Hepatitis C virus (HCV)–related liver disease is now the largest single indication for liver transplantation worldwide, and a growing disease burden is predicted over coming years. However, infection of the allograft is essentially universal, and it is often associated with accelerated disease progression, with recurrent cirrhosis occurring in approximately 30% of recipients within 5 years of transplantation. Transplantation for this indication is thus associated with reduced allograft and recipient survival,[3, 4] and this renders its outcomes inferior to those for transplantation performed for many other etiologies. A number of clinical risk factors, including increased levels of immunosuppression, increased donor age, posttransplant cytomegalovirus infections, and high levels of HCV viremia after transplantation, have been associated with more rapid disease progression.[5, 6] However, particularly in an age of increasing donor shortages, many of these factors are unmodifiable.
Despite the significant pathology associated with HCV recurrence after liver transplantation, outcomes can be highly variable, and the decision to proceed to antiviral therapy in an individual patient remains a difficult clinical challenge. Combination therapy with interferon-α (IFN-α) and ribavirin after liver transplantation is associated with response rates of generally less than 30% and entails the risk of inducing allograft rejection. In addition, toxicity-related dropout rates are high. In the longer term the development of IFN-free direct antiviral agent regimens may lead to significant improvements in our ability to treat this patient group. However, the availability of protease inhibitor therapy as an adjunct to IFN-based treatment presents not inconsiderable challenges, as evidenced by recent data indicating substantial morbidity in the treatment of individuals with decompensated liver disease before transplantation, and also because of issues of significant drug interactions with calcineurin inhibitors after transplantation.
In this setting, 2 significant issues have attracted much interest with respect to basic laboratory investigation within this field. First, the mechanisms underlying the accelerated recurrence of liver disease associated with HCV recurrence after transplantation remain poorly understood. Extensive research effort has thus been expended in gaining insights into these mechanisms, not only to inform future work into therapies to modify outcomes, but also to develop biomarkers predicting the development of aggressive fibrosis. The latter would be a major advance in guiding clinical decision making regarding the commencement of antiviral therapy. Second, the histopathological diagnosis of acute cellular rejection in the presence of recurrent HCV remains problematic: this is of critical importance because of the potential for increased immunosuppression to exacerbate HCV-related disease progression. Therefore, the development of biomarkers indicating the presence of acute cellular rejection in individuals with HCV recurrence has been an important experimental focus.
In order to explore these important areas, analyses have been performed at the genetic, transcriptomic, and proteomic levels. With respect to factors associated with rapid fibrosis progression, recent genetic analysis has indicated that the genetic cirrhosis risk score, a 7-gene signature predicting the development of progressive HCV-related liver disease promulgated in the non-immunosuppressed setting, is also predictive in HCV-infected liver transplant recipients. Among other genetic variations, the role of interleukin-28B polymorphisms has also been examined, although overall results remain inconclusive.[12, 13] Microarray analysis of allograft biopsy samples has also been applied to profile differential messenger RNA (mRNA) expression associated with variations in fibrosis progression rates, and this has implicated roles for genes related to myofibroblasts and stellate cell activation as well as genes related to infection, cellular development, and antigen presentation. A recent analysis of allograft transcriptomes has demonstrated characteristic mRNA expression patterns emerging within the first few months before the development of advanced fibrosis on biopsy or clinical disease progression, with changes occurring in multiple pathways. Serum biomarkers have also been sought, and a number of potential markers associated with rapid fibrosis progression have been identified.[17, 18] More recently, a proteomic approach has been applied, with recipients with rapid fibrosis progression demonstrating differential expression of proteins in a variety of immune, hepatoprotective, and fibrogenic pathways. An analysis of the serum metabolome was found to be consistent with this and suggested a role for oxidative stress in rapid fibrosis progression.
Transcriptional analysis has also been applied to explore potential pathways defining the presence of acute cellular rejection in the setting of recurrent HCV after liver transplantation. Microarray analysis has identified differential expression of genes associated with a variety of pathways, including apoptosis, as potentially distinguishing the presence of acute cellular rejection. Recently, a 15-gene set has been developed from microarray analysis to predict the presence of acute cellular rejection, although its sensitivity in a validation cohort was poor.
Despite hints derived from genomic, transcriptional, and proteomic analysis, much remains unknown about the mechanisms of disease progression in HCV after liver transplantation. In particular, reliable predictive biomarkers remain to be developed. There has been much recent interest in the exploration of microRNA (miRNA) expression to delineate disease mechanisms. These small noncoding RNA molecules bind to mRNAs and thereby modulate gene expression at the posttranscriptional level; individual miRNAs may regulate multiple gene products and thus diverse molecular pathways. Of particular interest for the development of disease biomarkers, miRNAs may be incorporated into exosomes, and hence their identification within the circulation may serve as a marker of events in tissues such as the liver.
The exploration of miRNAs in post–liver transplant HCV recurrence remains in its relative infancy. In a recent study, Farid et al. explored liver and serum expression of 3 hepatocyte-abundant miRNAs and detected elevated serum levels in ischemic liver injury and acute rejection. However, such focused analysis does not allow the exploration of less abundantly expressed miRNAs, which may, however, be important in disease pathogenesis. Two recent studies have analyzed miRNA expression in this setting in greater depth with microarray technology.[23, 24] In the current issue of Liver Transplantation, Joshi et al. analyze liver miRNA expression in carefully matched cohorts of individuals who previously had undergone transplantation for HCV-related liver disease, comparing those with slow versus rapid fibrosis progression, individuals with acute cellular rejection, and control subjects without viral hepatitis. A clear segregation of miRNA expression patterns was seen for all 4 groups. A pathway analysis that compared subjects with slow fibrosis and subjects with rapid fibrosis revealed differences in miRNA expression influencing antifibrotic, antiangiogenic, anti-inflammatory, and antiapoptotic pathways. A comparison of liver miRNA expression in individuals with rapid fibrosis progression and subjects with acute rejection also revealed disparities, with highly significant differences seen in 2 networks, one associated with insulin-like growth factor 1 receptor expression and another with proangiogenic pathways associated with the expression of vascular endothelial growth factor A. Intriguingly, an analysis of serum indicated differential expression of miRNA-19a and miRNA-20a between the rapid and slow fibrosis progression groups and revealed a potential noninvasive biomarker.
These results identify a number of potential pathways for further exploration with respect to the pathogenesis of recurrent HCV after liver transplantation, as well as potential biomarkers for aiding in the identification of rapid fibrosis progression and acute cellular rejection in this setting. However, some caveats concerning these data do arise. Analyses of individuals with slow or rapid fibrosis progression were performed with protocol liver biopsy samples taken 1 year after transplantation, at which time fibrosis progression was already well established. Another recent study has addressed this potential issue by the inclusion of serial samples, with earlier time points analyzed. In that study, a 9-miRNA signature was delineated that could potentially identify individuals at risk of rapid progression early after liver transplantation. Unsurprisingly, miRNAs regulating a number of similar pathways, including fibrosis, were identified in both studies. Interestingly however, only 2 miRNAs were apparently observed to be differentially expressed in common between the 2 studies.
A further potential limitation of the study by Joshi et al. is that analyzed biopsy samples from individuals with histologically diagnosed acute cellular rejection were obtained at the time of suspected acute rejection, and these were compared to protocol liver biopsy samples taken at 1 year from individuals with rapid fibrosis progression. It is thus not clear whether the observed changes in miRNA expression predict the development of a specific injury phenotype, or simply are the result of established differing patterns of injury within the allograft. To further explore the potential for the identified differentially expressed miRNAs to be used as biomarkers, further comparisons of serial samples taken from individuals during similar phases after transplantation will be required, and this will need to include early sampling before established injury.
It is to be hoped that further studies of miRNA expression in recurrent HCV after liver transplantation will help to clarify potential inconsistencies between the few studies that have been performed and to identify further pathogenic pathways of interest. Certainly, despite the aforementioned caveats, this nascent area of research possesses considerable potential to yield significant insights into the pathogenesis of aggressive recurrent liver disease in individuals undergoing liver transplantation for HCV. In addition, it holds the prospect of candidate biomarkers that can enhance our ability to more definitively identify the presence of acute cellular rejection and perhaps to predict the rapid progression of recurrent disease.