Prediction of sustained virological response in liver transplant recipients with recurrent hepatitis C virus following combination pegylated interferon alfa-2b and ribavirin therapy using tissue hepatitis C virus reverse transcriptase polymerase chain reaction testing
Guy W. Neff,
Department of Medicine, University of Miami, Miami, FL
Jackson Medical Towers, suite 1101, 1500 NW 12th Avenue, Miami, FL, 33136
The optimal duration of therapy for pegylated interferon combined with ribavirin in recurrent Hepatitis C virus (HCV) following liver transplantation is not known. We wanted to determine if testing for HCV in liver tissue by reverse transcriptase polymerase chain reaction (RT-PCR) was superior in predicting sustained virological response (SVR) in comparison to standard HCV ribonucleic acid (RNA) detection in the serum. All recipients received combination pegylated alpha-2b interferon (1.5 mcg / kg) and ribavirin (200–600mg / d) therapy for at least 48 weeks of therapy and were found to have nondetectable HCV RNA by PCR serum testing at the end of therapy. Sustained virological response (SVR) was defined as nondetectable serum HCV RNA at 6 months post treatment withdrawal. Ten liver transplant recipients were included in the study; mean time from transplantation was 29.2 months. All had nondetectable serum HCV RNA by RT-PCR. In hepatic tissue 7/10 patients HCV RNA was found to be positive by RT-PCR while 3/10 had nondetectable HCV RNA in their liver by RT-PCR. SVR was attained in all 3/10 that were hepatic tissue HCV PCR negative after 12 months of combination therapy. In conclusion, direct detection of HCV RNA by RT-PCR of liver tissue appears to more effectively predict SVR following pegylated interferon and ribavirin therapy than the conventional use of serum. (Liver Transpl 2004;10:595–598.)
Chronic Hepatitis C Virus (HCV) remains the most common indication for orthotopic liver transplantation (OLTx) in the United States.1, 2 In fact, the United Network of Organ Sharing (UNOS) reports that 42% of liver transplants performed in the United States in the year 2000 were a result of cirrhosis due to HCV.3 Unfortunately, there has been a universal recurrence of HCV infection post liver transplantation. Investigators have reported that the treatment of this recurrent HCV with combination interferon and ribavirin therapy in the post-transplant period has resulted in only limited success when compared to patients treated pretransplantation.4, 5
Our experience has been similar to that reported by others. We have found that sustained virological response has occurred in only a small percentage of our patients undergoing therapy for recurrent HCV post liver transplantation. The rate of relapse of HCV post discontinuation of antiviral treatment in our posttransplant patient population has been extraordinarily high in comparison to non-transplant liver patients. This has resulted in a decreased long-term survival for our patients transplanted for HCV in comparison to our patients transplanted for other indications.6
It may be that these patients could require longer treatment duration in comparison to pre-transplant patients. The ideal approach would include an accurate method to determine optimal therapy duration that could predict sustained virological response (SVR) and prevent this high relapse rate. Some authors have reported that direct detection of HCV ribonucleic acid (RNA) in liver tissue by reverse transcriptase polymerase chain reaction (RT-PCR) may be helpful in predicting SVR in nontransplant liver patients.7, 8 Our aim was to discover if direct testing for the presence of HCV RNA by RT-PCR in liver tissue was a more powerful predictor of SVR than conventional PCR testing of plasma for detectable HCV RNA after 48 weeks of combination pegylated interferon and ribavirin therapy for recurrent HCV in patients post liver transplantation.
We performed a retrospective review of the medical records of all patients with the diagnosis of recurrent hepatitis C post liver transplant seen at the University of Miami / Jackson Memorial Hospital's Transplant Center from January 1, 2000, to December 31, 2002. Patients were selected for therapy if they originally received a liver transplant for cirrhosis due to HCV and had evidence of progressive, recurrent HCV post liver transplant. All patients had been treated with pegylated interferon alfa-2b and ribavirin for at least 48 weeks and were HCV PCR nondetectable at the completion of the therapy. Those patients that had both standard RT-PCR testing of HCV RNA in the serum and hepatic tissue specimens were included in this analysis.
Liver biopsies were performed for HCV testing and histological review. Histological findings were graded and staged according to the Batts and Ludwig classification. Serum HCV testing was collected at 6 months following the cessation of therapy by the COBAS AMPLICOR™ Hepatitis C virus Test, version 2.0 (HCV RNA Qualitative PCR) and COBAS AMPLICOR™ HCV MONITOR TEST-version 2.0 (HCV RNA quantitative PCR). Tissue HCV PCR confirmation was done using Light Cycler (Roche) real-time PCR for HCV quantitation. The COBAS AMPLICOR HCV MONITOR Test, version 2.0 quantitation dynamic range: 600 IU / ml to 500,000 IU / ml. COBAS AMPLICOR HCV Qualitative (test): limit of detection 50 IU (approximately 100 copies / ml). The real-time PCR for liver tissue was 200 to 2,000,000 copies / mg (wet weight).
Our standard post-liver transplant immune suppression protocol consisted of a combination of tacrolimus and tapering methylprednisolone. Tacrolimus was started immediately before OLT at a dose of 0.05 mg / kg orally and continued postoperatively with dose adjustments to maintain whole blood 12-hour trough level at 15 ng / ml during the first postoperative week, 10–15 ng / ml for the first 3 months, and 8–10 ng / ml thereafter. Methylprednisolone was started immediately after the reperfusion of the hepatic graft with 1-gram intravenous bolus and then tapered to 200 mg per day (day 1), 160 mg per day (day 2), 120 mg per day (day 3), 80 mg per day (day 4), 40 mg per day (day 5), 20 mg per day (day 6), and then reduced to zero within 3–6 months post OLT.
All transplant recipients in this review were treated with a combination of pegylated interferon alfa-2b at a dose of 1.5 mcg / kg administered subcutaneously once a week combined with ribavirin in escalating dose fashion because of the known reduction in renal function due to tacrolimus. Patients were started on the reduced dose of 400 mg every day for patients with body weight less than 70 kg and 600 mg daily for patients with body weight greater than 70 kg. Ribavirin was increased by 200 mg increments on a monthly basis as tolerated by the patient's hemoglobin. Ribavirin was reduced by 200 mg or discontinued if hemoglobin decreased to less than 10 g / dl or less than 8 g / dl, respectively. We also adjusted the dose of both ribavirin and interferon as appropriate if the patient developed progressive renal impairment. The interferon dose was reduced 50% if platelets dropped to less than 50,000 and discontinued if less than 20,000. Filgrastim was given if there was a decrease in the absolute neutrophil count to less than 800 cells / dl and discontinued if less than 500 cells / dl.
There were 57 liver transplant recipients treated at our transplant center for recurrent HCV post liver transplant. Eighteen (31%) patients were female and 39 (69%) were male with a mean age 53.5 years (range 44 to 70 years) and a mean time from liver transplant of 31 months. Forty percent were Caucasian, 3% African American, and 57% Hispanic. HCV genotyping demonstrated 56 patients had HCV genotype 1, while 1 patient had genotype 3a. HCV RNA determination in the serum by RT-PCR revealed that 18 patients had undetectable HCV RNA viral loads in their serum at the end of treatment.
Of these 18 patients, 10 patients also had liver tissue HCV RNA PCR testing along with simultaneous HCV RNA detection in their serum and PCR both at the beginning and after 12 months of therapy. Three (30%) patients were female and 7 patients (70%) were male. The mean age of all 10 patients was 53 years (range, 45–71years), with a mean time from OLT of 29.2 months. Four patients (40%) were Caucasian and 6 (60%) patients were Hispanic.
Combination Antiviral Therapy
The mean dose of peginterferon alfa-2b therapy received over the course of the year was 1.0 mcg / kg. Most patients received a median dose of ribavirin of 400 mg per day. The dose of ribavirin was limited due to tacrolimus-associated reduction in glomerular filtration rate and the rapid development of significant anemia in this group of patients. It was standard practice in our medical center at the time to treat ribavirin-associated anemia with dose reduction rather than the use of recombinant erythropoietin alfa.
HCV RNA Quantitative PCR
The mean pre-treatment antiviral HCV PCR titer was 1.5 million IU/ml (range 500,000 to 10 million). All serum samples were serologically negative for HCV while 7/10 (70%) were HCV tissue PCR detectable and 3/10 (30%) were nondetectable. SVR was attained in 3/10 (30%); all 3 patients had nondetectable HCV RNA in both their serum and their liver by RT-PCR at the completion of 48 weeks of antiviral therapy.
In the 10 patients that had an end of treatment virological response and were HCV RNA nondetectable in their serum, biopsies at the end of treatment (48 weeks) revealed grade 1–2 inflammation in 6 patients and 4 had no inflammation present (grade 0). All 6 patients with low-level inflammation, grade 1–2, experienced HCV recurrence. Patients without histological inflammation, 3 of 4, did not experience HCV recurrence. The stage of fibrosis was consistent through this group at 1.5. The time to HCV recurrence in our 7 patients occurred on average 8 weeks after treatment was discontinued.
Depression occurred in 30 patients and 25 responded to antidepressant therapy, while treatment was discontinued in 4 patients as a result of failure to respond to antidepressant treatment. One discontinuation was not related to interferon therapy.
HCV recurrence following liver transplantation is universal. This tendency, unfortunately, can result in progressive liver disease that culminates in allograft failure with need for retransplantation.6, 9 Interferon therapy, both alone and in combination with ribavirin, has been used with limited success in an attempt to slow disease progression and improve histology.10, 11 In addition to the low SVR, interferon therapy of recurrent HCV post liver transplantation has been challenging due to many drug-related side effects from the interferon and / or concomitant immune suppression. Even more difficult are the unanswered questions such as what is the best time frame to initiate interferon therapy, what is the best dose, and, most important of all, what is the best treatment duration of therapy. Although pretransplant hepatitis C patients have had large multicenter trials that have clearly outlined the optimal therapeutic dose and treatment duration for the various HCV genotypes, there are no FDA consensus recommendations for treatment of recurrent HCV post liver transplantation. The 48 weeks time period in the non-transplant population is the standard of care for genotype 1 patients.12, 13 However, this may not be the optimal treatment duration for a patient treated for recurrent HCV post liver transplant. Therefore, more accurate end-of-treatment predictors for SVR are needed.
Several investigators have reported that the use of HCV RNA detection in the liver tissue by RT-PCR may be a better predictor of treatment duration to help predict SVR than the conventional use of HCV RNA detection in the serum.7, 8 These studies, however, were done in the nontransplant population and needed to be confirmed in patients treated for recurrent HCV post liver transplant. A potential downside to this approach is the requirement for serial liver biopsies to obtain specimens for analysis by RT-PCR. This problem is not so difficult in the liver transplant setting because many of the patient undergo repeat liver biopsies both as part of the posttransplant follow-up protocol and to investigate abnormal liver function tests and to rule out the presence of acute cellular rejection. We, therefore, wanted to examine whether this approach would be more accurate in prediction duration of treatment in our patients with recurrent HCV post liver transplant in comparison to standard HCV RNA detection in the serum by RT-PCR. The results of this current study demonstrated that only a small group (10/57; 17%) of our patients achieved an end-of-treatment virological response after 48 weeks of combination therapy with peginterferon alfa-2b and ribavirin. An even smaller group (3/57) was also HCV RNA nondetectable in their liver tissue by RT-PCR determination. Interestingly, all patients in this group with nondetectable HCV RNA in their liver tissue were also HCV RNA negative in their serum by RT-PCR at the time of treatment completion. Those patients with positive HCV RNA in liver tissue and nondetectable HCV RNA in their serum all had recurrence of the HCV after withdrawal of treatment.
The difference in sensitivity between tissue and serum may become less important as more sensitive serologic assays become available. However, the marked difference in viral titer detected between tissue and serum in our work presented at AASLD 2001, suggests that viral titer, within the liver tissue is significantly higher and may continue to provide the most accurate prediction of treatment duration (Fig. 1). Conversely, it is not clear whether sample errors may be important, as biopsy specimens are small and often not consistent, unintentionally resulting in lab error.
We conclude that the results of this study identify a possible predictor for SVR in the post-transplant phase by using tissue HCV PCR. Although the results are from a small number of patients with end-of-treatment response and a large trial is needed to better confirm tissue PCR testing, we can optimistically predict an SVR if the HCV PCR IHT testing is negative. It is interesting to speculate that the direct HCV RNA detection by RT-PCR of liver tissue may guide longer therapy durations, resulting in improved SVR, especially in the patients with genotype 1.