Volume 5 Supplement 1
Biomarkers of disease differentiation: HCV recurrence versus acute cellular rejection
© Gehrau et al; licensee BioMed Central Ltd. 2012
Published: 6 June 2012
The wound-healing process induced by chronic hepatitis C virus (HCV) infection triggers liver damage characterized by fibrosis development and finally cirrhosis. Liver Transplantation (LT) is the optimal surgical treatment for HCV-cirrhotic patients at end-stage liver disease. However, acute cellular rejection (ACR) and HCV recurrence disease represent two devastating complications post-LT. The accurate differential diagnosis between both conditions is critical for treatment choice, and similar histological features represent a challenge for pathologists. Moreover, the HCV recurrence disease severity is highly variable post-LT. HCV recurrence disease progression is characterized by an accelerated fibrogenesis process, and almost 30% of those patients develop cirrhosis at 5-years of follow-up. Whole-genome gene expression (WGE) analyses through well-defined oligonucleotide microarray platforms represent a powerful tool for the molecular characterization of biological process. In the present manuscript, the utility of microarray technology is applied for the ACR and HCV-recurrence biological characterization in post-LT liver biopsy samples. Moreover, WGE analysis was performed to identify predictive biomarkers of HCV recurrence severity in formalin-fixed paraffin-embedded liver biopsies prospectively collected.
Hepatic fibrogenesis is considered a model of the wound-healing response to chronic liver injury. This process is characterized by extra-cellular matrix (ECM) proteins accumulation as consequence of an imbalance between the deposition and degradation of ECM components [1–4]. Different stimuli such as cytokines and other extracellular signals, including reactive oxygen species (ROC) produced by parenchymal (mainly stellate cells) and non-parenchymal (mononuclear cellular infiltration) cells have been demonstrated to be involved in the fibrogenesis response to liver injury ( and references therein).
Unfortunately, HCV recurrence is universal. HCV RNA serum load dramatically decreases until almost undetectable level within 24-48 hours post-LT. However, it increments few days post-LT with a peak at 1-3 months, and reaching a 1-2 logs plateau higher than the pre-LT viral load after the first year post-LT . Of those patients, about 80-100% will develop HCV recurrence disease, and 25-30% of them will course with accelerated fibrosis progression and concomitant cirrhosis development within 5 years post-LT. These patients require liver re-transplantation or will develop liver failure . In parallel, acute cellular rejection (ACR) represents an additional cause of morbidity and allograft injury in HCV-infected recipients. Despite the rejection risk rate is controversial in HCV patients the incidence round 40% at 6-months post-LT as reported previously by large cohort studies . Both post-LT complications have clinical and histopathological overlapped features turning difficult the accurate differential diagnosis, which may threat the allograft and patient survival rate due to the opposite therapy [15–17].
The discovery of reliable biomarkers for differential diagnosis of both complications, and also for HCV recurrence disease surveillance constitutes the research endeavor. The implementation of whole-genome gene expression (WGE) studies using microarray technology represents an outstanding opportunity for biomarkers discovery. The present article is focused on performed WGE analysis aimed to differentiate ACR in the setting of HCV recurrence disease, and to predict liver fibrosis progression in HCV-infected recipients.
ACR and HCV recurrence differential diagnosis post-LT: the paradigm
The differential diagnosis of ACR in the setting of HCV recurrence disease remains an important cause of morbidity and late graft failure in liver-transplant recipients. The assessment of liver allograft biopsy is still considered to be the "gold standard" for proper differentiation between both post-LT conditions. However, subtle similarities in the histopathology and clinical course turn difficult and uncertain the pathological differentiation, even among experiences hepatologists [18, 19]. HCV recurrence disease have been described in three major forms: Acute or chronic recurrence and the less frequent and more aggressive primary cholestatic disease called fibrosing cholestatic hepatitis. All of these forms course with a characteristic lobular inflammatory cellular infiltrate . ACR occurs due to the attacks of the recipient immune system to the allograft and it is characterized by severe inflammatory infiltrate of the portal tract with the bile duct epithelial cells and the endothelium of hepatic arteries and veins as major targets. The appropriate clinical differentiation between both conditions directly impact in the associated therapy decision basically by the administration of steroid bolus for ACR treatment. Unfortunately, the ACR therapy results contradictory for HCV recurrence cases due to the induction of an exacerbation of the HCV infection accompanied by worse allograft and patient survival [16, 21].
The implementation of large-scale genomic analyses strategies has provided new insights into various disease processes and had assisted on elucidating genomic patterns for mechanism, diagnosis, prognosis, and treatment selection of complex and multi-factorial diseases . For instance, microarray technology represented a powerful tool for the molecular understanding and knowledge of involved gene networks and regulatory pathways for each particular condition.
In parallel, a LASSO model was fit with HCV recurrence vs. HCV-ACR as the dependent viable predicted, followed of N-fold cross validation to provide an unbiased estimate of generalization error. Interestingly, the best fitting-LASSO model included 15 genes with an accuracy of 100% for the training set, while the N-fold cross-validation accuracy was of 78.1%. Four out of those fifteen genes were also included into the exclusive gene list, and further validated in an independent set of 19 biopsy samples (validation set) .
The concluding results from the analysis of both experimental approaches using GeneChip® microarrays clearly demonstrate a differential gene expression signature between both conditions. Indeed, HCV recurrence disease is characterized by an adhesion and apoptosis of cytotoxic T cells profile regulated by canonical pathways related to IFN-γ and NFκB, while ACR is related to genes associated with an immediate hypersensitivity reaction . The histopathological features of both conditions are related with mottled hepatocytes apoptosis with a Th-1 type profiled lobular inflammation for HCV recurrence disease, and inflammation of the portal tract, bile ducts and hepatic vessel endothelium characterized by CD4+ and CD8+ T cells together with macrophages and eosinophil cells for ACR [16, 24, 25]. Thus, the genomic profiles identified by this study correlate well with the previously described cellular population that characterized both conditions [13, 23]. More importantly, it demonstrates the possibility to establish specific biomarker panels to be combined with the conventional histopathological assessment.
Prediction of fibrosis progression in HCV recurrence disease
Chronic HCV-related hepatic insufficiency is associated with decreased rates of patient and allograft survival in comparison with other indications for liver transplantation . Different factors inherent to the donor, recipient, and post-transplant variables have been associated with progression and severity of the liver allograft injury course in HCV recurrence disease [13, 27]. Nowadays any of those factors were established as independent predictors of HCV recurrence severity at early stage of the disease in HCV-infected recipients. For instance, it became critical the identification of reliable biomarker predictors of disease progression at the time of HCV recurrence diagnosis since the high variable aggressiveness of the disease.
Yet, the mechanisms involved in HCV recurrence development and progression are largely unknown. WGE analyses demonstrated to be a useful tool to either reveal the HCV recurrence molecular biology, and to encourage the identification of potential biomarkers to predict disease severity. A set of no invasive biomarkers have been proposed with promising results for the hepatic fibrosis progression assessment using specific peripheral blood serum proteins as biomarkers [30–32]. Up today, the established protein assay test demonstrated excellent utility for the identification of HCV-advanced cirrhosis, but slighter accuracy for earlier stages of the disease . Thus, only large cohort prospective studies in will contribute to optimize the analytical performance of those tests. At meantime, liver biopsy remains to be the gold standard for allograft fibrosis progression assessment in HCV recurrence despite its well-known no perfect accuracy [33–36]. The combined molecular markers detection and pathology characterization, together with reliable clinical data collected during the liver biopsy protocol may help to predict the severity of HCV recurrence to come.
The accurate follow-up and the differential diagnosis of post-transplant complications have been further struggled by no reliable and difficult pathology reports, essentially in post-transplanted HCV-infected patients . However, the implementation of WGE analyses permitted new insights about the molecular biology characterization of certain post-LT complications [13, 23, 28, 37, 38]. Furthermore, the extension of this technique to the follow-up of HCV recurrence patients might permit the diagnosis and surveillance of severity in the fibrosis progression, along with pathological evaluation . From this perspective, it is imperative the continuous study of different molecular aspects of mechanisms involved in HCV infection and ACR. The complete understanding of the biological process triggered in each condition will allow the identification of early predictors for disease differentiation and progression, and the implementation of them to the diagnostic arsenal. Importantly, it will impact directly in the identification and treatment success rates.
List of abbreviations used
Acute cellular rejection
copy Deoxyribonucleotide acid
CAP-GLY domain containing linker protein 4
copy Ribonucleotide acid
cDNA-mediated Annealing, Selection, Extension, and Ligation
Formalin-fixed paraffin embedded
Hepatitis C virus
Interleukin-28 receptor, alpha (interferon, lambda receptor)
Least absolute shrinkage and selection operator
Model of end-stage liver disease
Nuclear factor kappa-B
Reactive oxygen species
Suppressor of T cell receptor signaling 1
Whole-genome gene expression.
This article has been published as part of Fibrogenesis & Tissue Repair Volume 5 Supplement 1, 2012: Proceedings of Fibroproliferative disorders: from biochemical analysis to targeted therapies. The full contents of the supplement are available online at http://www.fibrogenesis.com/supplements/5/S1.
The current study was partially supported by a National Institute of Health (NIH) RO1 grant (RO1-DK069859).
- Schuppan D, Ruehl M, Somasundaram R, Hahn EG: Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis. 2001, 21 (3): 351-372. 10.1055/s-2001-17556.View ArticlePubMed
- Wallace K, Burt AD, Wright MC: Liver fibrosis. Biochem J. 2008, 411 (1): 1-18. 10.1042/BJ20071570.View ArticlePubMed
- Henderson NC, Forbes SJ: Hepatic fibrogenesis: from within and outwith. Toxicology. 2008, 254 (3): 130-135. 10.1016/j.tox.2008.08.017.View ArticlePubMed
- Iredale J: Defining therapeutic targets for liver fibrosis: exploiting the biology of inflammation and repair. Pharmacol Res. 2008, 58 (2): 129-136. 10.1016/j.phrs.2008.06.011.View ArticlePubMed
- Mas VR, Fisher RA, Archer KJ, Maluf DG: Proteomics and liver fibrosis: identifying markers of fibrogenesis. Expert Rev Proteomics. 2009, 6 (4): 421-431. 10.1586/epr.09.59.View ArticlePubMed
- Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP: The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol. 2006, 45 (4): 529-538. 10.1016/j.jhep.2006.05.013.View ArticlePubMed
- Friedman SL: Liver fibrosis -- from bench to bedside. J Hepatol. 2003, 38 (Suppl 1): S38-53.View ArticlePubMed
- Svegliati-Baroni G, De Minicis S, Marzioni M: Hepatic fibrogenesis in response to chronic liver injury: novel insights on the role of cell-to-cell interaction and transition. Liver Int. 2008, 28 (8): 1052-1064. 10.1111/j.1478-3231.2008.01825.x.View ArticlePubMed
- Moller S, Henriksen JH: Cardiovascular complications of cirrhosis. Gut. 2008, 57 (2): 268-278. 10.1136/gut.2006.112177.View ArticlePubMed
- Kim WR, Brown RS, Terrault NA, El-Serag H: Burden of liver disease in the United States: summary of a workshop. Hepatology. 2002, 36 (1): 227-242. 10.1053/jhep.2002.34734.View ArticlePubMed
- Gehrau R, Mas V, Archer KJ, Maluf D: Molecular classification and clonal differentiation of hepatocellular carcinoma: the step forward for patient selection for liver transplantation. Expert Rev Gastroenterol Hepatol. 2011, 5 (4): 539-552. 10.1586/egh.11.48.View ArticlePubMed
- Washburn K: Model for End Stage Liver Disease and hepatocellular carcinoma: a moving target. Transplant Rev (Orlando). 2010, 24 (1): 11-17. 10.1016/j.trre.2009.10.002.View Article
- Maluf DG, Archer KJ, Villamil F, Stravitz RT, Mas V: Hepatitis C virus recurrence after liver transplantation: biomarkers of disease and fibrosis progression. Expert Rev Gastroenterol Hepatol. 2010, 4 (4): 445-458. 10.1586/egh.10.39.View ArticlePubMed
- Garcia-Retortillo M, Forns X, Feliu A, Moitinho E, Costa J, Navasa M, Rimola A, Rodes J: Hepatitis C virus kinetics during and immediately after liver transplantation. Hepatology. 2002, 35 (3): 680-687. 10.1053/jhep.2002.31773.View ArticlePubMed
- Burton JR, Rosen HR: Acute rejection in HCV-infected liver transplant recipients: The great conundrum. Liver Transpl. 2006, 12 (11 Suppl 2): S38-47.View ArticlePubMed
- Knechtle SJ, Kwun J: Unique aspects of rejection and tolerance in liver transplantation. Semin Liver Dis. 2009, 29 (1): 91-101. 10.1055/s-0029-1192058.View ArticlePubMed
- Gelb B, Feng S: Management of the liver transplant patient. Expert Rev Gastroenterol Hepatol. 2009, 3 (6): 631-647. 10.1586/egh.09.58.View ArticlePubMed
- Regev A, Molina E, Moura R, Bejarano PA, Khaled A, Ruiz P, Arheart K, Berho M, Drachenberg CB, Mendez P, O'Brien C, Jeffers L, Tzakis A, Schiff ER: Reliability of histopathologic assessment for the differentiation of recurrent hepatitis C from acute rejection after liver transplantation. Liver Transpl. 2004, 10 (10): 1233-1239. 10.1002/lt.20245.View ArticlePubMed
- Unitt E, Gelson W, Davies SE, Coleman N, Alexander GJ: Minichromosome maintenance protein-2-positive portal tract lymphocytes distinguish acute cellular rejection from hepatitis C virus recurrence after liver transplantation. Liver Transpl. 2009, 15 (3): 306-312. 10.1002/lt.21680.View ArticlePubMed
- Gane EJ, Portmann BC, Naoumov NV, Smith HM, Underhill JA, Donaldson PT, Maertens G, Williams R: Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med. 1996, 334 (13): 815-820. 10.1056/NEJM199603283341302.View ArticlePubMed
- Kurian S, Grigoryev Y, Head S, Campbell D, Mondala T, Salomon DR: Applying genomics to organ transplantation medicine in both discovery and validation of biomarkers. Int Immunopharmacol. 2007, 7 (14): 1948-1960. 10.1016/j.intimp.2007.07.017.PubMed CentralView ArticlePubMed
- Mas VR, Maluf DG, Archer KJ, Yanek K, Williams B, Fisher RA: Differentially expressed genes between early and advanced hepatocellular carcinoma (HCC) as a potential tool for selecting liver transplant recipients. Mol Med. 2006, 12 (4-6): 97-104.PubMed CentralView ArticlePubMed
- Gehrau R, Maluf D, Archer K, Stravitz R, Suh J, Le N, Mas V: Molecular pathways differentiate hepatitis C virus (HCV) recurrence from acute cellular rejection in HCV liver recipients. Mol Med. 2011, 17 (7-8): 824-833.PubMed CentralView ArticlePubMed
- Demetris AJ: Evolution of hepatitis C virus in liver allografts. Liver Transpl. 2009, 15 (Suppl 2): S35-41.View ArticlePubMed
- Ramirez S, Perez-Del-Pulgar S, Forns X: Virology and pathogenesis of hepatitis C virus recurrence. Liver Transpl. 2008, 14 (Suppl 2): S27-35.View ArticlePubMed
- Bownik H, Saab S: The effects of hepatitis C recurrence on health-related quality of life in liver transplant recipients. Liver Int. 2010, 30 (1): 19-30. 10.1111/j.1478-3231.2009.02152.x.View ArticlePubMed
- De Martin E, Senzolo M, Gambato M, Germani G, Vitale A, Russo FR, Burra P: Fibrosis progression and the pros and cons of antiviral therapy for hepatitis C virus recurrence after liver transplantation: a review. Transplant Proc. 2010, 42 (6): 2223-2225. 10.1016/j.transproceed.2010.05.035.View ArticlePubMed
- Mas V, Maluf D, Archer KJ, Potter A, Suh J, Gehrau R, Descalzi V, Villamil F: Transcriptome at the time of hepatitis C virus recurrence may predict the severity of fibrosis progression after liver transplantation. Liver Transpl. 2011, 17 (7): 824-835. 10.1002/lt.22309.View ArticlePubMed
- Bedossa P, Poynard T: An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology. 1996, 24 (2): 289-293. 10.1002/hep.510240201.View ArticlePubMed
- Schmeding M, Dankof A, Krenn V, Krukemeyer MG, Koch M, Spinelli A, Langrehr JM, Neumann UP, Neuhaus P: C4d in acute rejection after liver transplantation--a valuable tool in differential diagnosis to hepatitis C recurrence. Am J Transplant. 2006, 6 (3): 523-530. 10.1111/j.1600-6143.2005.01180.x.View ArticlePubMed
- Schmeding M, Kienlein S, Rocken C, Neuhaus R, Neuhaus P, Heidenhain C, Neumann UP: ELISA-based detection of C4d after liver transplantation--a helpful tool for differential diagnosis between acute rejection and HCV-recurrence?. Transpl Immunol. 2010, 23 (4): 156-160. 10.1016/j.trim.2010.06.002.View ArticlePubMed
- Shaheen AA, Wan AF, Myers RP: FibroTest and FibroScan for the prediction of hepatitis C-related fibrosis: a systematic review of diagnostic test accuracy. Am J Gastroenterol. 2007, 102 (11): 2589-2600. 10.1111/j.1572-0241.2007.01466.x.View ArticlePubMed
- Afdhal NH: Diagnosing fibrosis in hepatitis C: is the pendulum swinging from biopsy to blood tests?. Hepatology. 2003, 37 (5): 972-974. 10.1053/jhep.2003.50223.View ArticlePubMed
- Dienstag JL: The role of liver biopsy in chronic hepatitis C. Hepatology. 2002, 36 (5 Suppl 1): S152-60.View ArticlePubMed
- Samuel D, Forns X, Berenguer M, Trautwein C, Burroughs A, Rizzetto M, Trepo C: Report of the monothematic EASL conference on liver transplantation for viral hepatitis (Paris, France, January 12-14, 2006). J Hepatol. 2006, 45 (1): 127-143. 10.1016/j.jhep.2006.05.001.View ArticlePubMed
- Benlloch S, Heredia L, Barquero C, Rayon JM, Pina R, Aguilera V, Prieto M, Berenguer M: Prospective validation of a noninvasive index for predicting liver fibrosis in hepatitis C virus-infected liver transplant recipients. Liver Transpl. 2009, 15 (12): 1798-1807. 10.1002/lt.21919.View ArticlePubMed
- Asaoka T, Kato T, Marubashi S, Dono K, Hama N, Takahashi H, Kobayashi S, Takeda Y, Takemasa I, Nagano H, Yoshida H, Ruiz P, Tzakis AG, Matsubara K, Monden M, Doki Y, Mori M: Differential transcriptome patterns for acute cellular rejection in recipients with recurrent hepatitis C after liver transplantation. Liver Transpl. 2009, 15 (12): 1738-1749. 10.1002/lt.21883.View ArticlePubMed
- Sreekumar R, Rasmussen DL, Wiesner RH, Charlton MR: Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation. Liver Transpl. 2002, 8 (9): 814-821. 10.1053/jlts.2002.35173.View ArticlePubMed
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