Interferon alfa has been increasingly used against recurrent hepatitis C (HCV) disease in post-liver transplant (LT) recipients. A serious potential adverse effect is acute rejection. We reviewed our experience using interferon-based therapy (interferon or pegylated interferon with or without ribavirin) for treating recurrent HCV in LT recipients. Forty-four LT recipients were treated with interferon for recurrent HCV. Five of the 44 patients developed acute rejection during interferon-based therapy. These 5 patients started treatment of 42.4 ± 33.89 months (mean ± SD) after LT. Mean (± SD) histological activity index and fibrosis scores before initiating antiviral therapy were 8.8 (± 1.92) and 2.6 (± 0.55), respectively. Patients were treated for 3.3 ± 2.28 months (mean ± SD) prior to rejection. At the time of rejection, HCV load was not detectable in 4 of the 5 recipients. All 5 patients had tolerated interferon therapy, and none had stopped therapy because of adverse effects. The rejection was successfully treated in 3 patients. In 2 of those 3 patients, cirrhosis eventually developed. In the 2 patients who did not respond to rejection treatment, immediate graft failure occurred, leading to re-LT in 1 patient and death from sepsis in the other. In conclusion, the results indicate that further studies are needed to assess the safety of interferon in LT recipients. Interferon-based therapy may lead to acute rejection and subsequent graft loss and should therefore be used with caution. Treated recipients may also develop progressive cirrhosis despite achieving a sustained virological response. (Liver Transpl 2004;10:859–867.)
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Hepatitis C (HCV) is the leading indication for liver transplantation in the United States.1–3 The number and proportion of individuals receiving transplants for HCV will likely grow as the cohort infected several decades ago start seeking medical care. Recurrent HCV viremia is universal, with approximately 90% of individuals undergoing transplantation for HCV having detectable virus soon after transplantation.4, 5 Histological disease is seen in 50% of recipients within 2 years.6 After 5 years, up to 30% of patients transplanted for HCV develop cirrhosis.4, 7, 8 The rate of decompensation after developing cirrhosis is approximately 42% after the first year in the posttransplant setting.9 Although earlier studies demonstrated no impact of recurrent HCV on patient survival, more studies suggest HCV may indeed negatively impact patient and graft survival.10, 11
As in nontransplant patients and the general population, the treatment of recurrent HCV disease after liver transplantation involves the use of interferon, which is believed to have both antiviral and immunomodulatory effects against HCV. However, in the posttransplant setting, antiviral therapy has been limited by relative ineffectiveness, poor tolerability, and adverse effects.12–15 Long-term response to combination interferon and ribavirin in transplant recipients is estimated to be 20% in patients treated for recurrent HCV.16 Efficacy is increased when pegylated interferon is used in combination with ribavirin, but it is still believed to be around 30%.17 Tolerance to interferon therapy after transplantation also appears to be less than in the general population, with exaggerated adverse effects including anemia and leukopenia.15, 18
A serious complication of interferon in organ transplant recipients is the precipitation of acute rejection and subsequent graft loss. Overall, the majority of acute rejection episodes after liver transplantation occur within 90 days of surgery and are seen in up to 75% of recipients, regardless of immunosuppressant regimen.19 However, acute rejection after liver transplantation in the setting of interferon therapy is not a well-documented phenomenon. This complication has been seen in renal transplant recipients and is believed to be due to the immunomodulatory effects of interferon.20 As a result, interferon is avoided in renal transplant recipients except in the setting of severe liver complications secondary to HCV.21 The incidence of serious acute rejection in renal transplant recipients treated with interferon ranges between 15 and 64%.22 Although interferon is used in liver transplant (LT) recipients to treat recurrent HCV, few data exist on its association with acute rejection. Feray et al. first reported chronic rejection in LT recipients treated with interferon for recurrent HCV.23
In the current study, we sought to examine the incidence and clinical outcome of patients who developed acute rejection while on interferon therapy.
HCV, hepatitis C; LT, liver transplant.
A chart review was performed on patients who underwent liver transplantation and were treated with interferon-based therapy for recurrent hepatitis C at the University of California Los Angeles Medical Center. We searched an administrative database and identified 44 recipients who received interferon, interferon and ribavirin, pegylated interferon, and pegylated interferon and ribavirin between January 1998 and May 2003. We collected data on recipient variables: sex, comorbidities, age at transplant, year of transplant, immunosuppressants, duration of treatment, and hematological, biochemical and virological results. We also collected data on treatment initiation date, date of cessation of interferon therapy, and reason for cessation (if course not completed), along with any interferon dose modifications made during treatment. In addition, the immunosuppression regimen, including corticosteroids, cyclosporine, tacrolimus, azathioprine, and mycophenolate mofetil, was followed along with any changes made over the course of interferon therapy. Side effects including anemia, leukopenia, and thrombocytopenia were recorded, along with any interferon dose modifications and/or use of growth factors such as erythropoietin and filgrastim. Psychological adverse effects such as depression and insomnia were also documented. All data was collected in accordance with institutional review board requirements.
The immunosuppression regimens after LT consisted of combinations of cyclosporine, prednisone, tacrolimus, azathioprine, and mycophenolate mofetil. According to our protocol, patients initially received 1 gram of intravenous methylprednisolone the day of transplantation; this was then tapered to 20 mg/day over 1 week. Oral prednisone was subsequently started at 20 mg/day and tapered as tolerated. Beginning in 1994, our institution established a regimen consisting of tacrolimus for maintenance immunosuppression. Generally, our protocol for posttransplantation target immunosuppression levels for tacrolimusconsists of 10 to 12 ng/mL at 1 to 3 months; 8 to 10 ng/mL at 3 to 6 months; 5 to 8 ng/mL at 6 to 12 months; and 2 to 5 ng/mL at greater than 12 months. For cyclosporine, the target values are 250 to 300 ng/mL at 1 to 3 months; 175 to 250 ng/mL at 3 to 6 months; 100 to 175 ng/mL at 6 to 12 months; and 50 to 100 ng/mL at greater than 12 months. Immunosuppression levels were checked bimonthly to monthly, with changes in doses made to reach target ranges. We collected immunosuppression values before and during antiviral therapy, as well as at the time of rejection.
The indication for performing liver biopsies in all transplant recipients was elevated liver-associated tests found on routine lab work. Protocol biopsies are not performed at our institution. Liver biopsies were read with an experienced LT pathologist. The histological activity index and fibrosis score, and the diagnosis of rejection, were determined according to established criteria.24–26 Criteria for beginning interferon therapy included detectable HCV RNA and a fibrosis score of 2 or more, and/or elevation of liver-associated tests at least twice the upper limit of normal.
Histological criteria used for the diagnosis of rejection included (1) portal inflammation composed of a mixed infiltrate (lymphocytes, plasma cells, eosinophils, and neutrophils); (2) bile duct inflammation and damage (lymphocytes within bile duct epithelium); and (3) venous endotheliitis demonstrated by lymphocytes infiltrating the endothelial and lifting up luminal endothelial cells.26 Rejection severity was based on the number of portal tracts affected by the infiltrate, the degree of damage, and the number of bile ducts damaged.26 There have been no changes in the histological diagnostic criteria for rejection over the study period.
Categorical variables were expressed as percentages, and continuous variables were presented as mean ± SD. All statistical analyses were performed using Microsoft Excel (Microsoft, Seattle, WA).
Between January 1998 and May 2003, 44 patients were treated with interferon-based therapy for recurrent HCV after liver transplantation at our institution. Of these patients, 33 were men and 11 were women. Four patients had received their second transplantat before initiation of antiviral therapy. Nine patients received living-related liver grafts, and 30 patients received cadaveric liver transplants. All patients were treated for biopsy-proven recurrent HCV; no patients had undergone renal transplantation prior to therapy. Sixteen patients were infected with HCV genotype 1a, 12 with 1b, 1 with genotype 2, and 1 with genotype 3. The genotypes of 14 patients were unknown. One patient was treated with interferon monotherapy, 10 with interferon and ribavirin, 3 with pegylated interferon, and 30 with pegylated interferon and ribavirin. The mean length of antiviral therapy was 11.2 ± 1.52 months (mean ± SD).
Of the 44 patients started on interferon-based therapy, 16 (36%) discontinued therapy because of adverse effects, 7 (16%) discontinued therapy because of nonresponse, and 13 (30%) patients were still on therapy as of the end of the study period. Of the 8 (18%) who completed therapy, 3 achieved a sustained virological response. HCV genotype was 1b in 1 patient and unknown in 2 patients. The mean treatment duration of the 3 patients was 20 ± 1 (±SD) months.
Of the 44 patients, 29 (66%) received filgrastim for leukopenia secondary to antiviral therapy, 16 (36%) were given erythropoietin for anemia, and 7 (16%) required dose reduction of interferon secondary to leukopenia. Interferon was discontinued early in 5 patients (11%) because of no demonstrable decrease in viral load and in a total of 18 patients (41%) secondary to adverse side effects. Treatment was terminated secondary to refractory leukopenia in 4 patients (9%), secondary to refractory anemia in 3 patients (7%), secondary to thrombocytopenia in 1 patient, and secondary to retinopathy in 1 patient. In addition, interferon was stopped in 4 patients secondary to depression (9%) and in 5 patients (11%) after the diagnosis of acute rejection was made. A total of 3 patients expired over the study period. The causes of death included lung cancer, cirrhosis, and sepsis.
Of the 44 patients treated with interferon therapy, 5 patients (11%) developed biopsy-proven acute cellular rejection: therefore, they define the current study group (Table 1 and Fig. 1). The age of the study group at initiation of antiviral therapy was 50 years ± 11.94 (mean ± SD). Three patients were men and 2 were women. One of the patients had an episode of rejection 5 years before antiviral initiation that responded to intravenous methylprednisolone and prednisone taper. All patients had detectable HCV RNA levels before treatment, as shown in Table 1. Two patients received pegylated interferon monotherapy, 2 received pegylated interferon and ribavirin, and 1 received interferon and ribavirin. All patients were hepatitis B surface antigen-negative and HIV-negative. No patient had undergone retransplantation before starting antiviral therapy. Dose modification of interferon was required in patient 1 because of cytopenia. Erythropoietin was not used in any of the patients, and filgrastim was used in 2 (patients 4 and 5). No patients discontinued therapy because of psychiatric adverse effects.
Table 1. Demographic Data, Laboratory Values, Treatment, and Outcome of Patients Developing Acute Rejection on Interferon Therapy
Alive: re-OLT for graft loss secondary to steroid-resistant rejection 4 mo after ACR
Alive: re-OLT for graft failure from cirrhosis 18 mo after ACR
Expired: sepsis 2 mo after steroid-resistant ACR
Alive: awaiting re-OLT for cirrhosis
Explant: chronic ductopenia rejection and vascular rejection with mild recurrent HCV and early cirrhosis
Explant: Moderate recurrent HCV and cirrhosis without evidence of rejection
Biopsy: (2 wk before death) marked cholestasis, extensive biliary injury consistent with previous ACR and early chronic rejection
Biopsy: (6 mo after ACR) moderate recurrent HCV and cirrhosis without evidence of rejection
The time from date of liver transplantation to date of initiation of interferon therapy was 42.4 ± 33.88 months (mean ± SD). Patients were treated for a mean time of 3.3 months (± 2.28) before the diagnosis of acute cellular rejection. Interferon was discontinued in all patients.
Of the 5 patients in the study group, 2 received cyclosporine-based regimens and 3 received tacrolimus-based regimens. All of the 39 patients who did not develop rejection were treated with tacrolimus. Patients in both groups had immunosuppression levels checked throughout the course of treatment, with modifications in dosage made to produce target levels as described. None of the study patients were on prednisone at the time of antiviral therapy initiation; however, 5 of the nonrejection patients were on prednisone during a portion of their treatment with interferon. With the exception of 1 patient (patient 2), immunosuppression levels were within protocol treatment range in study patients before and during antiviral therapy as well as at the time of acute rejection (Table 1). Of the 39 patients without rejection, 5 were below target tacrolimus levels prior to antiviral therapy, and 3 averaged a below-target level tacrolimus level over the course of interferon therapy. Of the patients with suboptimal immunosuppression levels without rejection, all were between 1 to 7 months posttransplantation, and 5 were on additional immunosuppression (prednisone and/or mycophenolate mofetil).
Of the 5 study patients with acute rejection, 3 responded to antirejection measures (Table 1). Two of the 3 patients who responded to these measures (patients 3 and 5) subsequently developed cirrhosis as a result of recurrent hepatitis C after 18 and 6 months, respectively. Their fibrosis stages were 3 before interferon initiation. Patient 3 received a second liver transplant; patient 5 is awaiting retransplantation. Patient 1 recovered completely from the episode of acute rejection. It is notable that this patient also recovered from an episode of acute rejection 5 years before starting antiviral therapy. Two of the 5 patients (2 and 4) in the study population developed severe acute rejection episodes that did not respond to intravenous methylprednisolone and/or prednisone or mycophenolate mofetil and subsequently experienced graft loss. Patient 2 successfully underwent a second liver transplantation and had evidence of chronic ductopenic rejection on biopsy of his explanted liver. Patient 4 expired from complications of sepsis and had evidence of early chronic rejection on biopsy performed 2 weeks before his death (Table 1).
Interferon therapy is a widely accepted treatment for LT recipients with recurrent hepatitis C. However, our study shows that interferon therapy in this population is not without serious risks. Acute rejection occurred in 5 of 44 patients (11.4%) treated with interferon at our institution. Two of the 5 patients developed graft loss as a result of steroid-resistant rejection, with 1 requiring a second LT and the other expiring from sepsis. The remaining 3 patients responded to steroid pulsation and/or additional immunosuppressant agents and recovered completely from the rejection episode. However, 2 of these 3 patients subsequently developed cirrhosis. This rapid progression of their recurrent HCV may be related to accelerated viral replication after intensified immunosuppressive therapy.27, 28
In the current era of liver transplantation, graft loss due to acute rejection is rare. Most episodes of acute rejection occur within 3 months of surgery and are responsive to steroids.29 In a cohort of 55 LT recipients treated for early acute cellular rejection, there were no deaths or retransplantation.30 Late acute rejection occurs after more than 180 days posttransplantation and has been reported at rates of 7 to 10% of all transplant patients.31, 32 Late acute cellular rejection tends to be refractory to medical treatment, particularly if the indication for transplantation was viral hepatitis.33, 34 Nevertheless, graft loss or death from late acute rejection in a series of 122 patients was still uncommon, occurring in 14 recipients.32 In the present study, acute rejection developed up to 83 months after liver transplantation, and 2 of the 5 patients who developed late acute rejection eventually experienced graft loss leading to retransplantation in 1 and death in the other.
Interferon has not previously been associated with an increased risk of acute rejection in post-LT patients. Several studies have supported the long-term efficacy and safety of interferon in this population.35 However, many of the published studies included small study groups.12, 18, 36–43 Jain et al. published a study of the safety and efficacy of interferon therapy.44 The study included LT recipients with either hepatitis B or C. They concluded that the risk of acute rejection was not increased by the introduction of interferon alfa. However, the patients who received interferon were given higher levels of prednisone during the trial than the controls.44 Feray et al. showed that chronic rejection can occur in up to 35% of patients treated with interferon.23
Although the relationship between interferon therapy and acute rejection in LT recipients has not been well documented, this association has been well established in the renal transplant population. Interferon was initially studied in these patients as prophylaxis against cytomegalovirus (CMV) infection after transplantation. In a study by Kovarik et al., there was a higher rate of rejection and a significantly higher number of HLA-DR mismatches in the group of patients who received interferon compared with the placebo group. The study was discontinued because of ethical concerns over the high rate of rejection after treatment with interferon in this population.45 Subsequent studies of renal transplant patients on interferon have also shown increased rates of steroid-resistant acute vascular rejection episodes.46, 47 As a result of this data, a recent review recommends that interferon therapy not be used in renal transplant recipients except those with fibrosing cholestatic hepatitis.21 It is unclear why renal transplant grafts appear to be more vulnerable to rejection episodes after interferon than LT grafts. HLA compatibility has been shown to influence the outcome of renal transplants to a greater degree than liver transplants.48, 49
As a result of the immunomodulatory and immunostimulatory effects of interferon, several mechanisms behind the induction of acute rejection have been postulated. One hypothesis is that interferon alfa may enhance the expression of HLA-DR antigens on donor hepatocytes, thus initiating a vigorous immune response and subsequent episode of rejection. This has been supported by in vivo studies showing that HLA-DR expression is enhanced in response to human interferon.20 Alterations in class II histocompatibility expression in transplanted tissue by interferon alfa have also been seen in a model of heart transplantation in rats.50 In an analysis of human liver grafts after transplantation, when rejection was the reason for graft failure, bile duct epithelial cells were found to be HLA-DR positive, as were hepatocytes in severe rejection episodes.51 The bile duct epithelia of donor specificity is thought to become the dominant class II antigenic stimulant and contribute to the vanishing bile duct syndrome seen in rejection. Dousset et al. reported the observation of acute vanishing bile duct syndrome in 2 of 5 patients treated with interferon: 1 of those 2 patients required retransplantation;the other expired.52 This hypothesis of enhancement in the expression of HLA-DR antigens by interferon can also be supported by recent evidence that pegylated interferon may be more likely to cause rejection than traditional interferon alfa.53, 54 In the present study, 4 of the 5 patients who experienced rejection received pegylated interferon. A possible mechanism for this increased rejection risk is related to the extended half-life and increased serum concentration of pegylated interferon. These properties could make it more likely to increase HLA expression, and therefore rejection, than interferon.
Another proposed mechanism for the increased risk of interferon-associated acute rejection may be decreased levels of immunosuppressants as a result of HCV clearance during antiviral therapy.55 It is suggested that antiviral therapy improves hepatocyte microsomal function, which leads to decreased immunosuppression levels. However, in our study, only 1 patient who developed rejection had a decreased mean immunosuppression level while on interferon. In addition, 3 patients who did not develop acute rejection had immunosuppression levels below our recommended protocol while on antiviral therapy. Decreased immunosuppression levels may be important; however, other factors are likely required for the development of acute rejection.
Although the exact mechanism is unknown, interferon is believed to lead to HCV clearance by inhibiting viral replication and enhancing the immune response against the hepatitis virus. The use of an immunomodulator such as interferon with immunosuppressants such as cyclosporine, tacrolimus, and prednisone raises concerns about possible interactions. These medications appear to inhibit T-cell activation and may inhibit interferon production. This has been suggested by the observation of low levels of circulating interferon alfa in renal transplant recipients on immunosuppressant therapy.56, 57 These studies raise concerns about the use of interferon in a transplant and immunosuppressed population. The effects of the additional use of growth factors such as filgrastim to improve tolerance of interferon therapy and avoid leukopenia also have yet to be studied. In addition, the possible protective effect of ribavirin against acute rejection as an adjunct to interferon remains to be explored. In the present study, 2 of three patients treated with pegylated interferon monotherapy experienced acute rejection. Early studies have suggested that ribavirin may have immunosuppressive effects.58 Ribavirin may therefore provide additional immunosuppressive qualities that provide protection from rejection in addition to its role in antiviral therapy.
Another important consequence of acute rejection after treatment with interferon observed in our study, aside from graft loss from resistant rejection, is the acceleration of the course of recurrent hepatitis C. Two patients who responded to increased immunosuppression and recovered from rejection subsequently experienced progressive cirrhosis and graft failure. With intensive immunosuppression, liver disease progression may be accelerated.27, 28 Therefore, the impact of treatment with interferon on graft survival after achieving serum virological response remains to be studied.
Also observed in our study were the features of mild acute rejection in the biopsies of 3 of the study patients prior to the initiation of interferon therapy. Two of these patients subsequently experienced graft failure after developing acute rejection while on interferon therapy. Distinguishing between graft hepatitis and acute cellular rejection can be difficult because the histological features of the 2 conditions can overlap. For example, portal infiltrate can be found in both rejection and recurrent HCV. However, in the setting of elevated quantitative HCV RNA levels and significant biopsy-proven elements of recurrent HCV after more than 7 months posttransplantation, the decision was made to treat with antiviral therapy. Gottschlich et al. suggest that quantitative HCV RNA levels can be useful in distinguishing between recurrent HCV and rejection.59 After treatment with interferon, the biopsy results of the 2 patients became diagnostic of acute rejection and interferon was discontinued. Therefore, it is difficult to determine whether or not interferon accelerated the process of acute rejection in these patients. However, while some histological features of recurrent hepatitis and rejection may overlap, it should be noted that definitive evidence for acute rejection, such as endotheliitis, was not seen prior to interferon therapy in any of the patients.
Limitations of our study include the lack of protocol biopsies accounting for possible unrecognized mild rejection and the retrospective nature of the analysis. An additional limitation is that the rejection associated with clearance of HCV infection may have been unrelated to interferon therapy. Indeed, Doughty et al. reported on a single patient who cleared the infection after being treated for rejection.60 However, in a series of over 500 recipients of transplants for HCV in our institution, spontaneous HCV clearance was not observed.61 Despite these limitations, our experience would argue that interferon therapy is not without risks. Because the exact mechanism of action of interferon in inducing liver allograft rejection remains obscure, prospective trials are needed in order to better assess the safety of such therapy in an immunosuppressed population.
In conclusion, with recurrent HCV becoming increasingly common in the transplant community, there will be a greater need to find safe and effective antiviral therapy. The risk of acute rejection and subsequent graft loss and the risk of accelerated recurrent HCV with subsequent cirrhosis in LT patients on interferon should be weighed heavily in choosing treatment for recurrent HCV, which itself can cause allograft failure if untreated. Treating physicians should therefore be aware of the risks and monitor patients accordingly.
The authors thank the post-liver transplant coordinators Anna Zafar, Kevin King, Alicia Ingemi, and Tina Smith for patient care. The authors also thank Yaeng Xayavong for critical review of the manuscript.