially explain discordant observations. The most frequently observed HCV-associated thyroid disorder involves circulating antithyroid peroxidase antibodies in female subjects [166]. Subclinical hypothyroidism was observed in 2% to 9% of patients who had chronic HCV infection and particularly in those who had MC [2,165,172]. Finally, a higher prevalence of papillary thyroid carcinoma in patients who had HCV was observed [173175]. Interferon alfa therapy may exacerbate or induce underlying latent thyroid disorders, and the relative contribution of the role played by HCV or by antiviral therapy in leading to such associations has been discussed [166,176178]. Recently, it was suggested that molecular mimicry between viral and self-antigens might be involved in the pathogenesis of HCV-associated autoimmune thyroid diseases [179]. A high prevalence of diabetes mellitus type 2 was observed in patients who had chronic HCV infection in several studies [180187], and it has been suggested that HCV acts as a risk factor independently of liver disease [188]. In patients who had HCV infection, the appearance of diabetes type 2 was associated with insulin resistance and was considered part of a complex virus-induced metabolic syndrome including both hepatic (steatosis) and extrahepatic manifestations. In agreement with this, an association between carotid atherosclerosis and HCV infection recently has been suggested [189,190]. Lung and cardiocirculatory disorders A pathogenetic link between HCV infection and idiopathic pulmonary brosis has been reported [191,192]. The fact that MC can be complicated by an involvement of pulmonary interstitium suggests that such association in some cases can be related to a pre-existing MC [193]. An association between HCV and hypertrophic cardiomyopathy was suggested by the observation of signicantly higher prevalence of anti-HCV in Japanese patients with such condition compared with controls [194]. Studies performed in Italy and in Greece by other authors, however, did not conrm this association. Regardless, the recent determination of a signicantly higher prevalence of carotid atherosclerosis in patients with HCV infection [189] is noteworthy; this association was mostly evident in case of active viral replication [190]. In a very recent study, the association between HCV chronic infection and carotid atherosclerotic lesions was conrmed also in a large Italian population. Further, HCV RNA sequence was determined in carotid plaques, strongly suggesting a local proatherogenetic action of the virus inside the plaque [195].

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Psychopathological disorders Patients who have HCV infection have a low quality of life and may experience excessive fatigue, decreased cognitive ability, and low mood tone [196198]. These symptoms are not related directly to liver damage, and it has been proposed that the virus may cause direct cerebral dysfunction by an unknown mechanism [197]. Recently, plasma tryptophan and kynurenine content in blood, together with indoleamine 2,3-dioxygenase activity in macrophages, was evaluated in a cohort of patients who had mild HCV-related chronic liver disease. Patients also underwent psychopathological evaluation. Serum tryptophan concentrations were lower than those of healthy subjects or patients who had chronic HBV infection, and were associated with high levels of anxiety and depression, strongly suggesting that these modications may be causally related. In addition, mechanisms involved in the pathogenesis of HCV-associated reduced tryptophan levels appeared dierent from those observed in other chronic infections, thus possibly representing a new model for viral-induced alterations of tryptophan metabolism [199201].

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[191] Ueda T, Ohta K, Suzuki N, et al. Idiopathic pulmonary brosis and high prevalence of serum antibodies to hepatitis C virus. Am Rev Respir Dis 1992;146:2668. [192] Ohta K, Ueda T, Nagai S, et al. Pathogenesis of idiopathic pulmonary brosisdis hepatitis C virus involved? Nihon Kyobu Shikkan Gakkai Zasshi 1993;315:325. [193] Ferri C, La Civita L, Fazzi P, et al. Interstitial lung brosis and rheumatic disorders in patients with hepatitis C virus infection. Br J Rheumatol 1997;36:3605. [194] Matsumori A, Ohashi N, Hasegawa K, et al. Hepatitis C virus infection and heart diseases: a multicenter study in Japan. Jpn Circ J 1998;62:38991. [195] Boddi M, Abbate R, Chellini B, et al. HCV infection facilitates asimptomatic carotid atherosclerosis: preliminary report of HCV RNA localization in human carotid plaques. Dig Liver Dis 2007;39:549. [196] Dwight MM, Kowdley KV, Russo JE, et al. Depression, fatigue, and functional disability in patients with chronic hepatitis C. J Psychosom Res 2000;49:3117. [197] Forton DM, Thomas HC, Murphy CA, et al. Hepatitis C and cognitive impairment in a cohort of patients with mild liver disease. Hepatology 2002;35:4339. [198] Hilsabeck RC, Perry W, Hassanein TI. Neuropsychological impairment in patients with chronic hepatitis C. Hepatology 2002;35:4406. [199] Cozzi A, Zignego AL, Carpendo R, et al. Low serum tryptophan levels, reduced macrophage IDO activity and high frequency of psychopathology in HCV patients. J Viral Hepat 2006;13:4028. [200] Korsmeyer SJ. BCL-2 gene family and the regulation of programmed cell death. Cancer Res 1999;59:1693s700s. [201] Tighe H, Warnatz K, Brinson D, et al. Peripheral deletion of rheumatoid factor B cells after abortive activation by IgG. Proc Natl Acad Sci U S A 1997;94:64651.

Clin Liver Dis 12 (2008) 637659

Hepatitis C and Liver Transplantation: Enhancing Outcomes and Should Patients Be Retransplanted
Elizabeth C. Verna, MDa, Robert S. Brown, Jr, MD, MPHb,*
a Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Medical Center, 622 West 168th Street, New York, NY 10032, USA b Division of Abdominal Organ Transplantation, Columbia University College of Physicians and Surgeons, 622 West 168th Street, PH14, New York, NY 10032, USA

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease (ESLD) worldwide and the most common indication for orthotopic liver transplantation (OLT) in the United States and Europe [1]. In contrast to most other leading indications for OLT, serologic and histologic recurrence of HCV after OLT is nearly universal. Death and allograft failure are more common in this population when compared with HCV-negative recipients [2], and HCV recurrence is the major source of graft failure. The natural history of HCV disease is clearly accelerated in the posttransplant setting, leading to cirrhosis in 10% to 25% of patients within 5 to 10 years [3]. Once cirrhosis has developed, complications are common because more than 40% of patients who have recurrent cirrhosis develop manifestations of decompensated disease within 1 year and less than 50% of patients survive 1 year after the onset of decompensation [3]. Compounding this problem, potent immunosuppressive agents and donor shortages leading to the use of older donors may be impeding improvements in outcomes and, some believe, worsening survival over time [4]. The donor pool has gradually expanded with the implementation of living donor liver transplantation (LDLT) and the use of extended criteria donation (ECD), including HCV-positive donors, older donors, steatotic

* Corresponding author. Center for Liver Disease and Transplantation, Columbia University Medical Center, 622 West 168th Street, PH14, NY 10032. E-mail address: rb464@columbia.edu (R.S. Brown). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.010 liver.theclinics.com

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livers, and donation after cardiac death (DCD). The use of these grafts remains controversial, particularly in HCV-positive recipients, in whom they could lead to accelerated brosis. Even with these attempts to increase organ availability, a profound discrepancy between supply and demand persists. Recurrent HCV and rejection remain the main obstacles in the posttransplant setting, and the balance between over- and underimmunosuppression to minimize the risk for rejection and recurrence remains dicult. Measures to prevent and control recurrent HCV with interferon (IFN) and ribavirin (RIBA) antiviral treatment continue to be disappointing, and data from properly designed randomized controlled trials to determine the best dosing and duration of treatment are lacking. In addition, although the risks of potent immunosuppression in the setting of recurrent HCV are now well documented, much remains unknown about the optimal induction and maintenance immunosuppression regimen in HCV-infected patients. Progressive allograft failure in this population is therefore common, and the debate continues as to who should be considered for retransplantation and when this evaluation should occur. Thus, optimizing outcomes in liver transplantation for HCV-related ESLD is of primary importance to the liver transplant community and to most of their patients. The strategies to improve outcomes discussed in this article include increasing access to adequate numbers of suitable liver grafts, improving antiviral treatment before and after transplantation, enhancing immunosuppressive strategies such that recurrent HCV is minimized, and regulating the use of retransplantation when these treatments fail.

Enhancing outcomes in transplantation for hepatitis C virus Optimizing the timing of transplantation Maximizing the benet that any patient derives from liver replacement depends on the timing of the procedure in many ways. This is especially important in the case of HCV infection; unlike the case in other liver diseases, progression is clearly more rapid in the posttransplant setting. The survival benet of transplantation may therefore be diminished when premature transplantation occurs such that the posttransplant and operative mortality outweighs the expected pretransplant liver disease-related mortality. Delaying transplantation, and therefore recurrent HCV, may add life-years and time for the development of new antiviral therapy, but this must be weighed against the risks for waiting, including the risks for complications, hepatocellular carcinoma (HCC), or becoming too sick for transplantation. Patients with Model for End-Stage Liver Disease (MELD) scores less than 15, and particularly with scores less than 12, do not derive a statistical survival benet in the rst year after transplantation [5]. The degree of survival benet increases with each increase in MELD for scores greater than 15. Although the posttransplant risk for dying is increased by 50%

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in patients with MELD scores greater than 30, these outcomes may still be reasonable, given the greater than 300-fold increase in pretransplant mortality in this population. Specic donor and recipient risk factors for poor outcomes are also taken into consideration, and the rate of waiting list removal is higher for the sickest patients. Transplant physicians and surgeons incorporate a wide variety of factors in the decision to transplant, and there is no absolute MELD score cuto for transplant futility. The optimal time for OLT is therefore when a patient achieves a MELD score of at least 15 or begins to show evidence of decompensation, manifested by synthetic dysfunction, malnutrition, or refractory ascites. This seems to be particularly true for the patient who has HCV, except perhaps in the setting of antiviral therapy with clearance of HCV. Safely expanding the donor pool The growing disparity between the number of deceased donor organs available and the patients awaiting transplantation has driven the expansion of the donor pool to include what has been termed extended criteria donation, the precise denition of which remains elusive. In general, ECD livers may have a wide range of characteristics traditionally thought to be undesirable for donation, including LDLT or split liver grafts, DCD, older donors, graft steatosis, prolonged cold ischemia time, human T-lymphotrophic virus (HTLV)-1 positivity, high-risk behavior in the donor, and HCV or hepatitis B core antibody (HBcAb) positivity. The use of these grafts varies widely between transplant centers, and because of limited data and the perceived risks of a potentially suboptimal graft, decisions are made on an individual basis. To date, long-term data in patients transplanted with specic types of extended-criteria grafts are lacking, and the available literature is dicult to interpret because the recipients frequently have less optimal characteristics before transplantation, and thus are not comparable to those who receive standard MELD-allocated grafts. When ECD grafts are grouped together, they generally have comparable outcomes to non-ECD grafts in HCV-infected recipients [6,7], although there is some evidence to the contrary [8]. One large single-center cohort tallied the number of unfavorable characteristics of each graft and showed that outcomes in patients who have all forms of liver disease are likely worse as the extended criteria score increases [8]. The specic aspect of each graft that makes it high-risk is extremely variable, however, and grouping these heterogeneous grafts and recipients is unlikely to reveal clinically useful information. Even in these large heterogeneous studies, using ECD grafts may maximize access to OLT, decrease wait time, and potentially decrease pretransplant mortality [6,7]. Of these factors, advanced donor age seems to be among the strongest predictors of poor outcomes in HCV-infected recipients. Although grafts from donors between the ages of 60 and 80 years function without any survival disadvantage in nonHCV-infected patients [9,10], several series report

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more severe recurrence and more rapid progression to cirrhosis when older donors are used in the setting of HCV [1114]. One report even suggests that the increased risk for graft dysfunction begins with donor age older than 40 years [11]. This has led some centers to try to avoid using grafts from donors with advanced age in HCV-infected recipients. The same may be true to a lesser extent with donor liver grafts that have excessive steatosis or prolonged ischemia time. Liver grafts from donors who have HCV and hepatitis B virus (HBV) infection have traditionally been considered contraindicated in OLT. Early data showed the risk for HBV transmission to be high in recipients of isolated HBcAb-positive grafts [1517]. More recent work in the era of hepatitis B immunoglobulin (HBIG) and HBV antiviral treatments, such as lamivudine, has shown that HBcAb-positive grafts have similar outcomes to those of patients who had HBV and were transplanted with HBVnegative grafts [1821]. These patients generally receive prophylaxis with lamivudine with or without HBIG, which, in most cases, can prevent graft infection [2225]. There is no consensus, however, on how long patients should remain on therapy, and this may depend on the recipients HBV antibody status [26,27]. HCV-positive grafts are also being used increasingly, almost universally in HCV-positive recipients, and no survival disadvantage has been seen when HCV-infected recipients receive HCV-positive grafts [2835]. The largest series to date is a retrospective study of the United Network for Organ Sharing (UNOS) database, including 96 HCVinfected patients who received HCV-positive grafts, and survival was equivalent to that of patients who received HCV-negative grafts. Grafts with HCV and HBcAb have also been used, and in one small series, also did not signicantly decrease patient survival, but this nding must be conrmed [29]. The use of HCV-positive liver grafts is an important additional graft source to HCV-positive patients on the transplant waiting list. DCD, previously known as nonheart-beating donation, was initially thought to be a risk factor for poor outcomes in HCV. It has regained favor, however, in the setting of new data reporting outcomes similar to those for traditional brain death donation [3638]. When compared in the UNOS database of all patients transplanted for any type of liver disease, there was a 3-year graft survival of 63% in the DCD group compared with 72% in the brain death group, which did not reach statistical signicance [36]. To date, there are no studies looking specically at survival or rate and severity of HCV recurrence in this population, and additional investigation is needed before rm conclusions on its impact on disease recurrence can be drawn. Adult-to-adult LDLT has been performed in the United States since 1998 [39]. Living donor organs may have theoretic advantages over deceased donor organs because they have signicantly less cold ischemia time and are from medically optimized donors. In addition, the reduced waiting period and optimal timing of transplantation aorded by living donation

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may decrease the risk for decompensation and death before transplantation and improve patient survival. The mortality in patients with potential donors for LDLT is half that of those listed for deceased donor liver transplantation (DDLT) only [40,41]. In addition, antiviral treatment may be administered before a deliberately timed transplantation, and it seems that LDLT while the recipient is HCV RNA-negative leads to a low rate of viral recurrence (10%20%) [42,43]. Despite these theoretic benets, LDLT grafts have been used with caution in HCV-infected recipients. Early data on the use of LDLT grafts in this population revealed a signicantly higher incidence of cholestatic hepatitis C, a rapidly progressive form of recurrent HCV [44]. In addition, an early study from Spain showed a statistically signicant increase in cirrhosis and clinical decompensation at 2 years in the LDLT group [45]. These ndings fueled concerns that the accelerated hepatocyte proliferation in split liver transplantation may predispose to more aggressive recurrence of HCV. Several recent large studies, however, have shown that there is no dierence in the incidence or severity of HCV recurrence when LDLT and DDLT are compared [4651]. Careful pathologic evaluation of protocol liver biopsies over 3 years after OLT in 23 LDLT patients and 53 DDLT patients found no signicant dierence in inammation and similar or less brosis in the LDLT group [47]. Most recently, data from the multicenter study named the Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL) on 275 transplants (181 LDLT and 94 DDLT) showed an overall statistically signicant survival advantage when DDLT was compared with LDLT (82% versus 74% at 3 years) [51]. Because of previous data suggesting worse outcomes in the rst 20 LDLTs done at a given center [52], however, these patients were then separated into three groups: DDLT, the rst 20 cases of LDLT at each center, and then all subsequent LDLT cases. There remained a statistically signicant dierence in survival between the DDLT group and the group comprising the rst 20 LDLT cases, but there was no dierence in survival or rate of progression to brosis between the DDLT and later LDLT cases (Fig. 1). It is therefore possible that the early reports of poor outcomes were a function of inexperience with the surgical and postoperative management and that LDLT is likely to be as safe as DDLT in HCV-infected patients. Currently, less than 5% of all adult liver transplants use living donor grafts, and this rate has declined because of the early concerns about severe recurrence, possible exhaustion of the eligible and interested donor and recipient pool, and the two highly publicized donor deaths that occurred between 2001 and 2003 [39,53,54]. Donor evaluation can also be costly and prolonged. In experienced centers, approximately one third of adult patients on the waiting list have a potential donor, and half of these actually donate, indicating that LDLT may be possible in up to 15% of patients evaluated [55,56]. LDLT may therefore be increasingly important in the expansion of the donor pool, but continued analysis is needed.

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Fig. 1. Graft survival after DDLT, LDLT of 20 cases or less (rst 20 cases at each center), and LDLT of greater than 20 cases (cases beyond the rst 20 at each center). Graft survival was signicantly lower in LDLT of 20 or less cases compared with LDLT of greater than 20 cases (P .0023) and DDLT (P .0007). There was no signicant dierence in graft survival between LDLT greater than 20 cases and DDLT, however (P .66, log rank test). (From Terrault NA, Shiman ML, Lok AS, et al. Outcomes in hepatitis C virus-infected recipients of living donor vs. deceased donor liver transplantation. Liver Transpl 2007;13:125; reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)

Antiviral therapy for the prevention and treatment of recurrent hepatitis C virus Recurrent HCV remains the leading cause of posttransplant graft failure. Histologic recurrence occurs in up to 90% of patients by 5 years [57], and although disease progression is variable, many cases rapidly progress to cirrhosis. Much work has been done to identify factors predictive of severe recurrence, and the donor characteristic with the strongest impact on outcomes seems to be donor age. Recipient factors, such as HCV viral load before transplantation [4,58], recipient cytomegalovirus (CMV) status [13,59,60], advanced recipient age, severe hyperbilirubinemia, and elevated international normalized ratio (INR) [59] before transplantation, also predict poor graft survival. Many of these risk factors, however, are not easily modiable, and treatment of HCV infection before and after OLT remains among the biggest challenges in liver transplantation. Three antiviral treatment strategies have been attempted, all with limited success: curative or preventive treatment before transplantation, preemptive therapy after transplantation but before documented histologic recurrence, and treatment of established recurrent disease. The ideal strategy would be curative therapy before transplant, which could prevent recurrent disease, but it is successful in few patients on the transplant waiting list. Data supporting a benet from preemptive therapy are limited. Thus, most current

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recommendations focus on treatment of established recurrent disease [61]. Unfortunately, enhanced viral replication in the setting of immunosuppression, the high proportion of patients who have genotype 1 or virus unresponsive to IFN-based therapies before transplant, and the poor tolerability of IFN and RIBA regimens render the treatment of recurrent HCV clinically challenging. There is currently no widely accepted and eective pre- or post-OLT treatment regimen, and additional investigation into more eective treatment dose and duration is required before universal recommendations can be made. Individualized tailoring of treatment based on the patients HCV viral load and genotype, the patients response to treatment, and side eects and comorbidities is likely to remain a predominant theme. Pretransplantation prophylactic antiviral treatment Because HCV titers may be a signicant predictor of graft and patient outcome after transplantation [58] and patients who achieve a sustained virologic response (SVR) or who are transplanted with undetectable viral loads on therapy have only an approximately 20% likelihood of recurrence, viral eradication while on the waiting list would be ideal [43,62,63]. There are limited prospective data or randomized trials comparing regimens, however, and the results of pre-OLT therapy have been generally disappointing. For patients who have compensated cirrhosis, pegylated (peg)-IFN and RIBA were studied in the National Institutes of Health (NIH)sponsored Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C) trial and yielded end-of-treatment (EOT) and SVR rates of 23% and 11%, respectively [64]. Treatment of patients who have decompensated liver disease, however, who comprise most potential transplant recipients, has been associated with exacerbations of encephalopathy, infections, and other serious adverse events, with up to a 10% treatment-related mortality rate and low rates of SVR [65]. Initial therapy with low-dose IFN and RIBA with slow-dose escalation may improve tolerability and ecacy in patient who have compensated cirrhosis [43,66]. In the largest low accelerating dose regimen (LADR) protocol, there was a lower adverse event rate but a discontinuation rate of 27% and EOT and SVR rates of 46% and 24%, respectively [43,66]. Fifteen patients with SVR were transplanted, and 12 (80%) remained free of viral recurrence at the end of follow-up. Pretransplant viral clearance would therefore be ideal, but because of the high risk for adverse events and limited ecacy, routine treatment of patients who have decompensated disease is not recommended outside of consultation with a transplant center or a clinical trial. The combination of the LADR protocol with deliberately timed LDLT if viral clearance is achieved is currently being studied in the NIHsponsored A2ALL cohort of living donor liver transplants and may lead to HCV cure in a small but signicant number of patients. Prophylactic treatment with HCV immunoglobulin (HCIG) formulations in the peritransplant period is still in early trials but has produced

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disappointing results to date. One formulation of human HCIG completed phase II clinical trials (Civacir; Nabi Biopharmaceuticals, Boca Raton, Florida) and has been found to be safe but caused only transient decreases in HCV viral load, without prevention of graft reinfection [67]. Concerns regarding the cost and potential infectivity of pooled human HCIG preparations may limit their use, even if proven ecacious. Recombinant monoclonal immunoglobulins are currently under investigation and one series with humanized monoclonal antibody was recently published by Schiano and colleagues [68], showing similarly disappointing results. Although all patients experienced a decrease in viral load, no patient had an undetectable viral load while on therapy. Some have postulated that these results may have at least partially been attributable to low dosing of the regimen [69]. Additional studies to identify the correct dosing and the most potent viral antigen target are needed, and perhaps these antibody preparations may be most ecacious when used in combination with other antiviral treatments. Currently, however, there is no role for HCIG therapy in the management of patients in the peritransplant period. Posttransplant treatment of recurrent hepatitis C virus Posttransplant preemptive treatment before histologic conrmation of liver injury has been attempted with the hope that treatment before hepatic dysfunction could improve ecacy and tolerability. High-dose immunosuppression and worsened leukopenia and anemia in this period usually negate these benets, however. In fact, in the rst 2 months after transplantation, fewer than half of patients have the clinical and cell count stability to start IFN and RIBA [70]. When studied, standard IFN and RIBA have achieved EOT response rates of 23% to 40% and an SVR rate of approximately 20%, but discontinuation rates of about 12% to 50% [7074]. A randomized trial of peg-IFN and RIBA versus placebo achieved an SVR of only 8% with treatment, however, with a withdrawal rate of 31% [75]. IFN, peg-IFN, and peg-IFN plus RIBA have also been compared directly in a small series [70], and pooled EOT and SVR rates were 14% and 9%, respectively, with most responders in the dual-therapy group. Dose reductions were required in 85% of patients, and therapy was discontinued prematurely in more than 40% of patients despite hematologic growth factor administration. In light of the variable course of recurrent infection, concerns about early immune modulation with IFN, and disappointing trial results, the International Liver Transplantation Society recommends antiviral therapy only in patients with signicant histologic recurrence (stage II brosis) [61]. Treatment in this setting is somewhat better studied, although the optimal treatment dose and duration are unknown and therapeutic regimens may need to be tailored to individual patients instead of universally applied (Fig. 2). For the treatment of established histologic recurrence, IFN monotherapy has displayed little ecacy [7577]. IFN in combination with RIBA leads to EOT response rates of 15% to 48% and SVR rates of 7% to 26%, with

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Protocol biopsies in all patients with HCV viremia post-OLT or biopsies for flares in LFTs

G1-2 inflammation AND < stage 2 fibrosis

G3-4 inflammation or stage 2 fibrosis

Cholestatic HCV

Minimize immunosuppression and avoid bolus steroid treatment for rejection

Treat with peg-IFN and RIBA

Treat with peg-IFN and RIBA Consider indefinite treatment given high rates of relapse

SVR

No SVR

Monitor

Consider maintenance therapy

Fig. 2. Algorithm for treatment of posttransplant HCV recurrence. LFT, liver function tests.

discontinuation rates of 30% to 50% [75,7882]. Results with peg-IFN and RIBA have generally been better, with overall EOT and SVR rates of 17% to 63% and 8% to 47%, respectively [75,78,79,81,8391], but regimen tolerance remains poor. Because of the increased incidence of side eects with the addition of RIBA, many have debated whether it really adds to treatment ecacy, and randomized controlled trials comparing the two regimens are limited. In one recent small series, Angelico and colleagues [92] showed no signicant dierence in SVR; however, this included a total of only 42 patients and may not have been powered to detect a dierence. More than 40 treatment trials looking at RIBA in combination with IFN or peg-IFN were recently pooled, and the composite SVR in patients treated with IFN and RIBA was 24% versus 27% in the peg-IFN plus RIBA group [93]. Pooled discontinuation rates were 24% and 26%, respectively. Despite these variable results, when SVR is achieved, it does seem to improve graft survival [94], and viral eradication should be attempted in patients who have signicant liver injury without contraindications. The most important predictors of treatment success are likely to be early response to therapy and dose adherence [89,91]. In the absence of SVR, treatment may achieve modulation of disease severity and prevent brosis, which remain important secondary goals. In one small trial, patients with mild recurrence (stage 12 brosis) were randomized to treatment with peg-IFN and RIBA or no treatment, and progression of brosis was signicantly more common in the untreated group [95]. Several investigators have demonstrated histologic benet of treatment in the absence of complete viral suppression, and despite the lack of

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controlled data showing improved patient and graft survival, some advocate the use of maintenance therapy. The expense of prolonged treatment in the setting of poor outcomes has led some to question the cost-eectiveness of antiviral therapy for recurrent HCV disease. Markov-based decision models have been used to model treatment with combination IFN and RIBA and found treatment to be cost-eective [96]. Treatment of 100 men with recurrent HCV disease would prevent 29 cases of recurrent cirrhosis and 7 deaths and have a cost-eectiveness ratio of more than $29,000 per year of life saved in this study. Therefore, patients with documented signicant histologic HCV recurrence should be considered for antiviral therapy, but additional work to nd the best regimen is needed and the emergence of the HCV enzyme inhibitors is eventually likely to alter treatment dramatically. Optimizing immunosuppression The delicate balance between adequate immunosuppression and control of HCV replication remains an area of controversy and research. Although the proportion of patients being transplanted for HCV is increasing, there is evidence that outcomes in this population have not improved with time and may even be worsening [4]. The increased potency of the immunosuppression routinely used has been implicated in this nding. In addition, because almost all patients have some degree of recurrent hepatitis with portal inammation, the conclusive dierentiation of rejection plus HCV from HCV alone is dicult and hinders accurate research conclusions and clinical decision making. This distinction is crucial, however, given the risk that treatment for each process poses to the other. Episodes of acute rejection clearly predict diminished survival in the setting of HCV, and treatment of acute rejection has been associated with diminished survival in HCV-positive but not HCV-negative recipients [97]. The impact of immunosuppression on HCV recurrence is most pronounced when high-intensity regimens are used to combat acute rejection [98101]. HCV-induced graft dysfunction, cirrhosis, and severe cholestatic hepatitis are all more common in recipients who receive high-dose bolus steroids, anti-lymphocyte, or interleukin (IL)-2 receptor antibody preparations for the treatment of acute rejection [58,102]. Pulsed intravenous steroid therapy is associated with a 4- to 100-fold increase in HCV RNA [103] and increased severity of histologic recurrence. As a result, many centers now initially treat mild acute rejection with modulation of immunosuppression by maximizing the dose of or substituting the calcineurin inhibitor or reintroduction of mycophenolic acid rather than bolus dose steroids. Data to support or discourage the use of any specic induction or maintenance regimen are less convincing. In fact, any association between clinical outcomes and one particular immunosuppression medication remains dicult to prove, given the multiple regimens used and the changes in regimen

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in an individual patient over time. In addition, most trials focus on rejection or short-term survival without meticulous documentation of HCV activity and detailed histologic data. In general, the standard immunosuppressive regimen typically includes a calcineurin inhibitor (tacrolimus or cyclosporine), a tapering dose of corticosteroids, and, in some cases, a lymphocyte antiproliferative agent (mycophenolate mofetil [MMF] or azathioprine). Much work has been done to identify the best choice of calcineurin inhibitor. Cyclosporine seems to have in vitro antiviral activity [104], but in vivo viral suppression is not evident when patients treated with cyclosporine or tacrolimus are compared [105]. Most studies have shown that the severity of HCV recurrence is similar in cyclosporine- and tacrolimus-based regimens [58,105111]. A recent meta-analysis of the topic conrmed that no dierence in patient or graft survival has been documented based on the choice of calcineurin inhibitor [112]. Data on HCV replication and histologic progression are lacking, however, emphasizing the need for future clinical trials to incorporate these factors as primary end points. Additional immunosuppressive agents and potent induction therapies have been less well studied. There is recent interest in the potential antiviral properties of MMF, but the data on the addition of MMF to the standard regimen have been conicting, with some studies reporting benet and others reporting diminished outcomes [113116]. Induction with anti T-cell or antiIL-2 receptor antibodies is infrequently used, predominantly in patients who are unlikely to tolerate calcineurin inhibitors because of renal failure or neurologic concerns. The data to support the use of these thymocyte-depleting regimens for induction in this setting are inconsistent, but in one prospective randomized trial comparing induction with rabbit antithymocyte globulin followed by tacrolimus and MMF versus tacrolimus, MMF and steroids showed no dierences in graft survival and recurrent HCV at 1 year [117]. AntiB-cell agents are also being used with limited data and may worsen HCV recurrence. For example, a retrospective analysis of patients who received induction with alemtuzumab revealed higher rates of HCV recurrence at 1 year [118]. These potent induction agents in the setting of HCV should therefore be used with caution and likely only in specic settings in which the standard regimen is not tolerated. The unifying theme throughout these trials may be that the overall intensity of immunosuppression is the most important predictor, with more intense immunosuppression leading to worse outcomes in HCV-infected patients. Rapid steroid tapers and steroid-free immunosuppression with or without induction antibodies have been proposed as strategies to mitigate the severity of recurrent HCV. Steroid-free regimens are theoretically preferable, because high-dose steroids are completely avoided and early data now show similar ecacy [117,119121]. These regimens may also lead to less new-onset diabetes [121123], a major risk factor for posttransplant renal dysfunction and severe recurrent HCV [124]. Steroid-free regimens, however, frequently use antiIL-2 receptor antibodies or lymphocyte-depleting agents, rendering

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the benets dicult to predict. It has even been postulated that because the impact of cellular rejection on liver grafts may be small when compared with other solid organ transplantations, perhaps tapering the immunosuppressive regimen o completely would be possible [125]. In this series, 34 patients on cyclosporine monotherapy had therapy discontinued, and although some were able to maintain avoidance of any immunosuppressive therapy and had improved biopsy ndings, 35% of patients experienced rejection while on the taper, 41% rejected the transplant after the taper was completed, and there was no control group. This approach cannot therefore currently be recommended. There is also emerging consensus that not only the absolute potency of the regimen but rapid changes in immunosuppressive strength are deleterious because they create alternating periods of increased viral replication followed by immune recognition and clearance of virally infected allograft cells during rapid immunosuppressive withdrawal. In fact, recent reports support the avoidance of rapid tapering of steroids and uctuation in immunosuppressive therapy [126,127]. Clear evidence to support any particular strategy therefore remains elusive. The authors low and slow approach is to administer what they anticipate to be the minimum sucient immunosuppression to minimize rejection, followed by a gradual taper and avoidance of intense rejection treatment with bolus steroids or antibodies. They use a calcineurin inhibitor (cyclosporine or tacrolimus) with a slow steroid taper over 6 to 12 months and MMF for the rst year. In addition, protocol biopsies at months 3, 12, and 24 are used to guide immunosuppressive and antiviral treatment decisions. Retransplantation When antiviral treatment and modulation of immunosuppression fail to halt the progression of recurrent HCV, the only denitive treatment for graft failure is retransplantation. The debate is ongoing, however, as to who should undergo retransplantation and when. No formal or widely accepted indications for retransplantation exist, and practices vary widely among institutions. When transplant centers in the United States were surveyed, most indicated that they oer retransplantation, but many were progressively less likely to perform the operation because of poor outcomes, and there was little uniformity in the perceived indications and contraindications for the operation [128]. When compared with primary transplantation, retransplantation for all indications is associated with longer hospital stays, greater cost, and decreased survival [129,130]. Patient and graft survival after retransplantation for HCV is worse than after primary transplants [131], and although data are limited, some believe that outcomes in retransplantation for HCV are worse than those for any other indication [132,133]. The Scientic Registry of Transplant Recipients database has provided one of the largest studied cohorts of retransplanted patients with more than 1700 patients,

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almost 500 of whom had HCV. This retrospective analysis found a 31% higher covariate adjusted mortality risk when HCV-infected recipients were compared with all other recipients combined [132]. The signicance of this dramatic dierence has been debated, however, given the heterogeneity of the nonHCV-infected group and the lack of data on the specic indications for retransplantation in this analysis. In addition, there is evidence to the contrary. One large European study and a large cohort from the University of California at Los Angeles found no signicant dierence in outcomes in patients who had and did not have HCV [130,134]. Furthermore, when the causes of liver disease are separated, although some groups of recipients have better outcomes (eg, HBV, autoimmune hepatitis), other groups, including cryptogenic and alcohol-related disease, have similar outcomes to recipients who have HCV [131,135]. There is also evidence that when indicators of severity of disease are controlled for, HCV may not independently predict poor outcomes [133,136]. Most recently, McCashland and colleagues [137] reported on data from 10 centers, and found no dierence in 1- and 3-year survival rates in HCV-infected and nonHCV-infected recipients. They also found that approximately one third of patients with HCV-related graft failure are not considered candidates for retransplantation and that only approximately one half of those evaluated are listed. Given the scarcity of organs available and the tremendous cost of transplantation, the possibility that HCV-infected recipients have poor outcomes has caused some centers to decline retransplantation in these patients. Given the ambiguity of the data, however, the suggestion that this dierence may not persist when the MELD score is controlled for, and the hope of improved outcomes as we learn more about modulation of antiviral and immunosuppressive regimens, most centers do not systematically exclude these patients. In fact, the proportion of patients retransplanted for HCV is rising [137,138]. Maximizing the benets of retransplantation by individual patient selection and appropriately timing the evaluation continues to be the goal, but this is hindered by the lack of prospective data and uncertainty as to acceptable post-retransplantation survival rates. Many studies have attempted to identify risk factors most predictive of poor outcomes, which include age, renal failure, hyperbilirubinemia, and poor conditioning (Table 1) [130,133,134,139141]. Patients with early aggressive recurrence, particularly cholestatic HCV, and graft failure within the rst year or with high MELD scores have poor outcomes with retransplantation [131,135,137,142144]. In addition, the time between rst and second transplants is a strong predictor, with the highest death rate in one series being in the intermediate period between 8 and 30 days (as compared with within the rst 7 days or after 30 days) [145]. These and other risk factors have been used to create models to predict who is likely to benet most and, perhaps more importantly, those in whom intervention may be futile. Rosen and colleagues [133] created one such model and used age, bilirubin, creatinine, UNOS status, and cause of graft failure to place patients into low-, medium- and high-risk groups.

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Table 1 Predictors of outcomes in liver retransplantation for hepatitis C virus Favorable outcomes Recipient factors MELD score !21 Portal HTN only Diminished outcomes Controversial eects

Advanced recipient age Renal failure Ventilator dependence MELD score O21 Donor factors Advanced donor age Short warm and Prolonged warm and cold ischemic times cold ischemic times Viral factors Antiviral response FCH HCV statusa Viral eradication Early recurrence Timing of !8 days or O30 days 830 days after transplant retransplantation after transplant
a

Abbreviations: FCH, brosing cholestatic HCV; HTN, hypertension. HCV status is unlikely to have a negative impact on retransplantation outcomes when the severity of illness is controlled for (see text).

Their model was highly predictive of survival but has not been rigorously validated. Markmann and colleagues [146] used a ve-point scoring system based on recipient age, creatinine, bilirubin, cold ischemic time, and ventilatory status, utilizing recipient and donor factors. Perhaps the most comprehensive model was created by Ghobrial and colleagues [136] for rst and second transplants, and it also included donor and recipient factors. The data included in the nal mortality index equation included donor age, recipient age, creatinine, bilirubin, prothrombin time, warm and cold ischemic times, and whether this was a second transplantation. The authors validated this model using the UNOS database with remarkable accuracy. Using this model, with MELD score substituted for creatinine, bilirubin, and prothrombin time and taking into account the duration of time between rst and second transplants, tables of estimated survival can be created that may be useful to estimate the outcomes of a selected patient [147]. In addition, the donor risk index originally derived from patients undergoing primary transplantation developed by Feng and colleagues [148] was recently used to look at retransplantation from the UNOS database [149]. These investigators added a cause of graft failure variable to the original model to create what they termed the retransplant donor risk index, which they found to be a strong predictor. Many of these models have been used by clinicians and researchers to characterize individual patient risk but are also important tools when comparing research cohorts. The group described by McCashland and colleagues [137], for example, was predominantly composed of lower risk patients, perhaps accounting for their impressive results. Although much remains unknown, several general lessons have been learned. Retransplantation should probably be avoided in patients who have early aggressive HCV recurrence [142]. Retransplantation should perhaps be immediate (within 7 days) in patients who have early graft failure

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because of technical complications, because intermediate durations of time may lead to worse outcomes. In addition, prolonged survival may be achieved by attempting retransplantation at a lower MELD score than would be considered in the initial transplantation. In fact, weighted utility curves by Burton and colleagues [142] indicate that the maximal value of retransplantation may be achieved at a MELD score of 21 for HCV-infected patients and 24 for all other patients. It is likely that these lessons have led to the evolution of patient selection, and perhaps a diminishing discrepancy between outcomes in HCV- and nonHCV-infected patients over time. As the focus of the debate regarding retransplantation for HCV shifts from deciding if it should occur to when it should occur and in which individuals, several crucial questions remain unanswered. Determining at what predicted survival rate the procedure becomes unacceptable is one major step, because prediction models are ineective when the survival rate they predict cannot be put into context and used in clinical decision making. In addition, the MELD system does not take into account several variables found to be highly predictive of outcomes specically in retransplantation, such as features of the graft and time since primary transplant, and may not always predict outcomes [137]. Widespread application of a more comprehensive model may therefore be required. Future directions OLT is a life-saving therapy to reverse the hepatic failure associated with chronic HCV infection. Its long-term success, however, remains limited by the profound shortage of organs available and diminished graft and patient survival in the face of HCV recurrence. Expansion of the donor pool is crucial through increasing public awareness of deceased donation, increased living donation, and using selected organs outside of widely accepted criteria that are shown to be safe in this population. Through this expansion of the donor pool alone, wait list mortality is likely to decrease. In addition, when HCV-infected patients undergo transplant surgery, every eort to prevent or diminish the severity of HCV recurrence must be made. Although great advances have occurred in the potency of our immunosuppressive armamentarium, in the case of HCV-related liver disease, we have yet to strike a predictable balance between suppression or treatment of rejection and avoiding uninhibited viral replication and progressive recurrent disease. Trials are required to optimize immunosuppression and are likely to involve protocols with minimal modulation in the potency of the regimen and lowdose steroids with slow tapers. Prevention of HCV reinfection of the graft with improved pretransplant prophylactic viral eradication and appropriate timing of transplantation in addition to better posttransplant treatment strategies are desperately needed. As the next generation of HCV treatment evolves to include HCV enzyme inhibitors, such as protease and polymerase inhibitors, much work is required to determine the regimen with the best

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ecacy and safety. It is conceivable that with these therapies, patients could be transplanted while on short courses of medications with suppressed or undetectable viral loads and perhaps be cured of HCV infection. Finally, when all these measures fail, additional research must be done to determine who should undergo retransplantation and when they are likely to derive the most benet from the second transplant. References
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[116] Zekry A, Gleeson M, Guney S, et al. A prospective cross-over study comparing the eect of mycophenolate versus azathioprine on allograft function and viral load in liver transplant recipients with recurrent chronic HCV infection. Liver Transpl 2004;10(1):527. [117] Eason JD, Nair S, Cohen AJ, et al. Steroid-free liver transplantation using rabbit antithymocyte globulin and early tacrolimus monotherapy. Transplantation 2003;75(8): 13969. [118] Marcos A, Eghtesad B, Fung JJ, et al. Use of alemtuzumab and tacrolimus monotherapy for cadaveric liver transplantation: with particular reference to hepatitis C virus. Transplantation 2004;78(7):96671. [119] Filipponi F, Callea F, Salizzoni M, et al. Double-blind comparison of hepatitis C histological recurrence rate in HCV liver transplant recipients given basiliximab steroids or basiliximab placebo, in addition to cyclosporine and azathioprine. Transplantation 2004;78(10):148895. [120] Belli LS, Alberti AB, Vangeli M, et al. Tapering o steroids three months after liver transplantation is not detrimental for hepatitis C virus disease recurrence. Liver Transpl 2003; 9(2):2012. [121] Llado L, Xiol X, Figueras J, et al. Immunosuppression without steroids in liver transplantation is safe and reduces infection and metabolic complications: results from a prospective multicenter randomized study. J Hepatol 2006;44(4):7106. [122] Navasa M, Bustamante J, Marroni C, et al. Diabetes mellitus after liver transplantation: prevalence and predictive factors. J Hepatol 1996;25(1):6471. [123] Boillot O, Mayer DA, Boudjema K, et al. Corticosteroid-free immunosuppression with tacrolimus following induction with Daclizumab: a large randomized clinical study. Liver Transpl 2005;11(1):617. [124] Baid S, Cosimi AB, Farrell ML, et al. Posttransplant diabetes mellitus in liver transplant recipients: risk factors, temporal relationship with hepatitis C virus allograft hepatitis, and impact on mortality. Transplantation 2001;72(6):106672. [125] Tisone G, Orlando G, Cardillo A, et al. Complete weaning o immunosuppression in HCV liver transplant recipients is feasible and favourably impacts on the progression of disease recurrence. J Hepatol 2006;44(4):7029. [126] Berenguer M, Aguilera V, Prieto M, et al. Signicant improvement in the outcome of HCV-infected transplant recipients by avoiding rapid steroid tapering and potent induction immunosuppression. J Hepatol 2006;44(4):71722. [127] Brillanti S, Vivarelli M, De Ruvo N, et al. Slowly tapering o steroids protects the graft against hepatitis C recurrence after liver transplantation. Liver Transpl 2002;8(10): 8848. [128] Burton JR Jr, Rosen HR. Liver retransplantation for hepatitis C virus recurrence: a survey of liver transplant programs in the United States. Clin Gastroenterol Hepatol 2005;3(7): 7004. [129] Biggins SW, Terrault NA. Should HCV-related cirrhosis be a contraindication for retransplantation? Liver Transpl 2003;9(3):2368. [130] Azoulay D, Linhares MM, Huguet E, et al. Decision for retransplantation of the liver: an experience- and cost-based analysis. Ann Surg 2002;236(6):71321. [131] Watt KD, Lyden ER, McCashland TM. Poor survival after liver retransplantation: is hepatitis C to blame? Liver Transpl 2003;9(10):101924. [132] Pelletier SJ, Schaubel DE, Punch JD, et al. Hepatitis C is a risk factor for death after liver retransplantation. Liver Transpl 2005;11(4):43440. [133] Rosen HR, Madden JP, Martin P. A model to predict survival following liver retransplantation. Hepatology 1999;29(2):36570. [134] Markmann JF, Markowitz JS, Yersiz H, et al. Long-term survival after retransplantation of the liver. Ann Surg 1997;226(4):40818. [135] McCashland TM. Retransplantation for recurrent hepatitis C: positive aspects. Liver Transpl 2003;9(11):S6772.

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[136] Ghobrial RM, Gornbein J, Steadman R, et al. Pretransplant model to predict posttransplant survival in liver transplant patients. Ann Surg 2002;236(3):31522. [137] McCashland T, Watt K, Lyden E, et al. Retransplantation for hepatitis C: results of a U.S. multicenter retransplant study. Liver Transpl 2007;13(9):124653. [138] Rosen HR, Martin P. Hepatitis C infection in patients undergoing liver retransplantation. Transplantation 1998;66(12):16126. [139] Ne GW, OBrien CB, Nery J, et al. Factors that identify survival after liver retransplantation for allograft failure caused by recurrent hepatitis C infection. Liver Transpl 2004; 10(12):1497503. [140] Rosen HR, Prieto M, Casanovas-Taltavull T, et al. Validation and renement of survival models for liver retransplantation. Hepatology 2003;38(2):4609. [141] Doyle HR, Morelli F, McMichael J, et al. Hepatic retransplantationdan analysis of risk factors associated with outcome. Transplantation 1996;61(10):1499505. [142] Burton JR Jr, Sonnenberg A, Rosen HR. Retransplantation for recurrent hepatitis C in the MELD era: maximizing utility. Liver Transpl 2004;10(10 Suppl 2):S5964. [143] Watt KD, Menke T, Lyden E, et al. Mortality while awaiting liver retransplantation: predictability of MELD scores. Transplant Proc 2005;37(5):21723. [144] Yao FY, Saab S, Bass NM, et al. Prediction of survival after liver retransplantation for late graft failure based on preoperative prognostic scores. Hepatology 2004;39(1):2308. [145] Busuttil RW, Farmer DG, Yersiz H, et al. Analysis of long-term outcomes of 3200 liver transplantations over two decades: a single-center experience. Ann Surg 2005;241(6): 90516. [146] Markmann JF, Gornbein J, Markowitz JS, et al. A simple model to estimate survival after retransplantation of the liver. Transplantation 1999;67(3):42230. [147] Zimmerman MA, Ghobrial RM. When shouldnt we retransplant? Liver Transpl 2005; 11(11 Suppl 2):S1420. [148] Feng S, Goodrich NP, Bragg-Gresham JL, et al. Characteristics associated with liver graft failure: the concept of a donor risk index. Am J Transplant 2006;6(4):78390. [149] Northup PG, Pruett TL, Kashmer DM, et al. Donor factors predicting recipient survival after liver retransplantation: the retransplant donor risk index. Am J Transplant 2007; 7(8):19848.

Clin Liver Dis 12 (2008) 661674

Hepatitis C Virus Infection and Hepatocellular Carcinoma


Wojciech Blonski, MD, PhDa,b, K. Rajender Reddy, MDa,*
Hospital of the University of Pennsylvania, 3 Dulles, 3400 Spruce Street, Philadelphia, PA 19104, USA b Department of Gastroenterology and Hepatology, Wroclaw Medical University, Borowska 213 Street, 50-556 Wroclaw, Poland
a

Epidemiology Primary liver cancer is the sixth most common cancer in the world and the third most common cause of death attributable to cancer [1]. Most primary liver cancers are hepatocellular carcinoma (HCC), accounting for 85% to 90% of cases [2]. There has been an increase in the number of cases of HCC in the United States over the past 2 decades of the previous century [3]. More than a 2-fold increase in the age-adjusted HCC incidence rate was observed between 1985 and 2002 in the United States [24]. This increase began in the mid-1980s, with the peak of proportional HCC increase in the late 1990s [2]. There was a marked and 2.5-fold increase in the average annual age-adjusted rate of HCC conrmed by histology or cytology, and this was from 1.3 per 100,000 population between 1978 and 1980 to 3.3 per 100,000 population between 1999 and 2001 [2,5]. An analysis of the Surveillance, Epidemiology, and End Results (SEER) database in the United States between 1976 and 2002 noted the greatest proportional increase in age-adjusted HCC incidence rate per 100,000 population among whites (including Hispanics and non-Hispanics); the lowest proportional increase was among Asians [2]. Between 1976 and 2002, an average annual age-adjusted HCC incidence rate per 100,000 population increased 2.5-fold in black Americans (1.0 versus 2.5), 2-fold in whites (2.5 versus 5.0), and 1.3-fold in Asians (6.0 versus 8.0) (Fig. 1) [2]. There was a shift in the distribution of patient age toward younger ages, with the greatest proportional increase

* Corresponding author. E-mail address: rajender.reddy@uphs.upenn.edu (K.R. Reddy). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.007 liver.theclinics.com

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Fig. 1. Average yearly age-adjusted incidence rates for HCC in men and women in the United States shown for 3-year intervals between 1975 and 2002. Whites include approximately 25% Hispanics, whereas those who report other race are predominantly (88%) Asian. Source: Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov.) SEER*Stat Database: IncidencedSEER 13 Regs Public-Use, Nov 2004 Sub (19732002 varying), National Cancer Institute, Division of Cancer Control and Population Sciences, Surveillance Research Program, Cancer Statistics Branch, released April 2005, based on the November 2004 submission. (From El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:2559; with permission.)

being in patients between 45 and 60 years old [2]. An average age at the time of diagnosis of HCC was 65 years, and 74% of HCCs were found in men [2]. Four large studies evaluating the risk factors for HCC in the United States observed the highest increase of HCC among patients who have hepatitis C virus (HCV) infection when compared with patients who have hepatitis B virus (HBV) [69]. Two of aforementioned studies performed in large single referral centers found that HCV was the major contributing factor to the increased incidence of HCC [6,7]. The rst study observed a threefold signicant increase in the age-adjusted rate of primary liver cancer in patients who had HCV infection (2.3 per 100,000 population during 19931995 versus 7.0 per 100,000 population during 19961998), with stable age-adjusted rates for primary liver cancer associated with HBV (2.2 versus 3.1 per 100,000 population), alcoholic cirrhosis (8.4 versus 9.1 per 100,000 population) or without risk factors (17.5 versus 19.0 per 100,000 population) [6]. The second single-center study observed a signicant increase in the number of patients who had HCC and HCV infection, from 18% between 1993 and 1995 to 31% between 1996 and 1998 (P .01), with an accompanying signicant decrease in the number of patients who had HCC and HBV infection (26% versus 17%; P .06) and a stable rate of patients who had HCC and negative HBV and HCV markers (56% versus 52%, P .5) [7]. A population-based study using SEER-Medicarelinked data among patients 65 years of age and older noted a marked increase in HCV-related HCC and

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a smaller increase in HBV-related HCC between 1993 and 1999 [8]. Overall, the risk for HCC in patients who had HCV increased by 226% relative to a 67% increase in patients who had HBV when adjusted for age, gender, and geographic region [8]. Finally, the study from the fourth largest indigent health care system in the United States observed an increase in HCC associated with HCV from 52% in 1992 to 1996 to 68% in 1997 to 2001 (P .2) [9] and a decrease in HCC associated with HBV from 37% to 34% (P 1.0) [9]. Therefore, chronic HCV infection has been considered the major risk factor for development of HCC. It is suggested that the current increase in HCC rates in young men has been caused by HCV infection acquired from intravenous drug use from the late 1960s to the early 1980s [10,11], recognizing that the natural history of HCV is of a slowly progressive nature, wherein it may take 2 to 3 decades or more to evolve to cirrhosis, often a requisite for HCC in HCV infection. The lower increase of HCC rates among the older population is likely explained by the fact that they may have acquired HCV infection through a blood transfusion, because these patients were more likely to receive blood transfusions [12] and did not live long enough to evolve to cirrhosis as a consequence of competing comorbid conditions being responsible for early nonliver diseaserelated mortality. There is geographic variability in the prevalence of markers of HCV infection in patients who have HCC; it has ranged from 27% in the United States [13], from 27% to 75% in Western Europe (France: 27%58%, Italy: 44%66%, and Spain: 60%75%) [1418], and up to 80% to 90% in Japan [19,20]. A meta-analysis of 32 case-control studies estimated that patients infected with HCV had nearly a 24-fold higher risk for developing HCC than those without HCV infection [21]. The risk for developing HCC in HCV-positive patients was much higher (31.2) in regions with relatively low endemicity for HBV infection, such as Japan and the Mediterranean countries, and much lower (11.5) in regions with a high endemicity of HBV infection, such as sub-Saharan Africa and southern Africa, China, Taiwan, South Korea, and Vietnam [21]. A large community-based prospective study from Taiwan that included 12,008 patients observed a 20-fold increased risk for developing HCC in antiHCV-positive patients when compared with antiHCV-negative subjects [22]. A systematic review of 21 studies of patients infected with HCV published between 1980 and 2001 observed that the pooled weighted incidence rates of end-stage liver disease (ESLD) and HCC were the highest in studies that included patients who had hemophilia and were infected by repeated transfusions of HCVinfected pooled clotting factor concentrates (7.9 and 1.0 per 1000 personyears, respectively) and transfusion-associated HCV (4.5 and 0.7 per 1000 person-years) in patients who received HCV-infected blood or blood products [23]. Conversely, the lowest incidence rates were found in studies that included patients who had community-acquired HCV infection (intravenous drug use, medical procedure-related, and nontransfusion-related causes of

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HCV infection) (1.9 and 0 per 1000 person-years) and women who received one-time HCV-contaminated antiD immune globulin (0.7 and 0 per 1000 person-years) [23]. Mechanisms of development of hepatitis C virusrelated hepatocellular carcinoma The exact mechanism of development of HCC in chronic HCV infection is unclear. Although it was initially suggested that HCV enhanced mutagenesis in hepatocytes as a result of chronic inammation and hepatocyte regeneration, it was questioned whether liver inammation itself is capable of causing HCC. The role of inammation alone in hepatocarcinogenesis is questionable because patients who have persistent severe inammation in the course of autoimmune hepatitis rarely develop HCC [24]. A transgenic mouse model has demonstrated that the HCV core protein plays a pivotal role in the development of HCC [25]. It was recently proposed that the HCV core protein may have oncogenic properties that allow omission of some of the usual multiple steps required during the process of carcinogenesis [24]. Therefore, the non-Vogelstein type process of hepatocarcinogenesis was suggested as the possible explanation of HCV-induced HCC (Fig. 2) [24]. In this process, the expression of the viral core protein would lead to the development of HCC even without the complete set of genetic aberrations usually required for carcinogenesis [24]. The HCV core protein has been shown to induce reactive oxygen species in the mouse liver in the absence of inammation [26]. Oxidative stress in the liver is caused by the direct eect of HCV core protein on mitochondria [27].

Fig. 2. Mechanism of HCV-associated hepatocarcinogenesis. Multiple steps are required in the induction of all cancers; it would be mandatory for hepatocarcinogenesis that genetic mutations accumulate in hepatocytes. In HCV infection, however, some of these steps might be skipped in the development of HCC in the presence of the core protein. The overall eects achieved by the expression of the core protein would be the induction of HCC, even in the absence of a complete set of genetic aberrations, required for carcinogenesis. By considering such a non-Vogelstein type process for the induction of HCC, a plausible explanation might be given for many unusual events happening in HCV carriers. CRC, colorectal cancer. (From Koike K. Molecular basis of hepatitis C virus-associated hepatocarcinogenesis: lessons from animal model studies. Clin Gastroenterol Hepatol 2005;3:S134; with permission.)

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Oxidative stress driven by HCV core protein may reduce mitochondrial metabolic processes, which, in turn, may induce the development of liver steatosis attributable to inhibition of b-oxidation and oxidative injury to mitochondrial and chromosomal DNA [27]. HCV core protein has been noted to induce liver steatosis in a transgenic mice model [2831] and in patients who have chronic hepatitis [28] by inhibiting microsomal triglyceride transfer protein activity [2830]. Based on the transgenic mice model, it is suggested that expression of the HCV structural proteins enhances a low background of liver steatosis, whereas additional low expression of nonstructural proteins increases the risk for HCC [31]. Further, HCV core protein may change the expression of cellular genes, interact with cellular proteins, or modulate intracellular signaling pathways. In addition, HCV core protein, in a transgenic mouse model, increased the expression of tumor necrosis factor-a and interleukin-1; enhances the activities of c-Jun N-terminal kinase and activator protein-1 [32]; and directly interacts with a transcriptional regulator retinoid X receptor-a that plays an important role in the control of cell proliferation, dierentiation, and lipid metabolism [33]. In a mouse liver model for HCV-related HCC, HCV core protein was shown to activate p38 mitogen-activated protein kinases and extracellular signal-regulated kinase together with ethanol; it also modulated the expression of several genes related to cell transformation, cell cycle, and antioxidants [34]. The specic interaction between the HCV core protein and proteasome activator PA28 in cell culture and in the livers of HCV-core transgenic mice and a patient who had chronic hepatitis C has also been observed [35]. HCV core protein may also modulate the intracellular signaling pathway, and thus may induce hepatocarcinogenesis by selective suppression of the expression of the suppressor of cytokine signaling SOCS-1 gene demonstrated in the liver tissues of animals and cultured cells [36]. Thus, there is experimental evidence to suggest that there might be interplay among several mechanisms that may lead to the development of HCC in an HCV-infected individual.

Risk factors for development of hepatocellular carcinoma in patients who have chronic hepatitis C virus infection HCV promotes the development of liver brosis and cirrhosis, thus increasing the risk for HCC [2]. It has been estimated that 1% to 3% of HCV-infected patients develop HCC after 30 years (Fig. 3) [2,7]. There are several factors that increase the likelihood of development of liver cirrhosis and HCC in patients who have chronic HCV infection, and these include older age, older age at the time of the onset of HCV infection, male gender, accompanying infection with HBV or HIV, heavy consumption of alcohol, diabetes mellitus, obesity, transfusion-acquired HCV infection, and genotype of HCV (Table 1) [13].

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Fig. 3. Proportion of patients who have HCC related to viral hepatitis. In this study, all USborn patients who were seen at MD Andersen Medical Center in Houston, Texas were tested for serologic evidence of HCV and HBV. Only HCV-positive HCC increased during the study period. (From El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132:255776; and Hassan MM, Frome A, Patt YZ, et al. Rising prevalence of hepatitis C virus infection among patients recently diagnosed with hepatocellular carcinoma in the United States. J Clin Gastroenterol 2002;35:2562,2669; with permission.)

Alcohol consumption Several studies report a synergistic interaction between alcohol consumption and chronic HCV infection on the development of HCC. Both factors actively promote liver cirrhosis [13]. Two hospital-based case-control studies observed an additive interaction between lifetime daily alcohol intake (LDAI) less than 50 g/d and HCV infection and multiplicative interaction for LDAI greater than 125 g/d and HCV infection on development of liver cirrhosis [23]. The eect of alcohol on the risk for development of liver cirrhosis was dose related in antiHCV-positive (odds ratio [OR] 9.2 for LDAI 0 g/d and OR 147.2 for LDAI R175 g/d) and antiHCV-negative (OR 1.0 for LDAI 0 g/d and OR 15.0 for LDAI R175 g/d) patients [23]. The eect of daily alcohol intake on the risk for HCC in patients who have HCV was found also to be dose related with a 2-fold and 4-fold increased risk in patients consuming alcohol at a rate of 41 to 80 g/ d or more than 80 g/d, respectively [37]. A history of heavy alcohol drinking at a rate of more than 80 g/d for at least 5 years was shown to have a 23-fold increase for the risk for HCC among patients who have chronic HCV infection [38]. These data were further supported by Donato and colleagues [39], who found a 2-fold increased risk for HCC in those infected with HCV and drinking alcohol at a rate of more than 60 g/d. Overall, consumption of alcohol in patients who have HCV infection at dosages of 0 to 60 g/d or more than 60 g/d was associated with a 55-fold and 109-fold increase in the risk for HCC, respectively, when compared with controls drinking similar amounts of alcohol but without HCV infection [39]. A case-control study from Texas found that heavy alcohol drinkers (R80 mL/d) infected with HCV had a 54-fold increase in the risk for HCC [40].

Table 1 Risk factors associated with development of hepatocellular carcinoma in patients who have hepatitis C virus infection Study Corrao and Arico [23] Tagger et al [37] Donato et al [38] Donato et al [39] Hassan et al [40] Yuan et al [41]a Donato et al [21] Tagger et al [37] Chiaramonte et al [42] Yuan et al [41] Yuan et al [41]a Komura et al [43] Ohata et al [44] Pekow et al [45] Kumar et al [46] Bruno et al [47] Tagger et al [37] Donato et al [38] Silini et al [48] Lopez-Labrador et al [49] Factor Alcohol Risk OR 9.2 (95% CI: 2.043.2) OR 147 (95% CI: 42.1514.3) Synergy index: 2.4 Synergy index: 4.0 RR 29.8 (95% CI: 13.366.5) RR 66.3 (95% CI: 20.5214) OR 55.0 (95% CI: 29.9101.0) OR 109 (95% CI: 50.9233.0) OR 53.9 (95% CI: 7.0415.7) Synergy index: 5.5 (95% CI: 3.97.0) OR 135 (95% CI: 79.7242) Synergy index: 2.4 HR 2.3 (95% CI: 5 1.14.6) OR 63.9 (95% CI: 8.6475.3) Synergy index: 4.8 (95% CI: 2.76.9) HR 2.9 (95% CI: 1.55.5) RR OR OR OR OR RR OR OR 2.81 (95% CI: 1.246.37) 6.39 (95% CI: 1.04, 39.35) 1.0 (95% CI: 0.81.3; P .9) 6.14 (95% CI: 1.7721.37) 34.2 (95% CI: 18.064.7) 34.3 (95% CI: 13.984.2) 1.7 (95% CI: 1.062.9) 4.286 (95% CI: 1.43712.82) Comment LDAI 0 g LDAI 175 g Alcohol: 4180 g/d Alcohol: O80 g/d Alcohol: 080 g/d Alcohol: O80 g/d Alcohol: 060 g/d Alcohol: O60 g/d Alcohol: O80 mL/d Alcohol: O4 drinks per day d d d d d Risk for recurrence of HCVor HBV-related HCC d d d d d d d d

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HBV

Diabetes

Liver Liver Liver HCV

steatosis steatosis steatosis genotype 1b

Abbreviations: CI, condence interval: HR, hazard ratio; RR, relative risk, LDAI, lifetime daily alcohol use. a Includes patients who had HCV, HBV, or both.

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Overall, the synergic interaction between HCV infection and heavy alcohol consumption on the risk for HCC was observed with synergy indices ranging from 2.0 to 5.5 [37,38,40,41]. Coinfection with hepatitis B virus Several studies observed the synergism between HBV and HCV infection with respect to the increased risk for HCC. A meta-analysis of 32 case-control studies found that infection with both HBV and HCV was associated with a 135-fold increased risk for HCC, whereas infection with HBV or HCV was associated with a 20-fold and 24-fold increased risk, respectively [21]. Another study observed a 2.4 synergy index between infection with both HCV and HBV in increasing the risk for HCC [37]. An Italian experience observed that patients who had liver cirrhosis and were infected with both HBV and HCV were at signicantly higher risk for developing HCC than patients who had a single viral infection [42]. Cumulative appearance rates for HCC at 5, 10, and 13 years were the highest in cirrhotic patients who had dual HBV/HCV infection (23%, 45%, and 55%, respectively) when compared with patients who had HBV infection (10%, 16%, and 16%, respectively) or HCV infection (21%, 28%, and 40%, respectively) alone [42]. Coinfection with HIV A meta-analysis of eight studies evaluating the eect of HIV coinfection on progressive liver disease in patients who had HCV infection observed that patients who had dual HIV/HCV infection had a signicantly elevated risk for severe liver disease when compared with those infected only with HCV [50]. Overall, the combined relative risk for developing decompensated liver disease was 6.14, whereas the relative risk for developing histologic cirrhosis was 2.07 in patients infected with both HIV and HCV when compared with those infected only with HCV [50]. HCC was likely to occur at a younger age and after a shorter duration of HCV infection in patients coinfected with HIV [51]. A recent multicenter retrospective study has shown that patients infected with both HIV and HCV developed HCC signicantly faster than those infected only with HCV (26 versus 34 years; P .002) [52]. Diabetes mellitus The recent meta-analysis of 13 case-control studies and 13 cohort studies showed a signicant association between diabetes and development of HCC in 9 case-control studies and in 7 cohort studies, respectively [53]. The mechanisms by which diabetes may increase the risk for HCC include predisposition to development of nonalcoholic steatohepatitis with progression to cirrhosis in up to 5% cases [54] or association with increased levels of potential carcinogenic insulin-like factors [55].

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A large case-control hospital-based study including 823 patients and 3459 controls observed that diabetes mellitus signicantly increased the risk for HCC only in the presence of other risk factors, such as hepatitis C, hepatitis B, or alcohol cirrhosis [56]. Another population-based case-control study observed the synergic interaction (synergy index of 5.5) between HCV infection and diabetes mellitus on the risk for HCC [41]. Overall, a meta-analysis of studies that adjusted for the association between diabetes and HCC for HCV or unspecied viral hepatitis showed no change or minimal change in the associated risk for HCC in those individuals with diabetes and HCV [53]. A recent study suggested that diabetes was a risk factor for the recurrence of HCV-related HCC, and thus decreased the overall survival rates after surgical treatment [43]. Obesity Obesity may increase the risk for development of liver steatosis and brosis in patients who have HCV [47,57]. Patients who have chronic HCV infection and a high grade of liver steatosis, particularly with steatohepatitis, might be at risk for more accelerated progression to liver cirrhosis [57]. Liver steatosis has been noted to be an independent and signicant risk factor for HCC in patients who have HCV [44,45]. A linear increase in the odds ratio for HCC with increased grades of liver steatosis from 1.61 for grade 1, to 3.68 for grade 2, and to 8.02 for grade 3 or 4 when compared with grade 0 has been reported [45]. Thus, steatosis might be an additional risk for HCC, and increased vigilance should be practiced in surveillance of HCV-infected patients who have liver steatosis [44,45]. Conversely, Kumar and colleagues [46] did not nd liver steatosis to be a risk factor for HCC in patients who have HCV infection in a prospective analysis of HCC risk derived from a rigorously matched cohort of HCV-infected patients. Genotype of hepatitis C virus The relation between the HCV genotype and the risk for HCC has been controversial. Several studies found that patients infected with HCV genotype 1b had from a twofold to sixfold increased risk for HCC and had more aggressive progression of associated liver dysfunction [37,38,4749,5860]. It has been suggested that an increased incidence of HCC in those with a background of genotype 1b might have been because of overrepresentation of HCV genotype 1b in patients who were of advanced age and had more advanced liver disease, however [48,49,59]. Prevention of hepatocellular carcinoma in patients who have hepatitis C virus infection Evidence has shown that therapy with interferon might be an ecacious prophylactic approach in patients who have chronic HCV infection. A recent

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analysis of eight cohort studies in Japan demonstrated that therapy with interferon improved the prognosis of HCV-positive patients who did not have liver cirrhosis and who did have liver cirrhosis and of patients who had cirrhosis and HCC [61]. Such therapy with interferon reduced the risk for HCC by 50% in patients who had chronic HCV infection without liver cirrhosis, with a further reduction to 20% among patients with a sustained virologic response [61]. Patients with a sustained virologic response treated with interferon were further shown to have an improvement in liver brosis histology [61]. Therapy with interferon also increased survival rates because of a reduction in the incidence of HCC and hepatic failure [61]. Therapy with interferon in patients who had compensated HCV-related liver cirrhosis caused a signicant reduction in the cumulative incidence of HCC when compared with patients who had HCV-related cirrhosis not receiving interferon with an adjusted relative risk of 0.54 [61]. Sustained responders presented with the strongest eect, with a relative risk of 0.05 when compared with untreated patients [61]. Finally, none of patients with a sustained viral response died from liver-related causes compared with 26% of untreated patients [61]. A meta-analysis of 11 studies that included 2178 patients who had HCVrelated liver cirrhosis observed that patients not receiving interferon had a threefold higher risk for HCC than those treated with interferon, regardless of achieving or not achieving a sustained virologic response [62]. The benet was greater in those who had a sustained virologic response [62]. These data were further supported by a recent study by Hung and colleagues [63], which has shown that a sustained virologic response induced by combined therapy with interferon-a-2b and ribavirin may decrease the incidence of HCC in patients who have HCV-related cirrhosis. A recent prospective randomized controlled study that evaluated the eect of therapy with interferon on the incidence of HCC in chronic active hepatitis C with advanced brosis or cirrhosis recently concluded in the United States [64]. Summary Primary liver cancer is the sixth most common cancer in the world and the third most common cause of death attributable to cancer. Most primary liver cancers are HCC, accounting for 85% to 90% of cases. There is a trend of growing incidence of HCC in the United States. One of the most important risk factors for developing HCC is chronic HCV infection. Such patients have been shown to have a greater than 20-fold increased risk for HCC than those without HCV infection. HCV increases the risk for HCC by promoting the development of liver brosis and cirrhosis. It has been estimated that 1% to 3% of HCV-infected patients develop HCC after 30 years. The risk factors for developing HCC among patients who have HCV-related cirrhosis include older age, older age at the time of the onset

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of HCV infection, male gender, accompanying infection with HBV or HIV, heavy consumption of alcohol, diabetes mellitus, obesity, transfusionacquired HCV infection, and genotype of HCV. Although several studies suggested the preventive eect of interferon from developing HCC in HCV-infected individuals, these ndings need to be validated in large prospective and randomized trials. References
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[40] Hassan MM, Hwang LY, Hatten CJ, et al. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus. Hepatology 2002;36: 120613. [41] Yuan JM, Govindarajan S, Arakawa K, et al. Synergism of alcohol, diabetes, and viral hepatitis on the risk of hepatocellular carcinoma in blacks and whites in the U.S. Cancer 2004;101:100917. [42] Chiaramonte M, Stroolini T, Vian A, et al. Rate of incidence of hepatocellular carcinoma in patients with compensated viral cirrhosis. Cancer 1999;85:21327. [43] Komura T, Mizukoshi E, Kita Y, et al. Impact of diabetes on recurrence of hepatocellular carcinoma after surgical treatment in patients with viral hepatitis. Am J Gastroenterol 2007;102:193946. [44] Ohata K, Hamasaki K, Toriyama K, et al. Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Cancer 2003;97:303643. [45] Pekow JR, Bhan AK, Zheng H, et al. Hepatic steatosis is associated with increased frequency of hepatocellular carcinoma in patients with hepatitis C-related cirrhosis. Cancer 2007;109: 24906. [46] Kumar D, Farrell GC, Kench J, et al. Hepatic steatosis and the risk of hepatocellular carcinoma in chronic hepatitis C. J Gastroenterol Hepatol 2005;20:1395400. [47] Bruno S, Silini E, Crosignani A, et al. Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study. Hepatology 1997;25:7548. [48] Silini E, Bottelli R, Asti M, et al. Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a case-control study. Gastroenterology 1996;111:199205. [49] Lopez-Labrador FX, Ampurdanes S, Forns X, et al. Hepatitis C virus (HCV) genotypes in Spanish patients with HCV infection: relationship between HCV genotype 1b, cirrhosis and hepatocellular carcinoma. J Hepatol 1997;27:95965. [50] Graham CS, Baden LR, Yu E, et al. Inuence of human immunodeciency virus infection on the course of hepatitis C virus infection: a meta-analysis. Clin Infect Dis 2001;33:5629. [51] Garcia-Samaniego J, Rodriguez M, Berenguer J, et al. Hepatocellular carcinoma in HIVinfected patients with chronic hepatitis C. Am J Gastroenterol 2001;96:17983. [52] Brau N, Fox RK, Xiao P, et al. Presentation and outcome of hepatocellular carcinoma in HIV-infected patients: a U.S.-Canadian multicenter study. J Hepatol 2007;47:52737. [53] El-Serag HB, Hampel H, Javadi F. The association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence. Clin Gastroenterol Hepatol 2006;4:36980. [54] Reid AE. Nonalcoholic steatohepatitis. Gastroenterology 2001;121:71023. [55] Moore MA, Park CB, Tsuda H. Implications of the hyperinsulinaemia-diabetes-cancer link for preventive eorts. Eur J Cancer Prev 1998;7:89107. [56] El-Serag HB, Richardson PA, Everhart JE. The role of diabetes in hepatocellular carcinoma: a case-control study among United States veterans. Am J Gastroenterol 2001;96:24627. [57] Adinol LE, Gambardella M, Andreana A, et al. Steatosis accelerates the progression of liver damage of chronic hepatitis C patients and correlates with specic HCV genotype and visceral obesity. Hepatology 2001;33:135864. [58] Tanaka K, Ikematsu H, Hirohata T, et al. Hepatitis C virus infection and risk of hepatocellular carcinoma among Japanese: possible role of type 1b (II) infection. J Natl Cancer Inst 1996;88:7426. [59] Murase J, Kubo S, Nishiguchi S, et al. Correlation of clinicopathologic features of resected hepatocellular carcinoma with hepatitis C virus genotype. Jpn J Cancer Res 1999;90: 1293300. [60] Zein NN, Poterucha JJ, Gross JB Jr, et al. Increased risk of hepatocellular carcinoma in patients infected with hepatitis C genotype 1b. Am J Gastroenterol 1996;91:25602. [61] Omata M, Yoshida H, Shiratori Y. Prevention of hepatocellular carcinoma and its recurrence in chronic hepatitis C patients by interferon therapy. Clin Gastroenterol Hepatol 2005;3:S1413.

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Clin Liver Dis 12 (2008) 675692

Hepatitis C and Innate Immunity: Recent Advances


Gyongyi Szabo, MD, PhD*, Angela Dolganiuc, MD, PhD
Department of MedicineLRB215, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 016052324, USA

Innate immunity: the rst line of defense in hepatitis V virus infection Innate immunity is the rst line of defense in the host response to invading viral, bacterial, or fungal pathogens, and hepatitis C virus (HCV), a single-stranded RNA virus, is no exception [1,2]. Cells participating in the innate immune response include monocytes, macrophages, dendritic cells (DCs), polymorphonuclear (PMN) leukocytes, natural killer (NK) cells, and natural killer T (NKT) cells, which are all equipped with pathogen-sensing receptors and are present in the liver (Fig. 1) [1]. Monocytes, macrophages, and PMN leukocytes are eectors and regulators of inammation because of their capacity to take up pathogens and produce reactive oxygen radicals and pro- and anti-inammatory cytokines. DCs are sophisticated in antigen presentation and induction of T-cell activation through their expression of costimulatory molecules and cytokine production, whereas NK cells provide interaction with virus-infected cells, T lymphocytes, and DCs [1,3]. Recognition of viral pathogens by a coordinated interaction of the cells of the innate immune system leads to activation of adaptive immunity targeting viral-specic antigens for pathogen elimination. Of the various pattern recognition receptors, Toll-like receptors (TLRs) and RNA helicase receptors play an important role in sensing viral RNA and in induction of the initial type I interferon (IFN) production. Double-stranded (ds) RNA is recognized by TLR3 expressed in the endosomes and retinoic acidinducible gene-I (RIG-I) and melanoma

This work was supported by Public Health Service grants AA014372 and AA008577 to G. Szabo and grant AA016571 to A. Dolganiuc. * Corresponding author. E-mail address: gyongyi.szabo@umassmed.edu (G. Szabo). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.003 liver.theclinics.com

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Fig. 1. Schematic of immune cells in the liver. Hepatocytes (H) are lined with biliary endothelial cells (EC) on the portal facet and stellate cells (SC) in the space of Disse (SD). EC separate the SD from the blood ow. Dendritic cells of plasmacytoid (PDC) and myeloid (MDC) origin, monocytes (Mo), macrophages (Mf), and Kuper cells (KC) are located in close proximity to EC. Blood monocytes are immature and can give rise to Mf or MDC, depending on the environment. On encountering pathogens derived from the bloodstream or infected hepatocytes, MDC and PDC are primarily responsible for antigen presentation to adaptive immune cells and creation of a favorable milieu for antigen presentation, including production of cytokines and availability of costimulatory molecules. In turn, adaptive immune cells, including T and B lymphocytes, NK cells, and NKT cells, react with antigen-specic proliferation, cytotoxicity, and production of soluble mediators, such as antibodies or cytokines. Collectively, these events lead to pathogen elimination. Mo, Mf, and KC also recognize pathogens; however, in contrast to dendritic cells, they have a less pronounced eect on the adaptive immunity. Mo, Mf, and KC are potent producers of inammatory and immunoregulatory cytokines and are powerful sources of free radicals with oxidative capacity, thus leading to initiation and maintenance of tissue inammation. Chronic inammation, in addition to direct pathogen recognition, activates SC, which, in turn, produce collagen and favor development of liver brosis. At endstage liver disease, chronic inammation and brosis drive progression to cirrhosis and possibly favor neoplastic transformation and liver cancer.

dierentiationassociated gene 5 (MDA5) localized in the cytosol, whereas single stranded (ss) RNA is sensed by TLR7 and TLR8 and by RIG-I in some viruses (Figs. 2 and 3) [35]. Toll-like receptor 3 Ligand engagement of TLR3 results in recruitment of the adapter molecule, TLR domain-containing adapter inducing IFNb (TRIF) (see Fig. 2) [6,7]. TRIF interacts with several signaling molecules, including tumor necrosis factor (TNF) receptor-associated factor (TRAF)6, which, in turn, activate nuclear regulatory factor kB (NF-kB) and mitogen-activated protein kinase (MAPK). Important to viral infection, TLR3 stimulation leads to activation of TNFR-associated factor family member-associated NF-kB activator (TANK)-binding kinase (TBK) 1 and IkB kinase (IKK)3, which

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Fig. 2. TLR3 and TLR7/8 signaling. After stimulation with ssRNA, TLR7/8 recruits myeloid dierentiation primary response gene 88 (MyD88), interleukin-1 receptorassociated kinases (IRAKs), and tumor necrosis factor (TNF) receptor-associated factor (TRAF)6 to activate two distinct pathways. When MyD88, IRAKs, and TRAF6 recruit Ubc13/TAK, the transforming growth factor (TGF)-b-activated kinase 1 (TAK1) complex then activates the IkB kinase (IKK) complex, composed of IKKa, IKKb, and IKKg/nuclear factor kB essential modulator, which catalyzes phosphorylation of inhibitory kB proteins and subsequent translocation of nuclear regulatory factor-kB (NF-kB) to the nucleus. TAK1 also activates the mitogen-activated protein kinase (MAPK) pathway, which mediates activator protein 1 (AP-1) activation. NF-kB and AP-1 control the expression of genes encoding inammatory cytokines. When MyD88, IRAKs, and TRAF6 recruit TRAF3, IRAK1, and IKK3 into a complex, interferon regulatory factor (IRF) 7 is directly phosphorylated by IRAK1 and IKK3 and is then translocated to the nucleus to induce expression of IFNa and IFN-inducible genes. On interaction with dsRNA, TLR3 recruits TLR domain-containing adapter inducing IFNb (TRIF) and interacts with TNFR-associated factor family member-associated NF-kB activator (TANK)-binding kinase (TBK) 1 and IKK3 or activates the PI3K/Akt complex, which both mediate phosphorylation of IRF3. The phosphorylated IRF3 dimerizes and is translocated to the nucleus to induce expression of IFNb and IFN-inducible genes, including interferon g inducible protein 10 kDa. TRIF also interacts with TRAF6 and receptor interacting protein 1, which mediate NF-kB activation. TLR3 and TLR7/8 are located in endosomes, and all signaling events occur in the cytoplasm, whereas activated transcription factors IRF3, IRF7, NF-kB, and AP-1 act on the genes within the nucleus. The cellular compartments are not scaled.

phosphorylates interferon regulatory factor (IRF) 3, allowing its dimerization and nuclear translocation. Activated IRF3 binds to the promoter of type I IFNs and triggers antiviral innate immune activation [6,7]. RNA helicases Recently, three homologous DExD/H box RNA helicases emerged as cytoplasmic sensors of virally derived RNA (see Fig. 3). On activation with viral dsRNA, two helicases (RIG-I [also called DDX58] and MDA5 [also called Helicard]) cooperate in induction of antiviral type I IFN [5,8]. The third helicase, LGP2, prevents viral-induced activation, most likely through sequestration of dsRNA from RIG-I or MDA5 [810]. Although

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Fig. 3. Intracytoplasmic viral recognition and antiviral pathways. The RNA derived from virions is recognized at the helicase domain of the RIG-I, MDA5, or LPG2 helicases. On ligand interaction, the tandem caspase activation and recruitment (CARD) domains of RIG-I and MDA5 engage the CARD domain of mitochondria-bound interferon promoter stimulator (IPS-1) and trigger activation of a series of kinases, including IkB kinase (IKK)a, IKKb, IKK3, and TNFR-associated factor family member-associated NF-kB activator (TANK)-binding kinase 1. These signaling events activate tumor necrosis factor (TNF) receptor-associated factor 3, nuclear regulatory factor-kB (NF-kB), and interferon regulatory factor (IRF), leading to their translocation to the nucleus and initiation of type 1 IFN production. LPG2 is CARDless and does not trigger the signaling events; however, it modulates the activity of RIG-I by blocking RIG-I self-association by disrupting homotypic CARD/helicase domain or C terminus interactions, by disrupting assembly of the RIG-Icontaining signaling complex on IPS-1, and by sequestering the viral dsRNA.

RIG-I and MDA5 seem to share structural and functional similarities, their recognition capacity of viral-derived RNA is distinct. RIG-I but not MDA5 senses Japanese encephalitis virus, Newcastle disease virus, vesicular stomatitis virus (VSV), Sendai virus, and inuenza virus, whereas MDA5 mounts antiviral responses against the picornavirus encephalomyocarditis virus [11]. To date, it is unknown how RIG-I undergoes activation only with viral RNA and not with cellular RNA. The direct implication of RIG-I or MDA5 in recognition of ssRNA hepatotropic viruses, such as HCV, has been suggested [12]. Of interest, RIG-I deciency strongly aects antiviral responses in conventional DCs and broblasts; however, no dierences are observed in plasmacytoid DCs, suggesting tissue preference of these helicases. RIG-I, MDA5, and LGP2 are all expressed in hepatocytes [13,14]. Interestingly, decreased expression levels of RIG-I and MDA5 were detected in Huh7 and Huh7.5 cells, which are permissive to HCV replicons and in vitro infection with the replication-ecient JFH1 and JFH/J6 HCV strains [15]. RIG-I and MDA5 share structural similarity in the caspase recruitment domains (CARDs) and RNA helicase domains, whereas LGP2 lacks the

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CARD domain [10,11]. RIG-I and MDA5 use their CARDs to signal downstream events by means of an adaptor named CARD adaptorinducing IFNb (Cardif), mitochondrial antiviral signaling protein (MAVS), interferon promoter stimulator (IPS-1), or virus-induced signaling adaptor (VISA) [16 19]. IPS-1 is anchored with its C terminus to the outer mitochondrial membrane, whereas its N-terminal CARD domain interacts with RIG-I and MDA5. Mitochondrial localization renders IPS-1 functional, because the cytoplasmic domain of the endoplasmic reticulum-bound IPS-1 no longer mediates downstream IRF and NF-kB activation [18]. The details of the signaling pathway downstream of IPS-1 are currently under scrutiny. Once activated by dsRNA, the IPS-1 most probably recruits appropriate signaling intermediates, such as IKKs (namely IKKa, IKKb, IKK3, and TBK1) to activate NF-kB, TRAF3, and IRF transcription factors [20]. All these pathways induce production of type 1 IFNs; however, the kinetics of the dierential production of IFNa and IFNb on retinoic acid-inducible gene I (RIG-I)like helicases (RLHs) activation is yet to be dened. It has recently been demonstrated that RIG-I but not MDA5 eciently binds to secondary structured HCV RNA to confer induction of IFNb expression [12]. LGP2 is a functional negative regulator of host defense, and it binds HCV [12]. In resting cells, RIG-I is maintained as a monomer in an autoinhibited state, but during virus infection and RNA binding, it undergoes conformational changes that promote self-association and CARD interactions with the IPS-1 adaptor protein to signal IRF3 and NF-kB responsive genes [9,10,12]. This interaction is regulated by an internal repressor domain, which controls RIG-I multimerization and recruitment of IPS-1. An analogous regulatory domain in LGP2 interacts with RIG-I to ablate self-association and signaling. Thus, RIG-I is a cytoplasmic sensor of HCV and is governed by regulatory domain interactions in that area shared with LGP2 as an on-o switch controlling innate defenses [12]. Toll-like receptors 7 and 8 TLR7 and TLR8 are expressed in the endosome and recognize several ssRNA viruses (see Fig. 2) [21]. TLR7 was initially identied as a receptor able to recognize imidazoquinolone derivatives with antiviral activity [22]. Subsequently, guanosine or uridine-rich ssRNA derived from HIV-1 and inuenza virus, synthetic poly U RNA, and certain small interfering RNAs were identied as ligands for TLR7 [22,23]. TLR7 is expressed in plasmacytoid DCs, and TLR7 mRNA was detected in hepatocytes [2427]. TLR8 is functional in humans but not in mice, and it is expressed in myeloid DCs, monocytes, macrophages, and regulatory T cells [28,29]. Human TLR8 mediates recognition of HIV-derived ssRNA and chemical ligand R848, and its role in HCV infection is currently unknown [30]. Recent studies revealed functional dierences between human TLR7 and TLR8, wherein TLR7

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agonists primarily activated plasmacytoid dendritic cells (PDCs), whereas TLR8 agonists activated myeloid dendritic cells (MDCs), monocytes, and macrophages [31]. In addition, the cytokine production prole of TLR7 was dominated by IFNa induction, whereas TLR8 triggered predominantly the proinammatory cytokines and chemokines, such as tumor necrosis factor-a (TNFa), interleukin (IL)-12, and macrophage inammatory protein (MIP)-1a [31]. TLR7/8 agonists impair monocyte-derived DC dierentiation and maturation; it is intriguing that the phenotype of TLR7/8 ligandtreated DCs is similar to DC defects found in HCV-infected patients [32,33]. The pattern recognition site composed of leucine-rich repeats of the TLR7/8 molecule is contained within the endosome, whereas the Toll/interleukin-1 receptor (TIR) domain is exposed to the cytoplasm, wherein it transduces intracellular signals by recruitment of the myeloid dierentiation primary response gene 88 (MyD88), a common TLR adaptor protein [3,6]. MyD88 further forms complexes with members of the IL-1 receptorassociated kinase (IRAK) family (IRAK1 and IRAK4) and TRAF6, which, in turn, activate transforming growth factor (TGF)-b-activated kinase 1 (TAK1) and result in NF-kB activation. Type I IFN induction after TLR7 activation is independent of IRF3, suggesting the possible involvement of other IRF family members in this pathway. IRF7 is structurally similar to IRF3, and although its expression is low in most cell types, it is constitutively expressed in PDCs [22,34]. IRF7 is able to form a signaling complex with MyD88, IRAK1, IRAK4, and TRAF6, wherein IRAK1 is capable of phosphorylating IRF7 [35]. Activated IRF7 homodimerizes, allowing this complex to translocate into the nucleus and bind to the interferon-stimulated response element (ISRE) promoter site [36]. Type I IFNs, composed of IFNa and IFNb, are released in response to TLR7 or TLR8 signaling.

Immune response and the outcome of hepatitis C virus infection Studies relating to innate immune response changes during viral clearance are limited. Nevertheless, it is clear that the interaction between HCV viral components and the immune system ultimately determines whether the balance tilts in the favor of the virus, leading to chronic infection, or in favor of the host, conditioning viral clearance. Evidence points to innate and adaptive immune responses as key determinants of the outcome. A strong, multispecic, T-lymphocyte response, including CD4 and CD8 cells, produced early in infection is associated with viral clearance and disease resolution, whereas a narrowly focused and delayed response is associated with chronic infection [3741]. Even in patients who develop chronic infection, however, T-cell responses were briey or episodically vigorous rather than absent at the onset of infection, indicating that a substantive CD4 T-cell response must also be maintained to facilitate clearance [42]. Further, CD8 T cells lose recognition of one or more HCV epitopes

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originally targeted in primary infection in patients who progressed to chronic infection, thus leading to almost a three-quarters reduction in the magnitude of the response [43]. Importantly, no new epitope specicity is developed in T cells of patients who have persistent HCV infection [43,44]. These results indicate that adaptive responses cannot overcome HCV aggressiveness alone and suggest that the adaptive immune system fails to receive support from innate immune cells to provide a long-lasting defense. The information in regard to the function of innate immunity during early HCV infection is scarce. In one study, resolution of acute HCV infection was associated with a single nucleotide polymorphism in the haplotype region of IL-10 and IL-19/IL-20 genes in African Americans but not in European Americans [45].

Hepatitis C virus interferes with innate immune recognition Increasing evidence suggests that HCV can interfere with innate immune activation at multiple levels. The nonstructural proteins of HCV, particularly NS3-4A, have been found to interact with various host adaptor molecules to disrupt type I IFN induction pathways. Foy and colleagues [46] found that NS3-4A serine protease blocked HCV-induced activation of IRF3 in the human hepatoma cell line, Huh7. It has been reported that NS3-4A protease also targets and cleaves the IPS-1 adaptor protein from the mitochondria to ablate signaling to IFNa/b immune defenses [16,47]. Inhibition of RIG-Idependent signaling to the IFN pathway has been found in HCV infection by the HCV NS3/4A protease activity [48,49]. This inhibition was localized to upstream of the noncanonical IKK-related kinases IKK3 and TBK1, which phosphorylate IRF3, at the level of the TLR adapter protein TRIF [48,49] and intercellular adhesion molecule 1 (ICAM1). In the replicon system IKK3 overexpression inhibited HCV expression even in the presence of neutralizing antibodies to IFN receptors or in the presence of a dominant negative specically targeted antiviral therapy (STAT) mutant [50], suggesting that IKK3 expression is important for rapid activation of the cellular antiviral response in HCV infection. In the liver biopsies of patients who have HCV, expression of IKK3 and the RNA helicases RIG-I, MDA5, and LGP2 was signicantly reduced, whereas expression of TBK1 and Cardif was not signicantly altered [50]. These observations support the contention that HCV interferes with host pathways directed at viral elimination. Although the HCV NS3-4Amediated cleavage of the adaptor molecules TRIF (adaptor to TLR3) and Cardif and IPS-1 (adaptor to RIG-I and MDA5) has been reported in the human hepatoma cells Huh7 and Huh7.5, recent studies performed in the nonneoplastic human hepatocyte PH5CH8 cells showed a retained and robust TRIF- and Cardif-mediated pathway activation. Dansako and colleagues [14] found that more robust

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induction of IFNb in PH5CH8 cells compared with Huh7 and NS3-4A failed to suppress TRIF-mediated IFNb production in these cells. Cardifmediated IFNb production was still suppressed by NS3-4A by cleaving Cardif at the Cys508 residue. Further analysis of the HCV NS3-4A protease dened the site of its interaction with host targets. NS3 mutants lacking the helicase domain retained the ability to control virus signaling initiated by RIG-I or MDA5 and suppressed downstream activation of IRF3 and NF-kB through the targeted proteolysis of IPS-1 [51]. Truncation of the NS3 protease domain or point mutation ablated the protease activity, however, providing evidence for the active site of NS3 interaction with the host immune recognition pathways. A recent study found modulation of TLR-mediated signaling in a macrophage cell line expressing HCV proteins [52]. Various genotypes of NS5A protein bound the common TLR adaptor MyD88, and thereby inhibited the recruitment of IRAK1 to MyD88 and impaired cytokine production in response to TLR ligands. Transfection of NS3, NS3/4, NS5B, or NS5A into mouse macrophages resulted in inhibition of IL-6 induction by various TLR ligands [52]. Although this observation indicates a novel possible way of interaction of HCV with innate immunity, results of this in vitro study are in contrast with previous observations from human monocytes and macrophages from individuals who had chronic HCV infection, wherein increased proinammatory cytokine induction was observed in response to TLR4 or TLR2 stimulation [53,54]. An additional consideration is whether macrophages are infected with HCV or whether these cells support HCV replication. Positive-strand RNA has been detected in peripheral blood mononuclear cells (PBMCs) and blood monocytes by several groups; however, observation of negative-strand RNA, which would indicate active viral replication, in monocytes and macrophages has been found [55]. A recent study found the presence of HCV genomic RNA in circulating DCs and at even higher expression level in monocytes [56]. In this study, infection of DCs with HCV was associated with impaired expression of IL-12 and TNFa [56]. In the liver, DCs and resident and recruited macrophages play an important role in elimination of damaged cells. In in vitro studies, engulfment of apoptotic blood mononuclear cells expressing HCV proteins resulted in differential chemokine expression and STAT signaling in DCs [57], suggesting that virus-infected hepatocytes may modulate phagocytic cell functions even in the absence of their direct infection with HCV. Increased expression of TLR2, TLR3, and TLR6 mRNA was found in PBMCs of patients who have chronic HCV infection that correlated with sustained virologic response [58]. In HCV treatment trials, the number of genes that were up- or downregulated by pegylated IFN and ribavirin treatment was fewer in patients with a poor response than in those with an intermediate or marked viral response [59]. The induction of IFN-inducible genes (20 50 -oligonucleotide synthetase), MX1, IRF7, and TLR7 genes was lower in patients with a poor response compared with patients with a marked or intermediate

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response, suggesting that blunted IFN signaling and TLR signaling is associated with the lack of response to IFN therapy [59]. A recent study found an association between TLR7 single nucleotide polymorphism (SNP) and protection from advanced inammation and brosis in male patients who had chronic HCV infection [60]. The C1-120G TLR7 allele was found to oer protection from the development of inammation and brosis [60].

Hepatitis C virus interferes with activation of adaptive immune responses by innate immune cells Eects on dendritic cells In addition to recognition of invading pathogens, DCs play a central role in activation of naive T lymphocytes to initiate virus-specic T-cell responses. DCs, including circulating MDCs, monocyte-derived DCs, and PDCs, have been studied in chronic HCV infection by several groups of investigators. PDCs are the major producers of IFNa and are specically equipped to sense viral nucleic acids by means of their expression of TLR7 and TLR9 [61]. Most investigators found decreased frequency, reduced IFNa production, and impaired T-cell stimulatory capacity of circulating PDCs [62,63]. Interestingly, the expression of CD123 and BDCA2, markers of PDCs, were expressed at higher levels in livers of HCV-infected patients, raising the possibility of sequestration of PDCs in HCV-infected livers [63]. Indeed, enrichment for DCs within the intrahepatic compartment was recently reported [64], possibly attributable to HCV E2/CD81-mediated induction of regulated on activation, normal T-cell expressed and secreted (RANTES) [65]. Despite the immunotolerogenic environment in the liver [66,67], MDCs from HCV-infected liver demonstrated higher expression of major histocompatibility complex (MHC) class II, CD86, and CD123; were more ecient stimulators of allogeneic T cells; and secreted less IL-10 compared with controls [68,69]. In contrast, some researchers nd that PDCs were present at lower frequencies in HCV-infected liver; however, they expressed higher levels of the regulatory receptor BDCA2 [68,69] and showed increased ability to prime T cells compared with controls [70]. Although the active HCV infection of DCs is uncertain [71,72], the HCV quasispecies sequences cloned from DCs bear an internal ribosome entry site with poor eciency for translation in cells of liver, lymphoid, or DC origin [73], suggesting that passage through DCs signicantly aects the function of the HCV virion. There are clear indications that HCV-derived proteins expressed in vivo aect immune functions. Studies from mice expressing HCV nonstructural protein showed decreased capacity of a mixed PDC and MDC population to activate T cells [74], whereas overexpression of structural proteins leads to impaired MHC class I presentation during DC maturation [75].

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In human studies, ndings related to myeloid DC functions are controversial. Complex defects, namely, decreased T-cell stimulatory capacity, overproduction of the immunoregulatory cytokine IL-10, and deciency in costimulatory molecules, were detected in MDCs of patients who had chronic HCV infection by some investigators [33,7680], whereas others failed to identify any MDC abnormalities [8185]. Such discrepancies most possibly derive from dierent patient cohorts but also from assessment of nonhuman primate models of HCV infection, dierent experimental approaches, and distinct read-outs. Anti-HCV treatment also may aect DC functions. Combination a-IFNribavirin therapy alters the cytokine prole of maturing DCs by suppressing IL-10 production but maintaining IL-12 (p70) and TNFa production, a pattern that would favor viral elimination through downstream eects on T cells [86]. MDCs from HCV-infected patients are impaired in their ability to drive T helper 1 (Th1) in response to IFNa [87]. Natural killer cells NK cells constitute a potent rapid part of the innate immune response to viral infections but also to neoplastic cells and also participate in priming of adaptive immunity [88]. NK cells are capable of performing cytolysis in addition to cytokine and chemokine release [88]. NK cells mount an anti-HCV response and can be triggered by HCV-derived proteins or HCV-infected cells. In vitro, NK cells were capable of inducing an HCV-associated or perforin- or granzyme-dependent lysis of human hepatoma cells in a direct cellular contact-dependent but MHC class Iindependent manner [89,90]. Such a potent cytolytic eect is only observed in cytokine-primed NKs, however, suggesting that HCV-infected cells are poor triggers of NK activity or that such eect is more potent in the context of established HCV-specic immune response. Further, inhibition of NK-cell CD16-mediated cytotoxicity after engagement of CD81 molecules on NK cells with HCV E2 protein was reported [91,92]. Impaired NK-cell triggering attributable to reduced expression of NKG2D ligands (major histocompatibility complex [MHC] class I chain-related A and B) on mature DCs and increased NK-cell inhibition through increased CD94/ NKG2A expression and transforming growth factor-b (TGFb) and IL10 production have been suggested to occur in vivo during HCV infection [9396]. More recently, increased expression of NKp30 and NKp46, the specialized NK-triggering receptors involved in nonMHC-restricted natural cytotoxicity, was identied in NK cells from HCV-infected patients [97]. Freshly separated NK cells from HCV-infected patients showed a signicant production of IL-10 and normal concentrations of IFNg on direct cell contactmediated triggering [97]. Thus, skewed NK receptor expression during HCV infection combined with increased production of immunoregulatory cytokines could contribute to inecient NK-DC and

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NKT-cell cross-talk, leading to inecient subsequent adaptive immune responses and lack of virus control [98].

Hepatitis C virus infection results in inammatory cell activation Chronic HCV infection is associated with activation of the inammatory cell and cytokine cascade, including recruitment of inammatory cells to the HCV-infected liver, increased liver and serum levels of proinammatory cytokines, and evidence of monocyte or macrophage activation [99]. Several mechanisms may account for this inammatory activation, including pattern recognition receptor activation as a result of HCV infection and amplication of the cytokine cascade by endogenous mediators or HCV-derived products. In addition to TLRs that recognize viral nucleic acid sequences, surfaceexpressed TLRs have been shown to sense HCV viral proteins, and thereby induce proinammatory pathways in inammatory cells. TLR2-mediated activation of monocytes and macrophages is induced by HCV core and NS3 proteins to result in activation of the inammatory cascade, including activation of IRAK1 kinase, NF-kB, MAPK, and TNFa production [33,54,63,100]. In addition, monocytes of patients who have chronic HCV infection respond to TLR4 stimulation with an augmented proinammatory response compared with noninfected controls [33,63]. Increased levels of TLR2 and TLR4 expression were observed in PBMCs of patients who had chronic HCV infection, and increased expression of TLR2 was particularly associated with increased circulating TNFa levels and hepatic inammatory activity [101]. In support of the role of TLR2 in HCV infection, a recent study found an association between a homozygous TLR2 Arg753Gln polymorphism and allograft failure and mortality after liver transplantation for chronic HCV, whereas there was no association found for TLR4 (Asp299Gly and Thr399Ile) polymorphism [102]. It has recently been proposed that the increased activation of inammatory cascade activation in HCV could be related to a loss of TLR tolerance in chronically HCV infected patients monocytes by means of multiple TLR signals, such as circulating HCV core proteins that stimulate TLR2, low levels of circulating endotoxin in these patients (TLR4), and the presence of increased levels of IFNg that can amplify inammatory cell activation and promote loss of TLR tolerance [54,103]. In addition to HCV proteins, some investigators found cell activation by HCV lipopeptides by means of TLR2 and TLR4 [53]. In children, chronic HCV infection was associated with increased expression of TLR2 and TLR4 in neutrophil leukocytes compared with HCVnegative controls or HCV antibody-positive individuals who spontaneously cleared HCV infection [104]. TLR2 and TLR4 mRNA and protein expression were also increased in the liver of children with chronic HCV infection compared with controls without viral infection [105].

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In adult HCV-infected cirrhotic livers, gene expression changes involved activation of the innate antiviral immune response genes that was in contrast with the gene activation pattern in livers with alcoholic cirrhosis [106]. The link between activation of inammatory cells and mediators and stellate cell activation and brosis is well established [107,108]. Recent evidence suggests that persistent inammation also predisposes to cancer. Consistent with this, biomarkers of oxidative stress and inammation were associated with hepatocellular carcinoma (HCC) in HCV-infected livers [109]. Another study found that chronic inammation associated with HCV infection shifts hepatocytic TGFb signaling from tumor suppression to brogenesis, which can accelerate liver brosis and the risk for HCC [110]. These observations lend support to the role of inammation in the increased frequency of HCC in HCV-infected livers. Innate immunity as a therapeutic target in hepatitis C virus infection Potent activation of antiviral immune pathways though selective TLR activation provides an attractive therapeutic target in HCV treatment. In support of this contention, recent studies found promising results with TLR7 and TLR9 agonists. The TLR7 and TLR9 activation strategy is based on increasing endogenous IFNa production in DCs; however, additional immunomodulatory eects of TLR9 or TLR7 are yet to be evaluated. Isatoribine, an agonist of TLR7, reduced plasma virus concentrations in chronic HCV infection [111]. Oral resiquimod, a TLR7 and TLR8 agonist, showed promising antiviral eects in phase IIa safety and ecacy trials [112,113]. IFNa levels correlated with decreases in viral titer and lymphocyte counts in the treatment group [112,113]. In another study, administration of CPG10101, a TLR9 agonist, showed safety and ecacy in normal volunteers [114]. Although u-like symptoms were the reported side eects, CPG10101 induced IFNs, cytokines, and chemokines in vivo, suggesting its potential in HCV therapy. At this time, however, further clinical trials with the TLR7 and TLR9 agonists are on hold because of concerns related to some of their side eects. Analysis of various TLR ligands on induction of antiviral molecules in human PBMCs revealed that agonists of TLR3, TLR4, TLR7, TLR8, and TLR9 were potent inducers of antiviral activity, including induction of IFNa and IFN-induced 20 ,50 -oligoadenylate synthase [115]. TLR4 and TLR8 stimulation also induced high levels of TNFa and IL-1b [115]. Recent localization of the functional NS3-4A protease cleavage domain on IPS-1, which is essential for blocking RIG-I signaling to IRF3 and NF-kB, provided another potential therapeutic target for HCV [51]. In vitro studies suggest that TLR ligands may overcome some of the immune defects associated with HCV infection. Yonkers and colleagues [62] reported that TLR3, TLR7/8, and TLR9 ligands could enhance MDC and PDC activation of naive CD4 T cells. PDCs from HCV-infected patients had reduced

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expression of activation markers (human leukocyte antigen D-related [HLA-DR]) and IFNa production on TLR7/8 stimulation, however, and showed decreased activation of CD4 T cells [62]. These data indicate that stimulation of certain TLRs may have benet on restoration of innate and adaptive immunity in chronic HCV infection. Summary Increasing evidence suggests that HCV can interfere with innate immune activation at multiple levels. First, HCV, through its viral proteins, can undermine viral recognition by cleaving pivotal adaptor proteins in TLR and RIG-I or MDA5 signaling. Second, HCV directly or indirectly modulates key antigen-presenting functions of various DC types, contributing to impaired virus-specic T-cell activation. Third, IFNa production by PDCs, the main cell type producing IFNa, is drastically reduced in chronic HCV infection. Fourth, chronic HCV infection results in activation of proinammatory pathways and mediators in inammatory cells that contribute not only to aberrant innate-adaptive immune interactions but to activation of liver brosis and a microenvironment that may support cancer formation. Therapeutic strategies to counteract innate immune alterations in chronic HCV provide a promising target and need further investigation.

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[77] Kanto T, Hayashi N, Takehara T, et al. Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus-infected individuals. J Immunol 1999;162(9):558491. [78] Bain C, Fatmi A, Zoulim F, et al. Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gastroenterology 2001;120(2):51224. [79] Gelderblom HC, Nijhuis LE, de Jong EC, et al. Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine prole. Liver Int 2007;27(7):94453. [80] MacDonald AJ, Semper AE, Libri NA, et al. Monocyte-derived dendritic cell function in chronic hepatitis C is impaired at physiological numbers of dendritic cells. Clin Exp Immunol 2007;148(3):494500. [81] Larsson M, Babcock E, Grakoui A, et al. Lack of phenotypic and functional impairment in dendritic cells from chimpanzees chronically infected with hepatitis C virus. J Virol 2004; 78(12):615161. [82] Longman RS, Talal AH, Jacobson IM, et al. Normal functional capacity in circulating myeloid and plasmacytoid dendritic cells in patients with chronic hepatitis C. J Infect Dis 2005;192(3):497503. [83] Longman RS, Talal AH, Jacobson IM, et al. Presence of functional dendritic cells in patients chronically infected with hepatitis C virus. Blood 2004;103(3):10269. [84] Piccioli D, Tavarini S, Nuti S, et al. Comparable functions of plasmacytoid and monocytederived dendritic cells in chronic hepatitis C patients and healthy donors. J Hepatol 2005; 42(1):617. [85] Rollier C, Drexhage JA, Verstrepen BE, et al. Chronic hepatitis C virus infection established and maintained in chimpanzees independent of dendritic cell impairment. Hepatology 2003; 38(4):8518. [86] Barnes E, Salio M, Cerundolo V, et al. Impact of alpha interferon and ribavirin on the function of maturing dendritic cells. Antimicrob Agents Chemother 2004;48(9):33829. [87] Miyatake H, Kanto T, Inoue M, et al. Impaired ability of interferon-alpha-primed dendritic cells to stimulate Th1-type CD4 T-cell response in chronic hepatitis C virus infection. J Viral Hepat 2007;14(6):40412. [88] Lee SH, Miyagi T, Biron CA. Keeping NK cells in highly regulated antiviral warfare. Trends Immunol 2007;28(6):2529. [89] Larkin J, Bost A, Glass JI, et al. Cytokine-activated natural killer cells exert direct killing of hepatoma cells harboring hepatitis C virus replicons. J Interferon Cytokine Res 2006; 26(12):85465. [90] Larkin J, Jin L, Farmen M, et al. Synergistic antiviral activity of human interferon combinations in the hepatitis C virus replicon system. J Interferon Cytokine Res 2003;23(5): 24757. [91] Crotta S, Ronconi V, Ulivieri C, et al. Cytoskeleton rearrangement induced by tetraspanin engagement modulates the activation of T and NK cells. Eur J Immunol 2006;36(4):91929. [92] Crotta S, Stilla A, Wack A, et al. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med 2002;195(1):3541. [93] Jinushi M, Takehara T, Tatsumi T, et al. Negative regulation of NK cell activities by inhibitory receptor CD94/NKG2A leads to altered NK cell-induced modulation of dendritic cell functions in chronic hepatitis C virus infection. J Immunol 2004;173(10):607281. [94] Jinushi M, Takehara T, Tatsumi T, et al. Autocrine/paracrine IL-15 that is required for type I IFN-mediated dendritic cell expression of MHC class I-related chain A and B is impaired in hepatitis C virus infection. J Immunol 2003;171(10):54239. [95] Lechmann M, Woitas RP, Langhans B, et al. Decreased frequency of HCV core-specic peripheral blood mononuclear cells with type 1 cytokine secretion in chronic hepatitis C. J Hepatol 1999;31(6):9718. [96] Godkin A, Jeanguet N, Thursz M, et al. Characterization of novel HLA-DR11-restricted HCV epitopes reveals both qualitative and quantitative dierences in HCV-specic

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CD4 T cell responses in chronically infected and non-viremic patients. Eur J Immunol 2001;31(5):143846. De Maria A, Fogli M, Mazza S, et al. Increased natural cytotoxicity receptor expression and relevant IL-10 production in NK cells from chronically infected viremic HCV patients. Eur J Immunol 2007;37(2):44555. Golden-Mason L, Rosen HR. Natural killer cells: primary target for hepatitis C virus immune evasion strategies? Liver Transpl 2006;12(3):36372. Szabo G, Dolganiuc A. HCV immunopathogenesis: virus-induced strategies against host immunity. Clin Liver Dis 2006;10(4):75371. Chang S, Dolganiuc A, Szabo G. Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins. J Leukoc Biol 2007; 82(3):47987. Riordan SM, Skinner NA, Kurtovic J, et al. Toll-like receptor expression in chronic hepatitis C: correlation with pro-inammatory cytokine levels and liver injury. Inamm Res 2006;55(7):27985. Eid AJ, Brown RA, Paya CV, et al. Association between toll-like receptor polymorphisms and the outcome of liver transplantation for chronic hepatitis C virus. Transplantation 2007;84(4):5116. Dolganiuc A, Oak S, Kodys K, et al. Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inammatory activation. Gastroenterology 2004;127(5):151324. Wisniewska-Ligier M, Wozniakowska-Gesicka T, Glowacka E, et al. Involvement of innate immunity in the pathogenesis of chronic hepatitis C in children. Scand J Immunol 2006;64(4):42532. Mozer-Lisewska I, Sluzewski W, Kaczmarek M, et al. Tissue localization of Toll-like receptors in biopsy specimens of liver from children infected with hepatitis C virus. Scand J Immunol 2005;62(4):40712. Lederer SL, Walters KA, Proll S, et al. Distinct cellular responses dierentiating alcoholand hepatitis C virus-induced liver cirrhosis. Virol J 2006;3:98. Iredale JP. Models of liver brosis: exploring the dynamic nature of inammation and repair in a solid organ. J Clin Invest 2007;117(3):53948. DeMinicis S, Bataller R, Brenner DA. NADPH oxidase in the liver: defensive, oensive, or brogenic? Gastroenterology 2006;131(1):2725. Maki A, Kono H, Gupta M, et al. Predictive power of biomarkers of oxidative stress and inammation in patients with hepatitis C virus-associated hepatocellular carcinoma. Ann Surg Oncol 2007;14(3):118290. Matsuzaki K, Murata M, Yoshida K, et al. Chronic inammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma. Hepatology 2007;46(1):4857. Horsmans Y, Berg T, Desager JP, et al. Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection. Hepatology 2005;42(3):72431. Pockros PJ, Schi ER, Shiman ML, et al. Oral IDN-6556, an antiapoptotic caspase inhibitor, may lower aminotransferase activity in patients with chronic hepatitis C. Hepatology 2007;46(2):3249. Pockros PJ, Guyader D, Patton H, et al. Oral resiquimod in chronic HCV infection: safety and ecacy in 2 placebo-controlled, double-blind phase IIa studies. J Hepatol 2007;47(2): 17482. Vicari AP, Schmalbach T, Lekstrom-Himes J, et al. Safety, pharmacokinetics and immune eects in normal volunteers of CPG 10101 (ACTILOM), an investigational synthetic tolllike receptor 9 agonist. Antivir Ther 2007;12(5):74151. Thomas A, Laxton C, Rodman J, et al. Investigating Toll-like receptor agonists for potential to treat hepatitis C virus infection. Antimicrob Agents Chemother 2007; 31(8):296978.

Clin Liver Dis 12 (2008) 693712

Hepatitis C Virus Entry and Neutralization


Zania Stamataki, PhD, Joe Grove, BSc, Peter Balfe, PhD, Jane A. McKeating, PhD*
Division of Immunity and Infection, Institute for Biomedical Research, University of Birmingham, Edgbaston B15 2TT, United Kingdom

Between 2 and 3% of the worlds population is infected with hepatitis C virus (HCV) [1]. Ten to 15% of infected individuals develop liver disease, with a signicant fraction requiring long-term health care for the treatment of brosis, cirrhosis, and hepatocellular carcinoma. HCV is one of the major indicators for liver transplantation, with current predictions suggesting that 70% of transplants are likely to be for HCV- related disease by 2010. The only available therapy, pegylated interferon (IFN) and ribavirin, is toxic, costly, complex to administer and in some cases ineective, with up to 50% of subjects infected with genotype 1 viruses failing to respond to treatment [2]. Although alterations to therapeutic regimens may increase the success of IFN-based therapies [3], there is an urgent need for new approaches. The recent development of systems to propagate HCV in cell culture (HCVcc) have allowed studies on the complete viral life cycle and oer new targets for the development of antiviral agents. The observation that infected individuals can spontaneously resolve HCV infection oers hope for the development of vaccine(s) that can mimic the natural clearance process and reduce the incidence of HCV-related disease. In this review, the authors summarize the current understanding of the mechanism(s) dening HCV entry and the role of neutralizing antibodies (nAbs) in controlling HCV replication. Tools to study hepatitis C virus entry Currently, there are two methods for studying HCV entry. The rst is based on the capacity of retroviruses lacking endogenous glycoproteins
* Corresponding author. E-mail address: j.a.mckeating@bham.ac.uk (J.A. McKeating). 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.008 liver.theclinics.com

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(gps) to incorporate foreign envelope proteins. The resulting pseudoparticles infect cells in a manner that is dened by the heterologous encoded gps. HCV encodes two gps, E1 and E2, and their detailed processing and biogenesis have been reviewed elsewhere [4]. The pseudotyping approach has allowed the characterization of viruses bearing a range of HCV gps of diverse genotypes and has established that E1 and E2 are both required to generate infectious HCV pseudoparticles (HCVpp) [57]. HCVpp entry is largely restricted to cells of the hepatocyte lineage, suggesting that HCV tropism for the liver is partially dened at the level of virus-receptor interaction(s) [5,810]. The second method for studying viral entry uses the recently described JFH-1 (Japanese fulminant hepatitis-1) strain of HCV that generates infectious particles in cell culture [1113]. Although restricted in the repertoire of E1E2 gps that can be studied, many of the entry characteristics of HCVpp have been conrmed in the HCVcc system, with respect to cellular tropism and the sensitivity of virus to neutralizing ligands. HCV E1 and E2 have been reported to form heterodimers [4,14], and recent data suggest that E1 may trimerize and the functional gp unit may represent a trimer of E1E2 heterodimers [15]. These observations support a physical model of HCV analogous to that of the tickborne encephalitis virion [16].

Elucidation of the receptors dening hepatitis C virus attachment and internalization HCVpp entry into hepatoma cell lines and primary human hepatocytes is via pH-dependent clathrin-mediated endocytosis [17,18]. Current evidence suggests that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81 [5,9,13,19], scavenger receptor class B member I (SR-BI) [9,2022], and the tight junction (TJ) protein claudin-1 (CLDN1) [23]. Other factors, such as glycosaminoglycans (GAGs) [24,25] and low-density lipoprotein (LDL) receptor [26], have been implicated in HCV entry, although their role is less well established. The authors review the evidence supporting a role for each of these molecules in the viral entry process. Tetraspanin CD81 Since the primary report describing the interaction of a soluble truncated form of HCV E2 (sE2) with CD81 [19], investigators have sought to establish the role of CD81 in the viral entry process. Several studies have reported that antibodies specic for CD81 [10,27] and soluble recombinant forms of the second extracellular loop (EC2) of CD81 (sCD81) inhibit HCVpp and HCVcc infection [27]. Further experiments demonstrating that transduction of HepG2 and HH29 human hepatoma cells, which lack CD81, to express CD81 rendered them permissive for HCV infection [8,10,13,27,28]. Furthermore, siRNA silencing of CD81 reduced viral infection [10,13,27] conrmed

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the essential role of CD81 in HCV entry. Diverse strains of HCV sE2 were reported to interact with CD81 with diering anities, with the genotype 1a strain H77 demonstrating the highest anity [10,2931]. The infectivity of HCVpp expressing a diverse panel of E1E2 gps depends on CD81; with all viruses showing comparable levels of sensitivity to neutralization by anti-CD81 monoclonal antibodies (mAbs) [28]. These data suggest important dierences in the interaction of E1E2 and sE2 gps with CD81 and that care should be taken in extrapolating data with sE2 for virus-receptor interactions [8]. Studies to assess whether CD81 is the primary receptor dening viral attachment have been inconclusive, and experiments have been unable to demonstrate viral attachment to cells that were known to be fully permissive for viral entry when compared with nonpermissive cell lines [23,32]. Nevertheless, studies designed to address whether anti-CD81 or sCD81 neutralize viral infection before or after viral adsorption to cells suggest that they mediate their eects after binding, and that CD81 is most likely to act as a coreceptor after viral attachment [8,33,34]. Several reports have suggested that the level(s) of CD81 expressed at the cell surface dene the susceptibility of cells to infection [3537]. The identication or mapping of amino-acid (aa) residues in E2 and CD81 that are critical for the interaction has been complicated by the involvement of multiple residues located throughout CD81 EC2 and the E2 gp [32,3842]. The critical regions of E2 were initially identied by screening E2-specic mAbs for their ability to inhibit sE2 binding to cell surfaceexpressed CD81 or sCD81 [5,30,43,44] and by the mutagenesis of putative CD81 interacting domains in E2 [42,4548]. These studies identied aas within the hypervariable region (HVR: aa 384401), a region immediately adjacent to the HVR [5,38,43,45,47], aa 480493 [49], aa 522 through 551 [30,43,47,50], and a region encompassing aa 613 through 618 [46]. Among mammals, CD81 is well conserved and comparison of sequences from several species that are nonpermissive for HCV infection enabled the identication of aa residues that are critical for CD81 interaction with sE2 [8,51,52]. Expression of these CD81 variants in HepG2 cells identied their ability to confer viral entry, albeit at reduced eciencies, highlighting once again the dierence in the interaction of sCD81 and full-length cell surfaceexpressed CD81 with HCV gps [8]. Tetraspanins are fourtransmembrane proteins that typically reside at the cell surface and assemble with themselves and other proteins to form tetraspanin-enriched microdomains (TEMs) (reviewed in [53]). Palmitoylation and multiple regions within the extracellular and cytoplasmic tails of tetraspanins have been reported to be important for oligomerization and association with TEMs [5456]. Mutagenesis studies identied that deletion of N- and C-terminal cytoplasmic tails and sites of palmitoylation resulted in variant CD81 molecules that could still allow HCV entry into HepG2 cells, suggesting that CD81 does not need to associate with TEMs to function as a viral coreceptor [41].

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Scavenger receptor class B member I The rst evidence for a role of SR-BI in HCV entry was the demonstration that sE2 bound to CD81-negative HepG2 cells by means of SR-BI [20]. SR-BI is the major receptor for high-density lipoprotein (HDL) and is involved in the tracking of cholesterol into hepatocytes by means of selective uptake from cholesterol-rich lipoproteins and by the uptake of HDL particles into endosomes. The role of SR-BI in lipid physiology was recently reviewed [57]. Unlike CD81, which is expressed ubiquitously, SR-BI expression is not relatively restricted from what we know, but it is higher in the adrenal glands and liver. Antibodies specic for SR-BI inhibit HCVcc infection, supporting a role for the molecule in HCV entry [9,21,22,58]. Experiments to validate an essential role for SR-BI in viral entry have proved dicult, however, because all cell types studied to date express SR-BI and siRNA silencing has been reported to have variable eects on HCVpp infectivity [9,10,34]. The interaction of sE2 with SR-BI has been shown to involve residues within and adjacent to the HVR [45,59,60], wherein deletion of the HVR ablates sE2-SR-BI interaction and limits HCVpp infection [9,20,45]. It has been suggested that this interaction is largely attributable to the overall conformation of the HVR rather than to specic sequences, because replacement of conserved or charged residues within the HVR modulated SR-BI binding in a predictable manner [45,60]. Alternative splicing of SR-BI mRNA yields the SR-BII isoform, which has a distinct C-terminal intracellular region lacking the PDZ domain present in SR-BI [61]. Although most SR-BI is found at the cell surface, 85% of SR-BII is expressed intracellularly [62]. SR-BII is capable of selective cholesterol uptake and receptor-mediated lipoprotein endocytosis [62], with the latter occurring by means of clathrin-coated vesicles dened by a dileucine motif in the C-terminal region [63]. Overexpression of both isoforms enhanced HCVcc infection, suggesting that both molecules can mediate HCV entry [21]. Several SR-BI ligands have been shown to inuence HCV infection, acting to enhance (HDL [64,65]) or inhibit (oxidized LDL [34], serum amyloid A [66,67]) viral entry. LDL and HDL have been reported to bind to dierent sites on SR-BI [68], suggesting that the site of ligand engagement has consequences for HCV interactions with SR-BI. Additional agents involved in lipoprotein tracking have been shown to exert pleiotropic eects on HCV infection. Very low-density lipoprotein (VLDL) particles containing apolipoprotein B (ApoB) and ApoE have been shown to associate with HCV during viral secretion from hepatoma cells [6971], suggesting that HCV particles are complexed with lipoproteins [72,73]. The addition of exogenous ApoB has been shown to inhibit HCV entry [59,74], whereas lipid-associated ApoC1 enhances viral entry [59]. Antibodies to ApoB have been reported to inhibit HCV infection [59,75]. Lipoprotein lipase (LPL) [75,76], which

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enhances the tracking of triglyceride-rich lipoproteins, including VLDL, LDL and oxidized LDL, in the liver, has been reported to neutralize HCV infection [75]. The role of lipid metabolism and cholesterol in the HCV life cycle has recently been reviewed [77]. Overall, this series of observations suggests that the uptake of HCV by target cells is intimately linked with the normal hepatocellular processes of lipid transport and agents that interfere with this process modulate HCV infection.

Tight junction claudin proteins An additional host cell molecule reported to be important for HCV entry was recently identied as the TJ protein CLDN1 [78]. TJs are continuous intercellular contacts at the apical poles of lateral cell membranes that form a barrier to regulate the paracellular transit of solutes across a cell layer and to establish cell polarity [79]. The CLDN family of transmembrane proteins extends into the paracellular space, wherein they form homo- and heteroassociations with CLDNs on apposing cells [80]. In an analogous manner to the approaches taken to validate the role of CD81 as a coreceptor for HCV, the following experiments conrmed the CLDN1 dependence of HCV entry: expression of CLDN1 in several nonpermissive cell lines allowed HCVpp and HCVcc entry [78], siRNA silencing of CLDN1 expression in permissive hepatoma cells reduced HCV infection and mutagenesis, and antibody blocking studies with tagged versions of CLDN1 demonstrated that the rst extracellular loop (EC1) is an essential coreceptor during late stage(s) of the HCV entry process [78]. CLDN1 is a member of a large family of related proteins. Of the 20 CLDN family members identied in humans to date, CLDN1, CLDN3, CLDN4, CLDN6, CLDN7 CLDN12, CLDN15, and CLDN23 have been detected in Serial Analysis of Gene Expression (SAGE) libraries derived from liver [81]. Recently, CLDN6 and CLDN9 were reported to act as coreceptors allowing HCV entry [82,83]. Mutagenesis studies of CLDN6 and CLDN9 [82] suggest that the EC1 of these molecules is important for coreceptor activity, involving a region thought to be involved in intercellular interactions [84]. Several TJ proteins have been reported to act as primary receptors for a range of viruses, including junctional adhesion molecule (JAM) for reovirus [85] and feline calicivirus [86] and coxsackie and adenovirus receptor (CAR) for coxsackievirus and adenovirus [87]. Recent work detailing the complex mechanism(s) underlying coxsackie virus group B virus (CBV) highlight the dynamic properties of intercellular junctions [88,89]. CBV binds to a primary receptor, decay accelerating factor (DAF), expressed on the luminal surface of polarized intestinal epithelial cells. CBV interaction with DAF initiates a signaling cascade that triggers an actin-dependent relocalization of the virion-DAF complex to lateral cell junctions, in which CAR is located and endocytosis can occur [89]. The authors recently reported that CLDN1 is

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predominantly expressed at the apical (canalicular) surface of hepatocytes in normal liver tissue consistent with its location at TJs; however, CLDN1 was also detected at the basolateral surface of hepatocytes [90]. CLDN1 colocalized with CD81 at apical and basolateral domains and with SR-BI at basolateral sites, supporting a model in which receptor complexes exist at the site(s) of HCV entry into the parenchyma by means of the sinusoidal blood. Role of cell polarization in hepatitis C virus infection Many tissues in the body contain polarized cells, and hepatocytes in the liver are known to be polarized, with TJs separating the apical (canalicular) and basolateral (sinusoidal) domains. Hepatic polarity is critical for normal liver function, with particular membrane domains performing specic tasks, such as biliary secretion from the canaliculi and serum protein secretion from the sinusoidal surface(s). To initiate infection, pathogens must breach the epithelial barrier to gain access to the body, and TJs can provide a natural defense against infection. Indeed, the poor ecacy of several viral vectors to deliver therapeutic genes to airway epithelium highlighted the role of TJs in the resistance of epithelia to viral infection [91,92]. The recent identication of CLDNs as a critical factor for HCV internalization [23,82,83] highlighted the importance of studying the role(s) of cell polarization in HCV entry. The authors used the colorectal adenocarcinoma Caco-2 cell line that is known to polarize to study the role of cellular polarity in HCV entry. Caco-2 cells express CD81, SR-BI, and CLDN1 proteins and support HCVpp entry [93]. Viral receptor expression levels increased on polarization and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins Zonula occludens-1 (ZO-1) and occludin-1 were unchanged on polarization. HCVpp infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry into polarized cells occurred predominantly by means of the apical surface. Disruption of TJs signicantly increased HCV entry, supporting a model in which TJs provide a physical barrier restricting viral access to receptors expressed at the lateral and basolateral cellular domains. Humoral immune response in hepatitis C virus infection HCV-infected individuals often have detectable RNA levels as early as 1 week after infection; however, antibodies against the virus are not detected until much later. Chen and colleagues [94] noted the delayed appearance, low titer, and restricted isotype of antibodies to structural and nonstructural HCV proteins. The contribution of the humoral response to disease outcome remains unclear; in contrast, it has been well documented that a potent multi-specic and long-lasting cellular immune response during the acute phase of HCV infection contributes to resolution [95105].

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A potential role for antibodies in controlling disease progression was rst demonstrated in subjects with primary hypogammaglobulinemia, demonstrating rapid disease progression and poor responses to IFN treatment [106]. An independent study reported that 43% of individuals who spontaneously resolved infection had antibodies specic to E2 HVR within the rst 6 months of infection, compared with only 13% of patients who failed to clear the infection [107]. In contrast, there were no signicant dierences between the two patient groups with respect to the time of emergence of antibodies to HCV core or nonstructural proteins [108]. Other investigators reported an early emergence of HVR-specic antibodies in a small cohort of subjects infected during hemodialysis who resolved infection [109]. Because the HVR is proposed to be a target for nAbs [110,111], several studies have suggested a role for anti-HVR antibodies in selecting viral variants to escape the humoral response [112115]. Although many studies have explored the specicity of the humoral immune response, until recently, functional assays were not available to determine the neutralizing capacity of serum antibodies. In vitro systems for the measurement of neutralizing antibodies Before the development of in vitro infection systems, the neutralization potential of HCV-specic antibodies was tested indirectly using neutralization of binding (NOB) assays, in which antibodies were screened for their ability to prevent the binding of sE2 to mammalian cells [116]. Baumert and colleagues [117] used a recombinant baculovirus system to express the HCV structural proteins that formed viral-like particles (VLPs) to study antibody reactivity and inhibition of VLP-cell interactions [118]. The VLPs elicited cellular and humoral immune responses in mice, generating anti-E2 antibody responses that were reactive against E2 from diverse HCV genotypes [119,120]. Once HCVpp became available [5,6], it was possible to assess antibodies for their ability to inhibit viral infection of hepatoma cell lines. HCVpp neutralization titers correlated well with the eectiveness of antibodies in HCV challenge protection studies in chimpanzees [121], validating the system as a model for assessing functional antibody responses. Indeed, the HCVpp system has allowed the testing of monoclonal antibodies and polyclonal antibodies from diverse sources (ie, from sera derived from patients or immunized animals) to inhibit infection [121123]. The recent discovery that the JFH-1 strain of HCV generates infectious particles in cell culture has allowed investigations into the sensitivity of authentic particles to antibody-dependent neutralization [12,13]. To date, HCVcc has been reported to be neutralized by E2-specic antibodies derived from human sera [12,13], E1-specic mouse antisera [124], and by an array of mAbs directed to the E1 and E2 gps. Polyclonal immunoglobulin (Ig) preparations derived from mice and guinea pigs immunized with E2 and E1E2 gps eciently neutralized HCVcc [37]. It is worth noting that the neutralization titers of polyclonal immune sera, E2-specic nAbs [5,49],

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and human-derived antibodies were consistently lower for HCVcc-expressing strain H77 (genotype 1a) and J6 (genotype 2a) gps than for HCVpp bearing the same gps (Z. Stamataki, unpublished data, 2008) [37], which may reect dierences in the level of gps incorporated into pseudoparticles compared with native particles. HCVcc engineered to express a diverse range of envelope gps should become available in the future and will prove invaluable for the characterization of neutralizing humoral immunity [13,124,125]. Detection of autologous and heterologous neutralizing antibodies Dissecting the humoral immune response to HCV at an individual level is a challenging endeavor; readily available gp sequences may represent certain viral genotypes but dier considerably from a patients autologous viral quasispecies. Most studies are limited to establishing the heterologous cross-reactivity of nAbs and do not measure their ecacy to neutralize autologous circulating strains in the subject under test. Several studies have demonstrated that chronically infected subjects present with high-titer antibodies capable of neutralizing heterologous HCVpp, suggesting that the neutralizing response targets epitopes that are conserved across diverse genotypes [10,11,115,121,123,126129]. In a case report, the authors recently characterized the strain-specic and cross-reactive nAb responses elicited in a chronically infected patient when the time of infection was known and sequential samples were available over the course of infection [115]. Serum antibodies were compared for their ability to neutralize HCVpp, expressing autologous gps cloned over time and heterologous gp strains. An antibody response to autologous virus was rst detected at seroconversion (8 weeks after infection), whereas cross-reactive nAb responses were not evident until after 33 weeks [115,123]. Although the cross-reactive gp-specic nAb titer and breadth increased during disease progression, HVR-specic responses detected at 9 weeks after infection decreased to undetectable levels by week 33. Serum antibodies failed to neutralize HCVpp expressing timematched autologous gps, suggesting the emergence of neutralization escape variants [115]. This delay in nAb emergence has been reported to associate with a failure to resolve infection spontaneously in a cohort of subjects accidentally infected with the same strain of HCV [130]. In this cohort, the rapid induction of nAbs early in the course of acute infection was directly linked to disease clearance. In agreement with analogous reports describing the genesis of HIV escape variants [131,132], these studies suggest that HCV can also escape the humoral nAb immune response. The role of the humoral response in selecting viral diversity, particularly in the HVR, was reinforced by reports that HCV-infected subjects with hypogammaglobulinemia showed reduced rates of nucleotide substitution in the HVR compared with controls [112,133]. The authors proposed that the HVR serves as a viral decoy, directing the immune system away from viral epitopes potentially less capable of rapid change and playing

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an accessory role for viral evolution [115,134,135]. Assuming that nAbs contribute to selecting the HCV quasispecies and to the reported association between the emergence of quasispecies and failure to clear acute infection [134,136138], one may question the benet of nAbs to HCV-infected individuals. Conversely, in chronic infection, the viral quasispecies complexity has been associated with milder clinical symptoms [139,140], suggesting that immune responses limiting liver damage may enhance viral antigenic variation [140144]. Further doubts regarding the ecacy of nAbs in controlling HCV replication have been reported, with conicting data regarding the clinical impact of anti-HCV therapies [145,146].

Epitope specicity of the neutralizing antibody response nAbs can exert their eect(s) by binding directly to virus particles and blocking subsequent interaction(s) with receptors or by inhibiting post-entry events, such as viral uncoating and subsequent replication (reviewed by Burton [147]). The former may occur by inducing conformational changes in the viral envelope that disable infection or by steric hindrance, physically shielding important viral interaction sites. The modes of action of HCV-specic nAbs have yet to be dened; however, several early studies suggested that serum antibodies from chronically infected individuals inhibited sE2CD81 association, leading these investigators to conclude that the CD81 binding site is immunogenic and that antibodies neutralize infectivity by inhibiting HCV interaction with CD81 [121,148]. Mapping of neutralizing mAbs generated in response to recombinant gp immunization has identied a series of linear epitopes within the N-terminal region of E2 [5]. In contrast, neutralizing human mAbs obtained from HCVinfected subjects generally recognize conformation-dependent epitopes [149153] (recently reviewed by Houghton and Abrignani [154] and Zeisel and colleagues [155]). Johansson and colleagues [153] reported on the broad reactivity of two human monoclonal antibodies that recognize overlapping yet distinct epitopes involving aas in the 523535 region of E2, known to be important for the E2-CD81 interaction. Similarly, an independent study characterized a panel of human monoclonal antibodies to three immunogenic conformational domains (designated A, B, and C) within E2, in which antibodies specic for domains B and C neutralized HCV and those recognizing domain A failed to neutralize viral infectivity. These investigators concluded that HCV E2 contains three immunogenic domains with distinct functions and that domains B and C may be involved in interactions with CD81. Finally, one needs to consider the generation of non-neutralizing HCV-specic antibodies, which may compete with nAbs and reduce their effectiveness. Such antibodies have been reported in other viral infections in which highly immunogenic non-neutralizing epitopes mislead the humoral immune response [156].

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Eect of lipoproteins on the sensitivity of virus to neutralizing antibodies Recent in vitro studies highlight the role of the VLDL pathway in the assembly and release of HCVcc particles [6971]. These data support earlier observations reporting on the density and sedimentation properties of human plasma-derived virus, concluding that the virus exists in association with lipoproteins or immunoglobulins [73,157159]. Thosmsen and colleagues [160] reported that HCV RNA was associated with b-lipoproteins in sera, a nding that was later conrmed by others [159,161,162]. These observations led several researchers to hypothesize that lipoprotein-associated virus may have reduced sensitivity to antibody-dependent neutralization. In an attempt to model the virus lipoprotein interactions, several investigators studied the eect of lipoproteins on the sensitivity of HCVpp to nAbs. These studies demonstrated that HDL promotes HCVpp infection and reduces the sensitivity of the virus to nAbs [60,64,163]. The lipoproteins seemed to exert their eect by means of interactions with the target cells, specically by means of promoting virus internalization in an SR-BIdependent manner [65]. Dreux and colleagues [64] reported that HDL increased the infectivity of HCVpp bound to hepatoma cell lines in vitro, suggesting that HDL modulates SR-BI cholesterol uptake.

Inducing protective immunity in vivo The propensity of HCV to establish chronic infection, to reinfect previously exposed individuals [164], to transmit directly by cell-cell routes in vitro [165], and to evolve neutralization escape variants [166] makes the development of an HCV vaccine a major challenge. Vaccine development has been hampered by the lack of a convenient small animal model [154,167]. The existence of natural immunity to infection in some humans [168] and in chimpanzees [142,169171] is encouraging, however, and suggests that the immune system can eliminate infection. Immunization of chimpanzees with HCV gps has protected the animals from challenge with autologous virus [172]; furthermore, this protection correlated with the induction of nAbs that inhibited E2-CD81 interactions [116]. In animals with reduced nAb responses [172] and those challenged with heterologous virus [154], sterilizing immunity was not achieved [173,174] and the animals failed to progress to a chronic state of infection [175]. Several immunized chimpanzees controlled infection without mounting any signicant humoral responses, however, demonstrating that humoral immunity is not required for clearing HCV in the chimpanzee model [176]. Reports on the immunogenicity of HCV structural proteins in mice and non-human primates are variable [120,176179]. Rodents vaccinated with E2 and E1E2 gps can generate cross-reactive nAb responses, with higher titers in guinea pigs than in mice [37]. Furthermore, vaccination of nine healthy volunteers with HCV-1 E1E2 gps elicited serum antibody responses that were

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able to neutralize heterologous HCVpp and HCVcc in vitro, and these responses were detectable 1 year after vaccination [180]. Immunization of rats with HCV E1E2 gps yielded poor responses against E1, with most antibodies recognizing E2 (J.A. McKeating, unpublished data, 1998). Human subjects immunized with E1 mounted a specic response, the neutralizing activity of these antibodies was not assessed [181,182]. E1 may be poorly immunogenic because of the presence of N-linked glycosylation sites [183]. Similarly, E2 immunogenicity is also aected by glycans [48,184,185]. Recent data studying HCV infection of the severe combined immunodeciency (SCID)chimeric mouse model demonstrated that transfusion of mice with high concentrations of Ig puried from a chronic HCV-infected subject, with demonstrable neutralizing activity in vitro, protected most animals from homologous virus challenge [21]. Overall, these data suggest that eliciting a nAb response is a valid objective of prophylactic vaccination.

Summary The processes of HCV entry and antibody-mediated neutralization are intimately linked. The high frequency of nAbs that inhibit E2-CD81 interaction(s) suggests that this is a major target for the humoral immune response. The observation that HCV can transmit to naive cells by means of CD81dependent and -independent routes in vitro awaits further investigation to assess the signicance in vivo but may oer new strategies for HCV to escape nAbs. The identication of CLDNs in the entry process highlights the importance of cell polarity in dening routes of HCV entry and release, with recent experiments suggesting a polarized route of viral entry into cells in vitro. Recent data showing the important role of lipoproteins in HCV assembly, release, and entry support earlier observations of the presence of virallipoprotein complexes in human plasma. There is a real urgency to study HCV infection of primary hepatocyte and mixed liver cell cultures to conrm the mechanisms of entry observed in nonpolarized hepatoma cell lines. Future experiments need to assess the receptor dependency of apical and basolateral routes of infection and whether they share similar sensitivities to neutralization by antibodies and small molecules targeting various steps in the entry process. Recent developments oer the prospect of a more complete understanding of the molecules dening HCV entry and the discovery of new targets for the development of novel antiviral agents.

Acknowledgments The authors thank all their colleagues for stimulating discussions over the years. Research in the McKeating laboratory is currently supported by Public Health Service grants AI40034 and AI50798, the Medical Research Council, and The Wellcome Trust.

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[152] Allander T, Forns X, Emerson SU, et al. Hepatitis C virus envelope protein E2 binds to CD81 of tamarins. Virology 2000;277(2):35867. [153] Johansson DX, Voisset C, Tarr AW, et al. Human combinatorial libraries yield rare antibodies that broadly neutralize hepatitis C virus. Proc Natl Acad Sci U S A 2007;104(41): 1626974. [154] Houghton M, Abrignani S. Prospects for a vaccine against the hepatitis C virus. Nature 2005;436(7053):9616. [155] Zeisel MB, Fa-Kremer S, Fofana I, et al. Neutralizing antibodies in hepatitis C virus infection. World J Gastroenterol 2007;13(36):482430. [156] Zhang P, Wu CG, Mihalik K, et al. Hepatitis C virus epitope-specic neutralizing antibodies in Igs prepared from human plasma. Proc Natl Acad Sci U S A 2007;104(20): 844954. [157] Hijikata M, Shimizu YK, Kato H, et al. Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. J Virol 1993;67(4):19538. [158] Kanto T, Hayashi N, Takehara T, et al. Density analysis of hepatitis C virus particle population in the circulation of infected hosts: implications for virus neutralization or persistence. J Hepatol 1995;22(4):4408. [159] Prince AM, Huima-Byron T, Parker TS, et al. Visualization of hepatitis C virions and putative defective interfering particles isolated from low-density lipoproteins. J Viral Hepat 1996;3:117. [160] Thomssen R, Bonk S, Propfe C, et al. Association of hepatitis C virus in human sera with beta-lipoprotein. Med Microbiol Immunol 1992;181(5):293300. [161] Agnello V, Abel G, Elfahal M, et al. Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci U S A 1999;96(22):1276671. [162] Wunschmann S, Medh JD, Klinzmann D, et al. Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor. J Virol 2000; 74(21):1005562. [163] Lavillette D, Morice Y, Germanidis G, et al. Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection. J Virol 2005;79(10):602334. [164] Kizhatil K, Albritton LM. Requirements for dierent components of the host cell cytoskeleton distinguish ecotropic murine leukemia virus entry via endocytosis from entry via surface fusion. J Virol 1997;71(10):714556. [165] Timpe JM, Stamataki Z, Jennings A, et al. Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies. Hepatology 2008;47(1):1724. [166] Shimizu YK, Hijikata M, Iwamoto A, et al. Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses. J Virol 1994;68(3):1494500. [167] Lechmann M, Liang T. Vaccine development for hepatitis C. Semin Liver Dis 2000;20(2): 21126. [168] Mehta SH, Cox A, Hoover DR, et al. Protection against persistence of hepatitis C. Lancet 2002;359(9316):147883. [169] Bassett SE, Guerra B, Brasky K, et al. Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection. Hepatology 2001; 33(6):147987. [170] Lanford R, Guerra B, Chavez D, et al. Cross-genotype immunity to hepatitis C virus. J Virol 2004;78(3):157581. [171] Weiner A, Paliard X, Selby M, et al. Intrahepatic genetic inoculation of hepatitis C virus RNA confers cross-protective immunity. J Virol 2001;75(15):71428. [172] Choo QL, Kuo G, Ralston R, et al. Vaccination of chimpanzees against infection by the hepatitis C virus. Proc Natl Acad Sci U S A 1994;91(4):12948. [173] Puig M, Major ME, Mihalik K, et al. Immunization of chimpanzees with an envelope protein-based vaccine enhances specic humoral and cellular immune responses that delay hepatitis C virus infection. Vaccine 2004;22(8):9911000.

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[174] Youn J, Park S, Lavillette D, et al. Sustained E2 antibody response correlates with reduced peak viremia after hepatitis C virus infection in the chimpanzee. Hepatology 2005;42(6): 142936. [175] See LB. Natural history of chronic hepatitis C. Hepatology 2002;36(5 Suppl 1):S3546. [176] Elmowalid G, Qiao M, Jeong S, et al. Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees. Proc Natl Acad Sci U S A 2007;104(20):842732. [177] Jeong SH, Qiao M, Nascimbeni M, et al. Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates. J Virol 2004; 78(13):69957003. [178] Murata K, Lechmann M, Qiao M, et al. Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection. Proc Natl Acad Sci U S A 2003;100(11):67538. [179] Qiao M, Murata K, Davis AR, et al. Hepatitis C virus-like particles combined with novel adjuvant systems enhance virus-specic immune responses. Hepatology 2003;37(1):529. [180] Stamataki Z, Coates S, Frey S, et al. Vaccination with recombinant hepatitis C virus antigen elicits cross-neutralizing antibodies in human volunteers. Presented at the 13th International Meeting on Hepatitis C Virus and Related Viruses. Cairns, Australia, December 2006 [Abstract 514]. [181] Leroux-Roels G, Batens A, Desombere I, et al. Immunogenicity and tolerability of intradermal administration of an HCV E1-based vaccine candidate in healthy volunteers and patients with resolved or ongoing chronic HCV infection. Hum Vaccin 2005;1(2):615. [182] Nevens F, Roskams T, Van Vlierberghe H, et al. A pilot study of therapeutic vaccination with envelope protein E1 in 35 patients with chronic hepatitis C. Hepatology 2003;38(5): 128996. [183] Fournillier A, Wychowski C, Boucreux D, et al. Induction of hepatitis C virus E1 envelope protein-specic immune response can be enhanced by mutation of N-glycosylation sites. J Virol 2001;75(24):1208897. [184] Helle F, Goard A, Morel V, et al. The neutralizing activity of anti-hepatitis C virus antibodies is modulated by specic glycans on the E2 envelope protein. J Virol 2007;81(15): 810111. [185] Heo TH, Chang JH, Lee JW, et al. Incomplete humoral immunity against hepatitis C virus is linked with distinct recognition of putative multiple receptors by E2 envelope glycoprotein. J Immunol 2004;173(1):44655.

Clin Liver Dis 12 (2008) 713726

Host Genetic Factors and Antiviral Immune Responses to Hepatitis C Virus


Chloe L. Thio, MD
Department of Medicine, Division of Infectious Diseases, Johns Hopkins University, 855 N. Wolfe Street, Suite 520, Baltimore, MD 21205, USA

Infection with hepatitis C virus (HCV) results in a gamut of clinical outcomes ranging from viral elimination to the development of end-stage liver disease or hepatocellular carcinoma. Viral elimination, which occurs in a minority (w20%) of acutely HCV-infected individuals, is the result of eective immune control of HCV replication [1]. The 80% of people who do not eliminate HCV progress to a chronic HCV infection. Not all chronic infections are created equal, because some have minimal liver disease, whereas others develop cirrhosis or hepatocellular carcinoma. Epidemiologic factors associated with these dierent clinical outcomes include age, ethnicity, other viral coinfections (especially HIV), and gender [24]. Even in a relatively epidemiologically homogeneous population, there are marked dierences in the ability to eliminate the virus that are not related to viral characteristics. One example is a cohort of 704 Irish women who were accidentally infected with the same viral inoculum (contaminated antiD immune globulin), and 314 (45%) of them cleared their infection [5]. Likewise, 43% of 152 German women cleared their infection after being exposed to the same contaminated lot of antiD immune globulin [6]. Such data suggest that it is not the virus but the interactions between the virus and the host immune response that are important for determining the natural history of an HCV infection [7]. Studies demonstrate that a strong broad immune response favors viral clearance compared with one that is weak or narrowly focused [8,9]. Likewise, once a chronic infection is established, HCV is not cytopathic to the hepatocytes; rather, it is the immune response to the virus that is believed to be responsible for the liver damage. A signicant barrier to dissecting the components of the immune response that result in viral clearance or

This work was supported by National Institutes of Health grant DA 13324 and the Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases Award. E-mail address: cthio@jhmi.edu 1089-3261/08/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cld.2008.03.002 liver.theclinics.com

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that lead to hepatocyte damage is the lack of small animal models or cell culture systems. Because such models are not available, an alternative approach to understanding the immune response is to analyze whether host genetic variants or polymorphisms in immune-response genes account for some of the heterogeneity in outcome. With the recent development of more rapid cost-eective genotyping procedures, such large-scale studies are possible and have already improved our understanding of HCV pathogenesis. This review briey summarizes the immune response to HCV to give some background to understand the candidate genes that are discussed. Associations with polymorphisms in various immune-response genes that aect the ability to achieve HCV clearance or aect HCV brosis progression are then reviewed.

Immune response to an acute hepatitis C virus infection After an HCV infection, the innate immune response is initially important for controlling viral replication (see the article by Szabo in this issue), with the adaptive immune response peaking at 8 to 10 weeks after infection. Ultimately, a coordinated eort between the innate and adaptive immune responses is necessary to eliminate HCV from the liver. The innate immune system recognizes single-stranded and double-stranded HCV RNA through its pattern recognition receptors, Toll-like receptor 3 (TLR3) on the hepatocyte cell surface and retinoic acidinducible gene I (RIG-1) in the cytoplasm of the hepatocyte. Engagement of either of these receptors activates a cascade of signals, including interferon-regulatory factor 3 (IRF-3), that culminates in the induction of type 1 interferons (IFNs), such as IFNa and IFNb. These interferons bind receptors that activate the Janus kinase (Jak)specifically targeted antiviral therapy (STAT) pathway, which then turns on hundreds of interferon-stimulated genes to control viral replication (reviewed Lloyd and colleagues [10]). The innate immune response also controls replication by means of its effector cells, the natural killer (NK) cells. Because NK cells comprise approximately 30% of all T cells in the liver [11], they likely are an important component to the anti-HCV immune response. Their role includes lysing infected cells, producing IFNg to control viral replication, and directing inammatory cells to HCV-infected hepatocytes. This initial innate immune response is an important host defense against the virus, but viral clearance also depends on an eective adaptive immune response. The onset of the cellular immune response is clinically detected by an increase in serum transaminases marking immune-mediated liver injury. A strong, broad CD4 and CD8 T-cell response is more likely to lead to HCV clearance than a weak narrowly focused response [8,9]. Evidence also suggests that a CD4 response that elicits T helper 1 (Th1) cytokines is

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more likely to result in viral clearance. Those with a weak CD4 response seem to have a functional impairment of these cells from exhaustion or anergy rather than a depletion of CD4 T cells. Similarly, acutely infected individuals who do not clear have CD8 T cells that are functionally impaired compared with those who clear the virus. Not all individuals with a robust response ultimately clear HCV, which, in some instances, may be attributable to the development of viral escape mutants from immune pressure on the virus. Thus, HCV must balance viral tness with immune escape. Several studies have documented viral escape mutations in human leukocyte antigen (HLA)restricted CD8 T-cell epitopes [1214]. In the cohort of Irish women who received the same viral inoculum, Ray and colleagues [12] demonstrated that those with amino acid substitutions in known HLA epitopes directed these mutations away from consensus in persons with the HLA allele associated with that epitope and toward consensus in those lacking the allele.

Polymorphisms in immune-response genes and outcome of acute hepatitis C virus infection As described above, the innate and adaptive immune responses are important for clearance of an acute HCV infection. Published studies to date have not examined polymorphisms in the innate immune-response pathway genes or the type I IFN genes to determine if they aect the ability to clear HCV. There is genetic epidemiologic evidence to support the importance of the NK cells, the eector cell of the innate immune response, in HCV outcome. Khakoo and colleagues [15] examined polymorphisms in the genes for the killer immunoglobulin-like receptors (KIRs), which are receptors on NK cells. As the name implies, these receptors have two or three immunoglobulin-like domains, a transmembrane domain, and a long or short cytoplasmic tail. Those with the long tails (2DL, 3DL) inhibit the NK cells when bound and those with short tails (2DS, 3DS) activate NK cells on binding. The ligands for the inhibitory KIRs are HLA class I molecules, whereas the ligands for the activating KIRs have not been dened. The study by Khakoo and colleagues [15] focuses on the inhibitory KIRs 2DL1, 2DL2, and 2DL3, of which the latter two are alleles of each other. These three KIRs bind HLA-C alleles, which are divided into two groups based on the amino acid at position 80 of the HLA allele: HLA-C1 alleles have an asparagine at position 80, whereas HLA-C2 alleles have an asparagine at position 80. The HLA-C1 alleles bind KIR2DL2/2DL3 and the HLA-C2 alleles bind KIR2DL1. The strongest inhibitory signal is transduced to the NK cell when KIR2DL1 binds an HLA-C2 allele, whereas the weakest signal is from KIR2DL3 binding an HLA-C1 allele (Table 1). Khakoo and colleagues [15] found that in individuals with low-titer HCV inocula (ie, injection drug users), homozygosity for the combination with

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Table 1 Strength of inhibitory signal for selected killer immunoglobulin-like receptors important in hepatitis C virus clearance Inhibitory KIR 2DL1 2DL2 2DL3
a

Liganda HLA-C2 HLA-C1 HLA-C1

Relative strength of NK cell inhibitory signal Strong Intermediate Weak

HLA-C1 and HLA-C2 are HLA-C alleles with asparagine or lysine at position 80, respectively.

the weakest inhibitory signal (KIR2DL3 and HLA-C1) favored HCV clearance compared with those without this compound genotype (odds ratio [OR] 2.33; P .001). Presumably, this association is attributable to those individuals with the protective genotype having the least inhibition of the NK cells giving rise to increased NK-cell activity. These investigators also found an independent association with the presence of the activating KIR3DS1 and its putative ligand, a group of HLA-B alleles known as HLA-Bw4. Immune-response genes of the adaptive immune response have been studied more extensively than those of the innate immune response in acute HCV infection. Several studies have tested the hypothesis that certain HLA class I alleles may present epitopes that lead to a more robust CD8 T-cell response to acute HCV infection. In the largest study, Thio and colleagues [16] examined individuals from three dierent cohorts that included 231 individuals with clearly documented HCV recovery and 444 matched persistently HCV-infected individuals. They found that HLAA*1101 (OR 0.49, 95% condence interval [CI]: 0.270.89), B*57 (OR 0.62, 95% CI: 0.391.0), and Cw*0102 (OR 0.43, 95% CI: 0.210.89) were associated with viral clearance. HLA-Cw*04 (OR 1.78, 95% CI: 1.212.59) and A*2301 (OR 1.78, 95% CI: 1.013.11) were associated with HCV persistence. The HLA-C data are consistent with the HLA-KIR data cited previously, because HLA-Cw*0102 is an HLA-C1 allele and HLA-Cw*04 is an HLA-C2 allele. This study did not nd an association between HLA class I homozygosity and viral persistence, which is interesting, because it has been hypothesized that heterozygosity would be advantageous for recognition of a broader array of epitopes. HLA-B*57 has also been associated with HCV clearance in an African population [17] and with slower HIV progression [18], but whether it is a gene linked to this allele or an immune characteristic about this allele itself that is protective in these chronic viral infections is not known. The other large study with class I HLA data is from the cohort of Irish women described previously who received the same viral inoculum. They found that the 86 subjects with viral clearance were more likely to have HLA-A*03 (OR 2.8), B*27 (OR 7.5), B*07 (OR 2.0), or Cw*01 (OR 7.1) compared with the 141 chronically infected women [19]. HLA-B*08 (OR 0.4) and

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B*18 (OR 0.2) were more common in women with viral persistence. These results (except for the HLA-Cw*01) are dierent from the study by Thio and colleagues [16], which may be attributable to baseline dierences between the cohorts, such as ethnicity, gender, and viral inoculum. Because the CD4 T-cell response is important for viral clearance, several groups have tested the hypothesis that HLA class II molecules, which present CD4 T-cell epitopes, are associated with HCV outcome. Several results are consistent, and two meta-analyses demonstrated that DQB1*0301 (estimates of 3.0 [20] and 2.4 [21]) and DRB1*11 (estimates of 2.5 and 2.0) are associated with HCV recovery. A bias of these meta-analyses is that studies without any HLA associations are not published or studies in which these alleles are not signicant do not report their specic ORs. The allele HLA-DRB1*01 was protective in the Irish cohort and in the whites in the study by Thio and colleagues [16]; thus, it may not be protective in nonwhite ethnic groups. Cytokines are small proteins secreted by a variety of T cells in response to an immune stimulus and have several roles, including mediating the immune response to infectious agents, such as HCV. Thus, several studies have examined polymorphisms in a variety of cytokine genes to determine their role in HCV clearance (Table 2). Chemokines are a specic set of cytokines that attract leukocytes to sites of infection. Several chemokines bind to the receptor CCR5, and polymorphisms in this receptor have been examined in clearance of HCV infection. A 32 base-pair deletion in the CCR5 gene (CCR5D32) leads to loss of CCR5 expression on the surface of the T cell, which is protective against HIV infection because CCR5 is an essential coreceptor for HIV [22]. CCR5 also inuences T-cell tracking and the

Table 2 Associations between polymorphisms in chemokine genes and hepatitis C virus clearance or persistence Chemokine gene CCR5D32 IFNg -764G (promoter mutation) IL-10 Outcome Clearance Clearance Clearance Comments Deletion may lead to increased T-cell response Increases promoter activity Several polymorphisms lower IL-10 expression which can increase Th1 response Homozygosity for the A allele is associated in some but not all studies Two separate polymorphisms leading to lower TGFb, which can increase NK cell activity

IL-12 1188 A/A

Persistence

TGFb

Clearance

TNFa(238, 308)

No associations

Abbreviations: IFNg, interferon-g; IL, interleukin; TGFb, transforming growth factor-b; TNFa, tumor necrosis factor-a.

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immune response. A study of women with genotype 1b found CCR5D32 to be more common in those with HCV clearance [23]. This association could be attributable to an increased T-cell response to the HCV antigens because ccr5 knockout mice have increased T-cell responses to a variety of antigens [24,25]. The chemokines that bind CCR5 have not been extensively studied with regard to HCV clearance, but polymorphisms in one of them, regulated on activation, normal T-cell expressed and secreted (RANTES), have been associated with HCV treatment response [26,27]. IFNg is produced by eector T cells and NK cells and is critical to the defense against HCV infection because it can inhibit HCV replication in the replicon system [28]. This gene does not have any coding region polymorphisms, but several noncoding region polymorphisms have been described [29,30]. In one study, nine noncoding region polymorphisms were genotyped in an HCV treatment cohort and an HCV recovery cohort, and the uncommon G variant at position 764 in the IFNg promoter was associated with recovery from HCV (OR 3.51, 95% CI: 0.98 12.49) and with sustained response to HCV therapy (OR 3.37, 95% CI: 1.159.83) [31]. This variant is more common in white Americans consistent with the ndings that spontaneous recovery is more common in whites [4]. The 764G variant has a higher binding anity to the nuclear regulatory factor (NF)-kB motif, resulting in higher levels of promoter activity and thus perhaps explaining the association with HCV recovery and treatment response [31]. Interleukin (IL)-10 is a cytokine important to the immune response to HCV through its downregulation of the Th1 response and suppression of secretion of proinammatory cytokines, such as tumor necrosis factor-a (TNFa) and IFNg. Several studies have implicated that polymorphisms in this region of the genome are important in HCV clearance. One study found that a particular promoter haplotype (1117A, 854T, 627A), which is associated with lower IL-10 expression, was more frequent in those with recovery (36%) than in persistent infection (23%) [32]. A promoter polymorphism at 1082 that is associated with higher IL-10 levels has also been associated with HCV persistence in women [33]. Oleksyk and colleagues [34] tested polymorphisms in the IL-10 gene region and found associations with HCV outcome in African Americans. Several other studies examined the IL-10 haplotypes and polymorphisms but did not nd associations with HCV recovery [3538]. IL-12 is a cytokine important for the generation of a Th1 response, which favors HCV clearance. A polymorphism in the IL-12 p40 gene (IL-12B) at position 1188 has been associated with increased and decreased levels of IL12 secretion [39,40]. Homozygosity for the A allele has been associated with HCV persistence in Chinese patients (OR 0.34; P .014) [41]. This was also found in a second study from the United Kingdom, in which 66% of the persistently infected people were A/A homozygous compared with 50% of those with spontaneous recovery [42]. This was not conrmed in

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a dierent population of German patients, however [43]. The author and her colleagues also failed to conrm this in their study from North America of 188 patients with HCV recovery matched to 360 persistently infected persons with HCV persistence. The A allele was found in 72.5% and 71% of persons with HCV recovery and persistence, respectively (C. Thio, unpublished data, 2007). Transforming growth factor-b (TGFb) is a cytokine that inhibits the immune response by suppressing NK cell activity and inhibiting IFNg and IL12 production. The C allele at 509 in the promoter, which leads to lower levels of TGF-b1, was associated with HCV recovery in a study of Japanese patients [44]. Similarly, Barrett and colleagues [35] studied two coding region polymorphisms in TGFb and found that the haplotype associated with low TGFb was associated with HCV clearance. In this study, these investigators also found that homozygosity for the low-producing IL-6 promoter 174 variant was associated with HCV recovery. Although TNFa is an important proinammatory cytokine with the bestknown functional polymorphisms at positions 308 and 238 in the promoter, none of the studies to date have found an association with these polymorphisms and HCV recovery or persistence [32,35]. Although the importance of the humoral immune response to HCV is just beginning to be uncovered, there is one study showing that certain immunoglobulin GM and KM allotypes aect HCV outcome. GM and KM allotypes are antigenic markers of the immunoglobulin G (IgG) heavy chains and k-light chains, respectively. GM allotypes are strongly associated with IgG subclass concentrations. In a study of African-American injection drug users, Pandey and colleagues [45] found that subjects with GM 1, 17 5, 13 and KM 1,3 phenotypes are more than three times as likely to clear HCV as those without those phenotypes.

Polymorphisms in immune-response genes and hepatitis C virus disease progression Of the individuals with a persistent HCV infection, approximately 30% progress to end-stage liver disease or cirrhosis. The mean time to development of cirrhosis is 20 to 30 years [2]; however, in some individuals, it occurs more quickly. Such variation in brosis progression rates has been associated with epidemiologic factors, such as age, gender, HIV status, and alcohol use [2]. Another factor is the strength of the immune response, which is initially stimulated to eliminate HCV. In a persistent infection, however, the continued inammatory state leads to hepatocyte necrosis and deposition of extracellular matrix (ECM) proteins by hepatic stellate cells. Activators of hepatic stellate cells include the Th1 response [46], the family of proteolytic enzymes known as matrix metalloproteases (MMPs), and the tissue inhibitors (TIMPs) of MMPs. Because the continued immune response generates

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an inammatory state, genetic variation of the immune system may aect brosis progression rates. One major limitation to genetic epidemiologic studies of HCV disease progression is accurately dening the severity and progression of brosis. The gold standard for determining the amount of brosis, which is often used in genetic epidemiology studies, is a liver biopsy. Unfortunately, this gold standard is problematic because it is imprecise as a result of dierences in size of the biopsy and regional dierences of brosis. Furthermore, there is reader error, with studies showing inadequate inter- and intrareader concordance [47]. A second limitation is that the studies are cross-sectional; thus, a liver biopsy at one particular point is used along with an estimated date of infection to determine the progression rate. This is problematic because the classication of disease state or progression rate can be unreliable due to the dependence on an estimated date. A third limitation is that some genetic epidemiology HCV studies dene disease stage based on liver enzyme elevations, but it is known that liver enzymes do not correlate well with the stage of disease. An advantage of this approach is that repeated measurements are more easily obtained than with a liver biopsy; thus, the problems associated with a cross-sectional study design are less applicable. Taken together, these limitations result in misclassication that can contribute to false associations, especially in small studies. With these caveats in mind, the author reviews the immune-response genes that have been studied in liver disease progression. HLA has been studied as a candidate gene in the disease progression hypothesis. In a study by Asti and colleagues [48], DRB1*1103 and DRB1*1104 were associated with normal alanine aminotransferase (ALT), whereas DRB1*1101 was more common in those with more chronic hepatitis, especially those with more advanced disease, as dened by liver biopsy. A French study by Renou and colleagues [49] examined 83 patients with normal ALT levels over a 6-month period and 233 patients with elevated ALT levels and found HLA-DRB1*11 to be overrepresented in those with a normal ALT level (43% versus 24%, OR 2.36). In this study, liver biopsies were performed and milder disease was also associated with HLADRB1*11. Subtyping of the HLA-DRB1*11 was not performed as in the study by Asti and colleagues [48]. A Polish study also found HLADRB1*11 to be more common in those with milder disease [50], and a German study found that it protected from cirrhosis [51]. The German study also found protection from HLA-DQB1*03. A study from Japan examined HLA class I and II antigens in those with normal ALT levels compared with those without and found that haplotypes with HLA-B*54 are more common in those with high ALT levels [52]. In this study, HLA haplotype DRB1*1302-DQB1*0604 was also associated with normal ALT levels. Several other studies have yielded inconsistent results [5355]. Patel and colleagues [56] also tested the hypothesis that HLA allelic diversity aects brosis progression rates, but they did not nd dierences in median

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progression rates in patients heterozygous or homozygous at all three HLA class I loci. They did not nd any individual class I associations, but they used a serologic assay to dene HLA types, making it more dicult to discover weaker associations. An approach to examining the inherited genetic variability of the presence or severity of diseases, which has become possible with the HapMap (http://www.hapmap.org/) and with the decrease in genotyping cost, is performing a genome-wide scan [57,58]. The advantage of such an approach is that it obviates the need for a priori knowledge of candidate genes. For HCV, early ndings from a genome-wide scan of disease severity have been published [59]. This scan included 433 patients from one center; significant results were then validated in 483 patients from a second center. The DNA from the rst center was pooled based on the following brosis stages from a baseline biopsy: no or minimal brosis (stage 0 or 1), mild (stage 2), and advanced (stage 3 or 4). The advanced brosis group was compared with no or mild disease. Of the 24,823 single nucleotide polymorphisms (SNPs) genotyped, 1609 (6.5%) had a twofold association with liver disease stage and 100 of these have been tested in samples from the second center. Two SNPs that remained associated with disease stage were Dead box polypeptide 5 (DDX5) and carnitine palmitoyltransferase 1A (CPT1A). The DDX5 SNP was a nonsynonymous change that was associated with more advanced brosis, and on analysis of SNPs in close proximity, two SNPs in the POLG2 gene (polymerase DNA-directed g2) were also associated with advanced brosis. DDX5 is an RNA helicase that is expressed in the liver and may interact with HCV RNA to activate stellate cells. The CPT1A SNP was also a nonsynonymous change, and it was more frequent in subjects with milder disease. This SNP (A275T) may lead to less oxidative stress, potentially explaining the association with decreased brosis. Cytokines are believed to be important in brogenesis, and rodent models conrm that they can aect brosis (reviewed by Bataller and colleagues [60]). TNFa is a proinammatory Th1 cytokine that is upregulated in chronic HCV, so it is a probrogenic candidate [61]. Yee and colleagues [62] compared polymorphisms in TNFa in 33 patients who had cirrhosis and 114 who did not have cirrhosis, although the precise stage of the noncirrhotic patients was not stated. The 308A and 238A promoter variants increased the risk for cirrhosis in this study. A Japanese study had similar results and found that these variants were associated with increased type IV collagen 7S, which is a marker of hepatic brosis [63], but they were not associated with liver enzyme elevations. A second Japanese study also found that these promoter variants were not associated with liver enzyme elevations [55]. One study examined seven CC chemokines and their receptors and found an association between a polymorphism in monocyte chemoattractant protein 2 (MCP-2), Q46K, and severe brosis (OR 2.29; P .018) [64]. In addition, CCR5D32 was associated with reduced portal inammation but

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increased brosis (OR 1.97; P .015). It is not clear why reduced portal inammation and more brosis are associated with the same genotype, especially because in autoimmune hepatitis mouse models and infectious models, CCR5D32 is associated with an increased inammatory response [65,66]. Studies from Spain and India also contradicted this result, because there was no association between CCR5D32 and brosis stage [67,68]. IL-10 is a Th1 cytokine that mediates its eects through the IL-10 receptor (IL-10R), which is a heterodimer consisting of IL-10R1, required for binding, and IL-10R2, required for signaling. IL-10 is an anti-inammatory cytokine that downregulates collagen 1 expression and upregulates collagenase. One study found that the minor allele in G330R IL-10R1 was associated with cirrhosis but not with inammation, suggesting that the probrotic eect of this allele was independent of the inammatory response [69]. TGF-b1 is one of the major probrogenic cytokines and has been implicated in disease progression. The proline-to-arginine switch at codon 25 in TGFB1 increases production of the cytokine and leads to increased brosis (P .023) [70]. Angiotensinogen II is another probrogenic cytokine. The promoter G-to-A SNP at 6 leads to increased transcription of the gene and is also associated with increased brosis. Together, SNPs in these two genes have a dose-response eect, because individuals with polymorphisms in both of these genes have more brosis progression than those with either alone. IL-12 is a cytokine that induces production of IFNg. A SNP in the gene for one of the subunits of IL-12 (IL-12p40) has been associated with immune-mediated diseases. Homozygosity for the minor allele of this SNP, 1188C/C, was more common in patients who had mild brosis (score %2) compared with severe brosis (score O2) (23.7% versus 6.25%; P .004), but there was no association with inammation [71]. Solute carrier family 11 member 1 (SLC11A1) protein is involved in macrophage function and in upregulation of chemokines important in the immune response to HCV (TNFa, IL-1b, and inducible nitric oxide synthase). Several mutations result in variable mRNA expression, including a GT tandem repeat promoter polymorphism. Four functionally dierent alleles have been described in the population, which are designated as 1 through 4. These alleles have been associated with other diseases, such as tuberculosis [72]. Individuals who were homozygous for allele 2 had lower inammation, less brosis (2.4% versus 18.1%, OR 8.85), and lower HCV RNA levels (336 versus 1290 103 IU/mL) compared with the other alleles [73]. Further work is needed to understand the biologic basis for this association. Myeloperoxidase (MPO) is an oxidant-generating enzyme found in macrophages and neutrophils. A functional promoter polymorphism (463A) in this gene has less MPO production but was associated with more brosis [74]. The reason for this association is unclear, but MPO has been associated with inhibition of NK activity and T-cell proliferation [75].

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Summary This review has concentrated on summarizing several published HLA and non-HLA associations in immune-response genes with various HCV outcomes. Some have been repeated in more than one study or have a biologic basis for the association. The associated SNPs increase our understanding of HCV clearance and liver disease progression. A limitation of this review, and of the current published studies, is that studies in which associations are not found often go unreported. To maximize the utility of such studies to oer insight into how the immune response may account for the heterogeneity in HCV outcomes, studies that do not nd associations should also be published. Another limitation is that epistatic interactions among polymorphisms in dierent genes are often not assessed. In the future, as large-scale genotyping becomes more aordable, there should be an increasing number of studies examining polymorphisms in various genes. As the ability to scan the genome improves, such studies should also oer clues to important genes involved in HCV pathogenesis, including ones that may not be obvious candidates. Nevertheless, such studies would still not be able to evaluate interactions of variants in dierent genes. Insights into the immune response from human genetic studies can lead to rational drug development to improve therapeutics for chronic hepatitis C. In addition, human genetic variations can also aect response to HCV therapies; thus, future therapeutic trials may involve studying polymorphisms and treatment response.

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