Therapy of Chronic Delta Hepatitis With Peginterferon Alfa
Therapy of Chronic Delta Hepatitis With Peginterferon Alfa
Patients with chronic delta hepatitis with anti-HDV in serum and HDV antigen in liver tissue were eligible for this prospective, open-label, nonrandomised study of peginterferon treatment for up to 260 weeks. Inclusion criteria included: age ≥18 years, serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) above the upper limit of normal (ALT >41 or AST >31 U/L) on an average of three determinations taken within 6 months before treatment, and a liver biopsy obtained within 12 months with a necroinflammatory score of at least 5 (out of 18) and at least 1 for hepatic fibrosis (out of 6) as scored using the Ishak modification of the HAI scoring system. All liver biopsies were interpreted by a single hepatopathologist (DEK) who was blinded to the clinical data. All patients had to have histological staining for HDV antigen. Hepatic venous pressure gradient (HVPG) measurements were obtained at the time of liver biopsy via the transjugular route with rigorous adherence to methodology as previously described. Exclusion criteria included evidence of other forms of liver disease, hepatocellular carcinoma, decompensated liver disease, human immunodeficiency virus (HIV) co-infection, active drug or alcohol abuse, any contraindication to peginterferon, and pregnancy or refusal to use adequate contraception during therapy.
All patients provided written, informed consent, and the study protocol and consent forms were approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health Clinical Center. The study was registered in ClinicalTrials.Gov (#NCT00023322).
Pegylated interferon alfa-2a (Peginterferon) was provided by Hoffman La Roche (Genentech) under a Clinical Trial Agreement with the NIDDK. Genentech did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Administration of peginterferon was conducted under an Investigational New Drug Application (IND # BB-10101) held by JHH.
The primary histological endpoint of the study was defined as improvement in hepatic histology after 144 weeks of peginterferon therapy. Histological response was defined as a decrease in disease activity by ≥3 points using the modified histological activity index (HAI) scale without worsening of fibrosis (Ishak).
A virological response (VR) to peginterferon therapy was defined as the inability to detect HDV RNA in serum by quantitative measurements. The primary virological endpoint of the study was a complete virological response (CVR) and was defined as the combination of HDV virological response with HBsAg seroconversion. HBsAg seroconversion was defined as loss of HBsAg (negative on qualitative test or <1 IU/mL on quantitative test) and appearance of anti-HBs using a qualitative test.
Secondary endpoints included (i) improvement in histology after 48 weeks of peginterferon therapy, (ii) loss of HDV RNA from serum (undetectable = <100 genome equivalents (GE)/mL) at weeks 48, 144 and 260 (maintained virological response), (iii) loss of HBsAg from serum at weeks 48, 144 and 260, (iv) loss of detectable HDV antigen staining in the liver at weeks 48, 144 and 260, (v) normalisation of serum ALT (<41 U/mL) at weeks 48, 144 and 260 (maintained biochemical response), (vi) changes in hepatic venous pressure gradient (HVPG) measurements and their relationship with hepatic histology.
Patients were admitted to the NIH Clinical Center to start treatment with peginterferon 180 μg/week and had blood samples drawn at 6, 12, 18, 24, 48, 72 h after treatment initiation, followed by weekly out-patient visits for the first month and visits every 4–8 weeks thereafter. Serum samples were stored in −80°C. Transjugular liver biopsies with measurement of HVPG were performed at weeks 0, 48, 144 and 260.
During treatment, the peginterferon dose could be increased or decreased based on effect and tolerance. Following the initial 24 weeks, the dose could be escalated up to 360 μg/week if an effect was not seen at a lower dose, or decreased as needed for toxicity and tolerance. Efficacy was assessed by ALT and changes were in increments or decrements of 45 or 90 μg/week every 16–24 weeks (Figure S1). ALT was chosen to represent response as serum HDV RNA results were not available in real time. At the week 48 biopsy, patients without a ≥3 point improvement in the HAI inflammatory score (without worsening of fibrosis score) or a 1 point improvement in Ishak fibrosis score were considered nonresponders. Therapy was discontinued for: (i) intolerance to peginterferon, (ii) lack of histological response at 1, 3 and 5 years (a maintained histological response) or (iii) a complete virological response (defined as the loss of HDV RNA and HBsAg seroconversion). Those with complete virological response or virological response were followed after therapy in a protocolised manner for a year after stopping therapy.
(Enlarge Image)
Figure S1.
This figure describes the peginterferon dose modification schedule while patients were on therapy.
In patients with concurrent active HBV infection (HBV DNA >10,000 copies/mL), nucleoside analogue therapy was instituted to suppress hepatitis B.
Quantitative measurement of serum HDV RNA level was performed by qPCR on stored samples with a lower limit of detection of 100 GE/mL (National Genetics Institute, Los Angeles, CA, USA).
Qualitative HBsAg testing was performed with the FDA approved VITROS® HBsAg assay (Ortho-Clinical Diagnostics, Raritan, NJ, USA) per manufacturer's recommendations with a signal/cut-off ratio of 1.0. Quantitative HBsAg (qHBsAg) levels were measured on stored serum samples in a single batch, using the Elecsys HBsAg II Quant assay (Roche Diagnostics, Indianapolis, IN, USA) with a lower limit of detection of 0.055 IU/mL.
Genomic DNA was extracted from whole blood using the Qiagen Flexigene DNA Kit (Qiagen, Valencia, CA, USA) for 10 patients who provided consent for DNA analysis. Allelic discrimination for the IL28B-associated rs12989760 SNP, associated with interferon responsiveness in hepatitis B and C, was performed utilising the Taqman Custom SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) on an ABI 7500 Real-Time PCR System (Applied Biosystems).
The slope of decline of HDV and HBsAg levels was calculated by linear regression using all available log-transformed data points from baseline to week 4. The week 12 decline was calculated by subtracting week 12 results from baseline. When week 12 results were not available, they were estimated using data from the previous and subsequent visits. Statistical analyses were performed using SPSS Statistics v. 19 (IBM, Armonk, NY, USA). Statistical significance was assessed using nonparametric tests.
Methods
Patients
Patients with chronic delta hepatitis with anti-HDV in serum and HDV antigen in liver tissue were eligible for this prospective, open-label, nonrandomised study of peginterferon treatment for up to 260 weeks. Inclusion criteria included: age ≥18 years, serum alanine aminotransferase (ALT) or aspartate aminotransferase (AST) above the upper limit of normal (ALT >41 or AST >31 U/L) on an average of three determinations taken within 6 months before treatment, and a liver biopsy obtained within 12 months with a necroinflammatory score of at least 5 (out of 18) and at least 1 for hepatic fibrosis (out of 6) as scored using the Ishak modification of the HAI scoring system. All liver biopsies were interpreted by a single hepatopathologist (DEK) who was blinded to the clinical data. All patients had to have histological staining for HDV antigen. Hepatic venous pressure gradient (HVPG) measurements were obtained at the time of liver biopsy via the transjugular route with rigorous adherence to methodology as previously described. Exclusion criteria included evidence of other forms of liver disease, hepatocellular carcinoma, decompensated liver disease, human immunodeficiency virus (HIV) co-infection, active drug or alcohol abuse, any contraindication to peginterferon, and pregnancy or refusal to use adequate contraception during therapy.
All patients provided written, informed consent, and the study protocol and consent forms were approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health Clinical Center. The study was registered in ClinicalTrials.Gov (#NCT00023322).
Pegylated interferon alfa-2a (Peginterferon) was provided by Hoffman La Roche (Genentech) under a Clinical Trial Agreement with the NIDDK. Genentech did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Administration of peginterferon was conducted under an Investigational New Drug Application (IND # BB-10101) held by JHH.
Definition of Endpoints and Response
The primary histological endpoint of the study was defined as improvement in hepatic histology after 144 weeks of peginterferon therapy. Histological response was defined as a decrease in disease activity by ≥3 points using the modified histological activity index (HAI) scale without worsening of fibrosis (Ishak).
A virological response (VR) to peginterferon therapy was defined as the inability to detect HDV RNA in serum by quantitative measurements. The primary virological endpoint of the study was a complete virological response (CVR) and was defined as the combination of HDV virological response with HBsAg seroconversion. HBsAg seroconversion was defined as loss of HBsAg (negative on qualitative test or <1 IU/mL on quantitative test) and appearance of anti-HBs using a qualitative test.
Secondary endpoints included (i) improvement in histology after 48 weeks of peginterferon therapy, (ii) loss of HDV RNA from serum (undetectable = <100 genome equivalents (GE)/mL) at weeks 48, 144 and 260 (maintained virological response), (iii) loss of HBsAg from serum at weeks 48, 144 and 260, (iv) loss of detectable HDV antigen staining in the liver at weeks 48, 144 and 260, (v) normalisation of serum ALT (<41 U/mL) at weeks 48, 144 and 260 (maintained biochemical response), (vi) changes in hepatic venous pressure gradient (HVPG) measurements and their relationship with hepatic histology.
Study Design and Dosing of Peginterferon
Patients were admitted to the NIH Clinical Center to start treatment with peginterferon 180 μg/week and had blood samples drawn at 6, 12, 18, 24, 48, 72 h after treatment initiation, followed by weekly out-patient visits for the first month and visits every 4–8 weeks thereafter. Serum samples were stored in −80°C. Transjugular liver biopsies with measurement of HVPG were performed at weeks 0, 48, 144 and 260.
During treatment, the peginterferon dose could be increased or decreased based on effect and tolerance. Following the initial 24 weeks, the dose could be escalated up to 360 μg/week if an effect was not seen at a lower dose, or decreased as needed for toxicity and tolerance. Efficacy was assessed by ALT and changes were in increments or decrements of 45 or 90 μg/week every 16–24 weeks (Figure S1). ALT was chosen to represent response as serum HDV RNA results were not available in real time. At the week 48 biopsy, patients without a ≥3 point improvement in the HAI inflammatory score (without worsening of fibrosis score) or a 1 point improvement in Ishak fibrosis score were considered nonresponders. Therapy was discontinued for: (i) intolerance to peginterferon, (ii) lack of histological response at 1, 3 and 5 years (a maintained histological response) or (iii) a complete virological response (defined as the loss of HDV RNA and HBsAg seroconversion). Those with complete virological response or virological response were followed after therapy in a protocolised manner for a year after stopping therapy.
(Enlarge Image)
Figure S1.
This figure describes the peginterferon dose modification schedule while patients were on therapy.
In patients with concurrent active HBV infection (HBV DNA >10,000 copies/mL), nucleoside analogue therapy was instituted to suppress hepatitis B.
Virological Assays
Quantitative measurement of serum HDV RNA level was performed by qPCR on stored samples with a lower limit of detection of 100 GE/mL (National Genetics Institute, Los Angeles, CA, USA).
Qualitative HBsAg testing was performed with the FDA approved VITROS® HBsAg assay (Ortho-Clinical Diagnostics, Raritan, NJ, USA) per manufacturer's recommendations with a signal/cut-off ratio of 1.0. Quantitative HBsAg (qHBsAg) levels were measured on stored serum samples in a single batch, using the Elecsys HBsAg II Quant assay (Roche Diagnostics, Indianapolis, IN, USA) with a lower limit of detection of 0.055 IU/mL.
Genetic Analysis
Genomic DNA was extracted from whole blood using the Qiagen Flexigene DNA Kit (Qiagen, Valencia, CA, USA) for 10 patients who provided consent for DNA analysis. Allelic discrimination for the IL28B-associated rs12989760 SNP, associated with interferon responsiveness in hepatitis B and C, was performed utilising the Taqman Custom SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) on an ABI 7500 Real-Time PCR System (Applied Biosystems).
Statistical Analysis
The slope of decline of HDV and HBsAg levels was calculated by linear regression using all available log-transformed data points from baseline to week 4. The week 12 decline was calculated by subtracting week 12 results from baseline. When week 12 results were not available, they were estimated using data from the previous and subsequent visits. Statistical analyses were performed using SPSS Statistics v. 19 (IBM, Armonk, NY, USA). Statistical significance was assessed using nonparametric tests.
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