Liver transplant complications in hepatitis C infected recipients: recurrence versus rejection

Abstract

Despite improvement on outcomes post liver transplantation (LT), complications such as HCV recurrence (HCV-rec) and acute cellular rejection (ACR) continue to be a challenge for transplant physicians. Accurate diagnostic tools to better dissect between those complications post-LT are crucial for prompt and correct diagnosis and treatment. It is well known that the overlapping features of clinical and histo-pathological characteristics between these conditions turn difficult the appropriate differential diagnosis. Recently, new technological advances had supported the field of biomarker discovery in many diseases. Disease biomarkers capable to differentiate ACR versus HCV-rec post-LT is a long waited task in the transplant community. This editorial describes and discusses potential biomarkers of disease differentiation including recent reports in the field of genomics, proteomics, immunohistochemistry among other technologies.

Keywords::

• acute rejection
• biomarkers
• gene expression
• HCV recurrence
• liver transplantation
• microarray
• miRNA

Accurate diagnostic tools that can better distinguish between hepatitis C virus recurrence (HCV-rec) and acute cellular rejection (ACR) post-liver transplantation (LT) are crucial for prompt and accurate treatment.

Although ACR and HCV-rec may have different onset periods [1], it is still a problem in the clinical setting as a consequence of overlapping clinical and histological features. Late ACR still is a frequent event in patients post-LT often associated with non-compliance, recipient age, primary biliary cirrhosis and previous graft loss [2]. The organ procurement and transplantation network (OPTN/HRTR) 2011 liver report [1] showed close to 20% of the ACR incidence at 1-year post-LT. Steroids are widely utilized for ACR treatment; however, its usage has been controversial in HCV-positive LT recipients [3]. Therefore, steroid-free immunosuppression protocols and monoclonal antibody-based immunosuppressant induction therapy have been intensely investigated [3,4].

Liver biopsy post-LT still remains as the ‘gold standard’ to diagnose ACR and to distinguish it from HCV-rec. Distinctive histological characteristics are described in the former study by Petrovic et al. [5], where histological features associated with ACR included bile duct injury with overlapping nuclei, lymphocytic infiltrates and necrosis, endothelialitis, portal inflammatory infiltrates containing eosinophils and polymorphonuclear leukocytes, hepatocyte mitoses and zone 3 canalicular cholestasis. In contrast, the only statistically significant feature associated with early HCV-rec was sinusoidal dilatation. Stepwise discriminant analysis showed that the presence of eosinophils in the portal inflammatory infiltrates, bile duct necrosis and bile duct lymphocytic infiltrates were independently associated with ACR. However, serial biopsy specimens from patients with recurrent HCV infection showed statistically significant progression in scores for portal inflammation, portal lymphoid aggregates and lobular inflammation. Issues associated with liver sampling and histological accuracy continue to be a challenge. Regev et al. [6] evaluated more than 100 LT biopsies conducted by five liver expert pathologists and concluded that the histopathological differentiation of HCV-rec and ACR post-LT had relatively low inter-observer and intra-observer agreement rates, showing concerning low reliability.

Disease biomarkers capable of differentiating ACR from HCV-rec post-LT are long awaited and identifying them is a challenging task in the transplant community. Perhaps the wisest strategy would be the result of a combination of histological, clinical assessments and molecular markers independently associated with each condition. In an effort to identify non-invasive biomarkers, Massoud et al. [7] performed a proteomic analysis in plasma samples from non-HCV LT recipients histologically diagnosed with ACR. Seven of the 41 proteins identified as differentially abundant (serum amyloid A, complement component 4 [C4], fibrinogen, complement component 1q, complement component 3, heat shock proteins 60 and 70) were measured with ELISA-based assays in a validation LT cohort consisting of patients with and without ACR. Furthermore, the combination of aC4 level ≤0.31 g/l and an ALT level ≥70 IU/ml together had a sensitivity of 96% and a specificity of 81%. Additionally, a report by Schmeding et al. [8] clearly demonstrated that humoral components, represented by C4d deposition, play an important role in ACR post-LT, and may consequently be helpful to distinguish between ACR and HCV-rec post-LT.

These findings were further supported by Jain et al. [9] in a study that aimed to examine the differences between distribution of CD4⁺, CD8⁺, CD56⁺ lymphocytes and C4d deposits in patients with ACR and HCV-rec post-LT. The authors found that mean CD4⁺, CD8⁺ and CD56⁺ lymphocytes were similar for ACR and HCV-rec. Moreover, 80% of patients with ACR showed moderate-to-strong staining for C4d and all HCV-rec patients showed none-to-mild C4d staining. Furthermore, Schmeding et al. [10] evaluated the role and potential value of C4d as a diagnostic marker in a prospective analysis by ELISA measurement in cryopreserved liver biopsies of LT patients, and noticed that C4d concentration may not be determined using ELISA-based tissue evaluation. In contrast with previous reports, Fayek [11] studied 70 liver tissues in ‘for-cause’ biopsies for C4d deposition. The author reported that C4d staining was not specifically associated with ACR and did not differentiate it from HCV-rec despite that it was associated with a trend toward poorer outcome.

Nowadays, the most common technique used for C4d detection in liver tissue is immunohistochemistry (Autostainer Link48, Dako Inc., Denmark), which requires 24–36 h since tissue harvest. Other molecular markers assessed by RT-qPCR technique may require less time (24 h) and may soon become more suitable for utilization in clinical practice.

Reported immunohistochemistry-based studies include myxovirus-resistance A protein expressed in hepatocyte and macrophages, and minichromosome maintenance protein-2 (Mcm-2) expressed in liver-parenchymal infiltrated lymphocytes. Increased hepatocellular myxovirus-resistance A expression was strongly associated with HCV-rec [12], whereas the increased number of Mcm-2-positive portal tract infiltrating lymphocytes was associated with ACR, suggesting that lymphocyte Mcm-2 expression is a useful adjunct to histology in differentiating between these entities [13].

Cabrera et al. [14] tested blood samples from 42 LT HCV patients with abnormal transaminases, with the commercially available immune function test (ImmuKnow^®, Cylex, Inc., Columbia, MD, USA) to differentiate ACR and HCV-rec. The report showed that in patients with histological evidence of ACR, the immune response was noted to be very high, whereas in those with active HCV-rec, the immune response was found to be very low. This finding suggested differential immune activation in each entity.

More recently, the advent of microarray technology has allowed researchers to explore and better understand molecular profiles and regulatory mechanisms associated with determined pathological conditions. Gene expression analyses demonstrated that deregulation of molecular pathways involved in inflammatory responses, apoptosis and complement system were found to be ACR specific over HCV-rec [15]. In a recent study, we identified from a cohort of 53 LT patients a signature of 71 differentially expressed genes, 59 genes specifically associated with HCV-rec and 12 genes uniquely associated with ACR. More importantly, the particular biological analysis of both gene sets strongly linked HCV-rec profile with T-cell-mediated hepatocyte cytotoxicity involving IFN-γ and NF-κB signaling pathways, while ACR genes were associated with immediate hypersensitivity reaction inducing graft damage mediated by eosinophil and neutrophil cell degranulation [16]. Previously, Sreekumar et al. [17] studied a small number of HCV LT patients with and without steroid-responsive ACR. Microarray analysis identified 25 genes relatively overexpressed, and 15 genes relatively underexpressed by more than twofold in the ACR when compared with the HCV-rec group. The authors noted that ACR was most notably associated with the relative overexpression of genes associated with MHC I and II, IGF-1 expression, apoptosis induction and T-cell activation.

Asaoka et al. [18] tested 22 LT biopsies from 21 HCV LT patients. Samples included nine biopsies with and 13 without histological evidence of ACR. A total of 2206 genes were significantly modulated in ACR. The study results suggested a novel transcriptome patterns for ACR with concurrent HCV infection, and that may be useful in the diagnosis of ACR in HCV LT recipients.

Following a similar analytical and biological rationale, miRNA had also been the focus of investigation. In an independent study, Joshi et al. [19] introduced the importance and potential role of miRNAs in the discrimination of these two conditions. LT patients with only HCV-rec (n = 9) and HCV with ACR (n = 5) were studied using microarray technology, the authors identified a profile of 190 miRNAs differentially expressed between conditions, mostly being associated with fibrogenesis through regulation of IGF-1 receptor and VEGFA-related molecular signaling pathways. However, as stated by the authors, a larger prospective cohort is needed to support this pioneer study. Recently, Farid et al. [20] investigated serum hepatocyte-derived miRNAs (HDmiRs) as markers of hepatic injury and ACR in LT. The authors reported that during episodes of ACR, serum HDmiRs were elevated up to 20-fold (miR-122, miR-148a and miR-194), and their levels appeared to rise earlier than aminotransferase levels, concluding that HDmiRs are promising candidate biomarkers of ACR and hepatic injury after LT.

As consequence of the availability of human genome data and the new technology supporting advances in genomics, proteomics and metabolomics techniques, translational research has recently presented attractive approaches for discovering biomarkers for health issues including transplantation. There is a clear need for better diagnostic and prognostic tools to monitor liver graft function and differentiation of complex conditions. An ideal biomarker would be non-invasive (e.g., serum biomarkers) and be able to predict outcomes and the severity of specific events such as ACR and HCV-rec before the damage is already evident at the histological level. Despite important advances achieved so far in the identification of several potentially useful biomarkers of ACR and HCV-rec, validation and demonstration of their clinical utility still needs to be tested. Unfortunately, studies correlating tissue and serum molecular biomarkers are lacking. Eventually in order to facilitate the accomplishment of such important goal, efforts should be made in the context of multi-institutional cooperative networks and transferring the results from high-throughput technology to limited ‘set of markers’ that can be tested easily and introduced in the clinical setting. Contrary to most of the described retrospective studies discussed here, powered studies will need to be prospective and to include training sets (for biomarker discovery in well-defined patients) as well as validation sets (independent group of patients with similar demographic and clinical characteristics) to evaluate accurate disease biomarkers for both disease diagnosis and prediction.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

No writing assistance was utilized in the production of this manuscript.