An ectopically expressed serum miRNA signature is prognostic, diagnostic, and biologically related to liver allograft rejection
Authors: James F. Trotter
Journal: Hepatology (2016)

As its efficacy has continually improved over the past three decades, liver transplantation (LT) has become an established therapy for selected patients with end-stage liver disease. Long-term survival rates are excellent, with approximately 60% of patients surviving beyond 10 years. In fact, there are more LT recipients alive than ever before, estimated at more than 65,000 by the Scientific Registry of Transplant Recipients.1 However, with this success comes new challenges. As more liver recipients survive well into the second decade, their exposure to immunosuppression and its side effects accumulate over the years. While required to sustain graft function, immunosuppression is also the source of complications primarily responsible for graft loss and death. Specifically, recurrent progressive hepatitis C, malignancy, and renal failure, all a direct result of immunosuppression, are the three greatest contributors to long-term graft loss.2 Consequently, one of the greatest challenges for transplant physicians is optimizing immunosuppression for individual patients. The goal is providing a sufficient level to prevent graft loss, but not so much as to create significant side effects, although the current means of accomplishing this is imprecise and slightly risky. Clinicians typically lower doses, usually to avoid side effects such as renal insufficiency, until either the liver function tests become elevated, an indirect indication of immune-mediated graft damage, or the side effect is mitigated. One potential means to meet this challenge would be studying operationally tolerant patients, that is, safely withdrawn from immunosuppression without graft injury, to discover genetic markers predictive of this phenotype in other patients.3 In fact, tolerant patients have identified gene expression profiles associated with natural killer and gamma-delta T-cell function as well as iron metabolism.4 While these genetic signatures could identify candidates for minimization of immunosuppression, none of these markers has yet proven clinical applicable. Another means to develop optimization of immunosuppression was undertaken by Shaked et al. in a report from the A-WISH study (Gradual Withdrawal of Immune System Suppressing Drugs in Patients Receiving a Liver Transplant) sponsored by the Immune Tolerance Network and the National Institute of Allergy and Infectious Diseases.5 They assessed outcomes of LT recipients who were studied during a carefully designed protocol of immunosuppression minimization. Liver biopsy and serum samples were acquired during different clinical phases of immunosuppression minimization: clinical stability, acute cellular rejection (ACR) as well as elevated liver function tests (LFTs) without ACR. Analyses of microRNA (miRNA), small noncoding RNA segments involved in RNA silencing and posttranscriptional regulation of gene expression, were performed in serum and liver tissue and correlated with clinical outcomes. Through complex hierarchical clustering and expression heatmap analysis, the identification of selected or clusters of miRNAs may potentially reflect the very earliest changes in the immune reaction to the transplanted graft, perhaps even before graft injury has occurred. In fact, similar studies in kidney and heart transplantation have previously been reported.6, 7 Using these carefully obtained samples, these investigators performed a series of studies. First, they analyzed paired biopsies and miRNA samples from 69 patients during 130 episodes of LFT elevations with or without rejection. They identified 31 miRNAs that were significantly associated with ACR. Ultimately, they found two miRNAs associated with ACR with a high degree of sensitivity (84%) and specificity (87%). Next, they evaluated another group of 27 patients who underwent immunosuppression minimization, during which blood samples were drawn at predetermined intervals. During minimization, some patients predictably developed LFT elevations attributed to ACR (or other causes). Their serum was analyzed to determine whether miRNA markers were present before rejection. In fact, the miRNA signature was identified up to 50 days preceding clinically evident ACR, suggesting that they may identify impending rejection before clinical evidence of allograft injury. Next, they provided evidence suggesting a mechanistic link to the transplanted hepatic graft, by associating the identified serum miRNAs to genes involved in immune/inflammatory and liver function. Finally, some of the messenger RNA targets of the miRNA were genes recognized to be active in rejection, including NR3C1 and PPP3CA. The conclusions of this study are potentially important and could yield obvious benefits in the management of LT recipients on several fronts. First, their results suggest that serum miRNAs could identify ACR without a liver biopsy. Consequently, these markers could supplant or, more likely, add to the diagnostic value of the current diagnostic gold standard for rejection, the liver biopsy. More important, immunosuppression could be personalized for individual patients by measuring miRNA during protocolized immunosuppression minimization. The identification of serum markers of rejection could predate clinical rejection thereby avoiding this problem altogether. Therefore, these findings could provide more-precise guidance of immunosuppression minimization, converting from its current state of a clumsy dose-finding expedition into more-exact science based on mechanistically linked molecular serum markers. However, there are important considerations that may temper these conclusions. The A-WISH cohort is a very highly selected group of patients. In fact, almost all LT recipients were excluded from the study. The excluded group of patients includes a wide spectrum of underlying conditions (approximate % of liver recipients) including: hepatitis C (50%); autoimmune disease etiology (10%); multiorgan recipient (8%); retransplantation (5%); fulminant liver failure (5%); hepatitis B (5%); and living-donor recipient (3%). Besides these underlying conditions, the patients had to survive for 1 year (excluding an additional 10% who died in that interval) and maintain a stable clinical course without significant renal impairment. Underscoring this point, the A-WISH trial enrolled patients for 10 years (between 2005 and 2015) at eight large transplant centers, and only 48 patients were ultimately entered into the minimization protocol. This would correspond to only 1 patient enrolled per center approximately every 2 years. Consequently, the cohort was ultimately composed of very stable, uncomplicated patients with alcoholic liver disease or cryptogenic cirrhosis. The restrictive exclusion criteria were intended to minimize the risk associated with immunosuppression minimization. In particular, hepatitis C patients were deemed at the highest risk, given that (before the advent of highly effective all-oral therapy) inducing rejection significantly increased the risk of graft injury or loss from severe hepatitis C virus recurrence. Autoimmune patients are typically younger, healthier, and more rejection prone during immunosuppression minimization. In fact, other studies of immunosuppression minimization found that the patients most successful in complete immunosuppression withdrawal were older, male, further removed from transplant, and without autoimmune liver disease.3 Given the very restrictive cohort included in the A-WISH trial, its findings may not be applicable to a more representative and diverse cohort of patients. The total number of patients in the A-WISH cohort is also relatively small. There were only 69 patients and the subgroup analysis including even smaller numbers of patients. The small "n" obviously decreases the power of these observations. Nonetheless, this study provides a provocative attempt at identifying molecular markers associated with rejection. These findings, as well as results from similar studies,4, 6, 7 could serve as a basis for trials to demonstrate their efficacy in immunosuppression optimization in LT. James F. Trotter, M.D. Baylor University Medical Center Dallas, TX