Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR

<p>Abstract</p> <p>Background</p> <p>Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a systematic evaluation of six putative reference genes for gene expression studies in human hepatocellular carcinoma (HCC) is presented.</p> <p>Methods</p> <p>Six genes, beta-2-microglobulin (<it>B2M</it>), glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>), hydroxymethyl-bilane synthase (<it>HMBS</it>), hypoxanthine phosphoribosyl-transferase 1 (<it>HPRT1</it>), succinate dehydrogenase complex, subunit A (<it>SDHA</it>) and ubiquitin C (<it>UBC</it>), with distinct functional characteristics and expression patterns were evaluated by qRT-PCR. Inhibitory substances in RNA samples were quantitatively assessed and controlled using an external RNA control. The stability of selected reference genes was analyzed using both <it>geNorm </it>and <it>NormFinder </it>software.</p> <p>Results</p> <p><it>HMBS </it>and <it>GAPDH </it>were identified as the optimal reference genes for normalizing gene expression data between paired tumoral and adjacent non-tumoral tissues derived from patients with HCC. <it>HMBS, GAPDH </it>and <it>UBC </it>were identified to be suitable for the normalization of gene expression data among tumor tissues; whereas the combination of <it>HMBS, B2M</it>, <it>SDHA </it>and <it>GAPDH </it>was suitable for normalizing gene expression data among five liver cancer cell lines, namely Hep3B, HepG2, HuH7, SK-HEP-1 and SNU-182. The determined gene stability was increased after exclusion of RNA samples containing relatively higher inhibitory substances.</p> <p>Conclusion</p> <p>Of six genes studied, <it>HMBS </it>was found to be the single best reference gene for gene expression studies in HCC. The appropriate choice of combination of more than one reference gene to improve qRT-PCR accuracy depends on the kind of liver tissues or cells under investigation. Quantitative assessment and control of qRT-PCR inhibitors using an external RNA control can reduce the variation of qRT-PCR assay and facilitate the evaluation of gene stability. Our results may facilitate the choice of reference genes for expression studies in HCC.</p

Paclitaxel-eluting balloon dilation of biliary anastomotic stricture after liver transplantation

AIM: To investigate the safety and effectiveness of endoscopic therapy with a paclitaxel-eluting balloon (PEB) for biliary anastomotic stricture (AS) after liver transplantation (LT). METHODS: This prospective pilot study enrolled 13 consecutive eligible patients treated for symptomatic AS after LT at the University Hospital of Münster between January 2011 and March 2014. The patients were treated by endoscopic therapy with a PEB and followed up every 8 wk by endoscopic retrograde cholangiopancreatography (ERCP). In cases of re-stenosis, further balloon dilation with a PEB was performed. Follow-up was continued until 24 mo after the last intervention. RESULTS: Initial technical feasibility, defined as successful balloon dilation with a PEB during the initial ERCP procedure, was achieved in 100% of cases. Long-term clinical success (LTCS), defined as no need for further endoscopic intervention for at least 24 mo, was achieved in 12 of the 13 patients (92.3%). The mean number of endoscopic interventions required to achieve LTCS was only 1.7 ± 1.1. Treatment failure, defined as the need for definitive alternative treatment, occurred in only one patient, who developed recurrent stenosis with increasing bile duct dilatation that required stent placement. CONCLUSION: Endoscopic therapy with a PEB is very effective for the treatment of AS after LT, and seems to significantly shorten the overall duration of endoscopic treatment by reducing the number of interventions needed to achieve LTCS

Orthotopic liver transplantation in human-immunodeficiency-virus-positive patients in Germany

Objectives: This summary evaluates the outcomes of orthotopic liver transplantation (OLT) of HIV-positive patients in Germany. Methods: Retrospective chart analysis of HIV-positive patients, who had been liver-transplanted in Germany between July 1997 and July 2011. Results: 38 transplantations were performed in 32 patients at 9 German transplant centres. The reasons for OLT were end-stage liver disease (ESLD) and/or liver failure due to hepatitis C (HCV) (n = 19), hepatitis B (HBV) (n = 10), multiple viral infections of the liver (n = 2) and Budd-Chiari-Syndrome. In July 2011 19/32 (60%) of the transplanted patients were still alive with a median survival of 61 months (IQR (interquartile range): 41-86 months). 6 patients had died in the early post-transplantation period from septicaemia (n = 4), primary graft dysfunction (n = 1), and intrathoracal hemorrhage (n = 1). Later on 7 patients had died from septicaemia (n = 2), delayed graft failure (n = 2), recurrent HCC (n = 2), and renal failure (n = 1). Recurrent HBV infection was efficiently prevented in 11/12 patients; HCV reinfection occurred in all patients and contributed considerably to the overall mortality. Conclusions: Overall OLT is a feasible approach in HIV-infected patients with acceptable survival rates in Germany. Reinfection with HCV still remains a major clinical challenge in HIV/HCV coinfection after OLT

Analysis of Bile Colonization and Intestinal Flora may Improve Management in Liver Transplant Recipients Undergoing ERCP

Background: Immunosuppression, denervation of biliary tract, and presence of biliary strictures favor colonization of bile with microorganisms after liver transplantation. Little is known about spectrum and antibiotic susceptibility of this colonization. Material and Methods: Bile and feces were collected prospectively from 38 patients who underwent endoscopic retrograde cholangiopancreaticography after liver transplantation. Samples were analyzed for colonization and antibiotic susceptibility. Results: From the 38 tested bile samples, 86.6% tested positive. Of those, 26 (78.8%) were polymicrobial. Of isolated bile samples, 52 (64.2%) were gram-positive, 22.2% were gram-negative, and 13.6% revealed Candida albicans. Most detectable gram-positive bacteria were Enterococcus faecium. Most detectable gram-negative bacteria were E. coli and Klebsiella pneumonia. Our analyses revealed high resistance rates of the isolates. Only 55.6% of isolates were sensitive to ciprofloxacin, 54% were sensitive to piperacillin/tazobactam, and 60.3% were sensitive to imipenem. High susceptibility rates were found for linezolid and vancomycin (72.9% and 72.6%, respectively). We found a high correlation between microorganisms found in bile and those isolated from stool. Conclusions: Bile of liver transplant recipients is frequently colonized with microorganisms. The starting point of this colonization is usually the intestine. Systematic analysis of bile colonization during endoscopic interventions on biliary tracts of liver transplant recipients might help to select effective prophylactic antibiotic regimes as well as to facilitate the choice of suitable antimicrobial therapy in case of septic complications

Neurologic complications in adult living donor liver transplant patients: an underestimated factor?

Liver transplantation is the only curative treatment in patients with end-stage liver disease. Neurological complications (NC) are increasingly reported to occur in patients after cadaveric liver transplantation. This retrospective cohort study aims to evaluate the incidence and causes of NC in living donor liver transplant (LDLT) patients in our transplant center. Between August 1998 and December 2005, 121 adult LDLT patients were recruited into our study. 17% of patients experienced NC, and it occurred significantly more frequently in patients with alcoholic cirrhosis (42%) and autoimmune hepatitis (43%) as compared with patients with hepatitis B or C (9/10%, P = 0.013). The most common NC was encephalopathy (47.6%) followed by seizures (9.5%). The choice of immunosuppression by calcineurin inhibitor (Tacrolimus or Cyclosporin A) showed no significant difference in the incidence of NC (19 vs. 17%). The occurrence of NC did not influence the clinical outcome, since mortality rate, median ICU stay and length of hospital stay were similar between the two groups. Most patients who survived showed a nearly complete recovery of their NC. NCs occur in approximately 1 in 6 patients after LDLT and seem to be predominantly transient in nature, without major impact on clinical outcome

Long-Term Renal Function in Liver Transplant Recipients After Conversion From Calcineurin Inhibitors to mTOR Inhibitors

BACKGROUND: Renal dysfunction often occurs in liver transplant (LT) recipients receiving calcineurin inhibitor (CNI)-based immunosuppressive regimens, increasing morbidity and mortality rates. Replacement of CNIs by mTOR inhibitor-based immunosuppressive protocols may prevent renal impairment in LT recipients. MATERIAL AND METHODS: Outcomes in patients who underwent LT between 1996 and 2010 at our center and who were switched from CNI-based to mTOR inhibitor-based immunosuppression were retrospectively analyzed. Renal course, hyperlipidemia, and graft rejection were assessed in patients maintained on this CNI-free regimen for at least 24 months. RESULTS: Of the 85 patients switched from CNI-based to mTOR inhibitor-based, CNI-free immunosuppression, 78 met the inclusion criteria. Within the first 6 weeks after switching, the covariable adjusted estimated glomerular filtration rate (eGFR) increased 5.6 mL/min [95% confidence interval 2.6–8.7 mL/min, p<0.001], but there were no further statistically noticeable changes in eGFR. Concentrations of cholesterol and triglycerides increased statistically, noticeable within the first 12 months after drug conversion. Histologically proven graft rejection was observed in 4 patients (5.1%) after conversion. CONCLUSIONS: Conversion from CNI-based to CNI-free, mTOR inhibitor-based immunosuppression after LT is safe and can result in significant renal recovery. CNI-free, mTOR inhibitor-based immunosuppression is a potential option for patients with contraindications for CNIs and for LT recipients with rapid reduction in kidney function due to CNIs

A prospective randomised, open-labeled, trial comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing liver transplantation for hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>The potential anti-cancer effects of mammalian target of rapamycin (mTOR) inhibitors are being intensively studied. To date, however, few randomised clinical trials (RCT) have been performed to demonstrate anti-neoplastic effects in the pure oncology setting, and at present, no oncology endpoint-directed RCT has been reported in the high-malignancy risk population of immunosuppressed transplant recipients. Interestingly, since mTOR inhibitors have both immunosuppressive and anti-cancer effects, they have the potential to simultaneously protect against immunologic graft loss and tumour development. Therefore, we designed a prospective RCT to determine if the mTOR inhibitor sirolimus can improve hepatocellular carcinoma (HCC)-free patient survival in liver transplant (LT) recipients with a pre-transplant diagnosis of HCC.</p> <p>Methods/Design</p> <p>The study is an open-labelled, randomised, RCT comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing LT for HCC. Patients with a histologically confirmed HCC diagnosis are randomised into 2 groups within 4-6 weeks after LT; one arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol and the second arm is maintained on a centre-specific mTOR-inhibitor-free immunosuppressive protocol for the first 4-6 weeks, at which time sirolimus is initiated. A 2^1/2 -year recruitment phase is planned with a 5-year follow-up, testing HCC-free survival as the primary endpoint. Our hypothesis is that sirolimus use in the second arm of the study will improve HCC-free survival. The study is a non-commercial investigator-initiated trial (IIT) sponsored by the University Hospital Regensburg and is endorsed by the European Liver and Intestine Transplant Association; 13 countries within Europe, Canada and Australia are participating.</p> <p>Discussion</p> <p>If our hypothesis is correct that mTOR inhibition can reduce HCC tumour growth while simultaneously providing immunosuppression to protect the liver allograft from rejection, patients should experience less post-transplant problems with HCC recurrence, and therefore could expect a longer and better quality of life. A positive outcome will likely change the standard of posttransplant immunosuppressive care for LT patients with HCC.</p> <p>Trial Register</p> <p>Trial registered at <url>http://www.clinicaltrials.gov</url>: NCT00355862</p> <p>(EudraCT Number: 2005-005362-36)</p

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes