The persistence of liver disease related to HCV infection as the most common indication for LT, and recurrence of HCV infection as the most common cause of posttransplant graft loss and mortality, has resulted in a high level of interest in factors that affect posttransplant outcomes. This study of predictors of key outcomes after LT for HCV infection has several potentially important new observations.
A central aspect of our current analysis is that the predictive usefulness of IL28B genotype for SVR is independent of other known predictors of response, including viral genotype. We observed that the relative predictive usefulness of the IL28B genotype for SVR, although highly significant, is one quarter that of the HCV genotype. Our study also confirms the association of both R-IL28B and D-IL28B CC genotypes with SVR in a larger and more complete cohort than reported previously (14–18). It is important to consider how our findings compare with those of others. Our findings contrast those of Coto-Llerena et al. (18) who reported that, although recipients with a CC genotype demonstrated a higher frequency of SVR when compared with non-CC recipients, a significant difference was not observed when the donor genotype was assessed (41% and 28%, respectively, P=0.179). The magnitude of the effect of donor CC genotype is, however, similar to that observed in our study, suggesting the lack of significance may be based on power. Two other studies have noted a positive association between the D-IL28B favorable genotype and successful AVT (14, 17). On balance, in magnitude and direction of effect, both recipient and donor IL28B genotypes seem to influence the frequency of SVR, with the response being enhanced when both R- and D- have the favorable genotype.
The mechanism through which the IL28B SNP genotype influences response to AVT is yet to be determined and merits some consideration. IFN-λ3, the product of IL28B gene, belongs to the type III interferon family, causing induction of interferon-stimulated genes (ISGs), maturation and differentiation of dendritic cells, modulation of TH1 and TH2 immune responses, and inhibition of Treg cells (19–22). Thus, IFN-λ3 is a proinflammatory cytokine, linking innate and adaptive immune responses (23). Favorable IL28B SNPs are associated with decreased levels of intrahepatic ISG (24, 25). Lower pretreatment ISG levels are predictive of ultimate SVR in the nontransplant setting (26).
A further potentially important aspect of our study is the differential predictivity of donor and recipient IL28B genotype for key clinical and histologic outcomes. We observed that recipients with a CC genotype have relatively slower histologic recurrence, with decreased ALT levels and viral load when compared with non-CC genotype, with the opposite association for donor CC genotype. Others have reported a significantly lower ALT and viral load in association with recipient CC genotype and significantly higher ALT among CC genotype donors (15–17). The clinical relevance of this paradoxical influence of IL28B CC genotype according to origin was evident when assessing the histologic data at 1 year after LT, when it was seen that recipient CC genotype was associated with lesser degrees of fibrosis than non-CC recipients, and the converse for donors. The survival analysis was consistent with histologic results, with a negative association of donor CC genotype with progression to cirrhosis, liver-related death, and retransplantation. Because of the suggestion of proinflammatory roles for the CC genotype, we explored associations of IL28B with risk for ACR. Although a higher frequency of ACR was seen in association with donor CC, the difference was not statistically significant. Although ACR was predictive of fibrosis in univariate modeling, its usefulness was lost in the multivariate analysis. It is possible, however, that an independent effect of ACR on propensity for more advanced fibrosis would be seen in a larger study and that part of the risk for more advanced fibrosis associated with donor CC and recipient non–CC genotype is conferred through an increased risk of ACR.
Taken together, these results suggest that a more aggressive course of recurrence of HCV is associated with a donor CC genotype in patients who do not receive AVT. The overall superior posttransplant outcomes associated with donor CC genotype occur by virtue of greater frequency of attainment of SVR during AVT. Because of a substantially increased risk of more severe histologic recurrence and greater likelihood of responding favorably to AVT, it is particularly important that AVT is administered when possible to patients who receive an allograft from a donor with a CC IL28B genotype.
Studies of variation in fibrosis progression with IL28B genotype have been inconsistent. In the nontransplant setting, Marabita et al. (27) found no significant difference in the frequency of advanced fibrosis between CC, CT, and TT genotypes (26%, 25%, and 15%, P=0.46). A study including patients with HCV/human immunodeficiency virus coinfection observed a higher rate of cirrhosis in CC when compared with CT/TT (24% and 13%, P=0.01) (28). It is possible that the effect of IL28B on liver fibrosis is only observed in populations with accelerated fibrosis such as human immunodeficiency virus coinfection or LT. Two studies in patients who underwent LT have examined the possibility of associations of IL28B genotype and histologic outcomes. Eurich et al. (16), in a study in which only recipient IL28B genotype was determined, did not find significant differences in fibrosis progression between genotypes. Similarly, Lange et al. (17) did not observe associations with either recipient or donor IL28B genotype and cirrhosis. The authors noted, however, that they lacked power to detect a meaningful difference in histologic outcomes. There are other important differences between our present study and those of Eurich et al. and Lange et al. in design (biopsies being or not mandatory at every time point during follow-up), patient numbers (91 transplants in the study by Lange et al. vs. 272 in our study), IL28B genotyping method (rs8099917 vs. rs12979860), approach to AVT, and prevalence of other risk factors for fibrosis. In the study by Eurich et al., IL28B genotyping was performed only in recipients, making an interaction between donor and recipients IL28B genotype impossible to examine (the interaction was very important in our analysis).
The basis for a fibrosing phenotype in association with recipient and donor IL28B genotypes cannot, of course, be determined from our study. Greater expression of IL28B messenger RNA has been documented in both explanted and allograft livers with the favorable antiviral genotype (14). Persistence of CC genotype donor dendritic and lymphocytic cells within the allograft could theoretically create an immunologic environment favoring inflammation and fibrogenesis in the absence of viral eradication.
There are several limitations to our study, including the fact that it was not possible, for example, to characterize the IL28B genotype in 27% of our patients. There were, however, no baseline differences between patients who underwent LT with and without available IL28B genotype. In addition, the distribution of IL28B genotypes among donors showed a trend for deviation from the Hardy-Weinberg equilibrium. The C-allele frequency was similar, however, to that reported among other liver transplant recipient populations and is likely a reflection of an enrichment of nonresponders to AVT and failure to clear HCV during acute infection among liver transplant recipients.
In conclusion, the clinical evolution of posttransplant HCV infection varies with the origin of the CC genotype. We also confirm the favorable association of both recipient and donor CC genotype on response to AVT. In the absence of viral eradication, donor CC genotype is associated with a greater frequency of a composite endpoint including progression to cirrhosis, liver-related death, and retransplantation. It is particularly important that patients who receive an allograft from a donor with a CC IL28B genotype receive AVT. The interplay between donor and recipient genotypes is clearly complex in liver transplant recipients. Studies of the relative impact of donor and recipient IL28B genotype on ISG and IFN-λ1-3 expression are eagerly awaited.
MATERIALS AND METHODS
The study included patients with CHC who underwent LT between January 1995 and July 2010 at our center. All patients were followed up according to a standard protocol. DNA was collected from recipients and donors. Available liver biopsies included mandatory biopsies at day 7 and at 4 and 12 months after LT and yearly thereafter and those requested by the treating physician for clinical reasons. Quantification of HCV RNA in the serum was performed with COBAS AMPLICOR HCV Test, version 2.0 assay (Roche Molecular Systems). The study protocol was approved by the institutional review board of the Mayo Clinic.
DNA Extraction and IL28B Genotyping
DNA was extracted from stored paraffin-fixed liver tissue blocks using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) assay. Donor and recipient DNA was tested for the polymorphism rs12979860 using the ABI TaqMan allelic discrimination kit and the ABI7900HT Sequence Detection System (Applied Biosystems, Carlsbad, CA).
Hepatitis C Recurrence and Allograft Histology
Recurrent hepatitis C infection was defined as a panlobular or portal lymphocytic infiltrate, with detection of quantifiable HCV RNA in the serum, and in the absence of any alternative cause. All liver biopsies were reviewed for evidence of ACR (29). Fibrosis stage and inflammation grade were evaluated in a standard fashion at years 1, 3, and 5 after LT and at the last biopsy (if >5 years) (30).
A subset of patients received AVT with peginterferon ± ribavirin. In these, SVR was assessed and defined as undetectable HCV RNA at 24 weeks after the end of treatment.
A composite endpoint consisting of liver-related death and/or allograft failure (retransplantation or progression to F4) was used as the primary endpoint. Patients with death or retransplantation during the first 90 days after LT, hepatic artery thrombosis, or biliary strictures at anytime during follow-up were excluded.
Continuous variables are summarized as mean (SD) or median (interquartile range). Comparisons were made with t test and analysis of variance or Mann-Whitney U and Kruskal-Wallis tests according to the distribution of data. Categorical variables were compared by means of the chi-square test. Analyses were performed considering all genotypes for IL28B (CC, CT, and TT) and grouping them as favorable versus nonfavorable (CC vs. nonCC). Univariate (multiple linear, logistic, and Cox) regression analyses were applied to build final multivariate models assessing risk factors for time to recurrence of HCV, fibrosis progression, and SVR, respectively. Factors in the univariate analysis showing P<0.2 were entered in the multivariate analyses. The final model included the covariates with the best fit to the data. All statistical analyses were performed with Stata (version 9.2; Stata Corp, College Station, TX).
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Keywords:Copyright © 2012 Wolters Kluwer Health, Inc. All rights reserved.
Acute cellular rejection; Fibrosis; Histologic grade; Interferon λ; Survival; Sustained virological response