The challenge of progressive hepatitis C following liver transplantation

[Guest guest]

The challenge of progressive hepatitis C following liver

transplantation

Editorial

Liver Transplantation

Volume 12, Issue 1, Pages 19-21

Jan 2006

L.

Baylor Regional Transplant Institute, Baylor University Medical

Center, Dallas, TX

The complications of cirrhosis due to chronic hepatitis C are the

leading indications for liver transplantation in the United States

and currently account for more than 40% of cases.[1] This proportion

has risen steadily over the 15 yr since the hepatitis C virus (HCV)

was identified and it is projected that the number will continue to

rise for another 10 to 20 yr.[1-3] The overwhelming majority of

patients who present for liver transplantation have either never been

treated with antiviral therapy, have been unable to tolerate it, or

have failed to respond. Thus, viremia persists after transplantation

in nearly all patients and commonly results in recurrent liver injury.

[4] Progression of liver injury after transplant is frighteningly

rapid in comparison to immune competent patients with cirrhosis

developing in 10 to 25% within 5 yr.[4][5] In perhaps the most

distressing report, Berenguer et al.[6] reported that half of

patients have bridging fibrosis or cirrhosis within 5 yr of the date

of transplant. A large proportion of cirrhotic patients will develop

decompensation and death within 1 yr.[6] In contrast, only 20% of

immune competent patients develop cirrhosis after 20 yr and only 3 to

6% of these decompensate each year; most remain well compensated

without significant threat to their survival.[7] Given the number of

patients and the dire implications of disease recurrence, chronic

hepatitis C has become perhaps the most difficult problem currently

facing physicians who manage liver transplant recipients.

Management of persistent HCV infection and recurrent chronic

hepatitis is difficult. Hyperimmune anti-HCV immune globulin

preparations have not yet been shown to be effective in this setting

as they have been in hepatitis B.[8][9] Antiviral therapy with

interferon-based regimens, as expected, is relatively ineffective in

these patients since many have previously failed treatment, most are

genotype 1 with high viral levels, and many have significant

cytopenia due to hypersplenism and medication effects.[10-12]

Preemptive therapy with interferon-based therapy administered shortly

after transplantation is especially difficult because of these

issues, the debility of many patients, and the potential for drug

interactions.[12] Furthermore, this requires treatment of all HCV

infected patients including the half who are not destined to have

rapidly progressive graft injury. Thus, most hepatologists prefer to

wait at least 6 to 12 months until patients have recovered from

transplant and surveillance liver biopsies have identified those with

progressive disease before considering antiviral treatment.

Nonetheless, treatment still remains a substantial commitment for

both patient and physician. Approximately 70% of cases require dose

reduction; ribavirin, in particular, is poorly tolerated in these

patients, who almost always have some degree of renal impairment from

their calcineurin inhibitor.[10][12] Despite these challenges, many

centers have reported a surprisingly high 20 to 30% sustained viral

clearance rate.[10][12] Of course, considering the difficulty of

treatment in this setting, these responses are far from what we would

hope for. Considerable effort is being invested in investigating

other ways to improve response to antiviral therapy in these

patients. Recent reports of potent HCV protease and polymerase

inhibitors raise the possibility that these might increase the

effectiveness of interferon-based treatment in the transplant

setting, though these studies are undoubtedly some distance in the

future.

Another potential way to influence the course of posttransplant HCV

infection might be to alter the immunosuppressive regimen. The more

rapid progression of HCV recurrence in recent years has been

attributed to more potent immunosuppressive drugs that are employed

at and around the time of transplantation.[4][5] Indeed, both OKT3

and high-dose corticosteroid pulse therapy used to treat acute

cellular rejection have been shown to accelerate HCV recurrence and

graft injury.[13][14] However, it does not appear that chronic

steroid administration has a similar effect.[5] A pilot study and a

more recent large multicenter randomized study have reported that

steroid-free immunosuppression does not appear to reduce HCV

recurrence in short-term follow-up.[15][16] Neither mycophenolate

mofetil nor azathioprine have been shown to have a consistent effect

on HCV recurrence.[17][18] Although it has been suggested that the

change in preference from cyclosporine to tacrolimus by most liver

transplant centers in the late 1990s might explain the apparent

rapidity of HCV disease in recent years, the data do not appear to

support this. The choice of calcineurin inhibitor has not been shown

to significantly influence HCV levels, severity of disease

recurrence, or the course of recurrent infection in either

prospective or retrospective reports.[5][18-21] Indeed, one might

imagine that the higher chance of early rejection with cyclosporine

might require more steroid boluses and thereby lead to more rapid

progression of HCV disease,[22] though this has not been reported.

Despite the absence of clinical data to support one calcineurin

inhibitor over another in HCV patients, recent in vitro studies have

shown that cyclosporine inhibits HCV replication, independent of

interferon induction, in a cell-based replicon model.[23] This has

led to renewed interest in studying the potential benefit of primary

cyclosporine-based immunosuppression following liver transplantation,

particularly in its ability to facilitate the response to antiviral

therapy. In this issue, Firpi et al.[24] from the University of

Florida confirm the in vitro effect of cyclosporine on HCV

replication in the replicon. They also report that patients with HCV

recurrence who were receiving cyclosporine were almost twice as

likely to clear HCV with interferon-based therapy as those receiving

tacrolimus. These results are intriguing, but are preliminary and

must be interpreted with caution. First, previous studies, including

a prior report from the same authors, have not shown such a

difference in responses to antivirals or have found low responses in

cyclosporine treated patients.[10-12][25-28] However, the studies are

highly selected and any difference might have been masked by this and

by the high proportion of patients who discontinue therapy.

Furthermore, many studies have not looked for a potential effect of

the immunosuppressive regimen on treatment response.[29-31] Second,

the current study was retrospective and the cyclosporine and

tacrolimus groups were neither selected nor treated concurrently.[24]

Third, there are some clinically significant differences in the

treatment groups that make interpretation of these results difficult.

Nonetheless, these results deserve attention and need to be addressed

in a prospective study.

Hepatitis C is the major indication for liver transplantation today

and it will become even more common in the future. Recurrent chronic

hepatitis C is a frequent and serious problem after transplant,

causes significant morbidity, and results in graft loss in about 10%

of patients. Effective treatment of HCV infection in these patients

is extremely difficult and time-consuming, and not often effective in

altering the course of recurrent disease. The need for more a more

effective way to prevent reinfection of the graft or to at least

reduce progression of liver injury is absolutely critical.

Prospective studies are urgently needed to optimize donor selection

to minimize the severity of recurrence, delineate the best tolerated

immunosuppressive regimens, clarify the timing and role of antiviral

therapy, and investigate new therapies such as hyperimmune anti-HCV

immune globulin, HCV protease or polymerase inhibitors, and

antifibrotic agents. We have a long way to go.

Cyclosporine suppresses hepatitis C virus in vitro and increases the

chance of a sustained virological response after liver transplantation

Liver Transplantation

Volume 12, Issue 1, Pages 51-57

Jan 2006

o J. Firpi 1 *, Haizhen Zhu 2, Giuseppe Morelli 1, Manal F.

Abdelmalek 1, Consuelo Soldevila-Pico 1, Victor I. Machicao 1, Roniel

Cabrera 1, Alan I. 3, Chen Liu 2, R. 1

1Division of Gastroenterology, Hepatology and Nutrition Section of

Hepatobiliary Diseases, University of Florida, Gainesville, FL

2Department of Pathology, Immunology, and Laboratory Medicine,

University of Florida, Gainesville, FL

3Department of Surgery, University of Florida, Gainesville, FL

Abstract

Cyclosporine is an immunosuppressive agent widely used in the

management of liver transplant recipients. Cyclosporine has been

shown to have antiviral activities against HIV, herpes simplex, and

vaccinia viruses. The aim of this study was to determine the effect

of Cyclosporine in viral clearance in the liver transplant recipients

during therapy with combination of interferon and ribavirin, and to

determine the anti-viral potential of Cyclosporine in vitro.

Immunosuppression consisted of either Cyclosporine or Tacrolimus-

based therapy. Both groups received therapy with interferon and

ribavirin for 48 weeks when evidence of progressive histologic

disease was determined. We found that subjects on Cyclosporine-based

immunosuppression (n = 56) had a higher sustained virological

response of 46% compared to 27% in the patients on Tacrolimus-based

therapy (n=59, P = 0.03). In vitro studies were performed to evaluate

the antiviral effect of Cyclosporine in the replicon system. These

studies showed that Cyclosporine inhibits hepatitis C viral

replication in a dose-dependent manner. Combination of Cyclosporine

with interferon showed additive effect, and its function is

independent of interferon signaling pathways. In conclusion,

Cyclosporine may offer an advantage to Tacrolimus in those patients

undergoing interferon-based therapy and should be studied in a

prospective randomized trial.

Viral Response

The study population consisted of 115 patients that were treated with

interferon-based therapy. From the 115 patients, 56 patients received

CsA and 59 patients received TAC-based immunosuppression. The mean

duration of the treatment was 48 weeks. The end-of-treatment response

(ETR) was 40% in the TAC group compared to the CsA group that

achieved an ETR of 60%. In the CsA group, 26 out of 56 (46%) patients

achieved a sustained virological response; while 16 out of 59 (27%)

patients on TAC achieved a SVR (P = 0.03). The patients who were

treated with CsA-based immunosuppression achieved a higher SVR

compared to those treated with TAC-based therapy. The pre-treatment

viral load was slightly higher in TAC group, but this did not reach

statistical significance (2.0 × 106 IU/ml vs. 1.5 × 106 IU/ml)

(NS). The percent of patients with genotypes 2 and 3 was also not

significantly different as seen in Table 1. The rate of SVRs based on

genotypes for the TAC and CsA groups is shown per each group,

respectively: 16% and 44 % for Genotypes 1, 80 % and 67% for

Genotypes 2, 80% and 40% for Genotypes 3, and no patients for the

genotypes 4. Dose reduction of IFN or ribavirin due to cytopenias was

necessary in 66% of the patients on CsA and in 82% on TAC (P =0.1).

Twenty patients (6 on CsA and 14 on TAC) had to stop therapy due to

side effects including cytopenias and depression (P = 0.3). Sixty-one

percent of the CsA patients and 50% in the TAC group received 80%

interferon or peginterferon plus 80% ribavirin for more than 80% of

the expected duration of therapy (P = 0.2). Thirteen patients of the

56 on CsA (23%) and 6 patients of the 59 (10%) have died, mostly due

to infections and HCV complications. However, although only one death

was thought related to IFN treatment side effect (chronic rejection)

(see Table 2). This difference in survival between the 2 groups was

statistically significant (P = 0.05), although was likely related to

the longer follow-up in the CsA group.

Article Text

End-stage liver disease associated with hepatitis C (HCV) infection

is the most common indication for liver transplantation (LT) in the

United States.[1] In contrast to the other leading indications for

LT, recurrence of HCV infection, as measured by detection of HCV RNA

by PCR, is nearly universal.[2][3] Although patients undergoing

transplantation for HCV have been reported to have patient and graft

survival comparable to most other indications,[4][5] recurrence of

HCV is a substantial source of morbidity, mortality, and graft loss.

[6][7] In a recent retrospective cohort study of over 11,000

transplant recipients, HCV infection as an indication for LT was

associated with significantly impaired patient and allograft survival.

[8] Viral recurrence occurs universally; however, recurrence is

apparent histologically in only about 50% of HCV-infected grafts and

progression to allograft failure leading to death or graft loss

occurs in approximately 10% by the fifth post-operative year.[9-11]

When recurrent HCV leads to decompensated cirrhosis,

retransplantation is often denied due to very poor survival.[12] HCV-

related disease progression is accelerated in immunocompromised

compared to immunocompetent patients with a progressive increase in

patients who have recently undergone LT, although the reasons for

this worsening outcome are under question.[13] Possible reasons for

this disturbing trend include the use of more potent

immunosuppressive agents and more marginal donors (older).[14] The

two most frequently used immunosuppressive drugs are Cyclosporin

(CsA) and Tacrolimus (TAC). Of interest, the usage of TAC has

increased from 0% before 1996 to nearly 80% after 1999. It is

intriguing to hypothesize that alterations in immunosuppressive

regimens may impact on disease recurrence and response to antiviral

therapy.

Limitations in treatment of patients with chronic HCV have led to

multiple trials to understand the role of immunosuppression in

disease progression and response to antiviral therapy. OKT3, IL-2r

antibodies (Abs) steroids, and others have been reported to

accelerate HCV, but there is no apparent relationship with the

primary immunosuppressive regimen of TAC or CsA.[15-17] Currently,

TAC is the primary immunosuppression agent used in the majority of

liver transplant recipients. However, CsA may have some theoretical

benefits in the HCV population. CsA has been shown to have antiviral

activities against human immunodeficiency virus type I,[18] herpes

simplex virus,[19] and vaccinia virus.[20] A recent report by

Wastashi et al. showed that CsA has a strong suppressive effect on

HCV replication using the HCV replicon cell culture system.[21] This

reduction was not observed with other immunosuppression like TAC.

This CsA effect was independent of its immunosuppresant function. In

another recent report, CsA appears to have a beneficial impact on HCV

antiviral therapy when combined with interferon-based regimens.[22]

There is also indirect evidence that CsA may augment the activity of

interferon against HCV.[23] However, several conflicting reports

regarding the efficacy of CsA in the liver transplant population have

also been published and there remains significant uncertainty

regarding the potential role of CsA in the HCV transplant recipient.

[24-26]. A better understanding of immunotherapy and its relation to

resolution of the infection may help in the design of better

therapies for the control of HCV infection in this population.

In this study, we have analyzed the impact of CsA on HCV replication

and response to interferon-based therapy within out liver transplant

population. Our data confirms an in-vitro antiviral effect for CsA

and suggests a role for its use for liver transplant patients

undergoing antiviral therapy for HCV.

Interferon-Based therapy

Combination therapy with interferon and ribavirin was utilized in

those patients with significant fibrosis (Ishak fibrosis stage >/=3)

on protocol or indication liver biopsy. Therapy was initiated at half

dose [1.5 MU interferon alfa-2b SQ thrice weekly (tiw)/PEG interferon

alfa-2a 135 ug weekly and ribavirin 400-600 mg daily] for 2 weeks,

and if tolerated, the dose was increased to full dose (interferon

alfa-2b 3 MU SC tiw/PEG interferon alfa-2a 180 ug weekly and

ribavirin 800-1,200 mg daily). Ribavirin dosage was based on weight;

patients < 75 kg received 800 mg and > 75 kg 1,000 mg. Hemoglobin,

white blood cell count, and platelet count were monitored weekly for

the first four weeks and then monthly thereafter. Dose reduction was

performed as follows: if PMN < 750 or platelets < 50,000/uL, the

interferon was reduced to 1.5 MU three times a week/PEG interferon to

135 g/week; if PMN < 500 or platelets < 30,000, therapy was stopped;

hemoglobin < 10 mg/dl, ribavirin was reduced to 600 mg per day; if

hemoglobin < 8 mg/dl the ribavirin was discontinued. Erythropoietin

was used to prevent ribavirin discontinuation when possible.

Granulocyte colony stimulating factor was not used to treat

cytopenias. Therapy was discontinued in any patient who developed

moderate to severe rejection, systemic bacterial infection, severe

neuropsychiatric symptoms, or symptomatic anemia. The intended

duration of treatment was 48 weeks for genotype 1 and 24 weeks for

genotype 2 and 3.

HCV RNA Testing

Serum HCV RNA values were measured six months after completion of

interferon-based therapy to assess for the presence of a sustained

virological response (SVR) in those patients with a negative HCV at

the end of treatment. Subsequently, an HCV RNA titer was obtained

annually. The HCV RNA titer was determined using a branched DNA

signal amplification assay (Quantiplex HCV RNA; Chiron Corporation,

Emeryville, CA). Serum samples that tested negative by the branched

DNA assay were analyzed using RT-PCR with a sensitivity of about 10-

200 copies (Qualitative Amplicore HCV Test; Roche, Mississauga,

Ontario, Canada).

Histology

Protocol liver biopsies were performed in our institution at month 4,

yearly, or when clinically indicated after liver transplantation.

Recurrent HCV disease was scored for inflammation and fibrosis, using

the modified Knodell scoring system of Ishak.[16] Patient treated

were those with Ishak score >/=3.

End Point

The primary end points were to determine the effect of CsA in viral

clearance in the liver transplant recipients with recurrence HCV

during therapy with combination of interferon and ribavirin, and to

determine the anti-HCV potential of CsA in the replicon cell culture

system.

DISCUSSION

HCV infection is of major concern after LT due to universal

recurrence, more rapid fibrosis progression, and potential graft

failure. Given the dismal outcomes with retransplantation for HCV,

all efforts to limit HCV recurrence and liver injury need to be

aggressively pursued after LT. The impact of the primary

immunosuppression regimen on disease and treatment outcomes has not

been well defined. Our non-concurrent cohort study suggests that CsA

may play a beneficial role as primary immunosuppression for patients

transplanted for HCV infection and may offer an advantage to TAC in

those patients undergoing IFN-based therapy. A recent study by

et al. showed no significant differences between TAC and CsA on

histologic HCV recurrence after LT.[17] Kakumu and colleagues

reported that the administration of CsA monotherapy can suppress

aminotransferases levels but not HCV RNA. Unlike patients on

corticosteroids, an increase of HCV RNA was not observed.[27] Our in-

vitro data suggests that CsA may confer a patient advantage from an

HCV replication standpoint, although there is yet no clinical data to

support these in-vitro findings. However, it may also be that the

clinical and/or viral benefit may be confined to those patients

undergoing combination therapy with interferon and ribavirin, which

was not addressed in this former study.

There is some evidence that combination of IFN and CsA may increase

the activity of IFN against the HCV infection, but there is no

published data about the effect of CsA on HCV infected patients

receiving treatment post-LT. In our analysis, patients receiving

combination therapy with interferon and ribavirin on CsA as basic

immunosuppression achieved a higher SVR than those patients on TAC-

based immunosuppression. There is no doubt that this SVR is higher

compared to the literature, but this study represents a preliminary

pilot study. The high SVR may be related to an antiviral effect of

CsA or other unknown factors. We were unable to find any significant

differences in patients characteristics between the two groups,

including dose reductions, viral load, genotype, BMI, or percentage

of patients who had failed interferon-based therapies before

transplantation.

In addition, we were able to confirm that CsA has antiviral activity

in cell culture systems and that the mechanism does not appear to act

via standard interferon pathways. We demonstrated that CsA inhibits

HCV RNA replication in a dose-dependent manner. At comparable

therapeutic levels of 250 ng/ml, CsA can suppress viral replication

by 20%. Of note, there was no evidence of cell toxicity at these

doses, as determined by both morphology and cell count. Combination

of CsA and IFN achieve better antiviral effect than either CsA or IFN

alone, suggesting an additive or synergistic effect. Analysis of the

IFN-stimulated gene G1P3 showed that CsA had no effect on this

classic IFN antiviral pathway. This data indicates that the CsA

antiviral pathway is different from classic IFN pathways. This

represents a novel antiviral property of CsA that requires further

investigation. Two recent Japanese studies have also reported an anti-

viral effect of CsA using the replicon cell culture system.[9][18]

Nakagawa treated HCV replicon cells with CsA showing suppression of

viral replication in a dose-dependent manner. In another study,

Watashi and colleagues treated HCV replicon cells with CsA and

decreased specific HCV proteins and HCV RNA levels. Together, these

studies demonstrate anti-HCV activity in vitro.

Owing to the accelerated rate of disease progression and graft

failure after LT in HCV patients, it will be very important to

determine the ideal immunosuppression regimen. Our data suggests a

potential advantage of CsA vs. TAC in HCV patients undergoing

antiviral therapy. A prospective randomized comparative trial between

CsA and TAC is warranted to further evaluate these observations.

RESULTS

In-Vitro Analysis

Anti-HCV Activity of CsA

The HCV replicon cell line was used to assess the effects of

different doses of CsA on the intracellular replication of HCV. GSB1

cells were treated with different concentration of CsA for 48 hours.

Figure 1 depicts the HCV replicon RNA replication inhibition by CsA

in a dose-dependent manner. By this in-vitro assay, 250 ng/ml of CsA

(comparable to therapeutic levels achievable in patients) leads to a

20% reduction in HCV replication. No effect on viral replication was

observed when the HCV replicon cells were treated with comparable

concentrations of TAC (see Fig. 2).

CsA effect on IFN Antiviral Activity

To test the effect of CsA on IFN antiviral activity, the replicon

cells were treated with both CsA and IFN-a, followed by real time PCR

analysis. As shown in Figure 3, addition of CsA and IFN- shows an

additive antiviral effect, but not synergistic effect, suggesting an

independent antiviral mechanism. To test this hypothesis, examination

of the IFN-stimulated gene G1P3 expression was performed after cells

were treated with IFN- and G1P3, either alone or in combination. As

shown in Figure 4, the CsA did not have any significant effect on IFN-

induced antiviral pathway.

In-Vivo Analysis

HCV Transplant Population

Between 1991 and 2002, 842 adult liver transplants were performed at

the University of Florida. Of a total of 842 LT, 358 (40%) liver

transplants were performed secondary to HCV cirrhosis. Of the 358 HCV

infected LT recipients, 107 (30%) received interferon-based therapy

before transplant. The median time from LT to initiation of therapy

was 2.6 ± 1.2 yr for the TAC group and 4.9 ± 1.1 yr for the CsA

group. Antiviral therapy after transplant was initiated for Ishak

fibrosis stage >/=3 or HCV-related cholestasis. A total of 115 (32%)

patients received treatment with interferon-based therapy after LT.

Characteristics of Study Groups

The general characteristics of the two study groups are shown in

Table 1. There were no statistically significant differences between

the CsA and TAC group.