LIVER TRANSPLANTATION 20:673–678, 2014
ORIGINAL ARTICLE
MicroRNAs in the Bile of Patients With Biliary Strictures After Liver Transplantation 1,2 € Tim O. Lankisch,1,2* Torsten Voigtlander, * Michael P. Manns,1,2 Angelika Holzmann,2,3 3 2,3,4 Seema Dangwal, and Thomas Thum 1 Department of Gastroenterology, Hepatology, and Endocrinology, 2Integrated Research and Treatment Center–Transplantation, 3Institute of Molecular and Translational Therapeutic Strategies, and 4Excellence Cluster of Regenerative Biology and Reconstructive Therapies, Hannover Medical School, Hannover, Germany
Biliary complications after liver transplantation remain a major cause of morbidity and reduced graft survival. Ischemic-type biliary lesions (ITBLs) are common and difficult to treat. The pathophysiology of ITBLs remains unclear, and diagnostic markers are still missing. The analysis of microRNA (miRNA) profiles is an evolving field in hepatology. Our aim was to identify specific miRNA patterns in the bile of patients with ITBLs after liver transplantation. Liver transplant patients with biliary complications were included in a cross-sectional study. Patients with ITBLs (n 5 37), anastomotic strictures (ASs; n 5 39), and bile duct stones (BDSs; n 5 12) were compared. Patients with ITBLs were categorized by disease severity. The miRNA concentrations in bile were determined with global miRNA profiling and subsequent miRNA-specific polymerase chain reaction–mediated validation. The concentrations of microRNA 517a (miR-517a), miR-892a, and miR-106a* in bile were increased for patients with ITBLs versus patients with ASs or BDSs (P < 0.05). Categorization by ITBL severity showed higher median concentrations in patients with intrahepatic and extrahepatic strictures (P > 0.05). miR-210, miR-3375p, miR-577, and miR-329 displayed no statistical differences. In conclusion, miR-517a, miR-892a, and miR-106a* are increased in the bile fluid of patients with ITBLs versus patients with ASs or BDSs. An analysis of miRNA profiles may be useful in the diagnosis and management of patients with ITBLs. Future studies are needed to prove the potential prognostic C 2014 AASLD. value of these miRNAs. Liver Transpl 20:673-678, 2014. V Received October 5, 2013; accepted February 17, 2014.
See Editorial on Page 637 The detection of novel biomarkers for the prediction of disease severity and clinical outcomes and for new insights into the pathogenesis of diseases plays a crucial role in current research. Recently, the presence of
extracellular microRNAs (miRNAs) in the blood and bile has been associated with various liver diseases, such as fibrosis, hepatocellular carcinoma, and biliary tract cancer, and they thus may be novel biomarkers with diagnostic and/or prognostic relevance.1-3 Biliary complications after orthotopic liver transplantation are a major cause of morbidity and
Additional Supporting Information may be found in the online version of this article. Abbreviations: AS, anastomotic stricture; BDS, bile duct stone; ERC, endoscopic retrograde cholangiography; ITBL, ischemic-type biliary lesion; miR, microRNA; miRNA, microRNA; PCR, polymerase chain reaction. This work was supported by a grant from the German Federal Ministry of Education and Research (reference number 01EO0802). The authors have no conflicts of interest to disclose. *These authors contributed equally to this work and are both first authors. Address reprint requests to Tim O. Lankisch, M.D., Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. Telephone: 149-511-532-2406; FAX: 149-511-532-3351; E-mail: [email protected] or Thomas Thum, M.D., Ph.D., Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. Telephone: 149-511-532-5272; FAX: 149-511-532-5274; E-mail: [email protected] DOI 10.1002/lt.23872 View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases
C 2014 American Association for the Study of Liver Diseases. V
€ 674 LANKISCH, VOIGTLANDER ET AL.
reduced graft survival.4,5 Anastomotic strictures (ASs) are usually related to surgical techniques and are generally well treated by stent placement during endoscopic retrograde cholangiography (ERC).6,7 In contrast, the pathogenesis of nonanastomotic strictures or ischemic-type biliary lesions (ITBLs) after orthotopic liver transplantation remains unclear. A role for ischemic events has been postulated, but ITBLs are believed to have a multifactorial origin.5 The treatment of ITBLs is difficult and includes multiple endoscopic procedures and, in some patients, even retransplantation. Therefore, a predictive marker for future ITBL development would be beneficial for treating ITBLs at an early stage. Because the process of biliary stricture formation takes place at the biliary epithelium, we hypothesized that miRNAs may be detectable directly in bile and are associated with biliary stricture formation in patients after orthotopic liver transplantation.
PATIENTS AND METHODS Patients Consecutive liver transplant patients with ASs (39 patients) or ITBLs (37 patients) were examined with ERC in the gastrointestinal endoscopy unit of Hannover Medical School (2008-2011). All liver transplants used donation after brain death donors. The indications for ERC were elevated cholestatic laboratory parameters, dilated bile ducts, and/or strictures detected by ultrasound. Vascular patency was assessed in all patients by duplex ultrasound before ERC. Twelve patients with bile duct stones (BDSs) served as the control group. Bile was aspirated through the placement of a 5-Fr, single-use, standard ERC catheter (without flushing or guidewire cannulation) into the bile duct before contrast material injection as described before.8 Approximately 0.5 to 5 mL of bile was collected and transferred to a sterile tube. Bile samples were stored at 280 C. The disease severity of ITBLs was categorized as follows: extrahepatic lesions alone (type 1), intrahepatic lesions alone (type 2), or multiple intrahepatic and extrahepatic lesions (type 3).9,10 The study protocol was approved by the local institutional ethics review board and was in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.
miRNA Isolation and Quantification The RNA isolation of bile samples was performed with a MasterPure RNA purification kit (MRC85102, Epicentre) according to the manufacturer’s protocol. As an internal spiked-in control, Caenorhabditis elegans microRNA 39 (miR-39) was added during the isolation process (after the addition of the ribonucleaseinhibiting isolation buffer). Values were normalized to the individual protein contents of the samples to adjust for the various sample viscosities. The protein
LIVER TRANSPLANTATION, June 2014
content was measured with the Bradford assay. An miRNA polymerase chain reaction (PCR) array analysis, including 905 miRNAs from RNA pools from 4 patients with ITBLs and 4 patients with ASs, was performed with a QuantiMir miRNome miRNA profiler human PCR array (RA660A-1, version 14, BioCat, Heidelberg, Germany). Screening patients were matched by sex, age, and laboratory values and were representative of the whole cohort. All subtypes of ITBLs were included in the screening cohort [ITBL types 1 and 2 (n 5 2) and ITBL type 3 (n 5 2)]. Poly-A (adenosine)-tailing of miRNAs, annealing of a deoxythymidine oligomer adaptor, and then universal reverse transcription were performed. SYBR Green– based real-time PCR was performed with an miRNAspecific primer and a universal primer. On the basis of the strength of the regulation [including a presence at the highest levels (low cycle thresholds) in only 1 of the 2 investigated groups, the top 1-2 highest up- or down-regulated miRNAs; see also Supporting Table 1 for selection criteria] in the array analysis or on the basis of evidence from the literature, the following miRNAs were chosen for further validation: miR-3375p, miR-517a, miR-892a, miR-106a*, miR-577, miR329, and miR-210 (Supporting Table 1). RNA was isolated from 88 bile samples (37 ITBL patients, 39 AS patients, and 12 BDS patients) and reverse-transcribed with a TaqMan miRNA reversetranscription kit (4366597, Applied Biosystems) and specific miRNA reverse-transcription primers (4427975, Applied Biosystems). Real-time PCR analysis was performed with iQ Supermix (170-8864, BioRad) and specific miRNA TaqMan probes (4427975, Applied Biosystems). Individual messenger RNA measurements were normalized to spike-in C. elegans controls, and quantification was performed with the difference-in-cycle-thresholds method.
Statistical Analysis Data were expressed as numbers and percentages or as medians and interquartile ranges. Noncontinuous parameters were analyzed with the v2 test or Fisher’s exact test as appropriate, and continuous parameters were analyzed with the Mann-Whitney U test. For correlations, Spearman’s rank correlation was used. The optimal diagnostic points of the markers were assessed at cutoff values with the largest Youden index (sensitivity 1 specificity 2 1). P values < 0.05 were considered statistically significant. The software used was the SPSS statistical package (version 19.0, SPSS, Inc., Chicago, IL).
RESULTS Levels of miR-517a, miR-892a, and miR-106a* in Bile Are Increased in Patients With ITBLs Demographic characteristics, clinical features, and laboratory values are presented in Table 1. The main indication for liver transplantation in all patients was viral hepatitis (34.2%, n 5 26), which was followed by
26/11 50 (40-58) 606 (544-666) 45 (37-50) 8 4 5 6 4
(21.6) (10.8) (13.5) (16.2) (10.8) 0 (0) 3 (8.1) 2 (5.4) 0 (0) 0 (0) 5 (13.5) 24.8 (12.6-44.4) 34 (20-56) 42 (24-54) 147 (114-368) 105 (64-346) 14 (10-35) 5 (3-25) 6.8 (3.9-8.9) 177 (154-199) 384.52 (158.84-981) 25.12 (9.87-41.94) 1.95 (0.62-5.22) 0.37 (0.17-0.87) 0.13 (0.05-0.34) 1.32 (0.68-3.15) 0.10 (0.02-0.25)
22/17 55 (35-60) 588 (428-675) 45 (39-55) 7 (18) 7 (18) 6 (15.4) 1 (2.6) 2 (5.1) 4 (10.3) 1 (2.6) 2 (5.1) 3 (7.7) 3 (7.7) 3 (7.7) 18.3 (7-54.8) 39 (20-111) 38 (24-74) 110 (77-230) 111 (30-245) 13 (9-20) 5 (2-12) 6.1 (4-7.5) 203 (167-225) 330.04 (136.06-608.56) 15.33 (9.49-21.69) 0.73 (0.39-1.41) 0.14 (0.08-0.37) 0.06 (0.03-0.15) 1.38 (0.7-3.23) 0.06 (0.01-0.11)
ITBL Group (n 5 37)
– – 0.11 (0.08-0.3) 0.06 (0.04-0.24) 0.03 (0.01-0.05) – –
31 (22-75) 27 (26-80) 106 (69-189) 110 (37-318) 9 (6-14) 7 (4-10) 5.4 (5.2-5.7) 212 (160-237)
– – – – – – – – – – – –
5/7 64 (56-69) – –
BDS Group (n 5 12)
– – – – – – –
ORIGINAL ARTICLE
MicroRNAs in the Bile of Patients With Biliary Strictures After Liver Transplantation 1,2 € Tim O. Lankisch,1,2* Torsten Voigtlander, * Michael P. Manns,1,2 Angelika Holzmann,2,3 3 2,3,4 Seema Dangwal, and Thomas Thum 1 Department of Gastroenterology, Hepatology, and Endocrinology, 2Integrated Research and Treatment Center–Transplantation, 3Institute of Molecular and Translational Therapeutic Strategies, and 4Excellence Cluster of Regenerative Biology and Reconstructive Therapies, Hannover Medical School, Hannover, Germany
Biliary complications after liver transplantation remain a major cause of morbidity and reduced graft survival. Ischemic-type biliary lesions (ITBLs) are common and difficult to treat. The pathophysiology of ITBLs remains unclear, and diagnostic markers are still missing. The analysis of microRNA (miRNA) profiles is an evolving field in hepatology. Our aim was to identify specific miRNA patterns in the bile of patients with ITBLs after liver transplantation. Liver transplant patients with biliary complications were included in a cross-sectional study. Patients with ITBLs (n 5 37), anastomotic strictures (ASs; n 5 39), and bile duct stones (BDSs; n 5 12) were compared. Patients with ITBLs were categorized by disease severity. The miRNA concentrations in bile were determined with global miRNA profiling and subsequent miRNA-specific polymerase chain reaction–mediated validation. The concentrations of microRNA 517a (miR-517a), miR-892a, and miR-106a* in bile were increased for patients with ITBLs versus patients with ASs or BDSs (P < 0.05). Categorization by ITBL severity showed higher median concentrations in patients with intrahepatic and extrahepatic strictures (P > 0.05). miR-210, miR-3375p, miR-577, and miR-329 displayed no statistical differences. In conclusion, miR-517a, miR-892a, and miR-106a* are increased in the bile fluid of patients with ITBLs versus patients with ASs or BDSs. An analysis of miRNA profiles may be useful in the diagnosis and management of patients with ITBLs. Future studies are needed to prove the potential prognostic C 2014 AASLD. value of these miRNAs. Liver Transpl 20:673-678, 2014. V Received October 5, 2013; accepted February 17, 2014.
See Editorial on Page 637 The detection of novel biomarkers for the prediction of disease severity and clinical outcomes and for new insights into the pathogenesis of diseases plays a crucial role in current research. Recently, the presence of
extracellular microRNAs (miRNAs) in the blood and bile has been associated with various liver diseases, such as fibrosis, hepatocellular carcinoma, and biliary tract cancer, and they thus may be novel biomarkers with diagnostic and/or prognostic relevance.1-3 Biliary complications after orthotopic liver transplantation are a major cause of morbidity and
Additional Supporting Information may be found in the online version of this article. Abbreviations: AS, anastomotic stricture; BDS, bile duct stone; ERC, endoscopic retrograde cholangiography; ITBL, ischemic-type biliary lesion; miR, microRNA; miRNA, microRNA; PCR, polymerase chain reaction. This work was supported by a grant from the German Federal Ministry of Education and Research (reference number 01EO0802). The authors have no conflicts of interest to disclose. *These authors contributed equally to this work and are both first authors. Address reprint requests to Tim O. Lankisch, M.D., Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. Telephone: 149-511-532-2406; FAX: 149-511-532-3351; E-mail: [email protected] or Thomas Thum, M.D., Ph.D., Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Carl-Neuberg Strasse 1, 30625 Hannover, Germany. Telephone: 149-511-532-5272; FAX: 149-511-532-5274; E-mail: [email protected] DOI 10.1002/lt.23872 View this article online at wileyonlinelibrary.com. LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases
C 2014 American Association for the Study of Liver Diseases. V
€ 674 LANKISCH, VOIGTLANDER ET AL.
reduced graft survival.4,5 Anastomotic strictures (ASs) are usually related to surgical techniques and are generally well treated by stent placement during endoscopic retrograde cholangiography (ERC).6,7 In contrast, the pathogenesis of nonanastomotic strictures or ischemic-type biliary lesions (ITBLs) after orthotopic liver transplantation remains unclear. A role for ischemic events has been postulated, but ITBLs are believed to have a multifactorial origin.5 The treatment of ITBLs is difficult and includes multiple endoscopic procedures and, in some patients, even retransplantation. Therefore, a predictive marker for future ITBL development would be beneficial for treating ITBLs at an early stage. Because the process of biliary stricture formation takes place at the biliary epithelium, we hypothesized that miRNAs may be detectable directly in bile and are associated with biliary stricture formation in patients after orthotopic liver transplantation.
PATIENTS AND METHODS Patients Consecutive liver transplant patients with ASs (39 patients) or ITBLs (37 patients) were examined with ERC in the gastrointestinal endoscopy unit of Hannover Medical School (2008-2011). All liver transplants used donation after brain death donors. The indications for ERC were elevated cholestatic laboratory parameters, dilated bile ducts, and/or strictures detected by ultrasound. Vascular patency was assessed in all patients by duplex ultrasound before ERC. Twelve patients with bile duct stones (BDSs) served as the control group. Bile was aspirated through the placement of a 5-Fr, single-use, standard ERC catheter (without flushing or guidewire cannulation) into the bile duct before contrast material injection as described before.8 Approximately 0.5 to 5 mL of bile was collected and transferred to a sterile tube. Bile samples were stored at 280 C. The disease severity of ITBLs was categorized as follows: extrahepatic lesions alone (type 1), intrahepatic lesions alone (type 2), or multiple intrahepatic and extrahepatic lesions (type 3).9,10 The study protocol was approved by the local institutional ethics review board and was in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.
miRNA Isolation and Quantification The RNA isolation of bile samples was performed with a MasterPure RNA purification kit (MRC85102, Epicentre) according to the manufacturer’s protocol. As an internal spiked-in control, Caenorhabditis elegans microRNA 39 (miR-39) was added during the isolation process (after the addition of the ribonucleaseinhibiting isolation buffer). Values were normalized to the individual protein contents of the samples to adjust for the various sample viscosities. The protein
LIVER TRANSPLANTATION, June 2014
content was measured with the Bradford assay. An miRNA polymerase chain reaction (PCR) array analysis, including 905 miRNAs from RNA pools from 4 patients with ITBLs and 4 patients with ASs, was performed with a QuantiMir miRNome miRNA profiler human PCR array (RA660A-1, version 14, BioCat, Heidelberg, Germany). Screening patients were matched by sex, age, and laboratory values and were representative of the whole cohort. All subtypes of ITBLs were included in the screening cohort [ITBL types 1 and 2 (n 5 2) and ITBL type 3 (n 5 2)]. Poly-A (adenosine)-tailing of miRNAs, annealing of a deoxythymidine oligomer adaptor, and then universal reverse transcription were performed. SYBR Green– based real-time PCR was performed with an miRNAspecific primer and a universal primer. On the basis of the strength of the regulation [including a presence at the highest levels (low cycle thresholds) in only 1 of the 2 investigated groups, the top 1-2 highest up- or down-regulated miRNAs; see also Supporting Table 1 for selection criteria] in the array analysis or on the basis of evidence from the literature, the following miRNAs were chosen for further validation: miR-3375p, miR-517a, miR-892a, miR-106a*, miR-577, miR329, and miR-210 (Supporting Table 1). RNA was isolated from 88 bile samples (37 ITBL patients, 39 AS patients, and 12 BDS patients) and reverse-transcribed with a TaqMan miRNA reversetranscription kit (4366597, Applied Biosystems) and specific miRNA reverse-transcription primers (4427975, Applied Biosystems). Real-time PCR analysis was performed with iQ Supermix (170-8864, BioRad) and specific miRNA TaqMan probes (4427975, Applied Biosystems). Individual messenger RNA measurements were normalized to spike-in C. elegans controls, and quantification was performed with the difference-in-cycle-thresholds method.
Statistical Analysis Data were expressed as numbers and percentages or as medians and interquartile ranges. Noncontinuous parameters were analyzed with the v2 test or Fisher’s exact test as appropriate, and continuous parameters were analyzed with the Mann-Whitney U test. For correlations, Spearman’s rank correlation was used. The optimal diagnostic points of the markers were assessed at cutoff values with the largest Youden index (sensitivity 1 specificity 2 1). P values < 0.05 were considered statistically significant. The software used was the SPSS statistical package (version 19.0, SPSS, Inc., Chicago, IL).
RESULTS Levels of miR-517a, miR-892a, and miR-106a* in Bile Are Increased in Patients With ITBLs Demographic characteristics, clinical features, and laboratory values are presented in Table 1. The main indication for liver transplantation in all patients was viral hepatitis (34.2%, n 5 26), which was followed by
26/11 50 (40-58) 606 (544-666) 45 (37-50) 8 4 5 6 4
(21.6) (10.8) (13.5) (16.2) (10.8) 0 (0) 3 (8.1) 2 (5.4) 0 (0) 0 (0) 5 (13.5) 24.8 (12.6-44.4) 34 (20-56) 42 (24-54) 147 (114-368) 105 (64-346) 14 (10-35) 5 (3-25) 6.8 (3.9-8.9) 177 (154-199) 384.52 (158.84-981) 25.12 (9.87-41.94) 1.95 (0.62-5.22) 0.37 (0.17-0.87) 0.13 (0.05-0.34) 1.32 (0.68-3.15) 0.10 (0.02-0.25)
22/17 55 (35-60) 588 (428-675) 45 (39-55) 7 (18) 7 (18) 6 (15.4) 1 (2.6) 2 (5.1) 4 (10.3) 1 (2.6) 2 (5.1) 3 (7.7) 3 (7.7) 3 (7.7) 18.3 (7-54.8) 39 (20-111) 38 (24-74) 110 (77-230) 111 (30-245) 13 (9-20) 5 (2-12) 6.1 (4-7.5) 203 (167-225) 330.04 (136.06-608.56) 15.33 (9.49-21.69) 0.73 (0.39-1.41) 0.14 (0.08-0.37) 0.06 (0.03-0.15) 1.38 (0.7-3.23) 0.06 (0.01-0.11)
ITBL Group (n 5 37)
– – 0.11 (0.08-0.3) 0.06 (0.04-0.24) 0.03 (0.01-0.05) – –
31 (22-75) 27 (26-80) 106 (69-189) 110 (37-318) 9 (6-14) 7 (4-10) 5.4 (5.2-5.7) 212 (160-237)
– – – – – – – – – – – –
5/7 64 (56-69) – –
BDS Group (n 5 12)
– – – – – – –
