Kai-Henrik Peiffer

Clinical utility of the ARCHITECT HCV Ag assay for early treatment monitoring in patients with chronic hepatitis C genotype 1 infection

BACKGROUND Virologic response-monitoring is essential for determining therapy duration in patients with chronic hepatitis C virus (HCV) infection. This is usually performed using highly sensitive HCV-RNA assays. However, HCV-RNA assays are time-consuming, expensive and require highly trained personnel. Quantitative determination of HCV core-antigen (HCVAg) levels may be used to supplement treatment monitoring. OBJECTIVES The clinical utility of the ARCHITECT HCV Ag assay (Abbott Diagnostics) for response-guided therapy was investigated. STUDY DESIGN We analyzed serum from 160 patients with HCV genotype 1 infection who had been treated with peg-interferon alfa-2b/ribavirin. HCVAg levels were determined at baseline, weeks 1, 2, 4 and 12. HCVAg levels were compared to those obtained with HCV-RNA assays: VERSANT HCV Quantitative 3.0 (bDNA) and Qualitative (TMA, both Siemens Healthcare) assay and the Abbott RealTime HCV assay (ART; Abbott Diagnostics). RESULTS Baseline HCVAg levels correlated well with HCV-RNA as assessed by bDNA (r=0.91; p<0.0001) and ART (r=0.92; p<0.0001), respectively. Patients with undetectable HCVAg levels at week 1 had a 90.9% probability (positive predictive value) to achieve a rapid virologic response (HCV-RNA undetectable at week 4) based on TMA and 86.4% based on ART, respectively. Patients with less than 1 log(10) reduction in HCVAg between baseline and week 12 had a 90% probability (negative predictive value) to achieve a nonresponse (<2 log(10) decline in HCV-RNA between baseline and week 12) based on bDNA and 100% based on ART, respectively. CONCLUSIONS Determination of HCVAg may be useful for antiviral response-monitoring in patients with HCV genotype 1 infection.

Interferon lambda 4 genotypes and resistance‐associated variants in patients infected with hepatitis C virus genotypes 1 and 3

Single‐nucleotide polymorphisms (SNPs) in the interferon lambda 4 (IFNL4) gene are predictors for treatment success in patients with hepatitis C virus (HCV) infection. For direct‐acting antiviral combinations only weak association with IFNL4 SNPs was observed. Little is known about potential selections of resistance‐associated variants (RAVs) by the IFNL4 genotype. This study analyzed the prevalence of RAVs to currently approved direct‐acting antivirals in a large European population in correlation to SNPs in IFNL4. Samples of 633 patients chronically infected with HCV genotypes 1a (n = 259), 1b (n = 323), and 3 (n = 51) were genotyped for rs12979860 (formerly known as IL28B) and rs368234815. RAVs in NS3, NS5A, and NS5B were detected by population‐based sequencing. In addition, IFNL4 SNPs and NS5A RAVs were analyzed including deep sequencing (n = 109) in an independent replication cohort of HCV genotype 1‐infected patients (n = 201). No significant correlation was found between IFNL4 SNPs and rare and common RAVs within NS3 and NS5B. In contrast, the NS5A RAV Y93H was detected frequently in HCV genotype 1b (14%) and significantly associated with the beneficial IFNL4 SNPs (P < 0.001 and P = 0.002, respectively). Moreover, the presence of Y93H in HCV genotype 1b patients was significantly associated with the second site variant T83M (P < 0.001). Independent factors significantly associated with the presence of Y93H were IFNL4 genotype and high baseline viral load. Conclusion: The NS5A RAV Y93H is significantly associated with the presence of beneficial IFNL4 SNPs and a high baseline viral load in HCV genotype 1‐infected patients, which may explain a lack of correlation or even an inverse correlation of treatment response with IFNL4 genotype in some NS5A inhibitor containing IFN‐free regimens. (Hepatology 2016;63:63–73)

IFN lambda 4 genotypes and resistance-associated variants in HCV genotype 1 and 3 infected patients.

Single‐nucleotide polymorphisms (SNPs) in the interferon lambda 4 (IFNL4) gene are predictors for treatment success in patients with hepatitis C virus (HCV) infection. For direct‐acting antiviral combinations only weak association with IFNL4 SNPs was observed. Little is known about potential selections of resistance‐associated variants (RAVs) by the IFNL4 genotype. This study analyzed the prevalence of RAVs to currently approved direct‐acting antivirals in a large European population in correlation to SNPs in IFNL4. Samples of 633 patients chronically infected with HCV genotypes 1a (n = 259), 1b (n = 323), and 3 (n = 51) were genotyped for rs12979860 (formerly known as IL28B) and rs368234815. RAVs in NS3, NS5A, and NS5B were detected by population‐based sequencing. In addition, IFNL4 SNPs and NS5A RAVs were analyzed including deep sequencing (n = 109) in an independent replication cohort of HCV genotype 1‐infected patients (n = 201). No significant correlation was found between IFNL4 SNPs and rare and common RAVs within NS3 and NS5B. In contrast, the NS5A RAV Y93H was detected frequently in HCV genotype 1b (14%) and significantly associated with the beneficial IFNL4 SNPs (P < 0.001 and P = 0.002, respectively). Moreover, the presence of Y93H in HCV genotype 1b patients was significantly associated with the second site variant T83M (P < 0.001). Independent factors significantly associated with the presence of Y93H were IFNL4 genotype and high baseline viral load. Conclusion: The NS5A RAV Y93H is significantly associated with the presence of beneficial IFNL4 SNPs and a high baseline viral load in HCV genotype 1‐infected patients, which may explain a lack of correlation or even an inverse correlation of treatment response with IFNL4 genotype in some NS5A inhibitor containing IFN‐free regimens. (Hepatology 2016;63:63–73)

The importance of HCV RNA measurement for tailoring treatment duration.

The introduction of telaprevir and boceprevir in the treatment of chronically HCV genotype 1 infected patients has led to substantially improved sustained virologic response rates and shorter treatment duration for a growing group of patients. Management and monitoring of patients receiving protease inhibitor-based triple therapy is of major importance and has become more complicated. Close monitoring of HCV RNA levels for patients on protease inhibitor-based therapy to identify subjects who are eligible for shortening of treatment duration, are virological non-responders or are in danger of experiencing a viral breakthrough is strongly recommended. Several virological tools including qualitative and quantitative HCV RNA assays for detection and quantification of HCV RNA are commercially available. We review these methods and their implications for HCV therapy as well as current sustained virologic response definition, stopping rules and recommendations for protease inhibitor-based treatment durations.

Intracellular accumulation of subviral HBsAg particles and diminished Nrf2 activation in HBV genotype G expressing cells lead to an increased ROI level

BACKGROUND & AIMS Hepatitis B virus genotype G (HBV/G) is characterized by a lack of HBeAg secretion and very low HBsAg secretion. This study aimed at (1) comparing HBV genotype G and A2 with respect to morphogenesis and release of HBV-derived particles, (2) characterizing factors contributing to HBV/G-associated pathogenesis. METHODS HBV/G- and HBV/A-expressing hepatoma cells and infected HepaRG cells were analyzed by confocal laser scanning microscopy, Western blot, real-time PCR, density gradient centrifugation, and electron microscopy. Modulation of the transcription factors Nrf2 and AP-1 was analyzed. RESULTS While the release of viral particles is not affected in HBV/G replicating cells, the secretion of subviral particles is impaired, although they are produced in high amounts. These subviral particles, which display an increased density and a predominantly filamentous morphology, accumulate at the endoplasmic reticulum. The PreS1PreS2 domain of genotype G, which forms aggregates, causes the block of HBsAg-secretion at the ER and leads to decreased transcriptional activator function of LHBs. Intracellular accumulation of HBsAg and impaired induction of the cytoprotective transcription factor Nrf2 lead to an elevated level of ROIs. This results in activation of JNK and as a consequence in Ser-phosphorylation of IRS-1, which is known to impair insulin signaling, a key factor for liver regeneration. CONCLUSIONS Although competent for release of viral particles, secretion of subviral particles is impaired in HBV/G expressing cells leading to ER-stress. In parallel, HBV-induced Nrf2 activation diminishes, which causes a decrease of the capacity to inactivate ROIs. This might be related to genotype-specific pathogenesis.

Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes

For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.

Risk of de novo Hepatocellular Carcinoma after HCV Treatment with Direct-Acting Antivirals

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

Interferon-free treatment choice according to baseline RASs leads to high SVR rates in HCV genotype 1 infected patients

AIM Different combinations of direct antiviral agents (DAA) lead to high SVR rates in HCV genotype 1 infected patients. However, presence of baseline resistance-associated substitutions (RASs) represents a major risk factor for treatment failure. It is unknown whether choice of treatment based on RASs has the potential to decrease virologic failure rates. METHODS Population-based sequencing of NS3 and NS5A genes was performed in HCV genotype 1 infected patients at a German university hospital. Treatment was individually selected based on resistance analyses. RESULTS In total, 319 patients (50% treatment-experienced and 30% with cirrhosis) were included. With the treatment choice based on the baseline NS3 and NS5A resistance profile SVR rates between 96 and 100% were observed in all subgroups, including treatment-experienced patients with cirrhosis and HCV genotype 1a infected cirrhotic patients. CONCLUSIONS The choice of treatment based on the RASs status at baseline may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure.

Investigation of NS3 Protease Resistance-Associated Variants and Phenotypes for the Prediction of Treatment Response to HCV Triple Therapy.

Triple therapy of chronic hepatitis C virus (HCV) infection with boceprevir (BOC) or telaprevir (TVR) leads to virologic failure in many patients which is often associated with the selection of resistance-associated variants (RAVs). These resistance profiles are of importance for the selection of potential rescue treatment options. In this study, we sequenced baseline NS3 RAVs population-based and investigated the sensitivity of NS3 phenotypes in an HCV replicon assay together with clinical factors for a prediction of treatment response in a cohort of 165 German and Swiss patients treated with a BOC or TVR-based triple therapy. Overall, the prevalence of baseline RAVs was low, although the frequency of RAVs was higher in patients with virologic failure compared to those who achieved a sustained virologic response (SVR) (7% versus 1%, P = 0.06). The occurrence of RAVs was associated with a resistant NS3 quasispecies phenotype (P<0.001), but the sensitivity of phenotypes was not associated with treatment outcome (P = 0.2). The majority of single viral and host predictors of SVR was only weakly associated with treatment response. In multivariate analyses, low AST levels, female sex and an IFNL4 CC genotype were independently associated with SVR. However, a combined analysis of negative predictors revealed a significantly lower overall number of negative predictors in patients with SVR in comparison to individuals with virologic failure (P<0.0001) and the presence of 2 or less negative predictors was indicative for SVR. These results demonstrate that most single baseline viral and host parameters have a weak influence on the response to triple therapy, whereas the overall number of negative predictors has a high predictive value for SVR.

Iron deficiency anaemia and cataracts in a patient with haemochromatosis

A 55-year-old woman presented to our hepatology outpatient clinic with symptoms of anaemia. Her medical history included hereditary haemochromatosis type 1, psoriatic arthropathy and bilateral cataracts since early adulthood. There was a family history of early-onset cataracts. Haemochromatosis was treated by frequent phlebotomies (2–4 per month) since diagnosis 2.5 years earlier, limited by several episodes of severe symptomatic anaemia (minimal haemoglobin: 7.0 g/dL). Despite anaemia, she presented with constantly high levels of ferritin (>1000 ng/mL) throughout the period of phlebotomies so that she was referred to our clinic for further diagnostics and therapy. At presentation, physical examination showed signs of anaemia but no signs of liver cirrhosis. The levels for γ-glutamyltransferase, alanine transaminase, bilirubin, international normalised ratio (INR) and thrombocytes were within the normal …