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Supplementary Fig. S3. Doxycyline-induced overexpression of Dicer in HCC cells. HepG2 (A) and Huh-7 (B) cells stably transfected with the inducible Dicer construct were incubated with or without doxycycline (Dox) for 24 h. C: HepG2 cells with the inducible Dicer construct were incubated for the indicated duration with or without doxycycline. A-C: The lysates were analyzed by immunoblotting using anti-Dicer and anti-β-actin antibodies. D: Western blot analysis of Dicer protein expression in Dicer-transgenic HepG2 xenografts from mice treated for 7 days with doxycycline (n = 3) or vehicle (n = 4), that were used for the experiment displayed in Fig. 5F. A-D: β-actin was used as endogenous control.

Table S1. Clinicopathological parameters of the patients with HCC. HBV, hepatitis B virus; HCV, hepatitis C virus

Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown.Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial–mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs.Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), HIF2α, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration.Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin Cancer Res; 23(14); 3896–905. ©2017 AACR.

Supplementary Information

Supplementary Figure S3: Body weight of HepG2 (A) and Huh-7 (B) xenograft-bearing mice treated with iRGD, control (Ctl.) peptide, PBS and sorafenib (Sora).

In the last decade, both hepatitis B virus (HBV)prevalence and mortality related to HBV infection have decreased promptly.Worldwide HBV vaccination programs, precautions against HBV transmission andeffective anti-viral drugs on market play crucial role for this encouragingresult. Besides stopping or reversing the hepato-fibrogenesis induced by HBVinfection, fighting against HBV related acute severe hepatitis are alsoimproved recently. HBV associated cirrhosis is still the major cause ofLTx, particularly in developing countries,whereas in developed countries, therate of LTx due to HBV induced cirrhosis has declined over time. With theexpanding use of NUCs before LTx, and the use of NUCs and HBIg even after LTx,HBV recurrence after LTx is no longer an important reason for graft loss orpatient death. However, this positive impact is not yet reflecting survival,probably because of increasing recipient and donor ages. On the other hand, inthe era of Milan criteria, overall hepatocellular carcinoma (HCC) survival hasso increased that the number of transplanted HCC cases has almost doubled.However tumor recurrence is still the major cause of death, and treatment isstill problematic., Hepatit B virus (HBV) enfeksiyonu prevelansındaki veHBV ilişkili mortalitesindeki azalma son yıllarda oldukça dikkat çekicidir. Dünyaçapında yaygın olarak uygulanan HBV aşı programları ve HBV’ye karşı kullanımda olananti-viral ilaçların etkinliği bu başarıda başat rol oynamaktadır. HBV ile mücadelede,sadece HBV’ye bağlı karaciğer fibrozunun ilerlemesi veya geriye döndürülmesi değil,aynı zamanda HBV ilişkili şiddetli akut hepatit tablosunun tedavisinde güzel sonuçlaralınmaktadır. Gelişmekte olan ülkelerde HBV ilişkili siroz karaciğer nakli konusundahalen esas sebep iken, gelişmiş ülkelerde zaman içinde HBV nedenli karaciğer nakilsıklıkları ciddi oranda düşüş göstermiştir. Anti-viral ilaçların karaciğer nakliöncesi etkin kullanımı, HBIg tedavisinin nakil sonrasında yaygın olarak kullanımısayesinde karaciğer nakli sonrası HBV nüksü, mortalite ve greft kaybı konusundaeskisi kadar sorun olmaktan çıkmıştır. Bu başarının nakil sonrası sürviler üzerinebariz bir yansıması henüz olmamıştır. Buradaki esas sebep ise, alıcı ve verici yaşınınson yıllarda önemli oranda artış göstermesi olarak gösterilmektedir. Ekolarak,Milan kriterlerinin yaygın olarak klinik pratiğe girmesiyle karaciğer nakli yapılanhepatosellüler kanser (HCC) hasta sayısı da neredeyse ikiye katlamıştır. Ancak,HCC rekürrensi, nakil sonrası en önemli ölüm nedeni olarak devam etmektedir.

Acute pancreatitis after ERCP (post-ERCP pancreatitis, PEP) occurs in 3–10% of patients. PEP is defined as clinical pancreatitis with amylase at least three times the upper limit of normal at more than 24 h after the procedure, requiring hospital admission or a prolongation of planned admission. Most patients have a mild or moderate self-limiting course of pancreatitis. In severe cases hemorrhagic pancreatitis, phlegmon, pseudocyst, infection, and multi-organ failure may develop. Mechanic and hydrostatic injury are regarded as major risk factors. Long cannulation time, several attempts for cannulation, precut, and injection of contrast material increase the risk for PEP. Patient-related risk factors include prior PEP, female sex, previous recurrent pancreatitis, suspected sphincter of Oddi dysfunction, and normal serum bilirubin level. Chronic pancreatitis has been demonstrated to be protective against PEP. Attenuation of the inflammatory reaction by nonsteroidal anti-inflammatory drugs; replacement of complete electrolyte infusion before, during, and after ERCP; and placement of pancreatic stents were shown to reduce the risk for PEP. Management of PEP depends on the severity of pancreatitis. In mild to moderate cases fluid replacement and pain management are sufficient. In severe cases with hemodynamic instability, systemic reactions, and organ failure, intensive care management is necessary.

Common bile duct stones (CBDS) are the most frequentcause of benign biliary obstruction requiring endoscopic treatment. Most CBDS are white to yellowish cholesterol stones or black pigment stones. They may appear as single or multiple calculi causing varying degrees of cholestasis or even biliary pancreatitis. The size ranges from small microconcrements to large stones in the order of several centimeters. CBDS smaller than the diameter of the bile duct migrate in the bile duct, and if they do not cause biliary obstruction, they may be asymptomatic and difficult to detect. Clinically relevant problems usually occur when stones get stuck and cholestasis develops. Stone extraction is required in patients with symptomatic CBDS and especially in those patients with biliary obstruction and with complications. Several methods for stone extraction exist including endoscopic, radiologic, and surgical approaches. Endoscopic retrograde cholangiopancreatography (ERCP) is less invasive than radiologic or surgical methods and therefore usually chosen as first-line treatment for stone extraction; however, ERCP bears the risk for severe procedure-associated complications such as pancreatitis, bleeding, cholangitis, and perforation. To reduce complications, unnecessary ERCPs should be avoided, and ERCP should only be performed for therapeutic stone extraction. This chapter describes diagnostic approaches and risk-based management options for CBDS.

Background and Aims: The aim of the study was to evaluate the risk of hepatocellular carcinoma (HCC) development after treatment with direct-acting antivirals (DAAs) and to compare HCC occurrence in these patients with that among patients treated with interferon (IFN)-based therapies. Methods: We analyzed a large cohort with chronic hepatitis C virus patients for the onset of new HCC after DAA treatment. A historical IFN-treated cohort was investigated for comparison. Results: A total of 819 patients were included in the DAA group. The median follow-up period was 263 days (0–1,001). Twenty-five patients (3.6 HCCs/100 person-years; 3.1%) were diagnosed with de novo HCC within the time of observation. No patient without cirrhosis had developed HCC. Patients with newly diagnosed HCC were mostly male, older, and treatment-experienced and had a lower 12-week sustained virologic response (SVR12) rate and higher levels of liver inflammation markers. The median time to HCC was 312 days (0–880). Investigation of the subcohort of 269 cirrhotic patients yielded an HCC rate of 8.9 HCCs/100 person-years. In this cohort, non-SVR12 was an independent risk factor for de novo HCC (HR 4.48; 95% CI 1.51–13.12; p = 0.007). Twenty-four patients (96%) with new HCC were Child-Pugh class A and 17 (68%) were diagnosed in early BCLC stage A. For the IFN-treated patients, we calculated an overall risk of HCC occurrence of 1.3/100 person-years (19 patients out of 351; 5.4%). The median time to diagnosis was 38.8 months (0–113). Conclusion: The de novo HCC rates did not differ between the DAA-treated patients and those who received IFN. Achievement of SVR is of utmost importance for HCC prevention.

Eine Cholezystolithiasis (CCL) ist haufig und betrifft 15–20 % der erwachsenen Bevolkerung. In 10–15 % der Patienten mit CCL kommt es zu einer Choledocholithiasis (CDL) als Folge einer Steinmigration aus der Gallenblase. Die endoskopische Therapie der CDL mittels endoskopischer retrograder Cholangiopankreatikographie (ERCP) hat weitgehend die chirurgische Steinentfernung abgelost und die Mortalitat bei endoskopisch behandelbaren Erkrankungen halbiert. Bei der Cholangitis sowie bei der biliaren Pankreatitis mit begleitender Cholangitis ist eine ERCP notfallmasig indiziert. Vor der Steinextraktion erfolgt in westlichen Landern standardmasig eine endoskopische Sphinkterotomie (EST), jedoch zeigt die in Asien favorisierte Sphinkteroplastie mittels hydrostatischer Ballondilatation vergleichbare Ergebnisse. Die Steinextraktion erfolgt mittels Steinextraktionskorbchen oder Steinextraktionsballonkatheter. Bei groseren Gallengangsteinen ist haufig eine Lithotripsie notwendig. Hierzu stehen die unterschiedlichen Verfahren der mechanischen oder elektrohydraulischen Lithotripsie bzw. Laserlithotripsie zur Verfugung. Der aktuelle Artikel behandelt die Indikationsstellung und technische Durchfuhrung der endoskopischen Therapie der CDL sowie das Komplikationsmanagement in besonderen Fallen.

Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown. Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial–mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs. Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), HIF2α, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration. Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin Cancer Res; 23(14); 3896–905. ©2017 AACR.

BACKGROUND & AIMS Hepatocyte death is an important factor in development and progression of cirrhosis. Cytokeratin 18-based serum markers reflecting apoptotic (M30) and overall epithelial cell death (M65 and M65EpiDeath) have been used as prognostic parameters for survival in patients with acute liver failure. However, there has been no trial investigating M30, M65 and M65EpiDeath as survival parameters in patients with cirrhosis and acute-on-chronic liver failure. METHODS Patients with cirrhosis were enrolled and followed until death, liver transplantation or last contact. M30, M65 and M65EpiDeath serum levels were quantified in patient's sera. RESULTS Three hundred and thirty-one patients were screened and 211 patients could be included in this study. The median duration of follow-up was 322 days with a range of 1-1382 days. All three cell death parameters correlated with the extent of the severity of the disease. However, M65EpiDeath was the only of the three parameters which was associated with the severe complications of cirrhosis including ascites, spontaneous bacterial peritonitis and hepatorenal syndrome. Additionally, M65EpiDeath was the only cell death parameter which was independently from liver function and its surrogate parameter such as Child-Pugh score and the model of end-stage liver disease associated with overall survival. CONCLUSIONS Epithelial cell death reflected by M65EpiDeath serum levels is an indicator for the severity of cirrhosis and a prognostic survival parameter in cirrhotic patients.

MicroRNAs (miRNAs) circulating extracellularly in the blood are currently intensively studied as novel disease markers. However, the preanalytical factors influencing the levels of the extracellular miRNAs are still incompletely explored. In particular, it is unknown, whether the incubation of blood samples as occurring in clinical routine can lead to a release of miRNAs from blood cells and thus alter the extracellular miRNA levels before the preparation of serum or plasma from the blood cells. Using a set of marker miRNAs and quantitative RT-PCR, we found that the levels of extracellular miRNA-1, miRNA-16, and miRNA-21 were increased in EDTA and serum collection tubes incubated for 1-3 hours at room temperature and declined thereafter; the levels of the liver-specific miRNA-122 declined monophasically. These events occurred in the absence of significant hemolysis. When the blood was supplemented with Ribonuclease A inhibitor, the levels of miRNA-1, miRNA-16, and miRNA-21 increased substantially during the initial 3 hours of incubation and those of miRNA-122 remained unchanged, indicating that the release of blood cell-derived miRNAs occurred during the initial 3 hours of incubation of the blood tubes, but not at later time points. Separation of 5-hour preincubated blood into vesicle and nonvesicle fractions revealed a selective increase in the portion of vesicle-associated miRNAs. Together, these data indicate that the release of vesicle-associated miRNAs from blood cells can occur in blood samples within the time elapsing in normal clinical practice until their processing without significant hemolysis. This becomes particularly visible on the inhibition of miRNA degradation by Ribonuclease A inhibitor.

We have recently shown that major alterations of serum sphingolipid metabolites in chronic liver disease associate significantly with the stage of liver fibrosis in corresponding patients. In the current study we assessed via mass spectrometry serum concentrations of sphingolipid metabolites in a series of 122 patients with hepatocellular carcinoma (HCC) compared to an age- and sex-matched series of 127 patients with cirrhosis. We observed a highly significant upregulation of long and very long chain ceramides (C16-C24) in the serum of patients with HCC as compared to patients with cirrhosis (P < 0.001). Accordingly, dihydro-ceramides, synthetic precursors of ceramides and notably sphingosine, sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) were upregulated in patients with HCC (P < 0.001). Especially the diagnostic accuracy of C16-ceramide and S1P, assessed by receiver operating curve (ROC) analysis, showed a higher area under the curve (AUC) value as compared to alpha fetoprotein (AFP) (0.999 and 0.985 versus 0.823, P < 0.001 respectively). In conclusion, serum levels of sphingolipid metabolites show a significant upregulation in patients with HCC as compared to patients with cirrhosis. Particularly C16-ceramide and S1P may serve as novel diagnostic markers for the identification of HCC in patients with liver diseases. Our data justify further investigations on the role of sphingolipids in HCC.

iRGD is a derivative of the integrin-binding peptide RGD, which selectively increases the penetrability of tumor tissue to various coadministered substances in several preclinical models. In this study, we investigated the ability of iRGD to improve the delivery of sorafenib and doxorubicin therapy in hepatocellular carcinoma (HCC) using established mouse models of the disease. A contrast-enhanced MRI method was developed in parallel to assess the in vivo effects of iRGD in this setting. We found that iRGD improved the delivery of marker substances to the tumors of HCC-bearing mice about three-fold without a parallel increase in normal tissues. Control peptides lacking the critical CendR motif had no effect. Similarly, iRGD also selectively increased the signal intensity from tumors in Gd-DTPA–enhanced MRI. In terms of antitumor efficacy, iRGD coadministration significantly augmented the individual inhibitory effects of sorafenib and doxorubicin without increasing systemic toxicity. Overall, our results offered a preclinical proof of concept for the use of iRGD coadministration as a strategy to widen the therapeutic window for HCC chemotherapy, as monitored by Gd-DTPA–enhanced MRI as a noninvasive, clinically applicable method to identify iRGD-reactive tumors. Cancer Res; 75(15); 3147–54. ©2015 AACR.

Die chronische Hepatitis-C-Virus(HCV)-Infektion ist die Hauptursache der Leberzirrhose, des hepatozellularen Karzinoms und der Lebertransplantation. Die Therapie unterliegt einer rasanten Entwicklung. Neue direkt antiviral wirksame Substanzen haben die Effektivitat gegenuber der zuvor verfugbaren Therapie mit pegyliertem Interferon und Ribavirin bei zugleich geringeren Nebenwirkungen deutlich verbessert. Mit den neuen Regimen lassen sich je nach Genotyp, Fibrosestadium und Vortherapie mehr als 90 % der Patienten heilen. Die Therapie der HCV ist meist interferonfrei und dauert 12 oder 24 Wochen, in ausgewahlten Fallen nur 8 Wochen. Fur die Behandlung des HCV-Genotyps 1 ist zum einen die Kombination des Polymeraseinhibitors Sofosbuvir mit dem Proteasehemmer Simeprevir oder den NS5A-Inhibitoren Daclatasvir bzw. Ledipasvir verfugbar, zum anderen die Dreierkombination von Paritaprevir, Ombitasvir und Dasabuvir. In besonderen Fallen kann die zusatzliche Gabe von Ribavirin erforderlich sein.

During endoscopic retrograde cholangiopancreatography (ERCP), a guidewire is used to cannulate biliary strictures and allow for therapeutic interventions. The aim of this study was to assess the success of stricture cannulation using a combination of a flexible guidewire and a stable nitinol wire vs. a novel, single, stiff-shaft, flexible-tip guidewire.Consecutive patients who were scheduled for ERCP for biliary obstruction were randomized to undergo the procedure with either a 260-cm long, angled-tip hydrophilic wire in combination with a nitinol wire as required (standard group), or a novel, 270-cm guidewire featuring a hyperflexible, hydrophilic tip with a stiff shaft (novel group). At unsuccessful negotiation of the stricture, patients in the standard group were switched to the novel guidewire and vice versa ("crossover"). Successful cannulation (primary success: as assigned; final success: after "crossover"), procedure time, and total number of wires needed per procedure were compared.A total of 222 patients were randomized and 197 were included in the study (97 in the standard group and 100 in the novel group). The primary success rate was significantly higher in the novel group (94/100, 94 %) compared with the standard group (77/97, 79 %; P = 0.00041), and final success was similar. Mean time (median, interquartile range) to stricture cannulation was 11.2 minutes (6.3, 3.7 - 14.6) in the standard group and 8.1 minutes (2.5, 0.9 - 7.7) in the novel group (P 0.0001). The mean total procedure time was 31.2 minutes (24.6, 16.5 - 40.8) vs. 24.3 minutes (16.9, 10.0 - 31.5), respectively (P = 0.0011). There were no complications observed with either of the guidewires.A guidewire that features a flexible tip with a stable shaft could replace the use of a combination of flexible and stable guidewires and increase the success rate of stricture cannulation while decreasing the procedure time.ClinicalTrials.gov Identifier: NCT 01382680.

Soluble CD163 (sCD163), a marker for macrophage activation, was found to be associated with the severity of liver cirrhosis. The aim of the current study was to investigate whether serum sCD163 levels correlate with liver inflammation and fibrosis in patients with chronic hepatitis B virus (HBV) infection. In a retrospective cohort study, serum sCD163 levels were assessed by ELISA together with clinical and laboratory data in 186 patients with chronic HBV infection and 15 healthy controls. The relation between parameters for liver fibrosis and necroinflammation and sCD163 levels was analysed. Additionally, sCD163 was quantified in a subset of follow-up serum samples after initiation of antiviral treatment. sCD163 levels differed among phases of chronic HBV infection (P < 0.0001), and sCD163 concentrations were associated with inflammatory activity and fibrosis in the liver. sCD163 levels ≥ 1961 ng/l had a high specificity in the identification of subjects with substantial fibrosis (F ≥ 2). sCD163 concentrations decreased significantly after initiation of antiviral treatment. The correlation of sCD163 levels with necroinflammation and fibrosis and the sCD163 decline under treatment indicates that macrophage activation plays a role in HBV-related liver pathogenesis.

Background & Aims Recurrence of hepatitis C virus (HCV) infection after orthotopical liver transplantation (OLT) is common and associated with reduced graft and patient survival. The protease inhibitor telaprevir may enhance virological response rates in patients after OLT in combination with pegylated interferon-alfa and ribavirin. Pharmacokinetic studies have shown significant drug–drug interactions between telaprevir and immunosuppression (IS), but telaprevir pharmacokinetics in OLT patients with IS are unknown. Aim of the present study was to analyse telaprevir plasma concentrations in patients with HCV genotype 1 infection after OLT in comparison to patients without OLT and IS. Methods Five patients with HCV genotype 1 infection after OLT and 37 HCV genotype 1-infected patients patients without prior OLT were treated with telaprevir 2250 mg daily, ribavirin 1000/1200 mg daily and pegylated interferon-alfa-2a 180 μg once weekly (triple therapy). Telaprevir plasma concentrations were analysed by liquid chromatography–electrospray-ionization-tandem mass spectrometry. HCV RNA was assessed by automatized reverse-transcription polymerase chain-reaction. Results Median (range) telaprevir plasma concentrations of TW 4, 8 and 12 were 3970 (1980–4430) ng/ml and 2520 (1870–8730) ng/ml in patients after OLT and ciclosporin- or tacrolimus-based IS, respectively, as compared to 2790 (1870–3140) in non-OLT patients (P = 0.3). In one patient with tacrolimus-based IS, telaprevir dose had to be adjusted to achieve virological response. Telaprevir plasma concentrations were steady at treatment weeks 4, 8 and 12 in patients with and without IS. Conclusions Telaprevir drug monitoring may be necessary in patients with tacrolimus-based IS in patients with HCV graft infection after OLT.

Summary Background Vitamin D is involved in many biological processes. The role of vitamin D in patients with hepatocellular carcinoma (HCC) remains inconclusive, although there is evolving evidence that vitamin D may modulate cancer development and progression. Aim To evaluate serum vitamin D as prognostic parameter in HCC, we performed a prospective cohort study. Methods HCC patients were prospectively recruited and 25-hydroxyvitamin D3 (25(OH)D3) levels were determined. 25(OH)D3 levels were compared to stages of cirrhosis and HCC stages with nonparametric Kruskal–Wallis tests and Spearman correlations in 200 HCC patients. The association of the 25(OH)D3 levels and overall survival (OS) was assessed in uni- and multivariate Cox regression models. Results Two-hundred patients with HCC were included. The mean follow-up time was 322 ± 342 days with a range of 1–1508 days. Nineteen patients underwent liver transplantation and 60 patients died within the observation time. The mean serum 25(OH)D3 concentration was 17 ± 13 ng/mL with a range of 1–72 ng/mL. 25(OH)D3 serum levels negatively correlated with the stage of cirrhosis as well as with stages of HCC. Patients with severe 25(OH)D3 deficiency had the highest mortality risk (hazard ratio 2.225, 95% confidence interval 1.331–3.719, P = 0.002). Furthermore, very low 25(OH)D3 levels were associated with mortality independently from the MELD score and high alpha-Fetoprotein levels (>400 ng/mL) in a multivariate Cox regression model. Conclusions We conclude that 25(OH)D3 deficiency is associated with advanced stages of hepatocellular carcinoma and it is a prognostic indicator for a poor outcome.

Background & Aims: Soluble CD163 (sCD163) is shed in the blood circulation by activated macrophages, correlates strongly with the hepatic venous pressure gradient (HVPG) and is thereby a good indicator of portal hypertension. It is unknown whether sCD163 correlates with the risk of variceal bleeding and overall survival (OS) in patients with liver cirrhosis. We performed a prospective study to investigate if sCD163 serum levels correlate with the risk of variceal bleeding and OS in cirrhotic patients. Methods: Patients with liver cirrhosis were prospectively enrolled and followed until death or last contact. At the day of inclusion in the study, blood samples were taken and sCD163 serum levels were assessed by ELISA (enzyme-linked immunosorbent assay). The time until the end points death and variceal bleeding was assessed and the risks of death or variceal bleeding were calculated with uni- and multivariate Cox regression analyses. Results: High sCD163 levels (>4100 ng/L) were associated with death independently of the MELD (model of end stage liver disease) score, CRP (C-reactive protein), age and gender. Furthermore, high sCD163 levels were associated with gastrointestinal bleeding independently of the variceal stage and red spots. Conclusions: The sCD163 serum level is a new independent noninvasive risk factor for death and variceal bleeding in cirrhotic patients. 2013 European Association for the Study of the Liver. Published

Immunotherapy in cancer is a recent and very promising approach, namely the inhibition of the PD/programmed death-ligand 1 (PD-L1) axis. Here we aimed to investigate the prognostic value of a soluble form of PD-L1 in hepatocellular carcinoma (HCC) patients.HCC patients were prospectively recruited and soluble programmed death-ligand 1 (sPD-L1) levels were determined. sPD-L1 levels were compared to stages of cirrhosis and HCC. The association of the sPD-L1 levels and overall survival (OS) was assessed.Two hundred fifteen patients with HCC were prospectively included. The median serum sPD-L1 concentration in patients with HCC was 0.5 ng/ml (range 0.03-6.04). Soluble PD-L1 levels positively correlated with the stage of cirrhosis and with stages of HCC. Furthermore, sPD-L1 correlated positively with a marker of macrophage activation (sCD163) and inflammation (C-reactive protein). The cut-off for high-level sPD-L1 (0.8 ng/ml) was defined by sPD-L1 levels determined in a healthy control cohort. Patients with high serum sPD-L1 concentrations had an increased mortality risk (hazard ratio 3.340, 95 % confidence interval 1.609-6.934, P0.001), while very low PD-L1 levels seem to come along with better prognosis. High sPD-L1 levels were associated with mortality independently from cirrhosis stage, alpha-fetoprotein and sCD163 levels in a multivariate Cox regression model.We conclude that a high sPD-L1 level is a possible prognostic indicator for a poor outcome in HCC patients. The predictive value of sPD-L1 levels for a successful anti-PD1/PD-L1 therapy should be investigated in the future.

Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.

Background MicroRNA-122 is a liver specific microRNA and is elevated in the sera of patients with chronic hepatitis C virus infection. Hepatic microRNA-122 levels have been described to be reduced in patients with non-response to antiviral treatment with pegylated interferon-α and ribavirin. Aim Assessment of differences in serum microRNA-122 levels in patients with sustained virological response and non-response. Methods RNA was extracted from pretreatment serum samples and microRNA-122 and microRNA-16 levels were measured by quantitative PCR and compared in patients with sustained virological response and non-response. Results The levels of microRNA-122 and microRNA-16 in the sera did not differ between patients with sustained virological response and non-response. Conclusion Serum microRNA-122 is not a suitable marker for treatment response prediction to combination therapy with pegylated interferon-α and ribavirin in patients with chronic hepatitis C virus infection.

Summary. About half of the patients with chronic hepatitis C are still not cured by treatment with the current standard of care, peginterferon α/ribavirin. Direct antiviral drugs may overcome the limitations of standard antiviral therapy. The most promising new agents are inhibitors of the NS3/4A protease, the NS5B polymerase and the NS5A protein. Several compounds against these targets have entered clinical evaluation. Early clinical trials have emphasized the high potential for selecting resistant Hepatitis C virus variants. Furthermore, development of several new direct antivirals was stopped because of concerns over tolerability and safety. Then, in 2010, two phase III trials with the NS3/4A protease inhibitors boceprevir (SPRINT-2) and telaprevir (ADVANCE) showed that the combination of these compounds with standard care increases sustained virologic response rates in treatment-naive genotype 1 patients from 38–44% to 66–75%. Future goals of therapy with direct antiviral agents are to improve tolerability, shorten the duration of therapy and overcome the issue of resistance. Several studies have been initiated that combine different novel therapies, with and without interferon α/ribavirin.

a b s t r a c t Background: Reduction of necroinflammatory activity is a major goal of antiviral therapy of patients with chronic hepatitis B. Serum ALT does not detect all forms of cell death. Objectives: To analyze dynamics of novel serum cell death markers for apoptosis and necrosis in associ- ation with virologic response to nucleos(t)ide (Nuc) analogue treatment. Study design: Quantification of the M30-apoptosis neoepitope and the cytokeratin-18 (M65-necrosis) serum levels before and during treatment of patients with chronic hepatitis B with Nuc (n = 26). Results: Before treatment, M30-apoptotic activity was significantly correlated with M65-necrosis and fibrosis but not with serum ALT. During therapy with Nucs, cell death parameters M30-apoptosis, M65- necrosis, and ALT declined in association with virologic response. The most frequent cell death pattern was simultaneous decline of ALT and M30-apoptosis which occurred more frequently in patients with HBs- Antigen decline than in patients with HBs-Antigen increase during treatment (87.5% vs. 40.0%; p = 0.024). ALT decline in association with increase of M30 apoptosis was frequent in patients with HBs-Antigen increase during treatment (36.3%) but was not observed in patients with HBs-Antigen decline during treatment. Conclusion: Decline of cell death parameters in association with decline of HBV-DNA and HBs-Antigen indicates a reduction in overall cell death activity during Nuc treatment supporting the concept that response to Nuc therapy reduces necroinflammatory activity and progression of liver disease. © 2011 Elsevier B.V. All rights reserved.

miR-122 is a liver-specific microRNA, which also circulates in the blood. The levels of miR-122 in serum and plasma correlate with hepatic necroinflammation in patients with hepatitis B virus (HBV) infection. Here, we investigated whether miR-122 levels correlate with surrogate markers for viral replication and translation. Furthermore, we examined whether miR-122 levels differ in the different groups of HBV-infected patients and whether miR-122 levels may be useful to identify patients with higher or lower risk for liver disease progression. Therefore, RNA was extracted from sera of therapy-naive patients with HBV infection (n = 89) and from healthy volunteers (n = 19). The concentration of miR-122 was assessed by quantitative real-time reverse-transcription PCR. HBs antigen and HBV DNA levels were quantified as surrogate parameters for HBV replication and translation. Liver biopsies were examined according to the histological activity index and the degree of fibrosis was assessed. We found that the miR-122 serum concentration correlated with the level of ALT, HBV DNA and HBs antigen (r = 0.259, P 3500 IU/mL) or low (

The liver contains large amounts of microRNA-122 (miR-122), whereas other tissues contain only marginal amounts of this miRNA. MicroRNAs have also been found to circulate in the blood in a cell-free form; their potential as readily accessible disease markers is currently evaluated. Here, we investigated if the serum levels of miR-122 might be useful as disease parameter in patients with chronic hepatitis C virus (HCV) infection.RNA was extracted from sera of patients with chronic HCV infection (CHC) and healthy controls and was analyzed for miR-22 content by quantitative real-time reverse-transcription polymerase chain reaction. miR-122 serum levels were correlated with standard parameters of liver function. Liver biopsies from the same patients were examined for the histologic activity index (HAI) and the degree of fibrosis.Sera from patients with CHC contained higher levels of miR-122 than sera from healthy controls. Serum miR-122 levels correlated well with markers of liver inflammatory activity, that is, the serum levels of alanine leucine transaminase (ALT) and aspartate transaminase, and the HAI score. In patients with persistently normal ALT levels, serum miR-122 levels did not differ from healthy controls. There was no correlation of serum miR-122 levels with serum albumin, international normalized ratio, liver fibrosis, or serum HCV RNA.The serum level of miR-122 strongly correlates with serum ALT activity and with necroinflammatory activity in patients with CHC and elevated ALT levels, but not with fibrosis stage and functional capacity of the liver.

Background & Aims: Thrombopoietin receptor agonists are a new class of compounds licenced for the treatment of immune thrombocytopenic purpura. They are currently being studied for patients with thrombopenia in advanced liver disease or under therapy for hepatitis C. There are indications that the risk for development of portal vein thrombosis in patients with advanced liver cirrhosis might be increased under therapy with thrombopoietin receptor agonists. We report a case of a patient with Child class B liver cirrhosis with concurrent immune thrombocytopenic purpura that developed portal vein thrombosis under therapy with the thrombopoietin receptor agonist romiplostim. Methods: A 50-year-old woman with hepatitis C virus associated immune thrombocytopenic purpura and Child class B liver cirrhosis presented in our emergency with rapidly evolving hydropic decompensation and general malaise. For immune thrombocytopenic purpura, the patient was started on the thrombopoietin receptor agonist romiplostim nine months ago. Results: During hospitalization, the platelet count was measured above 330,000/ll and partial portal vein thrombosis was diagnosed by imaging studies. The thrombotic event was assumed to be associated with the romiplostim treatment for immune thrombocytopenic purpura via excessive elevation of platelet count. After anticoagulation with heparin and cessation of romiplostim treatment, complete recanalisation of the portal vein was achieved. Conclusions: We conclude that romiplostim should be used with precaution in patients with hepatitis C-associated immune thrombocytopenic purpura and advanced liver cirrhosis as the risk for thrombotic complications may increase significantly. 2011 European Association for the Study of the Liver. Published

Summary. The aim of the present study was to investigate the variability of hepatitis C virus (HCV) CD81 binding regions (CD81-1/2) in peripheral blood mononuclear cells (PBMC)-derived and serum-derived HCV-RNA samples. HCV-RNA was isolated from PBMC (104 cells) and serum samples from 37 patients chronically infected with HCV genotype 1a/1b (n = 21/16). The hypervariable regions 1/2 (amino acid 384–410, amino acid 474–482) and regions CD81-1/2 (amino acid 474–494, amino acid 522–551) were analysed. Mutational frequency of amino acid sequences was compared between PBMC-derived and serum-derived HCV variants as well as local accumulation of mutations. Furthermore, CD81 was quantified on PBMC. Mutational frequency was not different between PBMC-derived and serum-derived HCV variants. A trend to lower mutational frequency in genotype 1a PBMC variants compared with serum-derived variants was observed in region CD81-2 (5%vs 10%). Smoothed mutational frequency analysis showed a significantly lower variability within genotype 1a CD81-2 in PBMC-derived compared to serum-derived HCV-RNA (P = 0.026). CD81 expression on PBMC was not correlated with the number of mutations within the CD81 binding regions. Conclusion: A higher conservation was observed in region CD81-2 in PBMC-derived versus serum-derived HCV-RNA indicating selection of HCV variants on PBMC. The variability in the CD81 binding regions appeared to be independent from CD81 expression.

Introduction Interferon alpha (IFN) down regulates CD81 expression on peripheral blood mononuclear cells (PBMC) in patients with chronic hepatitis C virus (HCV) infection. Aim of our study was to investigate whether amantadine alters IFN associated down regulation of CD81 expression on PBMC in patients with chronic hepatitis C. Methods Nineteen patients with chronic HCV infection received peginterferon alpha-2a/ribavirin (SOC) for 48 weeks. Patients were randomised to 12 weeks amantadine therapy ( n = 12) or no additional treatment ( n = 7). FACS analysis of CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was performed at baseline, week (TW) 4, TW12, and TW24 of antiviral therapy. Results A significant decline of CD81 expression was observed on CD4(+), CD8(+), and CD56(+) cells ( p = 0.011, p p = 0.015, respectively) but not on CD19(+) cells ( p > 0.2). CD81 expression on CD4(+), CD8(+), CD19(+), and CD56(+) cells was not different between patients treated with SOC plus amantadine and patients treated with SOC alone. Conclusion The current study confirms that CD81 expression is down regulated by SOC on CD4(+), CD8(+) and CD56(+) cells. Amantadine treatment was not associated with CD81 expression. Interaction between amantadine and CD81 is unlikely to be involved in potential antiviral activity of amantadine in chronic HCV infection.

The hepatitis C virus (HCV) nonstructural (NS) protein 4B is known for protein-protein interactions with virus and host cell factors. Only little is known about the corresponding protein binding sites and underlying molecular mechanisms. Recently, we have predicted a putative basic leucine zipper (bZIP) motif within the aminoterminal part of NS4B. The aim of this study was to investigate the importance of this NS4B bZIP motif for specific protein-protein interactions. We applied in silico approaches for 3D-structure modeling of NS4B-homodimerization via the bZIP motif and identified crucial amino acid positions by multiple sequence analysis. The selected sites were used for site-directed mutagenesis within the NS4B bZIP motif and subsequent co-immunoprecipitation of wild-type and mutant NS4B molecules. Respective interaction energies were calculated for wild-type and mutant structural models. NS4B-homodimerization with a gradual alleviation of dimer interaction from wild-type towards the mutant-dimers was observed. The putative bZIP motif was confirmed by a co-immunoprecipitation assay and western blot analysis. NS4B-NS4B interaction depends on the integrity of the bZIP hydrophobic core and can be abolished due to changes of crucial residues within NS4B. In conclusion, our data indicate NS4B-homodimerization and that this interaction is facilitated by the aminoterminal part containing a bZIP motif.

356 Background: The aim of the study was to evaluate the risk of HCC development after treatment with direct-acting antivirals (DAA) and to compare hepatocellular carcinoma (HCC) occurrence to patients treated with interferon (IFN)-based therapies. Methods: We analyzed a large cohort with chronic HCV patients for the onset of new hepatocellular carcinoma after DAA treatment. A historic interferon treated cohort was investigated for comparison. Results: 819 patients were included in the DAA group, 269 (32.8%) had established cirrhosis. Median follow up was 263 days (0-1001). 25 patients (3.1%) were diagnosed with de novo HCC within the observation time. All of these patients had established cirrhosis. Patients with new HCC were mostly male, older, treatment experienced, had a lower SVR12 rate and higher levels of liver inflammation markers. Investigation of the subcohort of 269 cirrhotic patients yielded a HCC rate of 9.3% during the follow-up of approximately one year. Non-SVR12 was an independent risk factor for de novo HCC (OR 4.983, 1.39-17.88, 0.014). Most HCCs were diagnosed in early stage BCLC A. In the historical cohort of 351 IFN treated patients the rate of de novo HCC was 5.4% overall and 11.8% in patients with already established cirrhosis (n = 68). In the multivariate analysis failed SVR (OR 5.386, 1.155-25.108, p = 0.032) remained an independent risk factor for de novo HCC. In a combined analysis of all patients (DAA and IFN treated) in a multivariate approach male gender, failed SVR and cirrhosis were independent factors for HCC development. Conclusions: DAA treatment in cirrhotic patients does not seem to reduce the risk of HCC development in the short term. HCC rates were not different between DAA-treated patients and those who received interferon. Achievement of SVR seems to be the most important aim to prevent HCC development.