Real-life experience with first generation HCV protease inhibitor therapy in Germany: The prospective, non-interventional PAN cohort.

BACKGROUND AND AIMS: The efficacy and safety of peginterferon alfa-2a (PEG-IFN) plus ribavirin (RBV) and either boceprevir (BOC) or telaprevir (TVR), and physician adherence to treatment algorithms were evaluated in patients included in an ongoing non-interventional study (PAN) enrolling adults with chronic hepatitis C virus (HCV) infection managed in German office-based practices. METHODS: The analysis included HCV genotype 1-infected, treatment-naïve and treatment-experienced patients treated with BOC or TVR. Demographic, treatment history, virological response, safety, and patient management data were collected. RESULTS: Of a total 1087 patients, 58.1 % achieved sustained virological responses (SVR). Response rates were higher in treatment-naïve (BOC 55 %; TVR 63.4 %) and prior relapse patients (BOC 63.2 %; TVR 74.5 %) versus previous null-responders (BOC 14.3 %; TVR 25 %). The most commonly reported adverse event overall was fatigue (60.6 %); 45.8 % patients experienced hemoglobin < 10 g/dL. Patients with cirrhosis had lower rates of SVR versus those without (42.9 % vs. 60.7 %, respectively), and had a higher incidence of serious adverse events (SAEs) (16.7 % vs. 8.6 %, respectively) and treatment discontinuation (44.6 % vs. 25.2 %, respectively). According to recommended response-guided treatment algorithms, about 70 % of patients were managed appropriately, 11/10 % (BOC/TVR) received unnecessarily extended therapy, and 19/7 % (BOC/TVR) received inappropriately shortened therapy. CONCLUSIONS: The efficacy and safety of BOC- and TVR-based triple therapy in this large, "real-world" cohort were largely comparable to that reported in pivotal clinical trials, although SVR rates were lower overall. Recommended futility or treatment extension rules were violated in a substantial proportion of patients with potential implications for response, adverse events and costs.

The number needed to treat to prevent mortality and cirrhosis-related complications among patients with cirrhosis and HCV genotype 1 infection.

Cirrhotic patients with chronic hepatitis C virus (HCV) infection remain at risk for complications following sustained virological response (SVR). Therefore, we aimed to evaluate treatment efficacy with the number needed to treat (NNT) to prevent clinical endpoints. Mortality and cirrhosis-related morbidity were assessed in an international multicentre cohort of consecutively treated patients with HCV genotype 1 infection and cirrhosis. The NNT to prevent death or clinical disease progression (any cirrhosis-related event or death) in one patient was determined with the adjusted (event-free) survival among patients without SVR and adjusted hazard ratio of SVR. Overall, 248 patients were followed for a median of 8.3 (IQR 6.2-11.1) years. Fifty-nine (24%) patients attained SVR. Among patients without SVR, the adjusted 5-year survival and event-free survival were 94.4% and 80.0%, respectively. SVR was associated with reduced all-cause mortality (HR 0.15, 95% CI 0.05-0.48, P = 0.002) and clinical disease progression (HR 0.16, 95% CI 0.07-0.36, P < 0.001). The NNT to prevent one death in 5 years declined from 1052 (95% CI 937-1755) at 2% SVR (interferon monotherapy) to 61 (95% CI 54-101) at 35% SVR (peginterferon and ribavirin). At 50% SVR, which might be expected with triple therapy, the estimated NNT was 43 (95% CI 38-71). The NNT to prevent clinical disease progression in one patient in 5 years was 302 (95% CI 271-407), 18 (95% CI 16-24) and 13 (95% CI 11-17) at 2%, 35% and 50% SVR, respectively. In conclusion, the NNT to prevent clinical endpoints among cirrhotic patients with HCV genotype 1 has declined enormously with the improvement of antiviral therapy.

Dynamics of hepatitis B virus quasispecies heterogeneity and virologic response in patients receiving low-to-moderate genetic barrier nucleoside analogs.

We characterized the early dynamics of hepatitis B virus (HBV) quasispecies evolution during the first weeks of antiviral therapy with low-to-moderate genetic barrier antiviral drugs and associated these data with antiviral response patterns. Fifteen chronic hepatitis B patients (men, 10; mean age, 34; HBeAg positive, 6) who received lamivudine or telbivudine for at least 52 weeks were included. HBV DNA was extracted from serum, and a 910-bp fragment covering domains A-F of the reverse transcriptase region was amplified, cloned and sequenced. Parameters of quasispecies heterogeneity, genetic diversity and complexity were calculated and were correlated with complete virologic response, defined as undetectable HBV DNA at week 52. Nine patients achieved complete virologic response during the observational period. While baseline HBV DNA levels and HBeAg status were associated with virologic response, baseline quasispecies complexity and diversity of responders showed no significant difference to those of nonresponders (P > 0.05). However, at week 4, quasispecies complexity of nonresponders was significantly higher compared with that of responders on the nucleotide level (P = 0.01) and the aa level (P = 0.04). The number of synonymous substitutions per synonymous site dropped significantly in responders at week 4 (P = 0.04), while there was no difference in nonresponders. The HBV quasispecies complexity at the early stage of antiviral therapy (week 4) with the low-to-moderate genetic barrier nucleoside analogs lamivudine or telbivudine was associated with subsequent virologic response. Further studies are needed to confirm HBV quasispecies evolution as additional predictive marker for beneficial treatment outcome.

Dysregulation of global microRNA expression in splenic marginal zone lymphoma and influence of chronic hepatitis C virus infection.

The precise molecular pathogenesis of splenic marginal zone lymphoma (SMZL) is still unknown. Clinical and epidemiological data suggest that chronic hepatitis C virus (HCV) infection may have an etiological role in a subset of cases.We performed a large-scale microRNA (miRNA) expression profiling analysis of 381 miRNAs by quantitative reverse transcription PCR (Q-RT-PCR) of 26 microdissected splenic tissue samples (7 HCV(+) SMZL; 8 HCV(-) SMZL and 11 non-neoplastic splenic controls). Single assay Q-RT-PCR and miRNA in situ hybridization (miRNA-ISH) were used to confirm the results in an independent cohort. Unsupervised hierarchical clustering of miRNA expression profiles demonstrated a distinct signature of SMZL compared with the normal splenic marginal zone. Supervised analysis revealed differentially expressed miRNAs, including miRNAs with previously recognized tumor suppressive or oncogenic potential. Five miRNAs were found significantly overexpressed in SMZL, including miR-21, miR-155 and miR-146a, whereas seven miRNAs showed significantly reduced expression, including miR-139, miR-345, miR-125a and miR-126. Furthermore, we identified miR-26b, a miRNA known to have tumor suppressive properties, as significantly downregulated in SMZL arising in HCV-positive patients (P=0.0016). In conclusion, there is a characteristic dysregulation of miRNA expression in SMZL with a possible implication in its molecular tumorigenesis.

Comparison of envelope 2 CD81 binding regions in PBMC-derived versus serum-derived hepatitis C virus isolates: higher conservation of CD81 region 2 in PBMC isolates.

UNLABELLED: 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.

[ALT screening for chronic liver diseases: scrutinizing the evidence]. / ALT als Screeningparameter für Lebererkrankungen: eine kritische Evaluation der Evidenz.

Elevated serum amino-transferase levels may be associated with liver injury. Testing for aspartate aminotransferase (AST) or alanine aminotransferase (ALT) is part of many routine screening approaches. The aim of this manuscript was to scrutinize the evidence for using ALT testing as a primary screening parameter for liver diseases. We conclude that (i) elevated serum ALT levels indicate a high specificity and a reasonable sensitivity liver injury, (ii) 10 - 25 % of German adults have elevated ALT levels, (iii) ALT values are increased in the majority but not all patients with acute and chronic liver disease (iv) elevated ALT-values are associated with an increased risk of liver-specific mortality, (v) elevated ALT values are also a risk factor for non-hepatic diseases including diabetes mellitus type 2, metabolic syndrome, cardiovascular diseases and malignancies, (vi) many liver diseases identified by an ALT screening can be treated successfully including prevention of development of clinical endpoints, (vii) an ALT-screening is very likely to be cost-effective although studies are needed for Germany to support this conclusion.

Effect of ribavirin on the frequency of RNase L cleavage sites within the hepatitis C viral genome.

The mechanisms of synergy in antiviral activity of interferon-alpha and ribavirin in treating chronic hepatitis C virus (HCV) infection are still unknown. Interferon-alpha indirectly induces cleavage of viral RNA by RNase L at UU/UA dinucleotides. There is evidence that HCV genomes with a higher number of UU/UA dinucleotides are more sensitive to interferon-alpha. As a guanosine analogue, ribavirin exerts a mutagenic effect promoting G-to-A and C-to-U transitions. This study investigates whether ribavirin-induced mutagenesis causes a higher frequency of UU/UA dinucleotides in the viral progeny sequences. Increased mutational frequencies in favour of G-to-A and C-to-U transitions during ribavirin treatment was reported by Hofmann et al. (Gastroenterology 2007;132:921-930). Overall, 937 nucleotide sequences from that publication were reanalysed for RNase L cleavage sites. These included HCV NS3 quasispecies from three patients with ribavirin monotherapy and NS5B quasispecies from patients who received ribavirin alone (n = 7) or in combination with interferon-alpha (n = 7) at baseline and during treatment; NS5B quasispecies from a subgenomic HCV replicon system after 24, 48 and 72 h of cultivation with or without ribavirin or with levovirin. For NS3 quasispecies during ribavirin monotherapy and NS5B quasispecies from patients who received ribavirin alone or in combination with interferon-alpha, analysis of RNase L cleavage sites did not reveal changes during treatment or differences between treatment regimes. Similarly, RNaseL cleavage sites from NS5B quasispecies of the HCV replicon did not differ significantly between time points or treatments. In conclusion, Ribavirin-induced mutagenesis did not increase RNase L cleavage sites (UU/UA dinucleotides) within the HCV NS3 or NS5B encoding regions.

Primary actinomycosis of the liver mimicking malignancy.

A 71-year old women presented with fever, a significant loss of body weight and abdominal pain in the upper right quadrant since approximately six months. Abdominal ultrasonography and magnetic resonance imaging (MRI) showed an irregularly shaped, inhomogeneous and hypointense lesion of the right liver lobe (6 x 8 cm in segment 7 and 8) with multiple satellite lesions. Irregular shape, hypovascular presentation during gadolinium enhancement, hypointensity in T 1-weighted images and dilation of peripheral bile ducts were suggestive for cholangiocarcinoma or metastasis. However, histological investigations revealed a rare case of primary actinomycosis of the liver which was successfully treated with antibiotics.

[Prediction of perioperative mortality in patients with advanced liver disease and abdominal surgery by the use of different scoring systems and tests]. / Prädiktion der perioperativen Mortalität bei Patienten mit fortgeschrittener Lebererkrankung und abdominalchirurgischen Eingriffen anhand unterschiedlicher Score- und Testsysteme.

Patients with advanced liver disease show increased morbidity and mortality after hepatic resection and non-hepatic digestive surgery. Furthermore, postoperative liver failure is associated with a poor outcome, representing an important clinical problem. For evaluation of the perioperative mortality and the hepatic function, several scoring systems, clinical parameters, and static and dynamic tests are available. Recently, the Model for End-Stage Liver Disease (MELD) has been shown to provide a complementary predictive value to the widely used Child Turcotte Pugh score. Patients with Child Turcotte Pugh class C cirrhosis and MELD scores >14 are generally not considered for surgical intervention. Patients with Child Turcotte Pugh class B cirrhosis and MELD scores >8-14 have an increased perioperative risk and the indication for surgery should be assessed carefully. In patients with Child Turcotte Pugh class A cirrhosis and MELD scores of

Prospective study of bone mineral density and metabolism in patients with chronic hepatitis C during pegylated interferon alpha and ribavirin therapy.

The importance of osteoporosis as a complication of end-stage liver disease is well known. However, significant osteopenia may occur in earlier stages of chronic hepatitis C (CHC). Furthermore, antiviral therapy may influence bone metabolism. Thirty patients with CHC genotype 1 infection and without established cirrhosis were treated with peginterferon-alfa and ribavirin. Dual-energy x-ray absorptiometry was performed at baseline, after 48 weeks of therapy, and by the end of a 24-week follow-up period. Bone mineral density (BMD), T-scores, and Z-scores were assessed. Serum C-terminal propeptide of type I collagen (CICP) and osteocalcin levels were measured. Thirteen patients had osteopenia (43%) and osteoporosis was present in four patients (13%). Antiviral therapy led to significant on-treatment increases of lumbar spine and hip BMD (P < or = 0.05) as well as T-scores (P < or = 0.05) and Z-scores (P < or = 0.01) irrespective of subsequent treatment response. Further analyses showed that in patients with sustained virological response (n = 19) most parameters remained highly above baseline values by the end of the 24-week follow-up period, while patients with virological relapse (n = 11) had decreases of BMD, T-scores and Z-scores thereafter that did not differ from baseline. Serum CICP and osteocalcin levels decreased during therapy. Osteocalcin levels remained below baseline in sustained responder, but showed an increase in relapsers by the end of the 24-week follow-up (P < or = 0.05). Osteopenia is detectable in a substantial proportion of CHC patients without established cirrhosis. Antiviral therapy leads to an on-treatment increase of BMD, which may last in those patients who achieve a sustained virological response.

Somatic hypermutation and mRNA expression levels of the BCL-6 gene in patients with hepatitis C virus-associated lymphoproliferative diseases.

Chronic hepatitis C virus (HCV) infection leads to mixed cryoglobulinaemia (MC) and B-cell non-Hodgkin lymphoma (B-NHL). Aberrant somatic hypermutation and deregulation of the oncogene BCL-6 is associated with lymphomagenesis. Recently, HCV was shown to induce BCL-6 mutations in vitro. The BCL-6 gene (area B) was cloned and sequenced from peripheral blood mononuclear cells (PBMC) of 21 chronically HCV-infected patients with or without MC and B-NHL, and six healthy controls. Mutational frequencies, genetic complexity and diversity were calculated. BCL-6 mRNA from PBMC was quantified by real-time polymerase chain reaction, and additional sustained virologic responders to antiviral therapy and HBV patients served as controls. The overall/recurrent mutational frequencies tended to be lower in MC and B-NHL patients when compared with controls (P = 0.15 and 0.06, respectively). Genetic complexity was significantly lower in MC and B-NHL patients (P = 0.025). BCL-6 mRNA concentration was decreased in all HCV patients when compared with healthy controls, sustained virologic responder and HBV patients (P = 0.005). Although HCV can induce BCL-6 mutations in vitro, lower mutational frequencies and decreased BCL-6 mRNA expression in vivo suggest no major role of aberrant somatic hypermutation in HCV-associated MC and B-NHL.

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

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 log(10) 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.

[Effectiveness of antiviral therapy in patients with chronic hepatitis C treated by private practice gastroenterologists]. / Effektivität der antiviralen Behandlung von Patienten mit chronischer Hepatitis C in der gastroenterologischen Facharztpraxis.

The standard treatment for patients with chronic hepatitis C consists of pegylated interferon (PegIFN) alpha in combination with ribavirin. Information on treatment effectiveness outside clinical trials is sparse. To study community-based health care, a regional network supported by the German network of competence for hepatitis (Hep-Net) was created between gastroenterologists in private practice and a tertiary referral centre. A treatment register containing evidence-based guidelines was established and 212 consecutive patients who were treated with either PegIF Nalpha 2a/ribavirin (n = 126) or PegIFNalpha2b/ribavirin (n = 86) for 24 weeks (HCV genotype 2, 3) and 48 weeks (HCV genotype 1, 4, 5), respectively, were included and followed prospectively. Twenty-four weeks after cessation of antiviral treatment a sustained virological response was achieved in 54 % of the patients. By univariate analyses, infection with HCV genotypes 2 or 3 (p < 0.0001), younger age (p < 0.0001), normal gamma-glutamyltransferase levels before initiation of treatment (p = 0.003), and absence of language communication problems (p = 0.023) were associated with a sustained virological response. The presence of liver cirrhosis in patients with HCV genotype 1, 4, 5 infection was associated with lower sustained response rates (p = 0.025). Patients infected with HCV genotype 1 in whom the PegIFNalpha dose was reduced had higher virological relapse rates (p = 0.049). With regard to the treating physician, sustained virological response rates ranged from 26 - 67 % in patients infected with HCV genotype 1. Our study shows that virological response rates similar to those in international randomised clinical trials can be achieved by private practice gastroenterologists. The presented network allows characterization of the treatment outcome in chronic hepatitis C not only with regard to virus- and host-related factors but also on an individual physician basis.

Mutations in the putative HCV-E2 CD81 binding regions and correlation with cell surface CD81 expression.

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.

[Hypertransaminasaemia and impaired liver function in a patient with oligosymptomatic celiac disease]. / Leberfunktionsstörung als Leitsymptom einer einheimischen Sprue.

Hypertransaminasaemia and impaired liver function in a patient with oligosymptomatic celiac disease. We describe the case of a 45-year-old man who was referred for evaluation of elevated aminotransferases. One year before referral the patient developed an ischemic stroke followed by a subdural hematoma three months later. In our outpatient clinic the patient presented with a malabsorption syndrome including diminished vitamin-K-dependent clotting factors. Serologic testing was positive for IgA antigliadin antibodies and IgA antiendomysial antibodies. Celiac disease was confirmed by an upper endoscopy examination and biopsies obtained from the distal duodenum. Histological examination showed villous atrophy, crypt hyperplasia and an increase in intraepithelial lymphocyte count consistent with celiac disease. After initiation of a gluten-free diet the malabsorption syndrome as well as liver dysfunction improved. Serum aminotransferase levels normalized within 6 months. The clinical course demonstrates involvement of the liver in patients with celiac disease.