Search

The diagnostic value of liver biopsy in patients with acute liver injury or acute liver failure (ALI/ALF) was investigated.Data from the initial event and follow-up visits were retrospectively analyzed in all patients with a liver biopsy during ALI/ALF from January 2010 to May 2020 at the University Hospital Frankfurt, Germany.The cohort comprised 66 patients. Post-biopsy hemorrhage occurred in 2 of 66 but was self-limited. In five patients suspected liver involvement by a systemic extrahepatic disease was confirmed and excluded in eight patients. In 4 of 66 patients, the etiology of ALI/ALF remained unknown. Liver biopsy hinted at the etiology of ALI/ALF in 2 of 6 patients with rare diagnoses (hemophagocytic lymphohistiocytosis: 2 of 66; ischemic liver injury: 1 of 66, ALI/ALF due to a systemic infection: 3 of 66). In 31 of 34 patients with drug-induced liver injury (DILI), histopathology suggested DILI; in further 2 patients, DILI was among the differential diagnoses. However, DILI was also the histopathologically preferred diagnosis in 12 of 15 patients with autoimmune hepatitis (AIH). Only in 3 of 15 patients, histopathology was considered compatible with AIH. Serum immunoglobulin G (IgG) and autoantibodies during ALI/ALF were higher in patients with AIH than with DILI. Patients with AIH did not show a more pronounced biochemical response to corticosteroids in the first 10 days of treatment than patients with DILI.Liver biopsy is indispensable when liver involvement by an extrahepatic disease is suspected. To distinguish AIH from DILI in ALI/ALF, serum IgG, and autoantibodies seem more helpful than liver biopsy; long-term follow-up is needed in these patients.

Bulevirtide is a first-in-class peptidic entry inhibitor for hepatitis B virus (HBV) and hepatitis D virus infection. In July, 2020, bulevirtide 2 mg received conditional marketing authorisation by the European Medical Agency for treatment of chronic hepatitis D virus infection. We investigated the antiviral activity of bulevirtide in patients chronically infected with HBV and hepatitis D virus.MYR202 (ClinicalTrials.gov, NCT03546621; EudraCT, 2016-000395-13) was a multicentre, parallel-group, randomised, open-label, phase 2 trial. Adults (aged 18-65 years) with chronic hepatitis D virus infection, including patients with cirrhosis and patients who had contraindications to PegIFNα treatment or for whom treatment did not work, were eligible and were enrolled from four hospitals in Germany and 12 hospitals in Russia. Patients were randomly assigned (1:1:1:1) to receive 2 mg (n=28), 5 mg (n=32), or 10 mg (n=30) subcutaneous bulevirtide once per day with tenofovir disoproxil fumarate (TDF; 245 mg once per day orally) or TDF alone (245 mg once per day orally; n=30) for 24 weeks. Randomisation was done using a digital block scheme with stratification, consisting of 480 randomisation numbers separated into 30 blocks. The primary endpoint was undetectable hepatitis D virus RNA or 2 logBetween Feb 16, 2016, and Dec 8, 2016, 171 patients with chronic hepatitis D virus infection were screened; 51 were ineligible based on the exclusion criteria and 120 patients (59 with cirrhosis) were enrolled. At week 24, 15 (54%, 95% CI 34-73) of 28 patients achieved undetectable hepatitis D virus RNA or a 2 logBulevirtide induced a significant decline in hepatitis D virus RNA over 24 weeks. After cessation of bulevirtide, hepatitis D virus RNA concentrations rebounded. Longer treatment durations and combination therapies should be investigated.Hepatera LLC, MYR GmbH, and the German Centre for Infection Research, TTU Hepatitis.

The urgent need in HCV-infected patients with liver disease mandated the rapid implementation of IFN-free DAA combination therapies following their regulatory approval in 2014 and 2015 without full knowledge of the optimal combinations and regimens. Investigating the evolution of the DAA utilization patterns and treatment outcomes could provide learnings for future situations. This was an analysis of a prospective observational database from the German Hepatitis C Registry (DHC-R) covering a period from May 2014 to September 2015. Adult patients had evidence of chronic HCV GT1 or GT4 infection and were treated with an IFN-free combination regimen of simeprevir (SMV) + sofosbuvir (SOF) or other IFN-free regimens: daclatasvir + sofosbuvir (DCV + SOF), ledipasvir/sofosbuvir (SOF/LDV), paritaprevir/r + ombitasvir ± dasabuvir (PrOD), with or without ribavirine (R). A total of 5496 subjects were followed during the period. During this period, clinical recommendations and treatment patterns evolved rapidly in response to new evidence from clinical trials and clinical routine and regulatory approval of additional regimens. High SVR12 rates were seen in this cohort, even in hard-to-treat patient subgroups. In the multivariate analysis, gender, age, advanced cirrhosis, and intensified treatment for cirrhotics were associated with treatment outcome. Despite limited knowledge of the optimal utilization of the newly approved DAA combinations and treatment durations as well as their comparative efficacy and safety profiles, high SVR rates were achieved regardless of the DAA combination. These outcomes were facilitated by the rapid adaptation of clinical recommendations. Future situations with high unmet medical need may follow a similar approach. Die dringende Behandlungsbedürftigkeit HCV-infizierter Patienten mit Lebererkrankung verlangte die rasche Einführung IFN-freier DAA-Kombinationstherapien nach ihrer Zulassung in 2014 und 2015 trotz unvollständigem Wissen über deren optimalen Einsatz. Die Untersuchung der Evolution des Einsatzes und der Ergebnisse könnte wichtige Erkenntnisse für zukünftige Situationen bergen. Diese Analyse einer prospektiven Kohorte des Deutschen Hepatitis C-Registers (DHC-R) deckt den Zeitraum von Mai 2014 bis September 2015 ab. Erwachsene Patienten mit chronischer HCV-GT1 oder GT4-Infektion wurden mit einer IFN-freien Therapie aus Simeprevir (SMV) + Sofosbuvir (SOF), oder anderen IFN-freien Regimen: Daclatasvir + Sofosbuvir (DCV + SOF), Ledipasvir/Sofosbuvir (SOF/LDV), Paritaprevir/r + Ombitasvir ± Dasabuvir (PrOD), mit oder ohne Ribavirin (R) behandelt. 5496 Patienten wurden während des Zeitraums beobachtet. In dieser Periode wandelten sich die klinischen Leitlinien und Behandlungsmuster rasch in Folge neuer Evidenz aus klinischen Studien und klinischer Erfahrung und der Zulassung von neuen Regimen. Hohe SVR12-Raten wurden in dieser Kohorte, selbst in schwierig zu behandelnden Patienten-Subgruppen, beobachtet. Die multivariate Analyse zeigte, dass Geschlecht, Alter, fortgeschrittene Zirrhose und die intensivierte Therapie bei Patienten mit Zirrhose mit dem Behandlungserfolg assoziiert waren. Trotz begrenzten Wissens um den optimalen Einsatz von neu zugelassenen DAA-Kombinationen und deren Behandlungsdauer sowie dem Vergleich ihrer Wirksamkeits- und Sicherheits-Profile wurden hohe SVR12-Raten mit verschiedenen DAA-Kombinationen erzielt. Diese Ergebnisse wurden unterstützt von einer raschen Adaption klinischer Therapie-Empfehlungen. Zukünftige Szenarien mit einem erheblichen ungedeckten medizinischen Bedarf könnten einem ähnlichen Ansatz folgen.

Carboxylesterase 2 (CES2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. Novel treatment strategies are exceedingly needed for cholangiocarcinoma (CCA) patients. Here, we assessed CES2 expression by immunohistochemistry in a CCA cohort comprising 171 non-liver fluke associated, intrahepatic (n = 72) and extrahepatic (perihilar: n = 56; distal: n = 43) CCAs. Additionally, 80 samples of high-grade biliary intraepithelial neoplastic tissues and 158 corresponding samples of histological normal, non-neoplastic biliary tract tissues were included. CES2 expression was highest in non-neoplastic biliary tissue and significantly decreased in CCA. Patients showing any CES2 expression in tumor cells had a significantly better overall survival compared to negative cases (p = 0.008). This survival benefit was also maintained after stratification of CES2-positive cases, by comparing low, medium and high CES2 expression levels (p-trend = 0.0006). Evaluation of CCA subtypes showed the survival difference to be restricted to extrahepatic tumors. Correlation of CES2 expression with data of tumor-infiltrating immune cells showed that particularly CD8+ T cells were more frequently detected in CES2-positive CCAs. Furthermore, treatment of CCA cell lines with the prodrug Irinotecan reduced cell viability, increased cytotoxicity and modulated inflammatory gene expression. In conclusion, reduced CES2 expression is associated with poor outcome and low CD8+ T cell infiltration in CCA patients. Further clinical studies could show, whether CES2 expression may serve as a predictive marker in patients treated with prodrugs converted by CES2.

Virologic failure to approved combinations of direct antiviral agents (DAA) in patients with chronic hepatitis C virus (HCV) infection is rare. Mostly it involves difficult to treat patients with advanced liver disease and prior interferon-experience. Before approval of VOX/VEL/SOF, a restricted number of patients received rescue treatment, and the choice of DAA combinations for re-treatment were selected on an individual basis. In the present analysis, patient characteristics and rescue-regimens after virologic failure mainly based on first generation DAAs are described. Data were obtained from the German Hepatitis C-Registry (DHC-R), which is a national multicenter real-world cohort currently including about 16 500 patients recruited by more than 250 centers. The present analysis is based on 6683 patients who initiated a DAA therapy and for whom follow-up data (per-protocol analysis) were available. Among the patients, 188 (2.8 %) experienced a virologic relapse. Compared to SVR-patients, relapse patients were significantly more often male (77.7 % versus 56.9 %, respectively, p 0.001), showed cirrhosis significantly more (48.4 % versus 28.1 %, respectively, p 0.001) and a prior interferon-containing therapy (46.3 % versus 39.0 %, respectively, p = 0.049). The majority of patients who relapsed were infected with genotype 1 (47.4 %) followed by genotype 3 (29.8 %), and 95 relapse patients started DAA re-treatment. Characteristics of patients with rescue-treatment are similar to these of patients with relapse after initial DAA treatment. Thirty-one of 39 patients with complete follow-up data achieved SVR (79.5 %), and 8 patients had a relapse again (20.5 %). Patients who received rescue treatment including a new DAA class according to guidelines, except patients who received VOX/VEL/SOF, showed higher SVR rates than the entire group (21/25, 84 %). All patients who received VOX/VEL/SOF achieved SVR (n = 4, 100 %). Patients with failure with DAA combination therapies are a difficult but urgent to treat population with the frequent presence of cirrhosis and prior treatment failure with interferon-based therapies. Rescue therapy with inclusion of a new DAA class leads to high SVR rates, but multiple targeted therapy with VOX/VEL/SOF seems to be most effective. Virologisches Versagen nach einer zugelassenen Kombinationstherapie aus direkt antiviral wirkenden Medikamenten (DAA) bei Patienten mit chronischer Hepatitis-C-Virus(HCV)-Infektion ist sehr selten. Zumeist handelt es sich, aufgrund von fortgeschrittener Lebererkrankung und vorangegangenen interferonbasierten Therapien, um schwierig zu behandelnde Patienten. Vor Zulassung von VOX/VEL/SOF erhielt eine geringe Zahl von Patienten eine Rescue-Therapie, und die Wahl der DAA-Kombination zur Re-Therapie unterlag individuellen Entscheidungskriterien. Die vorliegende Arbeit beschreibt Charakteristika und Rescue-Therapien, überwiegend basierend auf DAAs der ersten Generation, bei Patienten nach virologischem Versagen. Die Daten entstammen dem Deutschen Hepatitis C-Register (DHC-R). Dieses ist eine nationale, multizentrische, Real-World-Kohorte, die derzeit über 16 500 Patienten aus über 250 Zentren einschließt. Die aktuelle Analyse basiert auf 6683 Patienten mit initialer DAA-Therapie und kompletten Follow-up-Daten (Per-Protocol(PP)-Analyse). 188 Patienten (2,8 %) aus der PP-Population hatten einen virologischen Relapse. Die Relapse-Patienten waren verglichen zur SVR-Population signifikant häufiger männlich (77,7 % versus 56,9 %, p 0,001), hatten signifikant häufiger eine Zirrhose (48,4 % versus 28,1 %, p 0,001) und eine interferonbasierte Vortherapie (46,3 % versus 39,0 %, p = 0,049). Die meisten Patienten mit virologischem Versagen waren mit Genotyp 1 (57,4 %) gefolgt von Genotyp 3 (29,8 %) infiziert. 95 der Relapse-Patienten erhielten eine DAA-Re-Therapie. Die Charakteristika von Patienten mit Rescue-Therapie sind vergleichbar mit denen von Patienten mit Relapse nach initialer DAA-Therapie. 31 von 39 Patienten mit kompletten Follow-up-Daten erreichten SVR (79,5 %) und 8 Patienten erlitten einen erneuten Relapse (20,5 %). Patienten, die eine den Leitlinien entsprechende Rescue-Therapie mit einer neuen DAA-Klasse erhalten haben, ausgenommen VOX/VEL/SOF, zeigten höhere SVR-Raten als die Gesamtgruppe (21/25, 84 %). Alle Patienten, die VOX/VEL/SOF erhielten, erreichten einen SVR (n = 4, 100 %). Versager nach DAA-Kombinationstherapie entsprechen einer schwierig und zugleich dringlich zu behandelnden Patientengruppe mit häufigem Vorliegen einer Zirrhose und Versagen einer interferonbasierten Vortherapie. Rescue-Therapie mit einer neuen DAA-Klasse führt zu hohen SVR-Raten, eine Multi-Target-Therapie mit VOX/VEL/SOF scheint am effektivsten zu sein.

Suppression of fatty acid absorption is one goal to fight obesity. However, the responsible molecular mechanism is poorly understood. Aim of the present study was the search for the key regulator of the overall fatty acid absorption mechanism and its pharmaceutical modulation. As experimental tool we employed the polarized human intestinal tumor derived cell line CaCo2. Here we showed that influx of fatty acids is mediated by an apical heterotetrameric plasma membrane protein complex of which the calcium-independent membrane phospholipase A2 (iPLA2s) is one constituent. The newly synthesized bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) blocked iPLA2s, which structurally disrupted the fatty acid-uptake complex. Furthermore, the inhibition of iPLA2s lead to reduction of cytosolic lysophosphatidylcholine (LPC) production which suppressed p-JNK1, as a central regulator of metabolism. In a concerted action low p-JNK1 levels prohibited synthesis of the members of the fatty acid uptake complex as well as of apolipoprotein B and the connected members of the basolateral vesicular chylomicron excretion machinery, thereby inhibiting cellular lipid excretion. The basolateral chylomicron release was shown to determine the overall fatty acid-absorption capacity as rate limiting step, whereas apical uptake replenishes the cellular stores, enabling continuous transcellular movement of fatty acids. In conclusion, the UDCA-LPE mediated inhibition of p-JNK1 represents a powerful tool to control intestinal absorption of fatty acids and, thus may be employed as a drug to treat obesity.

Distal cholangiocarcinoma (dCCA) is a biliary tract cancer with a dismal prognosis and is often preceded by biliary intraepithelial neoplasia (BilIN), representing the most common biliary non-invasive precursor lesion. BilIN are histologically well defined but have not so far been characterised systematically at the molecular level. The aim of this study was to determine miRNA-regulated genes in cholangiocarcinogenesis via BilIN. We used a clinicopathologically well-characterised cohort of 12 dCCA patients. Matched samples of non-neoplastic biliary epithelia, BilIN and invasive tumour epithelia of each patient were isolated from formalin-fixed paraffin-embedded tissue sections by laser microdissection. The resulting 36 samples were subjected to total RNA extraction and the expression of 798 miRNAs was assessed using the Nanostring® technology. Candidate miRNAs were validated by RT-qPCR and functionally investigated following lentiviral overexpression in dCCA-derived cell lines. Potential direct miRNA target genes were identified by microarray and prediction algorithms and were confirmed by luciferase assay. We identified 49 deregulated miRNAs comparing non-neoplastic and tumour tissue. Clustering of these miRNAs corresponded to the three stages of cholangiocarcinogenesis, supporting the concept of BilIN as a tumour precursor. Two downregulated miRNAs, i.e. miR-451a (-10.9-fold down) and miR-144-3p (-6.3-fold down), stood out by relative decrease. Functional analyses of these candidates revealed a migration inhibitory effect in dCCA cell lines. Activating transcription factor 2 (ATF2) and A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) were identified as direct miR-451a target genes. Specific ATF2 inhibition by pooled siRNAs reproduced the inhibitory impact of miR-451a on cancer cell migration. Thus, our data support the concept of BilIN as a direct precursor of invasive dCCA at the molecular level. In addition, we identified miR-451a and miR-144-3p as putative tumour suppressors attenuating cell migration by inhibiting ATF2 in the process of dCCA tumorigenesis. © The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Background Cholangiocarcinoma is a rapidly fatal cancer entity with a median survival of less than one year. In contrast to many other malignancies, no substantial therapeutic breakthrough has been made in the past few decades, thereby limiting the treatment to cytotoxic chemotherapy with little beneficial effect for most patients. Targeted therapy tailored to the individual has shown substantial success in the recent past as a promising avenue for cancer therapy. Methods In this study, we determined the frequency of amplification of the HER2 gene in a comprehensive and well-characterized European cholangiocarcinoma cohort encompassing 436 patients including intrahepatic (n = 155), proximal (n = 155) and distal (n = 126) cholangiocarcinoma by strict application of a combined immunohistochemical and in situ hybridization algorithm following the current guidelines for HER2 assessment in gastric cancer. Results We identified a proportion of 1.4% (n = 6) patients that demonstrated HER2 gene amplification, with the highest rate among the distal cholangiocarcinoma patients (2.4%). None of the patients with equivocal (2+) immunohistochemical staining results exhibited gene amplification molecularly. In four of the five patients with HER2 positivity, gene amplification was already present in concomitantly tested high-grade biliary intraepithelial neoplasia (80%). HER2 gene amplification was not significantly associated with other clinical parameters, including survival. Conclusions This study identifies HER2 gene amplification as a rare event in cholangiocarcinoma of the Western population, occurring already in high-grade BilIN in a subset of patients. Furthermore, we provide a robust testing algorithm that may be used prior to therapy administration in future clinical trials evaluating the role of HER2 as a predictive marker in cholangiocarcinoma.

Background DNA mismatch repair (MMR) deficiency is a major pathway of genomic instability in cancer. It leads to the accumulation of numerous mutations predominantly at microsatellite sequences, a phenotype known as microsatellite instability (MSI). MSI tumors have a distinct clinical behavior and commonly respond well to immune checkpoint blockade, irrespective of their origin. Data about the prevalence of MSI among gallbladder cancer (GBC) have been conflicting. We here analyzed a well-characterized cohort of 69 Western-world GBCs. Methods We analyzed the mononucleotide MSI marker panel consisting of BAT25, BAT26, and CAT25 to determine the prevalence of MMR deficiency-induced MSI. Results MSI was detected in 1/69 (1.4%) of analyzed GBCs. The detected MSI GBC had a classical histomorphology, i.e. of acinar/tubular/glandular pancreatobiliary phenotype, and showed nuclear expression of all four MMR proteins MLH1, MSH2, MSH6, and PMS2. The MSI GBC patient showed a prolonged overall survival, despite having a high tumor stage at diagnosis. The patient had no known background or family history indicative of Lynch syndrome. Conclusions Even though the overall number of MSI tumors is low in GBC, the potentially therapeutic benefit of checkpoint blockade in the respective patients may justify MSI analysis of GBC. Electronic supplementary material The online version of this article (10.1186/s13000-019-0813-5) contains supplementary material, which is available to authorized users.

Liver function tests (alanine aminotransferase, ALT; gamma-glutamyltransferase, GGT) not always normalize after elimination of hepatitis C virus (HCV) by direct acting antivirals (DAAs), possibly indicating concomitant non-viral liver diseases. We analysed factors determining the biochemical response (normalized ALT/GGT) of DAA therapy in a large real-world cohort.The German Hepatitis C-Registry is a national multicenter registry study. Normal ALT was defined ≤35 U/L (female) and ≤50 U/L (male) or, according to AASLD, ≤19 U/L (female) and ≤30 U/L (male), normal GGT ≤40 U/L (female) and ≤60 U/L (male).At baseline, ALT was elevated in 3705/4946 (74.9%), ALT (AASLD) in 4669/4946 (94.4%) and GGT in 3018/4906 (61.5%). In this study, 97% of patients achieved SVR12. At week 12 after end of therapy, ALT was elevated in 451/4946 (9.1%), ALT according to AASLD in 1906/4946 (38.5%) and GGT in 863/4879 (17.7%). Persistently elevated ALT after DAA therapy was independently associated with high body mass index (BMI), age70 years, liver cirrhosis, diabetes, alcohol consumption and not achieving SVR12. Using the stricter AASLD criteria, opioid substitution and male sex were additional predictors. Higher GGT at week 12 was associated with high BMI, age70 years, liver cirrhosis, diabetes, alcohol consumption, opioid substitution and non-SVR. Importantly, persistently elevated liver tests after treatment, particularly GGT, were associated with hepatic decompensation and mortality during 4-years follow-up.Risk factors at baseline (obesity, diabetes, liver cirrhosis, alcohol consumption) are independently associated with persistently elevated liver function tests after SVR, indicating that these patients warrant further hepatological follow-up.German Clinical Trials Register (DRKS; ID DRKS00009717).

Background Cholangiocarcinoma (CCA) may arise in the intra- or extrahepatic biliary tract and is associated with a poor prognosis. Despite recent advances, to date there is still no established targeted therapeutic approach available. Non-surgical therapeutic agents are urgently needed, as most patients are non-eligible to surgical resection. Anti-PD-L1 therapy prevents cancer cells from evading the immune system and has emerged as a new treatment option in several cancer entities. Recently, PD-L1 expression has been analyzed in comparably small CCA patient cohorts. However, a systematic validation of different PD-L1 antibodies has not been performed in CCA so far. Methods We stained a tissue microarray consisting of 170 patients, including 72 intrahepatic cholangiocarcinomas (iCCAs), 57 perihilar cholangiocarcinomas (pCCAs) and 41 distal cholangiocarcinomas (dCCAs) by immunohistochemistry and evaluated PD-L1 positivity in tumor and stromal cells. We analyzed three different PD-L1 antibodies (clones 28–8, SP142, and SP263) that are frequently used and recommended for predictive diagnostic testing in other cancer types. Results For PD-L1 antibody clone SP263, 5% of iCCAs, 4% of pCCAs and 3% of dCCAs exhibited PD-L1 expression on tumor cells, thereby showing the highest frequencies of PD-L1 positivity. Accordingly, highest PD-L1 positivity rates of stromal cells with 31% in iCCA, 40% in pCCA and 61% in dCCA were detected for clone SP263. Agreement of PD-L1 positivity in tumor cells was moderate for clone 28–8 and SP263 (κ = 0.44) and poor between 28-8 and SP142 (κ = 0.13), as well as SP142 and SP263 (κ = 0.11), respectively. Statistical analyses of PD-L1 expression (clone SP263) on tumor cells with clinicopathological data revealed a positive correlation with shortened overall survival in CCA patients. Conclusions Selection of appropriate PD-L1 antibodies and careful evaluation of immunohistochemical staining patterns have a significant impact on PD-L1 testing in CCA. Clinical trials are necessary to investigate the putative beneficial effects of PD-L1 targeted immunotherapy in CCA patients. Electronic supplementary material The online version of this article (10.1186/s12885-018-5254-0) contains supplementary material, which is available to authorized users.

Norursodeoxycholic acid is an orally administered side chain-shortened homologue of ursodeoxycholic acid that undergoes hepatic enrichment with hepatoprotective, anti-inflammatory, and antifibrotic activity. We assessed the efficacy of two doses of norursodeoxycholic acid versus placebo for the treatment of non-alcoholic fatty liver disease.We did a multicentre, double-blind, placebo-controlled, randomised, phase 2 dose-finding clinical trial in tertiary referral hospitals and medical centres in Austria (n=6) and Germany (n=23) for patients with non-alcoholic fatty liver disease with or without diabetes. Patients with a clinical diagnosis of non-alcoholic fatty liver disease and serum alanine aminotransferase (ALT) concentrations of more than 0·8 times the upper limit of normal were randomly assigned (1:1:1) using a computer-generated central randomisation. Patients were randomly assigned to receive either norursodeoxycholic acid capsules at 500 mg per day or 1500 mg per day, or placebo, for 12 weeks with a subsequent 4-week follow-up period. All individuals involved in the trial were masked to treatment allocation. The primary efficacy endpoint was the mean relative percentage change in ALT concentrations between baseline and end of treatment assessed in the intention-to-treat population. This trial is registered with EudraCT, number 2013-004605-38.Between March 30, 2015, and Sept 20, 2016, of 198 individuals included in the analysis, 67 patients were randomly assigned to receive 500 mg norursodeoxycholic acid, 67 to 1500 mg norursodeoxycholic acid, and 64 to placebo. A dose-dependent reduction in serum ALT between baseline and end of treatment was observed with norursodeoxycholic acid versus placebo, with a significant effect in the 1500 mg group (mean change -27·8%, 95% repeated CI -34·7 to -14·4; p0·0001). Serious adverse events (n=6) and treatment-emergent adverse events (n=314) were reported in a similar proportion of patients across groups. 112 treatment-emergent adverse events occurred in the 1500 mg group, 99 in the 500 mg group, and 103 in the placebo group. The most frequent adverse events were headache, gastrointestinal disorders, and infections (eg, diarrhoea, abdominal pain, or nasopharyngitis).Norursodeoxycholic acid at 1500 mg resulted in a significant reduction of serum ALT within 12 weeks of treatment when compared with placebo. Norursodeoxycholic acid was safe and well tolerated encouraging further studies.Dr Falk Pharma GmbH.

Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a synthetic bile acid-phospholipid conjugate with profound hepatoprotective and anti-fibrogenic functions in vitro and in vivo. Herein, we aimed to demonstrate the inhibitory effects of UDCA-LPE on pro-fibrogenic integrin signalling. UDCA-LPE treatment of human embryonic liver cell line CL48 and primary human hepatic stellate cells induced a non-classical internalization of integrin &beta, 1 resulting in dephosphorylation and inhibition of SRC and focal adhesion kinase (FAK). Signalling analyses suggested that UDCA-LPE may act as a heterobivalent ligand for integrins and lysophospholipid receptor1 (LPAR1) and co-immunoprecipitation demonstrated the bridging effect of UDCA-LPE on integrin &beta, 1 and LPAR1. The disruption of either the UDCA-moiety binding to integrins by RGD-containing peptide GRGDSP or the LPE-moiety binding to LPAR1 by LPAR1 antagonist Ki16425 reversed inhibitory functions of UDCA-LPE. The lack of inhibitory functions of UDCA-PE and UDCA-LPE derivatives (14:0 and 12:0, LPE-moiety containing shorter fatty acid chain) as well as the consistency of the translocation of UDCA-LPE and integrins, which co-fractionated with LPE but not UDCA, suggested that the observed UDCA-LPE-induced translocation of integrins was mediated by LPE endocytic transport pathway.

Chronic hepatitis C virus infection is associated with a significant health burden. Long-term consequences are the development of liver cirrhosis and hepatocellular carcinoma. The introduction of direct-acting antivirals (DAA) has led to an increase in sustained virologic response rates (SVR), but is accompanied by higher treatment costs. The aim of this study was to assess the outcomes and costs of treating hepatitis C virus infected patients with DAAs in clinical practice in Germany.Data were derived from a noninterventional study including a pharmacoeconomic subset of 2673 patients with genotypes 1 and 3 who initiated and completed treatment between February 2014 and February 2017. Sociodemographic and clinical parameters as well as resource utilization were collected using a web-based data recording system. Costs were calculated using official remuneration schemes.The mean age of the patients was 54.6 years; 48% were men. 93.5% of all patients achieved an SVR. The average total treatment costs wereOV0556;67 979 (OV0556;67 131 medication costs,OV0556;824 ambulatory care,OV0556;24 hospital costs). The average costs per SVR ofOV0556;72 705 were calculated. Differences in SVR and costs according to genotype, treatment regimen, treatment experience, and cirrhosis were observed. Quality-of-life data showed no or a minimal decrease during treatment.This analysis confirms high SVR rates for newly introduced DAAs in a real-world setting. Costs per SVR estimated are comparable to first-generation DAA. Given the fact that the costs for the currently used treatment regimens have declined, it can be assumed that the costs per SVR have also decreased. Our insight into real-world outcomes and costs can serve as a basis for a comparison with the mentioned newly introduced treatment regimens.

Background A major molecular pathway of genetic instability in cancer is DNA mismatch repair deficiency. High-level microsatellite instability (MSI-H) is currently the best predictor of responsiveness towards immune checkpoint blockade. Data about the prevalence of high-level microsatellite instability in cholangiocarcinoma (CCA) has been conflicting. Methods We employed a cohort comprising 308 Western-world, non-liver fluke-associated CCAs (159 intrahepatic, 106 perihilar, and 43 distal). We analysed the mononucleotide microsatellite instability marker panel consisting of BAT25, BAT26, and CAT25 and detected MSI-H in 4/308 CCAs (1.3%). Results Patients affected by MSI-H CCA had mostly an atypical histomorphology (p = 0.004), showed a longer overall survival, although having a high tumour stage, and were of younger age. Correlation analysis of microsatellite instability status with tumour-infiltrating immune cells, MHC I, and PD-L1 expression in the same cholangiocarcinoma cohort showed higher numbers of CD8 + T cells, FOXP3 + regulatory T cells, CD20 + B cells and high or at least moderate MHC I expression levels in MSI-H CCAs. Conclusions Even though the overall number of MSI-H CCAs is low, the dismal prognosis of the disease and the therapeutic option of immune checkpoint blockade in the respective patients justify MSI testing of cholangiocarcinoma, particularly in younger patients showing an atypical histomorphology.

Background: Non-alcoholic fatty liver disease (NAFLD) is associated with inefficient macro- and micronutrient metabolism, and alteration of circulating phospholipid compositions defines the signature of NAFLD. This current study aimed to assess the pattern of serum phospholipids in the spectrum of NAFLD, and its related comorbidities and genetic modifications. Methods: 97 patients with diagnosed NAFLD were recruited at a single center during 2013–2016. Based on histological and transient elastography assessment, 69 patients were divided into non-alcoholic steatohepatitis (NASH) and non-alcoholic fatty liver (NAFL) subgroups. 28 patients served as healthy controls. Serum phospholipids were determined by liquid-chromatography mass spectrometry (LC-MS/MS). Results: The total content of phosphatidylcholine (PC) and sphingomyelin in the serum was significantly increased in NAFL and NASH patients, compared to healthy controls. In addition, serum lysophospatidylethanolamine levels were significantly decreased in NAFL and NASH individuals. Circulating PC species, containing linoleic and α-linolenic acids, were markedly increased in NAFLD patients with hypertension, compared to NAFLD patients without hypertension. The pattern of phospholipids did not differ between NAFLD patients with diabetes and those without diabetes. However, NAFLD patients with hyperglycemia (blood glucose level (BGL) >100 mg/dL) exhibited significantly a higher amount of monounsaturated phosphatidylethanolamine than those with low blood glucose levels. In addition, NAFLD patients with proven GG-genotype of PNPLA3, who were at higher risk for the development of progressive disease with fibrosis, showed lower levels of circulating plasmalogens, especially 16:0, compared to those with CC- and CG-allele. Conclusions: Our extended lipidomic study presents a unique metabolic profile of circulating phospholipids associated with the presence of metabolic risk factors or the genetic background of NAFLD patients.

ObjectiveHomozygous alpha1-antitrypsin (AAT) deficiency increases the risk for developing cirrhosis, whereas the relevance of heterozygous carriage remains unclear. Hence, we evaluated the impact of the two most relevant AAT variants (‘Pi*Z’ and ‘Pi*S’), present in up to 10% of Caucasians, on subjects with non-alcoholic fatty liver disease (NAFLD) or alcohol misuse.DesignWe analysed multicentric case–control cohorts consisting of 1184 people with biopsy-proven NAFLD and of 2462 people with chronic alcohol misuse, both cohorts comprising cases with cirrhosis and controls without cirrhosis. Genotyping for the Pi*Z and Pi*S variants was performed.ResultsThe Pi*Z variant presented in 13.8% of patients with cirrhotic NAFLD but only in 2.4% of counterparts without liver fibrosis (pConclusionThe Pi*Z variant is the hitherto strongest single nucleotide polymorphism-based risk factor for cirrhosis in NAFLD and alcohol misuse, whereas the Pi*S variant confers only a weak risk in alcohol misusers. As 2%–4% of Caucasians are Pi*Z carriers, this finding should be considered in genetic counselling of affected individuals.

Background: Biliary tract cancers (BTC) are rare malignant tumours with a poor prognosis. Previously, we have presented a detailed characterisation of the inflammatory infiltrate in BTC. Here, we analysed the impact of the expression of major histocompatibility complex class I (MHC I) on patient survival and the quantity, as well as the quality of tumour-infiltrating immune cell types in BTC. Methods: MHC I expression was assessed semi-quantitatively in 334 BTC, including extrahepatic (n=129) and intrahepatic cholangiocarcinomas (n=146), as well as adenocarcinomas of the gallbladder (n=59). In addition, 71 high-grade biliary intraepithelial lesions (BilIN 3) were included. Results were correlated with data on antitumour inflammation and investigated with respect to their association with clinicopathological variables and patient survival. Results: BTC showed a wide spectrum of different MHC I expression patterns ranging from complete negativity in some tumours to strong homogenous expression in others. In BilIN 3, significantly higher MHC I expression levels were seen compared to invasive tumours (P=0.004). Patients with strong tumoural MHC I expression had a significantly higher overall survival probability (median survival benefit: 8 months; P=0.006). MHC I expression strongly correlated with the number of tumour-infiltrating T-lymphocytes (CD4+ and CD8+) and macrophages. Conclusions: Differences of MHC I expression predict patient outcome and show correlations with specific components of the inflammatory infiltrate in BTC. These findings contribute to a better understanding of immune response and immune escape phenomena in cholangiocarcinogenesis.

The bile acid-phospholipid conjugate ursodeoxycholyl oleoyl-lysophophatidylethanolamide (UDCA-18:1LPE) is an anti-inflammatory and anti-fibrotic agent as previously shown in cultured hepatocytes and hepatic stellate cells as well as in in vivo models of liver injury. We hypothesize that UDCA-18:1LPE may directly inhibit the activation of immune cells. We found that UDCA-18:1LPE was capable of inhibiting the migration of phorbol ester-differentiated human THP-1 cells. We examined anti-inflammatory activity of UDCA-18:1LPE during activation of THP1-derived macrophages. Treatment of these macrophages by bacterial lipopolysaccharide (LPS) for 24 h induced the release of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β. This release was markedly inhibited by pretreatment with UDCA-18:1LPE by ~ 65–90%. Derivatives with a different fatty-acid chain in LPE moiety also exhibited anti-inflammatory property. Western blotting and indirect immunofluorescence analyses revealed that UDCA-18:1LPE attenuated the expression of phosphorylated p38, MKK4/MKK7, JNK1/2, and c-Jun as well as nuclear translocation of NF-κB by ~ 22–86%. After LPS stimulation, the Toll-like receptor adaptor proteins, myeloid differentiation factor 88 and TNF receptor associated factor 6, were recruited into lipid rafts and UDCA-18:1LPE inhibited this recruitment by 22% and 58%, respectively. Moreover, LPS treatment caused a decrease of the known cytoprotective lysophosphatidylcholine species containing polyunsaturated fatty acids by 43%, and UDCA-18:1LPE co-treatment reversed this decrease. In conclusion, UDCA-18:1LPE and derivatives inhibited LPS inflammatory response by interfering with Toll-like receptor signaling in lipid rafts leading to an inhibition of MAPK and NF-κB activation. These conjugates may represent a class of lead compounds for development of anti-inflammatory drugs.

Non-alcoholic fatty liver disease (NAFLD) is frequent among obese individuals with metabolic syndrome. Variants PNPLA3 p.I148M, TM6SF2 p.E167K and MBOAT7 rs641738 are associated with higher liver fat contents. Here we analyzed 63 biopsied non-obese, non-diabetic patients with NAFLD (39 men, age: 20-72 years) recruited within the German NAFLD CSG program. The frequencies of the PNPLA3, TM6SF2 and MBOAT7 polymorphisms were compared with the remaining patients in the NAFLD CSG cohort and with a control population (n = 174). Serum CK18-M30 was measured by ELISA. In non-obese NAFLD patients, the frequency of the PNPLA3 p.I148M allele (74.6%), but not of the TM6SF2 or MBOAT7 polymorphisms, was significantly (P < 0.05) higher as compared to the other patients in the NAFLD CSG cohort (54.9%) or controls (40.2%). The presence of the minor PNPLA3 p.I148M risk allele increased the risk of developing NAFLD (OR = 3.29, P < 0.001) and was associated with higher steatosis, fibrosis, and serum CK18-M30 levels (all P < 0.05). According to the population attributable fraction (PAF), 49.8% of NAFLD cases could be eliminated if the PNPLA3 mutation was absent. The MBOAT7 polymorphism was more frequent (P = 0.019) in patients with severe hepatic steatosis. In conclusion, PNPLA3, and to a lesser extent, MBOAT7 variants are associated with NAFLD risk and modulate liver injury in non-obese patients without diabetes.

Aims Pulmonary (ADC) and colorectal (CRC) adenocarcinomas are frequent entities in pathological routine diagnostics. Whereas the differential diagnosis is usually straightforward based on histomorphology, it can be challenging in small biopsies. In general, CDX-2, CK20, Napsin-A and TTF-1 are recommended immunohistological markers in this scenario. Hepatocyte nuclear factor 4 alpha (HNF4-α) and special AT-rich sequence-binding protein 2 (SATB2) were described recently as promising additional markers, but comprehensive large-scale data are lacking so far. Therefore, we analysed the expression of these six markers in 1021 non-small-cell lung cancers (NSCLC), including 472 ADC as well as in 80 pulmonary metastases of CRC. Methods and results Tissue microarrays of NSCLC and pulmonary metastases of CRC were stained for CDX-2, CK20, HNF4-α, Napsin-A, SATB2 and TTF-1 and staining results were correlated with clinicopathological variables. ADC exhibited expression of CDX-2, CK20, HNF4-α, Napsin-A, SATB2 and TTF-1 in nine (2%), 21 (4%), 17 (4%), 345 (73%), 35 (7%) and 408 (86%) samples, while 80 CRC were positive in 79 (99%), 74 (93%), 77 (96%), no (0%), 78 (98%) and five (6%) cases, respectively. Conclusions In addition to conventional immunomarkers, HNF4-α and particularly SATB2 may be helpful in the differential diagnosis of pulmonary ADC and metastases of CRC.

AIMS: Deleted in malignant brain tumours 1 (DMBT1) exerts functions in the regulation of epithelial differentiation and inflammation and has been proposed as a tumour suppressor. Because chronic inflammation is a hallmark of cholangiocarcinogenesis, the aim of this study was to investigate the expression of DMBT1 in biliary tract cancer (BTC) and to correlate this expression with clinicopathological data.METHODS AND RESULTS: The expression of DMBT1 protein was examined immunohistochemically in 157 BTC patients [41 intrahepatic (ICC), 60 extrahepatic cholangiocarcinomas (ECC) and 56 adenocarcinomas of the gallbladder (GBAC)]. Additionally, 56 samples of high-grade biliary intraepithelial neoplasia (BilIN 3) and 92 corresponding samples of histological non-neoplastic biliary tract tissues were included. DMBT1 expression was increased significantly in BilIN 3 compared to normal tissue (P < 0.0001) and BTC (P < 0.0001). BTC showed no significant difference in DMBT1 expression compared to non-neoplastic biliary tissue (P = 0.315). Absent DMBT1 expression in non-neoplastic biliary tissue of BTC patients was associated with poorer survival (P = 0.027). DMBT1 expression was correlated significantly with patients' age (P < 0.001).CONCLUSION: DMBT1 is expressed differently in cholangiocarcinogenesis and poorer patients' survival rates are associated with absent DMBT1 expression in non-neoplastic biliary tissue, suggesting a tumour-suppressive role of DMBT1 in early cholangiocarcinogenesis.

Excess hepatic fat accumulation leads to nonalcoholic steatohepatitis (NASH), a serious threat to health for which no effective treatment is available. However, the mechanism responsible for fatty acid uptake by hepatocytes remains unclear. Using the human hepatocyte-derived tumor cell line HepG2, we found that fatty acid influx is mediated by a heterotetrameric plasma membrane protein complex consisting of plasma membrane fatty acid-binding protein, caveolin-1, CD36, and calcium-independent membrane phospholipase A2 (iPLA2β). Blocking iPLA2β with the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) caused the dissociation of the complex, thereby inhibiting fatty acid influx (IC50 47 μM), and suppressed the synthesis of all subunits through a reduction in lysophosphatidylcholine from 8.0 to 3.5 μmol/mg of protein and corresponding depletion of phosphorylated c-Jun N-terminal kinase. These findings were substantiated by an observed 56.5% decrease in fatty acid influx in isolated hepatocytes derived from iPLA2β-knockout mice. Moreover, steatosis and inflammation were abrogated by UDCA-LPE treatment in a cellular model of NASH. Thus, iPLA2β acts as an upstream checkpoint for mechanisms that regulate fatty acid uptake, and its inhibition by UDCA-LPE qualifies this nontoxic compound as a therapeutic candidate for the treatment of NASH.-Stremmel, W., Staffer, S., Wannhoff, A., Pathil, A., Chamulitrat, W. Plasma membrane phospholipase A2 controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis.

Background & Aims Ledipasvir/sofosbuvir (LDV/SOF) for 8 to 24 weeks is approved for the treatment of chronic hepatitis C virus infection (HCV). In the ION-3 study, 8 weeks of LDV/SOF was non-inferior to 12 weeks in previously untreated genotype 1 (GT1) patients without cirrhosis. According to the Summary of Product Characteristics (SmPC), 8-week treatment may be considered in naive non-cirrhotic GT1-patients. However, there are only limited data on the effectiveness of an 8-week regimen of LDV/SOF under real-world conditions. The aim of the present study was to characterise patients receiving 8 weeks of LDV/SOF compared to those receiving 12 weeks of LDV/SOF, and to describe therapeutic outcomes in routine clinical practice. Methods The German Hepatitis C-Registry is a large national real-world cohort that analyses effectiveness and safety of antiviral therapies in chronic HCV. This data set is based on 2,404 patients. Treatment with SOF/LDV (without RBV) for 8 or 12 weeks was initiated on or before September 30, 2015. Results Overall, 84.6% (2,034/2,404) of the safety population (intention-to-treat-1 [ITT1]) and 98.2% (2,029/2,066) of the per protocol (PP) population achieved sustained virological response at week 12 (SVR12). In the 8-week group, 85.1% (824/968) of ITT1 and 98.3% (821/835) of PP patients achieved SVR12, while in the 12-week group, 85.5% (1,210/1,415) of ITT1, and 98.1% (1,208/1,231) of PP patients achieved SVR12. When treated according to the SmPC, 98.7% (739/749) of the patients achieved SVR12 (PP). Relapse was observed in 9.5% (2/21) of cirrhotic patients treated for 8 weeks (PP). Conclusions Under real-world conditions a high proportion of eligible patients receiving 8-week LDV/SOF treatment achieved SVR12. Relapse occurred more frequently in patients who did not meet the selection criteria according to the SmPC. Lay summary In a large real-world cohort of patients mainly treated by physicians in private practice in Germany, shorter HCV treatment (8-week) resulted in equivalent cure rates to 12-week treatment in genotype 1 HCV-infected patients. Thus, shorter treatment can be recommended in these patients which would substantially reduce costs of therapy. Clinical Trial number: DRKS00009717 (German Clinical Trials Register, DRKS)

Patients with sepsis frequently present with acute renal failure requiring intermittent renal replacement therapy and haemodynamic monitoring. To date, the effect of haemodialysis on PiCCO monitoring has not been determined.To determine the effect of haemodialysis on the measurement of haemodynamic variables using transpulmonary thermodilution.Prospective observational study.Medical ICU in a university hospital.Thirty patients with sepsis and acute renal failure undergoing intermittent haemodialysis.None.Cardiac index, global end-diastolic volume index and extravascular lung water index measured with or without haemodialysis.Significant correlations were found for all variables measured with and without haemodialysis (r = 0.55 to 0.91, P0.01). Measurements of cardiac index without and with haemodialysis were significantly different [4.71 vs. 4.18 lmin(-1)m(-2), difference -0.54 (SD 0.70), 95% confidence interval (CI) -0.80 to -0.28; P0.01], as were values of global end-diastolic volume index without and with haemodialysis [864.8 vs. 775.3 ml m(-2), difference -89.5 (SD 191.8), 95% CI -161.2 to -17.9; P = 0.02]. Measurements of extravascular lung water index without and with haemodialysis did not differ significantly [10.3 vs. 10.0 ml kg(-1), difference -0.3 (SD 2.0), 95% CI -1.1 to 0.5; P = 0.42].Although significant correlations were found for cardiac index, global end-diastolic volume index and extravascular lung water index with and without haemodialysis, cardiac index and global end-diastolic volume index were significantly reduced during haemodialysis, but not extravascular lung water index, when measured by the PiCCO system in patients with septic shock. Although differences were small, the variability of within-patient differences may be clinically important and care should be taken in relying solely on such measurements.

Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.

Summary Background Early treatment of acute hepatitis C virus (HCV) infection with interferon alfa is highly effective, but can be associated with frequent side-effects. We investigated the safety and efficacy of an interferon-free regimen for treatment of acute HCV infection. Methods In this prospective, open-label, multicentre, single-arm pilot study, we enrolled adults (≥18 years) with acute HCV genotype 1 monoinfection from ten centres in Germany. Patients were given ledipasvir (90 mg) plus sofosbuvir (400 mg) as a fixed-dose combination tablet once daily for 6 weeks. The primary efficacy outcome was the proportion of patients with sustained virological response (defined as undetectable HCV RNA 12 weeks after the end of treatment; other primary outcomes were safety and tolerability of ledipasvir plus sofosbuvir. The primary analysis population consisted of all patients who received at least one dose of study drug. Safety was also assessed in all patients who received at least one dose of the study drug. This trial is registered with ClinicalTrials.gov, number NCT02309918. Findings Between Nov 19, 2014, and Nov 10, 2015, we enrolled 20 patients. Median HCV RNA viral load at baseline was 4·04 log 10 IU/mL (1·71–7·20); 11 patients were infected with HCV genotype 1a and nine patients with genotype 1b. All patients achieved a sustained virological response 12 weeks after the end of treatment (20 [100%] of 20 patients). Treatment was well tolerated; there were no drug-related serious adverse events. Up to 12 weeks after treatment, 22 possible or probable drug-related adverse events were reported. There was one serious adverse event, which was judged unrelated to the study drug; one patient was admitted to hospital for surgery of a ruptured cruciate ligament. Interpretation Treatment for 6 weeks with ledipasvir plus sofosbuvir was well tolerated and highly effective in patients with acute HCV genotype 1 monoinfection. Short-duration treatment of acute hepatitis C might prevent the spread of HCV in high-risk populations. Funding Gilead Sciences, HepNet Study-House/German Liver Foundation, and German Centre for Infection Research (DZIF).

Objectives The aim of this multicenter retrospective study was to investigate safety and efficacy of direct acting antiviral (DAA) treatment in the rare subgroup of patients with HCV/HIV-coinfection and advanced liver cirrhosis on the liver transplant waiting list or after liver transplantation, respectively. Methods When contacting 54 German liver centers (including all 23 German liver transplant centers), 12 HCV/HIV-coinfected patients on antiretroviral combination therapy were reported having received additional DAA therapy while being on the waiting list for liver transplantation (patient characteristics: Child-Pugh A (n = 6), B (n = 5), C (n = 1); MELD range 7–21; HCC (n = 2); HCV genotype 1a (n = 8), 1b (n = 2), 4 (n = 2)). Furthermore, 2 HCV/HIV-coinfected patients were denoted having received DAA therapy after liver transplantation (characteristics: HCV genotype 1a (n = 1), 4 (n = 1)). Results Applied DAA regimens were SOF/DAC (n = 7), SOF/LDV/RBV (n = 3), SOF/RBV (n = 3), PTV/r/OBV/DSV (n = 1), or PTV/r/OBV/DSV/RBV (n = 1), respectively. All patients achieved SVR 12, in the end. In one patient, HCV relapse occurred after 24 weeks of SOF/DAC therapy; subsequent treatment with 12 weeks PTV/r/OBV/DSV achieved SVR 12. One patient underwent liver transplantation while on DAA treatment. Analysis of liver function revealed either stable parameters or even significant improvement during DAA therapy and in follow-up. MELD scores were found to improve in 9/13 therapies in patients on the waiting list for liver transplantation; in only 2 patients a moderate increase of MELD scores persisted at the end of follow-up. Conclusion DAA treatment was safe and highly effective in this nation-wide cohort of patients with HCV/HIV-coinfection awaiting liver transplantation or being transplanted.

Background: The combination of sofosbuvir (SOF), ribavirin (RBV) and peg-interferon-alfa-2a (peg-IFN-alfa-2a) as well as the combination of SOF and RBV for the treatment of patients infected with hepatitis c virus (HCV) has improved rates of sustained virological response (SVR) considerably in recent trials. However, there is only limited data concerning the efficacy and safety in a “real-life” cohort. Methods: We analyzed a cohort of 119 patients with chronic HCV infection treated at four investigational sites in Germany. All patients received either a combination treatment of SOF, RBV and peg-IFN-alfa-2a or SOF and RBV. Results: The rates of SVR at 12 weeks after end of treatment (SVR 12) were as follows: Among 76 patients with genotype 1 infection the SVR 12 rate was 74 % (n = 56), among 14 patients with genotype 2 infection the SVR 12 rate was 79 % (n = 11), among 24 patients with genotype 3 infection the SVR 12 rate was 92 % (n = 22) and among 5 patients with genotype 4 infection the SVR 12 rate was 80 % (n = 4). Of all 26 patients with a relapse in our cohort, 69 % (n = 18) of these patients presented with liver cirrhosis and 58 % (n = 15) were treatment experienced. Notably, the level of HCV-RNA after 4 weeks of treatment was a significant predictor of treatment response in genotype 1 patients. Patients with HCV-RNA levels ≥ 12 IU ml-1 after 4 weeks of treatment achieved SVR 12 only in 30 % (n = 17/56, p

Natural killer (NK) cells as components of the innate immunity substantially contribute to antitumor immune responses. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. An exception are the MHC class I chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes. A therapeutic induction of NKG2D ligands that primes cancer cells for NK cell lysis has not yet been achieved. By microarray studies, we found evidence that treatment of human hepatocellular carcinoma cells with the histone deacetylase inhibitor (HDAC-I) sodium valproate (VPA) mediates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. VPA induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. No significant changes in the expression of the NKG2D ligands ULBP1-3 were observed. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by NK cells which was abolished by addition of a blocking NKG2D antibody. Importantly, in primary human hepatocytes, VPA treatment did not induce MIC protein expression. Taken together, our data show that the HDAC-I VPA mediates specific priming of malignant cells for innate immune effector mechanisms. These results suggest the clinical evaluation of HDAC-I in solid tumors such as hepatocellular carcinoma, especially in combination with immunotherapy approaches employing adoptive NK cell transfer.

Chronic hepatic inflammation results in liver fibrosis. As effective anti-fibrogenic agents are lacking, we investigated ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE), a synthetic bile acid-phospholipid conjugate with anti-inflammatory and anti-apoptotic properties for tis effects on hepatic fibrogenesis.To stimulate fibrogenesis, LX2 hepatic stellate cells were cultured with conditioned medium from CL48 liver cells after exposure to stress-inducing conditions - methionine-choline-deficient (MCD) medium or TNFα/cycloheximide (CHX) - with or without UDCA-LPE preincubation. Anti-fibrogenic effects of UDCA-LPE were further studied in CL48 and LX2 cells and in primary human hepatic stellate cells (HHStec) directly exposed to TGF-β1. To test UDCA-LPE in vivo, C57BL/6 mice were fed a MCD diet for 11 weeks followed by 30 mg·kg(-1) UDCA-LPE 3× per week for 2.5 weeks.Expression of α-smooth muscle actin (α-SMA), α1-collagen, vimentin and TGF-β1 was down-regulated by up to 93% by UDCA-LPE in LX-2 cells cultured with conditioned medium. Also, UDCA-LPE inhibited Smad3 phosphorylation in CL48 cells incubated with MCD medium or TNFα/CHX and in LX2 cells exposed to conditioned medium. UDCA-LPE also decreased phosphorylated Smad3 and Smad2 directly induced by TGF-β1. Inhibition of TGF-β1/Smad2/3 signalling with down-regulation of target genes was confirmed in HHStec. In vivo, UDCA-LPE decreased hepatic α-SMA, α1-collagen and TGF-β1 expression and markedly lowered α-SMA protein and collagen deposition in MCD mice.By blocking TGF-β1/Smad2/3 signalling, UDCA-LPE suppressed key mediators of hepatic fibrogenesis. Thus, UDCA-LPE could be suitable for prevention of fibrotic progression of chronic liver disease.

The bile acid-phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a promising novel compound with profound hepatoprotective functions in vitro and in vivo. Because of high costs of LPE synthesis from hydrolysis of phosphatidylethanolamide (PE), costs for UDCA-LPE synthesis for in vivo and human use can become quite high. In this study, we evaluated whether ursodeoxycholyl phosphatidylethanolamide (udca-pe), which is more cost-effective, could replace udca-lpe in terms of protection from hepatocellular injury.Anti-apoptotic and anti-inflammatory properties of UDCA-PE and UDCA-LPE were compared in TNFα/cyclohexamide (CHX)-treated HepG2 cells as well as in a mouse model of d-galactosamine/lipopolysaccharide (Gal/LPS)-induced acute liver injury.Ursodeoxycholyl lysophosphatidylethanolamide inhibited TNFα/CHX-induced apoptosis in HepG2 cells in a dose-dependent manner and markedly ameliorated Gal/LPS-mediated fulminant hepatitis in mice. In contrast, UDCA-PE showed weaker hepatoprotective functions at low concentrations, and protection was lost at higher dosage. Analysis of hepatic gene expression showed that both conjugates significantly reduced Gal/LPS-mediated expression of chemoattractants, such as monocyte chemotactic protein 1 (MCP1) and RANTES. These inhibitory effects by UDCA-PE were transient while those by UDCA-LPE were sustained in attenuating expression of inflammatory MCP1 and RANTES expression.Our data underline the superiority of UDCA-LPE compared to UDCA-PE in ameliorating acute liver inflammation. This indicates the significance of the lyso-functional group of bile acid conjugate for optimal hepatoprotection and reduction in inflammation in vivo.

BACKGROUND. Innovative epigenetic therapeutics comprise histone deacetylase inhibitors (HDAC-I) and demethylating agents (DA). It was recently found that HDAC-I compounds exhibit profound therapeutic activities against hepatocellular carcinoma (HCC). A comprehensive preclinical investigation was performed on the potential of a combined HDAC-I/DA epigenetic regimen for the highly chemotherapy-resistant HCC entity. METHODS. Human HCC-derived cell lines or primary human hepatocytes (PHH) were treated with HDAC-I compound suberoylanilide hydroxamic acid (SAHA) or DA compound 5-aza-2′-deoxycytidine (5-aza-dC) or both and examined for cellular damage, proliferation, histone acetylation pattern, and DNA methylation. In vivo activities were investigated in a xenograft hepatoma model. RESULTS. Monotherapeutic application of SAHA or 5-aza-dC was found to induce substantial antiproliferative effects in HCC-derived cells, strongly enhanced by combined SAHA and 5-aza-dC treatment. PHH from different human donors did not exhibit any relevant cellular damage even when applying high doses of the combination regimen, whereas HCC-derived cell lines showed a dose-dependent damage. In vivo testing demonstrated a statistical significant inhibition of hepatoma cell growth for the combined treatment regime. CONCLUSIONS. Because the combined HDAC-I/DA epigenetic approach was found to produce significant antitumor effects in HCC model systems and did not impair cellular integrity of untransformed hepatocytes, this combination therapy is now considered for further investigation in clinical trials. Cancer 2007;109:2132–41. © 2007 American Cancer Society.

Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs or innovative tumor cell apoptosis-inducing agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Recently, we found 2 histone deacetylase inhibitors (HDAC-I), valproic acid and ITF2357, exhibiting inherent therapeutic activity against HCC. In TRAIL-sensitive cancer cells, the mechanism of HDAC-I-induced cell death has been identified to be TRAIL-dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC-derived cells, a prototype of TRAIL-resistant tumor cells, we found a HDAC-I-mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC-I and TRAIL, increasing the fraction of apoptotic cells two- to threefold compared with HDAC-I treatment alone, whereas any premature HDAC-I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell-specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC-I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC-I. Importantly, combination HDAC-I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC-I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC.

BACKGROUND/AIMS: Due to a particular resistance against conventional chemotherapeutics, palliative treatment of hepatocellular carcinomas (HCC) is highly ineffective. Recent demonstration of both proliferation-inhibition and apoptosis of hepatoma cells by a histone deacetylase inhibitor (HDAC-I) treatment opens up a promising new approach. However, little is known about tumor cell death mechanisms and HDAC-I influences on healthy hepatocytes. METHODS: HDAC-I substances with favourable in vivo profiles, valproate (VPA) and ITF2357, were investigated on HCC cell lines and primary human hepatocytes (PHH). Histone acetylation and apoptosis-modulating proteins were investigated by western-blotting, proliferation by sulforhodamin B binding, toxicity by enzyme release, apoptosis by FACS analysis. RESULTS: VPA and ITF2357 inhibited proliferation in HCC cell lines. Both substances induced considerable cellular damage in HCC-derived cells, but PHH tolerated these substances well. A downregulation of anti- and upregulation of proapoptotic factors was found. Moreover, Bcl-X(L) transfection into HCC cells abrogated apoptosis induced by both substances, indicating that modulation of intracellular pro- and anti-apoptotic proteins is a key event in VPA or ITF2357 induced tumor-cell death. CONCLUSIONS: Preferential induction of cell death in HCC-derived cell lines, without toxicity in PHH, demonstrates the potential of VPA and ITF2357 to become promising new tools in the fight against HCC.

Background Aquaporins (AQPs) have been recognized to promote tumor progression, invasion, and metastasis and are therefore recognized as promising targets for novel anti-cancer therapies. Potentially relevant AQPs in distinct cancer entities can be determined by a comprehensive expression analysis of the 13 human AQPs. Methods We analyzed the presence of all AQP transcripts in 576 different normal lung and non-small cell lung cancer (NSCLC) samples using microarray data and validated our findings by qRT-PCR and immunohistochemistry. Results Variable expression of several AQPs (AQP1, -3, -4, and -5) was found in NSCLC and normal lung tissues. Furthermore, we identified remarkable differences between NSCLC subtypes in regard to AQP1, -3 and -4 expression. Higher transcript and protein levels of AQP4 in well-differentiated lung adenocarcinomas suggested an association with a more favourable prognosis. Beyond water transport, data mining of co-expressed genes indicated an involvement of AQP4 in cell-cell signalling, cellular movement and lipid metabolism, and underlined the association of AQP4 to important physiological functions in benign lung tissue. Conclusions Our findings accentuate the need to identify functional differences and redundancies of active AQPs in normal and tumor cells in order to assess their value as promising drug targets.