SEARCH

SEARCH BY CITATION

Keywords:

  • IL28B;
  • interferon treatment;
  • liver transplantation;
  • virological response;
  • waiting list

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

IL28B gene polymorphisms are associated with the response to antiviral therapy in hepatitis C patients. We investigated the influence of IL28B polymorphisms on the response to therapy before and after liver transplantation (LT). Genotyping of SNPs rs8099917 and rs12979860 was performed in 128 HCV-infected liver transplant recipients and in their donors; all patients underwent antiviral treatment after LT. The prevalence of genotypes rs12979860CC and rs8099917TT was higher in donors than in recipients (50% vs.19%, p < 0.001 and 67% vs. 38%, p < 0.001, respectively). Response to antiviral therapy was significantly higher for recipient genotype rs12979860CC as compared to rs12979860CT/TT both before (100% vs. 48% p = 0.013) and after LT (59% vs. 25% p = 0.002). The figures were almost identical for SNP rs8099917. Sustained virological response after LT was particularly high in patients with favorable recipient and donor genotypes (p < 0.01 for both SNPs). In a subgroup of 34 patients treated while awaiting LT, a favorable donor IL28B genotype was associated with an improved virological response after LT. Our results support a major role of recipient IL28B genotype in the response to antiviral treatment for hepatitis C recurrence. Interestingly, donor genotype also seems to influence the response pattern, especially in recipients who have a favorable IL28B genotype.


Abbreviations: 
AH

acute hepatitis

CH

cholestatic hepatitis

CyA

cyclosporine A

EVR

early virological response

FCH

fibrosing cholestatic hepatitis

TAC

tacrolimus

HCV

hepatitis C virus

LT

liver transplantation

PBMC

peripheral blood mononuclear cells

pegIFN

pegylated interferon

SNP

single nucleotide polymorphism

SVR

sustained virological response

VL

viral load

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

Liver disease caused by chronic hepatitis C virus (HCV) infection is the main indication for liver transplantation (LT) in the Western World and Japan. Currently, the available strategies to prevent HCV infection of the graft after LT remain limited. Hepatitis C recurs universally in patients with detectable HCV–RNA at the time of LT, and chronic hepatitis and cirrhosis develop in a significant proportion of patients over time (1). Treatment with pegylated interferon (pegIFN) plus ribavirin in patients in the waiting list is typically restricted to those individuals with less advanced cirrhosis (Child A and B) and it is fully effective in only around 20% of patients (2,3). Treatment after LT is only successful in one-third of treated individuals (4,5).

Formerly, it was generally assumed that in HCV-infected patients the pattern of response to antiviral therapy remained unaltered after LT. However, Feliu et al. showed that the sensitivity to interferon-based therapy may change following LT: some patients who were IFN plus ribavirin nonresponders prior to LT, became responders after LT and vice versa (6). Theoretically, such changes in the pattern response could be attributed to variations in viral quasi-species composition following LT and/or to the genetic background of the newly allocated grafts (7,8). Indeed, mutations in NS5a (ISDR, PKRbd and V3), HVR1 and core regions have been postulated to influence the response to interferon therapy (8). However, the predictive value of such mutations is still controversial. Host factors including genetics (e.g. polymorphisms in HLA, MAPKAPK3, etc), age, sex, race, obesity and the degree of fibrosis progression have also been associated with the response to pegIFN plus ribavirin therapy.

Three independent genome-wide association studies have recently identified several single nucleotide polymorphisms (SNPs) around the IL28B gene that are strongly associated with treatment outcomes in genotype-1 HCV-infected patients (9–11). Two of these SNPs are rs12979860, located 3 kb upstream of the IL28B gene and rs8099917, situated in the intergenetic region between IL28B and IL28A. The rs12979860 CC genotype has been linked to higher rates of spontaneous HCV infection resolution and SVR following antiviral therapy compared to the CT/TT genotypes in different cohorts of hepatitis C patients (9,12). In addition, the T allele of rs8099917 has been associated with SVR to antiviral treatment (10,11). On the other hand, the minor G allele has been shown to be over-represented among patients with chronic hepatitis C, compared with those experiencing spontaneous recovery and those who achieved an SVR (12).

Due to the aggressive course of hepatitis C recurrence in the wake of LT, it is very important to evaluate the potential influence of IL28B SNP genotypes in this population. Moreover, it would be worthwhile to determine whether IL28B polymorphisms are related to the observed changes in response patterns to IFN-based therapy (6). For these reasons, the aim of our study was to investigate the influence of IL28B polymorphisms on the response to therapy both before and after LT. In addition, we explored the possible relationship between the change of IL28B SNP genotype and changes in sensitivity to antiviral therapy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

Patients, antiviral treatment and response definitions

Between 2001 and 2009, 426 patients received a liver transplant in our institution due to HCV-related end-stage liver disease. Among them, 136 recipients underwent at least 12 weeks of antiviral therapy in our center to treat hepatitis C recurrence. A subgroup of them (43 patients) had received at least 4 weeks of combined antiviral treatment while awaiting LT. All patients provided written informed consent and our study was approved by the Ethics Committee of Hospital Clinic of Barcelona in accordance with the guidelines set forth in the 1975 Declaration of Helsinki.

Antiviral treatment in the pretransplant setting consisted of IFNα2b 3 MU/day or PegIFN α2a 180 mcg/week and ribavirin 800–1200 mg/day (3). Subsequent to transplantation, patients were treated with PegIFNα2b 1.5 mcg/kg/week and ribavirin 800–1200 mg/day based on body weight for 48 weeks. In both treatment courses, the dose of interferon or ribavirin was reduced when necessary and erythropoietin and granulocyte-colony stimulating factor were used as clinically indicated (2–4).

Due to the fact that sustained virological response (SVR) cannot be assessed by standard definitions in patients undergoing treatment while awaiting LT, we used early virological response (EVR, a decrease ≥2 log10 during the first 12 weeks of therapy) as a marker of response to therapy before LT. In the posttransplant setting, we assessed SVR, defined as negative viremia persisting 6 months after completion of therapy.

Genotyping of IL28B polymorphisms

Genomic DNA from LT recipients and their donors was obtained from peripheral blood mononuclear cells (PBMC) using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). In those cases in which PMBC were not available, genomic DNA was extracted from formalin-fixed, paraffin-embedded biopsies of liver explants (recipients) and grafts (donors) obtained at the moment of LT using the QIAamp DNA FFPE Tissue Kit (Qiagen).

Genotyping of rs8099917 was performed using TaqMan® Predesigned SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) as recommended by the manufacturer. The genotype of SNP rs8099917 was defined as TT, GT or GG. Genotyping of rs12979860 was performed using a 5′nuclease assay with allele-specific TaqMan probes, as previously described by McCarthy et al. (13). Rs12979860 was defined as CC, CT or TT. Genotyping was conducted in a blind fashion relative to sample origin (donor or recipient) and response to antiviral therapy.

Statistical analysis

Quantitative variables are expressed as median (range). For categorical variables, differences between groups were calculated by Fisher's exact test. For quantitative variables, differences between groups were analyzed using a nonparametric test (Mann–Whitney for unpaired groups and Wilcoxon for paired groups). A two-sided p-value of less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

Patient characteristics and antiviral treatment

Patient characteristics:  Among the 136 liver transplant recipients selected for this study, we excluded eight patients who were infected with genotypes 2 or 3. Thus, the final cohort was composed of 128 HCV genotype one-infected patients. IL28 genotyping was successful for both SNPs in 113 recipients, as well as in 96 and 99 donors for rs8099917 and rs12979860, respectively. Both donor and recipient genotypes were available in 93 patients for rs8099917 and in 96 patients for rs12979860. In the subgroup of patients that were also treated before transplantation (n = 43), two individuals were excluded for non-1 HCV infection and seven for sample unavailability. Therefore, this subgroup was finally comprised of 34 patients. Baseline characteristics of the patients included are shown in Table 1.

Table 1.  Baseline characteristics of patients at the start of antiviral therapy for hepatitis C recurrence after LT
 Whole cohort (n = 128)Patients treated before and after LT (n = 34)
  1. Quantitative variables are shown as median (range) and qualitative variables in n (%). VL, Viral load; AH, acute hepatitis; CH, cholestatic hepatitis; FCH, fibrosing cholestatic hepatitis; CyA, cyclosporine A; FK, tacrolimus.

Sex (males)87 (68)24 (70)
Age (years)58 (30–70)60 (36–67)
Baseline VL (log10 UI/mL)6.53 (4.48–9.03)6.71 (4.88–9.03)
Genotype (1a/1b)12 (9)/116 (91)4 (12)/30 (88)
Fibrosis stage
 F119 (15)4 (12)
 F239 (30)9 (26)
 F320 (16)1 (3)
 F45 (4)3 (9)
 AH15 (12)5 (15)
 CH/FCH30 (23)12 (35)
Time between LT and treatment14 (2–88)10 (2–31)
Duration of treatment (months)48 (12–72)48 (12–72)
Immunosuppression at treatment (CyA/TAC/antimetabolites)43 (34)/78 (61)/7 (5)13 (38)/20 (59)/1 (5)

Antiviral treatment before LT:  In those patients that received antiviral treatment prior to transplantation, therapy was initiated while awaiting LT and its median duration was 17 weeks (range 4–48). Twenty patients (59%) achieved an EVR, while four (12%) were serum HCV-negative at the moment of LT. We did not find any association between virological response and sex, type of interferon used, dose reduction, age, MELD score, baseline viral load or length of treatment.

Antiviral treatment after LT:  Antiviral treatment subsequent to LT was indicated in cases of acute hepatitis with significant necroinflammatory activity, cholestatic/fibrosing cholestatic hepatitis or when liver fibrosis was F ≥ 1 (4,5). Therapy was initiated at a median time of 13 months after LT (range 2–87) and its median duration was 49 weeks (range 12–72).

Eighty-four patients (66%) achieved an EVR, while SVR was achieved by 41 (32%) treated individuals. Among those 34 patients treated before and after LT, 23 (68%) achieved an EVR and nine patients (26%) finally attained SVR.

Distribution of IL28B genotypes in LT recipients and their donors

The distribution of IL28B polymorphisms is shown in Figure 1. The prevalence of rs12979860 CC, CT and TT genotypes in recipient samples was 19%, 62% and 19%, respectively, whereas rs12979860 CC was the most frequent genotype in donors (50%), followed by genotypes CT (43%) and TT (7%) (p < 0.001). Similarly, for SNP rs8099917, recipient samples were significantly enriched in the heterozygous genotype (GT, 50%), while the prevalence of rs8099917 TT exceeded 60% in donors (p < 0.001).

image

Figure 1. Frequency of the IL28B polymorphisms (A) rs1279860 and (B) rs8099917 in recipients and their corresponding donors. Numbers in parentheses indicate the number of patients.

Download figure to PowerPoint

Overall, SNP distribution differed significantly between recipients and their donors. The homozygous genotypes rs12979860 CC and rs8099917 TT, which are associated with SVR and spontaneous HCV clearance, were most frequently found in donor samples. These favorable genotypes were rarely recorded in recipients, in whom the heterozygous genotypes rs12979860 CT and rs8099917 GT were predominant.

Pattern of response to antiviral treatment

Consistent with previous data (9,10), virological responses (EVR and/or SVR) occurred more frequently in patients with either rs12979860 CC or rs8099917 TT genotypes (favorable) than in those with rs12979860 CT or TT and rs8099917 GT or GG (unfavorable). For this reason, we used a simplified model (rs12979860 CC vs. CT/TT and rs8099917 TT vs. GT/GG) in subsequent analyses. As shown in Figure 2A, in those patients who received antiviral therapy prior to LT (n = 34), an EVR was achieved in 100% of patients with genotype rs12979860 CC and in 48% of patients with rs12979860 CT/TT (p = 0.013). Similarly, EVR was significantly higher in patients with the rs8099917 TT genotype than in those with rs8099917 GT/GG (90% vs. 46%, respectively, p = 0.017).

image

Figure 2. Virological response before and after LT in association with IL28B genotypes in recipients and donors: (A) recipient genotype and EVR before LT, (B) recipient genotype and SVR after LT and (C) donor genotype and SVR after LT. IL28B genotyping was successful for both SNPs in 113 recipients, as well as in 96 and 99 donors for rs8099917 and rs12979860. Numbers in parentheses indicate the number of patients.

Download figure to PowerPoint

After LT, SVR was significantly higher in recipients with IL28 genotypes rs12979860 CC or rs8099917 TT, as compared with those carrying genotypes rs12979860 CT/TT or rs8099917 GT/GG (p = 0.002 and p = 0.009, respectively; Figure 2B). In regards to donor genotype, SVR was higher in recipients receiving grafts from rs12979860 CC and rs8099917 TT donors, although the association between the IL28B donor genotype and SVR following LT did not reach statistical significance (Figure 2C).

We also evaluated the combined effect of IL28 genotypes from recipients and their corresponding donors in response to treatment after LT. As shown in Figure 3, SVR was significantly increased when both recipients and donors carried the homozygous genotype rs12979860 CC (83%), compared with those recipients who either carried or received at least one unfavorable IL28B genotype (27%, p < 0.001). Similar results were obtained for SNP rs8099917: SVR was higher in TT recipients of TT grafts (56%) versus the other genotype combinations (26%, p = 0.006).

image

Figure 3. Combined analysis of IL28B genotypes from recipient and donor pairs versus SVR after LT. SVR was significantly increased when both recipients and donors carried the homozygous genotypes (83% for rs12979860 CC and 56% for rs8099917TT), compared with the other genotype combinations (27% and 26% for rs12979860 and rs8099917, respectively). Both donor and recipient genotypes were available in 93 patients for rs8099917 and in 96 patients for rs12979860, which explains the slight differences in the denominators between Figures 2 and 3. Numbers in parentheses indicate the number of patients.

Download figure to PowerPoint

Finally, we analyzed whether or not sensitivity to antiviral treatment changed following LT. For this purpose, we examined EVR in the subgroup of 34 patients who had received antiviral treatment before and after LT. The pattern of EVR remained unaltered after LT in 22 of the 34 patients (64%; Table 2). Sixteen of these 22 patients achieved an EVR during both treatment courses, while the remaining six did not respond either before of after LT. On the other hand, the response pattern changed in 12 patients (35%) after LT (Table 3): four EVR became non-EVR, whereas eight non-EVR then became EVR. These eight non-EVR before LT were not carriers of any of the favorable SNPs, though interestingly they became EVR after receiving grafts from either rs12979860 CC or rs8099917 TT donors (p < 0.001). However, only two of these patients achieved an SVR after LT.

Table 2.  Distribution of recipient and donor IL28B genotypes and pattern of response to antiviral therapy both before and after LT
SNP genotype Pattern of response1
SNPRecipient–donorNon-EVR/Non-EVRNon-EVR/EVREVR/Non-EVREVR/EVR
  1. 1The pattern of response was analyzed in a subgroup of 34 patients that had been treated both before and after LT: Non-EVR/Non-EVR, non-EVR before and after LT; Non-EVR/EVR, non-EVR before LT and EVR after LT; EVR/Non-EVR, EVR before LT and non-EVR after LT; EVR/EVR, EVR before and after LT; n, number of patients.

rs12979860CC–CCn = 40 (0%)0 (0%)0 (0%)4 (100%)
CC–CT/TTn = 30 (0%)0 (0%)1 (33%)2 (67%)
CT/TT–CCn = 161 (6%)6 (37%)2 (13%)7 (44%)
CT/TT–CT/TTn = 115 (46%)2 (18%)1 (9%)3 (27%)
rs8099917TT–TTn = 80 (0%)0 (0%)1 (12%)7 (88%)
TT–GT/GGn = 21 (50%)0 (0%)1 (50%)0 (0%)
GT/GG–TTn = 204 (20%)7 (35%)2 (10%)7 (35%)
GT/GG–GT/GGn = 41(25%)1(25%)0 (0%)2 (50%)
Table 3.  Recipient and donor IL28B genotypes versus changes in the response pattern to antiviral therapy both before and after LT
Pattern of response1 rs8099917rs12979860
RecipientDonorRecipientDonor
  1. 1The pattern of response was analyzed in a subgroup of 34 patients that had been treated both before and after LT. The response pattern changed in 12 patients after LT: EVR/Non-EVR, EVR before LT and non-EVR after LT; Non-EVR/EVR, non-EVR before LT and EVR after LT.

  2. Homozygous favorable genotypes (rs12979860 CC and rs8099917 TT) are highlighted in bold.

  3. n, number of patients; nT, total number of patients in the nonEVR–EVR group.

EVR/non-EVRn = 4GTTTCTCC
 TTTTCTCT
 TTGTCCCT
 GGTTTTCC
n = 2GTTTTTCC
n = 2GTTTCTCC
Non-EVR/EVR (nT= 8)n = 1GTTTCTCT
n = 1GGTTCTCC
n = 1GGGTTTCC
n = 1GTTTTTCT

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

Recent studies have provided genetic evidence for the role of IL28B in spontaneous and treatment-related recovery form HCV chronic infection (9–14). To date, however, there is little information on the influence of IL28B genetic variants of LT recipients and their donors in response to therapy either before or after LT. The LT setting offers a unique opportunity to study whether the genetic background of the graft can impact the response to antiviral therapy in previously treated patients. In the present study, we investigated not only the influence of IL28B polymorphisms on the response to antiviral therapy after LT, but also the possible association of donor and recipient IL28B genotype with a change in treatment response following LT. To this end, we genotyped two SNPs near the IL28B gene, rs12979860 and rs8099917, in a cohort of LT recipients (and their donors) infected with HCV genotype 1, who had been treated with pegIFN plus ribavirin for recurrent hepatitis C after LT.

The frequency of the homozygous genotypes rs12979860 CC and rs8099917 TT was significantly lower in recipients than in donors, indicating that the population of HCV-related LT recipients seems to have been enriched in heterozygous genotypes. This observation may be explained by the fact that a proportion of hepatitis C patients with the homozygous favorable genotypes might have either spontaneously cleared the virus or responded to antiviral therapy, thus ultimately being excluded from the final LT cohort. This hypothesis is in agreement with genetic data from recent studies reporting the critical role IL28B plays in both natural and treatment-induced control of HCV infection (9,12–15).

Consistent with recent studies, homozygous rs12979860 CC or rs8099917 TT genotypes were associated with a higher percentage of response to antiviral treatment than the other genotypes (10,15). In our study, we demonstrate that the recipient genotype was independently associated with SVR following LT. Although SVR was achieved in approximately 40% of patients receiving grafts from rs12979860 CC or rs8099917 TT donors, we did not find a significant association between the IL28B donor genotype and SVR (p = 0.179 and p = 0.171, respectively). However, the allocation of grafts with favorable genotypes in recipients with similarly favorable genotypes increased the rate of SVR significantly, indicating that the genetic background of the graft may exert a positive effect on sensitivity to treatment following LT. Two recent studies involving a LT setting reported that the IL28B polymorphisms in both recipients and donors were significantly associated with SVR (16,17). Increased expression of ISGs at baseline has been associated with nonresponsiveness to antiviral treatment of chronic hepatitis C (18). Recently, a link between unfavorable IL28B genotypes and a preactivated state of ISGs has also been reported (19). Although we could not measure ISGs expression in our cohort, we believe it is possible that ISG activation status in the donor graft may partially explain the synergistic effects that both donor and recipient IL28B genotypes have on SVR. Further studies should be performed in order to confirm this hypothesis. On the other hand, SVR was achieved in about 20–30% of recipient/donor pairs lacking any of the favorable genotypes, which clearly suggests that other host or virological factors are involved in the response to IFN-based treatment. In this regard, the presence of mutations in the core and NS5A regions has been shown to be an accurate predictor of the response to IFN therapy, not only in immunocompetent chronic hepatitis C patients, but also in those in a LT setting (20).

From a clinical point of view, it is interesting that the sensitivity to antiviral therapy changed in 12 of the 34 patients: four EVR became non-EVR, whereas eight non-EVR became EVR. Those eight patients, who were not carriers of any of the SNPs associated with response to therapy, achieved EVR after receiving grafts from rs12979860 CC or rs8099917 TT donors. Despite the small numbers and the fact that only two of these patients achieved SVR, our findings further support a potential influence of donor genotype in the treatment-induced clearance of HCV.

In conclusion, these data support that recipient IL28B genotype may play an important role in the successful response to antiviral treatment of HCV recurrence after LT. We also provide evidence that the allocation of grafts from donors with a favorable genetic background may improve sensitivity to antiviral therapy following LT. However, translating these results into clinical practice remains difficult, as the allocation of grafts according to IL28B genotype seems unfeasible and there is still too high a proportion of patients with unfavorable donor and recipient genotypes that respond to therapy. Nevertheless, determining donor and recipient IL28B genotypes before initiating antiviral therapy may be of importance, as patients can then be categorized in terms of those with the best chances of responding to therapy, thus assisting both patients and physicians in better making treatment decisions. Further prospective studies are needed to evaluate the predictive value of IL28B genotypes in response to combination therapies utilizing PegIFN plus ribavirin with novel small molecules that target the nonstructural proteins of HCV.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

We thank Dr. Dolors Colomer for assistance in IL28B genotyping and Patricia Gonzalez for technical assistance.

Financial Support

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

X. Forns received support in part by grants from the Instituto de Salud Carlos III (PI080239) and the VIRGIL European Network of Excellence in Antiviral Drug Resistance (LSHM-CT-2004–503359). The other authors were supported by grants from the following institutions: M. Coto-Llerena from the Ministerio de Asuntos Exteriores y Cooperación (Agencia Española de Cooperación Internacional), G. Crespo from Hospital Clínic (Ajut a la Recerca Josp Font) and the Fundación BBVA, and J.A. Carrión from the Fundación BBVA.

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. X Forns has grant support from Roche and Schering-Plough.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Financial Support
  9. Disclosures
  10. References