Martin-Walter Welker

Serum microRNA-1 and microRNA-122 are prognostic markers in patients with hepatocellular carcinoma

BACKGROUND The identification of new biomarkers to predict the aggressiveness of hepatocellular carcinoma (HCC) and supplement the current set of prognosis and treatment algorithms is an important clinical need. Extracellular microRNAs (miRNAs) circulating in the blood are a new class of highly promising disease markers. AIM Here we investigated the prognostic potential of miR-1 and miR-122 in sera from patients with HCC. METHODS RNA was extracted from 195 sera of HCC patients and 54 patients with liver cirrhosis, obtained at the time of study enrolment. miR-1 and miR-122 levels were correlated with overall survival (OS), Cancer of the Liver Italian Program (CLIP) score, Barcelona Clinic Liver Cancer stage, clinical chemistry parameters and tumor specific treatment. RESULTS Patients with higher miR-1 and miR-122 serum levels showed longer OS than individuals with lower miR-1 and miR-122 serum concentrations (hazard ratio [HR] 0.440, 95% confidence interval [CI] 0.233-0.831, P=0.011 for miR-1 and HR 0.493, 95% CI 0.254-0.956, P=0.036 for miR-122, respectively). Serum miR-1 and miR-122 concentrations did not differ significantly between patients with HCC and liver cirrhosis. An age-, sex-, tumor stage and treatment-adjusted multivariate Cox regression analysis revealed that miR-1 serum levels (HR 0.451, 95% CI 0.228-0.856, P=0.015) were independently associated with OS, whereas serum miR-122 was not. miR-1 serum levels showed no relevant correlation with clinical chemistry liver parameters, whereas serum miR-122 correlated with clinical chemistry parameters of hepatic necroinflammation, liver function and synthetic capacity. CONCLUSION Our data indicate that serum miR-1 is a new independent parameter of OS in HCC patients and may therefore improve the predictive value of classical HCC staging scores.

Recurrent hepatocellular carcinoma after liver transplantation – an emerging clinical challenge

In western countries, hepatocellular carcinoma (HCC) is a major reason for orthotopic liver transplantation (OLT) with estimated recurrence rates between 15% and 20%. This selective literature review addresses follow‐up care after OLT in HCC and current treatment options. Recurrence prediction is based on pathological tumor stage, vascular invasion, serum alfafetoprotein levels, and histological differentiation, but further advances are expected by molecular profiling techniques. Lower levels of immunosuppressive agents are associated with a lower risk for HCC recurrence. Retrospective studies indicate that mammalian target of rapamycin (mTOR) inhibitors as immunosuppression reduce the risk of HCC recurrence. However, prospective studies supporting this potential advantage of mTOR inhibitors are still lacking, and higher rejection rates were reported for sirolimus after OLT in HCC. Prognosis is poor in recurrent HCC, a longer interval between OLT and recurrence and feasibility of surgical resection are associated with improved survival. Systemic treatment with sorafenib is the current standard of care in patients with advanced‐stage HCC not suitable for locoregional therapy. After OLT, combination of an mTOR inhibitor with sorafenib is feasible and frequently used in clinical practice. As safety and efficacy data are still limited, close clinical monitoring is mandatory.

Deep Sequencing Reveals Mutagenic Effects of Ribavirin during Monotherapy of Hepatitis C Virus Genotype 1-Infected Patients

ABSTRACT The preeminent mode of action of the broad-spectrum antiviral nucleoside ribavirin in the therapy of chronic hepatitis C is currently unresolved. Particularly under contest are possible mutagenic effects of ribavirin that may lead to viral extinction by lethal mutagenesis of the hepatitis C virus (HCV) genome. We applied ultradeep sequencing to determine ribavirin-induced sequence changes in the HCV coding region (nucleotides [nt] 330 to 9351) of patients treated with 6-week ribavirin monotherapy (n = 6) in comparison to placebo (n = 6). Baseline HCV RNA levels maximally declined on average by −0.8 or −0.1 log10 IU/ml in ribavirin- versus placebo-treated patients. No general increase in rates of nucleotide substitutions in ribavirin-treated patients was observed. However, more HCV genome positions with high G-to-A and C-to-U transition rates were detected between baseline and treatment week 6 in ribavirin-treated patients in comparison to placebo-treated patients (rate of 0.0041 transitions per base pair versus rate of 0.0022 transitions per base pair; P = 0.049). Similarly, the sensitive detection of low-frequency minority variants by statistical filtering indicated significantly more positions with G-to-A and C-to-U transitions in ribavirin-treated patients than in placebo-treated patients (rate of 0.0331 transitions versus rate of 0.0186 transitions per G/C-containing position at baseline; P = 0.018). In contrast, non-ribavirin-associated A-to-G and U-to-C transitions were not enriched in the ribavirin group (P = 0.152). We conclude that ribavirin exerts a mutagenic effect on the virus in patients with chronic hepatitis C by facilitating G-to-A and C-to-U nucleotide transitions.

Occult hepatitis C: How convincing are the current data?

Sustained virologic response (SVR), defined as undetectable hepatitis C virus (HCV) RNA in serum 24 weeks after withdrawal of (pegylated)-interferonalfa (PEG-IFN)-based antiviral therapy, has become the primary goal for therapy of patients infected with HCV. Recent reports about “occult” persistence of HCV infection despite constantly negative serum HCV RNA1-14 have led to uncertainty in patients and physicians. This review gives a summary of available data regarding “occult” HCV infection.

Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment

BACKGROUND & AIMS Sofosbuvir (SOF) based interferon-alfa free antiviral therapy has become the treatment of choice for patients with chronic hepatitis C virus (HCV) infection. Little is known about safety of drug combinations using two nucleos(t)ide polymerase inhibitors in patients with HCV associated advanced cirrhosis. Here, we report frequent occurrence of lactic acidosis associated with acute-on-chronic hepatic decompensation during ribavirin (RBV) plus SOF based antiviral therapy. METHODS Thirty-five patients with chronic hepatitis C and advanced fibrosis, compensated cirrhosis, and decompensated cirrhosis without and after liver transplantation were treated with SOF based antiviral therapy with and without RBV. Adverse events including lactic acidosis (pH <7.35, lactate >20 mg/dl) were recorded 24 weeks before and during (mean ±SD, 18±11 weeks) antiviral therapy. Efficacy was determined by assessment of serum HCV RNA. RESULTS We observed severe adverse events in 15/35 (43%) patients before (24 weeks) and in 12/35 (34%) patients during antiviral therapy, the majority in association with acute-on-chronic hepatic decompensation. Lactic acidosis occurred in 5/35 (14%) patients during therapy, while no event of lactic acidosis was observed prior to therapy. Lactic acidosis was associated with hepatic decompensation including renal failure and infection, and was severe (pH <7.3) in two patients. CONCLUSIONS RBV in combination with SOF based antiviral therapy in patients with HCV associated advanced cirrhosis may be associated with the development of lactic acidosis. Impaired renal function, and higher MELD/Child-Pugh scores were identified as potential risk factors.

Clinical value of on-treatment HCV RNA levels during different sofosbuvir-based antiviral regimens.

BACKGROUND & AIMS The European Association for the Study of the Liver (EASL) guidelines recommend HCV RNA measurements at specific time points during sofosbuvir(SOF)-therapy. However, it remains unclear, how these results should be interpreted. We aimed to analyze whether on-treatment HCV RNA levels predict relapse comparing the CobasAmpliPrep/CobasTaqMan v2.0 (CAP/CTM) and Abbott RealTime HCV (ART) assays. METHODS Samples were collected from 298 patients (HCV genotypes; GT1-5) at weeks (w) 0, 1, 2, 4, 8, 12, 16, 20 and 24 during SOF-based therapy at two university clinics and tested for HCV RNA level by CAP/CTM and ART. Patients were treated with SOF/ribavirin (RBV) 12/24 w (n=99), pegylated-interferon-alfa (PegIFN)/SOF/RBV 12 w (n=51), SOF/simeprevir (SMV)±RBV 12 w (n=69) or SOF/daclatasvir±RBV 12/24 w (n=79). RESULTS HCV RNA levels during the first 4weeks of SOF/RBV therapy were significantly lower in GT3 patients who achieved SVR compared with those who relapsed. All GT3 patients with a week 2 result <45IU/ml by CAP/CTM achieved SVR but only 33% of those with ⩾45IU/ml (p=0.0003). Similar results were documented with ART and 60IU/ml as cut-off (SVR: 100% vs. 29%; p=0.0002). In contrast, HCV RNA levels during early treatment phases were not significantly related to relapse in patients treated with other SOF-based regimens. Residual HCV RNA was frequently detected by ART at later stages of therapy. However, SVR rates remained high in these patients. At the end of SOF/SMV±RBV therapy HCV RNA was detectable with ART in 20% of patients, of whom 92% achieved SVR. CONCLUSIONS HCV RNA levels assessed at week 2 of SOF/RBV therapy can predict relapse in GT3-patients. Detectable HCV RNA results at later stages during SOF-based therapy may occur frequently with the more sensitive ART. However, this should not lead to treatment extension. LAY SUMMARY We analyzed the predictive value of hepatitis C virus (HCV) RNA levels measured at different time points for treatment efficacy. We found that the level of HCV RNA measured at week 2 of antiviral therapy can be used to predict treatment success in patients with HCV genotype 3 infection treated with sofosbuvir and ribavirin but not in patients treated with other sofosbuvir-based regimens. Low level HCV RNA is frequently detected by the RealTime HCV assay during later stages of antiviral therapy. However, this is not associated with reoccurrence of HCV RNA after the end of treatment.

Regression of fibrosis and portal hypertension in HCV-associated cirrhosis and sustained virologic response after interferon-free antiviral therapy

It is still controversial, whether and to what amount cirrhosis and portal hypertension are reversible in patients with hepatitis C virus (HCV)‐associated cirrhosis and sustained virologic response (SVR) after interferon‐free antiviral therapy. In this study, we prospectively evaluated dynamics of liver and spleen stiffness in HCV‐infected patients with advanced liver disease and SVR after interferon‐free treatment. A total of 54 patients with HCV‐associated cirrhosis and SVR were included. Liver and spleen stiffness was measured at therapy baseline (BL), end of treatment (EOT) and 24 weeks after EOT (FU24) by transient liver elastography (L‐TE) as well as by acoustic radiation force impulse of the liver (L‐ARFI) and spleen (S‐ARFI), as well as biochemical, virologic and clinical data. Improvement of liver and spleen stiffness was found in 44 of 50 (88%), 31 of 54 (57%) and 25 of 54 (46%) of patients assessed by L‐TE, L‐ARFI and S‐ARFI between baseline and FU24. Liver stiffness assessed by L‐TE improved between BL [median (range), 32.5 (9.1–75) kPa] and EOT [median (range), 21.3 (6.7–73.5) kPa; (P<.0001)], and between BL and FU24 [median (range), 21.2 (5.4–70) kPa; (P<.0001)]. Liver stiffness assessed by L‐ARFI improved between BL [median (range), 2.7 (1.2–4.1) m/s] and FU24 [median (range), 2.4 (1.2–3.9) m/s; P=.002), while spleen stiffness remained unchanged. Our data suggest that improvement of liver stiffness may be rather due to reduced necroinflammation and may be due to a less extent to regression of cirrhosis, as dynamics of liver stiffness improvement was more pronounced between BL and EOT than BL and FU24.

Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

Aim Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity. Patients and Methods Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry. Results Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027). Conclusion CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis.

12 DETECTION OF RESISTANT VARIANTS IN THE HEPATITIS C VIRUS NS3 PROTEASE GENE BY CLONAL SEQUENCING AT LONG-TERM FOLLOW-UP IN PATIENTS TREATED WITH BOCEPREVIR

M. Manns1, H. Reesink2, C. Moreno3, T. Berg4, Y. Benhamou5, Y. Horsmans6, G. Dusheiko7, R. Flisiak8, P. Meyvisch9, O. Lenz9, V. Sekar10, G. van ’t Klooster9, K. Simmen9, R. Verloes9. 1Medizinische Hochschule Hannover, Hannover, Germany; 2Amsterdam Medical Center, Amsterdam, The Netherlands; 3Erasme Hospital, Universite Libre de Bruxelles, Brussel, Belgium; 4Charite-Universitatsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany; 5Centre Hospitalier Universitaire Pitie-Salpetriere Paris, Paris, France; 6Saint-Luc Universite Catholique de Louvain, Brussel, Belgium; 7Royal Free Hospital, London, UK; 8Medical University of Bialystok, Bialystok, Poland; 9Tibotec BVBA, Mechelen, Belgium; 10Tibotec Pharmaceuticals, Yardley, PA, USA E-mail: rverloes@tibbe.jnj.com

Correlation of amino acid variations within nonstructural 4B protein with initial viral kinetics during interferon-alpha-based therapy in HCV-1b-infected patients

Summary.  Chronic hepatitis C is a major cause of liver cirrhosis leading to chronic liver failure and hepatocellular carcinoma. Different hepatitis C virus (HCV) proteins have been associated with resistance to interferon‐alpha‐based therapy. However, the exact mechanisms of virus‐mediated interferon resistance are not completely understood. The importance of amino acid (aa) variations within the HCV nonstructural (NS)4B protein for replication efficiency and viral decline during the therapy is unknown. We investigated pretreatment sera from 42 patients with known outcome to interferon‐based therapy. The complete NS4B gene was amplified and sequenced. Mutational analyses of predicted conformational, functional, structural and phylogenetic properties of the deduced aa sequences were performed. The complete NS4B protein was highly conserved with a median frequency of 0.015 ± 0.009 aa exchanges (median ± SD, 4.00 ± 2.31). Especially within the predicted transmembranous domains of the NS4B protein, the mean number of aa variations was low (median frequency, 0.013 ± 0.013). Neither the number of aa variations nor specific aa exchanges were correlated with HCV RNA serum concentration at baseline. A rapid initial HCV RNA decline of ≥1.5 log10 IU/mL at week 2 of interferon‐based therapy was associated with a higher frequency of nonconservative aa exchanges within the complete NS4B protein in comparison with patients with a nonrapid HCV RNA decline (median frequency, 0.011 ± 0.005 vs 0.004 ± 0.003, P = 0.006). Overall, the aa sequence of the NS4B protein was highly conserved, indicating an important role for replication in vivo. Amino acid variations with relevant changes of physicochemical properties may influence replication efficiency, associated with a rapid early virological response.