Genetic profiling using plasma-derived cell-free DNA in therapy-naïve hepatocellular carcinoma patients: a pilot study.

Background: Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine whether plasma-derived cell-free DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy. Patients and methods: Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was carried out using a panel targeting 46 coding and non-coding genes frequently altered in HCCs. Results: In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) of the patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) of the patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5 cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) of the cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations. Conclusion: In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients.

In vivo analysis at the cellular level reveals similar steatosis induction in both hepatitis C virus genotype 1 and 3 infections.

Steatosis is a frequent histological feature of hepatitis C virus (HCV) infection. Cohort studies of patients with chronic hepatitis C identified HCV genotype 3 (HCV GT3) as the prevalent steatotic genotype. Moreover, Huh-7 cells over-expressing HCV GT3 core protein accumulate more triglyceride in larger lipid droplets than cells expressing core proteins of other HCV genotypes. However, little is known about the relationship of steatosis and HCV infection at the cellular level in vivo. In this study, we used highly sensitive multiplex in situ hybridization methodology together with lipid staining to investigate HCV-induced lipid droplet accumulation at the cellular level in liver biopsies. Consistent with previous reports, histological steatosis grades were significantly higher in GT3 compared to GT1 infected livers, but independent of viral load. Using nile red lipid stainings, we observed that the frequency of lipid droplet containing cells was similar in HCV GT1- and HCV GT3-infected livers. Lipid droplet formation preferentially occurred in HCV-infected cells irrespective of the genotype, but was also observed in noninfected cells. These findings demonstrate that the main difference between GT1- and GT3-induced steatosis is the size of lipid droplets, but not the number or relative distribution of lipid droplets in infected vs uninfected hepatocytes.

Hepatitis B virus covalently closed circular DNA homeostasis is independent of the lymphotoxin pathway during chronic HBV infection.

Current treatment options for patients with chronic hepatitis B virus (HBV) infection are not curative as they are not effective in eliminating covalently closed circular DNA (cccDNA). cccDNA is a stable template for HBV transcription in the nucleus of hepatocytes and is thought to be one of the main factors responsible for HBV persistence. Recently, activation of the lymphotoxin beta receptor (LTßR) has been shown to trigger degradation of cccDNA through induction of cytidine deaminases of the APOBEC3 family in HBV cell culture model systems. To assess the presence and relevance of such mechanisms in the liver of chronically HBV-infected patients, we compared intrahepatic cccDNA levels with the expression levels of lymphotoxins and some of their target genes (eg APOBEC deaminases) in liver biopsy tissue. Our results confirm elevated gene expression levels of components of the lymphotoxin pathway including lymphotoxin alpha (LTα), lymphotoxin beta (LTß), APOBEC3B (A3B) and APOBEC3G (A3G) in the chronically HBV-infected liver compared to uninfected liver. Furthermore, expression levels of the genes of the APOBEC deaminase family were correlated with those of LTα and LTß gene expression, consistent with lymphotoxin-mediated upregulation of APOBEC gene expression. However, intrahepatic cccDNA and HBV replication levels were not correlated with LTα, LTß and APOBEC gene expression. In conclusion, these results suggest that although the lymphotoxin pathway is activated in the chronically HBV-infected liver, it has no major impact on HBV cccDNA metabolism in chronic HBV infection.

Impact of common risk factors of fibrosis progression in chronic hepatitis C.

OBJECTIVE: The natural course of chronic hepatitis C varies widely. To improve the profiling of patients at risk of developing advanced liver disease, we assessed the relative contribution of factors for liver fibrosis progression in hepatitis C. DESIGN: We analysed 1461 patients with chronic hepatitis C with an estimated date of infection and at least one liver biopsy. Risk factors for accelerated fibrosis progression rate (FPR), defined as ≥ 0.13 Metavir fibrosis units per year, were identified by logistic regression. Examined factors included age at infection, sex, route of infection, HCV genotype, body mass index (BMI), significant alcohol drinking (≥ 20 g/day for ≥ 5 years), HIV coinfection and diabetes. In a subgroup of 575 patients, we assessed the impact of single nucleotide polymorphisms previously associated with fibrosis progression in genome-wide association studies. Results were expressed as attributable fraction (AF) of risk for accelerated FPR. RESULTS: Age at infection (AF 28.7%), sex (AF 8.2%), route of infection (AF 16.5%) and HCV genotype (AF 7.9%) contributed to accelerated FPR in the Swiss Hepatitis C Cohort Study, whereas significant alcohol drinking, anti-HIV, diabetes and BMI did not. In genotyped patients, variants at rs9380516 (TULP1), rs738409 (PNPLA3), rs4374383 (MERTK) (AF 19.2%) and rs910049 (major histocompatibility complex region) significantly added to the risk of accelerated FPR. Results were replicated in three additional independent cohorts, and a meta-analysis confirmed the role of age at infection, sex, route of infection, HCV genotype, rs738409, rs4374383 and rs910049 in accelerating FPR. CONCLUSIONS: Most factors accelerating liver fibrosis progression in chronic hepatitis C are unmodifiable.

Hepatitis C virus dysregulates glucose homeostasis by a dual mechanism involving induction of PGC1α and dephosphorylation of FoxO1.

The maintenance of glucose homeostasis is a complex process in which the insulin signalling pathway plays a major role. Disruption of insulin-regulated glucose homeostasis is frequently observed in chronic hepatitis C (CHC) infection and might potentially contribute to type 2 diabetes mellitus (T2DM) development. Presently, the mechanism that links HCV infection to insulin resistance remains unclear. Previously, we have reported that HCV protein expression in HCV transgenic mice (B6HCV) leads to an overexpression of protein phosphatase 2A (PP2A) through an ER stress response. In the present work, we describe an association of FoxO1 hypophosphorylation and upregulation of both PGC-1α and G6Pase to phenotypic hyperglycaemia and insulin resistance in B6HCV mice. In vitro, we observed that PGC1α is concomitantly induced with PP2A. Moreover, we show that the enhanced PP2A expression is sufficient to inhibit insulin-induced FoxO1 phosphorylation via blockade of insulin-mediated Akt activation or/and through direct association and dephosphorylation of pS-FoxO1. Consequently, we found that the gluconeogenic gene glucose-6-phosphatase is upregulated. These observations were confirmed in liver biopsies obtained from CHC patients. In summary, our results show that HCV-mediated upregulation of PP2A catalytic subunit alters signalling pathways that control hepatic glucose homeostasis by inhibiting Akt and dephosphorylation of FoxO1.

Effect of antiviral therapy on circulating cytokeratin-18 fragments in patients with chronic hepatitis C.

Hepatocellular apoptosis plays a major role in the pathogenesis of chronic hepatitis C. It can be measured noninvasively by determining the circulating levels of cytokeratin-18 fragments. We hypothesized that the effect of antiviral therapy on this parameter will be different in patients with a sustained virological response, relapse (REL) and nonresponse (NR). We quantified cytokeratin-18 fragments in plasma of patients participating in the Swiss Hepatitis C cohort, who received antiviral therapy without stopping because of sides effects. A total of 315 patients were included, 183 with a sustained response, 64 with NR and 68 who relapsed. Mean levels ±SD of circulating cytokeratin-18 fragments before therapy were 174 ± 172 U/L for responsders, 188 ± 145 for nonresponders and 269 ± 158 U/L for patients who relapsed. The values were significantly higher in the REL group (ANOVA P < 0.006). A sustained response was associated with a significant improvement of the plasma levels (94 ± 92 U/L, paired test P < 0.000001), whereas there was no improvement in the nonresponder group (183 ± 158 U/L) and in the relapser group (158 ± 148 U/L). There was a weak correlation between alanine aminotransferase (ALT) and cytokeratin-18 fragment levels (r² = 0.35, P < 0.000001) before therapy but not after therapy and none with hepatitis C virus (HCV) viremia. Successful antiviral therapy results in a significant decrease in circulating levels of cytokeratin-18 fragments arguing for a reduction in hepatocellular apoptosis after clearance of the HCV. Baseline cytokeratin-18 fragment levels are higher in relapsers. Correlations with ALT are weak, suggesting that these two tests measure different but related processes.

Hepatitis with fibrin-ring granulomas.

We describe a 66-year-old woman hospitalized with fever, fatigue and hepatopathy. In her medical history arterial hypertension (treated with propranolol and lisinopril), diabetes mellitus type 2 (no treatment before admission) and a gout arthropathy were noted wherefore a therapy with allopurinol 300 mg per day has been started 4 months before. Liver biopsy revealed fibrin-ring granulomas, compatible with allopurinol-induced hepatitis. Because of persistence of high fever after stopping allopurinol, steroids (1 mg/kg) were started. Under this treatment, she developed pancytopenia and fever. The bone marrow aspiration revealed Leishmania infantum. A second liver biopsy showed amastigotes and a disappearance of the granulomas. The history revealed a travel to Malta 2 years earlier. Despite adequate treatment with liposomal amphotericin B the patient deteriorated and finally died in septic shock.

[Autoimmune hepatitis]. / Autoimmunhepatitis.

Autoimmune hepatitis (AIH) is chronic, predominantly periportal hepatitis with hypergammaglobulinemia and tissue autoantibodies. It is a relative rare disorder, with a preponderance of female patients, that can present at any age. Its diagnosis relies on the exclusion of viral, metabolic, genetic and toxic etiologies of chronic hepatitis or hepatic injury. There are no pathognomonic features, but the presence of antinuclear and smooth muscle antibodies or liver-kidney microsomal antibodies together with typical histological features in liver biopsy allows the diagnosis with good confidence. Corticosteroid therapy is effective in most patients. Because of a favorable side effect profile, the combination therapy with azathioprine should be used whenever possible. Relapses after initial treatment responses are frequent, but in the majority of patients the disease can be controlled. Liver transplantation is effective, and shows good 10-year survival rates.

Hepatitis C: a concise review.

Chronic hepatitis C is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Current treatment options are limited, but recent progress in the understanding of the molecular virology of hepatitis C has led to the identification of novel targets for antiviral intervention. In addition, gene and immunotherapeutic strategies to inhibit hepatitis C virus (HCV) replication or gene expression and to enhance the cellular immune response against HCV are being explored. These and other novel antiviral strategies may eventually complement existing therapeutic modalities. Here, we briefly review current concepts of the epidemiology, molecular virology, pathogenesis, natural history, diagnosis, therapy, and prevention of hepatitis C.

Hepatitis C: an update.

Hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. While current therapeutic options for hepatitis C are limited, recent progress in the understanding of the biology of HCV led to the identification of novel targets for antiviral intervention. In addition, molecular and immunotherapeutic strategies to inhibit HCV replication or gene expression and to enhance the cellular immune response against HCV are being explored. These and other novel antiviral strategies may eventually complement existing therapeutic modalities. Here, we briefly review current concepts of the epidemiology, molecular virology, pathogenesis, natural history, diagnosis, therapy, and prevention of hepatitis C.

Intracellular signalling and antiviral effects of interferons.

More than 4 decades after their discovery, interferons are used now in daily clinical practice for the treatment of chronic viral hepatitis, multiple sclerosis, chronic granulomatous disease, and malignant disease such as hairy cell leukaemia, chronic myeloid leukaemia, Kaposi's sarcoma, multiple myeloma and malignant melanoma. In general, treatment with interferons is successful in only a fraction of the patients suffering from these diseases. The reasons for treatment failures in many patients are not understood a present. The discovery of the Jak-Stat pathway as the principal signalling pathway for interferons opens new research options for a better understanding of interferon resistance in various diseases. Defective Jak-Stat signal transduction has now been described in cells expressing HBV proteins, in cells expressing HCV proteins, and in cell lines derived from malignant melanomas. A better understanding of these signalling defects might lead to new therapeutic strategies making interferons more effective in a larger percentage of patients.

Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response.

CD66a is an adhesion molecule member of the carcinoembryonic antigen immunoglobulin-like family present on the surface of epithelial cells, granulocytes and IL-2 activated T cells. We studied whether CD66a is expressed in vivo by T lymphocytes and whether it affects TCR-mediated activation. CD66a was detected by histochemistry, flow cytometry analysis, reverse transcription PCR and Western blot on fresh colon biopsies and T cell clones. A fraction of T cells in the lamina propria express CD66a, which is induced by IL-7 and IL-15 cytokines. T cells express four different CD66a splice variants and at least two forms of the protein are glycosylated in a cell type-specific manner. Triggering of CD66a on T cells with physiological ligands or with specific mAb increases TCR-mediated lymphokine release, in an antigen dose-independent manner. This effect requires the presence of the CD66a intracytoplasmic domain, which contains two immunoglobulin receptor family tyrosine-based inhibitory motif-like domains, as shown by stimulation of Jurkat cells transfected with different CD66a isoforms and is associated with increased induction of AP1 and NFkappaB transcription factors. These data indicate that CD66a amplifies T cell activation and thus could facilitate crosstalk between epithelial cells and T lymphocytes in intestinal immune response.

Defective Jak-STAT signal transduction pathway in melanoma cells resistant to growth inhibition by interferon-alpha.

Advanced malignant melanoma is an aggressive malignancy with poor prognosis. Current therapeutic strategies have a modest success rate. The most promising treatment consists of a combination of chemotherapy with interferon-alpha, but complete response rates remain less than 15%. Interferon-alpha is also effective in adjuvant therapy for non-advanced melanoma treated surgically. The molecular mechanisms leading to loss of growth restraints and gain of growth-promoting functions during carcinogenesis of malignant melanoma are not understood in detail. Here, we studied 9 human melanoma cell lines with regard to growth inhibition by interferon-alpha and defects in intracellular signal transduction through the Jak-STAT pathway. In 3 cell lines, we found a complete loss of growth restraint by interferon-alpha. In all of them, different components of the Jak-STAT pathway were defective. Since signal transduction through the Jak-STAT pathway is necessary for antiviral and antiproliferative effects of interferons, we conclude that defects in this pathway may be one of the mechanisms that lead to cancer progression through loss of growth-restraining functions. Moreover, our results indicate that a subgroup of melanomas could be completely resistant to interferon-alpha and should therefore not be treated with this cytokine.

Expression of hepatitis C virus proteins inhibits signal transduction through the Jak-STAT pathway.

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. Alpha interferon (IFN-alpha) therapy of chronic hepatitis C leads to a sustained response in 10 to 20% of patients only. The mechanisms of viral persistence and the pathogenesis of hepatitis C are poorly understood. We established continuous human cell lines, allowing the tightly regulated expression of the entire HCV open reading frame under the control of a tetracycline-responsive promoter. Using this in vitro system, we analyzed the effect of HCV proteins on IFN-induced intracellular signaling. Expression of HCV proteins in these cells strongly inhibited IFN-alpha-induced signal transduction through the Jak-STAT pathway. Inhibition occurred downstream of STAT tyrosine phosphorylation. Inhibition of the Jak-STAT pathway was not restricted to IFN-alpha-induced signaling but was observed in leukemia inhibitory factor-induced signaling through Stat3 as well. By contrast, tumor necrosis factor alpha-induced activation of the transcription factor NF-kappaB was not affected. Interference of HCV with IFN-alpha-induced signaling through the Jak-STAT pathway could contribute to the resistance to IFN-alpha therapy observed in the majority of patients and may represent a general escape strategy of HCV contributing to viral persistence and pathogenesis of chronic liver disease.

The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus.

The Jak-STAT pathway was originally discovered through the study of interferon induced intracellular signal transduction. Meanwhile, a large number of cytokines, hormones and growth factors have been found to activate Jaks and STATs. Jaks (Janus Kinases) are a unique class of tyrosine kinases that associate with cytokine receptors. Upon ligand binding, they activate members of the Signal Transducers and Activators of Transcription (STAT) family through phosphorylation on a single tyrosine. Activated STATs form dimers, translocate to the nucleus, bind to specific response elements in promotors of target genes, and transcriptionally activate these genes. Both positive and negative regulations of the Jak-STAT pathway have been identified. In a positive feedback loop, interferons transcriptionally activate the genes for components of the interferon stimulated gene factor 3 (ISGF3). A number of cytokines that activate the Jak-STAT pathway, e.g. IL-6, IL-4, LIF, G-CSF, have been shown to upregulate the expression of SOCS-JABs-SSIs, a recently discovered class of STAT inhibitors. Targeted disruption of genes for a number of Jaks and STATs in mice have revealed specific biological functions for many of them. Although most of the STATs are activated in cell culture by many different ligands, STAT knockout mice mostly show defects in a single or a few cytokine dependent processes. STAT1 knockout mice have an impaired interferon signalling, STAT4 knockouts impaired IL-12 signalling, STAT5a knockouts impaired prolactin signalling, STAT5b knockouts impaired growth hormone signalling, and STAT6 knockout impaired IL-4 and IL-13 signalling. Defects in the Jak-STAT pathway have already been identified in a number of human diseases. Prominent amongst them are leukaemias, lymphomas and inherited immunodeficiency syndromes. It can be expected that additional Jak-STAT related diseases will be identified over the next years. To date, specific STAT inhibitory drugs are not known, but a number of specific protein-protein interactions in the Jak-STAT pathway are potential targets for pharmaceutical interventions.

Interferon-alpha activates signal transducers and activators of transcription 5 and 6 in Daudi cells.

In most cells studied so far, interferon-alpha (IFN-alpha) activates signal transducer and activator of transcription (Stat1), Stat2 and Stat3, whereas interferon-gamma (IFN-gamma) induces Stat1 only. In general, each of the several dozens of cytokines, growth factors and hormones that signal through the Janus kinases-signal transducers and activators of transcription (Jak-STAT) pathway activates a distinct subset of STATs, and this selectivity is thought to be essential for the specificity of the cellular responses toward these ligands. Here, we have studied the pattern of STAT activation in the human lymphoblastoid cell line Daudi in response to IFN-alpha. In addition to Stat1, Stat2 and Stat3 activation, IFN-alpha was found to directly induce activation of Stat5 and Stat6. Cell-type-specific activation of additional STATs could be responsible for cell-type-specific responses to IFN-alpha.

Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection.

The aim of this study was to characterize the interferon induced intracellular signals in duck hepatocytes and to investigate the effects of duck interferon on virus replication in duck hepatitis B virus (DHBV) infected ducks. Interestingly, duck interferon was found to activate intracellular signal transduction pathways similar to those of its mammalian counterparts. An interferon stimulated gel shift activity like that of gene factor 3 is induced, as well as serum inducible element binding factors homologous to serum inducible factor A (SIF-A), SIF-B and SIF-C. Duck interferon induced signal transducer and activator of transcription activation is not inhibited by DHBV infection of hepatocytes. DHBV infected ducks treated for 10 days with recombinant duck interferon show a decrease in viral DNA in hepatocytes, and in many cases disappearance of viraemia. These findings confirm the usefulness of the DHBV infection model for the study of human hepatitis B virus infection.

Growth effects of alpha-interferon but not of bombesin or angiotensin II are mediated by activation of STAT proteins.

BACKGROUND: The recently discovered Jak/STAT signal transduction pathway is associated with cytokine or growth factor receptors; whether members of the G protein-coupled receptor superfamily also activate this pathway is not yet clear. As a first member, the angiotensin (AT)1A receptor has been demonstrated to phosphorylate Jak and STAT proteins. Bombesin, a neurotransmitter and growth factor in many cells and tissues, activates its G protein-coupled receptor and in addition phosphorylates proteins that might be members of the Jak/STAT family. This study investigated whether bombesin- or angiotensin-mediated growth effects are associated with STAT protein activation. METHODS: Functional receptors were characterized using ligand-binding studies, second-messenger activation and determination of ligand-mediated growth effects. STAT protein activation was analysed by electrophoretic mobility shift assay (EMSA) using labelled DNA response elements recognizing all known STAT proteins. RESULTS: Functional bombesin receptors mediating mitogenic effects were demonstrated on Swiss 3T3 fibroblasts, human melanoma cells (A375-6) and primary human lung fibroblasts; however, bombesin-related STAT protein activation was not observed by EMSA. Interferon-alpha typically activated a STAT1-STAT2-p48 heterotrimer, as well as STAT1-3 hetero- and homodimers in human melanoma cells and significantly inhibited growth of this cell line in vitro. Functional AT1A receptors on primary rat cardiac fibroblasts mediated angiotensin-stimulated growth effects but, in contrast to recently published data, did not activate any known STAT protein. CONCLUSION: Interferon alpha-stimulated growth inhibition is mediated by activation of the Jak/STAT pathway, whereas bombesin or AT1A receptor-mediated effects on cellular proliferation do not involve phosphorylation of STAT proteins.

Interferon-gamma receptor signaling is not required in the effector phase of the alloimmune response.

BACKGROUND: Gene transcripts for the Thl cytokines interleukin (IL)-2 and interferon-gamma (IFN-gamma) are frequently detected during allograft rejection. The relative importance of these cytokines in facilitating allograft rejection is unclear. Recently, we have shown that IL-2-deficient mice reject islet allografts. In the IL-2-deficient system, IFN-gamma gene transcripts are abundantly expressed. METHODS: To determine the relative importance of IFN-gamma-dependent effector mechanisms in mediating allograft rejection, the present study utilized IFN-gamma receptor-deficient mice as islet allograft recipients. Grafts were analyzed by immunohistology, and cytokine expression was measured by competitive template reverse transcriptase polymerase chain reaction. RESULTS: IFN-gamma receptor-deficient mice reject islet allografts by a process that is T cell-dependent. Although IFN-gamma receptor signaling is absent, these mice do not show a clear Th2 type response. CONCLUSION: Although the signals evoked through the IFN-gamma receptor may play a role, they are not essential to allograft rejection.