ClinIics in Liver Disease - August 2008

Preface

Clin Liver Dis 12 (2008) xiii–xiv

1089-3261/08

doi:10.1016/

/$ -

j.cld.

K. Rajender Reddy, MD

see front matter � 2008 Elsev

2008.03.014

David E. Kaplan, MD, MSc

Guest Editors

Despite major advances in the understanding of viral pathogenesis andsignificant evolution in antiviral therapies, chronic infection with the hepa-titis C virus (HCV) remains a highly prevalent and burdensome diseaseworldwide. It affects approximately 3% of individuals worldwide and isthe cause for approximately 7,500 annual deaths in the United States alone.While the current treatments offer reasonable response rates, there remainenormous challenges that include therapy-related adverse events, the risingpopulation of non-responders, and special populations such as those withcirrhosisdparticularly decompensated liver disease. Novel HCV-specific an-tiviral agents loom on the horizon, bringing with them the hope of majorimprovements in cure rates and dramatic alterations of the natural historyof HCV infection. Challenges undoubtedly are to come up in the searchfor treatment strategies that are better tolerated, have higher response rateswith shortened duration of therapy, while we maintain a high genetic barrierto prevent the phenotypic expression of viral resistance.

Therefore, in this issue of the Clinics in Liver Disease, we focus on topicsrelated to optimization of currently available therapies, evolution of nonin-vasive disease-staging techniques, and management of important conditionsassociated with or complicating chronic HCV infection while providing anupdate on emerging antiviral therapies and a snapshot of advances in theunderstanding of viral pathogenesis. Along with discussion of tailored

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xiv PREFACE

antivirals in the article by Bernd Kronenberger, Christoph Welsch, NicoleForestier, and Stefan Zeuzem, we present two articles discussing optimiza-tion of interferon-based approaches by Mitchell L. Shiffman and ThomasBerg. We examine the evolving state of non-invasive staging modalities inthe article by David S. Kotlyar, Wojciech Blonski, and Vinod K. Rustgi.Three ‘‘translational’’ articles focus on important host–virus interactionsthat contribute to innate and adaptive immune responses in the setting ofchronic viral infection of hepatocytes that are likely to impact the efficacyof novel antiviral therapies and antiviral vaccine approaches in the articlesby Gyongyi Szabo and Angela Dolganiuc; Zania Stamataki, Joe Grove, Pe-ter Balfe, and Jane A McKeating; and Chloe L. Thio. We conclude with fivearticles focusing on conditions frequently and concomitantly affecting HCVpatients (HIV co-infection, fatty liver disease), difficult-to-manage complica-tions (hepatocellular carcinoma, extrahepatic manifestations of HCV), andissues relating to liver transplantation as reviewed by Vincent Lo Re III, JayR. Kostman, and Valerianna K. Amorosa; Onpan Cheung and Arun J.Sanyal; Anna Linda Zignego and Antonio Craxı; Wojciech Blonski andK. Rajender Reddy; and Elizabeth C. Verna and Robert S. Brown.

We hope that these articles prove informative and provocative in theirchoice of both clinical and translational topics critically assessing the currentstate of the art and providing a glimpse of the future of hepatitis Cmanagement.

K. Rajender Reddy, MDDepartment of Hepatology

Hospital of the University of Pennsylvania3 Dulles

3400 Spruce StreetPhiladelphia, PA 19104

E-mail address: [email protected]

David E. Kaplan, MD, MScDivision of Gastroenterology

University of Pennsylvania 600 CRB415 Curie Boulevard

Philadelphia, PA 19104

Research SectionPhiladelphia Veterans Administration Medical Center

Research A402A3900 Woodland AvenuePhiladelphia, PA 19104


Optimizing the Current Therapyfor Chronic Hepatitis C Virus:

Peginterferon and Ribavirin Dosingand the Utility of Growth Factors

Mitchell L. Shiffman, MDHepatology Section, Virginia Commonwealth University Medical Center,

Box 980341, Richmond, VA 23298, USA

The primary goal when treating patients who have chronic hepatitis C vi-rus (HCV) is to achieve a sustained virologic response (SVR). For patientsto achieve this goal, however, three independent milestones must be sequen-tially achieved (Table 1). The first step is that the patient must achievea virologic response and become HCV RNA undetectable during treatment.It is fairly obvious but rarely stated that patients who do not achieve a viro-logic response cannot possibly achieve an SVR. The second step is that thepatient must maintain this response by remaining HCV RNA undetectablethroughout the duration of therapy. Patients who break through and be-come HCV RNA-positive during treatment also cannot achieve an SVR.The final step is that the patient must not relapse after treatment has beencompeted; thus: SVR ¼ (virologic response) � (breakthrough plus relapse).

The current treatment for chronic HCV infection is the combination ofpeginterferon and ribavirin [1,2]. These medications are effective for thetreatment of chronic HCV. Approximately 80% of patients who haveHCV genotype 1 and virtually all patents who have genotype 2 or 3 havea rapid and profound reduction in HCV RNA within the first 3 months afterinitiating treatment [1–4]. Unfortunately, not all patients with this early vi-rologic response (EVR) pattern achieve an SVR [1,4]. There are three majorreasons for this. Some patients do not become HCV RNA undetectable eventhough they achieved an EVR and had a 2-log reduction in HCV RNAwithin the first 12 weeks of treatment. In a previous review, this was referredto as a partial early virologic response [5]. Because these patients do not

Clin Liver Dis 12 (2008) 487–505


1089-3261/08/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.cld.2008.03.004 liver.theclinics.com

Table 1

Milestones for achieving a sustained virologic response

Virologic response

Patients must become HCV RNA undetectable

within 24 weeks after initiating treatment

Preventing breakthrough Patients must remain HCV RNA undetectable

throughout the duration of treatment

Preventing relapse Patients must remain HCV RNA undetectable after

treatment is discontinued

488 SHIFFMAN

become HCV RNA undetectable, they cannot achieve an SVR. Peginter-feron and ribavirin are associated with adverse events, which, at times,may be so severe that dosing with one or both of these medications has tobe adjusted, interrupted, or prematurely discontinued. These actions mayreduce the SVR by preventing an EVR from being achieved [6] or byenhancing breakthrough and relapse [7–9]. Finally, some patients havea slow response to treatment and only become HCV RNA undetectablelate during the course of treatment. These ‘‘slow to respond’’ patientshave an extremely low SVR because they experience a high rate of relapse[10–12].

This article focuses on how altering the doses of peginterferon and riba-virin can affect the three milestones leading to SVR, namely, virologicresponse, breakthrough, and relapse. This discussion first reviews the vari-ous virologic patterns and definitions of response, nonresponse, break-through, and relapse. The discussion will then address the roles thatpeginterferon and ribavirin play in achieving virologic response, how adjust-ing the doses of peginterferon or ribavirin could adversely affect these re-sponse patterns, and when it would be appropriate to use hematologicgrowth factors in the treatment of chronic HCV.

Patterns of virologic response

The term early virologic response refers to a 2-log decline in HCV RNAfrom the pretreatment baseline or being HCV RNA undetectable within12 weeks after the initiation of treatment [1,13,14]. Approximately 80% ofpatients who have genotype 1 and virtually all patients who have genotypes2 and 3 achieve an EVR [1,4,6,10,13,14]. Patients without an EVR rarely ifever achieve an SVR [1,6,10,13,14]. These patients are referred to as havinga null response [5]. At the Second National Institutes of Health ConsensusDevelopment Conference on the Management of HCV, it was recommendedthat all patients who have genotype 1 undergo HCV RNA testing at baselineand at week 12 [13,14]. Patients without an EVR (a null response) shouldstop treatment because they cannot achieve an SVR. It was furtherrecommended that all genotype 1–infected patients with an EVR continuetreatment for 48 weeks and that all patients who have genotypes 2 and 3simply be treated for 24 weeks [1,13,14]. Unfortunately, not all patients

489OPTIMIZING THE CURRENT THERAPY FOR CHRONIC HEPATITIS C VIRUS

with an EVR become HCV RNA undetectable, and continuing treatment inthese patients cannot lead to an SVR. It is therefore apparent that theserecommendations need to be modified.

Fig. 1 illustrates various virologic response patterns in patients with anEVR. It is apparent that an EVR constitutes a wide spectrum of virologicresponses that include patients who become HCV RNA undetectable within4 weeks to as late as 24 weeks after initiating treatment. Overall, 80% of pa-tients who have genotype 1 achieve an EVR, but only 65% actually becomeHCV RNA undetectable [1,10]. The remaining 15% of patients have a par-tial virologic response (Fig. 2). Approximately 10% of patients with a partialvirologic response eventually become HCV RNA undetectable if treatmentis continued past week 24 [10]. These patients rarely, if ever, achieve an SVRwhen treated for only 48 weeks. It is critically important that the partial vi-rologic response pattern be recognized and treatment be discontinued inthese patients at week 24 unless an alternative strategy is implemented(see section on management of partial responders).

Defining the time when the patient first becomes HCV RNA undetectableis critically important in the management of HCV treatment because this isdirectly related to the likelihood of an SVR [10]. Patients who become HCVRNA undetectable within 4 weeks of initiating treatment are referred to ashaving a rapid virologic response (RVR). As illustrated in Fig. 2, approxi-mately 15% of patients who have genotype 1 and 66% of patients whohave genotypes 2 and 3 achieve an RVR [4,10]. These patients are exqui-sitely sensitive to treatment; they have an SVR rate of 90% regardless oftheir genotype and the type of therapy they receive [4,10]. In a retrospectiveanalysis of a large clinical trial database, patients who had genotype 1 andan RVR had an SVR of 90% whether they were treated with peginterferon

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Peginterferon/Ribavirin

Limit of detectionPartial response

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, how-

ever. 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.

0

20

40

60

80

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4 12 24

Week Became HCV RNA (-)

% o

f Pat

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sGenotype 1Genotypes 2 and 3

PartialResponse

NonResponse

Early Virologic Response = 80%

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 Shiffman

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;357:124–34; 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;38:645–52.)

490 SHIFFMAN

and full-dose ribavirin, a lower dose of ribavirin, standard interferon and ri-bavirin, or even peginterferon monotherapy [10,15]. Shortening the durationof therapy from 48 to only 24 weeks also seemed to be effective in achievinghigh rates of SVR in genotype 1–infected patients with an RVR [15]. Theseobservations have significant implications for dosing peginterferon and riba-virin. Because patients with an RVR are so responsive to treatment, theprimary response to adverse events in these patients should be to reducethe doe of peginterferon or ribavirin and not to add growth factors or otheradjuvant therapies.

The later during treatment a patient becomes HCVRNAundetectable, thelower is the rate of SVR [10]. Patients who have genotype 1 who becomeHCVRNA undetectable between treatment weeks 4 and 12 have an SVR of ap-proximately 66%, but those who become HCV RNA undetectable betweenweeks 12 and 24 have an SVR of only 45%. The low SVR in slow respondersoccurs because of a higher rate of relapse. Genotype 1–infected patients whobecomeHCVRNAundetectable between weeks 12 and 24 have been referredto as ‘‘slow to respond,’’ and recent studies have demonstrated that prolong-ing the duration of treatment in these patients from 48 to 72 weeks can signif-icantly reduce the relapse rate [11,12]. This would obviously enhance theSVR. Patients who have genotypes 2 and 3 who do not become HCV RNAundetectable after week 4 have an SVR of only 49% [4], and a recent retro-spective analysis has suggested that prolonging treatment in these patientscould also reduce relapse [16]. Adjusting the duration of therapy in responseto on-treatment virologic response is discussed in a later chapter in this issueof Clinics in Liver Disease (see the article by Berg elsewhere in this issue).


Two additional patterns of virologic response are important to recognize:breakthrough and relapse (Fig. 3). Patients with breakthrough initiallybecome HCV RNA undetectable in serum during treatment but then havereappearance of HCV RNA in serum despite ongoing treatment. Patientswith relapse become and remain HCV RNA undetectable in serum through-out treatment but then have reappearance of HCV RNA in serum aftertreatment is discontinued. Common reasons for breakthrough and relapseinclude the premature termination or temporary interruption of peginter-feron or ribavirin [8].

Effect of interferon dosing on virologic response

It has long been recognized that the ability of interferon to reduce HCVRNA in serum is a dose-dependent process. Studies conducted at theauthor’s center longer than a decade ago demonstrated that increasing thedosage of standard interferon from 3 to 5 to 10 mU and then to 20 mU threetimes weekly every 3 months in those patients who remained HCV RNA-positive led to a stepwise increase in the overall percentage of patientsbecoming HCV RNA undetectable [17]. Up to 80% of patients treatedwith this approach eventually became HCV RNA undetectable. Increasingthe dosage of peginterferon alfa-2a from 45 to 180 mg/wk or peginterferonalfa-2b from 0.5 to 1.5 mg/kg/wk also led to a stepwise increase in virologicresponse [18,19]. High daily dosages of consensus interferon (9–15 mg/d) andhigher dosages of peginterferon, up to a peginterferon alfa-2a dose of360 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 undetect-able after treatment with standard doses of interferon or peginterferon[20–23].

The major limitation to using higher doses of interferon or peginterferonis the adverse events of these agents, which increase with increasing dose and

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HC

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2-log decline

Limit of detection

Peginterferon/RibavirinRelapse

BreakthroughNull response

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

492 SHIFFMAN

cause patients to discontinue treatment [20–23]. The current recommendedstarting doses for the two peginterferons were derived by balancingresponse and discontinuation rates to achieve the highest overall rates ofSVR. Because most patients respond to the current starting dosages ofpeginterferon (180 mg/wk for peginterferon alfa-2a and 1.5 mg/kg/wk for pe-ginterferon alfa-2b), it is not appropriate to initiate treatment with higherdoses of these medications. In contrast, it is quite rationale to considerescalating the dose of peginterferon or switching to high doses of dailyinterferon in patients with a suboptimal response who could tolerate sucha treatment strategy.

Increasing the interferon dose to overcome nonresponse

As depicted in Figs. 2 and 3, approximately 20% of patients who havegenotype 1 have a null response during treatment and fail to achieve anEVR [1,2,10]. Such patients are likely resistant to the antiviral effects ofinterferon. In vitro studies have demonstrated that interferon binds to itsmembrane receptor with the same affinity in nonresponders [24], but thedownstream immunologic response is downregulated [25]. As a result,increasing the dose of interferon or peginterferon is unlikely to lead toa virologic response in patients with a null response.

Approximately 15% of patients who have genotype 1 and up to 7% ofpatients 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 effects ofinterferon and ribavirin, and two previous studies have suggested thatmost nonresponders who responded to higher doses of interferon or pegin-terferon during retreatment are those with a previous partial virologicresponse [26,27]. In both of these studies, most patients, if not the onlypatients, who achieved an SVR during retreatment with a higher dose ofinterferon or peginterferon were those who had a decline in HCV RNAto less than 100,000 copies/mL (approximately 50,000 IU/mL) during theinitial course of treatment. It is therefore reasonable to consider escalatingthe interferon dose in patients with a partial virologic response as soon asthis response pattern is recognized. This typically occurs between treatmentweeks 12 and 24 (Fig. 4). If patients do not have a further decline in HCVRNA and become virus RNA undetectable with 3 additional months ofhigh-dose interferon therapy, it is highly unlikely that a virologic responseis going to occur and treatment thereafter should be discontinued. Patientswith a prior partial virologic response can also be retreated with higherdoses of interferon. If such patients do not achieve an EVR or becomeHCV RNA undetectable within 12 to 24 weeks of treatment, however,this approach should also be abandoned. In either situation, a higherdose of peginterferon or high doses of daily consensus interferon can beused.

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HC

V R

NA

(IU/m

L)

SVR

2-log decline

Limit of detection

Peginterferon/Ribavirin

Intensify interferon dose

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 intensification, treatment should be discontinued.


Effect of ribavirin dosing on virologic response

Ribavirin is an essential ingredient in the treatment of chronic HCV andaffects 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 serumHCV 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]. Addingribavirin to peginterferon also enhances virologic response [1]. Becausepeginterferon is more effective than standard interferon, however, thisrepresents only a 10% increase (from 59% to 69%) compared with thatobserved with peginterferon monotherapy.

Ribavirin enhances virologic response by accelerating the decline in HCVRNA over that observed with interferon alone [30]. Although the combina-tion of peginterferon and ribavirin does not increase the percentage ofpatients who achieve an RVR, it more than doubles (from 18% to 38%)the percentage of patients who become HCV RNA undetectable by treat-ment week 12 [10]. As noted previously, becoming HCV RNA undetectablesooner during treatment reduces relapse.

The starting dose of ribavirin may also play a role in enhancing virologicresponse. In several randomized controlled clinical studies a 3% to 9%higher virologic response rate was consistently observed when the startingribavirin dosage was increased by 200 to 400 mg/d [3,31,32]. It remains un-clear from these studies, however, if this difference is statistically or clinicallysignificant.

Ribavirin also helps to maintain the response and enhances the SVR bypreventing breakthrough and reducing relapse. Unfortunately, the adverseevents associated with ribavirin prevent higher doses of this medicationfrom being used and, in some patients, from maintaining adequate ribavirinexposure. Managing the adverse events of ribavirin has been the subject ofconsiderable debate.

494 SHIFFMAN

Impact of adverse events and dose reduction

The treatment of chronic HCV with peginterferon and ribavirin is asso-ciated with numerous adverse events [1,2]. The most common of these canbe classified as systemic flu-like symptoms, psychiatric manifestations, auto-immune reactions, and hematologic toxicities. The management of theseadverse events has been discussed previously [33]. Although adverse eventscan be successfully managed in many cases, approximately 20% to 40% ofpatients require that the dose of peginterferon or ribavirin be reduced ortemporarily interrupted. In 5% of patients, adverse events are so severethat treatment must be discontinued [1,2]. Higher doses of peginterferonand ribavirin have been associated with a greater incidence of adverse events[18–23,31,32,34].

The need to alter the dosing of peginterferon or ribavirin in response toadverse events may have a negative impact on all three milestones leading toan SVR. Two studies have clearly demonstrated that when the dose ofpeginterferon is reduced to less than 80% within the first 12 to 20 weeksof treatment, the ability to achieve an EVR and become HCV RNA unde-tectable is significantly impaired [6,7]. This is consistent with the observa-tions that achieving a virologic response highly depends on the dose ofinterferon [35] or peginterferon dose [18–23] and depends far less on thedose of ribavirin [3,31,32]. Because achieving a virologic response is the firstessential milestone leading to an SVR (see Table 1), any reduction in thisresponse has a negative impact on the SVR.

Prematurely discontinuing ribavirin leads to breakthrough and a higherrelapse rate, even if the peginterferon dose remains unaltered. In a recentstudy, patients who were HCV RNA undetectable at treatment week 24were randomly assigned to stop ribavirin and continue peginterferon aloneor to remain on both drugs [8]. Within 6 weeks of stopping ribavirin, break-through began to occur and this increased stepwise over time. At treatmentweek 48, 24 weeks after stopping ribavirin, breakthrough had developed in12% of patients. In contrast, breakthrough occurred in only 3% of patientsrandomized to continue peginterferon and ribavirin, and each of thesepatients prematurely discontinued ribavirin or both drugs in response toadverse events. It is therefore apparent that the primary, if not the only, rea-son for breakthrough is an interruption or permanent premature discontin-uation of ribavirin. Relapse was also significantly greater in patients whoprematurely stopped ribavirin: 42% compared with only 29% in patientswho remained on peginterferon and ribavirin. The only subgroup of patientswho did not develop breakthrough or have an increased relapse rate afterprematurely stopping ribavirin were those with an RVR. This strongly sug-gests that the primary response to adverse events in patients with an RVRshould be dose reduction rather than growth factor support.

As opposed to interrupting or prematurely stopping ribavirin, which en-hances breakthrough and relapse, recent data suggest that merely reducing


the dose of ribavirin in response to adverse events does not significantlyaffect these milestones, and therefore has little impact on the SVR. The firstreport to evaluate the impact of dose reduction on the SVR demonstratedthat only those patients whose total cumulative peginterferon or ribavirindose declined to less than 80% of expected after treatment week 12 had a sig-nificant decline in SVR [36]. So let us consider the following question: howmuch of a reduction in ribavirin dose was required to decrease to less than80% by week 12 in this study? The answered is depicted in Fig. 5. In thisstudy, the starting dosage of ribavirin was 800 mg/d [36]. The total cumula-tive expected ribavirin dosage was therefore: 800 mg/d � 7 d/wk � 48 wk oftherapy ¼ 268,800 mg. Fig. 5A illustrates the impact of reducing the ribavi-rin dosage from 800 to 600 mg/d, as was done in this study [36], at varioustime points during the 48 weeks of treatment. If the ribavirin dose reductionoccurred at treatment week 24, the patient still received 88% of the total

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Fig. 5. (A) Impact of dose reduction on the total cumulative dose of ribavirin. The starting dos-

age 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 figures 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 sus-

tained response in genotype-1-infected patients with chronic hepatitis C. Gastroenterology

2002;123:1061–9.)

496 SHIFFMAN

cumulative expected ribavirin dose. Moving the time at which this dose re-duction occurred to treatment week 12 or 6 failed to lower the cumulativeribavirin dose to less than 80% within the first 24 weeks of treatment.Even if dose reduction occurred at treatment week 4, the patient stillreceived 79% of the cumulative ribavirin dose by week 24. It is therefore ap-parent that patients could not have received less than 80% of their ribavirindose in this study through dose reduction alone. The only way this couldhave happened was for patients to have missed doses. The impact of inter-rupting ribavirin dosing is illustrated in Fig. 5B. In this figure, ribavirin dos-ing was interrupted at the start of treatment week 6 for variable periodsbefore the dosage was reduced from 800 to 600 mg/d. Missing just 3 daysof 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 ribavirindosing, from greater than 97% to less than 60% of the total expected cumu-lative dose, on virologic response and SVR [9]. It is important to note thatthis study included only those patients who remained on full-dose peginter-feron for 48 weeks, and therefore evaluated the impact of reducing only theribavirin dose. No significant impact of ribavirin dose on virologic responsewas observed. A significant decline in SVR (from 57% to 67% to only 34%)did occur but only when the total cumulative ribavirin dose declined to lessthan 60%. As illustrated in Fig. 5, this can only occur by interrupting orprematurely discontinuing ribavirin dosing. Because no significant declinein virologic response was observed in this analysis, the decline in SVRmust have been secondary to breakthrough and relapse.

The impact of dose reducing peginterferon or ribavirin independent ofeach other was examined in patients with prior nonresponse to interferon(with or without ribavirin) undergoing retreatment with peginterferon andribavirin in the Hepatitis C Antiviral Long-Term Treatment Against Cirrho-sis (HALT-C) clinical trial [7]. This study included more than 900 patientswho had genotype 1, making it the largest and most comprehensive analysisof dose reduction performed to date. Reducing the total cumulative dose ofpeginterferon to less than 80% led to a decline in virologic response andSVR. In contrast, reducing the dose of ribavirin had no impact on virologicresponse or SVR as long as patients remained on full-dose peginterferon andribavirin dosing was not interrupted or prematurely terminated. It is there-fore apparent that missed doses of ribavirin rather than dose reduction iswhat has an adverse impact on SVR and that this occurs by increasingbreakthrough and relapse.

Role of epoetin alfa in the treatment of chronic hepatitis C virus

One of the most common adverse events in patients receiving pegin-terferon and ribavirin for treatment of chronic HCV is anemia [1,2]. Thisis secondary to a dose-dependent hemolysis induced by ribavirin- and


interferon-induced bone marrow suppression, which inhibits a compensatoryreticulocytosis [37]. The mean decline in hemoglobin observed with peginter-feron and ribavirin in large registration trials is 2.5 to 3 g/dL [1,2]. Twenty per-cent of persons have a decline in hemoglobin of 4 g/dL or more, however [38].Anemia significantly lowers the quality of life and exacerbates the fatigue ex-perienced by patients receiving HCV therapy [39]. In general, it is recommen-ded that the ribavirin dose be reducedwhen the hemoglobin decreases bymorethan 4 g/dL from the pretreatment baseline or to less than 10 g/mL and that itbe 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 pegin-terferon and ribavirin is controversial. Several years ago, an open-label trialdemonstrated that epoetin alfa could reverse the amenia that developedduring treatment with peginterferon and ribavirin [40]. This observationled to a double-blind placebo-controlled trial demonstrating that epoetinalfa could reduce anemia, allow patients to remain on a higher mean doseof ribavirin [41], and improve quality of life during HCV treatment [42].A recent cost analysis suggested that using a hematologic growth factor inlieu of dose reduction would be cost-effective [43]. Unfortunately, this anal-ysis was based on the premise that using epoetin alfa would actually enhancethe SVR, without any data to support this claim. Despite these shortcom-ings, these reports were fueled with the belief that reducing the ribavirindose would have a negative impact on SVR and led to the widespread useof epoetin alfa and darbepoetin alfa in patients who developed anemia whilebeing treated with peginterferon and ribavirin [39].

The only randomized, prospective, controlled trial evaluating epoetin alfain patients receiving peginterferon and weight-based ribavirin for chronicHCV failed to demonstrate that this approach could enhance the SVR,even though this agent was administered at the onset of therapy beforeany patients developed anemia or required dose reduction [32]. There areseveral reasons why patients who received epoetin alfa in this study failedto demonstrate any improvement in SVR. First, ribavirin was reducedsequentially in 200-mg steps with the goal being not to interrupt or prema-turely discontinue dosing. Second, using epoetin-alfa did reduce but did notcompletely eliminate the need to dose reduce ribavirin. Finally, reducing thedose of ribavirin did not adversely affect the ability of all patients to achievean SVR, especially those with an RVR.

Another study conducted in patients who had HCV and were coinfectedwith HIV has also demonstrated that the routine use of epoetin alfa did notenhance the SVR during treatment with peginterferon and ribavirin [44]. Inthis study, no relation between ribavirin dose and anemia was apparent, andthe commonly used protease inhibitor zidovudine rather than ribavirin wasfound to be the major cause of anemia.

Although widely used and generally safe, hematologic growth factorsdo cause adverse events, including hypertension, headache, arthralgias, par-esthesias, and injection site erythema [45–47]. More recently, an increased

498 SHIFFMAN

rate of thromboembolic events has been observed in patients receiving theseagents, and this has led the US Food and Drug Administration to adminis-ter a warning regarding the overuse of these agents. Antibody-mediatedpure red blood cell aplasia has also been reported in an HCV-infected pa-tient receiving epoetin alfa during treatment with peginterferon and ribavi-rin [48]. It is therefore apparent that dose reduction rather than epoetin alfashould be the first strategy in a patient receiving peginterferon and ribavirinfor HCV treatment, especially if this patient has achieved an RVR or if ane-mia has developed after the patient has become HCV RNA undetectable. Asopposed to reducing ribavirin to 600 mg as recommended in previous stud-ies [1,2,36], the author’s group reduces ribavirin by 200-mg steps. This ap-proach maximizes total cumulative ribavirin exposure, as illustrated inFig. 6. In fact, the medical and nursing staff at the author’s institution fre-quently perform a one-step (200 mg) ribavirin reduction in a patient whohas developed severe fatigue even if the hemoglobin has not declined toless than 10 mg/dL. This small reduction in the ribavirin dose does nothave a significant impact on the total cumulative ribavirin exposure (seeFig. 6) and, in many cases, yields significant symptomatic improvementand allows the patient to be more enthusiastic about continuing therapy.The goal of dose reduction is to prevent the hemoglobin from decreasingso much that the patient must interrupt ribavirin dosing, which clearlyincreases the risks of breakthrough and relapse [7,8].

These observations do not mean that epoetin alfa should be abandonedas an adjuvant therapy in the treatment of chronic HCV. There is no doubtthat some patients develop anemia so rapid and severe in its onset that theycannot possibly remain on ribavirin, and therefore cannot be successfullytreated for chronic HCV without the use of epoetin alfa. Profound anemiaoccurs in approximately 5% to 10% of patients within the first 12 weeks ofinitiating HCV treatment [1,2,38], and most of these patients may need tostop or interrupt ribavirin dosing. Many physicians faced with this clinicalsituation initiate epoetin alfa and then restart ribavirin 1 to 2 weeks later,

40

50

60

70

80

90

100

110

0 6 12 18 24 30 36 42 48 54WEEKS

% M

AXIM

AL R

IBAV

IRIN

DO

SE

1000 mg/d

800 mg/d

600 mg/d

400 mg/d

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.


after the hemoglobin has increased to greater than 10 g/dL. Unfortunately,this is not an effective strategy, because these patients have a high rate ofbreakthrough and relapse. As a result, the strategy of the author and his groupis simply to discontinue therapy, allow the anemia to resolve, obtain approvalto use epoetin alfa, and then restart HCV treatment along with growth factorsupport from the onset. Severe anemia is more common during HCV treat-ment in patients who have advanced cirrhosis or chronic renal insufficiencyand in liver transplant recipients [39]. Most of these patients cannot possiblyremain 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 severeneutropenia [1,2]. This is most commonly observed when treating patientswho have cirrhosis or African Americans who have essential neutropenia[49]. Although it is recommended that the dose of peginterferon be reducedwhen the absolute neutrophil count decreases to 750 cells/mL, many physi-cians experienced in the care of patients who have cirrhosis and chronicHCV allow the absolute neutrophil count to decline to 500 cells/mL. A pre-vious study in patients who had cirrhosis has demonstrated that the risk ofinfection is not significantly increased in patients receiving peginterferon[50]. A controlled trial has demonstrated that granulocyte colony-stimulat-ing factor significantly increases the neutrophil count in these patients [51].Most patients who have chronic HCV, even those who have cirrhosis, donot develop significant neutropenia and are not at increased risk for severeinfections during treatment with peginterferon. The exceptions are patientswho 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 leadto SVR be balanced against the adverse event profile experienced by patientsreceiving peginterferon and ribavirin. This can only be accomplished if thespecific virologic response pattern achieved during treatment is recognized.It is therefore essential that HCV RNA be monitored at regular intervalsduring treatment until the patient becomes HCV RNA undetectable ora virologic nonresponse pattern is defined, as illustrated in Figs. 1 and 3.This is best accomplished by monitoring HCV RNA at monthly intervals.At the author’s institution, the medical and nursing staff treating patientswith chronic HCV measure HCV RNA at baseline and every month untilthe patient has become HCV RNA undetectable or a non-response virologicpattern is identified and the treatment is stopped. This allows them to bal-ance adverse events, virologic response, and dose modification as describedin Table 2.

Table 2

Balancing adverse events, virologic response, and dose modification

Timing of adverse

event Strategy

Weeks 1–4 Discontinue treatment, allow adverse event to recover, develop strategy

to prevent adverse event from recurring, and initiate retreatment

Weeks 4–12 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

Weeks 12–24 If HCV RNA undetectable: modify dose in small steps (see text and Fig. 6);

recheck HCV RNA 1 month after dose modification 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

After week 24 Modify dose in small steps (see text and Fig. 6)

500 SHIFFMAN

The patient with minimal or infrequent side effects during treatment iseasy to treat. Virologic response is monitored at monthly intervals, and ifthe patient becomes HCV RNA undetectable, treatment is continued forthe appropriate duration of therapy. This is based on genotype and thetime at which the patient becomes HCV RNA undetectable during treat-ment (see the article by Berg elsewhere in this issue). If the patient doesnot have a decline in HCV RNA by more than 2 log units, treatment shouldbe discontinued by week 12 or as soon as the null response pattern is recog-nized. In patients with a partial response, the dose of peginterferon could beescalated for an additional 3 months. Alternatively, high-dose daily consen-sus interferon could be used [23]. The goal of this maneuver is to render pa-tients HCV RNA undetectable. Thus, if the patient fails to become HCVRNA undetectable after 3 additional months of dose intensification, treat-ment should be discontinued.

The patient who is most difficult to treat develops adverse events so se-vere that the patient must temporarily interrupt or discontinue treatmentwithin the first few weeks to months after treatment is initiated. Dose mod-ification so early into treatment frequently leads to a null response. If thepatient does achieve an EVR or becomes HCV RNA undetectable, however,the interruption in treatment significantly increases the risk of breakthroughor relapse [7–9]. It is therefore preferable for such patients simply to discon-tinue treatment, allow the adverse effects to resolve, and then reinitiatepeginterferon and ribavirin with the knowledge and tools required to lessenthe severity or prevent these adverse events from recurring. If the adverseevent in question is severe anemia, the use of epoetin alfa during retreatmentis justified. Possibly the only exception to this recommendation would be in


the patient who has already achieved an RVR. In this case, simply complet-ing treatment even with lower doses of peginterferon and ribavirin hasa high likelihood of yielding an SVR [10]. It is important to realize that thesetreatment decisions cannot be made unless HCV RNA is monitoredfrequently and the virologic response pattern is clearly defined.

Most patients receiving HCV treatment develop adverse events of mod-erate severity, which progressively worsen and require that peginterferonor ribavirin be dose modified at some point after the patient has becomeHCV RNA undetectable. The primary reason for dose modification in thesepatients is progressive fatigue, myalgias, arthralgias, insomnia, and depres-sion. The management of these adverse events has been discussed previously[33]. At the author’s institution, it has been found that reducing the dose ofribavirin by 200 mg or the dosage of peginterferon by 20% (from 180 to135 mg for peginterferon alfa-2a or from 1.5 to 1.2 mg/kg for peginterferonalfa-2b) can significantly improve adverse events and enable many of thesepatients to complete treatment. Several studies [9,35,36] and the analysis inFigs. 5 and 6 clearly suggest that such a strategy is unlikely to have a nega-tive impact on the SVR. Nevertheless, whenever the dose of peginterferon orribavirin is modified, HCV RNA should be assessed 1 month later so thatbreakthrough can be identified. If breakthrough does occur, treatmentshould be discontinued, because continuing treatment in a patient withbreakthrough cannot lead to an SVR. Before treatment is abandoned, how-ever, the author advises that repeat HCV RNA testing be performed to con-firm that the previous result was not a false-positive value. False-positiveand false-negative values do occur during HCV RNA testing [53]. Discor-dant virologic results cannot be recognized unless HCV RNA testing is per-formed at regular intervals during treatment [5]. Pushing patients to remainon full-dose therapy when they are experiencing adverse events forces themto seek relief by skipping doses or prematurely stopping treatment on theirown. This clearly enhances the likelihood of breakthrough and relapse.

Summary

In summary, the ability to achieve an SVR depends on the patient achiev-ing the three milestones of treatment: virologic response, maintaining thevirologic response, and not relapsing. Achieving virologic response is pri-marily a function of the peginterferon dose, and some patients may requirethat the peginterferon dose be escalated to become HCV RNA undetectable.Ribavirin affects all three of the milestones that lead to an SVR. The majorimpact of ribavirin is to prevent breakthrough and reduce relapse, however.Interrupting or prematurely discontinuing ribavirin therapy increases thelikelihood of breakthrough and relapse, and this reduces the SVR. In con-trast, reducing the dose of ribavirin, especially after the patient has becomeHCV RNA undetectable, does not seem to affect virologic response, relapse,or the SVR. Ideally, dose reduction should be performed in 200-mg steps,

502 SHIFFMAN

because this maximizes total cumulative ribavirin exposure and often allevi-ates the adverse event. A randomized controlled trial has demonstrated thatusing epoetin alfa in lieu of this stepwise dose reduction strategy does not af-fect the SVR. Modifying the doses of peginterferon and ribavirin duringtreatment can only be accomplished when the virologic response pattern iswell defined. This demands that HCVRNAbemonitored at regular intervals,preferably monthly, until the patient has become HCV RNA undetectable ora nonresponse pattern has been recognized and treatment is discontinued.

Acknowledgments

The author acknowledges the support and insight of the medical andnursing staff at his institution (Sarah Hubbard, PA, Charlotte Hofmann,RN, April Long, NP, Jennifer Salvatori, RN, Denice Shelton, RN, PaulaSmith, RN and Kimberly Williams, RN) who have contributed to manyof the observations and concepts detailed in this article and have helpedto improve the treatment of chronic HCV for our patients. Dr. Shiffmanis a consultant for Roche Laboratories, Roche Molecular Systems, ValeantPharmaceuticals, and Wyeth Pharmaceuticals. He receives research supportfrom Schering-Plough, Roche Laboratories, Valeant Pharmaceuticals, Ver-tex Pharmaceuticals, and Human Genome Sciences and is a speaker forRoche Laboratories and Valeant Pharmaceuticals.

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[28] Bodenheimer HC Jr, Lindsay KL, Davis GL, et al. Tolerance and efficacy of oral ribavirin

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with ribavirin as initial treatment for chronic hepatitis C. N Engl J Med 1998;339:1485–92.

[30] Herrmann E, Lee JH, Marinos G, et al. Effect of ribavirin on hepatitis C viral kinetics in

patients treated with pegylated interferon. Hepatology 2003;37:1351–8.

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viral response in patients with chronic hepatitis C: final results of the WIN-R study, a US

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[32] ShiffmanML, Salvatore J, Hubbard S, et al. Treatment of chronic hepatitis C virus genotype

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[33] Shiffman ML. Side effects of medical therapy for chronic hepatitis C. Ann Hepatol 2004;3:

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[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

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[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. Gastro-

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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. De-spite considerable reduction of the incidence of new infections, the preva-lence of HCV is predicted to remain constant in the near future [1].Furthermore, the current combined interferon (IFN)-based therapy is effec-tive in only a fraction of the patients and is also plagued with adverse effects.There is therefore a need to develop new therapies. Early clinical trials havejust begun on some promising HCV-targeted compounds, such as the sub-strate specific NS3-4A protease inhibitors or the NS5B polymerase inhibi-tors that have been tested alone or in combination with pegylatedinterferon alfa (peg-IFNa) with or without ribavirin.

For the time being, however, the currently recommended therapy forchronic hepatitis C remains the combination of peg-IFNa and ribavirinfor 24 or 48 weeks. Several ongoing studies have recently been put forthto optimize therapeutic strategies by tailoring the duration of treatmentand also by evaluating the optimal dose of peg-IFNa and ribavirin. Also ex-plored were different baseline factors, which were shown to improve the pre-diction of the outcome, especially when correlated with viral kinetics.

This review therefore focuses on the analysis of current concepts andstrategies in favor of a more individualized treatment regimen in patientswho have HCV infection.

Clin Liver Dis 12 (2008) 507–528

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).

* Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Charite, Campus

Virchow-Klinikum, Universitatsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin,

Germany.




508 BERG

Principles of tailored treatment in hepatitis C virus type 1

Treatment response in patients who have chronic HCV infection is quiteheterogeneous and depends on host factors (ie, age, gender, alanine amino-transferase [ALT] levels, stage of fibrosis, insulin resistance) and viral fac-tors, such as serum concentration of HCV RNA at the time of initiationof antiviral therapy, including HCV genotypes [2–7]. Thus, virologic factorsand host factors must be considered if one tries to get sound information onthe individual likelihood of response. If one is aware of the influence of viraland host factors with respect to the possible consequences of therapeutic re-sponse, it indeed seems rather illogical to treat all patients with the samefixed therapeutic regimen.

Viral kinetic studies have further expanded our knowledge of the mech-anisms of IFNa action and have shown that the likelihood of achievinga sustained virologic response (SVR) clearly depends on the speed of the re-sponse [8–10]. It is well established that the rate of SVR is inversely corre-lated with the time on treatment that is necessary to clear HCV RNAfrom serum. Individual prognosis with respect to the long-term treatmentoutcome can therefore be best predicted by early viral kinetics, because allbaseline factors finally alter the speed of response (Fig. 1). Indeed, if one in-cludes viral kinetic parameters in multivariate models to predict SVR, mostof the baseline factors lose their predictive power. The rational backgroundof any individualized therapy is based on the concept that rapid respondersneed less therapy as compared with those patients who are slow responders.

Virologic factors

HCV genotypelevel of replicationmutational pattern

(ISDR)(HCV genetic mutations)

Host factors

agegender

stage of fibrosisinsulin resistance/steatosis

cholesterol levelALT levelsGGT levels

genetic polymorphisms

Early viral kinetics

Summarizing the individuallikelihood of response

Fig. 1. Individual prognosis of a patient with respect to treatment outcome is best reflected by

viral kinetics, because all known host and viral baseline predictors finally affect this type of

response.

509TAILORED TREATMENT FOR HEPATITIS C

Table 1 summarizes the available data on long-term treatment outcomeseen in HCV type 1 infection treated for 48 weeks with standard combina-tion therapy according to the early virologic response pattern.

In a previous article, Shiffman (see the article by Shiffman elsewhere inthis issue) discussed how to identify the optimal doses in an individual-basedstrategy to influence the efficacy of the response. Perhaps even more impor-tant to solve the problem is to try to influence the therapeutic response bymodifying treatment duration, thereby differentiating between slow andrapid virologic responders. Still, there remains the intriguing question asto what the critical criteria are to be sure that relapses do not occur evenwhen we can now anticipate (provided that sensitive tests have been applied)that the HCV RNA has become undetectable on treatment. Thus, a cleardefinition of the duration of the negative HCV RNA interval required toprevent 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 bystandard qualitative HCV RNA tests (detection limit of 50 IU/mL) doesnot, per se, indicate that the virus was completely eliminated from the se-rum. There is now emerging evidence that by applying more sensitive assays(eg, transcription-mediated amplification [TMA] or real-time polymerasechain reaction [PCR] assays with a detection limit !10 IU/mL), a significantproportion of patients who were shown to have undetectable HCV RNAlevels by standard assay are still viremic (Figs. 3 and 4) [11,12]. This impliesthat many patients considered so far to have had a relapse might, in fact,have been nonresponders with a minimal replication level (minimal residualviremia) (see Fig. 2B).

Quite importantly, the detection of minimal residual HCV RNA levels atweek 12 must be taken as a serious predictor for a possible later relapseevent. Morishima and colleagues [12] provided evidence that all patientsin whom a minimal residual viremia could be detected at week 12 and stillconfirmed at week 20 had a relapse. The application of extremely sensitiveHCV RNA tests to design individualized therapeutic strategies is thereforeindispensable.

Experiences with shortening treatment duration in hepatitis C virus type 1

infection

Treatment guidelines recommend that patients infected with HCV geno-type 1 should be treated for 48 weeks with the combination of peg-IFN plusribavirin [13,14]. The recommendation of this fixed treatment duration isbased on the outcome of a large randomized international trial publishedin 2004 by Hadziyannis and colleagues [6], in which patients were

Table 1

Overview of the predicted long-term outcome in hepatitis C virus type 1–infected patients treated for 48 weeks with pegylated interferon alfa plus ribavirin

combination therapy according to early virologic response patterns

Virologic response pattern

Frequency of response

(approximate % of patients) Definitions

Predicted long-term outcomePossible consequences to

improve outcomeSVR Relapse

Rapid virologic response 20% HCV RNA !50 IU/mL at

week 4

O90% !10% ‘‘Overtreatment’’ (shorten

treatment duration)

Early virologic response 40% HCV RNA O50 IU/mL at

week 4 but !50 IU/mL at

week 12

70%–80% !15% None, ‘‘adequate therapy’’

Slow virologic response 20% HCV RNA first !50 IU/mL

at week 24

!30% O70% ‘‘Undertreatment’’ (extending

treatment duration)

Nonresponse 20% !2-log10 HCV RNA decline

at week 12 or O50 IU/mL

at week 24

!5% d Treatment intensification

(dosage and duration)

510

BERG

0

1

2

3

4

5

6

7

Detection limit

SVR

Relapse

Time

HC

V R

NA

(log 1

0 IU

/mL)

Individually different on-treatmentHCV-RNA negative interval

0

1

2

3

4

5

6

7

Detection limit (standard assay)

SVR

Relapse

Time

HC

V R

NA

(log 1

0 IU

/mL)

Detection limit (real-time PCR or TMA)

B

A

Peg-IFNa + Ribavirin


Fig. 2. Current hypotheses for the reasons why patients with an end-of-treatment virologic re-

sponse 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 ribavirinat a dosage of 800 or 1000/1200 mg/d. SVR rates ranged from 29% to52% across the four treatment groups, and the highest SVR rates wereachieved with a 48-week treatment regimen plus ribavirin at a dosage of1000/1200 mg/d.

6

2027

37

0

20

40

60

80

100

Week 12 Week 20 Week 24 Week 48

Perc

enta

ge o

f Apm

licor

-neg

ativ

esa

mpl

es w

ith d

etec

tabl

e H

CV

RN

Aby

TM

A

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 (Deerfield, 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 long-

term treatment against cirrhosis (HALT-C) trial. Hepatology 2006;44:360–7.)

512 BERG

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 48weeks in patients who had low viremia (!800,000 IU/mL) yielded onlya moderate effect on SVR rates (52% versus 65%). These data indicatethat a subgroup of patients defined by pretreatment HCV RNA levels of

1

2

3

4

5

6

7

0 12 24 36 48Weeks

Detection limit < 50 IU/mL

HCV RNAdecline

> 2 log10

HCV RNA „negative“bystandard assay

+ + +

TaqMan-PCR positive (> 10 IU/mL)


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.


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 findings, Zeuzem and colleagues [15] performed a study toexamine whether patients who have HCV genotype 1 and low baseline HCVRNA levels (defined as !600,000 IU/mL) may require just 24 weeks of treat-ment with peg-IFNa plus ribavirin. Two hundred thirty-five treatment-naivepatients infected with HCV genotype 1 were treated for 24 weeks with peg-IFNa-2b at a dosage of 1.5 mg/kg/wk and weight-based ribavirin (800–1400mg/d). A comparable group of patientswho had type 1 infection and lowbase-line viremia who had been treated for 48 weeks within the Manns’ trial [3]served as a historical control. The end-of-treatment virologic response rateswere comparable between both studies (ie, 80% versus 74%). SVR rateswere significantly different (50% versus 71%), however, because of higher re-lapse 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 getsound information concerning tailoring of treatment duration.

These investigators then re-evaluated their findings and found that a sub-group of patients expressing a rapid virologic response (RVR) already atweek 4 (defined as HCV RNA levels !29 IU/mL by real-time PCR) hadan 89% chance to achieve an SVR even after only 24 weeks of combinationtherapy, which was comparable to the SVR rates observed in patients withan RVR treated for 48 weeks (85% in historical control group with RVRs).

Jensen and colleagues [16] aimed to explore the prognostic factors for anRVR (HCV RNA !50 IU/mL at week 4) and an SVR in type 1–infectedpatients treated in the study by Hadziyannis and colleagues [6] for 24 weekswith peg-IFNa-2a at a dose of 180 mg plus ribavirin at a dosage of 800 or1000 to 1200 mg/d by stepwise multiple logistic regression analysis. Theirfindings confirmed 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 demonstratesthat HCV RNA level was the only independent predictor of RVR.

These findings could also be confirmed in a prospective study by Ferenciand colleagues [17], in which type 1–infected patients and a small proportionof type 4–infected patients with an RVR (!50 IU/mL at week 4) weretreated 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 (de-fined as O600,000 IU/mL) achieved SVR rates lower as compared withthose with a low viral load of less than 600,000 IU/mL (74% versus93%). Stage of fibrosis also influenced SVR rates in patients defined as‘‘super-responders’’ (SVR rates were 90% with stage F0–F2 versus 79%with stage F3–F4). Also of interest are these investigators’ observationsthat frequency of relapse rates could be determined according to the assaysapplied to determine HCV RNA. In the case of a negative real-time PCRHCV RNA at week 4, the relapse rate was 10% as compared with relapserates of 30% in patients still positive with the sensitive assay at week 4.

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

End-of-treatment virologic

response rates (HCV

RNA !50 IU/mL) SVR rates

Patients with

RVR

Patients without

RVR

Patients with

RVR

Patients without

RVR

24 weeks; RBV, 800 mg 94% 63% 89% 16%

24 weeks; RBV, 1000–

1200 mg

97% 70% 88% 23%

48 weeks; RBV, 800 mg 73% 56% 73% 35%

24 weeks, 1000–1200 mg

RBV

93% 63% 91% 44%

RVR is defined by HCV RNA !50 IU/mL at week 4.

Abbreviation: RBV, ribavirin.

Data from Jensen DM, Morgan TR, Marcellin P, et al. Early identification of HCV geno-

type 1 patients responding to 24 weeks peginterferon alpha-2a (40 kd)/ribavirin therapy. Hep-

atology 2006;43:954–60.

514 BERG

Between February 2001 and November 2003, the Dynamically Individu-alized Treatment of Hepatitis C Infection and Correlates of Viral/Hostdynamics (DITTO-HCV) study group evaluated a dynamically individual-ized treatment schedule according to the early virologic response (comparedwith a standard-of-care peg-IFNa plus ribavirin combination therapy for48 weeks) [18]. Patients categorized as having an RVR (definition: totallog10 decline of HCV RNA during the first 4 weeks R2 and a second phasedecline R0.09 per day) were randomized to 24 or 48 weeks of treatment.SVR rates in HCV type 1–infected patients with an RVR who were treatedfor only 24 weeks were substantial (65%) but were lower than in thosetreated for 48 weeks (83%). No significant difference, however, wasobserved when patients with a low viral load (ie, !800,000 IU/mL) wereconsidered, in whom SVR rates were nearly identical regardless of whetherthey 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 fromGermany,HCVtype1–infectedpatients received an individualized treatment duration from 18 to 48 weeks tai-lored according to early viral kinetics. Patients with a low viral load (!800,000IU/mLandRVRdefined as!5.3 IU/mLbymeans ofTMAassay) achieved anSVRof 95%after the 18-week treatment duration, and these findingswere sim-ilar to the comparable control group treated for 48 weeks (94%SVR rate) [19].

Definition of a new hepatitis C virus RNA cutoff to predict sustained

virologic response

The determination of viral load has turned out to be of such importanceto predict SVR that a more refined definition of the cutoff level indicating


low versus high viral load is desired so as to tailor treatment duration moreaccurately. In the past, patients have been classified as having a ‘‘high’’ or‘‘low’’ viral load using a cutoff of 2 � 106 copies/mL (corresponding to600,000–800,000 IU/mL, depending on the assay). This was defined in theera of conventional IFNs, however. Several retrospective studies havenow been undertaken with the aim of determining the most effective cutoffto differentiate between high and low viral loads based on the probability ofan SVR with peg-IFNa plus ribavirin therapy [20–22]. Using the logistic re-gression model taking into account discrete (eg, gender, race, cirrhosis sta-tus) and continuous (eg, age, weight, baseline viral load, pretreatmentALT quotient) variables, HCV RNA turned out to be a strong independentpredictor of SVR, but this effect was not linear, indicating that at greaterthan a cutoff of 5.6 log10 IU/mL, the contribution of viral load in predictingan SVR was minimal. Thus, a baseline HCV RNA level of approximately400,000 IU/mL was found to be optimal for use as a cutoff for the proba-bility of achieving an SVR in patients who had type 1 HCV treated for 48weeks. Less than the limit of 400,000 IU/mL, there is a nearly linear corre-lation between the amount of HCV RNA and the chance to establish anSVR, whereas HCV RNA levels greater than 400,000 IU/mL showed no sig-nificant effect on the SVR rates [20]. The reason for the finding that a viralload greater than 400,000 IU/mL had no significant effect on SVR is farfrom clear. One might speculate, however, that at greater than this level,the chance to achieve complete HCV RNA elimination from serum is re-duced and many patients have minimal residual hepatitis C viremia.

Nevertheless, it still remains to be seen whether the now established cutoffbased on the standard PCR test holds true when the real-time PCR assaysare 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 weekshas significantly increased the SVR rate in patients who have HCV genotype1. Thus, the standard duration of therapy for patients who have chronicHCV is 48 weeks. The ability to achieve an SVR depends on the time atwhich HCV RNA becomes undetectable, however, and patients who havea slow virologic response have an increased risk for relapse after treatmenthas been discontinued, even when HCV RNA was undetectable at the end ofthe 48-week treatment period [8]. Obviously, the HCV RNA-negative inter-val was too short to clear HCV from the host tissue permanently. Extensionof treatment duration may be one possibility for improving SVR rates byreducing relapse rates in these patients [23]. This concept is, however, con-fronted with some limitations, considering the troublesome side effects asso-ciated with this combination therapy, which, as a consequence, may beresponsible for an increasing number of dropouts when treatment duration

516 BERG

is extended. In this situation, the intention-to-treat analysis may not be op-timal to demonstrate the potential benefit of extending treatment duration,because the higher withdrawal rates (when extending treatment duration)and higher relapse rates (when shortening treatment duration) may neutral-ize each other.

In two randomized and controlled multicenter studies from Germany andSpain, researchers analyzed whether the extension of treatment duration be-yond 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 treat-ment groups with active treatment for 48 weeks (group A, n ¼ 230) or 72weeks (group B, n ¼ 225) [24]. All patients received peg-IFNa-2a at a dosageof 180 mg/wk plus ribavirin at a dosage of 800 mg/d. After the end of treat-ment, patients from both groups were followed up for a further 24 weeks.The frequency of side effects was similar in both groups, but the on-treat-ment discontinuation rate between groups varied greatly (24% in group Aas compared with 41% in group B). The intention-to treat analysis revealedno major differences in SVR rates between the two groups (53% in group Aand 54% in group B). From a per-protocol analysis, however, by includingonly patients who received at least 80% of the peg-IFNa and ribavirin doseand who completed the planned (�4 weeks) treatment and follow-up period,a 10% difference in SVR rate in favor of group B became evident (68% ingroup A versus 78% in group B). This indicates that a subgroup of patientsmay profit from the 72-week regimen. It could be shown that patients whostill were HCV RNA-positive at week 12 had significantly higher SVR rateswhen treated for 72 weeks instead of 48 weeks (29% versus 17%). Relapserates were significantly higher in slow virologic responders, defined as be-coming HCV RNA undetectable for the first time at week 24 when treatedfor only 48 weeks instead of 72 weeks (relapse rate of 64% versus 40% ingroups A and B). In contrast, patients with an early virologic response inwhom HCV RNA levels were less than 50 IU/mL at weeks 4 and 12 had ex-cellent SVR rates, ranging between 76% and 84% at the end of the follow-up period independent of the treatment period.

In the study from Spain, a total of 510 HCV-infected patients (also in-cluding genotypes 2 and 3) were treated with peg-IFNa-2a at a rate of180 mg/wk plus ribavirin at a dose of 800 mg [25]. Only patients who haddetectable HCV RNA at week 4 were randomized to complete 48 (groupA, n ¼ 165) or 72 (group B, n ¼ 161) weeks of treatment, however. Theend-of treatment response rate was similar in both groups (61%). TheSVR rates were significantly higher in the 72-week group as comparedwith the 48-week group (45% versus 32%), however, and it also became ev-ident that the 72- week–treated patients demonstrated significantly lower re-lapse rates (13% versus 48% in the 48-week group). Analyzing only patientswith HCV genotype 1, SVR rates were 28% and 44% in group A (n ¼ 149)and group B (n ¼ 142), respectively.


Also, in two other studies by Perlman and colleagues [26] and Ferenciand colleagues [27] analyzing patients who had HCV genotype 1 witha slow virologic response, it became clear that extending the treatment du-ration with peg-IFNa and weight-based ribavirin from 48 weeks to 72 weekssignificantly improved SVR rates by reducing relapse rates. In Fig. 5, the ex-pected SVR rates are presented in those patients who had a slow virologicresponse 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 pre-dict whether patients may achieve an SVR or experience relapse. It was con-cluded that HCV type 1–infected patients require continuous absence ofdetectable of HCV RNA in serum for 36 weeks to attain 90% probabilitiesof an SVR (ie, relapse rate of 10%). These data confirm the previously men-tioned findings indicating that patients with an early virologic response atweek 12 hardly experienced relapse independent of treatment duration (ie,48 or 72 weeks), with an HCV RNA-negative interval on treatment of 36to 60 weeks. Late responders who first became HCV RNA-negative atweek 24 and were treated for 72 weeks (ie, who had undetectable HCVRNA 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 findingthat clearly contradicts the proposed model by Drusano and Preston [28].Obviously, the HCV RNA-negative phase required to prevent a relapse

182628

38

58

44

0

20

40

60

80

100

Berg et al. Pearlman et al.

SVR

Rat

e %

Sanchez-Tapias et al.

Fig. 5. Summary of three studies evaluating 72-week treatment duration inHCV 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 pa-

tients 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 800–1400 mg RBV/d.

518 BERG

must be calculated in a more exponential way and seems to depend on howearly 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 potentialcandidates for shortening treatment duration from 48 to 24 weeks. Around10% to 15% of all HCV type 1–infected patients reach these criteria. Theoptimal cutoff to define 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 sen-sitive real-time PCR assays or TMA tests at week 4, however, levels of lessthan 600,000 IU/mL seem to be appropriate (more conservative physiciansmay even choose 400,000 IU/mL). Because hepatitis C viremia may fluctu-ate in the natural course of the disease, however, it is desirable to have atleast two measurements of HCV RNA before starting therapy to confirmlow-level replication over time. To exclude to the best possible level the pres-ence of minimal residual viremia at week 4, which clearly increases the likeli-hood of experiencing a relapse, the most sensitive HCV RNA assays shouldbe used when shortening treatment duration is considered. Furthermore,

W 4 W 24

HCV RNA negativeinterval 20 weeks

W 48W 12

HCV RNA negativeinterval 36 weeks

SVR 70-80%Relapse rate ~ 20%

W 72W 24

HCV RNA negative interval 48 weeks

SVR > 90% (if < 800.000 IU/mL)Relapse rate < 10%

SVR 40-60%Relapse ~ 30%

detection limit

detection limit

detection limit

BL

BL

BL

Treatment period

Treatment period

Treatment period

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 vi-

rologic 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.


patients who have advanced fibrosis or established cirrhosis, or those whoneed a marked dose reduction within the first 24 weeks of treatment, shouldat present be excluded from abbreviated treatment regimens because of in-sufficient data for those cohorts.

In contrast, there is now emerging evidence that patients who have a slowvirologic response not reaching an undetectable HCV RNA level beforeweek 24 who are exposed to a 48-week standard treatment period runa high risk for relapse (reaching up to 70%). Extension of the treatment pe-riod up to 72 weeks can help to reduce relapse rates in these patients. In thiscontext, it should be pointed out that the thus far generally accepted stop-ping rule for patients with a less than 2-log10 decline of HCV RNA withinthe initial 12 weeks of therapy should be reconsidered, because the high neg-ative predictive value of this stopping rule of 98% to 100% has so far onlybeen established for the 48-week treatment duration. Therefore, this cutoffmay 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 geno-type 2 or 3 infection can be treated for 24 weeks with the combination ofpeg-IFNa plus ribavirin at a dosage of 800 mg/d [13,14]. This recommenda-tion is based on the outcome of a large randomized international trial inwhich patients were randomized to treatment for 24 or 48 weeks with peg-IFNa-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 inthis trial, with no significant differences between any combination of treat-ment duration and ribavirin dose.

In Fig. 7, the SVR rates and end-of-treatment response rates of the fourgroups are compared. Treatment duration up to 24 weeks was associatedwith higher end-of-treatment response rates than observed in patientstreated 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 insteadof 48 weeks (Fig. 8). More patients randomized to the 48-week group with-drew from treatment than in the abbreviated treatment group. Thus, thehigher relapse rates in the 24-week treatment groups and the higher dropoutrates in the 48-week groups tend to cancel each other out and create the ap-pearance that the outcomes are similar with the two strategies.

Based on these findings, it can again be deduced that the optimal ther-apeutic regimen still remains unsettled and that there are certainly also pa-tients who have HCV genotypes 2 and 3 who do not achieve an SVR after24 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 fibrosis, are associated with the risk forexperiencing relapse after fixed standard combination therapy given for

Viro

logi

cal r

espo

nse

(% H

CV

RN

A <5

0 IU

/mL)

24 weeks 48 weeks

RBV800 mg/day

RBV1000/1200 mg/day

RBV1000/1200 mg/day

RBV800 mg/day

84% 81% 79% 80%85%

82%90%

94%

0

20

40

60

80

100

n= 96 96 144 144 99 99 153 153

Fig. 7. End-of-treatment (EOT) response (black bars) and SVR rates (white bars) in HCV type

2– and type 3–infected patients treated for 24 or 48 weeks with low (800 mg/d) or weight-based

(1000–1200 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 ri-

bavirin dose. Ann Intern Med 2004;40:346–55.)

520 BERG

24 weeks [29]. For instance, relapse rates were nearly three times higher intype 3–infected patients with a high viral load (O600,000 IU/mL) ascompared with those with low viremia (23% versus 8%) after a 24-weekcourse of peg-IFNa-2b at a dose of 1.5 mg plus weight-based ribavirin(800–1400 mg).

Also in type 2 and 3 infection, viral kinetics (ie, time to HCV RNA neg-ative status) are of increasing importance to optimize antiviral treatment in-dividually. Compared with type 1 infection, viral decline is more rapid intype 2 and 3 infection [30]. Because most patients achieve an RVR at treat-ment week 4, this explains, analogous to the situation in type 1 infection,why most patients are sufficiently treated with a 24-week treatment course.In Table 3, it is outlined how viral kinetics influence the expected SVR ratesin patients who have genotypes 2 and 3.

Experiences with shortening treatment duration in hepatitis C virus type 2

and 3 infection

The finding that maximal SVR rates could be obtained with 24 weeks oftreatment has spurred researchers to investigate even shorter durations oftreatment for patients infected with HCV genotype 2 or 3. To date, five trials(four national investigator-initiated studies [31–34] and one large interna-tional F. Hoffmann-La Roche Ltd. (Basel, Switzerland)–sponsored study,

Relap

se

(%

)

10% 10%

4%

6%

0

5

10

15

20

25

24 weeks 48 weeks

RBV

800 mg/day

RBV

1000/1200 mg/day

RBV

800 mg/day

RBV

1000/1200 mg/day

RBV

800 mg/day

RBV

1000/1200 mg/day

RBV

800 mg/day

RBV

1000/1200 mg/day

13% 13%

3%

5%

24 weeks 48 weeks

Relap

se (%

)

0

5

10

15

20

25

A

B

Fig. 8. (A) Relapse rates in HCV type 2– and type 3–infected patients treated for 24 or 48 weeks

with low (800 mg/d) or weight-based (1000–1200 mg/d) ribavirin (RBV). Peg-IFNa-2a at a dos-

age 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 ran-

domized study of treatment duration and ribavirin dose. Ann Intern Med 2004;40:346–55.)


the ACCELERATE study [35]) have examined abbreviated 12- to 16-weektreatment regimens for genotypes 2 and 3. The various trials differ greatlyin study design, treatment regimen (especially ribavirin dosage), and patientcharacteristics with respect to the percentage of patients who have type 2 be-ing included; thus, great care is necessary when examining the results andespecially when attempting to compare results of studies or generalize thefindings to a broad population (Table 4). In three trials, treatment durationwas determined on the basis of RVR status at week 4 [31–33]. In contrast, twostudies randomized patients at baseline to abbreviated treatment or the stan-dard 24-week duration [34,35].

Table 3

Overview of the predicted long-term outcome in hepatitis C virus type 2– and type 3–infected

patients treated for 24 weeks with pegylated interferon alfa plus ribavirin combination therapy

according to early virologic response patterns

Virologic

response

pattern

Frequency of

response

(approximate) Definitions

Predicted long-term

outcomePossible

consequencesSVR Relapse

RVR O65% HCV RNA !50

IU/mL at week 4

R85% !10% ‘‘Overtreatment’’

(shorter duration

possible)

No RVR 30% HCV RNA O50

IU/mL at week 4

w50%

(36%–77%)

w50% ‘‘Undertreatment’’

(extending

treatment

duration)

No early

virologic

response

!5% HCV RNA O50

IU/mL at week

12

w5% Treatment

intensification

(dosage and

duration)

Data from Refs. [31–35].

522 BERG

Table 5 summarizes the principal findings of these four investigator-initiated 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. Differences from the 24-weektreatment period were marginal and statistically not significant, but overallrelapse rates were higher. In contrast, patients without an RVR had signif-icantly lower SVR rates even when treated for 24 weeks (36%–77%).

The largest trial to examine the merits of abbreviated therapy in patientswho have HCV genotype 2 or 3 infection was the ACCELERATE study,which randomized 1469 patients at baseline to 16 weeks or 24 weeks of treat-ment with peg-IFNa-2a at a dosage of 180 mg/wk plus ribavirin at a dosageof 800 mg/d [35].

In the intention-to-treat analysis, SVR rates were 70% versus 62% for 24weeks versus 16 weeks, respectively (P!.001). These findings were obtaineddespite a higher rate of withdrawal in patients randomized to 24 weeks com-pared with 16 weeks of treatment.

The findings demonstrate conclusively that it is not appropriate to recom-mend abbreviated treatment as a general strategy for patients who have ge-notype 2 or 3 infection. Nevertheless, closer inspection of data confirm thatRVR is a powerful predictor of outcomes in patients who have HCV geno-type 2 or 3 and also sheds light on the appropriateness of assigning patientsto abbreviated treatment based on their RVR status. Two thirds (67%) ofall treated patients achieved an RVR. The SVR rates in these patients beingtreated for 24 weeks were higher (90%) than in patients treated only for 16weeks (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

Study

No. patients

treated

% HCV

genotype 2

Treatment

duration, weeks

Stratification/

randomization

according to

week 4 response

Treatment

regimen

RBV dosage,

mg/d

Definition of

RVR, serum

HCV RNA level,

IU/mL

Randomized trials

Shiffman et al

[35]

1463 50% 16 versus 24 No Peg-IFNa-2a,

180 mg/wk þRBV

800 !50

Yu et al [34] 150 100% 16 versus 24 No Peg-IFNa-2a,

180 mg/wk þRBV

1000/1200 !50

von Wagner

et al [33]

153 27% 16 versus 24 Yes Peg-IFNa-2a,

180 mg/wk þRBV

800–1200 !600

Mangia

et al [32]

283 75% 12 versus 24 Yes Peg-IFNa-2b,

1.0 mg/kg/wk

þ RBV

1000/1200 !50

Nonrandomized

Dalgard

et al [31]

122 24% 14 versus 24 Yes Peg-IFNa-2b,

1.5 mg/kg/wk

þ RBV

800–1400 !50

Abbreviation: RBV, ribavirin.

523

TAIL

ORED

TREATMENTFOR

HEPATIT

ISC

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

Treatment

group

End-of-treatment

response SVR rate Relapse rate

Mangia et al [32] 12 weeks in RVR pts

(n ¼ 133)

95% 85% 10%

24 weeks regardless of

RVR status (n ¼ 70)

79% 76% 4%

24 weeks, no RVR

(n ¼ 80)

68% 64% 6%

Von Wagner et al [33] 16 weeks in RVR pts

(n ¼ 71)

94% 82% 13%

24 weeks in RVR pts

(n ¼ 71)

85% 80% 5%

24 weeks, no RVR

(n ¼ 11)

72% 36% n.a.

Dalgard et al [31] 14 weeks in RVR pts

(n ¼ 95)

n.a. 90% 10%

24 weeks, no RVR

(n ¼ 27)

n.a. 56% 26%

Yu et al [34] 16 weeks (n ¼ 50) 100% 94% 6%

24 weeks (n ¼ 100) 98% 95% 3%

16 weeks in RVR pts n.a. 100% d

24 weeks in RVR pts n.a. 98% n.a.

16 weeks, no RVR n.a. 57% 43%

24 weeks, no RVR n.a. 77% 9%

Shiffmann et al [35] 16 weeks (n ¼ 733) 89% 62% 31%

24 weeks (n ¼ 732) 82% 70% 18%

16 weeks in RVR pts

(n ¼ 489)

n.a. 79% n.a.

24 weeks in RVR pts

(n ¼ 470)

n.a. 85% n.a.

16 weeks, no RVR

(n ¼ 220)

n.a. 26% n.a.

24 weeks, no RVR

(n ¼ 247)

n.a. 45% n.a.

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

524 BERG

lower (49% in patients treated for 24 weeks versus 27% in patients treatedfor 16 weeks).

When baseline HCV RNA levels are superimposed on an RVR, it is ap-parent that these two factors exert a powerful influence on SVR rates. In pa-tients with low HCV RNA levels, defined as 400,000 IU/mL or less, the SVRrates were 95% in those treated for 24 weeks and 90% in those treated for 16weeks [35]. In contrast, SVR rates were 88% in patients with a baselineHCV RNA level greater than 800,000 IU/mL who were treated for 24 weeksand 78% in patients treated for 16 weeks.

67

26

67

13

0

20

40

60

Perc

enta

ge o

f pat

ient

s

SVR

65

7680

24

4

100

n= 21 34 30 37 19 29 23 27Relapse

Fig. 9. SVR and relapse rates in HCV type 2– and type 3–infected patients without an RVR

treated for 24 or 48 weeks with low (800 mg/d) or weight-based (1000–1200 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 dos-

age 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;40:346–55.)


Extending treatment duration in those who are slow to respond

There are data from randomized prospective trials that show prolongingthe duration of treatment increases SVR rates in genotype 1–infected pa-tients who are slow to respond [24–27]. This raises the question of whetherintensification of the therapeutic regimen could increase SVR rates in geno-type 2– or 3–infected patients who do not achieve an RVR. There are nodata from prospective trials available on this topic. This question hasbeen addressed through a retrospective analysis of data [36] from the trialby Hadziyannis and colleagues [6]. In patients who did not have RVRs,end-of-treatment response rates ranged from 65% to 76% across the fourtreatment groups (Fig. 9). Consistent with results observed in genotype 1-in-fected patients, relapse rates decreased in inverse proportion to the intensityof treatment and were lowest in those treated for 48 weeks with peg-IFNa-2a plus ribavirin at a dosage of 1000/1200 mg/d (4%) and highest in thosetreated for 24 weeks with ribavirin at a dosage of 800 mg/d (26%) [36]. Al-though these results must be confirmed in a prospective study, they suggestthat intensification of therapy may be effective in increasing SVR rates ingenotype 2– or 3–infected 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 ge-notype 1 and 4 are ‘‘difficult to cure’’ because of viral kinetic evidence to the

526 BERG

contrary. Conversely, it is no longer appropriate to think of all patients whohave HCV genotype 2 or 3 as ‘‘easy to cure.’’ Although genotype is an im-portant driver of response and is useful in designing the initial treatmentplan, it is clear that once treatment is initiated, RVR is the most importantand powerful predictor of SVR.

In genotype 2– or 3–infected patients, response-guided therapy usingmeasurement of the virologic response after 4 weeks of combination therapyallows the treatment regimen to be tailored to the individual.

At present, available data from five studies, including a total of morethan 2000 patients, demonstrate that genotype 2– or 3–infected patientswith an RVR can achieve high SVR rates with abbreviated therapy. Thereis a trade-off between slightly higher relapse rates in patients treated withabbreviated regimens and slightly higher withdrawal rates in those treatedfor the standard duration. The highest SVR rates and lowest relapse ratesare obtained in patients with an RVR who have low baseline HCV RNAlevels (%400,000 IU/mL) even when treated for only 16 weeks, and suchtherapy may be a reasonable option for these patients. In contrast, patientswithout an RVR run a high risk for relapse even when treated for 24 weeks.From retrospective analysis, however, there is evidence that prolongation oftreatment duration can reduce relapse rates.

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ment of chronic hepatitis C in previously untreated patients infected with HCV genotype

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[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):2609–17.

[33] vonWagnerM, HuberM, Berg T, et al. Randomized multicenter study comparing 16 vs. 24

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cally infected with HCV genotype 2 or 3. Gastroenterology 2005;129(2):522–7.

[34] YuML, Dai CY, Huang JF, et al. A randomised study of peginterferon and ribavirin for 16

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[35] ShiffmanML, 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):124–34.

[36] Willems B, Hadziyannis SJ, Morgan TR, et al. Should treatment with peginterferon plus ri-

bavirin be intensified in patients with HCV genotype 2/3 without a rapid virologic response?

J Hepatol 2007;46(Suppl 1):S6, [abstract no. 8].

Novel Hepatitis C Drugsin 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 curedwith the current standard treatment consisting of pegylated interferon-alfaand ribavirin. For those patients who have chronic hepatitis C and didnot respond to interferon-alfa–based antiviral therapy, there is currentlyno approved treatment option available. ‘‘Difficult-to-cure’’ patient popula-tions 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 develop-ment of a subgenomic replicon system [1] and, more recently, of a cell cul-ture infectious HCV clone [2,3] enables the development of a specificallytargeted antiviral therapy for hepatitis C (STAT-C). Many HCV-specificcompounds are under investigation in preclinical and clinical trials. It is an-ticipated that HCV-specific inhibitors can improve treatment opportunitiesfor patients who have chronic hepatitis C, especially in patients who are‘‘difficult to cure.’’

Clin Liver Dis 12 (2008) 529–555

Therapeutic approaches for specifically 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) flanking a single open reading frame en-coding for a polyprotein of approximately 3100 amino acids. Translation ofthe HCV polyprotein is initiated by an internal ribosome entry site (IRES)

* Corresponding author.

E-mail address: [email protected] (S. Zeuzem).



530 KRONENBERGER et al

located in the 50 UTR of the HCV genome (Fig. 1). The HCV polyprotein isco- and posttranslationally processed by host- and virally encoded proteasesinto structural (core, envelope E1, E2, and p7) and nonstructural (NS2,NS3, NS4A, NS4B, NS5A, and NS5B) proteins (Fig. 2). The functions ofthe HCV proteins and therapeutic approaches for specifically targeted anti-viral therapy are listed in Table 1.

The core protein binds and packages the viral RNA genome and formsthe viral nucleocapsid [4,5]. In addition, the core protein interacts with theenvelope protein E1. In infected cells, the core protein is found on mem-branes of the endoplasmic reticulum, in membranous webs, and on the sur-face of lipid droplets. The core protein interacts with various cellularproteins. The association with lipid droplets may have a role during viralreplication or virion morphogenesis. The deletion of the C-terminal hydro-phobic region of the core protein causes translocation to the nucleus. Be-cause of the various interactions with cellular proteins and the consecutiveeffects on signal transduction pathways and transcription, the core proteinhas been implicated in hepatocarcinogenesis and the alteration of apoptosis.Blocking of the core protein could alter viral morphogenesis, alter viral rep-lication, and reverse liver steatosis. To date, small-molecule inhibitors forthe core protein are not available.

The envelope proteins E1 and E2 are essential for host cell entry and arepotential targets of antiviral therapy. Blocking of HCV entry could beachieved by blocking the envelope proteins or by blocking cellular receptorsof HCV, including the tetraspanin CD81, the scavenger receptor class B type

1. binding and

internalization

2. release and

uncoating

3. IRES mediated

translation

4. polyprotein

processing

(+)RNA

(-)RNA

Endoplasmic reticulum5. membraneous

web formation

6. replication

(+)RNA7. assembly

and release

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.

C E1 E2 p7 NS2 NS3 NS4B NS5A NS5B

C E1 E2 p7 NS2 NS3 NS4A NS4B NS5A NS5B

Core EnvelopeGlycoproteins

CysteinProtease

SerineProtease

Helicase Mebr. web

IFN-resistance?

RNA-dependentRNA-Polymerase

Serine Proteaseco-factor

Ion Channel

NS3/4ASerine Protease

NS2/3Cysteine Protease

co- and posttranslationalpolyprotein processing

structuralproteins

non-structuralproteins

HCV polyprotein

NS4A

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.

531NOVEL HEPATITIS C DRUGS IN CURRENT TRIALS

I (SR-BI), heparan sulfate (HS), dentritic cell and liver and lymph node-spe-cific ICAM-3 grabbing non-integrin (DC-SIGN/L-SIGN), the low-densitylipoprotein (LDL) receptor, and claudin-1 [6–8]. Testing of entry inhibitorsis still at the preclinical stage.

P7 is a small hydrophobic structural protein [9,10] that homo-oligomer-izes into a circular hexamer and is believed to form an ion channel in itscenter [11]. The role of p7 in HCV replication is still unclear. In chimpan-zees, p7 has been shown to be important for infectivity. An antiviral com-pound that has been shown in one study to block the p7 ion channel invitro is amantadine [12]; however, the results on antiviral activity of aman-tadine in patients who have chronic hepatitis C are conflicting. A recentstudy of the antiviral effect of amantadine on cell culture infectiousHCV particles showed that across a spectrum of HCV isolates and geno-types, amantadine affected neither RNA replication nor the release or in-fectivity of HCV particles [13]. Furthermore, the same study showed thatthe p7 ion channel activity was not affected by amantadine. Overall, therecent results demonstrate that amantadine is not an HCV-selectiveantiviral.

The NS proteins include enzymes necessary for protein maturation (NS2/3 cysteine protease and NS3/4A serine protease) and viral replication (NS3helicase/nucleoside triphosphatase and NS5B RNA-dependent RNA-poly-merase). The NS2/3 protease mediates a single cleavage at the NS2/NS3junction and releases the mature NS3 protease. NS4A enhances the proteo-lytic activity of the NS3 serine protease domain (Fig. 3). The NS3/4Aprotease cleaves at four downstream sites in the polyprotein to generatethe N-termini of the NS4A, NS4B, NS5A, and NS5B proteins. The structure

Table 1

Hepatitis C virus structural and nonstructural proteins and potential targets for specifically tar-

geted antiviral therapy for hepatitis C virus

HCV

protein Function Therapeutic approach

50-UTR Contains the IRES necessary for

initiation of translation of the

HCV polyprotein

Inhibition of IRES blocks initiation of

translation; inhibitors are antisense

oligonucleotides, ribozymes, and

small molecules (eg, VGX-410C,

siRNA) in vitro

Core Nucleocapsid, viral morphogenesis,

RNA-binding and replication,

association with lipid droplets

Inhibitionmight influence viral replication

and viral morphogenesis and inhibit

the development of liver steatosis

E1/E2 Envelope, attachment, entry,

interferon-alfa resistance (?)

Blocking of envelope proteins or

corresponding receptors may inhibit

HCV entry and reduce de novo

infection of susceptible cells

p7 Contains an ion channel potentially

involved in viral infection

Blocking may reduce infectivity

NS2 Forms with NS3, a dimeric cysteine

protease, for cleavage of the NS2/3

junction

Inhibition blocks HCV polyprotein

processing

NS3 � NS2/3 cysteine protease

� NS3/4A serine protease

� Part of the viral RNA complex

� RNA helicase

� Nucleotide triphosphatase

� Inhibition of the innate antiviral

response

Inhibition blocks polyprotein procession

and viral replication; furthermore,

blocking may restore the innate

antiviral response; inhibitors

(telaprevir, boceprevir) are in phase 2

clinical trials

NS4A � Cofactor of the NS3/4A serine

protease

� Enhances proteolytic activity of

the NS3 protease

NS4B � Membrane protein localized at the

endoplasmatic reticulum

� Forms a membranous web potentially

harboring the HCV replication

complex

NS5A � Potential effect on interferon

resistance

� Involved in RNA replication

� Modulation of cellular signaling

pathways (apoptosis, cell growth)

Inhibitor in phase 1 trial; inhibition may

overcome interferon resistance and

reduce HCV replication

NS5B �RNA-dependent RNA-polymerase 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.


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 arepromising targets for the development of specific inhibitors.

The NS3/4A serine protease has also been shown to cleave and inactivatethe host proteins (Toll-like receptor domain-containing adapter inducingIFNb [Trif] and Cardif). Both proteins have important roles in the inter-feron response mediated by Toll-like receptor 3 (TLR3) and retinoic-acidinducible gene I (RIG-I), respectively [14,15]. Inactivation of both proteinsblocks the double-stranded (ds) RNA–dependent innate immune responseand indicates that the NS3/4A serine protease may have an importantrole in evasion of the innate immune response.

Furthermore, it has been shown that NS3 is not only a protease but anintegral part of the viral RNA replication complex and functions as anRNA helicase and a nucleotide triphosphatase (NTPase). Because of themultiple functions, it can be assumed that inhibitors against NS3 are highlyefficient in blocking HCV replication. Several antiviral drugs directedagainst the NS3/4A protease have been developed and are listed in Table 2.

NS4B is a membrane protein with four transmembrane regions localizedat the endoplasmatic reticulum membrane. The function of NS4B is not wellunderstood. NS4B is assumed to form a membranous web that is necessaryfor the HCV replication complex [16]. The functions of NS4B need to be un-derstood better before it can be considered a promising target of antiviraltherapy.

NS5A is a pleiotropic protein with key roles in viral RNA replication andmodulation of the physiology of the host cell. NS5A is mostly known be-cause of its potential effect on interferon-alfa signaling. Furthermore,NS5A has been shown to affect cell growth of target cells and apoptosis.The crystal structure of domain I of NS5A has recently been solved [17],and specific inhibitors are currently being developed.

Table 2

Emerging therapies against hepatitis C virus

Name Producer Study phase

Long-acting interferons

Albinterferon Human Genome Sciences/Novartis Phase 3

Pegamax Maxygen/Roche Preclinical/phase 1

(stopped)

Locteron (BLX-883) Biolex Phase 2

Interferon-omega Intarcia Therapeutics Phase 2

Oral interferon

Belerofon Nautilus Biotech

STAT-C

NS3/4 serine protease inhibitors

Ciluprevir (BILN 2061) Boehringer Ingelheim Stopped

Telaprevir (VX-950) Vertex Phase 2

Boceprevir (SCH 503034) Schering-Plough Phase 2

ITMN-191 InterMune Phase 1

GS9132/ACH-806 Gilead Sciences/Achillion Stopped

NS5B RNA-dependent RNA-polymerase inhibitors

Nucleoside analogues

Valopicitabine (NM283) Idenix/Novartis Stopped

R1626 (prodrug of R1479) Roche Phase 2

R1656/R7128 Pharmasset/Roche Phase 1

XTL-2125 XTL-Biopharmaceuticals Stopped

MK-608 Merck Preclinical

Nonnucleoside polymerase inhibitors

HCV-796 ViroPharma/Wyeth Stopped

BILB 1941 Boehringer Ingelheim Stopped

A-837093 Abbott Preclinical

GS-9190 Gilead Phase 1

NS5A inhibitors

A-831 Arrow Therapeutics/AstraZeneca Phase 1

A-689 Arrow Therapeutics/AstraZeneca Preclinical

Cyclophilin inhibitors

DEBIO-25 Debiopharm Phase 1 in HCV/HIV-

coinfected patients

NIM811 Novartis Preclinical


The NS5B RNA-dependent RNA-polymerase is another promising tar-get for the development of HCV-specific compounds. NS5B reveals the typ-ical polymerase structure, a classic ‘‘right-hand’’ shape of thumb, palm, andfinger domains encircling the active site (Fig. 4) [18]. To date, many inhib-itors against the NS5B RNA-dependent RNA-polymerase have been devel-oped, including nucleoside analog polymerase inhibitors and nonnucleosidepolymerase inhibitors (see Table 2).

The IRES located in the 50-UTR is required for the initiation of HCVpolyprotein translation. The IRES has been targeted by the antisense oligo-nucleotide inhibitor ISIS 14803 and the small-molecule organic drug mife-pristone (VGX 410C). The development of ISIS 14803 was stopped

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 in-

dicated for nucleoside and nonnucleoside compounds.


because of weak and highly variable antiviral activity and substantial in-creases of aminotransferases in a phase 1 trial in patients who had chronichepatitis C [19]. Mifepristone is currently being investigated in a phase 2trial in patients who have chronic hepatitis C, and data are pending [20].

The 30-UTR is necessary for RNA replication and amplification of IRES-dependent protein translation and also seems to be a promising antiviraltarget.

To date, most new antiviral drugs for HCV focus on the NS3/4A serineprotease and on the NS5B RNA-dependent RNA-polymerase. The resultsof clinical trials are presented in the following sections.

Current therapies for hepatitis C virus

Pegylated interferon-alfa and ribavirin are the current standard of carefor treatment of patients who have chronic hepatitis C. Great efforts havebeen made for the individualization of antiviral therapy to reduce adverseevents and to improve sustained virologic response rates. HCV genotypeand early virologic response during treatment are important factors for in-dividualization of antiviral therapy. An extended treatment duration of72 weeks has been shown to reduce relapse rates significantly in patientswho have chronic HCV genotype 1 infection and a slow virologic responsecompared with the standard duration of 48 weeks [21,22]. Conversely, HCVgenotype 1–infected patients with a low baseline HCV RNA concentrationwho become HCV RNA-negative at week 4 may be treated for 24 weekswithout compromising sustained virologic response rates [23].


In patients who have HCV genotype 2 or 3 infection, who have a betterresponse to interferon-alfa than patients infected with HCV genotype 1, thestandard treatment duration is 24 weeks. Several trials investigated whethera shorter treatment duration is possible in patients who have genotype 2 or3 infection without compromising the sustained virologic response. Smallertrials showed that a shorter treatment duration of 12 to 16 weeks is equallyeffective as the standard treatment duration in those patients infected withHCV genotype 2 or 3 who achieve a rapid virologic response after 4 weeksof therapy [24,25]. The large ACCELERATE trial comparing 16 versus24 weeks of treatment in patients who had HCV genotype 2 or 3 infectionshowed that a shorter treatment duration of 16 weeks results in reducedsustained virologic response rates compared with the standard treatmentduration, however [26]. In the ACCELERATE trial, a shorter course oftherapy over 16 weeks has been shown to be as effective as a 24-week coursein those patients who have genotype 2 or 3 infection, have a baseline viralload of 400,000 IU/mL or less, and achieve an early virologic response atweek 4 [26]. In patients who have genotype (2 and) 3 infection withouta rapid virologic response (!50 IU/mL) at week 4, a longer treatment du-ration may be necessary to optimize sustained virologic response rates [27].

The ribavirin dose may also influence the rate of sustained virologic re-sponse. The Weight-based dosing of pegINterferon alfa-2b and Ribavirin(WIN-R) trial investigated pegylated interferon alfa-2b plus weight-based ri-bavirin (800–1400 mg) or flat-dose ribavirin (800 mg) in patients who hadchronic hepatitis C [28]. Sustained virologic response but not end-of-treat-ment rates were significantly higher in patients treated with weight-basedribavirin than in patients treated with flat-dose ribavirin (44.2% versus40.5%). Sustained virologic response rates by intention-to-treat analysiswere 34.0% and 28.9%, respectively, in genotype 1–infected patients. In ge-notype 2– or 3–infected patients, sustained virologic response rates were notsignificantly different (61.8% and 59.5%, respectively), regardless of treat-ment duration [28]. From the current point of view, further individualiza-tion of standard therapy seems unlikely to improve the convenience andoutcome of antiviral therapy markedly. New interferons have been devel-oped, 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 resultsfrom the genetic fusion of interferon-alfa with human albumin. Albinter-feron has a longer half-life than pegylated interferon-alfa.

A recent phase 2b clinical trial investigated antiviral efficacy andtolerability of albinterferon in combination with ribavirin in patientswho had chronic hepatitis C genotype 1 infection and were naive to


interferon-alfa–based treatment regimens [29]. In this trial, the virologic re-sponse rates 12 weeks after treatment were 58% and 55% in patients treatedwith albinterferon, 900 mg and 1200 mg every 2 weeks, plus ribavirin versus58% in patients treated with pegylated interferon-alfa-2a, 180 mg onceweekly, plus ribavirin. The health-related quality-of-life scores were morefavorable in the albinterferon group than in the pegylated interferon-alfa-2a group. Overall, these interim results indicate that albinterferon plusribavirin may offer efficacy comparable to pegylated interferon-alfa-2aplus ribavirin with half of the injections and the potential for less impair-ment of quality of life.

Albinterferon is currently being investigated in two randomized, open-label, active controlled, multicenter, noninferiority phase 3 trials to evalu-ate the efficacy, safety, and impact on health-related quality of life ofalbinterferon in combination with ribavirin versus pegylated interferon-alfa-2a in combination with ribavirin. The ACHIEVE 1 trial is conductedin patients infected with HCV genotype 1 randomized into three treatmentgroups, including two groups with subcutaneously administered albinter-feron once every 2 weeks (900 or 1200 mg), and a control group withpegylated interferon-alfa-2a administered once every week at a dose of180 mg. All patients receive oral ribavirin (1000–1200 mg) concomitantlyand are treated for 48 weeks. The ACHIEVE 2/3 trial is being conductedin patients infected with HCV genotype 3. The ACHIEVE 2/3 trial hasthe same design as the ACHIEVE 1 trial with a shorter treatment dura-tion of 24 weeks and a lower ribavirin dose of 800 mg. The primary effi-cacy end point in both trials is a sustained virologic response, defined asan undetectable HCV RNA level (!10 IU/mL) at weeks 72 and 48,respectively.

Higher doses of albinterferon administered every 4 weeks, in combinationwith ribavirin, are to be explored in separate trials, which are expected tobegin in 2007.

Interferon-omega

Interferon-omega is a type 1 interferon that shares 70% homology ofthe amino-acid sequence to interferon-alfa and is derived from Chinesehamster ovary cells. Interferon-omega, which is fully glycosylated, hasbeen broadly studied in phase 1 and 2 clinical studies. These studieshave demonstrated that interferon-omega is safe and has potent anti-HCV activity. Preliminary results from a phase 2 study comparing inter-feron-omega (25 mg/d) with the combination of interferon-omega andribavirin (1000–1200 mg/d) have shown virologic response rates 12 weeksafter the end of treatment of 6% and 36%, respectively [30]. It is plannedto administer interferon-omega by an implantable osmotic minipump, re-quiring changes every 3 months, which delivers consistent drug levelsthrough the device outlet.


Locteron (BLX-883)

Locteron is a recombinant interferon-alfa that is released by a special bio-degradable polymeric drug delivery system. The polymeric drug delivery sys-tem consists of polyester or polyether copolymers that are degraded byhydrolysis and oxidation and enables linear release of compounds. Locteronis designed to be administered every 2 weeks. A phase 1 clinical study inves-tigating the safety, pharmacokinetics, and pharmacodynamics of locteron inhealthy volunteers was completed [31]. Results of this phase 1 clinical trialshowed that a single dose of locteron was safe and well tolerated. In partic-ular, groups receiving locteron reported fewer, less severe, and shorterlasting flu-like symptoms than those subjects receiving pegylatedinterferon-alfa-2b.

Maxy-alpha (R7025/RO5014583, pegamax)

Maxy-alpha (R7025) is an interferon-alfa variant that has been createdthrough a directed molecular evolution gene shuffling technology to havestronger antiviral activity against the hepatitis C virus and to be more effec-tive in stimulating immune responses than standard interferon-alfa. A pegy-lated form of maxy-alpha has been developed using the same pegylationtechnology as for pegylated interferon-alfa-2a. Preclinical data comparingmaxy-alpha with pegylated interferon-alfa-2a show that maxy-alpha has in-creased antiviral activity in the HeLa encephalomyocarditis virus assay andstronger immune stimulatory activity on T helper 1 (Th1) cytokine induc-tion and dendritic cell maturation than pegylated interferon-alfa-2a [32].A double-blind, dose-escalation, controlled phase 1 study of a single subcu-taneous administration of maxy-alpha has been initiated in healthy volun-teers [32], and data are pending.

Oral interferon (belerofon)

Belerofon is a variant of human interferon-alfa with a single amino-acidreplacement that has been designed to lower the susceptibility of interferon-alfa to proteolytic degradation and to make it longer lasting in serum [33].In animal models, appropriate oral doses have shown that belerofon can beabsorbed from the intestine into the bloodstream and reaches blood levelscomparable to those obtained by subcutaneously injected interferon-alfa[33]. Oral belerofon is formulated as enteric-coated tablets containing thelyophilized 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 ribavirinwith a distinct pharmacologic profile. Taribavirin is preferentially taken


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 efficacy and safety of taribavirin versus ribavirin combined with pegy-lated interferon in patients who have chronic hepatitis C was investigatedin a phase 2 open-label study [34]. Patients were stratified according toHCV genotype and randomized to receive pegylated interferon-alfa-2a ata dosage of 180 mg/wk plus taribavirin at a dosage of 800, 1200 or 1600mg/d or ribavirin at a dosage of 1000 or 1200 mg/d. Patients infectedwith HCV genotype 1, 4, 5, or 6; mixed genotypes; or an indeterminate ge-notype were treated for 48 weeks, and those infected with HCV genotype 2or 3 were treated for 24 weeks. All patients were followed for 24 weeks afterthe end of treatment to determine the rate of sustained virologic response.The rates of sustained virologic response were 23%, 37%, and 29% forthe different taribavirin dose groups, respectively, and 44% for pegylatedinterferon alfa-2a plus ribavirin. Fewer patients on any dose of taribavirinhad severe anemia (hemoglobin !10 g/dL) than on ribavirin (4% versus27%).

Two large-scale randomized phase 3 clinical trials comparing the safetyand efficacy of taribavirin plus pegylated interferon-alfa-2b (VISER-1)and pegylated interferon-alfa-2a (VISER-2) versus pegylated interferon-alfa-2b/2a plus ribavirin, respectively, were recently completed. In both tri-als, patients were stratified according to genotype, baseline viral load, andweight. The VISER-1 trial showed a significantly lower rate of anemiaamong patients treated with taribavirin compared with ribavirin; however,the overall rate of sustained virologic response in the VISER-1 trial waslower 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 dosesof taribavirin in combination with pegylated interferon [34].

Specifically targeted antiviral therapy for hepatitis C virus

Protease inhibitors

Ciluprevir (BILN 2061)Ciluprevir is the first potent and specific inhibitor of the NS3/4A serine

protease [37] that was tested within a randomized, multiple-dose, double-blind, placebo-controlled pilot study in patients who had chronic hepatitisC [38]. In this study, the oral administration of ciluprevir (25–500 mg twiceper day) to patients who had chronic hepatitis C genotype 1 infection for2 days resulted in viral RNA reductions of 2- to 3-log10 copies/mL inmost of the patients. The study provided proof of concept that HCVNS3/4A protease inhibitors are a therapeutic option for patients whohave chronic hepatitis C.


The HCV genotype is currently the most important baseline predictivefactor for virologic response to interferon-alfa–based treatment. The antivi-ral efficacy of ciluprevir was also investigated in patients who had chronicHCV genotype 2 or 3 infection [39]. The antiviral efficacy of ciluprevirwas less pronounced and more variable in HCV genotype 2– or genotype3–infected patients compared with HCV genotype 1–infected patients,showing that treatment response to protease inhibitors may also dependon HCV genotype [38,39].

Safety and tolerability are important issues in the development of new an-tiviral drugs. In rhesus monkeys treated with high doses of ciluprevir for4 weeks, histologic signs of cardiotoxicity were observed. Because of thiscardiotoxicity, the clinical development of ciluprevir was stopped.

The development of resistance against ciluprevir was studied in HCV ge-notype 1b replicon cells [40]. Several mutations in the NS3 protease wereidentified that were associated with resistance against ciluprevir (Table 3).Modeling studies indicate that all mutations are located in close proximityto 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 specific treatment of hepatitis C. Comparedwith ciluprevir, telaprevir exhibits a longer half-life of the bound enzyme in-hibitor complex [41].

The first placebo-controlled double-blind phase 1 study with telaprevirwas started in June 2004. In this study, 34 patients who had chronic geno-type 1 infection were randomized to receive placebo or telaprevir at a dosageof 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 failedprior interferon-based treatment. During treatment with telaprevir, all

Table 3

Mutations in the protease conferring resistance to various protease inhibitors

BILN 2061 Telaprevir Boceprevir ITMN-191

In vitro R155Q

A156V/T

D168V/A/Y

A156S/V/T T54A

A156S/T

V170A

V23A

Q41R

S138T

D168A/V/E

D168V/A156S/V

S489L

In vivo No data V36M/A

T54A

R155K/T

36/155

A156S/V/T

36/156

T54A No data

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


patients showed a decline in viral load of 2 log10 or greater. Twenty-six of28 patients had a decline in viral load of 3 log10 or greater. In the 750-mgdose group, the median reduction of HCV RNA was 4.4 log10 after14 days. In the 450-mg dose group and the 1250-mg dose group, the maxi-mum effect was seen between days 3 and 7 of dosing, followed by an increaseof HCV RNA [42]. The increase of HCV RNA between days 7 and 14 in-dicates the selection of variants with reduced sensitivity to telaprevir [42,43].

Several mutations conferring resistance to telaprevir were detected in thereplicon system and during monotherapy with telaprevir [43]. The develop-ment of resistant variants was investigated in patients during telaprevirmonotherapy [43]. The known in vitro and in vivo mutations conferring re-sistance to telaprevir are listed in Table 3. Mutations that confer low-levelresistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance(A156V/T, 36þ155, 36þ156) to telaprevir were detected and correlated withtelaprevir exposure and virologic response.

Viral fitness generally refers to the relative replication competence ofa virus under defined circumstances. Sarrazin and colleagues [43] generatedan algorithm to calculate viral fitness using the HCV RNA data and se-quence data at the end of treatment and at follow-up. Changes in the fre-quency of mutations after the end of dosing showed an inverse relationbetween in vivo viral fitness and resistance. In the absence of telaprevirselective pressure, most resistant variants were replaced by wild-type viruswithin 3 to 7 months.

The rapid development of resistance during telaprevir monotherapy indi-cates that combination therapy with pegylated interferon-alfa or other directantiviral drugs seems to be necessary to avoid the development of resistance[43]. Subsequently, telaprevir was investigated in combination with pegy-lated interferon-alfa-2a [44]. Treatment-naive patients who had chronic hep-atitis C genotype 1 infection were randomized for treatment with pegylatedinterferon-alfa-2a plus placebo (n ¼ 4), telaprevir monotherapy (n ¼ 8), ora combination of telaprevir and pegylated interferon-alfa-2a (n ¼ 8) for2 weeks. The median changes in HCV RNA level from baseline to day15 were �1.09 log10 IU/mL, �3.99 log10 IU/mL, and �5.49 log10 IU/mLin 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 antiviraleffects of telaprevir in combination with pegylated interferon-alfa-2a. Thetreatment was well tolerated, all patients completed dosing, and no seriousadverse events were reported.

A detailed kinetic analysis of variants in patients treated with telapreviralone or with telaprevir plus pegylated interferon-alfa-2a for 14 days wasperformed [45]. This analysis indicates that the initial antiviral response totelaprevir is attributable to a sharp reduction in wild-type virus, which un-covers preexisting telaprevir-resistant variants. The combination of telapre-vir and pegylated interferon-alfa-2a inhibited wild-type and resistant


variants, indicating that telaprevir-resistant variants are sensitive to pegy-lated interferon-alfa-2a.

Combination therapy with pegylated interferon-alfa-2a plus ribavirin wasoffered to the patients after the initial 14-day dosing period [44,45]. Nineteenof the 20 patients began standard therapy after completing the dosing period(1 patient of the previous telaprevir monotherapy group declined treat-ment). Twelve weeks after starting standard therapy, HCV RNA was unde-tectable in 5 patients of the previous telaprevir group and in all 8 patients ofthe previous telaprevir plus pegylated interferon-alfa-2a group. At 24 weeks,HCV RNA was undetectable in all patients who started standard therapy inthe previous telaprevir monotherapy group (n ¼ 7) and the previous telap-revir plus pegylated interferon-alfa-2a group (n ¼ 8) [44,45].

In consecutive studies, telaprevir is being combined with pegylated inter-feron-alfa-2a and ribavirin [46,47] and is currently being further investigatedin this combination in two phase 2 studies (PROVE-1 and -2). In thePROVE-1 trial (conducted in the United States), treatment-naive patientsinfected with HCV genotype 1 (n ¼ 260) have been randomized into fourgroups:

1. Twelve weeks of therapy with telaprevir (750 mg every 8 hours) in com-bination with pegylated interferon-alfa-2a and ribavirin (total durationof 12 weeks, n ¼ 20)

2. Twelve weeks of therapy with telaprevir (750 mg every 8 hours) in com-bination with pegylated interferon-alfa-2a and ribavirin, followed bypegylated interferon-alfa-2a and ribavirin alone for 12 weeks (totalduration of 24 weeks, n ¼ 80)

3. Telaprevir (750 mg every 8 hours) in combination with pegylated inter-feron-alfa-2a and ribavirin for 12 weeks, followed by pegylated inter-feron-alfa-2a and ribavirin alone for 36 weeks (total duration of48 weeks, n ¼ 80)

4. Control arm with pegylated interferon-alfa-2a and ribavirin for 48 weeks(total duration of 48 weeks, n ¼ 80)

Only patients in the 12- and 24-week treatment arms who achieve a rapidviral response (HCV RNA level !10 IU/mL) by the end of week 4 and whomaintain this status through to week 10 or 20, respectively, are planned tostop all treatment at the 12- or 24-week time point (according to the studyarms) and are to be followed after treatment to evaluate whether theyachieve a sustained virologic response. Patients in these treatment armswithout a rapid virologic response are required to continue on pegylatedinterferon-alfa-2a and ribavirin for a total of 48 weeks, however.

The PROVE-2 study is conducted in Europe (n ¼ 320, n ¼ 80 for eachtreatment arm) and has a similar study design as the PROVE-1 study. Inthe PROVE-2 trial, the 12-week treatment arm with telaprevir plus pegy-lated interferon-alfa-2a plus ribavirin followed by 36 weeks of treatmentwith pegylated interferon-alfa-2a plus ribavirin is replaced by 12 weeks of


treatment with telaprevir plus pegylated interferon-alfa-2a only. Anotherimportant difference is that patients within the PROVE-2 trial can stoptreatment according to study arm assignment independent of a rapid viro-logic response criterion.

The interim analysis of the PROVE-1 trial showed that a rapid virologicresponse after 4 weeks of treatment (HCV RNA level !10 IU/mL) wasachieved in 79% of patients in the triple-therapy arm, including telaprevir,and in 11% of patients in the standard combination arm. The virologic re-sponse rate after 12 weeks of treatment (HCV RNA level !10 IU/mL)was 70% in the triple-therapy arm and 39% in the standard combinationarm. Nine patients who were treated with pegylated interferon-alfa-2a,ribavirin, plus telaprevir in study arm A and achieved a rapid virologic re-sponse at week 4 (!10 IU/mL) discontinued triple therapy after 12 weeks.Twenty weeks after discontinuation of triple therapy, six of nine patientshad undetectable HCV RNA in serum. This is the first result indicatingthat specifically targeted antiviral therapy against HCV may shorten therequired treatment duration and improve the sustained virologic responserate.

In the PROVE-1 trial, the total incidence of adverse events in patientstreated with telaprevir, interferon-alfa-2a, and ribavirin was similar tothat of the control group. Discontinuation attributable to adverse eventswas more frequent in the telaprevir arm compared with the control arm(9% versus 3%), however. Gastrointestinal events, rashes (severe in severalcases), and anemia were more common in the triple-therapy arms, which in-cluded telaprevir, than in the standard double-combination treatment arm.

The efficacy of telaprevir in patients infected with HCV genotype 1 whohave not achieved a sustained virologic response with a previous interferon-based treatment is to be investigated in the PROVE-3 trial (n ¼ 400 with60% nonresponders and 40% relapsers to previous interferon-based treat-ment). The PROVE-3 trial is a double-blind randomized phase 2 study in-vestigating telaprevir or placebo with peginterferon-alfa-2a, with orwithout ribavirin. In the PROVE-3 trial, patients are to be randomizedinto four groups [48]:

1. Twelve weeks of therapy with telaprevir, pegylated interferon-alfa-2aand ribavirin, followed by 12 weeks of treatment with placebo, pegy-lated interferon-alfa-2a, and ribavirin (total duration of 24 weeks)

2. Twenty-four weeks of therapy with telaprevir and pegylated interferon-alfa-2a (total duration 24 weeks)

3. Twenty-four weeks of therapy with telaprevir plus pegylated interferon-alfa-2a and ribavirin, followed by 24 weeks of treatment with pegylatedinterferon-alfa-2a and ribavirin (total duration of 48 weeks)

4. Control arm with placebo plus pegylated interferon-alfa-2a and ribavi-rin for 24 weeks, followed by 24 weeks of treatment with pegylated in-terferon-alfa-2a and ribavirin only (total duration of 48 weeks)


All patients who do not achieve an early virologic response at week 4 dis-continue treatment. Patients in the control group 4 have the option to re-ceive telaprevir through a rollover study after week 24. Final data areawaited 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 interferon-alfa-2b [49].

In a phase 1 open-label combination study, boceprevir was evaluated incombination with pegylated interferon-alfa-2b versus either agent alone ina crossover design in adult patients who have HCV genotype 1 and wereprevious nonresponders to pegylated interferon-alfa-2b–based 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) pegylatedinterferon-alfa-2b (1.5 mg/kg/wk) as monotherapy for 14 days, and (3) boce-previr plus pegylated interferon-alfa-2b combination therapy for 14 days ina 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 mgor 400 mg, respectively, compared with �1.08 � 0.22 and �1.61 � 0.21 forboceprevir at a rate of 200 mg and 400 mg, respectively, and �1.08 � 0.22and �1.26 � 0.20 for pegylated interferon-alfa-2b alone in the groups withboceprevir administered at 200 mg and 400 mg, respectively [50].

Several mutations conferring resistance to boceprevir were observed inthe replicon system, but only a single mutation has to date been describedin one patient by direct sequencing (see Table 3). Based on the results ofthe phase 1 clinical program and extensive preclinical safety and pharmacol-ogy studies, a large randomized phase 2 dose-finding study has been initi-ated [51]. This study was designed to evaluate the safety and efficacy ofboceprevir in combination with pegylated interferon-alfa-2b, with and with-out added ribavirin, for 24 or 48 weeks in patients infected with chronicHCV genotype 1 who were nonresponders to previous pegylated interferonand ribavirin combination therapy. The primary objective of this study wasto determine the safe and effective dose range of boceprevir in combinationwith pegylated interferon-alfa-2b in this patient population. A secondaryobjective was to explore whether ribavirin provides an additional benefitwhen combined with boceprevir plus pegylated interferon-alfa-2b. Thestudy was completed in 2007, and the results are pending [51].

The safety and efficacy of boceprevir in treatment-naive patients whohave chronic HCV genotype 1 infection is currently being investigated ina phase 2, randomized, 5-arm, comparative, open-label safety and efficacystudy. The study compares treatment with pegylated interferon-alfa-2bplus ribavirin with treatment with boceprevir plus pegylated interferon-alfa-2b and ribavirin.


Optimizing pharmacokinetics: ritonavir boosting
Ritonavir is a protease inhibitor approved for treatment of HIV with po-
tent inhibitory effects against cytochrome P450 3A (CYP3A). Codosing withritonavir leads to pharmacokinetic enhancement (boosting) of peptidomi-metic HIV protease inhibitors, which are metabolized by CYP3A. The inter-action of the investigational HCV protease inhibitors telaprevir andboceprevir with ritonavir was studied in vitro and in vivo [52]. In rat andhuman liver microsomes, the metabolism of telaprevir and boceprevir wasstrongly inhibited by ritonavir. On codosing of telaprevir or boceprevirwith ritonavir in rats, plasma exposure of the HCV protease inhibitorswas increased by greater than 15-fold and plasma concentrations 8 hoursafter dosing were increased by greater than 50-fold.

ITMN-191
ITMN-191 is another inhibitor of the NS3/4A protease with potent anti-
viral activity in vitro [53]. Several mutations associated with resistance toITMN-191 were found in the replicon system (see Table 3). A phase 1a trialin healthy volunteers was completed in May 2007. The phase 1b study in pa-tients who have chronic hepatitis C is designed to assess the effect on viralkinetics, viral resistance, pharmacokinetics, safety, and tolerability of multi-ple ascending doses of ITMN-191 given as monotherapy for 14 days. Twiceper 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 inhib-itor with a distinct mechanism of action compared with the previously de-scribed NS3/4A protease inhibitors. ACH-806 prevents the formation ofthe replicase complex after viral protein processing, a necessary step in viralreplication that occurs before copying the viral RNA genome. This uniquemechanism may contribute to a lack of cross-resistance between ACH-806and other HCV NS3/4A protease inhibitors in vitro. Data from a phase1b trial indicated that ACH-806 has antiviral activity against HCV, validat-ing the novel anti-HCV mechanism. Based on elevations of serum creati-nine, which were reversible after completion of dosing, however, furtherdevelopment of ACH-806 was stopped [55]. ACH-1095 is one of a seriesof next-generation compounds with potent antiviral potency in the repliconsystem [56]. Results from clinical trials on this compound are not yetavailable.
Polymerase inhibitors

Two classes of polymerase inhibitors, nucleoside and nonnucleoside poly-merase inhibitors, have been developed. Nucleoside analog polymeraseinhibitors are converted to triphosphates by cellular kinases and


incorporated into the elongating RNA strand as chain terminators. Gener-ally, they show similar efficacy 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]. Valopici-tabine inhibits the viral polymerase directly and is incorporated into grow-ing strands of viral RNA, with subsequent termination of RNA chainextension. Human polymerases do not seem to be affected by valopicitabine.

Valopicitabine was investigated in patients who had chronic hepatitis Calone and in combination with pegylated interferon. Patients infected withHCV genotype 1 and prior nonresponse to interferon-based antiviral treat-ment showed a mean reduction of 0.15 to 1.21 log10 IU/mL after 14 daysof treatment with different doses of valopicitabine ranging from 50 to800 mg/d [58].

Combination therapy was generally well tolerated; however, higher dosesof valopicitabine were associated with gastrointestinal side effects that weresevere in some patients [59]. Therefore, the maximum dose of valopicitabinewas reduced from 800 to 400 mg/d by protocol amendment during the prog-ress of the phase 2 trials. An interim analysis showed that the combinationof pegylated interferon-alfa-2a plus valopicitabine in treatment-naive pa-tients who had chronic hepatitis C genotype 1 infection was associatedwith a mean decline of HCV RNA of 3.90 to 4.56 log10 IU/mL and 3.75to 4.41 log10 IU/mL after 24 and 36 weeks of treatment, respectively [59].

Valopicitabine in combination with pegylated interferon-alfa-2a was alsoinvestigated in patients with prior nonresponse to interferon-alfa–basedtreatment [60]. The interim analysis of this study after 24 weeks of treatmentshowed a significantly stronger decline of HCV RNA in the combination ofvalopicitabine (800 mg) and pegylated interferon-alfa-2a versus pegylated in-terferon-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 tobe reduced to 400 mg/d by protocol amendment [61]. After 48 weeks of treat-ment, the decline of HCV RNA in the previous high-dose valopicitabine pluspegylated interferon-alfa-2a arms was still 0.8 log10 IU greater than in the pe-gylated interferon-alfa-2a plus ribavirin combination arm; however, the dif-ference was not significant [61]. Based on the overall risk-benefit profileobserved in clinical testing, the development program of valopicitabine forthe 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 polymeraseshowed resistance to the active metabolite of valopicitabine [62,63]. Repli-con variants bearing this mutation showed reduced fitness compared withthe wild-type replicon.


Nonsynergistic interactions of antiviral drug combinations could bea problem for future therapies. It was found that ribavirin antagonizes thein vitro anti-HCV activity of 20-C-methylcytidine, the active metabolite ofvalopicitabine. These findings may have implications in future clinical stud-ies with specific 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 cul-ture [65]. R1626 is a prodrug of R1479 [66].

A multiple dose ascending phase 1 study was designed to evaluate thesafety, tolerability, pharmacokinetics, and antiviral activity of R1626 in pre-viously untreated patients infected with chronic hepatitis C genotype 1 [66].In this study, patients were randomized for treatment with different doses ofR1626 ranging from 500 to 4500 mg of R1626 or placebo twice daily. Pa-tients 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 observedwith R1626 at doses of 1500, 3000, and 4500 mg, respectively. A phase 2trial evaluates safety and efficacy of R1626 in combination with peginter-feron-alfa-2a and ribavirin.

Development of resistance to R1479 was investigated in the replicon sys-tem. Resistance was associated with the presence of amino-acid substitu-tions S96T and S96T/N142T in the NS5B polymerase [67].

PSI-6130 and R7128
R7128 is another nucleoside type polymerase inhibitor that has been de-
veloped for the treatment of chronic hepatitis C [68]. R7128 is a prodrug ofPSI-6130, an oral cytidine nucleoside analogue. In preclinical studies, notoxicity was observed in various human cells, including liver cells, bone mar-row cells, and white blood cells. When compared in laboratory studies withseveral other compounds in development for the treatment of HCV, PSI-6130 was found to be more active at low concentrations or less toxic. Incombination with interferon, PSI-6130 was active and additive to the activ-ity of interferon alone in these preclinical assays. Results from phase 1 stud-ies on R7128 are pending.

Nonnucleoside polymerase inhibitors
The mechanisms of action of nonnucleoside polymerase inhibitors are
different from those of nucleoside polymerase inhibitors. Therefore, cross-resistance between these two classes is unlikely to occur. Several structurallydistinct nonnucleoside inhibitors of the HCV RNA–dependent RNA-polymerase NS5B have been reported to date, including benzimidazole,benzothiadiazine, and disubstituted phenylalanine or thiophene or dihydro-pyranone derivatives. They target different sites within the thumb domain of


the polymerase. Nonnucleoside inhibitors based on the structure of a benz-imidazole or an indole core bind to an exposed guanosine triphosphate(GTP) site on the protein surface, whereas inhibitors with a phenylalanine,thiophene, or dihydropyranone scaffold bind 1.0–1.5 nm from that externalGTP site in the region of a hydrophobic cleft at the base of the thumb do-main (see Fig. 4). Different resistance profiles attributable to distinct targetsites can be expected for the class of nonnucleoside inhibitors. As with pro-tease inhibitors, however, a single mutation may already confer resistance tononnucleoside polymerase inhibitors. In contrast to nucleoside polymeraseinhibitors, a restricted spectrum of activity of nonnucleoside polymerase in-hibitors against different HCV genotypes and subtypes has been described.In addition to classic nonnucleoside analog inhibitors, several pyrophos-phate mimics have been described that interact with catalytic metal ionsin the active site of the enzyme.

HCV-796
HCV-796 is a nonnucleoside polymerase inhibitor of the NS5B RNA-
dependent RNA-polymerase that has demonstrated potent antiviral activityin vitro and in patients who have chronic hepatitis C. Monotherapy showeda maximum antiviral effect after 4 days of treatment with a mean reductionof HCV RNA of 1.4 log10 IU/mL. Viral load started to increase thereafter,however, indicating that resistance might be an issue. The emergence ofa C316Y amino-acid substitution in NS5B in isolates of patients treatedwith HCV-796 was associated with the development of resistance toHCV-796.

The combination of HCV-796 and pegylated interferon-alfa-2b was in-vestigated in treatment-naive patients who had chronic hepatitis C. Thecombination of HCV-796 and pegylated interferon-alfa-2b produceda mean viral reduction of 3.3 to 3.5 log10 IU/mL after 14 days of treatmentcompared with 1.6 log10 IU/mL with pegylated interferon-alfa-2b alone [69].The antiviral activity of HCV-796 differed by HCV genotype. Mean reduc-tions at day 14 for patients infected with HCV genotype 1 ranged from 2.6to 3.2 log10 IU/mL in the combination groups versus l.2 log10 IU/mL forpegylated interferon-alfa-2b alone. For HCV genotype non–1-infected pa-tients, the respective reductions of HCV RNA were 3.5 to 4.8 log10 IU/mLversus 2.6 log10 IU/mL. Common adverse events in all groups were thosetypically associated with interferons, including headache, chills, rash, andmyalgia [69].

In a consecutive phase 2 study evaluating HCV-796 in combination withpegylated interferon and ribavirin, clinically significant elevations of liverenzymes were observed in approximately 8% of patients receiving HCV-796, including two patients who experienced serious adverse events leadingto withdrawal from active therapy with HCV-796, pegylated interferon, andribavirin [70]. Because of this potential safety issue, HCV-796 treatment wasdiscontinued in the phase 2 program.


GS-9190
GS-9190 is another nonnucleoside polymerase inhibitor with potent anti-
viral activity in the replicon system [71]. The antiviral activity of GS-9190 ishigher in HCV genotype 1 replicons compared with HCV genotype 2 repli-cons. 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 com-pound exhibits potent and specific inhibition of the HCV RNA–dependentRNA-polymerase in enzymatic- and cell-based assays. In a phase 1 trial,BILB 1941 was given as monotherapy in a liquid formulation for 5 daysand demonstrated significant antiviral activity in patients infected withHCV genotype 1 [72]. Increased virologic response was limited by gastroin-testinal intolerance that precluded testing at higher doses. The contributionto the gastrointestinal side effects by BILB 1941 versus the constituents ofthe liquid formulation remains uncertain.

NS5A antagonists

A-831 and A-689
A-831 targets the NS5A protein and has shown potent activity in the re-
plicon assay. The drug has an excellent therapeutic index and good pharma-cokinetic properties. A-831 is currently being investigated in a phase 1 trial,and results are pending. A-689 is another NS5A inhibitor currently in pre-clinical development. A-689 has a different chemical structure from A-831and binds to the NS5A target at a different 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. Itwas shown that cyclophilin B binds to the HCV NS5B polymerase and stim-ulates its RNA-binding activity. Cyclophilin inhibitors show strong antiviralactivity in vitro and in vivo. Cyclophilin inhibitors may not only be effectiveagainst HCV but against HIV. The cyclophilin inhibitor DEBIO-025showed a strong dual antiviral activity against HCV and HIV in a phase1 trial with HCV/HIV-coinfected patients [74]. In this study, the mean max-imal decrease in HIV-1 viral load was 1.0 log10 IU/mL. A pronounced effecton 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 morethan 2 log10 IU/mL, with differences depending on genotype. DEBIO-025was well tolerated. Laboratory abnormalities observed were hyperbilirubi-nemia and low platelet count. Clinical data for another cyclophilin inhibitor,NIM811, are currently not available.


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 metabo-lite of celgosivir is castanospermine, which is a potent inhibitor of the alpha-glucosidase I that is a host enzyme required for viral assembly and release.Celgosivir is potentially synergistic with other mechanistically diverse anti-HCV drugs. A phase 2, multicenter, double-blind, controlled study on theefficacy of celgosivir in patients who had chronic hepatitis genotype 1 infec-tion with prior nonresponse to interferon-based antiviral treatment wasperformed [75]. In this study, virologic nonresponders to previous inter-feron-alfa–based antiviral treatment showed a 1.2-log10 decline of HCVRNA after treatment with celgosivir, pegylated interferon-alfa-2b, andribavirin compared with a 0.4-log10 decline after treatment with placeboplus pegylated interferon-alfa-2b and ribavirin (one-sided P!.05).

Summary

Research in the past years has focused on individualization of interferon-alfa–based treatment regimens for patients who have chronic hepatitis C.New long-acting interferons may improve convenience of application andpotentially improve the adverse event profile. Further modification of inter-feron therapy is unlikely to improve sustained virologic response ratesmarkedly.

Specific targeted antiviral therapy for HCV is a new perspective in thetreatment of chronic hepatitis C especially for patient populations that aredifficult to treat. The results from recent clinical trials indicate that severalcompounds have potent antiviral activity; however, not all were consideredas safe and well tolerated. A central question remains whether the new an-tiviral compounds not only increase the virologic response during treatmentbut increase the rate of sustained virologic response after treatment. A firstinterim analysis on sustained virologic response after treatment with telap-revir in combination with pegylated interferon-alfa and ribavirin has re-cently been presented, and the results are promising. The results suggestthat combination therapy with telaprevir with pegylated interferon-alfaplus ribavirin may not only increase the rate of sustained virologic responsecompared with standard therapy but may allow a reduction of treatmentduration.

The viral RNA-polymerase of HCV has a high error rate. As a result, dif-ferent HCV variants are continuously produced during replication. HCVdoes not integrate into host DNA. Therefore, every infected cell has thepotential of producing multiple drug-resistant mutants over time. The emer-gence of resistance might possibly limit the success of HCV-specific antiviralcompounds, and is therefore a highly clinically relevant issue.


Selection of drug-resistant HCV strains may occur when viral replicationcontinues while drugs are taken. Not all viral strains have the same ability toreplicate. The inherent ability of a virus to replicate is termed viral fitness.The fitness of variants present in a patient may lead to differences in viralresponse. The fitness of viral variants was investigated in patients whohad chronic hepatitis C after stopping monotherapy with telaprevir [43].In this study, high-level resistant strains were replaced more rapidly thanlow-level resistant strains by wild-type virus. The results indicate that telap-revir resistance is inversely correlated with viral fitness to replicate [43].

Innate and adaptive immune responses play an important role in thecontrol of viral diseases. In most patients infected with the hepatitis C vi-rus, the innate and adaptive immune responses are too weak for completeelimination of HCV-infected cells, which is the prerequisite for a cure fromHCV. The consequence of an impaired immune response is the persistenceof infected cells and the development of chronic hepatitis C. Several mech-anisms have been identified by which the hepatitis C virus attenuatesinnate and adaptive immune responses. It can be anticipated that inhibi-tion of HCV replication by new HCV-specific antiviral compounds leadsto a reconstitution of the innate and adaptive immune response againstHCV during antiviral therapy. A potential reconstitution of the immuneresponse against HCV could enhance the infected cell loss during treat-ment, and thereby improve the antiviral efficiency of HCV-specific com-pounds [50].

Primary or secondary resistance to HCV specific inhibitors is potentiallya serious problem. Combination of (non–cross-resistant) antiviral com-pounds inhibiting different viral and cellular mechanisms may reduce theproblem of resistance. Therefore, future research should not only focus onthe development of new compounds but on optimal drug combinations. Arecent study on cross-resistance of telaprevir and interferon-alfa indicatesthat telaprevir-resistant strains are sensitive to interferon-alfa [45]. Thisstudy supports the concept that combination therapy may reduce the devel-opment and selection of resistant strains. The development of agents in dif-ferent classes may allow construction of antiviral combinations that enhancethe effectiveness of antiviral treatment, reduce the duration of treatment,and, eventually, may even avoid the use of interferon-alfa.

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Noninvasive Monitoring of Hepatitis CFibrosis Progression

David S. Kotlyar, BSa, Wojciech Blonski, MD, PhDb,c,Vinod K. Rustgi, MD, FACPd,*

aUniversity of Pennsylvania School of Medicine, 3 Ravdin/3400 Spruce Street,

Philadelphia, PA 19104, USAbHospital of the University of Pennsylvania, 3 Dulles, 3400 Spruce Street,

Philadelphia, PA 19104, USAcDepartment of Gastroenterology and Hepatology, Wroclaw Medical University,

Borowska 213 Street, 50-556 Wroclaw, PolanddMetropolitan Liver Diseases/Gastroenterology Center, Georgetown University Medical

Center, 8316 Arlington Boulevard, Suite 515, Fairfax,

VA 22031, USA

Hepatitis C remains the most common cause of chronic liver disease inthe United States. Monitoring of the progression of fibrosis is an importanttool for the clinician in assessing the aggressiveness of infection and level ofhepatic injury. Historically, liver biopsy has been used as the ‘‘gold stan-dard’’ in determining the degree of fibrosis and the degree of hepatitic nec-roinflammatory injury [1,2]. The degree of inflammation and fibrosisgleaned from a liver biopsy also has been used as a vital tool in strategizingtreatment for hepatitis C [3].

Biopsy is not only an invasive and possibly painful procedure; it also car-ries a small risk for morbidity and death [3]. Further, there is the issue ofsampling error, defined as variable levels of fibrosis throughout the liver,with biopsy only examining a small (1/50,000) portion of the liver [4]. Inone study, several patients had minimal fibrosis on biopsy but by meansof transient elastography, a newly developed form of ultrasound, theywere found to have severe fibrosis and severely elevated portal pressures,as confirmed by hepatic-portal venous gradient measurement [4].

Biopsy has been shown to have significant inter- and intraobserver vari-ability among pathologists, with an average 20% error rate in the staging offibrosis [5]. One study with 124 patients noted that approximately 33% of

Clin Liver Dis 12 (2008) 557–571


E-mail address: [email protected] (V.K. Rustgi).



558 KOTLYAR et al

samples had different fibrosis scores among different pathologists, althoughonly 9.7% were deemed to be clinically significant if grade 3 and grade 4fibrosis were considered similar [6]. The amount of tissue procured affectsthe staging as well; in one study, staging was significantly underscoredwhen the sample was smaller [7]. Interpretation of a liver biopsy by expertpathologists with more than 10 years of experience in an academic centeralong with a minimum biopsy sample of 2 cm and 11 or more portal tractsmay have much improved interobserver agreement and less sample error[8,9]. Although biopsy remains an invaluable tool in determining liver in-jury, there remains a need to supplement biopsy with noninvasive methodsof assessing liver fibrosis.

Importance and pathophysiology of fibrosis

Fibrosis is usually secondary to hepatic injury and inflammation. Often,although transaminase levels may be normal in a patient who had chronichepatitis C infection, the patient may have significant levels of fibrosis,which suggests that transaminase levels are a suboptimal surrogate markerfor fibrosis progression [1]. Of those with moderate inflammation, mostdevelop cirrhosis within 20 years and those with bridging fibrosis or severeinflammation usually develop cirrhosis within 10 years [1].

Fibrosis is a dynamic process; in the healthy individual, although there isno change in the structure of the extracellular matrix (ECM) on histology,there are simultaneous catabolic and metabolic processes that reach equilib-rium with each other [10]. In the fibrotic state, there is excessive ECM pro-duction, which outstrips the catabolism of ECM elements [10]. It has beenshown that a major player in fibrosis is the hepatic stellate cell (HSC, alsoknown as Ito cells), which normally functions to store vitamin A and usuallyremains morphologically stable [11]. In liver injury, however, these cells be-come ‘‘activated,’’ wherein their morphology changes from spheroid cells tomore elongated and spindle-shaped cells reminiscent of myofibroblasts [11].They have a reduction in the amount of vitamin A and begin to secretedense forms of collagen, such as collagen I [11]. They also express matrixmetalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metal-loproteinases (TIMPs), which alter the makeup of the ECM [11]. TheHSCs also begin to proliferate, responding to increased levels of platelet-derived growth factor (PDGF) and increase production of collagen I andother proteins, which increases fibrosis production because of cytokines,such as transforming growth factor-b (TGFb) [11]. Progressive scarringmakes the entire liver more ‘‘stiff’’ (as measured by the shear modulus),which is highly significant for noninvasive testing [12].

The ECM may also affect fibrotic production of HSCs, making the path-ophysiology of fibrosis a vicious cycle [12]. Interestingly, there is also phos-phorylation of focal adhesion kinase (FAK) in HSCs, a key protein involvedin the activation of cell movement [13]. There is also increased expression of

559MONITORING OF HEPATITIS C FIBROSIS PROGRESSION

nuclear factor (NF)-kB, which promotes HSC proliferation and antiapop-totic properties [13]. FAK activation secondary to ECM (and extracellularlysyl oxidase) has also been observed in hypoxic metastases of tumors;this parallels fibrosis as a model in which the ECM greatly influences cellularbehavior [14]. Fig. 1 is a schematic showing a model of the progression offibrosis [12].

Transient elastography

Within the past 4 years, transient elastography has emerged as a highlyuseful noninvasive form of ultrasound that can help to assess the level offibrosis in a patient who has hepatitis C. Elastography is also known bythe name ‘‘FibroScan,’’ as developed in France. The scan was developedon the principle that livers with increasing degrees of scarring have decreas-ing elasticity and that a shear wave propagating through stiffer materialwould progress faster than in one with more elastic material [15]. The tran-sient nature of measuring the shear wave (w100 milliseconds) is ideal,

Fig. 1. Biology of injury, inflammation, and fibrogenesis. In most diseases leading to fibrosis,

injury and subsequent inflammation are prominent. Injury to the liver, typically involving

hepatocytes, leads to activation of immune cells and an inflammatory response. Inflammatory

mediators are an important stimulus for stellate cell activation, and thus fibrogenesis. Impor-

tantly, 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 fibrosis. Hepatology 2006;43(Suppl):S113; with

permission.)

560 KOTLYAR et al

because the liver’s position moves with breathing, and other reflected elasticwaves can be excluded from analysis by this method [15].

The elastography device is a vibrating probe with an ultrasound trans-ducer on its end. The transducer emits a shear wave of low frequency at50 Hz, and ‘‘pulse-echo ultrasound acquisitions’’ are used to measure theamount 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 is100 times the size of a standard biopsy [16]. Stiffness is measured by theshear modulus, which uses kilopascal (kPa) units [15]. Fig. 2 shows a sche-matic of how transient elastography acquires the data on the stiffness oftissue [17].

In one of the initial studies of elastography with 106 patients, the mean re-ceiver operating curves (ROC) for different METAVIR stages of fibrosiswere 0.9 for stage F1 or greater, 0.88 for stage F2 or greater, 0.91 for stageF3 or greater, and 0.99 for stage F4 [15]. Intraobserver and interobserver var-iability were 3.3% and 3.3%, respectively, which is significantly better thana liver biopsy [15]. A recent observation by Rigamonti and Fraquelli [18] de-scribes such variability as less than 1% after a 1-month training period.

The advantages of elastography include its relative low cost, noninvasiveapproach, rapid acquisition of information (within about 5 minutes), andease of use [15]. The limitations seen in the first study included one scanwith an interobserver variability of 18%, which was then attributed to het-erogenic distribution of fibrosis. Also, there was a measurement failure rateof 6%; this was likely attributable to patients who had ascites or who wereobese, because the low frequency shear wave can be dampened by fat or as-cites, and thus become not measurable [15]. Those with narrow intercostalspaces are also a challenge to assess with elastography [15].

A follow-up prospective study with 183 patients showed that elastogra-phy 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 onbiopsy for stage F2 or greater, stage F3 or greater, and stage F4, respectively[19]. Combining elastography with the FibroTest showed an AUC of 0.88for stage F2 or greater and 0.95 for stage F3 or F4 [19]. Diagnostic cutoffvalues for staging were somewhat different than in the original study; thesewere 7.1 kPa, 9.5 kPa, and 12.5 kPa for stage F2 or greater, stage F3 orgreater, 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 showedgood ROC results with an AUC of 0.81, 0.95, and 0.99 for stage F2 orgreater, stage F3 or greater, and stage F4, respectively, when comparedwith biopsies that were at least 2.5 cm in length [20]. Cutoff values for diag-nosis were again similar in range but different than in other studies by Ziolusing 8.8 kPa, 9.6 kPa, and 14.6 kPa for stage F2 or greater, stage F3 orgreater, and stage F4, respectively [20].

Fig. 2. Schematic of transient elastography (FibroScan). (Courtesy of Echosens SA, Paris,

France; with permission.)


562 KOTLYAR et al

Another trial with 711 patients showed good ROC results as well. TheROC AUC was 0.8, 0.9, and 0.96 for stage F2 or greater, stage F3 orgreater, and stage F4, respectively [21]. The investigators used cutoff valuesfor various METAVIR stages based on a positive predictive value of at least90%. Their cutoff values were 7.2 kPa, 12.5 kPa, and 17.6 kPa for stage F2or greater, stage F3 or greater, and stage F4, respectively [21]. Also of note,cutoff values for complications of cirrhosis were calculated for a negativepredictive value of greater than 90%. These were 27.5 kPa for esophagealvarices, 37.5 kPa for Child-Pugh B or C score, 49.1 kPa for a past historyof ascites, 53.7 kPa for hepatocellular carcinoma, and 62.7 kPa for esopha-geal bleeding [21].

Two trials from Japan also have validated the use of elastography. In onestudy of 237 patients who had chronic hepatitis C, elastography was foundto be highly superior to biochemical markers in distinguishing stages offibrosis [22]. Median stiffness values were 4.1 kPa (range: 3.5–4.9 kPa) for50 stage 0 patients, 6.3 kPa (range: 4.8–8.5 kPa) at stage 1, 8.8 (range:6.8–12.0 kPa) at stage 2, 14.6 (range: 10.5–18.6 kPa) at stage 3, and 22.2(range: 15.4–28.0) at stage 4 [22]. Patients who had attained a sustained vi-rologic response had a stiffness value of 3.8 kPa, 5.7 kPa, 6.8 kPa, and6.1 kPa for stages 1, 2, 3, and 4, respectively [22].

The other Japanese study examined resections of liver in 30 patients andcompared digital image analysis (DIA) of resections with elastography. TheROC AUC was 0.932 when DIA showed fibrotic areas of greater than10%in the liver, and it was 0.991 when DIA showed fibrotic areas of more than20% of the liver [23]. One limitation of the study was that it did not compareDIA and METAVIR scores.

Another analysis from Germany with 79 patients showed somewhatworse AUC values, but these became comparable when elastography wascombined with the aspartate transaminase–to-platelet ratio (APRI) [24].Instead of transient elastography, the investigators used ‘‘real-time’’ elastog-raphy, which is used in breast, thyroid, and prostate imaging and is a typeof 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 forMETAVIR stage F2 or greater, stage F3 or greater, and stage F4, respec-tively [24].

Accuracy in the diagnosis of cirrhosis was also examined in a separateanalysis of 775 patients [25]. The AUC was excellent at 0.95, with the highestsensitivity and specificity being at 9.4 kPa and 17.1 kPa, respectively, for thefinding of cirrhosis [25]. A cutoff value of 11.7 kPa had a sensitivity of 0.91and specificity of 0.87 for finding cirrhosis [25]. Patients with an elastogra-phy value of less than 9.4 kPa had a greater than 95% chance that theydid not have cirrhosis, with those having a value greater than 17.1 kPa hav-ing a 95% chance that they did indeed have cirrhosis [25]. Being male orhaving steatosis increased the risk for being among those having been mis-classified (which occurred in 80 [10.3%] of 775 patients) [25].


One trial quantified failure during elastographic measurement. Therewere 935 patients studied in this prospective trial, and on multivariate anal-ysis of 888 patients, it was found that physicians with more experience, spe-cifically with more than 100 elastography examinations on patients, weremore likely to be successful when measuring liver stiffness [26]. Male pa-tients who were obese were difficult to measure, having an odds ratio of0.11 of a successful measurement, and obese women had an odds ratio of0.16 of a successful measurement; older patients had a small decreasefrom baseline of 0.96, but it was statistically significant [26]. AUC valueswere also calculated in the study and were 0.79, 0.89, and 0.91 for stageF2 or greater, stage F3 or greater, and stage F4, respectively [26].

Radiology

Single photon emission computed tomography (SPECT) and in vivophosphorus 31 (31P) magnetic resonance spectroscopy (MRS) have beenproposed as valuable noninvasive methods of assessing severity of liver dis-ease in patients who have chronic hepatitis C [27,28]. It was observed thatthe two SPECT parameters, minimum spleen pixel count and maximumright hepatic lobe pixel count, identified an accurate fibrosis score whencompared with liver biopsy in 39 of 46 patients, yielding a positive and neg-ative predictive value of 86% [28]. A study of 48 patients who had chronichepatitis C–related liver disease demonstrated that 31P MRS can differenti-ate 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 and83% specificity of identifying mild hepatitis, a PME/PDE ratio of greaterthan 0.2 but less than 0.3 yielded 47% sensitivity and 87% specificity in iden-tifying moderate hepatitis, and a PME/PDE ratio of 0.3 or greater yielded82% sensitivity and 81% specificity in identifying liver cirrhosis [27]. Itwas proposed that 31P MRS might be useful in particular in evaluatingthe progression of liver disease in patients who have chronic hepatitis C [27].

The use of Doppler sonography in assessing the severity of liver disease inpatients who have chronic hepatitis C remains controversial. A recent studyof 65 patients who had liver disease related to hepatitis C demonstrated nosignificant differences in the Doppler indices with increasing severity of liverdisease [29]. In light of these findings, Doppler sonography cannot be rec-ommended as a reliable noninvasive method evaluating the severity of liverdisease related to chronic hepatitis C [29].

Direct serum markers of liver fibrosis

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

564 KOTLYAR et al

serum marker of fibrolysis MMP-1 might be helpful in evaluating liver fibro-sis in patients who have chronic hepatitis C (PIIINP/MMP-1 score) [30].The fibrosis index (PIIINP/MMP score) was recently shown to be a usefulmarker in analyzing liver fibrosis in patients who have chronic hepatitis Cduring and after treatment with interferon-a and ribavirin because of its sig-nificant correlation (r ¼ 0.68, P!.001) with the METAVIR fibrosis score[31]. A fibrosis index score less than 0.20 allowed for ruling out stage F3to F4 and stage F2 to F4 fibrosis with respective negative predictive valuesof 96% and 88%, whereas a score greater than 0.50 yielded 89% and 100%positive predictive values for identification of stage F3 to F4 and stage F2 toF4 fibrosis, respectively [31]. An early and regular decrease in the fibrosis in-dex was observed during treatment, with stable values in patients after treat-ment response, whereas patients who did not respond to treatmentdemonstrated no significant changes in the fibrosis index [31].

It was proposed that the FIBROSpect II, a fibrosis panel including sev-eral ECM remodeling proteins, such as serum hyaluronic acid (HA),TIMP-1, and a2- macroglobulin may separate patients who have moderateto severe liver fibrosis from those who do not have liver fibrosis or have onlymild liver fibrosis [32]. Among 696 patients who had chronic hepatitis C andmoderate to severe liver fibrosis (METAVIR stage F2–F4), a FIBROSpectII cutoff point greater than 0.36 demonstrated a positive predictive value of74.3%, negative predictive value of 75.8%, and accuracy of 75% [32]. An-other study also suggested that this index might also be a good alternativeto liver biopsy by excluding advanced liver fibrosis or cirrhosis (scoreO0.42) in patients who have chronic hepatitis C [33]. An analysis of 142liver specimens from 136 patients who had chronic hepatitis C and a 38%prevalence for advanced liver fibrosis observed a positive predictive valueof 63%, negative predictive value of 94%, and accuracy of 76% using a FI-BROSpect cutoff point greater than 0.42 [33]. It was suggested that patientswith FIBROSpect II scores between 0.42 and 0.8 should be referred for liverbiopsy, however, because of the decreased sensitivity and specificity in estab-lishing the stage of liver fibrosis for this range of values [33]. These data werefurther supported by a recent prospective study that validated the diagnosticvalue of FIBROSpect II in identifying patients who have chronic hepatitis Cwho do not have liver fibrosis [34]. Conversely, this method has not beenrecommended to identify and differentiate among intermediate stages ofliver fibrosis [34]. Among 108 consecutive patients who had chronic hepatitisC with a 36.1% prevalence of advanced liver fibrosis, a FIBROSpect II cut-off point of greater than 0.42 was characterized by a positive predictivevalue of 60.9%, negative predictive value of 82.3%, and accuracy of73.1% [34].

It was also suggested that serum levels of insulin-like growth factor-I(IGF-I) could serve as noninvasive marker of liver fibrosis in patients whohave chronic hepatitis C [35]. Patients who have chronic hepatitis C have ab-normal synthesis of IGF-1, and its serum levels were consistent with the


severity of liver fibrosis, demonstrating significantly lower values in patientswho had liver cirrhosis (METAVIR stage F4) when compared with otherdegrees of fibrosis (METAVIR stage F0–F3) [35]. In addition, a serum fi-brogenesis marker, YKL-40, was recently proposed as a noninvasive markerof identification of liver fibrosis in patients who have chronic hepatitis C[36]. YKL-40 was found to be superior to other fibrosis markers, such asHA or PIIINP, in separating severe fibrosis (stage F2–F4) from mild fibrosis(stage F0–F1) [36]. Among fibrosis markers, however, HA was the best pre-dictor of differentiating between liver cirrhosis (stage F4) and liver fibrosis(stage F0–F3) in patients who have chronic hepatitis C [36]. After therapywith interferon, YKL-40 was the only marker that significantly decreasedin responders and nonresponders [36].

Biochemical markers of liver fibrosis

Several noninvasive tests based on biochemical markers have been pro-posed as surrogates of invasive methods evaluating the severity of liver dis-ease related to chronic hepatitis C. The analyzed tests included theFibroTest score (calculated with a patient’s age; gender; and levels of serumhaptoglobin, a2-macroglobulin, apolipoprotein A1, g-glutamyl transpepti-dase [GGTP], and bilirubin), APRI, Forns score (calculated based on plate-let count, GGTP, cholesterol level, and age), and FIB-4 (combination ofplatelets, alanine aminotransferase [ALT], and aspartate aminotransferase[AST]) [37–39].

The FibroTest score was found to yield a 100% negative predictive valuefor the absence of significant liver fibrosis and cirrhosis and more than 90%positive predictive value for the presence of significant liver fibrosis and cir-rhosis [38]. It was observed that staging of liver fibrosis by the FibroTesthad a 5-year prognostic value comparable with that done by liver biopsy inpatients who have chronic hepatitis C [40]. Staging by the FibroTest was su-perior to liver biopsy in predicting complications and deaths related to chronichepatitis C [40].Moreover, it was suggested that the use of the FibroTest couldreduce the number of liver biopsies by 32% in patients who have chronic hep-atitis C and undergo hemodialysis and renal transplantation [41].

It was shown that the APRI can accurately identify the presence or ab-sence of liver fibrosis or liver cirrhosis in 51% and 81% of treatment-naivepatients who had chronic hepatitis C, respectively [39]. By determining ap-propriate cutoff values for the APRI to achieve predictive values of 90%,Snyder and colleagues [42], were able to identify the severity of liver diseaseaccurately in up to 59% of patients who had chronic hepatitis C when com-pared with their results of liver biopsies. Conversely, Lackner and colleagues[43] observed that the APRI identified significant liver fibrosis only in 24%of patients who had chronic hepatitis C and excluded liver cirrhosis in 80%of patients.

566 KOTLYAR et al

The Forns score yielded a 96% negative predictive value and 66% posi-tive predictive value for identifying significant liver fibrosis in patients whohad chronic hepatitis C in one study [37]. The optimal proposed cutoffpoints were less than 4.21 for absence of liver fibrosis and greater than 6.9for presence of significant liver fibrosis [37]. A recent study of 235 consecu-tive patients who had chronic hepatitis C observed that a combination of theFibroTest, APRI, and Forns score without liver biopsy identified 81.3% ofpatients who had liver fibrosis, whereas in the remaining 18.7% of patients,liver biopsy was required to identify liver fibrosis [44].

A prospective comparison of discordant results between the FibroTestand liver biopsy in 537 patients who had chronic hepatitis C demonstratedthat failure of liver biopsy was more than sevenfold more common than fail-ure of the FibroTest among patients with an identified reason for discordantresults [45].

A recent comparison between FIB-4, liver biopsy, and the FibroTestshowed that values less than 1.45 and more than 3.25 allow for accurateidentification of patients who have moderate or severe liver fibrosis, respec-tively [46]. Results of FIB-4 less than 1.45 and greater than 3.25 agreed withthose 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 de-tecting severity of liver disease in patients who have chronic hepatitis C.These include an AST/ALT ratio of 1 or greater, level of platelets lessthan 140,000 cells/mL, and globulin/albumin ratio greater than 1 [47]. TheAPRI and Forns score in 243 patients who had chronic hepatitis C and sig-nificant liver fibrosis proved by liver biopsy showed at least a 90% negativepredictive value in ruling out significant liver fibrosis, whereas only a plateletcount less than 140,000 cells/mL and a globulin/albumin ratio greater than 1yielded an acceptably similar positive predictive value in identifying signifi-cant liver fibrosis [47]. An analysis of these tests among 78 patients who hadbiopsy-confirmed hepatitis C–related liver cirrhosis demonstrated that al-though all had excellent performance in excluding liver cirrhosis with a neg-ative 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 scoringsystem (range: 0–9) for detecting severe liver fibrosis in 173 patients whohad chronic hepatitis C. This system incorporated the three most significantindependent predictors of severe liver fibrosis: platelet count, AST, and al-bumin [48]. That noninvasive scoring system was shown to yield a 95% neg-ative predictive value for a cutoff point of 2 and a 94% positive predictivevalue for a cutoff point of 4 [48]. Another scoring system recently developedand internally validated by Alsatie and colleagues [49] included five factorsindependently associated with advanced liver fibrosis in 286 patients whohad chronic hepatitis C, such as diabetes mellitus, platelet count less than150,000 cells/mL, AST of 65 IU/mL or greater, international normalized


ratio (INR) of 1.1 or greater, and bilirubin of 0.85 mg/dL or greater. Basedon the proposed scoring system, patients were subsequently divided intolow-risk (score of 0), intermediate-risk (score of 1–3), and high-risk groupsof advanced liver fibrosis (score R4) [49]. When the results of the scoringsystem were compared with the results of liver biopsies among analyzed pa-tients, 9% of patients in the low-risk group, 34% of patients in the interme-diate-risk group, and 92% of patients in the high-risk group had actualadvanced liver fibrosis [49].

Noninvasive and simple indices were also suggested as good predictors ofliver fibrosis in patients who underwent liver transplantation attributable tohepatitis C–related liver cirrhosis [50,51]. Benlloch and colleagues [50] devel-oped a fibrosis index based on four independent predictors of liver fibrosis,including prothrombin time, albumin/total protein ratio, AST, and timesince liver transplantation. The proposed threshold of 0.2 for separatingbridging fibrosis or cirrhosis from portal or no fibrosis yielded a positivepredictive value of 49% and negative predictive value of 95% in validatingthis test, which included 96 patients [50]. These investigators proposed thattheir fibrosis index might be accurately used to identify patients who haveposttransplant hepatitis C with a low risk for significant liver fibrosis,thus allowing them to avoid liver biopsy [50]. A subsequent study by Schmi-lovitz-Weiss and colleagues [51] suggested that serum levels of globulin andimmunoglobulin G (IgG) were the only predictors of extent of liver fibrosis,and thus could become noninvasive markers of liver fibrosis in patients whohad recurrent hepatitis C after liver transplantation. Table 1 summarizes thepredictive values for biochemical and serum markers.

Summary

Noninvasive approaches in the diagnosis and monitoring of fibrosis arestill evolving. Although, historically, liver biopsy has remained the test ofchoice in determining the level of fibrosis, there remains a need for supple-mental noninvasive options. Transient elastography is an inexpensive, rapid,and relatively accurate form of noninvasive monitoring, especially in severefibrosis (METAVIR stage F3 or F4) [25,26]. It is a nascent technology, how-ever, and there is no clear indication that elastography is better than biopsyfor less severe fibrosis; most ROC values are between 0.80 and 0.90 with ex-perienced pathologists and liver biopsy samples more than 2 cm in length[19–21,26]. With improved resolution and longer term data, it may becomea vital supplement. Radiologic tests, such as new forms of MRS, are intrigu-ing but require further studies and technological development beforeimplementation.

Biochemical tests are quite good at ruling out advanced fibrosis but notfor confirming fibrosis, especially intermediate forms [37,38,43]. Severalstudies have shown that combining serum markers with elastography im-prove results [19,24].

Table 1

Noninvasive fibrosis and biochemical markers differentiating between patients who have mild

and severe fibrosis

Name of test Variables Study

Positive

predictive

value

Negative

predictive

value

Fibrosis index

by Trocme

PIIINP, MMP-1 Trocme et al [31] 100% 96%

FibroSpect II serum HA, TIMP-1,

a2-macroglobulin

Patel et al [32] 74% 76%

Christensen et al [33] 63% 94%

Zaman et al [34] 61% 82%

YKL-40

Hyaluronic acid

PIIINP intravenous

collagen

d Saitou et al [36] 80%

79%

76%

67%

79%

76%

77%

66%

FibroTest Age, gender,

haptoglobin,

a2-macroglobulin,

apolipoprotein A1,

GGTP, bilirubin

Imbert-Bismut

et al [38]

O90% 100%

Bourliere et al [44] 76% 71%

APRI AST/PLT Wai et al [39]

(significant

fibrosis) (cirrhosis)

91%

65%

90%

100%

Snyder et al [42]

(retrospective)

(prospective)

90%

92%

88%

95%


Forns score Age, PLT, GGTP,

cholesterol

Forns et al [37] 66% 96%


FIB-4 Age, PLT, ALT,

AST

Vallet-Pichard et al

[46]

82% 95%

Fibrosis index

by Benlloch

(transplanted

liver)

PT, albumin/total

protein, AST,

time since liver

transplantation

Benlloch et al [50] 49% 95%

568 KOTLYAR et al

Direct markers of fibrosis are somewhat less useful. The exception, HA,has been shown to be a sensitive test at ruling out cirrhosis but is otherwisenonspecific [36]. The combined use of transient elastography and biochem-ical markers seems to be the most promising noninvasive technique; techno-logical improvements and increased data may help to inform the clinicianwhether a liver biopsy is necessary in some patients, and this can facilitateguidance of treatment.

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[31] Trocme C, Leroy V, Sturm N, et al. Longitudinal evaluation of a fibrosis index combining

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with chronic hepatitis C treated by interferon-alpha and ribavirin. J ViralHepat 2006;13:643.

[32] Patel K, Gordon SC, Jacobson I, et al. Evaluation of a panel of non-invasive serummarkers

to differentiatemild frommoderate-to-advanced liver fibrosis in chronic hepatitis C patients.

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[34] ZamanA, RosenHR, IngramK, et al. Assessment of FIBROSpect II to detect hepatic fibro-

sis in chronic hepatitis C patients. Am J Med 2007;120:280.

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2007;52:3245.

[36] Saitou Y, Shiraki K, Yamanaka Y, et al. Noninvasive estimation of liver fibrosis and

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out hepatic fibrosis by a simple predictive model. Hepatology 2002;36:986.

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with hepatitis C virus infection: a prospective study. Lancet 2001;357:1069.

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significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology 2003;38:

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biomarkers (FibroTest) in patients with chronic hepatitis C. Clin Chem 2006;52:1887.

[41] VarautA, FontaineH, Serpaggi J, et al. Diagnostic accuracy of the Fibrotest in hemodialysis

and renal transplant patients with chronic hepatitis C virus. Transplantation 2005;80:1550.

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in chronic hepatitis C. J Clin Gastroenterol 2006;40:535.

[43] Lackner C, Struber G, Liegl B, et al. Comparison and validation of simple noninvasive tests

for prediction of fibrosis in chronic hepatitis C. Hepatology 2005;41:1376.

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and cirrhosis prediction in chronic hepatitis C patients: proposal for a pragmatic approach

classification without liver biopsies. J Viral Hepat 2006;13:659.

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between biochemical markers and biopsy in patients with chronic hepatitis C. Clin Chem

2004;50:1344.

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invasive evaluation of liver fibrosis in HCV chronic hepatitis. Am J Gastroenterol 2005;100:

868.

[48] Metwally MA, Zein CO, Zein NN. Predictors and noninvasive identification of severe liver

fibrosis in patients with chronic hepatitis C. Dig Dis Sci 2007;52:582.

[49] Alsatie M, Kwo PY, Gingerich JR, et al. Amultivariable model of clinical variables predicts

advanced fibrosis in chronic hepatitis C. J Clin Gastroenterol 2007;41:416.

[50] Benlloch S, Berenguer M, Prieto M, et al. Prediction of fibrosis in HCV-infected liver trans-

plant recipients with a simple noninvasive index. Liver Transpl 2005;11:456.

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Clin Transplant 2007;21:391.

Hepatitis C Infection and NonalcoholicFatty Liver Disease

Onpan Cheung, MDa, Arun J. Sanyal, MBBS, MDb,*aDivision 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, USAbDivision of Gastroenterology, Hepatology, and Nutrition, Department of Internal Medicine,

Virginia Commonwealth University Medical Center, MCV Box 980341,

Richmond, VA 23298–0341, USA

Hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting170 million (3%) of the world’s population and approximately 2.7 millionAmericans; cirrhosis can occur in 20% of these patients [1–3]. There aresix HCV genotypes; of these, type 1 is the most common in the United Statesand is also the most difficult genotype for which to achieve a sustained viro-logic response (SVR) to standard antiviral therapy.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause ofchronic liver disease in North America and affects up to 30% of the generalpopulation [4]. Its clinical-histologic spectrum ranges from nonalcoholicfatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), which can prog-ress to cirrhosis in 15% to 20% of subjects [5]. NASH is distinguished fromNAFL by the presence of inflammation and cytologic ballooning with orwithout Mallory hyaline or pericellular fibrosis in addition to steatosis [6].NAFL is estimated to affect up to two thirds of obese adults and nearly50% of obese children. Approximately 30% to 50% of adult Americanswho are obese may have NASH [7,8]. Although NAFLD can occur in allages 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 andis considered the hepatic manifestation of the metabolic syndrome [10,11].

Given that hepatitis C and NAFLD occur commonly in the general pop-ulation, it is not surprising that these conditions often coexist in the samepatient. Also, emerging data indicate that HCV itself may promote the

Clin Liver Dis 12 (2008) 573–585


E-mail address: [email protected] (A.J. Sanyal).



574 CHEUNG & SANYAL

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

Mechanisms of hepatic steatosis

Overview

Hepatic steatosis develops when fat influx and synthesis exceed fat catab-olism and export [12]. The predominant lipid that accumulates in hepaticsteatosis is triglyceride [13,14], and this is derived by esterification of a glyc-erol moiety with free fatty acids (FFAs). FFA influx is derived from dietaryfat intake or from peripheral lipolysis. FFA synthesis in the liver is alsoreferred to as de novo lipogenesis (DNL) and is mediated by a series ofenzymes 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, suchas sterol regulatory element binding protein (SREBP). b-oxidation ofFFAs for energy generation occurs in the mitochondria and is regulatedby adenosine monophosphate–activated kinase (AMPK) and peroxisomeproliferator–activated receptor (PPAR-a). FFA can also be hydrolyzed bylipases and exported from the liver while bound to apolipoprotein (apo) Bas very-low-density lipoprotein (VLDL).

Factors that are known to be associated with increased circulating FFAare obesity and insulin resistance [15]. FFAs further interfere with insulinsignaling in striated muscle and adipocytes by reducing their ability totake up glucose [16]. This excess glucose is potentially available for conver-sion to FFA in the liver by means of acetyl CoA, a downstream product ofglucose metabolism. Simultaneously, there is an increase in DNL secondaryto retained sensitivity to insulin for this pathway.

Under conditions of FFA loading, there is a preferential use of FFAinstead of glucose for mitochondrial ATP generation. Thus, intrahepaticinsulin resistance attributable to FFA promotes mitochondrial fatty acidb-oxidation. There are, however, conflicting reports of the status of mito-chondrial fatty acid oxidation in NASH. The circulating beta-hydroxybuty-rate (b-OH butyrate) levels, a product of mitochondrial fatty acid oxidation,are increased in NASH. At the same time, there are reports of decreased mi-tochondrial fatty acid oxidation based on exhalation of deuterated hydrogenafter 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 b-oxidation induce a vicious cycle of hepatic insulin resistance by means offailure of phosphorylation of insulin receptor that ultimately leads to failureof phosphorylation of insulin receptor substrate (IRS)-2. As a result,

575HEPATITIS C INFECTION

activation of glycogen synthase (GSK3) and hepatocyte nuclear factor(FOXO) is downregulated. Failure to phosphorylate FOXO results in its nu-clear 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 in-creased activity results in increased hepatic glucose output and worsening ofhyperglycemia.

Despite the strong link between insulin resistance and NAFLD, there issome increasing evidence that NASH may occur in the absence of overtinsulin resistance [18–20]. Although poorly understood, a potential mecha-nism may be related to an underlying intrinsic alteration in the regulation offatty acid metabolism. Studies have suggested that hepatic steatosis inNAFLD can begin as overstorage of unmetabolized energy in hepatocytesin those who consume excess energy that exceeds the energy combustioncapability of the PPAR-a–mediated b-oxidation [18,20,21].

Mechanisms of steatosis in hepatitis C virus genotype 3

Hepatic steatosis is a common nonspecific histologic feature found ingreater than 50% of individuals infected with HCV [22]. Genotype 3a, inparticular, has been shown to link to steatosis more strongly than with othergenotypes [23]. Obesity is also a well-recognized risk factor for the develop-ment of steatosis and fibrosis in HCV-infected patients [24,25].

Genotype 3 is the only subtype that has been shown to correlate witha higher grade of steatosis independent of other host-related factors, suchas the presence of NAFLD [26]. The severity of steatosis in these patientsis directly related to the burden of the HCV RNA viral load, and resolutionof steatosis is often observed with the loss of viremia after antiviral treat-ment [27–29]. It has been postulated that HCV genotype 3 can cause steato-sis also by interfering with triglyceride secretion.

The exact mechanism of HCV-induced steatosis is not completely under-stood, although research studies have been slowly unraveling several potentialcontributors. In particular, HCV core protein has been studied in vitro and intransgenicmice. It seems that intracellular lipid accumulation occurs when theHCV core protein is strongly expressed [30] and that the core protein can befound on the surface of lipid droplets within the cytoplasm in cell culturestransfected with HCV, although it is absent in control cells [31]. In a recent re-port, HCV core protein and NS4b proteins derived from genotype 3a werefound to be capable of inducing proteolytic cleavage of SREBP, a transcrip-tion 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 met-abolic perturbations caused by activation of proinflammatory mechanismsand underlying obesity and insulin resistance. The degree of steatosis in

576 CHEUNG & SANYAL

this genotype is independent of HCV viral load, and the use of antiviraltherapy alone does not lead to improvement in steatosis in these patients.Similar data have been obtained for genotype 4 infection, whereas fewdata are available regarding genotype 2 infection [33].

HCV virus, regardless of genotype, has been shown to cause insulin resis-tance through direct effects on viral proinflammatory cytokines and suppres-sors of cytokine signaling [34–37]. Using HCV core gene transgenic mice,a recent study has demonstrated the presence of impaired glucose toleranceand marked insulin resistance in those animals [38]. Furthermore, the sametransgenic mice showed an increased level of tumor necrosis factor-a (TNFa)that has been previously shown, in vivo and in vitro, to induce insulin resistancebymeans of inhibition of tyrosine phosphorylation of IRS-1 and IRS-2 [39,40].

Additional studies, as shown in Table 1, have identified factors associatedwith hepatic steatosis in HCV. HCV entry into hepatocytes may be mediatedby low-density lipoprotein (LDL) receptor, and that HCV core protein mayinteract with apoA2, which is a major component of high-density lipopro-teins, and this interaction can lead to hepatocellular steatosis by inhibitingmicrosomal triglyceride transfer protein activity [41]. Another nonstructuralprotein, NS5A, has been found to interact with apoA1 and apoA2, and there-fore can lead to altered cholesterol trafficking [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 nonalcoholicfatty liver disease

The concomitant presence of HCV and NAFLD is inferred from thepresence of greater than 5% steatosis in the liver in subjects who do notconsume clinically relevant (O20 g/d for women and O30 g/d for men)amounts of alcohol. The diagnosis of steatohepatitis is somewhat more chal-lenging. Lobular inflammation can be seen in NASH and HCV, and its

Table 1

Summary of studies regarding hepatic steatosis in hepatitis C virus

Author, year Patients (n) Characteristics associated with steatosis (P)

Hourigan et al [75] 148 BMI (0.0001), age (0.002)

Rubbia-Brandt et al [23] 101 Genotype 3 (0.002)

Adinolfi et al [45] 180 Genotype 3 (!0.01), BMI in genotype 1 (!0.001),

visceral fat distribution (!0.001)

Hickman et al [76] 160 Genotype 3 (0.0001)

Sanyal et al [77] 144 BMI (!0.001), weight (!0.003)

Younossi et al [49] 120 BMI (0.03), genotype 3 (0.03)

Castera et al [27] 151 BMI O25 kg/m2 (0.02), genotype 3 (0.07)

Patton et al [29] 574 Genotype 3 (!0.03)

Liu et al [78] 95 Hyperglycemia (0.01), BMI O27 kg/m2 (!0.01)

Abbreviation: BMI, body mass index.


presence cannot therefore be taken as a feature of NASH in such patients.The presence of cytologic ballooning along with steatosis serves as the min-imal requirement to make the diagnosis of steatohepatitis in subjects whohave HCV. Although Mallory’s hyaline strengthens the case for steatohepa-titis, it is not always present and its absence does not exclude steatohepatitis.Similarly, pericellular fibrosis is not always present in steatohepatitis, and itsabsence does not exclude the diagnosis. It is always important to rememberthat the ‘‘nonalcoholic’’ nature of the disease has to be assessed clinically.

As shown in Table 2, steatosis secondary to metabolic reasons (genotype1, NAFLD) or HCV virus (genotype 3) worsens the sequence of events lead-ing to advanced fibrosis in patients who have HCV. Progression of HCVtoward cirrhosis is also accelerated by several factors, such as male gender,age, alcohol use, HIV coinfection, HCV duration and genotype, the pres-ence of diabetes mellitus, insulin resistance, and obesity [44–47].

In patients infected with HCV, NASH has been reported to be more com-monly seen in those who are obese, especially with central obesity, and inthose with advanced age, hyperglycemia, hypertriglyceridemia, elevatedtransaminases, and HCV genotype 3 [48]. These patients also tend to havemore advanced stages of fibrosis compared with those with superimposedsteatosis alone or no steatosis at all [49].

Mechanisms

As a pathophysiologic hallmark of NAFLD, insulin resistance is stronglylinked to oxidative stress and inflammatory cytokines [50,51]. As previouslymentioned, patients who have NAFLD have high circulating levels of lipidperoxidation products that are proinflammatory and profibrotic. Interest-ingly, the same observation has also been reported in patients who have

Table 2

Factors associated with advanced fibrosis in hepatitis C virus

Author, year Patients (n) Characteristics associated with fibrosis (P)

Mihm et al [79] 85 Hepatic steatosis (n/a)

Hourigan et al [44] 148 Hepatic steatosis (!0.03)

Adinolfi et al [45] 180 Hepatic steatosis (!0.001), age (!0.001)

Clouston et al [80] 80 Hepatic steatosis (0.001), age (0.003)

Hui et al [34] 260 HOMA-IR (!0.001)

Poynard et al [81] 1428 Hepatic steatosis (0.007)

Ratziu et al [82] 710 Hyperglycemia (!0.01), BMI (!0.01),

steatosis (!0.01)

Sanyal et al [77] 144 BMI (!0.003), cytologic ballooning (!0.003),

diabetes (!0.03)

Younossi et al [49] 120 Superimposed NASH (!0.001)

Rubbia-Brandt et al [26] 755 Hepatic steatosis (!0.001 in genotype 3)

Bugianesi et al [83] 132 HOMA-IR (0.02 in genotype 3)

Abbreviations:BMI, bodymass index; HOMA-IR, Insulin resistance was calculated using the

homeostasis model assessment (HOMA) method as per the following equation: insulin resistance

(HOMA-IR)¼ fasting insulin (mU/mL)� fasting glucose (mmol/L)/22.5; n/a, data not available.

578 CHEUNG & SANYAL

chronic HCV [52–54]. In general, steatosis leads to increased lipid peroxida-tion in hepatocytes, which, in turn, activates hepatic stellate cells (HSCs) toproduce extracellular matrix components and collagen deposition [55–57].The potential use of antioxidant therapy to halt hepatic fibrogenesis, reducelipid peroxidation, and attenuate HSC activation has been investigated.Early studies have shown that the use of vitamin E in patients who havechronic HCV results in a greater chance of obtaining a complete virologicresponse to interferon (IFN) therapy and a significantly greater reductionin viral load than in patients without vitamin E treatment [58]. There isalso evidence that the use of vitamin E in chronic HCV significantly reducesultrasonographic scores for liver steatosis and improves liver enzyme levels,hyperinsulinemia, and liver fibrosis [59]. Although the exact mechanism ofhow antioxidant improves these parameters is poorly understood, furtherlarge-scale studies are needed to investigate the impact of oxidative stresson 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 concurrenthepatic steatosis

Generally, the presence of hepatic steatosis reduces the likelihood ofachieving an SVR to pegylated IFN and ribavirin combination therapy,especially if steatosis is greater than 33% [60]. Some studies have implicatedinsulin resistance and cytologic ballooning as additional markers for failureof antiviral therapy. Although steatosis is an important cofactor in the pro-gression of fibrosis and necroinflammation in HCV, the exact mechanismwhereby steatosis may have a negative impact on the response to antiviraltherapy remains poorly understood. The current concepts related to thisare reviewed here.

Mechanisms

Hepatic steatosis is associated with obesity and insulin resistance. Obesity,insulin resistance, and HCV genotype have also been reported to affect anti-viral response (Table 3). In addition to causing steatosis and fibrosis progres-sion in HCV, insulin resistance has been shown to correlate with a poorresponse to antiviral therapy, particularly in HCV genotype 1 [61]. Obesity,per se, has been previously reported to be a risk factor for nonresponse toantiviral therapy independent of HCV genotype and the presence of cirrhosis.Obese patients have approximately an 80% lower chance of achieving anSVR to antiviral therapy compared with nonobese patients [62]. Althoughthe actual mechanism whereby obesity affects the response to antiviraltherapy is not completely understood, it is speculated that steatosis in obesepatients who have HCV has increased accumulation of lipid droplets withinhepatocytes, which can act as a functional barrier for the interaction between

Table 3

Factors associated with poor response to antiviral therapy in hepatitis C virus

Author, year Patients (n) Characteristics associated with nonresponse (P)

Akuta et al [84] 394 Hepatic steatosis, genotype 1 (n/a)

Bressler et al [62] 174 BMI O30 kg/m2 (0.01), cirrhosis (!0.01),

genotype 1 (!0.01)

Poynard et al [81] 1428 BMI, hepatic steatosis (!0.001)

Sanyal et al [77] 144 Presence of NAFLD (p!0.01)

Patton et al [29] 574 Genotype 1 (0.02)

Harrison et al [60] 231 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, thedrug may be preferentially absorbed through blood capillaries or the lym-phatic circulation. Because obese people are known to have poor lymphaticcirculation [64], this can potentially lead to suboptimal serum levels of pegy-lated IFN, thus reducing the likelihood of a successful antiviral response.

Another potential mechanism whereby obesity may affect antiviralresponse is through modulation of the interferon signaling pathway. Nor-mally, IFNa-activated cellular signaling is negatively regulated by inhibitoryfactors, 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 phosphor-

ylated 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 efficacy.

580 CHEUNG & SANYAL

As reported in a recent study, obese subjects who have HCV genotype 1 werefound to have increased mRNA expression of SOCS-3 compared with con-trols [65]. This potential association between obesity and altered cytokine-mediated regulation of interferon signaling may contribute to poor antiviralresponse 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 isan integral part of HCV replication, and it is believed that HCV virus entershepatocytes by means of LDL receptors [66]. Hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase) is associated with upregula-tion of LDL receptors [67], and its inhibitor (statins) has been shown to becapable of inhibiting HCV viral replication in vitro [68–70]. The same find-ing, however, has not been observed in vivo at a similar conventional doseof statins [71]. Ikeda and colleagues [68] have recently demonstrated in vitrothat fluvastatin, when compared with other statins, exhibits the strongestanti-HCV activity, with a much lower dose required to achieve a 50% reduc-tion in RL activity (median inhibitory concentration [IC50]). The group alsoreported that the use of a statin with IFNa seems to enhance the antiviraleffect of IFN well beyond the activity observed when the drug is used alone.The anti-HCV activity of statins results from the inhibition of geranylgera-nylation of HCV cellular proteins rather than from direct inhibition of cho-lesterol biosynthesis. Geranylgeranyl is a cholesterol intermediate that hasbeen shown in vitro to be crucial in HCV replication [69,70]. Statins mayseem to be a potential therapeutic option for HCV infection; however, be-cause of the lack of in vivo data supporting their antiviral efficacy and thefact that higher total cholesterol and LDL levels have been observed and re-ported to be associated with increased SVR rates after IFN-based therapy, itis suggested that lowering LDL levels may not necessarily be beneficial forantiviral therapy [72,73]. Further study is therefore required to define the in-teraction between statins and IFN response.

Increase the efficacy of antiviral therapy for hepatitis C virus–infectedpatients who have concomitant nonalcoholic steatohepatitis

Subjects infected with HCV genotypes 2 and 3 have a higher probability ofachieving an SVR to combination antiviral therapy, regardless of the presenceof hepatic steatosis, compared with those infected with genotype 1. In suchsubjects, it may not be necessary to treat the underlying NAFLD or NASHbefore anti-HCV therapy. Conversely, subjects who have genotype 1 infectionare at greater risk for becoming virologic nonresponders. There is thereforea need to modify HCV treatment to increase the response rate.


Although the presence of concomitant NAFLD increases the risk for anti-HCV treatment failure, there are no data on the efficacy of treating NAFLDas a way to improve virologic response. Despite this lack of evidence, there isstill considerable enthusiasm to treat NAFLD as a modifiable risk factorto improve the response to antiviral therapy in subjects who have HCV.Although optimal therapy for NAFLD remains to be defined, the focus oftreatment on the underlying risks (ie, obesity, insulin resistance) is a reason-able therapeutic goal on the basis of achieving improvement in cardiovascularrisks. It is therefore reasonable to treat obesity and the metabolic syndromewhen they are present. In the absence of any data on treating NASH toimprove virologic response, antiviral therapy may not need to be withheld,but in addition, one should consider instituting a diet and lifestyle planshortly before starting treatment. For those in whom a virologic response ismost urgently required, such as those who have severe inflammation or bridg-ing fibrosis, it may be necessary to treat obesity aggressively with the goal ofimproving insulin resistance and steatosis. There are emerging data suggest-ing that thiazolidinediones (eg, pioglitazone) may be effective in improvingsteatohepatitis [74]. Finally, the use of inhibitors of cholesterol and lipidbiosynthesis as adjuncts to HCV therapy, especially in patients who do nothave dyslipidemia, merits further evaluation.

Summary

Hepatitis C is a common chronic liver disease in theworld.Disease progres-sion and virologic response to antiviral therapy are greatly affected by severalcrucial host and viral factors, with two such important contributors being thepresence of NAFLD and insulin resistance. Obesity and insulin resistance aresignificant public health concerns in North America, and it has becomeincreasingly evident that NAFLD is a frequently encountered challenge inmanaging patients who have HCV in terms of clinical and histologic manifes-tations in addition to treatment efficacy. The presence of concomitant HCVand NAFLD is associated with greater fibrosis and a low rate of SVR toantiviral therapy.

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Management Complexitiesof 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

aDivision of Infectious Diseases, Department of Medicine, 502 Robert Wood Johnson Pavilion,

University of Pennsylvania School of Medicine, Philadelphia, PA 19104–6073, USAbDepartment of Biostatistics and Epidemiology and Center for Clinical Epidemiology

and Biostatistics, 423 Guardian Drive, Philadelphia, PA 19104-6021, USAcInfectious Diseases Section, Room 809A, Philadelphia Veterans Affairs Medical Center,

3900 Woodland Avenue, Philadelphia, PA 19104, USA

Because of shared risk factors, approximately one third of patients whohave HIV type 1 (HIV) are coinfected with chronic hepatitis C virus(HCV) infection [1,2]. HIV coinfection accelerates the course of HCV-asso-ciated liver disease, and compared with those infected with chronic HCValone (ie, HCV monoinfected), there is more rapid progression of hepaticfibrosis in HIV/HCV-coinfected individuals [3–5]. Thus, because HIV hasbecome a chronic illness as a result of the effectiveness of highly active anti-retroviral therapy (HAART), HCV-related liver disease has emerged asa major cause of morbidity and mortality among HIV-infected patients inthe developed world [6–8].

Because HIV/HCV coinfection is prevalent and increases the risk ofHCV-associated liver disease, effective anti-HCV therapy is critical for thelong-term survival of these patients. Among HIV-infected individuals,HCV therapy can lead to viral eradication [9–12]; may halt or regress he-patic fibrosis [13,14]; and has been shown to be cost-effective [15]. A varietyof complexities, including overall reluctance by patients and providers toinitiate HCV therapy, increased hepatotoxicity of antiretroviral therapy,

Clin Liver Dis 12 (2008) 587–609

This article was supported by National Institutes of Health research grant K01 AI070001-

01A1 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 19104–6073.

E-mail address: [email protected] (V. Lo Re).



588 LO RE et al

drug-drug interactions, and adverse effects of HCV therapy, have mademanagement of chronic HCV infection a major challenge in the HIV-in-fected population, however.

In this article, the authors review the (1) epidemiology of HCV amongHIV-infected individuals, (2) effect of HIV on the natural history ofchronic 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 efficiently by percutaneous exposure tocontaminated blood, through sexual intercourse, and from mother to infant.Because both viruses have similar routes of transmission, coinfection withHCV is common among HIV-infected individuals. In the United States,a cross-sectional analysis of two large HIV trials (n ¼ 1687 subjects) demon-strated that the overall prevalence of HCV coinfection was 16.1% (95%confidence interval [CI]: 14.3%–17.8%) [1]. Approximately 80% of these pa-tients were infected with HCV genotype 1, and 75% had high HCV RNAlevels (ie, O800,000 IU/mL) [1]. Similar HCV prevalence rates have beendemonstrated among HIV-infected populations in France [16], Germany[17], Switzerland [18], and Greece [19]. In contrast, the prevalence of HCVinfection in the general US population is 1.6% [20].

The prevalence of HCV infection varies with the mode of transmission ofHIV. As such, chronic HCV has been reported in up to 90% of HIV-infectedhemophiliacs [21–23] and 90% ofHIV-positive injection drug users [2,24–27].Transmission of HIV and HCV through blood products has been reducedmarkedly in the United States by screening of blood donations for both vi-ruses, however [28]. In contrast, the incidence of HCV infection amongHIV-infected homosexual men has increased recently, and unprotectedanal intercourse, traumatic sexual practices, and concomitant sexually trans-mitted diseases have been the main risk factors for HCV acquisition [29].

The risk of perinatal transmission of HCV is increased for infants bornto HIV/HCV-coinfected mothers. A recent meta-analysis of 10 studiesdemonstrated that the risk for HCV vertical transmission among HIV/HCV-coinfected mothers was 2.82 (95% CI: 1.8–4.5) compared with HCV-monoinfected mothers [30]. The cumulative incidence of HCV infection is17.1% in infants born to HIV/HCV-coinfected mothers compared with5.4% for those born to mothers with only HCV infection [31–33]. HigherHCV RNA levels are associated with increased perinatal transmission [32].

Effect of HIV on the natural history of hepatitis C virus

HIV infection adversely affects every aspect of the natural history ofchronic HCV. Although 14% to 45% of HCV-monoinfected individualsspontaneously clear HCV after acute infection, HCV clearance occurs in

589MANAGEMENT COMPLEXITIES OF HIV/HEPATITIS C VIRUS COINFECTION

only 5% of HIV/HCV-coinfected persons, and less often in those with lowerCD4 cell counts [34–36]. HIV/HCV coinfection is associated with higherHCV RNA levels compared with HCV-monoinfected patients [5,37–40],and HCV RNA levels increase as the CD4 cell count decreases, suggestingthat HIV-induced immune deficiency allows for increased HCV replication[5,38].

In addition, liver fibrosis progression is accelerated in HIV/HCV-coin-fected patients, with a more rapid progression to cirrhosis compared withHCV-monoinfected individuals [3,5]. HIV/HCV-coinfected persons are athigher risk for advanced hepatic fibrosis and cirrhosis compared withHCV-monoinfected individuals [3,5,37,41–44]. Among 67 HIV/HCV-coin-fected patients undergoing paired liver biopsies separated by a median of2.8 years, Sulkowski and colleagues [45] demonstrated that 28% of patientshad an increase of at least two modified Ishak stages of hepatic fibrosis.Among those with mild fibrosis on initial biopsy, 26% had a two-stage pro-gression on follow-up biopsy.

HIV/HCV coinfection increases the risk of hepatocellular carcinomacompared with HIV-monoinfected patients (adjusted hazard ratio [HR] ¼5.35, 95% CI: 2.34–12.20) [46] but not compared with HCV-monoinfectedpatients (adjusted HR ¼ 0.84, 95% CI: 0.55–1.27) [47]. This cancer presentssooner after cirrhosis develops [48] and more commonly as infiltrating andmultifocal lesions [49] in HIV/HCV-coinfected patients compared withthose with chronic HCV alone.

The effect of HIV on HCV-related end-stage liver disease has been exam-ined exclusively in patients who have hemophilia. These studies show thatthe cumulative incidence of hepatic failure is 11% to 35% over a mean fol-low-up of 10 to 24 years from initial factor concentrate exposure, corre-sponding to a yearly incidence of 1.5% per year [21–23,50–52]. Theresults also indicate a 3- to 21-fold increase in the risk of end-stage liver dis-ease in HIV/HCV-coinfected patients compared with HCV-monoinfectedpatients.

HIV coinfection decreased the median survival time of patients who hadHCV-associated end-stage liver disease compared with HCV-monoinfectedpatients (16 versus 48 months; P!.001) in a Spanish cohort [53]. The risk ofdeath in HIV-infected patients is substantially higher in HIV/HCV-coin-fected patients compared with those with chronic HCV alone (relative risk[RR] ¼ 2.26, 95% CI: 1.51–3.38) [53]. Predictors of death among HIV/HCV-coinfected patients were Child-Pugh score (HR ¼ 1.20, 95%CI: 1.08–1.37), CD4 count less than 100 cells/mm3 (HR ¼ 2.48, 95%CI: 1.52–4.06), and hepatic encephalopathy at the time of decompensation(HR ¼ 2.45, 95% CI: 1.41–4.27) [54].

With the increased longevity of HIV-infected patients as a result of po-tent antiretroviral therapy and the prophylaxis of traditional opportunisticpathogens, HCV-related liver disease has emerged as a major cause of mor-bidity and mortality in this population [6,7,55–58]. Results from the Data

590 LO RE et al

Collection on Adverse Events of Anti–HIV Drugs (D:A:D) Study, a collab-orative network of 11 HIV-infected cohorts from North America, Europe,and Australia, demonstrate that HCV-related liver disease is now the secondleading cause of death in the HIV-infected population [8]. Among 23,441HIV-infected patients enrolled in the study between December 1999 andFebruary 2004 and followed for a median of 3.5 years, 1246 deaths occurredand 181 (14.5%) were liver related. Among these liver-related deaths, 66%were attributable to chronic HCV, 17% were attributable to chronic hepa-titis B, and 7% were attributable to hepatitis B and HCV. In addition, re-cent data from the Strategies for Management of Antiretroviral Therapy(SMART) study demonstrate that mortality rates among HIV/viral hepati-tis-coinfected individuals are nearly four times those of HIV-monoinfectedpersons [59].

In summary, these data clearly show that chronic HCV is more severe inHIV/HCV-coinfected patients. A greater proportion of HIV/HCV-coin-fected patients develop chronic HCV, and HIV/HCV-coinfected patientsare at substantially higher risk for liver-related complications, such as cir-rhosis, end-stage liver disease, and hepatocellular carcinoma comparedwith HCV-monoinfected patients. In addition, HCV-associated liver diseaseis now the major cause of morbidity and mortality among HIV-infectedpatients in the developed world.

Impact of antiretroviral therapy on hepatitis C virus infection

Effect of antiretroviral therapy on the natural history of hepatitis C virus

Recent cohort studies have shown that use of HAART, particularlyachieving HIV suppression, is associated with better hepatic outcomes inHIV/HCV-coinfected patients. Mehta and colleagues [60] reported that de-tectable HIV RNA (adjusted odds ratio [OR] ¼ 3.4, 95% CI: 1.6–7.1) andshorter cumulative HAART exposure (adjusted OR per year of exposure ¼0.8, 95% CI: 0.70–0.96) were associated with necroinflammatory activityamong coinfected persons. Brau and colleagues [61] found that HIV/HCV-coinfected patients with HIVRNA suppression had a slower fibrosis progres-sion rate than those with detectable HIV RNA (0.122 versus 0.151 Ishakfibrosis units per year; P ¼ .013) but similar to that of HCV-monoinfectedpatients (0.122 versus 0.128 Ishak fibrosis units per year; P ¼ .52). Similarly,Verma and colleagues [62] reported that HIV/HCV-coinfected persons onHAART had comparable liver histology findings to those of HCV-monoin-fected persons. In a study of 162 HIV/HCV-coinfected individuals whounderwent liver biopsy, absence of protease inhibitor therapy as part ofthe antiretroviral regimen was an independent predictor of progression tocirrhosis (RR ¼ 4.74, 95% CI: 1.34–16.67) [63].

Use of HAART has also been shown to be associated with reduced liver-related mortality among HIV/HCV-coinfected individuals. A German


cohort study that followed coinfected patients over a 12-year period showedthat HAART was an independent predictor of liver-related survival (OR ¼0.106, 95% CI: 0.020–0.564) [64]. Among HIV/HCV-coinfected patientswith preexisting hepatic failure, HAART was associated with improved sur-vival (HR ¼ 0.5, 95% CI: 0.30–0.90) and 40% of HIV/HCV-coinfected pa-tients receiving HAART were alive after 3 years compared with 18% not onHAART [54]. Finally, the recent D:A:D study [8] showed that there was lit-tle change in the liver-related death rate with increasing cumulative durationof HAART use.

Taken together, the available data suggest that HAART favorably affectsthe course of HCV in HIV-infected patients, decreases the rate of death at-tributable to liver disease, and should not be withheld from HIV/HCV-coinfected persons because of fears regarding toxicity.

Hepatotoxicity of antiretroviral therapy in hepatitis C virus coinfection

Although the use of HAART has had enormous benefits for HIV/HCV-coinfected patients, liver toxicity associated with these medications remainsa concern for physicians who treat this population. Four main mechanismsof antiretroviral-related hepatotoxicity have been described in HIV-infectedindividuals: (1) mitochondrial toxicity, (2) hypersensitivity reactions involv-ing the liver, (3) direct drug toxicity, and (4) immune reconstitution afterHAART initiation in the presence of hepatitis coinfection [65]. More thanone 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 se-verity of liver injury [66]. Severe hepatotoxicity, the primary outcome inmost hepatotoxicity studies in the HIV-positive population, has been de-fined as a grade 3 (5.1–10 times the upper limit of normal) or grade4 (O10 times upper limit of normal) change in aspartate aminotransferase(AST) or alanine aminotransferase levels during HAART treatment ora greater than 3.5-fold increase in these levels higher than baseline if amino-transferases are elevated at HAART initiation [67].

Using these criteria, studies have shown that HCV coinfection increasesthe risk of severe hepatotoxicity in HIV-infected patients treated withHAART [67–75]. Discontinuation of HAART is more frequent and occursearlier among HIV/HCV-coinfected patients than for those with HIV alone[76]. Moreover, the risk of severe hepatotoxicity with HAART is increasedfor HIV/HCV-coinfected patients with advanced (METAVIR stage 3 or 4)fibrosis (RR ¼ 2.75, 95% CI: 1.08–6.97) [77]. Anxiety related to hepatotox-icity should not, as it often does, dissuade or delay patients and physiciansfrom initiating a therapy that may attenuate HCV-related liver disease andreduce liver-related mortality, however.

Given the increased risk of hepatotoxicity in the HIV/HCV-infected pop-ulation, eradication of HCV with antiviral therapy might improve tolerance

592 LO RE et al

to HAART. In a recent study among 132 HIV/HCV-coinfected patientstreated with interferon-based therapy in Spain, the yearly incidence of severehepatotoxicity was greater in patients who did not achieve a sustained viro-logic response (SVR; defined as an undetectable HCV RNA level at the endof treatment and 24 weeks later) than in those who did (12.9% versus 3.1%;P!.001) and in patients who had advanced liver fibrosis than in those whodid not have it (14.4% versus 7.6%; P ¼ .003) [78]. Failure to achieve SVR(OR ¼ 6.13, 95% CI: 1.83–37.45) and use of didanosine or stavudine as partof the antiretroviral regimen (OR ¼ 3.59, 95% CI: 1.23–10.42) were inde-pendent predictors of hepatotoxicity after interferon therapy. These datademonstrate that achieving SVR after anti–HCV treatment can reduce therisk of hepatotoxicity during antiretroviral therapy, which should furtherencourage the treatment of chronic HCV in HIV.

Certain antiretroviral medications or classes may be more likely to pro-duce elevated aminotransferases or lead to clinically apparent hepatotoxicity(Table 1). The use of the nucleoside analogues stavudine, didanosine, ortheir combination can lead to a higher rate of hepatic steatosis in the coin-fected population [79]. The nonnucleoside reverse transcriptase inhibitornevirapine has been shown to increase the risk of severe hepatotoxicity inHIV/HCV-coinfected patients, and elevations in aminotransferase levelsmay develop 4 to 6 months after initiation of the medication [67,80,81]. Ne-virapine use has also been associated with more advanced hepatic fibrosis(adjusted OR ¼ 2.56, 95% CI: 1.02–6.58) in a cross-sectional study of 152HIV/HCV-coinfected patients who underwent a liver biopsy [82]. Incontrast, most HIV/HCV-coinfected patients who initiate a proteaseinhibitor–containing regimen do not experience treatment-limiting hepato-toxicity. Although liver enzyme elevations may develop with any proteaseinhibitor, lopinavir-ritonavir, fosamprenavir-ritonavir, and nelfinavir seemto be less hepatotoxic [83] and tipranavir-ritonavir seems to be the mosthepatotoxic [84]. An unconjugated hyperbilirubinemia can occur duringatazanavir and indinavir therapy but does not reflect liver damage and isrelated to the inhibition of the uridine diphosphate glucuronosyl transferaseenzyme [85,86]. Dual protease inhibitor therapy does not increase the rate ofhepatotoxicity [87]. Data on the hepatotoxicity of fusion inhibitors, inte-grase inhibitors, and chemokine receptor antagonists in HIV/HCV-coin-fected patients are lacking.

Integrating hepatitis C virus care in HIV practice

Multidisciplinary approach

HIV/HCV-coinfected patients should be referred to a hepatologist orinfectious diseases physician with expertise in HIV/HCV coinfection toprovide more information about diagnosis, natural history, and therapeuticoptions. Clinical management should subsequently be multidisciplinary,

Table 1

Specific antiretroviral concerns in persons coinfected with HIV and hepatitis C virus

Drug Comment Recommendation

Abacavir � 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]

� Ensure adequate weight-based ribavirin dosing

� Emphasize ribavirin adherence

Didanosine � 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 [133–136]

� Increased risk of hepatic decompensation during

HCV therapy in coinfected patients who have

cirrhosis [137,138]

� Concomitant use with ribavirin contraindicated

Nevirapine � Increased rate of severe hepatotoxicity in

HIV/HCV-coinfected persons [67,80,81]

� May be associated with hepatic fibrosis in coinfected

individuals [82]

� Consider alternate antiretroviral agent in coinfected

persons

Stavudine � May be associated with hepatic steatosis in

HIV/HCV-coinfected persons [79]

� Consider avoiding use of stavudine, if possible

Zidovudine � Can potentiate ribavirin-induced anemia, possibly

through suppression of hematopoiesis [131]

� Avoid concomitant use with ribavirin

� Monitor hemoglobin levels closely if no other

antiretroviral options

593

MANAGEMENTCOMPLEXIT

IESOFHIV

/HEPATIT

ISC

VIR

USCOIN

FECTIO

N

594 LO RE et al

with input from advanced practice nurses, psychiatrists, pharmacists, dieti-cians, and addiction management experts [88].

Support and education are crucial to the management of chronic HCV inHIV-infected patients, and health care providers should provide additionaleducational materials and offer referral to support groups to those undergo-ing evaluation for established HCV infection. Patients should be counseledto 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 ofchronic HCV [89] and may compromise antiviral therapy by decreasingadherence or interfering with the antiviral action of interferon-based ther-apy [90]. Efforts to diagnose and treat alcohol abuse should be performedin all HIV/HCV-coinfected patients before beginning HCV therapy, andrelapse into drug and alcohol use should be repeatedly assessed. Treatmentfor drug and alcohol abuse should be made available to all who want andneed it. Safe levels of alcohol consumption in HIV/HCV-coinfected pa-tients remain unclear, but even moderate levels of consumption may accel-erate disease progression [91]. All HIV/HCV-coinfected patients shouldtherefore be advised to abstain from alcohol [92,93].

Treatment of neuropsychiatric disorders

Neuropsychiatric disorders, particularly depression, are common amongHIV/HCV-coinfected patients [94] and are a frequent adverse effect of pegy-lated interferon therapy [9–11]. Identification and treatment of neuropsychi-atric 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 tohepatitis A virus infection (anti–hepatitis A immunoglobulin G [IgG]antibody) and previous or concurrent hepatitis B virus infection (hepatitisB surface antigen, anti–hepatitis B core IgG antibody, and anti–hepatitisB surface antibody). Acute infection with hepatitis A or B in those withunderlying chronic HCV increases the risk for fulminant hepatitis and canresult in high morbidity [95,96]. Despite evidence of decreased response inHIV-infected persons, hepatitis A and B vaccination should be performedin those who are seronegative for these viruses [97].

Liver fibrosis assessment

Evaluation of liver histology with a liver biopsy is the best tool for assess-ing the likelihood of progression of hepatic injury and is a better predictor of


subsequent clinical events than hepatic aminotransferase elevations, HCVgenotype, or HCV RNA level [98,99]. In one study, 29% of HIV/HCV-coinfected patients with persistently normal alanine aminotransferase levelshad advanced fibrosis on liver biopsy [100]. As such, many experts recom-mend a liver biopsy in HIV/HCV-coinfected patients to assess the extentof underlying liver damage. The liver biopsy can help to guide HCV treat-ment decisions, permits direct determination of the degree of necroinflam-mation, and allows detection of other hepatic abnormalities (eg, steatosis,iron overload, concomitant infections) [101]. Evaluation of liver histologywith a liver biopsy can also inform the need for hepatocellular carcinomascreening, which is recommended once cirrhosis is present.

Despite the value of the liver biopsy, it possesses several limitations thathave contributed to low acceptance by HIV/HCV-coinfected patients, par-ticularly its invasive nature, occasional serious complications [101], samplingerror attributable to the small size of the extracted tissue and inherentheterogeneity of hepatic fibrosis [102,103], and high cost [104]. As a result,noninvasive modalities to evaluate hepatic fibrosis have been increasinglyexamined in the HIV/HCV-coinfected population [105]. These noninvasivetools are currently divided into two major categories: (1) serum biochemicalmarkers (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]) [106–109] and (2)imaging techniques, primarily elastometry (FibroScan, Echosens, Paris,France) [110,111]. These tools have high predictive value in identifyingadvanced hepatic fibrosis and cirrhosis, but they have been imprecise in dis-tinguishing among the intermediate stages of hepatic fibrosis [112]. In addi-tion, serum fibrosis markers have generally been less reliable in coinfectedpatients because of the inflammatory nature of HIV disease and coadminis-tration of medications that may interfere with test results.

Given the accelerated progression of HCV-related liver disease amongHIV-infected patients, improvements in the efficacy of HCV therapy inthe HIV-infected population, the high predictive value of the early virologicresponse to HCV therapy (at week 12 of treatment) to identify who does andwho does not respond, and the acknowledged limitations of the liver biopsy,current HIV/HCV coinfection management guidelines no longer requirethat 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 consideredin all coinfected patients [113]. Combination pegylated interferon plus riba-virin for 48 weeks represents the standard of care for treating chronic HCVin HIV-infected individuals, as it is for HCV-monoinfected persons. As with

596 LO RE et al

HCV-monoinfected patients, the primary goal of treatment is viral eradica-tion (ie, SVR) [91,112]. A second potential benefit of HCV therapy is a re-duction in the risk for liver-related complications. Among a Spanish cohortof antiretroviral-treated HIV/HCV-coinfected patients, receipt of HCVtherapy was associated with improved survival, and no episodes of hepaticdecompensation were noted among subjects who achieved an SVR [114]. Inan Italian retrospective cohort study, HIV/HCV-coinfected patients withcirrhosis who received a mean of 9 months of pegylated interferon plusribavirin without virologic response were less likely to develop adverseliver-related outcomes (ie, ascites, jaundice, encephalopathy, variceal bleed-ing, hepatocellular carcinoma, death) compared with age- and Child-Pughscore–matched 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 pegy-lated interferon plus ribavirin is the optimal therapy for chronic HCVamong HIV-infected patients [9–11]. There were notable differences intype of pegylated interferon use, dose regimen of ribavirin, and type ofcoinfected patients enrolled between the studies, and these are highlightedin Table 2. These differences make it impossible to compare overall resultsbetween these clinical trials.

In each study, the highest SVR rates were observed in the pegylatedinterferon plus ribavirin arms. SVR rates for HCV genotype 1–infected per-sons in these arms were 14% for ACTG Study 5071, 17% for the RIBAVICtrial, and 29% for the APRICOT [9–11]. For subjects infected with HCVgenotype 2 or 3, SVR rates were considerably higher: 44% (RIBAVIC trial),62% (APRICOT), and 72% (ACTG Study 5071) [9–11], emphasizing theimportance of HCV genotype as a predictor of SVR, as in studies ofHCV-monoinfected patients. Similarly, low pretreatment HCV RNA level(%800,000 IU/mL) is also associated with SVR [9]. This was highlightedin the APRICOT, in which SVR rates were 61% among persons who hadHCV genotype 1 and a baseline HCV RNA level of 800,000 IU/mL orless who were randomized to pegylated interferon plus ribavirin [9]. In con-trast, the SVR rate was only 18% for those who had HCV genotype 1 andan HCV RNA level greater than 800,000 IU/mL who received pegylatedinterferon plus ribavirin. Additional predictors of SVR that have beenreported include absence of prior history of injection drug use [10], detect-able HIV RNA [10], age of 40 years or younger [11], baseline alanine ami-notransferase level greater than three times the upper limit of normal [11],antiretroviral therapy with a nonnucleoside reverse transcriptase inhibitoror protease inhibitor [116], and non-black race [116]. The early virologicresponse, defined as a greater than 2-log IU/mL decrease in HCV RNA levelafter 12 weeks of therapy, has high predictive value in patients who havecoinfection [9–11]. Thus, if a patient has not had an early virologic response,

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 ACTG 5071 (n ¼ 133) APRICOT (n ¼ 868) RIBAVIC (n ¼ 412) PRESCO (n ¼ 389)

Site United States United States, Europe, Australia France Spain

Peginterferon formulation Peginterferon a-2a Peginterferon a-2a Peginterferon a-2b Peginterferon a-2a

Ribavirin dosage 600–1000 mg/d 800 mg/d 800 mg/d 1000–1200 mg/d

HCV genotype 1 (%) 77 61 48 51

Bridging fibrosis/cirrhosis (%) 11 12 39 27

On antiretroviral therapy (%) 85 84 83 67

CD4 count (cells/mm3) 453 (median) 520 (mean) 547 (median) 540 (mean)

Undetectable HIV RNA (%) 61 (!50 copies/mL) 60 (!50 copies/mL) 70 (!400 copies/mL) 72 (!50 copies/mL)

SVR rate, genotype 1 (%) 14 29 17 35

SVR rate, genotype 2/3 (%) 73 62 44 72

Withdrawal rate (%) 12 25 39 45

597

MANAGEMENTCOMPLEXIT

IESOFHIV

/HEPATIT

ISC

VIR

USCOIN

FECTIO

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598 LO RE et al

the likelihood of SVR is negligible. Extending therapy in patients who donot have an early virologic response does not increase SVR rates [117].

The SVR rates reported in the pivotal HIV/HCV treatment trials areconsiderably lower than those reported for HCV-monoinfected patients(42%–52% in HCV genotype 1 and 78%–84% in HIV genotypes 2 and3). Possible reasons for the poorer SVR rates among HIV/HCV-coinfectedpatients include (1) high HCV RNA levels in subjects with HIV coinfection,(2) qualitative defects in the cellular and innate immune response, (3) andlower doses of ribavirin or dose escalation administered in the treatment tri-als (because of concern for potential increased risk of hematologic toxicityin this population). Recently, the Spanish Pegasys Ribavirina Espana Coin-feccion (PRESCO) study examined whether administration of weight-basedribavirin (1000 mg/d if body weight !75 kg and 1200 mg/d if body weightO75 kg) in combination with pegylated interferon, as is used in HCV-mono-infected 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 3–infected patientsand 35% of genotype 1– or 4–infected patients. Only 3% of patients stoppedHCV therapy because of severe anemia. As such, recent HIV/HCV manage-ment guidelines now recommend weight-based ribavirin dosing in HIV-pos-itive persons undergoing combination HCV therapy.

When SVR is achieved, HCV therapy can halt or regress hepatic fibrosisin HIV/HCV-coinfected patients, even in the absence of SVR [13,14]. Datafrom the APRICOT show that 70% of patients who achieved SVR withpegylated interferon plus ribavirin had a two-stage improvement in Ishakfibrosis score on a repeat liver biopsy 24 weeks after the end of HCV treat-ment. Among those who did not achieve an SVR, 43% receiving pegylatedinterferon plus ribavirin also had this histologic response. CombinationHCV therapy has also been shown to decrease progression of HCV-relatedfibrosis in HIV-infected individuals [14]. For coinfected patients who havenot responded to HCV therapy or who have relapsed after treatment, clin-ical trials are currently examining whether the long-term administration ofinterferon can prevent liver fibrosis progression even in the absence ofSVR [120]. Preliminary results from ACTG 5178, which randomized coin-fected subjects who did not achieve early virologic response to pegylatedinterferon alfa-2a monotherapy or observation for 72 weeks, demonstratedno difference in fibrosis progression on paired liver biopsies between thegroups, although no significant fibrosis progression was observed in eitherarm [121]. The lack of fibrotic progression in the observation arm precludedthe ability to find efficacy in the pegylated interferon maintenance arm.Thus, since there was no improvement in hepatic fibrosis among thecoinfected non-responders who received pegylated interferon maintenancetherapy, this treatment modality cannot be recommended in routine clinicalpractice. New agents with specific anti-HCV activity are being tested [122],and clinical trials examining the efficacy and safety of these drugs in


coinfected patients should be prioritized without waiting for the final resultsof phase III trials conducted in HCV-monoinfected patients.

Adverse effects 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 treatmentdiscontinuation more frequently among HIV/HCV-coinfected patients(12%–17% withdrawal rates in randomized trials Refs. [9–11]) comparedwith HCV-monoinfected patients (14%–22% withdrawal rates in clinicaltrials Refs. [12,123]).

Results from the APRICOT show that pegylated interferon reduces HIVRNA levels approximately 1.0 log copies among subjects with detectableHIV RNA [9]. In the same study, HCV therapy precipitated a decrease in ab-solute CD4 cell counts throughout the 48-week treatment phase of the study,which then returned to baseline values by 24 weeks after completing HCVtherapy [9]. Despite the decrease in CD4 cell counts, the CD4 cell percentageis typically unchanged, and clinical progression to AIDS was not observed inany subject in the APRICOT during the study period. Cooper and colleagues[124] recently compared infection rates between HIV/HCV-coinfected andHCV-monoinfected patients receiving interferon-based therapy for chronicHCV. Rates of infection did not differ by HIV status, and HIV was not foundto be an independent predictor of infection during HCV therapy.

Leukopenia and thrombocytopenia are dose-related adverse effects of pe-gylated interferon. In particular, use of granulocyte colony-stimulating factorwas allowed in two of the pivotal HIV/HCV coinfection treatment trials toimprove leukopenia [9,10]. Anemia is also a common adverse effect duringcombination anti-HCV therapy [125]. It arises because of the suppressionof erythropoiesis induced by interferon [126] and the reversible hemolysisinduced by ribavirin [127,128]. Reduction of the ribavirin dose had beenrecommended if anemia developed during HCV therapy, but this is associ-ated with reduced SVR rates [123]. Recombinant human erythropoietin cancounteract the anemia associated with HCV therapy in HIV/HCV-coinfectedsubjects and helps to avoid ribavirin dose reduction [129], maximizing theeffectiveness of antiviral therapy. The use of hematopoietic growth factorsin HIV/HCV-coinfected patients has been associated with an improved clin-ical response to pegylated interferon plus ribavirin therapy [130].

A recent retrospective cohort study found that the incidence of significantweight loss (defined as loss of at least 5% of baseline body weight) was sub-stantially greater in dually treated HIV/HCV-coinfected subjects comparedwith treated HCV- or HIV-monoinfected subjects [131]. Among 192patients (n ¼ 63 HIV/HCV-coinfected, n ¼ 64 HCV-monoinfected, n ¼ 65HIV-monoinfected), significant 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 HRof 2.76 (95% CI: 1.67–4.55) and 38.5 (95% CI: 8.5–174.7), respectively.

600 LO RE et al

The degree of weight loss was also greater among the HIV/HCV-coin-fected cohort compared with both monoinfected cohorts. Body weightsfor HIV/HCV-coinfected and HCV-monoinfected subjects were stable be-fore initiation of HCV therapy, but both cohorts lost weight after HCVtreatment initiation, with the rate of weight loss being greater for duallytreated HIV/HCV-infected subjects. Receipt of more than two nucleosidereverse transcriptase inhibitors increased the risk of clinically significantweight loss (adjusted HR ¼ 8.17, 95% CI: 2.37–28.20), suggesting thatmitochondrial toxicity might play some role in weight loss during dualHIV/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 nucle-oside reverse transcriptase inhibitors included in the antiretroviral regimen(see Table 1). A retrospective cohort study among 217 HIV/HCV-coin-fected patients receiving pegylated interferon plus ribavirin found that zido-vudine use was associated with a greater mean hemoglobin decline at4 weeks of HCV therapy (3.1 versus 2.1 g/dL; P!.001) compared withnonusers [132]. By week 12 of HCV therapy, zidovudine use was morefrequently associated with ribavirin dose reduction (52% versus 18%;P!.001) and erythropoietin use (49% versus 23%; P!.001) comparedwith those who did not receive the drug. Zidovudine likely exacerbatesribavirin-related anemia by inhibiting the hematopoietic response to ribavi-rin-induced hemolysis. Thus, it is advisable to avoid zidovudine use duringHCV therapy, and providers should considering switching to an alternativenucleoside reverse transcriptase inhibitor before initiation of HCV treat-ment [112].

Because ribavirin increases the intracellular active metabolite of dida-nosine [133], concomitant use of both medications increases the likelihoodof mitochondrial toxicity, which may lead to pancreatitis and symptom-atic lactic acidosis [134–137]. In the APRICOT and RIBAVIC trial, dida-nosine use increased the risk of hepatic decompensation when used inconjunction with ribavirin [138,139]. As a result, ribavirin should notbe administered to persons taking didanosine as part of their HAARTregimen.

A recent retrospective substudy of the RIBAVIC trial reported that aba-cavir use increased the risk of early virologic failure to combination HCVtherapy (OR ¼ 4.9, 95% CI: 1.5–16.1), possibly because of the intracellularcompetition between ribavirin and abacavir, both guanosine analogues, foractivation through phosphorylation [140]. In a Spanish cohort that con-trolled for serum ribavirin levels, however, abacavir use was not associatedwith early virologic failure, suggesting that appropriate weight-based ribavi-rin dosing and adherence are important when abacavir is part of the antire-troviral regimen [141].


Advanced liver disease
The management of advanced liver disease in HIV/HCV-coinfected
patients is complex. Patients who have cirrhosis should have regular moni-toring for evidence of decompensation and hepatocellular carcinoma. Indi-viduals who have decompensated liver disease are generally not candidatesfor HCV therapy because treatment increases the risk of life-threateningcomplications [139]. HIV therapy may improve hepatic outcomes andsurvival in coinfected patients who have liver failure [54]. Administrationof these medications in this setting is challenging, however, because of alter-ation of hepatic metabolism and the risk of drug-induced liver injury. Ofnote, hepatic metabolism of nonnucleoside reverse transcriptase inhibitors,particularly efavirenz, is impaired in coinfected patients who have cirrhosis,but no similar effect is seen for protease inhibitors [142].

Orthotopic liver transplantation is an option for coinfected patients whohave decompensated liver disease. HIV coinfection is a major determinantof poor outcomes and death in HCV-infected persons undergoing livertransplantation, however [143]. de Vera and colleagues [144] demonstratedsubstantially reduced 5-year survival after liver transplantation amongHIV/HCV-coinfected patients compared with HCV-monoinfected patients(33% versus 72%; P ¼ .07). In addition, 22% of coinfected patients wereunable to tolerate HAART after transplantation, which was a major deter-minant of death. Early treatment of HCV after transplantation may bea potential strategy to reduce these adverse outcomes, but more study isneeded and ongoing in this area.

Summary

Because of shared routes of transmission, HCV coinfection is commonamong HIV-infected persons. Because of the effectiveness of antiretroviraltherapy, chronic HCV has now emerged as a major cause of morbidityand mortality in this population. Because chronic HCV is highly prevalentamong HIV-infected patients and has a rapid disease progression, antiviraltherapy with pegylated interferon plus ribavirin is critical for the long-termsurvival of HIV/HCV-coinfected patients. Additional studies are needed toexamine the natural history of chronic HCV in HIV/HCV-coinfected pa-tients, identify the appropriate treatment candidates, and identify additionalinterventions that can improve response to antiviral therapy.

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Extrahepatic Manifestationsof Hepatitis C Virus Infection

Anna Linda Zignego, MD, PhDa,*,Antonio Craxı, MDb

aCenter for Systemic Manifestations of Hepatitis Viruses (MaSVE), Department

of Internal Medicine, University of Florence, Viale GB Morgagni 85, 50134 Firenze, ItalybGastroenterologia and Epatologia, Di.Bi.M.I.S., University of Palermo, Piazza Marina,

34–90133 Palermo, Italy

Hepatitis C virus (HCV) is at the same time a hepatotropic and a lympho-tropic virus and may cause hepatic and extrahepatic diseases. Extrahepaticmanifestations linked to HCV range from disorders for which a significantassociation with viral infection is supported by epidemiologic data and bybiological plausibility, to anecdotal observations without clear proof of cau-sality. B cell lymphoproliferative disorders (ie, mixed cryoglobulinemia andnon-Hodgkin’s lymphoma) are the extrahepatic conditions most closelylinked to HCV, having been investigated extensively, and represent a modelfor both pathogenetic and clinico–therapeutic deductions. An associationbetween HCV infection and other morbid conditions, including dermato-logic, nephrological, neurologic, endocrinologic, cardiocirculatory, andlung disorders also has been suggested. Overlap syndromes characterizedby the presence in one patient of manifestations belonging to various path-ologic conditionsdtypically of autoimmune/lymphoproliferative naturedwould suggest that chronic HCV infection is a distinct systemic diseasewith a varying spectrum of clinical manifestations. Interferon-based antivi-ral therapy is considered, when feasible, the mainstay of treatment for mostHCV-linked extrahepatic diseases. Although its efficacy in curtailing a non-hepatic manifestation after obtaining viral clearance often is seen as a confir-mation of the key pathogenetic role played by HCV, clinical symptoms andviral persistence also may be disjointed, the former persisting even beyonda sustained viral response to therapy. Because of this fact and the intrinsicpotential inefficacy of interferon, which in some cases may even exacerbate

Clin Liver Dis 12 (2008) 611–636


E-mail address: [email protected] (A.L. Zignego).



612 ZIGNEGO & CRAXI

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

Classification of extrahepatic manifestations of hepatitis C virus

Extrahepatic manifestations of HCV infection (EHMs-HCV) range fromdisorders for which a significant association with HCV infection is sup-ported clearly by multiple lines of evidence to anecdotal observations with-out clear proof of causality [1,2]. A tentative classification of EHMs-HCV issuggested in Box 1. According to such classification, extrahepatic manifesta-tions of HCV infection are distinguished, taking into account the robustnessof available scientific data. It is thus likely that the nosography of someEHMs-HCV may change over time.

Group A includes EHMs-HCV characterized by a strong associationproven by both epidemiologic and pathogenetic evidence. This category in-cludes B-cell lymphoproliferative disorders (LPDs). Group B includes disor-ders for which a significant association with HCV infection is supported bysubstantial epidemiologic data and groups C and D associations still requireconfirmation and/or a more detailed characterization as opposed to obser-vations that are of similar pathologic nature but of different etiology, or id-iopathic 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 deposi-tion of circulating immune complexes in the small vessels and characterizedby multiple organ involvement, mainly skin, peripheral nerves, kidney, andsalivary glands, and less frequently associated with widespread vasculitisand malignant lymphoma [3–6]. The strong association between HCV andMC has been confirmed repeatedly by serologic and molecular investiga-tions [4,6,7]. Generally speaking, cryoglobulinemias are conditions charac-terized by the presence of serum immunoglobulins that become insolublebelow 37�C and can dissolve by warming serum (cryoglobulins, CGs). Ac-cording to Brouet and colleagues [8], CGs are classified on the basis of theirimmunoglobulin composition. In type I, they are composed of a pure mono-clonal 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 poly-clonal IgG and monoclonal IgM or polyclonal IgG and polyclonal IgM, re-spectively. In MC, the IgM represents an autoantibody bearing rheumatoidfactor (RF) activity. In type II MC (MC II), the IgM RF molecules mostfrequently display the WA cross-reactive idiotype [9]. MC II accounts for50% to 60%, and type III (MC III) for the remaining 40% to 50% of MC.

Box 1. Classification of extrahepatic manifestations of hepatitisC virus infection

Association defined on the basis of high prevalenceand pathogenesisMixed cryoglobulinemia (complete or incomplete clinical

syndrome)B-cell non-Hodgkin’s lymphoma

Association defined on the basis of higher prevalencesthan in controlsMonoclonal gammopathiesPorphyria cutanea tardaLichen planusDiabetes mellitus

Associations to be confirmed/characterizedAutoimmune thyroiditisThyroid cancerSicca syndromeAlveolitis–lung fibrosisNoncryoglobulinemic nephropathiesErectile dysfunctionsCarotid AtherosclerosisPsychopathological disorders

Anecdotal observationsPsoriasisPeripheral/central neuropathiesChronic polyarthritisRheumatoid arthritisPolyarthritis nodosaBechet’s syndromeMyositis/dermatomyositisFibromyalgiaChronic urticariaChronic pruritusKaposi’s pseudosarcomaVitiligoCardiomyopathiesMooren corneal ulcerNecrolytic acral erythema

613EXTRAHEPATIC MANIFESTATIONS OF HEPATITIS C VIRUS

614 ZIGNEGO & CRAXI

The prevalence of chronic HCV infection in patients who have CGs intheir serum ranges from 19% to more than 50% according to various stud-ies [10,11]. CGs, however, are generally present at low levels, and symptomsare generally absent or mild in chronically HCV-infected patients, whereasclinically overt MC syndrome (MCS) would be evident in 10% to 30% ofMC subjects [10–12].

Serum mixed CGs, high RF values, and reduced C4 values are the mostfrequent laboratory data. The most common symptoms of MCS are weak-ness, arthralgias, and purpura (Meltzer and Franklin triad). Raynaud’s phe-nomenondmicrocirculatory changes of the small vessels of the hands andfeet, identified as color changes in response to colddperipheral neuropathy,sicca syndrome, renal involvement, lung disorders, fever, and cytopeniasalso may be observed [3]. In a recent study involving 231 Italian MC pa-tients, peripheral neuropathy was observed in most cases, representing themost 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 assymmetric distal neuropathies, multiple mononeuritis, or mononeuropa-thies. Involvement of the central nervous system is unusual and generallypresents as transient dysarthria and hemiplegia. Pathologic findings showaxonal damage with epineural vasculitic infiltrates and endoneuralmicroangiopathy.

A sicca syndrome (xerostomia and xerophthalmia) caused by involve-ment of salivary and lacrimal glands is recognized in a large proportionof MC patients [14–18]. This syndrome close resembles primary Sjogren’ssyndrome; however it typically lacks antinuclear autoantibodies and antiepi-thelial neutrophil-activating peptide (ENA, SSA/Ro, SSB/La) [13]. Thepathogenetic role of HCV infection in sicca syndrome and the characteris-tics distinguishing classic Sjogren’s syndrome from those associated withHCV remain at issue [19]. It has been proposed that HCV infection is a crite-rion to exclude diagnosis of primary Sjogren’s syndrome, especially if mixedcryoglobulins and hypocomplementemia are present, and anti-SSA/Roantibodies are absent [20].

Significant renal involvement is present in up to one third of patientswho have MC, being observed in about 20% of patients upon clinical pre-sentation 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 subclin-ical features of renal involvement including microscopic hematuria, pro-teinuria below the nephrotic range (!3 g/24 h), and with normal oronly fairly reduced renal function (creatinine !1.5 mg%). Arterial hyper-tension 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 syndromemay represent the principal manifestation of MC. About 20% to 30%of cases present with an acute nephritic syndrome as their first renal


manifestation [4,23]. Presence of a significant renal involvement is amongthe worst prognostic indices in patients who have MC, even when itscourse is variable [13,23]. In a study of 231 patients who had MC, glomer-ulonephritis with subsequent renal failure was the main complication(33%), leading to death during long-term follow-up [13]. The typical his-tologic pattern of renal damage observed in patients who have MC type Iis membranoproliferative glomerulonephritis (MPGN) [13]. The presenceof capillary thrombi, made up of precipitated cryoglobulins and depositsof IgM in capillary loops typically differentiates the cryoglobulinemicform from idiopathic MPGN. In a minority of cases (generally type IIIMC), different pathologic findings have been described (ie, focal and me-sangioproliferative glomerulonephritis and membranous glomerulonephri-tis) [24,25].

The association between MC and severe liver damage has been discussedwidely [1,2,9,26–29]. Several studies have shown an epidemiologic associa-tion between MC and severe liver damage [10,30]. An association betweenMC and liver steatosis also has been suggested [31]. Overall, it seems thatboth MC and the stage of liver damage are more related to a long durationof infection.

The occurrence of MC generally has an important impact on the qualityof life and survival of patients who have MC. Survival analysis according tothe Kaplan-Meier method shows a cumulative 10-year survival, calculatedfrom time of diagnosis, to be significantly lower in patients who have MCas compared with an age- and sex-matched general population. Lowest sur-vival rates were observed in males and in subjects who had renal involve-ment, the main causes of death being nephropathy (33%), malignancies(23%), liver failure (13%), and diffuse vasculitis (13%) [13].

Because of its variable presentation, no standardized criteria for the diag-nosis and staging of MCS are available, even if classifications have been pro-posed [32]. In the presence of purpura, mixed CGs, reduced C4 values, andorgan involvement, the diagnosis of MCS is relatively easy. Quite fre-quently, however, it is suggested by abnormal laboratory data (RF test ormixed CGs or reduced C4 values) with or without mild symptoms like ar-thralgias or asthenia. Moreover, some subjects who have HCV may showclinically evident MCS, though incomplete from a serologic point of view,mainly with the temporary absence of circulating CGs. This may be ex-plained by the difficulty in a proper determination of the presence ofCGs, because of their thermolability and the variability of the rate ofCGs responsible for vasculitic damage [13,33].

Because some mixed CGs are present in low concentrations, the differen-tiation between type II and type III CGs often requires a more sensitivemethod for immunochemical characterization such as electroimmunofixa-tion or Western blot, than conventional immunoelectrophoresis [34]. Severaldata, including the presence of a clonal expansion of B-lymphocytes (BL) inperipheral blood or liver infiltrates [35–38], and the histopathological

616 ZIGNEGO & CRAXI

features of the bone marrow and liver lymphoid infiltrates, confirm the lym-phoproliferative 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-Hodgkin’s 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 lym-phoproliferation of uncertain significance (MLDUS) in subjects who haveclinico–laboratory features of MC II is typical [33,39–41]. MLDUS repre-sents oligoclonal proliferations of small BL, preferentially located in thebone marrow and liver. In these organs, MLDUS is generally presentwith phenotypic and histologic aspects comparable to indolent B-cell lym-phoma. Further immune–morphologic analysis reveals two different varie-ties: the first and more frequent variety, with analogous features to B-cellchronic lymphatic leukemia (CLL)/small cell lymphoma and a second, lessfrequent, lymphoplasmacytic-like form. The prevalence of these histologicpatterns has varied in different reports [40,42–48].

Lymphoma

HCV-associated lymphoid malignancies can be observed during thecourse 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-lastinginfection. According to recent data, MC patients had a 35 times higher riskof NHL than the general population [50].

An association between B-cell derived NHL and HCV infection initiallywas suggested by studies performed in populations from southern Europeand the southern United States, [46,51–55], whereas some northern Euro-pean and northern United States or Canadian surveys did not confirma higher prevalence of chronic HCV infection in patients with lymphomathan in the general population [56–61]. During the last decade, many studiesperformed at different latitudesdgenerally larger in size than initial onesdas well as meta-analysis, were able to confirm the existence of such associa-tion, even with a clear south/north gradient of prevalence. These differencesat least partly reflect how HCV is diffuse at different rates in the specificpopulations studied, but also suggest a likely contribution of environmentalor genetic factors [62] to lymphomagenesis.

Although virtually all types of lymphoid malignancy can be found in pa-tients with HCV infection, the strongest association is with B-cell derivedNHL [18,40,51,53,54,63–65]. The use of different classifications presentedin the various studies, however, may confound the evaluation of the actualincidences of each histotype [48,65–67]. Peripheral B-cell derived indolentNHL appears to be the most frequent HCV-associated lymphoma inmany studies. According to the Revised European-American Lymphoma


(REAL) classification/World Health Organization (WHO) Classification[68,69], the main HCV-related histotypes include B-cell chronic lymphocyticleukemia/small lymphocyte lymphoma, diffuse large B-cell lymphoma, fol-licular lymphoma, lymphoplasmacytic lymphoma, and marginal zone lym-phoma [40]. A recent European multicenter survey suggests a specific roleof HCV infection in the pathogenesis of diffuse large B-cell lymphoma[70]. Among marginal zone lymphomas, a special association with HCV in-fection was reported for the mucosa-associated lymphoid tissue (MALT)lymphoma [65,71,72] and the splenic forms. This is confirmed by reportsof regression of marginal splenic lymphoma after successful HCV clearancebecause of antiviral therapy, in spite of previous ineffective chemotherapy[73–75]. In general, the observations of a hematological regression ofsome HCV-associated NHL and expanded B-cell clones following effectiveantiviral therapy represent a consistent argument in favor of the pathoge-netic role played by HCV infection, even if a direct antineoplastic role of in-terferon cannot be excluded.

A serum monoclonal gammopathy, more frequently type IgM/K, hasbeen described among HCV-associated LPDs [76]. In most patients withHCV, however, MG was classified as MGUS (monoclonal gammopathiesof uncertain significance), whereas a few patients who have HCV withmonoclonal gammopathy can be considered as affected by myeloma accord-ing to their clinico–pathologic 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 mecha-nisms involved in the evolution of HCV infection to B-cell LPDs are avail-able, but in spite of interesting hypotheses, the exact mechanisms involvedare not known. Originally, the observation of HCV lymphotropism at thebeginning of the 1990s led to the hypothesis of a causal link between infec-tion of lymphatic cells and autoimmune–lymphoproliferative disorders [79].Earlier, it was observed that both HCV-positive strand (genomic) and -neg-ative strand (antigenomic ¼ replicative intermediates) could be detected inperipheral blood mononuclear cells (PBMC) taken from patients who hadchronic HCV infection [80]. During the past decade, ex vivo and in vitrostudies with techniques of increasing specificity and sensitivity have led tobetter characterization of HCV lymphotropism [81–87]. Nonetheless, noclear proof of a direct link between HCV lymphotropism and LPD patho-genesis has been obtained, despite the demonstration of a more extensive in-volvement of the lymphatic system by HCV infection in patients who haveB-cell LPDs than those who do not [5,88,89], and of an enhancing effect ofB-cell infection by HCV in promoting the proliferation of lymphoid cells[90]. By contrast, several studies have supported an indirect role of HCVthrough activation of the host’s immune response [37,91–94]. The

618 ZIGNEGO & CRAXI

importance of a sustained antigenic stimulation by viral epitopes and of thespecific binding between the HCV E2 protein and the CD81 molecule hasbeen stressed [95], supporting the key role played by the promotion ofa strong polyclonal B-cell response to chronic viral infection that progres-sively would favor lymphomagenesis until final malignant transformation.

Contrasting data, showing the possibility that HCV may favor mutationsof immunoglobulin genes and oncogenes by a ‘‘hit and run’’ mechanism, wereobtained in cell lines and in cultured cells taken from patients with HCV [96].This analysis originated from previous observations showing a significant as-sociation between Bcl-2 rearrangement (14;18 translocation) and chronicHCV infection, especially in those subjects who developed type 2 MC [97–102], and MALT lymphoma [103]. In patients who had type II MC, the anal-ysis of synchronous and metachronous blood samples showed the clonalexpansion of B-cells harboring this chromosomal rearrangement [99]. Inaddition, it was possible to demonstrate an overexpression of the antiapop-totic Bcl-2 protein with a higher bcl-2/bax ratio in t (14;18)-positive B-cellsamples [99], and a modification of t (14;18) B-cell clone detection followingantiviral treatment [104]. Only treatments leading to sustained clearance ofHCV RNA from serum led to the regression of clones, with consequentlack of t (14;18)-positive cells in PBMC at the end of treatment, whereasthis effect was not observed in nonresponder patients [104]. An extensive fol-low-up of HCV-positive MC patients after sustained virological response toIFN-based treatments has revealed the possibility of viral persistence in thelymphatic compartment in the absence of serumHCVRNAor liver infection.Interestingly, isolated lymphatic infection was associated strictly with the per-sistence of both MC-syndrome and t (14;18)-bearing B-cell clones.

These observations reaffirm, after more than a decade since the originalsuggestions, the strong likelihood of a role played by HCV lymphotropismin lymphomagenesis, but the exact mechanisms involved remain unclear. Onthe other hand, they strongly suggest that the pathogenesis of MC is notnecessarily related to the intrahepatic challenge between HCV epitopesand lymphoid infiltrates. Considering the complexity of LPDs and the het-erogeneity of HCV-associated LPDs, it is conceivable that MC and otherHCV-related LPDs may have different pathogenetic pathways. An accurateunderstanding of these different pathways and the frequency of their in-volvement in the pathogenetic mechanisms would be essential for the correctappraisal of both therapeutic and preventive measures [105,106]. An attemptto summarize current knowledge of the interrelations between HCV andLPDs is presented in Fig. 1.

Treatment of hepatitis C virus-related mixed cryoglobulinemiaand lymphoproliferative disorders

Before the identification of its viral etiology, MC treatment includeda variable combination of anti-inflammatory and immunosuppressive

+

HCVHCV

HCV E2 /CD81

binding

B-cell infection

Sustained B-

cell activation

Genetic and/ or

environmental factors

Prolonged BL

survival

t(14;18)/others?

Bcl-2 overexpression

B-cell apoptosis

inhibition

MC

Additional genetic aberrations

Malignant NHL

HCV-induced mutagenesis

Likelih

oo

d o

f reversio

n after H

CV

erad

icatio

n

-

+

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

based hypothesis.


strategies. Soon after linking chronic HCV infection to MC, several studieswere carried out to assess the effect of interferon (Table 1). Interferon mono-therapy initially was used, sometimes in association with corticosteroids(CS) [24,26,107–116], and resulted generally in effective improvement inMCS, even if associated with a very high relapse rate after discontinuationof therapy (see Table 1). In later studies, the combination of interferon withribavirin (RBV) offered 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)[121–123] (see Table 1). Additional controlled studies, however, are neededto gain definitive information.

Interestingly, all available studies show that clinico–immunologic and vi-rologic responses generally are related [112,117,119,122–124]. The persis-tence of isolated lymphatic infection after therapy was associated withpersistence of MC syndrome stigmata [105]. Disappearance of BL monoclo-nal infiltrate from bone marrow and BL expansion in peripheral blood fol-lowing IFN therapy also has been shown. In particular, the antiviralresponse was proven to be significantly associated with the absence of circu-lating B-cell clones bearing t (14;18) translocation [99,104,125]. The

Table 1

Antiviral therapy in hepatitis C virus-related mixed cryoglobulinemia

Author Year

Number

of patients Treatment

Treatment

duration (months)

End of

treatment

Response

sustained

Ferri 1993 15 Interferon 2 MIU/d (1 m)–2 MIU

three times weekly (5 m) (þCS)6 80%

Ferri 1993 26 Interferon 2 MIU /d (1 m)–2 MIU

three times weekly (5 m) (þCS)6 100% 0

Marcellin 1993 2 Interferon 3 MIU three times weekly 6 50%

Johnson 1993 4 Interferon 1–10 MIU 2–12 75%a

Misiani 1994 27 Interferon 1.5 MIU three times weekly

(1 w)–3 MIU three times weekly

(23 w)

6 60% 0

Dammacco 1994 15 Interferon 3 MIU three times weekly 12 53.3% 25%

16 Interferon 3 MIU three times weekly

(þCS)12 52.9% 33.3%

Johnson 1994 14 Variable interferon 0a

Mazzaro 1994 18 Interferon 3 MIU three times weekly 28%

Mazzaro 1995 18 Interferon 3 MIU three times weekly 6 28% 11%

18 Interferon 3 MIU three times weekly 12 39% 22%

Casaril 1996 25 Interferon 6 MIU three times weekly 6 52%b

Cohen 1996 20 Interferon 3 MIU three times weekly 60%c 9%c

Akriviadis 1997 20 Interferon 3–5 MIU three times weekly 6–12 65%b 33%b

Casato 1997 31 Interferon 3 MIU/d (3 m)–3 MIU

three times weekly (R9 m)

R12 62%

Durand 1998 5 NR RBV 10–36 100% 0%

Calleja 1999 18 Interferon 3 MIU three times weekly 12 55% 28%

8 NR Interferon 3 MIU three times weekly

þ RBV

12 63% 38%

Zuckerman 2000 9 NR Interferon 3 MIU three times weekly

þ RBV

6 78%

620

ZIG

NEGO

&CRAXI

Cacoub 2002 14 Variable interferon þ RBV

(þ variable CS)

6–56 71%

Mazzaro 2003 27 NR or Rel Interferon 3 MIU three times weekly

þ RBV

12 85%

Alric 2004 18 Interferon 3 MIU three times weekly

or pegylated interferon þ RBV

R18 70%

Cacoub 2005 9 Pegylated interferon 1.5 mg/kg/w þRBV

R10 88%

Saadoun 2006 32 Interferon 3 MIU three times weekly

þ RBV

R6 67.5%

40 Pegylated interferon þ RBV 56.3%

a Kidney function improvement.b 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 disord rs: an overview.World J Gastroenterol 2007;13:2467–78.

621

EXTRAHEPATIC

MANIF

ESTATIO

NSOFHEPATIT

ISC

VIR

US

,

e

622 ZIGNEGO & CRAXI

reappearance of circulating translocated BL clones after virological relapseat the end of treatment, and their persistent detection in subjects with un-modified viral load after antiviral therapy, strongly indicates that clonal ex-pansion of translocated cells depends on modifications of viral replicationinduced by antiviral treatment [104,125]. More recently, long-term analysisof patients with HCVand MCS showing SVR after therapy indicated thatoccult lymphatic infection and persistence of MCS stigmata also were asso-ciated with persistent determination of expanded t (14;18) carrying B-cellclones [105]. These data suggest the critical role played by a complete viraleradication and its possible role in preventing the evolution of LPD.

An algorithmic approach to the treatment of HCV-related MCS is out-lined in Fig. 2. Overall, available data show that combination antiviral treat-ment with pegylated interferon and RBV should be considered as the firstoption in subjects who have HCV-positive MCS. Interferon-based antiviraltreatment in HCV patients who have MCS is usually more complex to han-dle than in patients without MC for several reasons, including: the absenceof standardized treatment protocols, the frequent presence of contraindica-tions, and the difficulties in the accurate interpretation of results. Biochem-ical markers of MC response (cryocrit, RF, or complement values) may berelated less strictly to virological response than alanine aminotransferase(ALT) levels. This may confirm the importance of a multistage pathogenetic

Contraindications to IFNand/or severe MCS

Contraindications to IFNand/or severe MCS

Assessment of MCS and of contraindications to IFN-based therapyAssessment of MCS and of contraindications to IFN-based therapy

No contraindicationsand mild/moderate MCS

PEG IFN

plus ribavirin*

PEG IFN

plus ribavirin*

SR°SR° NRNR

LAC diet

Low-medium doses

CS +/- other sympto matic

treatment

NON

RESPONDERS

PE + CS +/-CF

RituximabRituximab

RESPONDERS

Antiviral

therapy ?

Moderate-severe MPGN, skin vasculitis

or

Severe-rapidly progressive MPGN

Severe sensomotor neuropathies

Widespread vasculitis

Mild-moderate

purpura Weakness

Arthralgias

Mild neuropathy

severe

Fig. 2. An algorithmic approach to the treatment of hepatitis C virus-positive mixed cryoglo-

bulinemia (MC). *Schedule as for non-MC patients. Abbreviations: CS, corticosteroids; CF, cy-

clophosphamide; NR, no MCs response; PE-plasma exchange; SR, sustained MCs response.


mechanism in MCS, suggesting the need for precise monitoring of this cat-egory of patients and the definition of predictive markers.

For patients in whom antiviral treatment is contraindicated or is nottolerated or in patients who are nonresponders, alternative therapeuticapproaches should be used, CS, immunosuppressive drugs, Non-SteroidalAnti-Inflammatory Drugs (NSAIDs), plasmapheresis (PE) and a hypoanti-genic diet (low antigen content [LAC] diet) administration [41,126]. Treat-ment should be tailored to the individual patient according to the severityof clinical symptoms, also considering the possible additional factors in-volved (age, renal failure, comorbidities), and the time (weeks or months)required for symptom remission. Corticosteroids represent the most com-monly used nonantiviral therapy for MCS and generally allow the controlof most MC symptoms even at low doses. They may favor HCV replica-tion, however, induce several adverse effects, and do not significantly mod-ify the natural history of the disease. Cytostatic–immunosuppressive drugs(ie, cyclophosphamide, chlorambucil, and azathioprine)may be used, espe-cially during the acute phases of MCS, but they may cause severe adverseeffects [127]. A special note must be made of new B-cell specific immuno-suppressive therapy based on the use of chimeric antibodies (rituximab)against the CD20, a B-cell specific surface antigen [128,129]. Rituximab iseffective in most patients who have MC, leading to marked improvementor resolution of the syndrome, especially of skin lesions, and to regressionof the expanded B-cell clones [128,129]. This therapeutic approach appearsto be very promising for managing patients who have MCS, but future con-trolled studies still are required to establish its ultimate role in treatingHCV-related MCS. It should be stressed that rituximab, as an immunosup-pressive agent, leads to an increase in HCV replication, but no significantreactivation of HCV-related liver disease has been reported. Its use in com-bination with direct antiviral molecules [41,128–131], when available, prob-ably 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) specifically isindicated in the presence of clinically significant acute manifestations (cryo-globulinemic nephritis, severe sensorimotor neuropathies, cutaneous ulcers,and hyperviscosity syndrome). The LAC diets, generally prescribed at the ini-tial stage of MCS, essentially act by reducing the antigen load to the reticulo–endothelial system, thus allowing a more efficient removal of CGs.

Recent studies, performed in specific subgroups of patients with HCV-associated NHLs, such as marginal zone lymphomas [73,132], support therationale for the use of antiviral therapy also in the setting of malignantHCV-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 ofHCV-negative cases following treatment with IFN. In addition, regressionof clonal proliferation in response to antiviral treatment was shown to be

624 ZIGNEGO & CRAXI

associated clearly with virological response [99,104,125]. Seemingly onlya proportion of HCV-associated lymphomas are cured with antiviral ther-apy. Also in responsive patients who had SLVL, the rearrangement of themonoclonal immunoglobulin genes persistently was detected in the bloodeven after a complete hematological response [75]. Such data suggestthat the multistep lymphomagenetic cascade may have points of no-return,making the LPD progressively independent from HCV infection (seeFig. 1). Although antiviral therapy appears to be an attractive therapeutictool for low-grade HCV-positive NHL, in intermediate and high-gradeNHL, chemotherapy is most likely to be necessary while antiviral treat-ment possibly could represent a maintenance therapy [133]. Further studiesare needed to standardize antiviral therapy better in HCV-associatedNHL. The use of rituximab in HCV-associated NHL, in monotherapyor in combination with antiviral treatment and/or chemotherapy, appearsvery promising, particularly in the setting of low-grade NHL, where ritux-imab monotherapy has been proposed as first-line treatment [134–136]. Inspite of the limited number of described cases, rituximab may be consid-ered a safe and effective therapy for HCV-related indolent B-celllymphoma.

Other extrahepatic disorders and overlap syndromes

Many and different disorders have been linked to HCV infection. In mostcases, because of possible methodological bias, mainly in patient selection inthe various studies, it is difficult to verify whether the suggested associationis coincidental or whether a real pathogenetic link exists. Several conditionsare observed more frequently in the context of an MC and quite rarely asidiopathic forms. This is the case of skin, kidney, salivary glands, or lungdisorders. In addition, a clinicoserological overlap between differentEHMs-HCV often is observed [32,137,138]. In spite of the heterogeneityof the morbid conditions that have been linked to HCV, two issues are quiteconstant: the pathogenetic involvement of the host’s immune system and thesymptom severity with consequent worsening of quality of life [139]. In ad-dition, extrahepatic manifestations may be observed variably in the presenceor absence of hepatic damage. On the whole, these observations support theview of HCV infection as a distinct systemic disease with widely variableclinical expressions.

Dermatopathologic manifestations

In addition to MC-related purpura, HCV infection also has been associ-ated with several cutaneous disorders, including the sporadic variant of por-phyria cutanea tarda (PCT) [1,140–143], a metabolic disorder characterizedby reduced hepatic activity of uroporphyrinogen decarboxylase, and withoral lichen planus (OLP) [144–154]. A strong association between the


sporadic form of PCT and HCV was suggested by the high prevalence(greater than 50%) of HCV markers in these patients, mainly in studiesfrom southern Europe [1,140–143]. In HCV-positive patients withoutPCT, however, no significant alteration in porphyrin metabolism was shown[142,155,156]. Epidemiologic studies also proved the existence of a correla-tion between OLP and chronic HCV infection [144]. The prevalence of HCVinfection in a large population of patients who had OLP was about 27%[145]. Data supporting this correlation essentially stem from studies per-formed in Japan [144] and southern Europe [145–148], but these were notconfirmed in other populations [149–152]. It generally is agreed that in thesecutaneous disorders, HCV infection plays an indirect role, probably actingas a triggering factor in genetically predisposed individuals. As a matter offact, attempts to cure these disorders with antiviral therapy led to discor-dant, but generally negative results with the possible induction of a clinicalmanifestation in previously unaffected patients or worsening of the disease[33].

Nephrological disorders

A causative association between HCV and non-MC related forms of re-nal damage (membranoproliferative glomerulonephritis without simulta-neous presence of cryoglobulins, or membranous nephropathy) has beensuggested, but requires confirmation [33].

Neurologic disorders

Neurologic complications have been associated with HCV infectionmostly in the context of MC, but also in the absence of this condition[157,158]. Only cryoglobulinemic neuropathy, however, has been associatedclearly with HCV infection.

Disorders of the joints, bones and muscles

HCV-related chronic polyarthritis can be observed in HCV-positive pa-tients both with and without MC [13,159]. True rheumatoid arthritis(RA), in keeping with classic criteria, seems to be uncommon in subjectswho have HCV. In patients who have HCV, tests are usually negative forantibodies to cyclic citrullinated peptides (anti-CCP), which may help to dif-ferentiate the two conditions (true RA and HCV-related RA-like form). Bycontrast, intermittent oligoarthritis, generally not erosive and involving thebig and middle-sized joints, is observed frequently [13,32,159]. An associa-tion of HCV infection with osteosclerosis and a correlation between suchmanifestation and the imbalance in the osteoprotegerin/RANKL (ReceptorActivator of NF-kB Ligand) system with a predominance of osteoprotegerinalso were observed in some studies [160,161]. HCV infection also was sug-gested to be associated with myositis and dermatomyositis [162].

626 ZIGNEGO & CRAXI

Endocrinologic disorders

Practically all known manifestations of a thyroid disorder have been de-scribed in patients who have HCV infection, but frequently with discordantdata [163–167]. Geographic, genetic, or environmental cofactors [168–171]and different methodological approaches partially explain discordant obser-vations. The most frequently observed HCV-associated thyroid disorder in-volves circulating antithyroid peroxidase antibodies in female subjects [166].Subclinical hypothyroidism was observed in 2% to 9% of patients who hadchronic HCV infection and particularly in those who had MC [2,165,172].Finally, a higher prevalence of papillary thyroid carcinoma in patientswho had HCV was observed [173–175]. Interferon alfa therapy may exacer-bate or induce underlying latent thyroid disorders, and the relative contribu-tion of the role played by HCV or by antiviral therapy in leading to suchassociations has been discussed [166,176–178]. Recently, it was suggestedthat molecular mimicry between viral and self-antigens might be involvedin the pathogenesis of HCV-associated autoimmune thyroid diseases [179].A high prevalence of diabetes mellitus type 2 was observed in patientswho had chronic HCV infection in several studies [180–187], and it hasbeen suggested that HCV acts as a risk factor independently of liver disease[188]. In patients who had HCV infection, the appearance of diabetes type 2was associated with insulin resistance and was considered part of a complexvirus-induced metabolic syndrome including both hepatic (steatosis) and ex-trahepatic manifestations. In agreement with this, an association betweencarotid atherosclerosis and HCV infection recently has been suggested[189,190].

Lung and cardiocirculatory disorders

A pathogenetic link between HCV infection and idiopathic pulmonary fi-brosis has been reported [191,192]. The fact that MC can be complicated byan involvement of pulmonary interstitium suggests that such association insome cases can be related to a pre-existing MC [193]. An association be-tween HCV and hypertrophic cardiomyopathy was suggested by the obser-vation of significantly higher prevalence of anti-HCV in Japanese patientswith such condition compared with controls [194]. Studies performed inItaly and in Greece by other authors, however, did not confirm this associ-ation. Regardless, the recent determination of a significantly higher preva-lence of carotid atherosclerosis in patients with HCV infection [189] isnoteworthy; this association was mostly evident in case of active viral repli-cation [190]. In a very recent study, the association between HCV chronicinfection and carotid atherosclerotic lesions was confirmed also in a largeItalian population. Further, HCV RNA sequence was determined in carotidplaques, strongly suggesting a local proatherogenetic action of the virus in-side the plaque [195].


Psychopathological disorders

Patients who have HCV infection have a low quality of life and may ex-perience excessive fatigue, decreased cognitive ability, and low mood tone[196–198]. These symptoms are not related directly to liver damage, and ithas been proposed that the virus may cause direct cerebral dysfunction byan unknown mechanism [197]. Recently, plasma tryptophan and kynureninecontent in blood, together with indoleamine 2,3-dioxygenase activity inmacrophages, was evaluated in a cohort of patients who had mild HCV-re-lated chronic liver disease. Patients also underwent psychopathological eval-uation. Serum tryptophan concentrations were lower than those of healthysubjects or patients who had chronic HBV infection, and were associatedwith high levels of anxiety and depression, strongly suggesting that thesemodifications may be causally related. In addition, mechanisms involvedin the pathogenesis of HCV-associated reduced tryptophan levels appeareddifferent from those observed in other chronic infections, thus possibly rep-resenting a new model for viral-induced alterations of tryptophan metabo-lism [199–201].

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Hepatitis C and Liver Transplantation:Enhancing Outcomes and Should

Patients Be Retransplanted

Elizabeth C. Verna, MDa,Robert S. Brown, Jr, MD, MPHb,*

aDivision of Digestive and Liver Diseases, Department of Medicine,

Columbia University Medical Center, 622 West 168th Street, New York, NY 10032, USAbDivision 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 livertransplantation (OLT) in the United States and Europe [1]. In contrast tomost other leading indications for OLT, serologic and histologic recurrenceof HCV after OLT is nearly universal. Death and allograft failure are morecommon in this population when compared with HCV-negative recipients[2], and HCV recurrence is the major source of graft failure. The naturalhistory 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 morethan 40% of patients who have recurrent cirrhosis develop manifestationsof decompensated disease within 1 year and less than 50% of patientssurvive 1 year after the onset of decompensation [3].

Compounding this problem, potent immunosuppressive agents anddonor shortages leading to the use of older donors may be impedingimprovements in outcomes and, some believe, worsening survival overtime [4]. The donor pool has gradually expanded with the implementationof living donor liver transplantation (LDLT) and the use of extended crite-ria donation (ECD), including HCV-positive donors, older donors, steatotic

Clin Liver Dis 12 (2008) 637–659

* Corresponding author. Center for Liver Disease and Transplantation, Columbia

University Medical Center, 622 West 168th Street, PH14, NY 10032.

E-mail address: [email protected] (R.S. Brown).



638 VERNA & BROWN

livers, and donation after cardiac death (DCD). The use of these grafts re-mains controversial, particularly in HCV-positive recipients, in whom theycould lead to accelerated fibrosis. Even with these attempts to increase organavailability, a profound discrepancy between supply and demand persists.Recurrent HCV and rejection remain the main obstacles in the posttrans-plant setting, and the balance between over- and underimmunosuppressionto minimize the risk for rejection and recurrence remains difficult. Measuresto prevent and control recurrent HCV with interferon (IFN) and ribavirin(RIBA) antiviral treatment continue to be disappointing, and data fromproperly designed randomized controlled trials to determine the best dosingand duration of treatment are lacking. In addition, although the risks ofpotent immunosuppression in the setting of recurrent HCV are now welldocumented, much remains unknown about the optimal induction andmaintenance immunosuppression regimen in HCV-infected patients. Pro-gressive allograft failure in this population is therefore common, and the de-bate continues as to who should be considered for retransplantation andwhen this evaluation should occur.

Thus, optimizing outcomes in liver transplantation for HCV-relatedESLD is of primary importance to the liver transplant community and tomost of their patients. The strategies to improve outcomes discussed in thisarticle include increasing access to adequate numbers of suitable liver grafts,improving antiviral treatment before and after transplantation, enhancingimmunosuppressive strategies such that recurrent HCV is minimized, andregulating the use of retransplantation when these treatments fail.

Enhancing outcomes in transplantation for hepatitis C virus

Optimizing the timing of transplantation

Maximizing the benefit that any patient derives from liver replacementdepends on the timing of the procedure in many ways. This is especially im-portant in the case of HCV infection; unlike the case in other liver diseases,progression is clearly more rapid in the posttransplant setting. The survivalbenefit of transplantation may therefore be diminished when prematuretransplantation occurs such that the posttransplant and operative mortalityoutweighs the expected pretransplant liver disease-related mortality. Delay-ing transplantation, and therefore recurrent HCV, may add life-years andtime for the development of new antiviral therapy, but this must be weighedagainst the risks for waiting, including the risks for complications, hepato-cellular carcinoma (HCC), or becoming too sick for transplantation.

Patients with Model for End-Stage Liver Disease (MELD) scores lessthan 15, and particularly with scores less than 12, do not derive a statisticalsurvival benefit in the first year after transplantation [5]. The degree ofsurvival benefit increases with each increase in MELD for scores greaterthan 15. Although the posttransplant risk for dying is increased by 50%

639HEPATITIS C AND LIVER TRANSPLANTATION

in patients with MELD scores greater than 30, these outcomes may still bereasonable, given the greater than 300-fold increase in pretransplant mortal-ity in this population. Specific donor and recipient risk factors for poor out-comes are also taken into consideration, and the rate of waiting list removalis higher for the sickest patients. Transplant physicians and surgeons incor-porate a wide variety of factors in the decision to transplant, and there is noabsolute MELD score cutoff for transplant futility. The optimal time forOLT is therefore when a patient achieves a MELD score of at least 15 orbegins to show evidence of decompensation, manifested by synthetic dys-function, malnutrition, or refractory ascites. This seems to be particularlytrue for the patient who has HCV, except perhaps in the setting of antiviraltherapy with clearance of HCV.

Safely expanding the donor pool

The growing disparity between the number of deceased donor organsavailable and the patients awaiting transplantation has driven the expansionof the donor pool to include what has been termed extended criteria donation,the precise definition of which remains elusive. In general, ECD livers mayhave a wide range of characteristics traditionally thought to be undesirablefor donation, including LDLT or split liver grafts, DCD, older donors, graftsteatosis, prolonged cold ischemia time, human T-lymphotrophic virus(HTLV)-1 positivity, high-risk behavior in the donor, and HCV or hepatitisB core antibody (HBcAb) positivity. The use of these grafts varies widely be-tween transplant centers, and because of limited data and the perceived risksof a potentially suboptimal graft, decisions are made on an individual basis.To date, long-term data in patients transplanted with specific types of ex-tended-criteria grafts are lacking, and the available literature is difficult to in-terpret because the recipients frequently have less optimal characteristicsbefore 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-in-fected recipients [6,7], although there is some evidence to the contrary [8].One large single-center cohort tallied the number of unfavorable characteris-tics of each graft and showed that outcomes in patients who have all forms ofliver disease are likely worse as the extended criteria score increases [8]. Thespecific aspect of each graft that makes it ‘‘high-risk’’ is extremely variable,however, and grouping these heterogeneous grafts and recipients is unlikelyto reveal clinically useful information. Even in these large heterogeneousstudies, using ECD grafts may maximize access to OLT, decrease waittime, and potentially decrease pretransplant mortality [6,7].

Of these factors, advanced donor age seems to be among the strongestpredictors of poor outcomes in HCV-infected recipients. Although graftsfrom donors between the ages of 60 and 80 years function without any sur-vival disadvantage in non–HCV-infected patients [9,10], several series report

640 VERNA & BROWN

more severe recurrence and more rapid progression to cirrhosis when olderdonors are used in the setting of HCV [11–14]. One report even suggests thatthe increased risk for graft dysfunction begins with donor age older than 40years [11]. This has led some centers to try to avoid using grafts from donorswith advanced age in HCV-infected recipients. The same may be true toa lesser extent with donor liver grafts that have excessive steatosis or pro-longed ischemia time.

Liver grafts from donors who have HCV and hepatitis B virus (HBV)infection have traditionally been considered contraindicated in OLT. Earlydata showed the risk for HBV transmission to be high in recipients ofisolated HBcAb-positive grafts [15–17]. More recent work in the era ofhepatitis B immunoglobulin (HBIG) and HBV antiviral treatments, suchas lamivudine, has shown that HBcAb-positive grafts have similar outcomesto those of patients who had HBV and were transplanted with HBV-negative grafts [18–21]. These patients generally receive prophylaxis withlamivudine with or without HBIG, which, in most cases, can prevent graftinfection [22–25]. There is no consensus, however, on how long patientsshould remain on therapy, and this may depend on the recipient’s HBVantibody status [26,27]. HCV-positive grafts are also being used increas-ingly, almost universally in HCV-positive recipients, and no survival disad-vantage has been seen when HCV-infected recipients receive HCV-positivegrafts [28–35]. The largest series to date is a retrospective study of theUnited Network for Organ Sharing (UNOS) database, including 96 HCV-infected patients who received HCV-positive grafts, and survival was equiv-alent to that of patients who received HCV-negative grafts. Grafts withHCV and HBcAb have also been used, and in one small series, also didnot significantly decrease patient survival, but this finding must be con-firmed [29]. The use of HCV-positive liver grafts is an important additionalgraft source to HCV-positive patients on the transplant waiting list.

DCD, previously known as non–heart-beating donation, was initiallythought 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 fortraditional brain death donation [36–38]. When compared in the UNOSdatabase of all patients transplanted for any type of liver disease, therewas a 3-year graft survival of 63% in the DCD group compared with72% in the brain death group, which did not reach statistical significance[36]. To date, there are no studies looking specifically at survival or rateand severity of HCV recurrence in this population, and additional investiga-tion is needed before firm conclusions on its impact on disease recurrencecan be drawn.

Adult-to-adult LDLT has been performed in the United States since 1998[39]. Living donor organs may have theoretic advantages over deceaseddonor organs because they have significantly less cold ischemia time andare from medically optimized donors. In addition, the reduced waitingperiod and optimal timing of transplantation afforded by living donation


may decrease the risk for decompensation and death before transplantationand improve patient survival. The mortality in patients with potentialdonors for LDLT is half that of those listed for deceased donor liver trans-plantation (DDLT) only [40,41]. In addition, antiviral treatment may beadministered before a deliberately timed transplantation, and it seems thatLDLT while the recipient is HCV RNA-negative leads to a low rate of viralrecurrence (10%–20%) [42,43].

Despite these theoretic benefits, LDLT grafts have been used withcaution in HCV-infected recipients. Early data on the use of LDLT graftsin this population revealed a significantly higher incidence of cholestatichepatitis C, a rapidly progressive form of recurrent HCV [44]. In addition,an early study from Spain showed a statistically significant increase incirrhosis and clinical decompensation at 2 years in the LDLT group [45].These findings fueled concerns that the accelerated hepatocyte proliferationin split liver transplantation may predispose to more aggressive recurrenceof HCV. Several recent large studies, however, have shown that there isno difference in the incidence or severity of HCV recurrence when LDLTand DDLT are compared [46–51]. Careful pathologic evaluation of protocolliver biopsies over 3 years after OLT in 23 LDLT patients and 53 DDLTpatients found no significant difference in inflammation and similar or lessfibrosis in the LDLT group [47]. Most recently, data from the multicenterstudy named the Adult-to-Adult Living Donor Liver TransplantationCohort Study (A2ALL) on 275 transplants (181 LDLT and 94 DDLT)showed an overall statistically significant survival advantage when DDLTwas compared with LDLT (82% versus 74% at 3 years) [51]. Because of pre-vious data suggesting worse outcomes in the first 20 LDLTs done at a givencenter [52], however, these patients were then separated into three groups:DDLT, the first 20 cases of LDLT at each center, and then all subsequentLDLT cases. There remained a statistically significant difference in survivalbetween the DDLT group and the group comprising the first 20 LDLTcases, but there was no difference in survival or rate of progression to fibro-sis between the DDLT and later LDLT cases (Fig. 1). It is therefore possiblethat the early reports of poor outcomes were a function of inexperience withthe surgical and postoperative management and that LDLT is likely to be assafe as DDLT in HCV-infected patients.

Currently, less than 5% of all adult liver transplants use living donorgrafts, and this rate has declined because of the early concerns about severerecurrence, possible exhaustion of the eligible and interested donor and recip-ient pool, and the two highly publicized donor deaths that occurred between2001 and 2003 [39,53,54]. Donor evaluation can also be costly and prolonged.In experienced centers, approximately one third of adult patients on the wait-ing list have a potential donor, and half of these actually donate, indicatingthat LDLT may be possible in up to 15% of patients evaluated [55,56].LDLTmay therefore be increasingly important in the expansion of the donorpool, but continued analysis is needed.

Fig. 1. Graft survival after DDLT, LDLT of 20 cases or less (first 20 cases at each center), and

LDLT of greater than 20 cases (cases beyond the first 20 at each center). Graft survival was

significantly lower in LDLT of 20 or less cases compared with LDLT of greater than 20 cases

(P¼ .0023) and DDLT (P¼ .0007). There was no significant difference in graft survival between

LDLT greater than 20 cases and DDLT, however (P ¼ .66, log rank test). (From Terrault NA,

Shiffman 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.)

642 VERNA & BROWN

Antiviral therapy for the prevention and treatment of recurrenthepatitis 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], andalthough disease progression is variable, many cases rapidly progress tocirrhosis. Much work has been done to identify factors predictive of severerecurrence, and the donor characteristic with the strongest impact on out-comes seems to be donor age. Recipient factors, such as HCV viral loadbefore transplantation [4,58], recipient cytomegalovirus (CMV) status[13,59,60], advanced recipient age, severe hyperbilirubinemia, and elevatedinternational normalized ratio (INR) [59] before transplantation, also pre-dict poor graft survival. Many of these risk factors, however, are not easilymodifiable, and treatment of HCV infection before and after OLT remainsamong the biggest challenges in liver transplantation.

Three antiviral treatment strategies have been attempted, all with limitedsuccess: curative or preventive treatment before transplantation, preemptivetherapy after transplantation but before documented histologic recurrence,and treatment of established recurrent disease. The ideal strategywould be cu-rative therapy before transplant, which could prevent recurrent disease, but itis successful in few patients on the transplant waiting list. Data supportinga benefit from preemptive therapy are limited. Thus, most current


recommendations focus on treatment of established recurrent disease [61].Unfortunately, enhanced viral replication in the setting of immunosuppres-sion, the high proportion of patients who have genotype 1 or virus unrespon-sive to IFN-based therapies before transplant, and the poor tolerability ofIFN and RIBA regimens render the treatment of recurrent HCV clinicallychallenging. There is currently no widely accepted and effective pre- orpost-OLT treatment regimen, and additional investigation intomore effectivetreatment dose and duration is required before universal recommendationscan be made. Individualized tailoring of treatment based on the patient’sHCV viral load and genotype, the patient’s response to treatment, and sideeffects and comorbidities is likely to remain a predominant theme.

Pretransplantation prophylactic antiviral treatment
Because HCV titers may be a significant predictor of graft and patient
outcome after transplantation [58] and patients who achieve a sustainedvirologic response (SVR) or who are transplanted with undetectable viralloads on therapy have only an approximately 20% likelihood of recurrence,viral eradication while on the waiting list would be ideal [43,62,63]. Thereare limited prospective data or randomized trials comparing regimens, how-ever, and the results of pre-OLT therapy have been generally disappointing.For patients who have compensated cirrhosis, pegylated (peg)-IFN andRIBA were studied in the National Institutes of Health (NIH)–sponsoredHepatitis C Antiviral Long-Term Treatment Against Cirrhosis (HALT-C)trial and yielded end-of-treatment (EOT) and SVR rates of 23% and11%, respectively [64]. Treatment of patients who have decompensated liverdisease, however, who comprise most potential transplant recipients, hasbeen associated with exacerbations of encephalopathy, infections, and otherserious adverse events, with up to a 10% treatment-related mortality rateand low rates of SVR [65].

Initial therapy with low-dose IFN and RIBA with slow-dose escalationmay improve tolerability and efficacy in patient who have compensated cir-rhosis [43,66]. In the largest ‘‘low accelerating dose regimen’’ (LADR) pro-tocol, 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]. Fifteenpatients with SVR were transplanted, and 12 (80%) remained free of viralrecurrence at the end of follow-up. Pretransplant viral clearance wouldtherefore be ideal, but because of the high risk for adverse events and limitedefficacy, routine treatment of patients who have decompensated disease isnot recommended outside of consultation with a transplant center or a clin-ical trial. The combination of the LADR protocol with deliberately timedLDLT if viral clearance is achieved is currently being studied in the NIH-sponsored A2ALL cohort of living donor liver transplants and may leadto HCV cure in a small but significant number of patients.

Prophylactic treatment with HCV immunoglobulin (HCIG) formulationsin the peritransplant period is still in early trials but has produced

644 VERNA & BROWN

disappointing results to date. One formulation of human HCIG completedphase II clinical trials (Civacir; Nabi Biopharmaceuticals, Boca Raton,Florida) and has been found to be safe but caused only transient decreasesin HCV viral load, without prevention of graft reinfection [67]. Concernsregarding the cost and potential infectivity of pooled human HCIG prepa-rations may limit their use, even if proven efficacious. Recombinant mono-clonal immunoglobulins are currently under investigation and one serieswith humanized monoclonal antibody was recently published by Schianoand colleagues [68], showing similarly disappointing results. Although allpatients experienced a decrease in viral load, no patient had an undetectableviral load while on therapy. Some have postulated that these results mayhave at least partially been attributable to low dosing of the regimen [69].Additional studies to identify the correct dosing and the most potent viralantigen target are needed, and perhaps these antibody preparations maybe most efficacious when used in combination with other antiviral treat-ments. Currently, however, there is no role for HCIG therapy in the man-agement of patients in the peritransplant period.

Posttransplant treatment of recurrent hepatitis C virus
Posttransplant preemptive treatment before histologic confirmation of
liver injury has been attempted with the hope that treatment before hepaticdysfunction could improve efficacy and tolerability. High-dose immunosup-pression and worsened leukopenia and anemia in this period usually negatethese benefits, however. In fact, in the first 2 months after transplantation,fewer than half of patients have the clinical and cell count stability to startIFN and RIBA [70]. When studied, standard IFN and RIBA have achievedEOT response rates of 23% to 40% and an SVR rate of approximately 20%,but discontinuation rates of about 12% to 50% [70–74]. A randomized trialof peg-IFN and RIBA versus placebo achieved an SVR of only 8% withtreatment, 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, withmost responders in the dual-therapy group. Dose reductions were requiredin 85% of patients, and therapy was discontinued prematurely in more than40% of patients despite hematologic growth factor administration.

In light of the variable course of recurrent infection, concerns about earlyimmune modulation with IFN, and disappointing trial results, the Interna-tional Liver Transplantation Society recommends antiviral therapy only inpatients with significant histologic recurrence (stage II fibrosis) [61]. Treat-ment in this setting is somewhat better studied, although the optimal treat-ment dose and duration are unknown and therapeutic regimens may need tobe tailored to individual patients instead of universally applied (Fig. 2).

For the treatment of established histologic recurrence, IFN monotherapyhas displayed little efficacy [75–77]. IFN in combination with RIBA leads toEOT response rates of 15% to 48% and SVR rates of 7% to 26%, with

Protocol biopsies in all patients withHCV viremia post-OLT

or biopsies for flares in LFTs

G1-2 inflammation AND< stage 2 fibrosis

G3-4 inflammation or≥ stage 2 fibrosis

Minimize immunosuppressionand avoid bolus steroidtreatment for rejection

Treat with peg-IFNand RIBA

SVR No SVR

Monitor Consider maintenancetherapy

Cholestatic HCV

Treat with peg-IFNand RIBA

Consider indefinite treatmentgiven high rates of relapse

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


discontinuation rates of 30% to 50% [75,78–82]. Results with peg-IFN andRIBA have generally been better, with overall EOT and SVR rates of 17%to 63% and 8% to 47%, respectively [75,78,79,81,83–91], but regimen toler-ance remains poor. Because of the increased incidence of side effects with theaddition of RIBA, many have debated whether it really adds to treatmentefficacy, and randomized controlled trials comparing the two regimens arelimited. In one recent small series, Angelico and colleagues [92] showedno significant difference in SVR; however, this included a total of only 42patients and may not have been powered to detect a difference. Morethan 40 treatment trials looking at RIBA in combination with IFN orpeg-IFN were recently pooled, and the composite SVR in patients treatedwith IFN and RIBA was 24% versus 27% in the peg-IFN plus RIBA group[93]. Pooled discontinuation rates were 24% and 26%, respectively. Despitethese variable results, when SVR is achieved, it does seem to improve graftsurvival [94], and viral eradication should be attempted in patients who havesignificant liver injury without contraindications. The most important pre-dictors of treatment success are likely to be early response to therapy anddose adherence [89,91].

In the absence of SVR, treatment may achieve modulation of diseaseseverity and prevent fibrosis, which remain important secondary goals. Inone small trial, patients with mild recurrence (stage 1–2 fibrosis) were ran-domized to treatment with peg-IFN and RIBA or no treatment, and pro-gression of fibrosis was significantly more common in the untreated group[95]. Several investigators have demonstrated histologic benefit of treatmentin the absence of complete viral suppression, and despite the lack of

646 VERNA & BROWN

controlled data showing improved patient and graft survival, some advocatethe use of maintenance therapy.

The expense of prolonged treatment in the setting of poor outcomes has ledsome to question the cost-effectiveness of antiviral therapy for recurrent HCVdisease. Markov-based decision models have been used to model treatmentwith combination IFN and RIBA and found treatment to be cost-effective[96]. Treatment of 100 men with recurrent HCV disease would prevent29 cases of recurrent cirrhosis and 7 deaths and have a cost-effectiveness ratioof more than $29,000 per year of life saved in this study. Therefore, patientswith documented significant histologic HCV recurrence should be consideredfor antiviral therapy, but additional work to find the best regimen is neededand the emergence of the HCV enzyme inhibitors is eventually likely to altertreatment dramatically.

Optimizing immunosuppression

The delicate balance between adequate immunosuppression and controlof HCV replication remains an area of controversy and research. Althoughthe proportion of patients being transplanted for HCV is increasing, there isevidence that outcomes in this population have not improved with time andmay even be worsening [4]. The increased potency of the immunosuppres-sion routinely used has been implicated in this finding. In addition, becausealmost all patients have some degree of recurrent hepatitis with portalinflammation, the conclusive differentiation of rejection plus HCV fromHCV alone is difficult and hinders accurate research conclusions and clinicaldecision making. This distinction is crucial, however, given the risk thattreatment for each process poses to the other.

Episodes of acute rejection clearly predict diminished survival in the set-ting of HCV, and treatment of acute rejection has been associated withdiminished survival in HCV-positive but not HCV-negative recipients [97].The impact of immunosuppression on HCV recurrence is most pronouncedwhen high-intensity regimens are used to combat acute rejection [98–101].HCV-induced graft dysfunction, cirrhosis, and severe cholestatic hepatitisare all more common in recipients who receive high-dose bolus steroids,anti-lymphocyte, or interleukin (IL)-2 receptor antibody preparations forthe treatment of acute rejection [58,102]. Pulsed intravenous steroid therapyis associated with a 4- to 100-fold increase in HCV RNA [103] and increasedseverity of histologic recurrence. As a result, many centers now initially treatmild acute rejection with modulation of immunosuppression by maximizingthe dose of or substituting the calcineurin inhibitor or reintroduction ofmycophenolic acid rather than bolus dose steroids.

Data to support or discourage the use of any specific induction or main-tenance regimen are less convincing. In fact, any association between clinicaloutcomes and one particular immunosuppression medication remains diffi-cult to prove, given the multiple regimens used and the changes in regimen


in an individual patient over time. In addition, most trials focus on rejectionor short-term survival without meticulous documentation of HCV activityand detailed histologic data. In general, the standard immunosuppressiveregimen typically includes a calcineurin inhibitor (tacrolimus or cyclospor-ine), a tapering dose of corticosteroids, and, in some cases, a lymphocyteantiproliferative agent (mycophenolate mofetil [MMF] or azathioprine).Much work has been done to identify the best choice of calcineurin inhibi-tor. Cyclosporine seems to have in vitro antiviral activity [104], but in vivoviral suppression is not evident when patients treated with cyclosporine ortacrolimus are compared [105]. Most studies have shown that the severityof HCV recurrence is similar in cyclosporine- and tacrolimus-based regi-mens [58,105–111]. A recent meta-analysis of the topic confirmed that nodifference in patient or graft survival has been documented based on thechoice of calcineurin inhibitor [112]. Data on HCV replication and histo-logic progression are lacking, however, emphasizing the need for future clin-ical trials to incorporate these factors as primary end points.

Additional immunosuppressive agents and potent induction therapieshave been less well studied. There is recent interest in the potential antiviralproperties of MMF, but the data on the addition of MMF to the standardregimen have been conflicting, with some studies reporting benefit andothers reporting diminished outcomes [113–116]. Induction with anti–T-cell or anti–IL-2 receptor antibodies is infrequently used, predominantlyin patients who are unlikely to tolerate calcineurin inhibitors because ofrenal failure or neurologic concerns. The data to support the use of thesethymocyte-depleting regimens for induction in this setting are inconsistent,but in one prospective randomized trial comparing induction with rabbitantithymocyte globulin followed by tacrolimus and MMF versus tacroli-mus, MMF and steroids showed no differences in graft survival and recur-rent HCV at 1 year [117]. Anti–B-cell agents are also being used with limiteddata and may worsen HCV recurrence. For example, a retrospective analy-sis of patients who received induction with alemtuzumab revealed higherrates of HCV recurrence at 1 year [118]. These potent induction agents inthe setting of HCV should therefore be used with caution and likely onlyin specific settings in which the standard regimen is not tolerated.

The unifying theme throughout these trials may be that the overall inten-sity of immunosuppression is themost important predictor, withmore intenseimmunosuppression leading to worse outcomes in HCV-infected patients.Rapid steroid tapers and steroid-free immunosuppression with or withoutinduction antibodies have been proposed as strategies to mitigate the severityof recurrent HCV. Steroid-free regimens are theoretically preferable, becausehigh-dose steroids are completely avoided and early data now show similarefficacy [117,119–121]. These regimens may also lead to less new-onset diabe-tes [121–123], a major risk factor for posttransplant renal dysfunction andsevere recurrent HCV [124]. Steroid-free regimens, however, frequently useanti–IL-2 receptor antibodies or lymphocyte-depleting agents, rendering

648 VERNA & BROWN

the benefits difficult to predict. It has even been postulated that because theimpact of cellular rejection on liver grafts may be small when comparedwith other solid organ transplantations, perhaps tapering the immunosup-pressive regimen off completely would be possible [125]. In this series, 34 pa-tients on cyclosporine monotherapy had therapy discontinued, and althoughsome were able to maintain avoidance of any immunosuppressive therapyand had improved biopsy findings, 35% of patients experienced rejectionwhile on the taper, 41% rejected the transplant after the taper was completed,and there was no control group. This approach cannot therefore currently berecommended. There is also emerging consensus that not only the absolutepotency of the regimen but rapid changes in immunosuppressive strengthare deleterious because they create alternating periods of increased viral rep-lication followed by immune recognition and clearance of virally infectedallograft cells during rapid immunosuppressive withdrawal. In fact, recentreports support the avoidance of rapid tapering of steroids and fluctuationin immunosuppressive therapy [126,127].

Clear evidence to support any particular strategy therefore remainselusive. The authors’ ‘‘low and slow’’ approach is to administer what theyanticipate to be the minimum sufficient immunosuppression to minimizerejection, followed by a gradual taper and avoidance of intense rejectiontreatment with bolus steroids or antibodies. They use a calcineurin inhibitor(cyclosporine or tacrolimus) with a slow steroid taper over 6 to 12months andMMF for the first year. In addition, protocol biopsies at months 3, 12, and 24are used to guide immunosuppressive and antiviral treatment decisions.

Retransplantation

When antiviral treatment and modulation of immunosuppression fail tohalt the progression of recurrent HCV, the only definitive treatment for graftfailure is retransplantation. The debate is ongoing, however, as to who shouldundergo retransplantation and when. No formal or widely accepted indica-tions for retransplantation exist, and practices vary widely among institu-tions. When transplant centers in the United States were surveyed, mostindicated that they offer retransplantation, but many were progressivelyless likely to perform the operation because of poor outcomes, and therewas little uniformity in the perceived indications and contraindications forthe operation [128].

When compared with primary transplantation, retransplantation for allindications is associated with longer hospital stays, greater cost, anddecreased survival [129,130]. Patient and graft survival after retransplanta-tion for HCV is worse than after primary transplants [131], and althoughdata are limited, some believe that outcomes in retransplantation forHCV are worse than those for any other indication [132,133]. The ScientificRegistry of Transplant Recipients database has provided one of the largeststudied cohorts of retransplanted patients with more than 1700 patients,


almost 500 of whom had HCV. This retrospective analysis found a 31%higher covariate adjusted mortality risk when HCV-infected recipientswere compared with all other recipients combined [132]. The significanceof this dramatic difference has been debated, however, given the heterogene-ity of the non–HCV-infected group and the lack of data on the specificindications for retransplantation in this analysis. In addition, there is evi-dence to the contrary. One large European study and a large cohort fromthe University of California at Los Angeles found no significant differencein outcomes in patients who had and did not have HCV [130,134]. Further-more, when the causes of liver disease are separated, although some groupsof recipients have better outcomes (eg, HBV, autoimmune hepatitis), othergroups, including cryptogenic and alcohol-related disease, have similar out-comes to recipients who have HCV [131,135]. There is also evidence thatwhen indicators of severity of disease are controlled for, HCV may not in-dependently predict poor outcomes [133,136]. Most recently, McCashlandand colleagues [137] reported on data from 10 centers, and found no differ-ence in 1- and 3-year survival rates in HCV-infected and non–HCV-infectedrecipients. They also found that approximately one third of patients withHCV-related graft failure are not considered candidates for retransplanta-tion and that only approximately one half of those evaluated are listed.

Given the scarcity of organs available and the tremendous cost of trans-plantation, the possibility that HCV-infected recipients have poor outcomeshas caused some centers to decline retransplantation in these patients. Giventhe ambiguity of the data, however, the suggestion that this differencemay notpersist when theMELD score is controlled for, and the hope of improved out-comes as we learn more about modulation of antiviral and immunosuppres-sive regimens, most centers do not systematically exclude these patients. Infact, the proportion of patients retransplanted for HCV is rising [137,138].Maximizing the benefits of retransplantation by individual patient selectionand appropriately timing the evaluation continues to be the goal, but this ishindered by the lack of prospective data and uncertainty as to acceptablepost-retransplantation survival rates.Many studies have attempted to identifyrisk factorsmost predictive of poor outcomes,which include age, renal failure,hyperbilirubinemia, and poor conditioning (Table 1) [130,133,134,139–141].Patients with early aggressive recurrence, particularly cholestatic HCV, andgraft failure within the first year or with high MELD scores have poor out-comes with retransplantation [131,135,137,142–144]. In addition, the timebetween first and second transplants is a strong predictor, with the highestdeath rate in one series being in the intermediate period between 8 and30 days (as compared with within the first 7 days or after 30 days) [145].

These and other risk factors have been used to create models to predictwho is likely to benefit most and, perhaps more importantly, those inwhom intervention may be futile. Rosen and colleagues [133] created onesuch model and used age, bilirubin, creatinine, UNOS status, and causeof graft failure to place patients into low-, medium- and high-risk groups.

Table 1

Predictors of outcomes in liver retransplantation for hepatitis C virus

Favorable outcomes Diminished outcomes Controversial effects

Recipient factors MELD score !21 Advanced recipient age

Portal HTN only Renal failure

Ventilator dependence

MELD score O21

Donor factors Advanced donor age

Short warm and

cold ischemic times

Prolonged warm and

cold ischemic times

Viral factors Antiviral response FCH HCV statusa

Viral eradication Early recurrence

Timing of

retransplantation

!8 days or O30 days

after transplant

8–30 days after transplant

Abbreviations: FCH, fibrosing cholestatic HCV; HTN, hypertension.a HCV status is unlikely to have a negative impact on retransplantation outcomes when the

severity of illness is controlled for (see text).

650 VERNA & BROWN

Their model was highly predictive of survival but has not been rigorouslyvalidated. Markmann and colleagues [146] used a five-point scoring systembased on recipient age, creatinine, bilirubin, cold ischemic time, and ventila-tory status, utilizing recipient and donor factors. Perhaps the most compre-hensive model was created by Ghobrial and colleagues [136] for first andsecond transplants, and it also included donor and recipient factors. Thedata included in the final mortality index equation included donor age,recipient age, creatinine, bilirubin, prothrombin time, warm and cold ische-mic times, and whether this was a second transplantation. The authorsvalidated 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 betweenfirst and second transplants, tables of estimated survival can be created thatmay be useful to estimate the outcomes of a selected patient [147]. In addi-tion, the donor risk index originally derived from patients undergoing pri-mary transplantation developed by Feng and colleagues [148] was recentlyused to look at retransplantation from the UNOS database [149]. Theseinvestigators added a cause of graft failure variable to the original modelto create what they termed the retransplant donor risk index, which theyfound to be a strong predictor. Many of these models have been used byclinicians and researchers to characterize individual patient risk but arealso important tools when comparing research cohorts. The group describedby McCashland and colleagues [137], for example, was predominantly com-posed of lower risk patients, perhaps accounting for their impressive results.

Although much remains unknown, several general lessons have beenlearned. Retransplantation should probably be avoided in patients whohave early aggressive HCV recurrence [142]. Retransplantation should per-haps be immediate (within 7 days) in patients who have early graft failure


because of technical complications, because intermediate durations of timemay lead to worse outcomes. In addition, prolonged survival may beachieved by attempting retransplantation at a lower MELD score thanwould be considered in the initial transplantation. In fact, weighted utilitycurves by Burton and colleagues [142] indicate that the maximal value ofretransplantation may be achieved at a MELD score of 21 for HCV-infectedpatients and 24 for all other patients. It is likely that these lessons have led tothe evolution of patient selection, and perhaps a diminishing discrepancybetween outcomes in HCV- and non–HCV-infected patients over time. Asthe focus of the debate regarding retransplantation for HCV shifts fromdeciding if it should occur to when it should occur and in which individuals,several crucial questions remain unanswered. Determining at what predictedsurvival rate the procedure becomes unacceptable is one major step, becauseprediction models are ineffective when the survival rate they predict cannotbe put into context and used in clinical decision making. In addition, theMELD system does not take into account several variables found to behighly predictive of outcomes specifically in retransplantation, such as fea-tures of the graft and time since primary transplant, and may not alwayspredict outcomes [137]. Widespread application of a more comprehensivemodel may therefore be required.

Future directions

OLT is a life-saving therapy to reverse the hepatic failure associated withchronic HCV infection. Its long-term success, however, remains limited bythe profound shortage of organs available and diminished graft and patientsurvival in the face of HCV recurrence. Expansion of the donor pool iscrucial through increasing public awareness of deceased donation, increasedliving donation, and using selected organs outside of widely accepted criteriathat are shown to be safe in this population. Through this expansion of thedonor pool alone, wait list mortality is likely to decrease. In addition, whenHCV-infected patients undergo transplant surgery, every effort to prevent ordiminish the severity of HCV recurrence must be made. Although greatadvances have occurred in the potency of our immunosuppressive arma-mentarium, in the case of HCV-related liver disease, we have yet to strikea predictable balance between suppression or treatment of rejection andavoiding uninhibited viral replication and progressive recurrent disease.Trials are required to optimize immunosuppression and are likely to involveprotocols with minimal modulation in the potency of the regimen and low-dose steroids with slow tapers. Prevention of HCV reinfection of the graftwith improved pretransplant prophylactic viral eradication and appropriatetiming of transplantation in addition to better posttransplant treatmentstrategies are desperately needed. As the next generation of HCV treatmentevolves to include HCV enzyme inhibitors, such as protease and polymeraseinhibitors, much work is required to determine the regimen with the best

652 VERNA & BROWN

efficacy and safety. It is conceivable that with these therapies, patients couldbe transplanted while on short courses of medications with suppressed orundetectable viral loads and perhaps be cured of HCV infection. Finally,when all these measures fail, additional research must be done to determinewho should undergo retransplantation and when they are likely to derive themost benefit from the second transplant.

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ical recurrence rate in HCVþ liver transplant recipients given basiliximab þ steroids or

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7(8):1984–8.

Hepatitis C Virus Infectionand Hepatocellular Carcinoma

Wojciech Blonski, MD, PhDa,b,K. Rajender Reddy, MDa,*

aHospital of the University of Pennsylvania, 3 Dulles, 3400 Spruce Street,

Philadelphia, PA 19104, USAbDepartment of Gastroenterology and Hepatology, Wroclaw Medical University,

Borowska 213 Street, 50-556 Wroclaw, Poland

Epidemiology

Primary liver cancer is the sixth most common cancer in the world andthe third most common cause of death attributable to cancer [1]. Most pri-mary liver cancers are hepatocellular carcinoma (HCC), accounting for 85%to 90% of cases [2]. There has been an increase in the number of cases ofHCC 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 ratewas observed between 1985 and 2002 in the United States [2–4]. This in-crease began in the mid-1980s, with the peak of proportional HCC increasein the late 1990s [2]. There was a marked and 2.5-fold increase in the averageannual age-adjusted rate of HCC confirmed by histology or cytology, andthis was from 1.3 per 100,000 population between 1978 and 1980 to 3.3per 100,000 population between 1999 and 2001 [2,5]. An analysis of the Sur-veillance, Epidemiology, and End Results (SEER) database in the UnitedStates between 1976 and 2002 noted the greatest proportional increase inage-adjusted HCC incidence rate per 100,000 population among whites (in-cluding Hispanics and non-Hispanics); the lowest proportional increase wasamong Asians [2]. Between 1976 and 2002, an average annual age-adjustedHCC incidence rate per 100,000 population increased 2.5-fold in blackAmericans (1.0 versus 2.5), 2-fold in whites (2.5 versus 5.0), and 1.3-foldin Asians (6.0 versus 8.0) (Fig. 1) [2]. There was a shift in the distributionof patient age toward younger ages, with the greatest proportional increase

Clin Liver Dis 12 (2008) 661–674


E-mail address: [email protected] (K.R. Reddy).



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: Sur-

veillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov.) SEER*Stat

Database: IncidencedSEER 13 Regs Public-Use, Nov 2004 Sub (1973–2002 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 submis-

sion. (From El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular

carcinogenesis. Gastroenterology 2007;132:2559; with permission.)

662 BLONSKI & REDDY

being in patients between 45 and 60 years old [2]. An average age at the timeof 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 UnitedStates observed the highest increase of HCC among patients who havehepatitis C virus (HCV) infection when compared with patients who havehepatitis B virus (HBV) [6–9]. Two of aforementioned studies performedin large single referral centers found that HCV was the major contributingfactor to the increased incidence of HCC [6,7]. The first study observeda threefold significant increase in the age-adjusted rate of primary liver can-cer in patients who had HCV infection (2.3 per 100,000 population during1993–1995 versus 7.0 per 100,000 population during 1996–1998), with stableage-adjusted rates for primary liver cancer associated with HBV (2.2 versus3.1 per 100,000 population), alcoholic cirrhosis (8.4 versus 9.1 per 100,000population) or without risk factors (17.5 versus 19.0 per 100,000 population)[6]. The second single-center study observed a significant increase in thenumber of patients who had HCC and HCV infection, from 18% between1993 and 1995 to 31% between 1996 and 1998 (P ¼ .01), with an accompa-nying significant decrease in the number of patients who had HCC and HBVinfection (26% versus 17%; P ¼ .06) and a stable rate of patients who hadHCC and negative HBV and HCV markers (56% versus 52%, P ¼ .5) [7]. Apopulation-based study using SEER-Medicare–linked data among patients65 years of age and older noted a marked increase in HCV-related HCC and

663HEPATITIS C VIRUS INFECTION AND LIVER CANCER

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 toa 67% increase in patients who had HBV when adjusted for age, gender,and geographic region [8]. Finally, the study from the fourth largest indigenthealth care system in the United States observed an increase in HCCassociated with HCV from 52% in 1992 to 1996 to 68% in 1997 to 2001(P ¼ .2) [9] and a decrease in HCC associated with HBV from 37% to34% (P ¼ 1.0) [9].

Therefore, chronic HCV infection has been considered the major riskfactor for development of HCC. It is suggested that the current increasein HCC rates in young men has been caused by HCV infection acquiredfrom intravenous drug use from the late 1960s to the early 1980s [10,11], rec-ognizing 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 req-uisite for HCC in HCV infection. The lower increase of HCC rates amongthe older population is likely explained by the fact that they may haveacquired HCV infection through a blood transfusion, because these patientswere more likely to receive blood transfusions [12] and did not live longenough to evolve to cirrhosis as a consequence of competing comorbidconditions being responsible for early non–liver disease–related mortality.

There is geographic variability in the prevalence of markers of HCV in-fection in patients who have HCC; it has ranged from 27% in the UnitedStates [13], from 27% to 75% in Western Europe (France: 27%–58%, Italy:44%–66%, and Spain: 60%–75%) [14–18], and up to 80% to 90% in Japan[19,20]. A meta-analysis of 32 case-control studies estimated that patientsinfected with HCV had nearly a 24-fold higher risk for developing HCCthan those without HCV infection [21]. The risk for developing HCC inHCV-positive patients was much higher (31.2) in regions with relativelylow endemicity for HBV infection, such as Japan and the Mediterraneancountries, and much lower (11.5) in regions with a high endemicity ofHBV infection, such as sub-Saharan Africa and southern Africa, China,Taiwan, South Korea, and Vietnam [21]. A large community-based prospec-tive study from Taiwan that included 12,008 patients observed a 20-foldincreased risk for developing HCC in anti–HCV-positive patients whencompared with anti–HCV-negative subjects [22]. A systematic review of21 studies of patients infected with HCV published between 1980 and2001 observed that the pooled weighted incidence rates of end-stage liverdisease (ESLD) and HCC were the highest in studies that included patientswho had hemophilia and were infected by repeated transfusions of HCV-infected pooled clotting factor concentrates (7.9 and 1.0 per 1000 person-years, respectively) and transfusion-associated HCV (4.5 and 0.7 per 1000person-years) in patients who received HCV-infected blood or blood prod-ucts [23]. Conversely, the lowest incidence rates were found in studies thatincluded patients who had community-acquired HCV infection (intravenousdrug use, medical procedure-related, and non–transfusion-related causes of

664 BLONSKI & REDDY

HCV infection) (1.9 and 0 per 1000 person-years) and women who receivedone-time HCV-contaminated anti–D immune globulin (0.7 and 0 per 1000person-years) [23].

Mechanisms of development of hepatitis C virus–related

hepatocellular carcinoma

The exact mechanism of development of HCC in chronic HCV infectionis unclear. Although it was initially suggested that HCV enhanced mutagen-esis in hepatocytes as a result of chronic inflammation and hepatocyte regen-eration, it was questioned whether liver inflammation itself is capable ofcausing HCC. The role of inflammation alone in hepatocarcinogenesis isquestionable because patients who have persistent severe inflammation inthe course of autoimmune hepatitis rarely develop HCC [24]. A transgenicmouse model has demonstrated that the HCV core protein plays a pivotalrole in the development of HCC [25]. It was recently proposed that theHCV core protein may have oncogenic properties that allow omission ofsome of the usual multiple steps required during the process of carcinogen-esis [24]. Therefore, the ‘‘non-Vogelstein’’ type process of hepatocarcinogen-esis 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 tothe development of HCC even without the complete set of genetic aberra-tions usually required for carcinogenesis [24].

The HCV core protein has been shown to induce reactive oxygen speciesin the mouse liver in the absence of inflammation [26]. Oxidative stress in theliver is caused by the direct effect 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 effects 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.)


Oxidative stress driven by HCV core protein may reduce mitochondrialmetabolic processes, which, in turn, may induce the development of liversteatosis attributable to inhibition of b-oxidation and oxidative injury tomitochondrial and chromosomal DNA [27]. HCV core protein has beennoted to induce liver steatosis in a transgenic mice model [28–31] and in pa-tients who have chronic hepatitis [28] by inhibiting microsomal triglyceridetransfer protein activity [28–30]. Based on the transgenic mice model, it issuggested that expression of the HCV structural proteins enhances a lowbackground of liver steatosis, whereas additional low expression of non-structural 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 theexpression of tumor necrosis factor-a and interleukin-1; enhances the activ-ities of c-Jun N-terminal kinase and activator protein-1 [32]; and directlyinteracts with a transcriptional regulator retinoid X receptor-a that playsan important role in the control of cell proliferation, differentiation, andlipid metabolism [33]. In a mouse liver model for HCV-related HCC,HCV core protein was shown to activate p38 mitogen-activated proteinkinases and extracellular signal-regulated kinase together with ethanol; italso modulated the expression of several genes related to cell transforma-tion, cell cycle, and antioxidants [34]. The specific interaction between theHCV core protein and proteasome activator PA28 in cell culture and inthe livers of HCV-core transgenic mice and a patient who had chronic hep-atitis C has also been observed [35]. HCV core protein may also modulatethe intracellular signaling pathway, and thus may induce hepatocarcinogen-esis by selective suppression of the expression of the suppressor of cytokinesignaling SOCS-1 gene demonstrated in the liver tissues of animals andcultured cells [36]. Thus, there is experimental evidence to suggest that theremight be interplay among several mechanisms that may lead to the develop-ment 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 fibrosis and cirrhosis, thusincreasing the risk for HCC [2]. It has been estimated that 1% to 3% ofHCV-infected patients develop HCC after 30 years (Fig. 3) [2,7]. Thereare several factors that increase the likelihood of development of livercirrhosis and HCC in patients who have chronic HCV infection, and theseinclude older age, older age at the time of the onset of HCV infection, malegender, accompanying infection with HBV or HIV, heavy consumption ofalcohol, diabetes mellitus, obesity, transfusion-acquired HCV infection,and genotype of HCV (Table 1) [13].

Fig. 3. Proportion of patients who have HCC related to viral hepatitis. In this study, all US-

born 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 molec-

ular carcinogenesis. Gastroenterology 2007;132:2557–76; 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;35:2562,266–9; with

permission.)

666 BLONSKI & REDDY

Alcohol consumption

Several studies report a synergistic interaction between alcohol consump-tion and chronic HCV infection on the development of HCC. Both factorsactively promote liver cirrhosis [13]. Two hospital-based case-control studiesobserved an additive interaction between lifetime daily alcohol intake(LDAI) less than 50 g/d and HCV infection and multiplicative interactionfor LDAI greater than 125 g/d and HCV infection on development of livercirrhosis [23]. The effect of alcohol on the risk for development of liver cir-rhosis was dose related in anti–HCV-positive (odds ratio [OR] ¼ 9.2 forLDAI ¼ 0 g/d and OR ¼ 147.2 for LDAI R175 g/d) and anti–HCV-nega-tive (OR ¼ 1.0 for LDAI ¼ 0 g/d and OR ¼ 15.0 for LDAI R175 g/d)patients [23]. The effect of daily alcohol intake on the risk for HCC in pa-tients who have HCV was found also to be dose related with a 2-fold and4-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 drinkingat a rate of more than 80 g/d for at least 5 years was shown to have a 23-foldincrease for the risk for HCC among patients who have chronic HCV infec-tion [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 anddrinking alcohol at a rate of more than 60 g/d. Overall, consumption ofalcohol in patients who have HCV infection at dosages of 0 to 60 g/d ormore than 60 g/d was associated with a 55-fold and 109-fold increase inthe risk for HCC, respectively, when compared with controls drinkingsimilar amounts of alcohol but without HCV infection [39]. A case-controlstudy from Texas found that heavy alcohol drinkers (R80 mL/d) infectedwith 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 Factor Risk Comment

Corrao and Arico [23] Alcohol OR ¼ 9.2 (95% CI: 2.0–43.2)

OR ¼ 147 (95% CI: 42.1–514.3)

LDAI ¼ 0 g

LDAI ¼ 175 g

Tagger et al [37] Synergy index: 2.4

Synergy index: 4.0

Alcohol: 41–80 g/d

Alcohol: O80 g/d

Donato et al [38] RR ¼ 29.8 (95% CI: 13.3–66.5)

RR ¼ 66.3 (95% CI: 20.5–214)

Alcohol: 0–80 g/d

Alcohol: O80 g/d

Donato et al [39] OR ¼ 55.0 (95% CI: 29.9–101.0)

OR ¼ 109 (95% CI: 50.9–233.0)

Alcohol: 0–60 g/d

Alcohol: O60 g/d

Hassan et al [40] OR ¼ 53.9 (95% CI: 7.0–415.7) Alcohol: O80 mL/d

Yuan et al [41]a Synergy index: 5.5 (95% CI: 3.9–7.0) Alcohol: O4 drinks per day

Donato et al [21] HBV OR ¼ 135 (95% CI: 79.7–242) d

Tagger et al [37] Synergy index: 2.4 dChiaramonte et al [42] HR ¼ 2.3 (95% CI: 5 1.1–4.6) d

Yuan et al [41] OR ¼ 63.9 (95% CI: 8.6–475.3) d

Yuan et al [41]a Diabetes Synergy index: 4.8 (95% CI: 2.7–6.9) d

Komura et al [43] HR ¼ 2.9 (95% CI: 1.5–5.5) Risk for recurrence of HCV-

or HBV-related HCC

Ohata et al [44] Liver steatosis RR ¼ 2.81 (95% CI: 1.24–6.37) d

Pekow et al [45] Liver steatosis OR ¼ 6.39 (95% CI: 1.04, 39.35) d

Kumar et al [46] Liver steatosis OR ¼ 1.0 (95% CI: 0.8–1.3; P ¼ .9) dBruno et al [47] HCV genotype 1b OR ¼ 6.14 (95% CI: 1.77–21.37) d

Tagger et al [37] OR ¼ 34.2 (95% CI: 18.0–64.7) d

Donato et al [38] RR ¼ 34.3 (95% CI: 13.9–84.2) dSilini et al [48] OR ¼ 1.7 (95% CI: 1.06–2.9) d

Lopez-Labrador et al [49] OR ¼ 4.286 (95% CI: 1.437–12.82) d

Abbreviations: CI, confidence interval: HR, hazard ratio; RR, relative risk, LDAI, lifetime daily alcohol use.a Includes patients who had HCV, HBV, or both. 6

67

HEPATIT

ISC

VIR

USIN

FECTIO

NAND

LIV

ER

CANCER

668 BLONSKI & REDDY

Overall, the synergic interaction between HCV infection and heavy alco-hol consumption on the risk for HCC was observed with synergy indicesranging from 2.0 to 5.5 [37,38,40,41].

Coinfection with hepatitis B virus

Several studies observed the synergism between HBV and HCV infectionwith respect to the increased risk for HCC. Ameta-analysis of 32 case-controlstudies found that infection with both HBV and HCV was associated witha 135-fold increased risk for HCC, whereas infection with HBV or HCVwas associated with a 20-fold and 24-fold increased risk, respectively [21]. An-other study observed a 2.4 synergy index between infection with both HCVand HBV in increasing the risk for HCC [37]. An Italian experience observedthat patients who had liver cirrhosis and were infected with both HBV andHCV were at significantly higher risk for developing HCC than patientswho had a single viral infection [42]. Cumulative appearance rates for HCCat 5, 10, and 13 years were the highest in cirrhotic patients who had dualHBV/HCV infection (23%, 45%, and 55%, respectively) when comparedwith 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 effect of HIV coinfectionon progressive liver disease in patients who had HCV infection observedthat patients who had dual HIV/HCV infection had a significantly elevatedrisk for severe liver disease when compared with those infected only withHCV [50]. Overall, the combined relative risk for developing decompensatedliver disease was 6.14, whereas the relative risk for developing histologiccirrhosis was 2.07 in patients infected with both HIV and HCV whencompared with those infected only with HCV [50]. HCC was likely to occurat a younger age and after a shorter duration of HCV infection in patientscoinfected with HIV [51]. A recent multicenter retrospective study hasshown that patients infected with both HIV and HCV developed HCCsignificantly faster than those infected only with HCV (26 versus 34 years;P ¼ .002) [52].

Diabetes mellitus

The recent meta-analysis of 13 case-control studies and 13 cohort studiesshowed a significant association between diabetes and development of HCCin 9 case-control studies and in 7 cohort studies, respectively [53]. The mech-anisms by which diabetes may increase the risk for HCC include predispo-sition to development of nonalcoholic steatohepatitis with progression tocirrhosis in up to 5% cases [54] or association with increased levels of poten-tial carcinogenic insulin-like factors [55].


A large case-control hospital-based study including 823 patients and 3459controls observed that diabetes mellitus significantly increased the risk forHCC only in the presence of other risk factors, such as hepatitis C, hepatitisB, or alcohol cirrhosis [56]. Another population-based case-control studyobserved the synergic interaction (synergy index of 5.5) between HCV infec-tion and diabetes mellitus on the risk for HCC [41].

Overall, a meta-analysis of studies that adjusted for the associationbetween diabetes and HCC for HCV or unspecified viral hepatitis showedno change or minimal change in the associated risk for HCC in those indi-viduals with diabetes and HCV [53]. A recent study suggested that diabeteswas a risk factor for the recurrence of HCV-related HCC, and thusdecreased the overall survival rates after surgical treatment [43].

Obesity

Obesity may increase the risk for development of liver steatosis and fibrosisin patients who have HCV [47,57]. Patients who have chronic HCV infectionand a high grade of liver steatosis, particularly with steatohepatitis, might beat risk for more accelerated progression to liver cirrhosis [57]. Liver steatosishas been noted to be an independent and significant risk factor for HCC inpatients who have HCV [44,45]. A linear increase in the odds ratio forHCC with increased grades of liver steatosis from 1.61 for grade 1, to 3.68for grade 2, and to 8.02 for grade 3 or 4 when compared with grade 0 hasbeen reported [45]. Thus, steatosis might be an additional risk for HCC,and increased vigilance should be practiced in surveillance of HCV-infectedpatients who have liver steatosis [44,45]. Conversely, Kumar and colleagues[46] did not find liver steatosis to be a risk factor for HCC in patients whohave HCV infection in a prospective analysis of HCC risk derived froma rigorously matched cohort of HCV-infected patients.

Genotype of hepatitis C virus

The relation between the HCV genotype and the risk for HCC has beencontroversial. Several studies found that patients infectedwithHCVgenotype1b had from a twofold to sixfold increased risk forHCC and hadmore aggres-sive progression of associated liver dysfunction [37,38,47–49,58–60]. It hasbeen suggested that an increased incidence ofHCC in thosewith abackgroundof genotype 1b might have been because of overrepresentation of HCV geno-type 1b in patients who were of advanced age and had more advanced liverdisease, 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 efficaciousprophylactic approach in patients who have chronic HCV infection. A recent

670 BLONSKI & REDDY

analysis of eight cohort studies in Japan demonstrated that therapy withinterferon improved the prognosis of HCV-positive patients who did nothave liver cirrhosis and who did have liver cirrhosis and of patients whohad cirrhosis and HCC [61]. Such therapy with interferon reduced therisk for HCC by 50% in patients who had chronic HCV infection withoutliver cirrhosis, with a further reduction to 20% among patients with a sus-tained virologic response [61]. Patients with a sustained virologic responsetreated with interferon were further shown to have an improvement inliver fibrosis histology [61]. Therapy with interferon also increased survivalrates because of a reduction in the incidence of HCC and hepatic failure[61].

Therapy with interferon in patients who had compensated HCV-relatedliver cirrhosis caused a significant reduction in the cumulative incidence ofHCC when compared with patients who had HCV-related cirrhosis notreceiving interferon with an adjusted relative risk of 0.54 [61]. Sustainedresponders presented with the strongest effect, with a relative risk of 0.05when compared with untreated patients [61]. Finally, none of patientswith a sustained viral response died from liver-related causes comparedwith 26% of untreated patients [61].

A meta-analysis of 11 studies that included 2178 patients who had HCV-related liver cirrhosis observed that patients not receiving interferon hada threefold higher risk for HCC than those treated with interferon, regard-less of achieving or not achieving a sustained virologic response [62]. Thebenefit 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 com-bined therapy with interferon-a-2b and ribavirin may decrease the incidenceof HCC in patients who have HCV-related cirrhosis. A recent prospectiverandomized controlled study that evaluated the effect of therapy with inter-feron on the incidence of HCC in chronic active hepatitis C with advancedfibrosis or cirrhosis recently concluded in the United States [64].

Summary

Primary liver cancer is the sixth most common cancer in the world andthe third most common cause of death attributable to cancer. Most primaryliver cancers are HCC, accounting for 85% to 90% of cases. There is a trendof growing incidence of HCC in the United States. One of the most impor-tant risk factors for developing HCC is chronic HCV infection. Suchpatients have been shown to have a greater than 20-fold increased risk forHCC than those without HCV infection. HCV increases the risk for HCCby promoting the development of liver fibrosis and cirrhosis. It has beenestimated that 1% to 3% of HCV-infected patients develop HCC after30 years. The risk factors for developing HCC among patients who haveHCV-related cirrhosis 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. Although several studiessuggested the preventive effect of interferon from developing HCC inHCV-infected individuals, these findings need to be validated in large pro-spective and randomized trials.

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Hepatitis C and Innate Immunity:Recent Advances

Gyongyi Szabo, MD, PhD*,Angela Dolganiuc, MD, PhD

Department of Medicine–LRB215, University of Massachusetts Medical School,

364 Plantation Street, Worcester, MA 01605–2324, USA

Innate immunity: the first line of defense in hepatitis V virus infection

Innate immunity is the first line of defense in the host response to invad-ing viral, bacterial, or fungal pathogens, and hepatitis C virus (HCV),a single-stranded RNA virus, is no exception [1,2]. Cells participating inthe innate immune response include monocytes, macrophages, dendriticcells (DCs), polymorphonuclear (PMN) leukocytes, natural killer (NK)cells, and natural killer T (NKT) cells, which are all equipped with patho-gen-sensing receptors and are present in the liver (Fig. 1) [1]. Monocytes,macrophages, and PMN leukocytes are effectors and regulators of inflam-mation because of their capacity to take up pathogens and produce reactiveoxygen radicals and pro- and anti-inflammatory cytokines. DCs are sophis-ticated in antigen presentation and induction of T-cell activation throughtheir expression of costimulatory molecules and cytokine production,whereas NK cells provide interaction with virus-infected cells, T lympho-cytes, and DCs [1,3]. Recognition of viral pathogens by a coordinated inter-action of the cells of the innate immune system leads to activation ofadaptive immunity targeting viral-specific antigens for pathogen elimina-tion. Of the various pattern recognition receptors, Toll-like receptors(TLRs) and RNA helicase receptors play an important role in sensing viralRNA and in induction of the initial type I interferon (IFN) production.Double-stranded (ds) RNA is recognized by TLR3 expressed in the endo-somes and retinoic acid–inducible gene-I (RIG-I) and melanoma

Clin Liver Dis 12 (2008) 675–692

This work was supported by Public Health Service grants AA014372 and AA008577 to

G. Szabo and grant AA016571 to A. Dolganiuc.


E-mail address: [email protected] (G. Szabo).



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 flow. Dendritic cells of plasmacytoid (PDC) and myeloid (MDC) origin,

monocytes (Mo), macrophages (Mf), and Kupffer 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 hepato-

cytes, 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 cyto-

kines and availability of costimulatory molecules. In turn, adaptive immune cells, including T

and B lymphocytes, NK cells, and NKT cells, react with antigen-specific proliferation, cytotox-

icity, 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 effect on the adaptive immunity. Mo,

Mf, and KC are potent producers of inflammatory and immunoregulatory cytokines and are

powerful sources of free radicals with oxidative capacity, thus leading to initiation and mainte-

nance of tissue inflammation. Chronic inflammation, in addition to direct pathogen recognition,

activates SC, which, in turn, produce collagen and favor development of liver fibrosis. At end-

stage liver disease, chronic inflammation and fibrosis drive progression to cirrhosis and possibly

favor neoplastic transformation and liver cancer.

676 SZABO & DOLGANIUC

differentiation–associated gene 5 (MDA5) localized in the cytosol, whereassingle stranded (ss) RNA is sensed by TLR7 and TLR8 and by RIG-I insome viruses (Figs. 2 and 3) [3–5].

Toll-like receptor 3

Ligand engagement of TLR3 results in recruitment of the adapter mole-cule, TLR domain-containing adapter inducing IFNb (TRIF) (see Fig. 2)[6,7]. TRIF interacts with several signaling molecules, including tumor ne-crosis factor (TNF) receptor-associated factor (TRAF)6, which, in turn, ac-tivate nuclear regulatory factor kB (NF-kB) and mitogen-activated proteinkinase (MAPK). Important to viral infection, TLR3 stimulation leads to ac-tivation of TNFR-associated factor family member-associated NF-kB acti-vator (TANK)-binding kinase (TBK) 1 and IkB kinase (IKK)3, which

Fig. 2. TLR3 and TLR7/8 signaling. After stimulation with ssRNA, TLR7/8 recruits myeloid

differentiation primary response gene 88 (MyD88), interleukin-1 receptor–associated 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 transform-

ing 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 nu-

clear 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 inflammatory 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 ex-

pression 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 ac-

tivation. TLR3 and TLR7/8 are located in endosomes, and all signaling events occur in the cy-

toplasm, whereas activated transcription factors IRF3, IRF7, NF-kB, and AP-1 act on the

genes within the nucleus. The cellular compartments are not scaled.

677HEPATITIS C AND INNATE IMMUNITY: RECENT ADVANCES

phosphorylates interferon regulatory factor (IRF) 3, allowing its dimeriza-tion and nuclear translocation. Activated IRF3 binds to the promoter oftype I IFNs and triggers antiviral innate immune activation [6,7].

RNA helicases

Recently, three homologous DExD/H box RNA helicases emerged ascytoplasmic sensors of virally derived RNA (see Fig. 3). On activationwith 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 likelythrough sequestration of dsRNA from RIG-I or MDA5 [8–10]. Although

Fig. 3. Intracytoplasmic viral recognition and antiviral pathways. The RNA derived from vi-

rions 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)-bind-

ing 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 CARD-

less 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-I–containing signaling complex on IPS-1, and

by sequestering the viral dsRNA.


RIG-I and MDA5 seem to share structural and functional similarities, theirrecognition capacity of viral-derived RNA is distinct. RIG-I but not MDA5senses Japanese encephalitis virus, Newcastle disease virus, vesicular stoma-titis virus (VSV), Sendai virus, and influenza virus, whereas MDA5 mountsantiviral responses against the picornavirus encephalomyocarditis virus [11].To date, it is unknown how RIG-I undergoes activation only with viralRNA and not with cellular RNA. The direct implication of RIG-I orMDA5 in recognition of ssRNA hepatotropic viruses, such as HCV, hasbeen suggested [12]. Of interest, RIG-I deficiency strongly affects antiviralresponses in conventional DCs and fibroblasts; however, no differencesare observed in plasmacytoid DCs, suggesting tissue preference of these hel-icases. RIG-I, MDA5, and LGP2 are all expressed in hepatocytes [13,14].Interestingly, decreased expression levels of RIG-I and MDA5 were de-tected in Huh7 and Huh7.5 cells, which are permissive to HCV repliconsand in vitro infection with the replication-efficient JFH1 and JFH/J6HCV strains [15].

RIG-I and MDA5 share structural similarity in the caspase recruitmentdomains (CARDs) and RNA helicase domains, whereas LGP2 lacks the


CARD domain [10,11]. RIG-I and MDA5 use their CARDs to signal down-stream events bymeans of an adaptor named CARD adaptor–inducing IFNb(Cardif), mitochondrial antiviral signaling protein (MAVS), interferon pro-moter stimulator (IPS-1), or virus-induced signaling adaptor (VISA) [16–19]. IPS-1 is anchored with its C terminus to the outer mitochondrialmembrane, whereas its N-terminal CARD domain interacts with RIG-Iand MDA5. Mitochondrial localization renders IPS-1 functional, becausethe cytoplasmic domain of the endoplasmic reticulum-bound IPS-1 no longermediates downstream IRF and NF-kB activation [18]. The details of the sig-naling pathway downstream of IPS-1 are currently under scrutiny. Once ac-tivated by dsRNA, the IPS-1 most probably recruits appropriate signalingintermediates, such as IKKs (namely IKKa, IKKb, IKK3, and TBK1) to ac-tivate NF-kB, TRAF3, and IRF transcription factors [20]. All these pathwaysinduce production of type 1 IFNs; however, the kinetics of the differentialproduction of IFNa and IFNb on retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) activation is yet to be defined.

It has recently been demonstrated that RIG-I but not MDA5 efficientlybinds to secondary structured HCV RNA to confer induction of IFNbexpression [12]. LGP2 is a functional negative regulator of host defense,and it binds HCV [12]. In resting cells, RIG-I is maintained as a monomerin an autoinhibited state, but during virus infection and RNA binding, itundergoes conformational changes that promote self-association andCARD interactions with the IPS-1 adaptor protein to signal IRF3 andNF-kB responsive genes [9,10,12]. This interaction is regulated by an inter-nal repressor domain, which controls RIG-I multimerization and recruit-ment of IPS-1. An analogous regulatory domain in LGP2 interacts withRIG-I to ablate self-association and signaling. Thus, RIG-I is a cytoplasmicsensor of HCV and is governed by regulatory domain interactions in thatarea shared with LGP2 as an ‘‘on-off’’ switch controlling innate defenses[12].

Toll-like receptors 7 and 8

TLR7 and TLR8 are expressed in the endosome and recognize severalssRNA viruses (see Fig. 2) [21]. TLR7 was initially identified as a receptorable to recognize imidazoquinolone derivatives with antiviral activity [22].Subsequently, guanosine or uridine-rich ssRNA derived from HIV-1 and in-fluenza virus, synthetic poly U RNA, and certain small interfering RNAswere identified as ligands for TLR7 [22,23]. TLR7 is expressed in plasmacy-toid DCs, and TLR7 mRNA was detected in hepatocytes [24–27]. TLR8 isfunctional in humans but not in mice, and it is expressed in myeloid DCs,monocytes, macrophages, and regulatory T cells [28,29]. Human TLR8 me-diates recognition of HIV-derived ssRNA and chemical ligand R848, and itsrole in HCV infection is currently unknown [30]. Recent studies revealedfunctional differences between human TLR7 and TLR8, wherein TLR7


agonists primarily activated plasmacytoid dendritic cells (PDCs), whereasTLR8 agonists activated myeloid dendritic cells (MDCs), monocytes, andmacrophages [31]. In addition, the cytokine production profile of TLR7was dominated by IFNa induction, whereas TLR8 triggered predominantlythe proinflammatory cytokines and chemokines, such as tumor necrosis fac-tor-a (TNFa), interleukin (IL)-12, and macrophage inflammatory protein(MIP)-1a [31]. TLR7/8 agonists impair monocyte-derived DC differentia-tion and maturation; it is intriguing that the phenotype of TLR7/8 ligand-treated DCs is similar to DC defects found in HCV-infected patients [32,33].

The pattern recognition site composed of leucine-rich repeats of theTLR7/8 molecule is contained within the endosome, whereas the Toll/inter-leukin-1 receptor (TIR) domain is exposed to the cytoplasm, wherein it trans-duces intracellular signals by recruitment of the myeloid differentiationprimary response gene 88 (MyD88), a common TLR adaptor protein [3,6].MyD88 further forms complexes with members of the IL-1 receptor–associ-ated kinase (IRAK) family (IRAK1 and IRAK4) and TRAF6, which, inturn, activate transforming growth factor (TGF)-b-activated kinase 1(TAK1) and result in NF-kB activation. Type I IFN induction after TLR7activation is independent of IRF3, suggesting the possible involvement ofother IRF family members in this pathway. IRF7 is structurally similar toIRF3, and although its expression is low in most cell types, it is constitutivelyexpressed in PDCs [22,34]. IRF7 is able to form a signaling complex withMyD88, IRAK1, IRAK4, and TRAF6, wherein IRAK1 is capable of phos-phorylating IRF7 [35]. Activated IRF7 homodimerizes, allowing this com-plex to translocate into the nucleus and bind to the interferon-stimulatedresponse element (ISRE) promoter site [36]. Type I IFNs, composed ofIFNa 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 clear-ance are limited. Nevertheless, it is clear that the interaction betweenHCV viral components and the immune system ultimately determineswhether the balance tilts in the favor of the virus, leading to chronic infec-tion, or in favor of the host, conditioning viral clearance. Evidence points toinnate and adaptive immune responses as key determinants of the outcome.A strong, multispecific, T-lymphocyte response, including CD4þ andCD8þ cells, produced early in infection is associated with viral clearanceand disease resolution, whereas a narrowly focused and delayed responseis associated with chronic infection [37–41]. Even in patients who developchronic infection, however, T-cell responses were briefly or episodically vig-orous rather than absent at the onset of infection, indicating that a substan-tive CD4þ T-cell response must also be maintained to facilitate clearance[42]. Further, CD8þ T cells lose recognition of one or more HCV epitopes


originally targeted in primary infection in patients who progressed tochronic infection, thus leading to almost a three-quarters reduction in themagnitude of the response [43]. Importantly, no new epitope specificity isdeveloped in T cells of patients who have persistent HCV infection[43,44]. These results indicate that adaptive responses cannot overcomeHCV aggressiveness alone and suggest that the adaptive immune systemfails to receive support from innate immune cells to provide a long-lastingdefense. The information in regard to the function of innate immunityduring early HCV infection is scarce. In one study, resolution of acuteHCV infection was associated with a single nucleotide polymorphism inthe haplotype region of IL-10 and IL-19/IL-20 genes in African Americansbut not in European Americans [45].

Hepatitis C virus interferes with innate immune recognition

Increasing evidence suggests that HCV can interfere with innate immuneactivation at multiple levels. The nonstructural proteins of HCV, particu-larly NS3-4A, have been found to interact with various host adaptor mole-cules to disrupt type I IFN induction pathways. Foy and colleagues [46]found that NS3-4A serine protease blocked HCV-induced activation ofIRF3 in the human hepatoma cell line, Huh7. It has been reported thatNS3-4A protease also targets and cleaves the IPS-1 adaptor protein fromthe mitochondria to ablate signaling to IFNa/b immune defenses [16,47].

Inhibition of RIG-I–dependent signaling to the IFN pathway has beenfound in HCV infection by the HCV NS3/4A protease activity [48,49].This inhibition was localized to upstream of the noncanonical IKK-relatedkinases IKK3 and TBK1, which phosphorylate IRF3, at the level of theTLR adapter protein TRIF [48,49] and intercellular adhesion molecule 1(ICAM1). In the replicon system IKK3 overexpression inhibited HCV ex-pression even in the presence of neutralizing antibodies to IFN receptorsor in the presence of a dominant negative specifically targeted antiviral ther-apy (STAT) mutant [50], suggesting that IKK3 expression is important forrapid activation of the cellular antiviral response in HCV infection. In theliver biopsies of patients who have HCV, expression of IKK3 and theRNA helicases RIG-I, MDA5, and LGP2 was significantly reduced,whereas expression of TBK1 and Cardif was not significantly altered [50].These observations support the contention that HCV interferes with hostpathways directed at viral elimination.

Although the HCV NS3-4A–mediated cleavage of the adaptor moleculesTRIF (adaptor to TLR3) and Cardif and IPS-1 (adaptor to RIG-I andMDA5) has been reported in the human hepatoma cells Huh7 andHuh7.5, recent studies performed in the nonneoplastic human hepatocytePH5CH8 cells showed a retained and robust TRIF- and Cardif-mediatedpathway activation. Dansako and colleagues [14] found that more robust


induction of IFNb in PH5CH8 cells compared with Huh7 and NS3-4Afailed to suppress TRIF-mediated IFNb production in these cells. Cardif-mediated IFNb production was still suppressed by NS3-4A by cleaving Car-dif at the Cys508 residue.

Further analysis of the HCV NS3-4A protease defined the site of its inter-action with host targets. NS3 mutants lacking the helicase domain retainedthe ability to control virus signaling initiated by RIG-I or MDA5 and sup-pressed downstream activation of IRF3 and NF-kB through the targetedproteolysis of IPS-1 [51]. Truncation of the NS3 protease domain or pointmutation ablated the protease activity, however, providing evidence for theactive site of NS3 interaction with the host immune recognition pathways.

A recent study found modulation of TLR-mediated signaling in a macro-phage cell line expressingHCVproteins [52]. Various genotypes ofNS5A pro-tein bound the common TLR adaptor MyD88, and thereby inhibited therecruitment of IRAK1 to MyD88 and impaired cytokine production in re-sponse to TLR ligands. Transfection of NS3, NS3/4, NS5B, or NS5A intomouse macrophages resulted in inhibition of IL-6 induction by variousTLR ligands [52]. Although this observation indicates a novel possible wayof interaction of HCV with innate immunity, results of this in vitro studyare in contrast with previous observations from human monocytes and mac-rophages from individuals who had chronic HCV infection, wherein in-creased proinflammatory cytokine induction was observed in response toTLR4 or TLR2 stimulation [53,54]. An additional consideration is whethermacrophages are infected with HCV or whether these cells support HCV rep-lication. Positive-strandRNAhas been detected in peripheral bloodmononu-clear cells (PBMCs) and blood monocytes by several groups; however,observation of negative-strand RNA, which would indicate active viral repli-cation, in monocytes and macrophages has been found [55]. A recent studyfound the presence of HCV genomic RNA in circulating DCs and at evenhigher expression level in monocytes [56]. In this study, infection of DCswith HCV was associated with impaired expression of IL-12 and TNFa [56].

In the liver, DCs and resident and recruited macrophages play an impor-tant role in elimination of damaged cells. In in vitro studies, engulfment ofapoptotic blood mononuclear cells expressing HCV proteins resulted in dif-ferential chemokine expression and STAT signaling in DCs [57], suggestingthat virus-infected hepatocytes may modulate phagocytic cell functions evenin the absence of their direct infection with HCV.

Increased expression of TLR2, TLR3, and TLR6 mRNA was found inPBMCs of patients who have chronic HCV infection that correlated with sus-tained virologic response [58]. In HCV treatment trials, the number of genesthat were up- or downregulated by pegylated IFNand ribavirin treatment wasfewer in patients with a poor response than in those with an intermediate ormarked viral response [59]. The induction of IFN-inducible genes (2050-oligo-nucleotide synthetase), MX1, IRF7, and TLR7 genes was lower in patientswith a poor response compared with patients with a marked or intermediate


response, suggesting that blunted IFN signaling and TLR signaling is associ-ated with the lack of response to IFN therapy [59]. A recent study found anassociation between TLR7 single nucleotide polymorphism (SNP) andprotection from advanced inflammation and fibrosis in male patients whohad chronic HCV infection [60]. The C1-120G TLR7 allele was found to offerprotection from the development of inflammation and fibrosis [60].

Hepatitis C virus interferes with activation of adaptive immune responses

by innate immune cells

Effects on dendritic cells

In addition to recognition of invading pathogens, DCs play a centralrole in activation of naive T lymphocytes to initiate virus-specific T-cell re-sponses. DCs, including circulating MDCs, monocyte-derived DCs, andPDCs, have been studied in chronic HCV infection by several groups of in-vestigators. PDCs are the major producers of IFNa and are specificallyequipped to sense viral nucleic acids by means of their expression ofTLR7 and TLR9 [61]. Most investigators found decreased frequency, re-duced IFNa production, and impaired T-cell stimulatory capacity of circu-lating PDCs [62,63]. Interestingly, the expression of CD123 and BDCA2,markers of PDCs, were expressed at higher levels in livers of HCV-infectedpatients, raising the possibility of sequestration of PDCs in HCV-infectedlivers [63]. Indeed, enrichment for DCs within the intrahepatic compartmentwas recently reported [64], possibly attributable to HCV E2/CD81-mediatedinduction 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 expressionof major histocompatibility complex (MHC) class II, CD86, and CD123;were more efficient stimulators of allogeneic T cells; and secreted lessIL-10 compared with controls [68,69]. In contrast, some researchers findthat PDCs were present at lower frequencies in HCV-infected liver; how-ever, 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 HCVquasispecies sequences cloned from DCs bear an internal ribosome entry sitewith poor efficiency for translation in cells of liver, lymphoid, or DC origin[73], suggesting that passage through DCs significantly affects the functionof the HCV virion. There are clear indications that HCV-derived proteinsexpressed in vivo affect immune functions. Studies from mice expressingHCV nonstructural protein showed decreased capacity of a mixed PDCand MDC population to activate T cells [74], whereas overexpression ofstructural proteins leads to impaired MHC class I presentation during DCmaturation [75].


In human studies, findings related to myeloid DC functions are contro-versial. Complex defects, namely, decreased T-cell stimulatory capacity,overproduction of the immunoregulatory cytokine IL-10, and deficiencyin costimulatory molecules, were detected in MDCs of patients who hadchronic HCV infection by some investigators [33,76–80], whereas othersfailed to identify any MDC abnormalities [81–85]. Such discrepanciesmost possibly derive from different patient cohorts but also from assessmentof nonhuman primate models of HCV infection, different experimentalapproaches, and distinct read-outs.

Anti-HCV treatment also may affect DC functions. Combination a-IFN-ribavirin therapy alters the cytokine profile of maturing DCs by suppressingIL-10 production but maintaining IL-12 (p70) and TNFa production, a pat-tern that would favor viral elimination through downstream effects on Tcells [86]. MDCs from HCV-infected patients are impaired in their abilityto drive T helper 1 (Th1) in response to IFNa [87].

Natural killer cells

NK cells constitute a potent rapid part of the innate immune responseto viral infections but also to neoplastic cells and also participate in prim-ing of adaptive immunity [88]. NK cells are capable of performing cytol-ysis in addition to cytokine and chemokine release [88]. NK cells mountan anti-HCV response and can be triggered by HCV-derived proteins orHCV-infected cells. In vitro, NK cells were capable of inducing anHCV-associated or perforin- or granzyme-dependent lysis of human hepa-toma cells in a direct cellular contact-dependent but MHC class I–indepen-dent manner [89,90]. Such a potent cytolytic effect is only observed incytokine-primed NKs, however, suggesting that HCV-infected cells arepoor triggers of NK activity or that such effect is more potent in the con-text of established HCV-specific immune response. Further, inhibition ofNK-cell CD16-mediated cytotoxicity after engagement of CD81 moleculeson NK cells with HCV E2 protein was reported [91,92]. Impaired NK-celltriggering attributable to reduced expression of NKG2D ligands (majorhistocompatibility complex [MHC] class I chain-related A and B) on ma-ture DCs and increased NK-cell inhibition through increased CD94/NKG2A expression and transforming growth factor-b (TGFb) and IL-10 production have been suggested to occur in vivo during HCV infection[93–96]. More recently, increased expression of NKp30 and NKp46, thespecialized NK-triggering receptors involved in non–MHC-restricted natu-ral cytotoxicity, was identified in NK cells from HCV-infected patients[97]. Freshly separated NK cells from HCV-infected patients showed asignificant production of IL-10 and normal concentrations of IFNg on di-rect cell contact–mediated triggering [97]. Thus, skewed NK receptor ex-pression during HCV infection combined with increased production ofimmunoregulatory cytokines could contribute to inefficient NK-DC and


NK–T-cell cross-talk, leading to inefficient subsequent adaptive immuneresponses and lack of virus control [98].

Hepatitis C virus infection results in inflammatory cell activation

Chronic HCV infection is associated with activation of the inflammatorycell and cytokine cascade, including recruitment of inflammatory cells tothe HCV-infected liver, increased liver and serum levels of proinflammatorycytokines, and evidence of monocyte or macrophage activation [99]. Severalmechanisms may account for this inflammatory activation, including patternrecognition receptor activation as a result of HCV infection and amplificationof the cytokine cascade by endogenous mediators or HCV-derived products.

In addition to TLRs that recognize viral nucleic acid sequences, surface-expressed TLRs have been shown to sense HCV viral proteins, and therebyinduce proinflammatory pathways in inflammatory cells. TLR2-mediatedactivation of monocytes and macrophages is induced by HCV core andNS3 proteins to result in activation of the inflammatory cascade, includingactivation of IRAK1 kinase, NF-kB, MAPK, and TNFa production[33,54,63,100]. In addition, monocytes of patients who have chronic HCVinfection respond to TLR4 stimulation with an augmented proinflammatoryresponse compared with noninfected controls [33,63]. Increased levels ofTLR2 and TLR4 expression were observed in PBMCs of patients whohad chronic HCV infection, and increased expression of TLR2 wasparticularly associated with increased circulating TNFa levels and hepaticinflammatory activity [101]. In support of the role of TLR2 in HCV infec-tion, a recent study found an association between a homozygous TLR2Arg753Gln polymorphism and allograft failure and mortality after livertransplantation for chronic HCV, whereas there was no association foundfor TLR4 (Asp299Gly and Thr399Ile) polymorphism [102].

It has recently been proposed that the increased activation of inflamma-tory cascade activation in HCV could be related to a loss of TLR tolerancein chronically HCV infected patients’ monocytes by means of multiple TLRsignals, such as circulating HCV core proteins that stimulate TLR2, lowlevels of circulating endotoxin in these patients (TLR4), and the presenceof increased levels of IFNg that can amplify inflammatory cell activationand promote loss of TLR tolerance [54,103]. In addition to HCV proteins,some investigators found cell activation by HCV lipopeptides by means ofTLR2 and TLR4 [53].

In children, chronic HCV infection was associated with increased expres-sion of TLR2 and TLR4 in neutrophil leukocytes compared with HCV-negative controls or HCV antibody-positive individuals who spontaneouslycleared HCV infection [104]. TLR2 and TLR4 mRNA and protein expres-sion were also increased in the liver of children with chronic HCV infectioncompared with controls without viral infection [105].


In adult HCV-infected cirrhotic livers, gene expression changes involvedactivation of the innate antiviral immune response genes that was in contrastwith the gene activation pattern in livers with alcoholic cirrhosis [106]. Thelink between activation of inflammatory cells and mediators and stellate cellactivation and fibrosis is well established [107,108]. Recent evidence suggeststhat persistent inflammation also predisposes to cancer. Consistent with this,biomarkers of oxidative stress and inflammation were associated with hepa-tocellular carcinoma (HCC) in HCV-infected livers [109]. Another studyfound that chronic inflammation associated with HCV infection shifts hep-atocytic TGFb signaling from tumor suppression to fibrogenesis, which canaccelerate liver fibrosis and the risk for HCC [110]. These observations lendsupport to the role of inflammation in the increased frequency of HCC inHCV-infected livers.

Innate immunity as a therapeutic target in hepatitis C virus infection

Potent activation of antiviral immune pathways though selective TLRactivation provides an attractive therapeutic target in HCV treatment. Insupport of this contention, recent studies found promising results withTLR7 and TLR9 agonists. The TLR7 and TLR9 activation strategy is basedon increasing endogenous IFNa production in DCs; however, additionalimmunomodulatory effects of TLR9 or TLR7 are yet to be evaluated. Isa-toribine, an agonist of TLR7, reduced plasma virus concentrations inchronic HCV infection [111]. Oral resiquimod, a TLR7 and TLR8 agonist,showed promising antiviral effects in phase IIa safety and efficacy trials[112,113]. IFNa levels correlated with decreases in viral titer and lympho-cyte counts in the treatment group [112,113]. In another study, administra-tion of CPG10101, a TLR9 agonist, showed safety and efficacy in normalvolunteers [114]. Although flu-like symptoms were the reported side effects,CPG10101 induced IFNs, cytokines, and chemokines in vivo, suggesting itspotential in HCV therapy. At this time, however, further clinical trials withthe TLR7 and TLR9 agonists are on hold because of concerns related tosome of their side effects.

Analysis of various TLR ligands on induction of antiviral molecules inhuman PBMCs revealed that agonists of TLR3, TLR4, TLR7, TLR8,and TLR9 were potent inducers of antiviral activity, including inductionof IFNa and IFN-induced 20,50-oligoadenylate synthase [115]. TLR4 andTLR8 stimulation also induced high levels of TNFa and IL-1b [115].

Recent localization of the functional NS3-4A protease cleavage domainon IPS-1, which is essential for blocking RIG-I signaling to IRF3 andNF-kB, provided another potential therapeutic target for HCV [51]. In vitrostudies suggest that TLR ligands may overcome some of the immune defectsassociated with HCV infection. Yonkers and colleagues [62] reported thatTLR3, TLR7/8, and TLR9 ligands could enhance MDC and PDC activa-tion of naive CD4þ T cells. PDCs from HCV-infected patients had reduced


expression of activation markers (human leukocyte antigen D-related[HLA-DR]) and IFNa production on TLR7/8 stimulation, however, andshowed decreased activation of CD4þ T cells [62]. These data indicatethat stimulation of certain TLRs may have benefit on restoration of innateand adaptive immunity in chronic HCV infection.

Summary

Increasing evidence suggests that HCV can interfere with innate immuneactivation at multiple levels. First, HCV, through its viral proteins, can un-dermine viral recognition by cleaving pivotal adaptor proteins in TLR andRIG-I or MDA5 signaling. Second, HCV directly or indirectly modulateskey antigen-presenting functions of various DC types, contributing to im-paired virus-specific T-cell activation. Third, IFNa production by PDCs,the main cell type producing IFNa, is drastically reduced in chronic HCVinfection. Fourth, chronic HCV infection results in activation of proinflam-matory pathways and mediators in inflammatory cells that contribute notonly to aberrant innate-adaptive immune interactions but to activation ofliver fibrosis and a microenvironment that may support cancer formation.Therapeutic strategies to counteract innate immune alterations in chronicHCV provide a promising target and need further investigation.

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Hepatitis C Virus Entryand 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 world’s population is infected with hepatitisC virus (HCV) [1]. Ten to 15% of infected individuals develop liver disease,with a significant fraction requiring long-term health care for the treatmentof fibrosis, cirrhosis, and hepatocellular carcinoma. HCV is one of the majorindicators for liver transplantation, with current predictions suggesting that70% of transplants are likely to be for HCV- related disease by 2010. Theonly available therapy, pegylated interferon (IFN) and ribavirin, is toxic,costly, complex to administer and in some cases ineffective, with up to50% of subjects infected with genotype 1 viruses failing to respond to treat-ment [2]. Although alterations to therapeutic regimens may increase the suc-cess 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 offernew targets for the development of antiviral agents. The observation that in-fected individuals can spontaneously resolve HCV infection offers hope forthe development of vaccine(s) that can mimic the natural clearance processand reduce the incidence of HCV-related disease. In this review, the authorssummarize the current understanding of the mechanism(s) defining HCV en-try and the role of neutralizing antibodies (nAbs) in controlling HCVreplication.

Clin Liver Dis 12 (2008) 693–712

Tools to study hepatitis C virus entry

Currently, there are two methods for studying HCV entry. The first isbased on the capacity of retroviruses lacking endogenous glycoproteins


E-mail address: [email protected] (J.A. McKeating).



694 STAMATAKI et al

(gps) to incorporate foreign envelope proteins. The resulting pseudoparticlesinfect cells in a manner that is defined by the heterologous encoded gps.HCV encodes two gps, E1 and E2, and their detailed processing and biogen-esis have been reviewed elsewhere [4]. The pseudotyping approach hasallowed the characterization of viruses bearing a range of HCV gps ofdiverse genotypes and has established that E1 and E2 are both requiredto generate infectious HCV pseudoparticles (HCVpp) [5–7]. HCVpp entryis largely restricted to cells of the hepatocyte lineage, suggesting that HCVtropism for the liver is partially defined at the level of virus-receptor inter-action(s) [5,8–10]. The second method for studying viral entry uses therecently described JFH-1 (Japanese fulminant hepatitis-1) strain of HCVthat generates infectious particles in cell culture [11–13]. Although restrictedin the repertoire of E1E2 gps that can be studied, many of the entry charac-teristics of HCVpp have been confirmed in the HCVcc system, with respectto cellular tropism and the sensitivity of virus to neutralizing ligands. HCVE1 and E2 have been reported to form heterodimers [4,14], and recent datasuggest that E1 may trimerize and the functional gp unit may represent a tri-mer of E1E2 heterodimers [15]. These observations support a physicalmodel of HCV analogous to that of the tickborne encephalitis virion [16].

Elucidation of the receptors defining hepatitis C virus attachment

and internalization

HCVpp entry into hepatoma cell lines and primary human hepatocytes isvia pH-dependent clathrin-mediated endocytosis [17,18]. Current evidencesuggests that at least three host cell molecules are important for HCV entryin vitro: the tetraspanin CD81 [5,9,13,19], scavenger receptor class B mem-ber I (SR-BI) [9,20–22], 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 inHCV entry, although their role is less well established. The authors reviewthe evidence supporting a role for each of these molecules in the viral entryprocess.

Tetraspanin CD81

Since the primary report describing the interaction of a soluble truncatedform of HCV E2 (sE2) with CD81 [19], investigators have sought to establishthe role of CD81 in the viral entry process. Several studies have reported thatantibodies specific for CD81 [10,27] and soluble recombinant forms of thesecond extracellular loop (EC2) of CD81 (sCD81) inhibit HCVpp andHCVcc infection [27]. Further experiments demonstrating that transductionof HepG2 and HH29 human hepatoma cells, which lack CD81, to expressCD81 rendered them permissive for HCV infection [8,10,13,27,28]. Further-more, siRNA silencing of CD81 reduced viral infection [10,13,27] confirmed

695HEPATITIS C VIRUS ENTRY AND NEUTRALIZATION

the essential role of CD81 in HCV entry. Diverse strains of HCV sE2 werereported to interact with CD81 with differing affinities, with the genotype1a strain H77 demonstrating the highest affinity [10,29–31]. The infectivityof HCVpp expressing a diverse panel of E1E2 gps depends on CD81; withall viruses showing comparable levels of sensitivity to neutralization byanti-CD81monoclonal antibodies (mAbs) [28]. These data suggest importantdifferences in the interaction of E1E2 and sE2 gps with CD81 and that careshould be taken in extrapolating data with sE2 for virus-receptor interactions[8].

Studies to assess whether CD81 is the primary receptor defining viralattachment have been inconclusive, and experiments have been unable todemonstrate viral attachment to cells that were known to be fully permissivefor viral entry when compared with nonpermissive cell lines [23,32]. Never-theless, studies designed to address whether anti-CD81 or sCD81 neutralizeviral infection before or after viral adsorption to cells suggest that they me-diate their effects after binding, and that CD81 is most likely to act as a cor-eceptor after viral attachment [8,33,34]. Several reports have suggested thatthe level(s) of CD81 expressed at the cell surface define the susceptibility ofcells to infection [35–37].

The identification or mapping of amino-acid (aa) residues in E2 and CD81that are critical for the interaction has been complicated by the involvement ofmultiple residues located throughout CD81 EC2 and the E2 gp [32,38–42].The critical regions of E2 were initially identified by screening E2-specificmAbs for their ability to inhibit sE2 binding to cell surface–expressed CD81or sCD81 [5,30,43,44] and by the mutagenesis of putative CD81 interactingdomains in E2 [42,45–48]. These studies identified aas within the hypervari-able region (HVR: aa 384–401), a region immediately adjacent to the HVR[5,38,43,45,47], aa 480–493 [49], aa 522 through 551 [30,43,47,50], and a regionencompassing aa 613 through 618 [46].

Among mammals, CD81 is well conserved and comparison of sequencesfrom several species that are nonpermissive for HCV infection enabledthe identification of aa residues that are critical for CD81 interaction withsE2 [8,51,52]. Expression of these CD81 variants in HepG2 cells identifiedtheir ability to confer viral entry, albeit at reduced efficiencies, highlightingonce again the difference in the interaction of sCD81 and full-lengthcell surface–expressed CD81 with HCV gps [8]. Tetraspanins are four-transmembrane proteins that typically reside at the cell surface and assemblewith themselves and other proteins to form tetraspanin-enriched microdo-mains (TEMs) (reviewed in [53]). Palmitoylation and multiple regions withinthe extracellular and cytoplasmic tails of tetraspanins have been reported tobe important for oligomerization and association with TEMs [54–56].Mutagenesis studies identified that deletion of N- and C-terminal cytoplas-mic tails and sites of palmitoylation resulted in variant CD81 molecules thatcould still allow HCV entry into HepG2 cells, suggesting that CD81 doesnot need to associate with TEMs to function as a viral coreceptor [41].

696 STAMATAKI et al

Scavenger receptor class B member I

The first evidence for a role of SR-BI in HCV entry was the demonstra-tion 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 isinvolved in the trafficking of cholesterol into hepatocytes by means of selec-tive uptake from cholesterol-rich lipoproteins and by the uptake of HDLparticles into endosomes. The role of SR-BI in lipid physiology was recentlyreviewed [57]. Unlike CD81, which is expressed ubiquitously, SR-BI expres-sion is not relatively restricted from what we know, but it is higher in theadrenal glands and liver. Antibodies specific for SR-BI inhibit HCVcc infec-tion, supporting a role for the molecule in HCV entry [9,21,22,58]. Experi-ments to validate an essential role for SR-BI in viral entry have proveddifficult, however, because all cell types studied to date express SR-BI andsiRNA silencing has been reported to have variable effects on HCVpp infec-tivity [9,10,34].

The interaction of sE2 with SR-BI has been shown to involve residueswithin and adjacent to the HVR [45,59,60], wherein deletion of the HVRablates sE2-SR-BI interaction and limits HCVpp infection [9,20,45]. It hasbeen suggested that this interaction is largely attributable to the overallconformation of the HVR rather than to specific sequences, because replace-ment of conserved or charged residues within the HVR modulated SR-BIbinding in a predictable manner [45,60].

Alternative splicing of SR-BI mRNA yields the SR-BII isoform, whichhas a distinct C-terminal intracellular region lacking the PDZ domain pres-ent in SR-BI [61]. Although most SR-BI is found at the cell surface, 85% ofSR-BII is expressed intracellularly [62]. SR-BII is capable of selective choles-terol uptake and receptor-mediated lipoprotein endocytosis [62], with thelatter occurring by means of clathrin-coated vesicles defined by a dileucinemotif in the C-terminal region [63]. Overexpression of both isoformsenhanced HCVcc infection, suggesting that both molecules can mediateHCV entry [21].

Several SR-BI ligands have been shown to influence HCV infection, act-ing to enhance (HDL [64,65]) or inhibit (oxidized LDL [34], serum amyloidA [66,67]) viral entry. LDL and HDL have been reported to bind to differentsites on SR-BI [68], suggesting that the site of ligand engagement has conse-quences for HCV interactions with SR-BI. Additional agents involved in li-poprotein trafficking have been shown to exert pleiotropic effects on HCVinfection. Very low-density lipoprotein (VLDL) particles containing apoli-poprotein B (ApoB) and ApoE have been shown to associate with HCVduring viral secretion from hepatoma cells [69–71], suggesting that HCVparticles are complexed with lipoproteins [72,73]. The addition of exogenousApoB has been shown to inhibit HCV entry [59,74], whereas lipid-associatedApoC1 enhances viral entry [59]. Antibodies to ApoB have been reported toinhibit HCV infection [59,75]. Lipoprotein lipase (LPL) [75,76], which


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

Tight junction claudin proteins

An additional host cell molecule reported to be important for HCV entrywas recently identified as the TJ protein CLDN1 [78]. TJs are continuousintercellular contacts at the apical poles of lateral cell membranes that forma barrier to regulate the paracellular transit of solutes across a cell layerand to establish cell polarity [79]. The CLDN family of transmembraneproteins extends into the paracellular space, wherein they form homo- andheteroassociations with CLDNs on apposing cells [80]. In an analogous man-ner to the approaches taken to validate the role of CD81 as a coreceptor forHCV, the following experiments confirmed the CLDN1 dependence of HCVentry: expression of CLDN1 in several nonpermissive cell lines allowedHCVpp and HCVcc entry [78], siRNA silencing of CLDN1 expression inpermissive hepatoma cells reduced HCV infection and mutagenesis, andantibody blocking studies with tagged versions of CLDN1 demonstratedthat the first extracellular loop (EC1) is an essential coreceptor during latestage(s) of the HCV entry process [78].

CLDN1 is a member of a large family of related proteins. Of the 20 CLDNfamily members identified in humans to date, CLDN1, CLDN3, CLDN4,CLDN6, CLDN7 CLDN12, CLDN15, and CLDN23 have been detectedin Serial Analysis of Gene Expression (SAGE) libraries derived from liver[81]. Recently, CLDN6 and CLDN9 were reported to act as coreceptors al-lowing HCV entry [82,83]. Mutagenesis studies of CLDN6 and CLDN9[82] suggest that the EC1 of these molecules is important for coreceptor activ-ity, involving a region thought to be involved in intercellular interactions [84].

Several TJ proteins have been reported to act as primary receptors fora 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 complexmechanism(s) underlying coxsackie virus group B virus (CBV) highlight thedynamic properties of intercellular junctions [88,89]. CBV binds to a primaryreceptor, decay accelerating factor (DAF), expressed on the luminal surfaceof polarized intestinal epithelial cells. CBV interaction with DAF initiatesa signaling cascade that triggers an actin-dependent relocalization of the vi-rion-DAF complex to lateral cell junctions, in which CAR is located and en-docytosis can occur [89]. The authors recently reported that CLDN1 is

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predominantly expressed at the apical (canalicular) surface of hepatocytes innormal liver tissue consistent with its location at TJs; however, CLDN1 wasalso detected at the basolateral surface of hepatocytes [90]. CLDN1 colocal-ized with CD81 at apical and basolateral domains and with SR-BI at basolat-eral 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 theliver are known to be polarized, with TJs separating the apical (canalicular)and basolateral (sinusoidal) domains. Hepatic polarity is critical for normalliver function, with particular membrane domains performing specific tasks,such as biliary secretion from the canaliculi and serum protein secretionfrom the sinusoidal surface(s). To initiate infection, pathogens must breachthe epithelial barrier to gain access to the body, and TJs can provide a nat-ural defense against infection. Indeed, the poor efficacy of several viralvectors to deliver therapeutic genes to airway epithelium highlighted therole of TJs in the resistance of epithelia to viral infection [91,92]. The recentidentification of CLDNs as a critical factor for HCV internalization[23,82,83] highlighted the importance of studying the role(s) of cell polariza-tion in HCV entry. The authors used the colorectal adenocarcinoma Caco-2cell line that is known to polarize to study the role of cellular polarity inHCV entry. Caco-2 cells express CD81, SR-BI, and CLDN1 proteins andsupport HCVpp entry [93]. Viral receptor expression levels increased on po-larization and CLDN1 relocalized from the apical pole of the lateral cellmembrane to the lateral cell-cell junction and basolateral domains. In con-trast, expression and localization of the TJ proteins Zonula occludens-1(ZO-1) and occludin-1 were unchanged on polarization. HCVpp infectedpolarized and nonpolarized Caco-2 cells to comparable levels, and entryinto polarized cells occurred predominantly by means of the apical surface.Disruption of TJs significantly increased HCV entry, supporting a model inwhich TJs provide a physical barrier restricting viral access to receptorsexpressed 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 as1 week after infection; however, antibodies against the virus are not detecteduntil much later. Chen and colleagues [94] noted the delayed appearance,low titer, and restricted isotype of antibodies to structural and nonstructuralHCV proteins. The contribution of the humoral response to disease out-come remains unclear; in contrast, it has been well documented that a potentmulti-specific and long-lasting cellular immune response during the acutephase of HCV infection contributes to resolution [95–105].


A potential role for antibodies in controlling disease progression was firstdemonstrated in subjects with primary hypogammaglobulinemia, demon-strating rapid disease progression and poor responses to IFN treatment[106]. An independent study reported that 43% of individuals who spontane-ously resolved infection had antibodies specific to E2 HVR within the first 6months of infection, compared with only 13% of patients who failed to clearthe infection [107]. In contrast, there were no significant differences betweenthe two patient groups with respect to the time of emergence of antibodies toHCV core or nonstructural proteins [108]. Other investigators reported anearly emergence of HVR-specific antibodies in a small cohort of subjectsinfected during hemodialysis who resolved infection [109]. Because theHVR is proposed to be a target for nAbs [110,111], several studies have sug-gested a role for anti-HVR antibodies in selecting viral variants to escape thehumoral response [112–115]. Although many studies have explored the spec-ificity of the humoral immune response, until recently, functional assays werenot 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 neutralizationpotential of HCV-specific antibodies was tested indirectly using ‘‘neutraliza-tion of binding’’ (NOB) assays, in which antibodies were screened for theirability to prevent the binding of sE2 to mammalian cells [116]. Baumert andcolleagues [117] used a recombinant baculovirus system to express the HCVstructural proteins that formed viral-like particles (VLPs) to study antibodyreactivity and inhibition of VLP-cell interactions [118]. The VLPs elicited cel-lular and humoral immune responses in mice, generating anti-E2 antibody re-sponses that were reactive against E2 from diverse HCV genotypes [119,120].

Once HCVpp became available [5,6], it was possible to assess antibodiesfor their ability to inhibit viral infection of hepatoma cell lines. HCVpp neu-tralization titers correlated well with the effectiveness of antibodies in HCVchallenge protection studies in chimpanzees [121], validating the system asa model for assessing functional antibody responses. Indeed, the HCVppsystem has allowed the testing of monoclonal antibodies and polyclonal an-tibodies from diverse sources (ie, from sera derived from patients or immu-nized animals) to inhibit infection [121–123].

The recent discovery that the JFH-1 strain of HCV generates infectiousparticles in cell culture has allowed investigations into the sensitivity of au-thentic particles to antibody-dependent neutralization [12,13]. To date,HCVcc has been reported to be neutralized by E2-specific antibodies derivedfrom human sera [12,13], E1-specific mouse antisera [124], and by an array ofmAbs directed to the E1 and E2 gps. Polyclonal immunoglobulin (Ig)preparations derived from mice and guinea pigs immunized with E2 andE1E2 gps efficiently neutralized HCVcc [37]. It is worth noting that theneutralization titers of polyclonal immune sera, E2-specific nAbs [5,49],

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and human-derived antibodies were consistently lower for HCVcc-expressingstrain H77 (genotype 1a) and J6 (genotype 2a) gps than for HCVpp bearingthe same gps (Z. Stamataki, unpublished data, 2008) [37], which may reflectdifferences in the level of gps incorporated into pseudoparticles comparedwith native particles. HCVcc engineered to express a diverse range of enve-lope gps should become available in the future and will prove invaluablefor 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 isa challenging endeavor; readily available gp sequences may represent certainviral genotypes but differ considerably from a patient’s autologous viralquasispecies. Most studies are limited to establishing the heterologouscross-reactivity of nAbs and do not measure their efficacy to neutralizeautologous circulating strains in the subject under test. Several studieshave demonstrated that chronically infected subjects present with high-titerantibodies capable of neutralizing heterologous HCVpp, suggesting thatthe neutralizing response targets epitopes that are conserved across diversegenotypes [10,11,115,121,123,126–129]. In a case report, the authors recentlycharacterized the strain-specific and cross-reactive nAb responses elicited ina chronically infected patient when the time of infection was known andsequential samples were available over the course of infection [115]. Serumantibodies were compared for their ability to neutralize HCVpp, expressingautologous gps cloned over time and heterologous gp strains. An antibodyresponse to autologous virus was first detected at seroconversion (8 weeksafter infection), whereas cross-reactive nAb responses were not evident untilafter 33 weeks [115,123]. Although the cross-reactive gp-specific nAb titerand breadth increased during disease progression, HVR-specific responsesdetected at 9 weeks after infection decreased to undetectable levels byweek 33. Serum antibodies failed to neutralize HCVpp expressing time-matched autologous gps, suggesting the emergence of neutralization escapevariants [115]. This delay in nAb emergence has been reported to associatewith a failure to resolve infection spontaneously in a cohort of subjectsaccidentally infected with the same strain of HCV [130]. In this cohort,the rapid induction of nAbs early in the course of acute infection wasdirectly linked to disease clearance. In agreement with analogous reportsdescribing the genesis of HIV escape variants [131,132], these studies suggestthat HCV can also escape the humoral nAb immune response.

The role of the humoral response in selecting viral diversity, particularlyin the HVR, was reinforced by reports that HCV-infected subjects withhypogammaglobulinemia showed reduced rates of nucleotide substitutionin the HVR compared with controls [112,133]. The authors proposed thatthe HVR serves as a ‘‘viral decoy,’’ directing the immune system awayfrom viral epitopes potentially less capable of rapid change and playing


an accessory role for viral evolution [115,134,135]. Assuming that nAbs con-tribute to selecting the HCV quasispecies and to the reported associationbetween the emergence of quasispecies and failure to clear acute infection[134,136–138], one may question the benefit of nAbs to HCV-infected indi-viduals. Conversely, in chronic infection, the viral quasispecies complexityhas been associated with milder clinical symptoms [139,140], suggestingthat immune responses limiting liver damage may enhance viral antigenicvariation [140–144]. Further doubts regarding the efficacy of nAbs in con-trolling HCV replication have been reported, with conflicting data regardingthe clinical impact of anti-HCV therapies [145,146].

Epitope specificity of the neutralizing antibody response

nAbs can exert their effect(s) by binding directly to virus particles andblocking subsequent interaction(s) with receptors or by inhibiting post-entryevents, such as viral uncoating and subsequent replication (reviewed by Bur-ton [147]). The former may occur by inducing conformational changes in theviral envelope that disable infection or by steric hindrance, physically shield-ing important viral interaction sites. The modes of action of HCV-specificnAbs have yet to be defined; however, several early studies suggested thatserum antibodies from chronically infected individuals inhibited sE2-CD81 association, leading these investigators to conclude that the CD81binding site is immunogenic and that antibodies neutralize infectivity by in-hibiting HCV interaction with CD81 [121,148].

Mapping of neutralizing mAbs generated in response to recombinant gpimmunization has identified a series of linear epitopes within the N-terminalregion of E2 [5]. In contrast, neutralizing human mAbs obtained from HCV-infected subjects generally recognize conformation-dependent epitopes[149–153] (recently reviewed by Houghton and Abrignani [154] and Zeiseland colleagues [155]). Johansson and colleagues [153] reported on the broadreactivity of two human monoclonal antibodies that recognize overlappingyet distinct epitopes involving aas in the 523–535 region of E2, known to beimportant for the E2-CD81 interaction. Similarly, an independent studycharacterized a panel of human monoclonal antibodies to three immuno-genic conformational domains (designated A, B, and C) within E2, in whichantibodies specific for domains B and C neutralized HCV and those recog-nizing domain A failed to neutralize viral infectivity. These investigatorsconcluded that HCV E2 contains three immunogenic domains with distinctfunctions and that domains B and C may be involved in interactions withCD81. Finally, one needs to consider the generation of non-neutralizingHCV-specific antibodies, which may compete with nAbs and reduce their ef-fectiveness. Such antibodies have been reported in other viral infections inwhich highly immunogenic non-neutralizing epitopes mislead the humoralimmune response [156].

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Effect of lipoproteins on the sensitivity of virus to neutralizing antibodies

Recent in vitro studies highlight the role of the VLDL pathway in theassembly and release of HCVcc particles [69–71]. These data support earlierobservations reporting on the density and sedimentation properties of humanplasma-derived virus, concluding that the virus exists in association with lipo-proteins or immunoglobulins [73,157–159]. Thosmsen and colleagues [160]reported that HCVRNAwas associated with b-lipoproteins in sera, a findingthat was later confirmed by others [159,161,162]. These observations ledseveral researchers to hypothesize that lipoprotein-associated virus mayhave reduced sensitivity to antibody-dependent neutralization. In an attemptto model the virus lipoprotein interactions, several investigators studied theeffect of lipoproteins on the sensitivity of HCVpp to nAbs. These studiesdemonstrated that HDL promotes HCVpp infection and reduces the sensitiv-ity of the virus to nAbs [60,64,163]. The lipoproteins seemed to exert theireffect by means of interactions with the target cells, specifically by means ofpromoting virus internalization in an SR-BI–dependent manner [65]. Dreuxand colleagues [64] reported that HDL increased the infectivity of HCVppbound to hepatoma cell lines in vitro, suggesting that HDL modulatesSR-BI cholesterol uptake.

Inducing protective immunity in vivo

Thepropensity ofHCV to establish chronic infection, to reinfect previouslyexposed individuals [164], to transmit directly by cell-cell routes in vitro [165],and to evolve neutralization escape variants [166] makes the development ofan HCV vaccine a major challenge. Vaccine development has been hamperedby the lack of a convenient small animal model [154,167]. The existence ofnatural immunity to infection in some humans [168] and in chimpanzees[142,169–171] is encouraging, however, and suggests that the immune systemcan eliminate infection. Immunization of chimpanzees with HCV gps hasprotected the animals from challenge with autologous virus [172]; further-more, this protection correlated with the induction of nAbs that inhibitedE2-CD81 interactions [116]. In animals with reduced nAb responses [172]and those challenged with heterologous virus [154], sterilizing immunity wasnot achieved [173,174] and the animals failed to progress to a chronic stateof infection [175]. Several immunized chimpanzees controlled infection with-out mounting any significant humoral responses, however, demonstratingthat humoral immunity is not required for clearing HCV in the chimpanzeemodel [176].

Reports on the immunogenicity of HCV structural proteins in mice andnon-human primates are variable [120,176–179]. Rodents vaccinated withE2 and E1E2 gps can generate cross-reactive nAb responses, with higher titersin guinea pigs than inmice [37]. Furthermore, vaccination of nine healthy vol-unteers with HCV-1 E1E2 gps elicited serum antibody responses that were


able to neutralize heterologous HCVpp and HCVcc in vitro, and these re-sponses were detectable 1 year after vaccination [180]. Immunization of ratswith HCV E1E2 gps yielded poor responses against E1, with most antibodiesrecognizing E2 (J.A. McKeating, unpublished data, 1998). Human subjectsimmunized with E1 mounted a specific response, the neutralizing activity ofthese antibodies was not assessed [181,182]. E1 may be poorly immunogenicbecause of the presence of N-linked glycosylation sites [183]. Similarly, E2 im-munogenicity is also affected by glycans [48,184,185]. Recent data studyingHCV infection of the severe combined immunodeficiency (SCID)–chimericmouse model demonstrated that transfusion of mice with high concentrationsof Ig purified from a chronic HCV-infected subject, with demonstrable neu-tralizing activity in vitro, protected most animals from homologous viruschallenge [21]. Overall, these data suggest that eliciting a nAb response isa valid objective of prophylactic vaccination.

Summary

The processes of HCV entry and antibody-mediated neutralization areintimately linked. The high frequency of nAbs that inhibit E2-CD81 interac-tion(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 CD81-dependent and -independent routes in vitro awaits further investigation toassess the significance in vivo but may offer new strategies for HCV to escapenAbs. The identification of CLDNs in the entry process highlights the impor-tance of cell polarity in defining routes of HCV entry and release, with recentexperiments 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 viral-lipoprotein complexes in human plasma. There is a real urgency to studyHCV infection of primary hepatocyte and mixed liver cell cultures to confirmthe mechanisms of entry observed in nonpolarized hepatoma cell lines.Future experiments need to assess the receptor dependency of apical andbasolateral routes of infection and whether they share similar sensitivitiesto neutralization by antibodies and small molecules targeting various stepsin the entry process. Recent developments offer the prospect of a more com-plete understanding of the molecules defining HCV entry and the discovery ofnew targets for the development of novel antiviral agents.

Acknowledgments

The authors thank all their colleagues for stimulating discussions over theyears. Research in the McKeating laboratory is currently supported by Pub-lic Health Service grants AI40034 and AI50798, the Medical ResearchCouncil, and The Wellcome Trust.

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Host Genetic Factors and AntiviralImmune Responses to Hepatitis C Virus

Chloe L. Thio, MDDepartment 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 out-comes ranging from viral elimination to the development of end-stage liverdisease or hepatocellular carcinoma. Viral elimination, which occurs in a mi-nority (w20%) of acutely HCV-infected individuals, is the result of effectiveimmune control of HCV replication [1]. The 80% of people who do noteliminate HCV progress to a chronic HCV infection. Not all chronic infec-tions are created equal, because some have minimal liver disease, whereasothers develop cirrhosis or hepatocellular carcinoma. Epidemiologic factorsassociated with these different clinical outcomes include age, ethnicity, otherviral coinfections (especially HIV), and gender [2–4]. Even in a relatively ep-idemiologically homogeneous population, there are marked differences inthe ability to eliminate the virus that are not related to viral characteristics.One example is a cohort of 704 Irish women who were accidentally infectedwith the same viral inoculum (contaminated anti–D immune globulin), and314 (45%) of them cleared their infection [5]. Likewise, 43% of 152 Germanwomen cleared their infection after being exposed to the same contaminatedlot of anti–D immune globulin [6]. Such data suggest that it is not the virusbut the interactions between the virus and the host immune response thatare important for determining the natural history of an HCV infection [7].

Studies demonstrate that a strong broad immune response favors viralclearance compared with one that is weak or narrowly focused [8,9]. Like-wise, once a chronic infection is established, HCV is not cytopathic to thehepatocytes; rather, it is the immune response to the virus that is believedto be responsible for the liver damage. A significant barrier to dissectingthe components of the immune response that result in viral clearance or

Clin Liver Dis 12 (2008) 713–726

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.




714 THIO

that lead to hepatocyte damage is the lack of small animal models or cellculture systems.

Because such models are not available, an alternative approach to under-standing the immune response is to analyze whether host genetic variants orpolymorphisms in immune-response genes account for some of the hetero-geneity in outcome. With the recent development of more rapid cost-effec-tive genotyping procedures, such large-scale studies are possible and havealready improved our understanding of HCV pathogenesis. This reviewbriefly summarizes the immune response to HCV to give some backgroundto understand the candidate genes that are discussed. Associations withpolymorphisms in various immune-response genes that affect the ability toachieve HCV clearance or affect HCV fibrosis progression are thenreviewed.

Immune response to an acute hepatitis C virus infection

After an HCV infection, the innate immune response is initially impor-tant 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 effort between the innate and adaptive immuneresponses is necessary to eliminate HCV from the liver. The innate immunesystem recognizes single-stranded and double-stranded HCV RNA throughits pattern recognition receptors, Toll-like receptor 3 (TLR3) on the hepato-cyte cell surface and retinoic acid–inducible gene I (RIG-1) in the cytoplasmof the hepatocyte. Engagement of either of these receptors activates a cas-cade of signals, including interferon-regulatory factor 3 (IRF-3), that culmi-nates in the induction of type 1 interferons (IFNs), such as IFNa and IFNb.These interferons bind receptors that activate the Janus kinase (Jak)–specif-ically targeted antiviral therapy (STAT) pathway, which then turns on hun-dreds of interferon-stimulated genes to control viral replication (reviewedLloyd and colleagues [10]).

The innate immune response also controls replication by means of its ef-fector cells, the natural killer (NK) cells. Because NK cells comprise approx-imately 30% of all T cells in the liver [11], they likely are an importantcomponent to the anti-HCV immune response. Their role includes lysinginfected cells, producing IFNg to control viral replication, and directing in-flammatory cells to HCV-infected hepatocytes.

This initial innate immune response is an important host defense againstthe virus, but viral clearance also depends on an effective adaptive immuneresponse. The onset of the cellular immune response is clinically detected byan increase in serum transaminases marking immune-mediated liver injury.A strong, broad CD4þ and CD8þ T-cell response is more likely to lead toHCV clearance than a weak narrowly focused response [8,9]. Evidence alsosuggests that a CD4þ response that elicits T helper 1 (Th1) cytokines is

715HOST GENETICS AND HEPATITIS C

more likely to result in viral clearance. Those with a weak CD4þ responseseem to have a functional impairment of these cells from exhaustion oranergy rather than a depletion of CD4þ T cells. Similarly, acutely infectedindividuals who do not clear have CD8þ T cells that are functionally im-paired compared with those who clear the virus. Not all individuals witha robust response ultimately clear HCV, which, in some instances, may beattributable to the development of viral escape mutants from immune pres-sure on the virus. Thus, HCV must balance viral fitness with immune escape.Several studies have documented viral escape mutations in human leukocyteantigen (HLA)–restricted CD8þ T-cell epitopes [12–14]. In the cohort ofIrish women who received the same viral inoculum, Ray and colleagues[12] demonstrated that those with amino acid substitutions in knownHLA epitopes directed these mutations away from consensus in personswith the HLA allele associated with that epitope and toward consensus inthose 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 im-portant for clearance of an acute HCV infection. Published studies todate have not examined polymorphisms in the innate immune-responsepathway genes or the type I IFN genes to determine if they affect the abilityto clear HCV. There is genetic epidemiologic evidence to support the impor-tance of the NK cells, the effector cell of the innate immune response, inHCV outcome. Khakoo and colleagues [15] examined polymorphisms inthe genes for the killer immunoglobulin-like receptors (KIRs), which are re-ceptors on NK cells. As the name implies, these receptors have two or threeimmunoglobulin-like domains, a transmembrane domain, and a long orshort cytoplasmic tail. Those with the long tails (2DL, 3DL) inhibit theNK cells when bound and those with short tails (2DS, 3DS) activate NKcells on binding. The ligands for the inhibitory KIRs are HLA class I mol-ecules, whereas the ligands for the activating KIRs have not been defined.The study by Khakoo and colleagues [15] focuses on the inhibitory KIRs2DL1, 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 groupsbased on the amino acid at position 80 of the HLA allele: HLA-C1 alleleshave an asparagine at position 80, whereas HLA-C2 alleles have an aspar-agine at position 80. The HLA-C1 alleles bind KIR2DL2/2DL3 and theHLA-C2 alleles bind KIR2DL1. The strongest inhibitory signal is trans-duced to the NK cell when KIR2DL1 binds an HLA-C2 allele, whereasthe weakest signal is from KIR2DL3 binding an HLA-C1 allele (Table 1).Khakoo and colleagues [15] found that in individuals with low-titer HCVinocula (ie, injection drug users), homozygosity for the combination with

Table 1

Strength of inhibitory signal for selected killer immunoglobulin-like receptors important in

hepatitis C virus clearance

Inhibitory KIR Liganda Relative strength of NK cell inhibitory signal

2DL1 HLA-C2 Strong

2DL2 HLA-C1 Intermediate

2DL3 HLA-C1 Weak

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

respectively.

716 THIO

the weakest inhibitory signal (KIR2DL3 and HLA-C1) favored HCV clear-ance compared with those without this compound genotype (odds ratio [OR]¼ 2.33;P¼ .001). Presumably, this association is attributable to those individ-uals with the protective genotype having the least inhibition of the NK cellsgiving rise to increased NK-cell activity. These investigators also found an in-dependent association with the presence of the activating KIR3DS1 and itsputative ligand, a group of HLA-B alleles known as HLA-Bw4.

Immune-response genes of the adaptive immune response have been stud-ied more extensively than those of the innate immune response in acuteHCV infection. Several studies have tested the hypothesis that certainHLA class I alleles may present epitopes that lead to a more robustCD8þ T-cell response to acute HCV infection. In the largest study, Thioand colleagues [16] examined individuals from three different cohorts thatincluded 231 individuals with clearly documented HCV recovery and 444matched persistently HCV-infected individuals. They found that HLA-A*1101 (OR ¼ 0.49, 95% confidence interval [CI]: 0.27–0.89), B*57(OR ¼ 0.62, 95% CI: 0.39–1.0), and Cw*0102 (OR ¼ 0.43, 95%CI: 0.21–0.89) were associated with viral clearance. HLA-Cw*04 (OR ¼1.78, 95% CI: 1.21–2.59) and A*2301 (OR ¼ 1.78, 95% CI: 1.01–3.11)were associated with HCV persistence. The HLA-C data are consistentwith the HLA-KIR data cited previously, because HLA-Cw*0102 is anHLA-C1 allele and HLA-Cw*04 is an HLA-C2 allele. This study did notfind an association between HLA class I homozygosity and viral persistence,which is interesting, because it has been hypothesized that heterozygositywould be advantageous for recognition of a broader array of epitopes.HLA-B*57 has also been associated with HCV clearance in an African pop-ulation [17] and with slower HIV progression [18], but whether it is a genelinked to this allele or an immune characteristic about this allele itself that isprotective in these chronic viral infections is not known. The other largestudy with class I HLA data is from the cohort of Irish women describedpreviously who received the same viral inoculum. They found that the 86subjects with viral clearance were more likely to have HLA-A*03 (OR ¼2.8), B*27 (OR ¼ 7.5), B*07 (OR ¼ 2.0), or Cw*01 (OR ¼ 7.1) comparedwith the 141 chronically infected women [19]. HLA-B*08 (OR ¼ 0.4) and


B*18 (OR ¼ 0.2) were more common in women with viral persistence. Theseresults (except for theHLA-Cw*01) are different from the study by Thio andcolleagues [16], which may be attributable to baseline differences betweenthe cohorts, such as ethnicity, gender, and viral inoculum.

Because the CD4þ T-cell response is important for viral clearance, sev-eral groups have tested the hypothesis that HLA class II molecules, whichpresent CD4þ T-cell epitopes, are associated with HCV outcome. Severalresults are consistent, and two meta-analyses demonstrated thatDQB1*0301 (estimates of 3.0 [20] and 2.4 [21]) and DRB1*11 (estimatesof 2.5 and 2.0) are associated with HCV recovery. A bias of these meta-anal-yses is that studies without any HLA associations are not published or stud-ies in which these alleles are not significant do not report their specific ORs.The allele HLA-DRB1*01 was protective in the Irish cohort and in thewhites in the study by Thio and colleagues [16]; thus, it may not be protec-tive in nonwhite ethnic groups.

Cytokines are small proteins secreted by a variety of T cells in response toan immune stimulus and have several roles, including mediating the immuneresponse to infectious agents, such as HCV. Thus, several studies have ex-amined polymorphisms in a variety of cytokine genes to determine theirrole in HCV clearance (Table 2). Chemokines are a specific set of cytokinesthat attract leukocytes to sites of infection. Several chemokines bind to thereceptor CCR5, and polymorphisms in this receptor have been examined inclearance 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 cor-eceptor for HIV [22]. CCR5 also influences T-cell trafficking and the

Table 2

Associations between polymorphisms in chemokine genes and hepatitis C virus clearance or

persistence

Chemokine gene Outcome Comments

CCR5D32 Clearance Deletion may lead to increased T-cell

response

IFNg -764G (promoter mutation) Clearance Increases promoter activity

IL-10 Clearance Several polymorphisms lower IL-10

expression which can increase Th1

response

IL-12 1188 A/A Persistence Homozygosity for the A allele is

associated in some but not all

studies

TGFb Clearance Two separate polymorphisms leading

to lower TGFb, which can increase

NK cell activity

TNFa(�238, �308) No associations

Abbreviations: IFNg, interferon-g; IL, interleukin; TGFb, transforming growth factor-b;

TNFa, tumor necrosis factor-a.

718 THIO

immune response. A study of women with genotype 1b found CCR5D32 tobe more common in those with HCV clearance [23]. This association couldbe attributable to an increased T-cell response to the HCV antigens becauseccr5 knockout mice have increased T-cell responses to a variety of antigens[24,25]. The chemokines that bind CCR5 have not been extensively studiedwith regard to HCV clearance, but polymorphisms in one of them, regulatedon activation, normal T-cell expressed and secreted (RANTES), have beenassociated with HCV treatment response [26,27].

IFNg is produced by effector T cells and NK cells and is critical to thedefense against HCV infection because it can inhibit HCV replication inthe replicon system [28]. This gene does not have any coding region poly-morphisms, but several noncoding region polymorphisms have been de-scribed [29,30]. In one study, nine noncoding region polymorphisms weregenotyped in an HCV treatment cohort and an HCV recovery cohort,and the uncommon G variant at position �764 in the IFNg promoterwas associated with recovery from HCV (OR ¼ 3.51, 95% CI: 0.98–12.49) and with sustained response to HCV therapy (OR ¼ 3.37, 95% CI:1.15–9.83) [31]. This variant is more common in white Americans consistentwith the findings that spontaneous recovery is more common in whites [4].The �764G variant has a higher binding affinity to the nuclear regulatoryfactor (NF)-kB motif, resulting in higher levels of promoter activity andthus perhaps explaining the association with HCV recovery and treatmentresponse [31].

Interleukin (IL)-10 is a cytokine important to the immune response toHCVthrough its downregulation of the Th1 response and suppression of secretionof proinflammatory cytokines, such as tumor necrosis factor-a (TNFa) andIFNg. Several studies have implicated that polymorphisms in this regionof the genome are important in HCV clearance. One study found thata particular promoter haplotype (�1117A, �854T, �627A), which isassociated with lower IL-10 expression, was more frequent in those withrecovery (36%) than in persistent infection (23%) [32]. A promoter poly-morphism at �1082 that is associated with higher IL-10 levels has alsobeen associated with HCV persistence in women [33]. Oleksyk and col-leagues [34] tested polymorphisms in the IL-10 gene region and found as-sociations with HCV outcome in African Americans. Several other studiesexamined the IL-10 haplotypes and polymorphisms but did not find asso-ciations with HCV recovery [35–38].

IL-12 is a cytokine important for the generation of a Th1 response, whichfavors HCV clearance. A polymorphism in the IL-12 p40 gene (IL-12B) atposition 1188 has been associated with increased and decreased levels of IL-12 secretion [39,40]. Homozygosity for the A allele has been associated withHCV persistence in Chinese patients (OR ¼ 0.34; P ¼ .014) [41]. This wasalso found in a second study from the United Kingdom, in which 66% ofthe persistently infected people were A/A homozygous compared with50% of those with spontaneous recovery [42]. This was not confirmed in


a different population of German patients, however [43]. The author and hercolleagues also failed to confirm this in their study from North America of188 patients with HCV recovery matched to 360 persistently infected per-sons with HCV persistence. The A allele was found in 72.5% and 71% ofpersons with HCV recovery and persistence, respectively (C. Thio, unpub-lished data, 2007).

Transforming growth factor-b (TGFb) is a cytokine that inhibits the im-mune response by suppressing NK cell activity and inhibiting IFNg and IL-12 production. The C allele at �509 in the promoter, which leads to lowerlevels of TGF-b1, was associated with HCV recovery in a study of Japanesepatients [44]. Similarly, Barrett and colleagues [35] studied two coding re-gion polymorphisms in TGFb and found that the haplotype associatedwith low TGFb was associated with HCV clearance. In this study, these in-vestigators also found that homozygosity for the low-producing IL-6 pro-moter �174 variant was associated with HCV recovery.

Although TNFa is an important proinflammatory cytokine with the best-known functional polymorphisms at positions �308 and �238 in thepromoter, none of the studies to date have found an association with thesepolymorphisms and HCV recovery or persistence [32,35].

Although the importance of the humoral immune response to HCV isjust beginning to be uncovered, there is one study showing that certain im-munoglobulin GM and KM allotypes affect HCV outcome. GM and KMallotypes are antigenic markers of the immunoglobulin G (IgG) heavychains and k-light chains, respectively. GM allotypes are strongly associatedwith IgG subclass concentrations. In a study of African-American injectiondrug 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 HCVas 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 develop-ment of cirrhosis is 20 to 30 years [2]; however, in some individuals, it occursmore quickly. Such variation in fibrosis progression rates has been associ-ated with epidemiologic factors, such as age, gender, HIV status, and alco-hol use [2]. Another factor is the strength of the immune response, which isinitially stimulated to eliminate HCV. In a persistent infection, however, thecontinued inflammatory state leads to hepatocyte necrosis and deposition ofextracellular matrix (ECM) proteins by hepatic stellate cells. Activators ofhepatic stellate cells include the Th1 response [46], the family of proteolyticenzymes known as matrix metalloproteases (MMPs), and the tissue inhibi-tors (TIMPs) of MMPs. Because the continued immune response generates

720 THIO

an inflammatory state, genetic variation of the immune system may affectfibrosis progression rates.

One major limitation to genetic epidemiologic studies of HCV diseaseprogression is accurately defining the severity and progression of fibrosis.The ‘‘gold standard’’ for determining the amount of fibrosis, which is oftenused in genetic epidemiology studies, is a liver biopsy. Unfortunately, thisgold standard is problematic because it is imprecise as a result of differencesin size of the biopsy and regional differences of fibrosis. Furthermore, thereis reader error, with studies showing inadequate inter- and intrareader con-cordance [47]. A second limitation is that the studies are cross-sectional;thus, a liver biopsy at one particular point is used along with an estimateddate of infection to determine the progression rate. This is problematic be-cause the classification of disease state or progression rate can be unreliabledue to the dependence on an estimated date. A third limitation is that somegenetic epidemiology HCV studies define disease stage based on liver en-zyme elevations, but it is known that liver enzymes do not correlate wellwith the stage of disease. An advantage of this approach is that repeatedmeasurements are more easily obtained than with a liver biopsy; thus, theproblems associated with a cross-sectional study design are less applicable.Taken together, these limitations result in misclassification that can contrib-ute to false associations, especially in small studies. With these caveats inmind, the author reviews the immune-response genes that have been studiedin liver disease progression.

HLA has been studied as a candidate gene in the disease progression hy-pothesis. In a study by Asti and colleagues [48], DRB1*1103 andDRB1*1104 were associated with normal alanine aminotransferase (ALT),whereas DRB1*1101 was more common in those with more chronic hepati-tis, especially those with more advanced disease, as defined by liver biopsy.A French study by Renou and colleagues [49] examined 83 patients withnormal ALT levels over a 6-month period and 233 patients with elevatedALT levels and found HLA-DRB1*11 to be overrepresented in those witha normal ALT level (43% versus 24%, OR ¼ 2.36). In this study, liver bi-opsies were performed and milder disease was also associated with HLA-DRB1*11. Subtyping of the HLA-DRB1*11 was not performed as in thestudy by Asti and colleagues [48]. A Polish study also found HLA-DRB1*11 to be more common in those with milder disease [50], and a Ger-man study found that it protected from cirrhosis [51]. The German studyalso found protection from HLA-DQB1*03. A study from Japan examinedHLA class I and II antigens in those with normal ALT levels compared withthose without and found that haplotypes with HLA-B*54 are more commonin those with high ALT levels [52]. In this study, HLA haplotypeDRB1*1302-DQB1*0604 was also associated with normal ALT levels. Sev-eral other studies have yielded inconsistent results [53–55]. Patel and col-leagues [56] also tested the hypothesis that HLA allelic diversity affectsfibrosis progression rates, but they did not find differences in median


progression rates in patients heterozygous or homozygous at all three HLAclass I loci. They did not find any individual class I associations, but theyused a serologic assay to define HLA types, making it more difficult to dis-cover weaker associations.

An approach to examining the inherited genetic variability of the pres-ence or severity of diseases, which has become possible with the HapMap(http://www.hapmap.org/) and with the decrease in genotyping cost, is per-forming a genome-wide scan [57,58]. The advantage of such an approach isthat it obviates the need for a priori knowledge of candidate genes. ForHCV, early findings from a genome-wide scan of disease severity havebeen published [59]. This scan included 433 patients from one center; signif-icant results were then validated in 483 patients from a second center. TheDNA from the first center was pooled based on the following fibrosis stagesfrom a baseline biopsy: no or minimal fibrosis (stage 0 or 1), mild (stage 2),and advanced (stage 3 or 4). The advanced fibrosis group was comparedwith no or mild disease. Of the 24,823 single nucleotide polymorphisms(SNPs) genotyped, 1609 (6.5%) had a twofold association with liver diseasestage and 100 of these have been tested in samples from the second center.Two SNPs that remained associated with disease stage were Dead box poly-peptide 5 (DDX5) and carnitine palmitoyltransferase 1A (CPT1A). TheDDX5 SNP was a nonsynonymous change that was associated with moreadvanced fibrosis, and on analysis of SNPs in close proximity, two SNPsin the POLG2 gene (polymerase DNA-directed g2) were also associatedwith advanced fibrosis. DDX5 is an RNA helicase that is expressed in theliver and may interact with HCV RNA to activate stellate cells. TheCPT1A SNP was also a nonsynonymous change, and it was more frequentin subjects with milder disease. This SNP (A275T) may lead to less oxidativestress, potentially explaining the association with decreased fibrosis.

Cytokines are believed to be important in fibrogenesis, and rodent modelsconfirm that they can affect fibrosis (reviewed by Bataller and colleagues[60]). TNFa is a proinflammatory Th1 cytokine that is upregulated inchronic HCV, so it is a profibrogenic candidate [61]. Yee and colleagues[62] compared polymorphisms in TNFa in 33 patients who had cirrhosisand 114 who did not have cirrhosis, although the precise stage of the non-cirrhotic patients was not stated. The �308A and �238A promoter variantsincreased the risk for cirrhosis in this study. A Japanese study had similarresults and found that these variants were associated with increased typeIV collagen 7S, which is a marker of hepatic fibrosis [63], but they werenot associated with liver enzyme elevations. A second Japanese study alsofound that these promoter variants were not associated with liver enzymeelevations [55].

One study examined seven CC chemokines and their receptors and foundan association between a polymorphism in monocyte chemoattractant pro-tein 2 (MCP-2), Q46K, and severe fibrosis (OR ¼ 2.29; P ¼ .018) [64]. Inaddition, CCR5D32 was associated with reduced portal inflammation but

722 THIO

increased fibrosis (OR ¼ 1.97; P ¼ .015). It is not clear why reduced portalinflammation and more fibrosis are associated with the same genotype, es-pecially because in autoimmune hepatitis mouse models and infectiousmodels, CCR5D32 is associated with an increased inflammatory response[65,66]. Studies from Spain and India also contradicted this result, becausethere was no association between CCR5D32 and fibrosis stage [67,68].

IL-10 is a Th1 cytokine that mediates its effects 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-inflammatory cytokinethat downregulates collagen 1 expression and upregulates collagenase. Onestudy found that the minor allele in G330R IL-10R1 was associated with cir-rhosis but not with inflammation, suggesting that the profibrotic effect of thisallele was independent of the inflammatory response [69].

TGF-b1 is one of the major profibrogenic cytokines and has been impli-cated in disease progression. The proline-to-arginine switch at codon 25 inTGFB1 increases production of the cytokine and leads to increased fibrosis(P ¼ .023) [70]. Angiotensinogen II is another profibrogenic cytokine. Thepromoter G-to-A SNP at �6 leads to increased transcription of the geneand is also associated with increased fibrosis. Together, SNPs in these twogenes have a dose-response effect, because individuals with polymorphismsin both of these genes have more fibrosis progression than those with eitheralone.

IL-12 is a cytokine that induces production of IFNg. A SNP in the genefor one of the subunits of IL-12 (IL-12p40) has been associated withimmune-mediated diseases. Homozygosity for the minor allele of thisSNP, 1188C/C, was more common in patients who had mild fibrosis(score %2) compared with severe fibrosis (score O2) (23.7% versus6.25%; P ¼ .004), but there was no association with inflammation [71].

Solute carrier family 11 member 1 (SLC11A1) protein is involved in mac-rophage function and in upregulation of chemokines important in the im-mune response to HCV (TNFa, IL-1b, and inducible nitric oxidesynthase). Several mutations result in variable mRNA expression, includinga GT tandem repeat promoter polymorphism. Four functionally differentalleles have been described in the population, which are designated as 1through 4. These alleles have been associated with other diseases, such as tu-berculosis [72]. Individuals who were homozygous for allele 2 had lower in-flammation, less fibrosis (2.4% versus 18.1%, OR ¼ 8.85), and lower HCVRNA levels (336 versus 1290 � 103 IU/mL) compared with the other alleles[73]. Further work is needed to understand the biologic basis for thisassociation.

Myeloperoxidase (MPO) is an oxidant-generating enzyme found in mac-rophages and neutrophils. A functional promoter polymorphism (�463A)in this gene has less MPO production but was associated with more fibrosis[74]. The reason for this association is unclear, but MPO has been associatedwith inhibition of NK activity and T-cell proliferation [75].


Summary

This review has concentrated on summarizing several publishedHLA andnon-HLA associations in immune-response genes with various HCV out-comes. Some have been repeated in more than one study or have a biologicbasis for the association. The associated SNPs increase our understanding ofHCV clearance and liver disease progression.

A limitation of this review, and of the current published studies, is thatstudies in which associations are not found often go unreported. To maxi-mize the utility of such studies to offer insight into how the immune responsemay account for the heterogeneity in HCV outcomes, studies that do notfind associations should also be published. Another limitation is that epi-static interactions among polymorphisms in different genes are often notassessed.

In the future, as large-scale genotyping becomes more affordable, thereshould be an increasing number of studies examining polymorphisms in var-ious genes. As the ability to scan the genome improves, such studies shouldalso offer clues to important genes involved in HCV pathogenesis, includingones that may not be obvious candidates. Nevertheless, such studies wouldstill not be able to evaluate interactions of variants in different genes. In-sights into the immune response from human genetic studies can lead to ra-tional drug development to improve therapeutics for chronic hepatitis C. Inaddition, human genetic variations can also affect response to HCV thera-pies; thus, future therapeutic trials may involve studying polymorphismsand treatment response.

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ClinIics in Liver Disease - August 2008