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Keywords:

  • Dual grafts;
  • hepatitis C;
  • IL28B;
  • liver transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

IL28B genetic polymorphism is related to interferon-sensitivity in chronic hepatitis C, but the significance of grafts carrying different genotypes from recipients is still unclear in liver transplantation. A 51-year-old Japanese male carrying a minor genotype underwent dual liver transplantation for liver cirrhosis due to hepatitis C virus (HCV). The left lobe graft carried a major genotype, and the right a minor genotype. He achieved virological response during the course of pegylated-interferon and ribavirin therapy against recurrent hepatitis C for 2 years, but HCV relapsed immediately at the end of the therapy. Two years after antiviral therapy, liver biopsy was performed from each graft. The specimens showed A1F0 in the left lobe graft and A2F2 in the right. Moreover, quantitative polymerase chain reaction was performed using RNA extracted from each specimen to see there was no HCV RNA in the left lobe whereas there was in the right. This case provides clear evidence that IL28B genetic variants determine interferon sensitivity in recurrent hepatitis C following liver transplantation, which could result in new strategies for donor selection or for posttransplant antiviral therapy to HCV positive recipients.


Abbreviations: 
HCV

hepatitis C virus

LT

liver transplantation

PEG-IFN

pegylated interferon

RBV

ribavirin

LDLT

living donor liver transplantation

SVR

sustained virological response

qRT-PCR

quantitative reverse transcription-polymerase chain reaction

SLV

standard liver volume

VR

virological response

AST

aspartate aminotransferase

ALT

alanine aminotransferase

ISG

interferon stimulated gene

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

Hepatitis C virus (HCV) and its related diseases are the leading cause of liver transplantation (LT) worldwide (1). Despite LT, graft reinfection by HCV occurs in almost all cases following transplantation and the outcome of posttransplant antiviral therapy is very poor (2). Although the combination of pegylated interferon and ribavirin (PEG-IFN/RBV) is the standard antiviral therapy for HCV, it is expensive and has some side effects such as inducing a flu-like syndrome, pancytopenia and autoimmune hepatitis, which can be extremely detrimental to an individual after LT. Precise prediction for IFN sensitivity in recurrent hepatitis C after LT is urgently required.

The IL28B genetic polymorphism has been reported to be significantly related with the outcome of PEG-IFN/RBV therapy for chronic hepatitis C (3). A low virological response rate has been demonstrated in patients who carry the minor genotype at rs8099917 (TG or GG), located approximately 8000 bp downstream from the IL28B gene, compared with those who carry the major genotype (TT). Recently, we demonstrated that this genetic variant was also related to the outcome of posttransplant PEG-IFN/RBV therapy for recurrent hepatitis C (4). The sustained virological response (SVR) rate is relatively high in recipients carrying the major genotype with grafts from donors carrying the major genotype. No recipients carrying the minor genotype achieved a SVR with a graft from a donor carrying the minor genotype. However, accumulation of a greater number or deceased-donor cases is necessary because that study included less than a 100 living-donor cases. Many factors other than IL28 genetic variants could affect the outcome of antiviral therapy, such as age, sex, fibrosis and viral mutations (5–7). The significance of the graft from donors carrying the major genotype in IL28B for the recipients carrying the minor genotype is not yet fully understood.

We previously reported the case of a living-donor liver transplantation (LDLT) using dual grafts from two donors (8). The recipient carrying the minor genotype (T/G) recently had liver biopsies performed from each graft after PEG-IFN/RBV therapy for recurrent hepatitis C. The donor of the right lobe carried the minor genotype (TG) at rs8099917, but the donor of the left lobe carried the major genotype (TT).

In this paper, we describe the outcomes of two liver grafts with different IL28B genetic variants in one individual.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

DNA extraction and direct sequencing

The recipient's and each donor's DNA was extracted from their exenterate liver tissues at transplantation and direct sequencing was performed using a BigDye Terminator v1.1 Cycle Sequence Kit (Applied Biosystems Inc., Tokyo, Japan). The PCR primers used to detect rs8099917 were 5′-CTT CTG CAA CAA ATC GTC CC-3′ (sense) and 5′-AGG AGC TTG CAC TAG CTC TT-3′ (antisense).

RNA extraction and quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from a piece of each donor's liver biopsy specimens using ISOGEN (Nippon Gene, Tokyo, Japan) and qRT-PCR was performed using TaqMan EZ RT-PCR Core Reagents (Applied Biosystems Inc.). A TaqMan probe (5′-CTG CGG AAC CGG TGA GTA CAC-3′) and specific primers (sense, 5′-CTG CGG AAC CGG TGA GTA CAC-3′; antisense, 5′-CAC TGG GAA GCA CCC TAT CA-3′) were used for quantification of HCV RNA.

Definition of viral response and liver histology

The SVR was defined as the absence of HCV RNA by qRT-PCR (PCR Cobas TaqMan system; Roche Diagnostics) at the end of the treatment and 24 weeks after the completion of therapy. Virological response (VR) was defined as the absence of HCV RNA as well as whether or not relapse occurred. The grade of inflammation and the stage of fibrosis were defined according to the Metavir grading score (9).

A Case Report

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

A 51-year-old Japanese male was referred to our department as an LDLT candidate. He suffered chronic hepatitis C (genotype 1b) and had failed two previous course of PEG-IFNα2b and ribavirin therapy. LDLT was performed using dual grafts from two donors. The left lobe was from his 21-year-old son and the right lobe from his 42-year-old wife, as previously described (8). Each HLA type is as follows: recipient; A11 A24 B35 B35 DR4 DR12, his son; A24 A31 B35 B59 DR4 DR12 and his wife; A24 A26 B35 B59 DR4 DR9. Both grafts were implanted by anastomosis of hepatic veins, portal veins and arteries from each side. The bile duct reconstruction was carried out by a duct–duct method for the right lobe graft and by a Roux-en Y hepatojejunostomy for the left lobe graft. The clinical course after transplantation went on without major surgical complications. Immunosuppression was induced by mycophenolate mofetil (Cellcept®; Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) with basiliximab (Simulect®; Novartis Pharma, Basel, Switzerland) and maintained with tacrolimus (Prograf®; Astellas, Tokyo, Japan), the dosages of which were adjusted to trough concentrations of 5–10 ng/mL.

His clinical course is summarized in Figure 1. HCV reinfection was detected 4 weeks post-LDLT and the viral load sharply increased up to 6.7 log IU/mL at 9 weeks post-LDLT. PEG-IFN/RBV therapy commenced 15 weeks post-LDLT with a weekly dose of 60 μg PEG-IFNα2b (Pegintron®; Schering-Plough Inc., Kenilworth, NJ, USA) and 600 mg RBV (Rebetol®; Schering-Plough Inc.) daily. The viral load decreased steadily and he achieved VR 60 weeks after the start of therapy. But 24 weeks later, HCV RNA was detected again and PEG-IFNα2b was switched to PEG-IFNα2a (Pegasys®; Hoffman-La Roche Inc, Switzerland) according to the report from Sherman et al. (10). However, his liver function gradually worsened and PEG-IFN/RBV therapy was terminated 96 weeks after commencement. Although the viral load increased sharply up to 6.0 log IU/mL, his liver function did not worsen with only liver supporting therapy. Seventy-two weeks after PEG-IFN/RBV therapy was completed, which was 4 years post-LT, liver biopsies were performed on each graft.

image

Figure 1. The clinical course after LT: the current case achieved transient VR, but did not achieve SVR with posttransplant PEG-IFN/RBV therapy for recurrent HCV.

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Prior to biopsy, the recipient's DNA was extracted from his exenterate liver tissues at transplantation and direct sequencing was performed to reveal that he carried the minor IL28B allele (rs8099917). Each donor's DNA was extracted from the liver biopsy at transplantation, and his son was identified as carrying the major genotype (TT), whereas his wife was identified as carrying the minor genotype (TG) (Figure 2). The participants of these studies were fully informed, and this work was approved by the ethical committee of Kyushu University.

image

Figure 2. Direct sequencing demonstrated that the recipient and the right lobe graft donor carried the heterozygous (T/G) allele at rs8099917 (indicated by red arrow), and the left lobe graft donor carried the major homozygous (T/T) allele.

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The histological findings of each specimen were completely different (Figure 3). The left lobe from the donor with the major genotype in IL28B displayed only mild hepatitis and no fibrosis with a Metavir grading score of A1F0. The right lobe from the donor with the minor genotype demonstrated moderate inflammation and bridging fibrosis (A2F2). Preserved bile ducts and a lack of endotheliitis were observed in both specimens, therefore the possibility of chronic rejection was denied. Moreover, qRT-PCR using total RNA extracted from each specimen revealed that no HCV RNA could be detected in the left lobe, whereas 15.3 copies/μg of HCV RNA were detected in the right lobe. Virological outcomes were also different in each graft, and these findings can possibly be explained by the different genotypes of IL28B.

image

Figure 3. The liver biopsies from each graft at 4 years post-LT: (1) the left lobe graft from the recipient's son carrying the major genotype had nine portal areas and demonstrated mild chronic inflammatory infiltrates in the portal areas and lobules, accompanied by mild lobular hepatitis without fibrosis. Moderate macrovesicular and microvesicular steatosis was seen. The number of bile ducts was preserved, and endotheliitis was not observed. These features indicated chronic hepatitis, A1F0, RAI P1B0V0. (2) The right lobe graft from the recipient's wife carrying the minor genotype had three portal areas and displayed moderate chronic inflammatory infiltrates in the portal areas and lobules, accompanied by interface hepatitis and bridging fibrosis. Endotheliitis was not observed. These features were indicative of chronic hepatitis with A2F2 and RAI P1B0V0.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

There is no longer any doubt regarding the significant association of IL28B genetic variations with the outcome of IFN therapy for HCV (3). We previously demonstrated that both IL28B genetic variants in recipients and donors are important in posttransplant IFN therapy, suggesting that both hepatocytes and host immune cells were associated with the relationship between IL28B genetic variants and IFN sensitivity (4). However, in cases of LDLT, many donors carry the same genetic traits as recipients. In addition, several factors other than IL28B genetic variants could influence IFN sensitivity. The significance of the grafts from donors who carry different genotypes in IL28B from the recipients is not fully understood.

In the current case, dual liver grafts from two donors carrying different IL28B genetic variants were transplanted, and specimens of liver biopsies from each graft at 2 years posttransplantation and post-PEG-IFN/RBV therapy were completely different. The left lobe graft showed displayed A1F0, which was from the recipient's 21-year-old son carrying the major genotype in IL28B, whereas the right lobe graft exhibited A2F2, which was from the recipient's 42-year-old wife carrying the minor genotype. The qRT-PCR assays revealed HCV RNA only in the graft carrying the minor genotype. It could never be concluded that left lobe graft really achieved ‘SVR’ because the amount of RNA extracted from each specimen was as low as about 500μg. In addition, the amount of HCV-RNA isolated in the right lobe is very low considering the high viral load in the serum, though that is inconsistent to Ramirez et al. (11). This is also due to the low amount of total RNA extracted from limited biopsy sample. We have priority over the pathological diagnosis, not over the research. We never try to perform liver biopsy again for just sampling considering patients’ safety. With limited samples, we believe that, at least, there was the difference in viral load between two grafts in addition to the histological changes 4 years after transplantation. Recently, the association of IL28B genetic variations with not only treatment-induced but spontaneous clearance of HCV was reported (12), suggesting IL28B major genotype possibly had some protective function against HCV and led to prevent the graft from the progression of fibrosis even after PEG-IFN/RBV treatment for 2 years.

The correlation of the molecular mechanism between IL28B genetic variants and IFN sensitivity has never been clarified in detail. The key role of hepatocytes or infiltrating host immune cells, is of great interest for posttransplant IFN therapy. Honda et al. (13) reported correlation between IL28B genetic variants and pre-treatment hepatic interferon stimulated gene (ISG) expression. Several reports have demonstrated that responders to IFN therapy have pretreatment low ISG expression in hepatocytes and high ISG expression in Kupffer cells (14,15). In transplanted liver grafts, Kupffer cells, although they are also called residential macrophages, are from recipients because their duration of life is a few months at longest, whereas hepatocytes are from donors. IL28B genotypes of both donors and recipients are still thought to correlate with hepatic ISG expression interacting each other. Sarasin-Filipowicz et al. (14) also demonstrated that hepatic ISG could not be induced above the pretreatment level by IFN therapy in patients with pretreatment high hepatic ISG expression, resulted in the lack of a VR. This suggests that the IL28B minor genotype may have some form of desensitization effect in IFN therapy in the liver by inducing pretreatment high hepatic ISG expression. On the other hand, ISG15, one of the most strongly induced ISGs, had been recently demonstrated to have a proviral function for HCV (16). Still, little is known about the relationship between IL28B genetic variants and cell-specific ISG expression in the liver. The reason why these alterations in ISG expression cause varying outcomes in IFN therapy requires further investigation. The current case, at least, strongly suggested that hepatocytes play a key role in the correlation between IL28B genetic variants and IFN-sensitivity.

All host and viral factors, other than grafts, that may influence IFN sensitivity are exactly the same in the current case. The different histological findings and viral replication of each graft suggests that IL28B genetic variant is a significantly important factor for IFN sensitivity. It is unknown whether only IL28B genetic variants affected both the histological and virological outcomes seen in the current case. Inflammation and fibrosis of the right lobe graft may have been caused by chronic rejection because of an unrelated blood donation. However, the preserved bile ducts and lack of endotheliitis seen in both grafts denies the possibility of chronic rejection. Cholestasis was never seen in both grafts. Biliary strictures following LT have been reported not to be associated with the type of biliary reconstruction; duct-to-duct or hepatojejunostomy (17). No report has demonstrated the association of the type of reconstruction with liver fibrosis. Females are widely shown to be a good prognostic factor for IFN sensitivity in chronic hepatitis C patients (5). However, Cescon et al. (18) reported that there was no relationship between gender of the donor and the SVR rate of posttransplant IFN therapy against recurrent hepatitis C. Wali et al. (19) described advancing donor age as a powerful determinant of rapid fibrosis after LT for hepatitis C, and several reports also described donor age as a predictor for the outcome of posttransplant anti-HCV therapy (18). However, these reports described that donor age of just more than 50 or 60 was unfavorable predictor for IFN outcome and progression of fibrosis. Moreover, in a recent report, Jimenez-Perez et al. (20) described that donor age was not the predictor for SVR after LT. It could be never concluded in the current case that the difference of ages between 21-year and 42-year resulted in different graft histological changes.

In conclusion, this is the first report of two livers with different IL28B genotypes demonstrating different outcomes of posttransplant IFN therapy for recurrent HCV in one individual. Although the mechanism of cell-specific antiviral potential associated with IL28B genetic variation is yet to be determined, the current case suggests the significance of hepatocytes carrying the major genotype in IL28B for IFN therapy against HCV. This could result in new protocols for recipients with HCV-related liver diseases. In recent future, grafts from donor carrying IL28B major genotype might be transplanted to HCV positive recipients, if they have several donor candidates and have enough time to check the genotype. In cases of unfavorable genotype donors, new strategies such as HCV protease inhibitor would be proposed.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors of this manuscript have no funding source regards to this report.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. A Case Report
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References