Clinical Trial Details
— Status: Recruiting
Administrative data
| NCT number |
NCT00164073 |
| Other study ID # |
122/03 |
| Secondary ID |
|
| Status |
Recruiting |
| Phase |
N/A
|
| First received |
September 13, 2005 |
| Last updated |
July 31, 2007 |
| Start date |
February 2004 |
| Est. completion date |
February 2007 |
Study information
| Verified date |
September 2005 |
| Source |
Bayside Health |
| Contact |
Mark D Berzsenyi, MBBS |
| Phone |
0417 574 015 |
| Email |
m.berzsenyi[@]alfred.org.au |
| Is FDA regulated |
No |
| Health authority |
Australia: Human Research Ethics Committee |
| Study type |
Observational
|
Clinical Trial Summary
The purpose of this study is to examine gene expression profiles by DNA microarray in
patients who are responders and non-responders to interferon and ribavirin treatment for
hepatitis C virus (HCV). Genes involved in inflammation and fibrosis and mediators of the
Th-1 lymphocyte response will be looked for. It is hoped that genetic targets for future
more effective and less toxic treatments will be identified.
Description:
Background and Research Plan:
Chronic HCV infection often follows a progressive course over many years, and can ultimately
result in cirrhosis and the need for liver transplantation or hepatocellular carcinoma. The
decision to treat patients with chronic hepatitis C infection is based upon several factors
including the natural history and stage of the disease and the efficacy and adverse effects
related to therapy. As a general rule patients who are considered for treatment should have
histologic and virologic evidence of chronic infection (i.e., HCV RNA detectable in serum)
and an elevated serum ALT (1). Combination treatment with Interferon and Ribavirin is
currently the best available treatment for chronic HCV infection. However, response to
therapy is suboptimal and there are several side effects.
Side effects seen during therapy of patients with Interferon and Ribavirin include flu-like
symptoms mostly due to interferon, cytopenias and haemolysis induced by Ribavirin.
Psychiatric changes due to interferon, occur in up to 50% and thyroid abnormalities
requiring therapy occur in about 1 to 5% treated with interferon. Autoimmune disease and
retinal haemorrhages have also been reported. In addition, combination therapy is associated
with significant risks in patients with renal dysfunction, cardiac disease and haemolytic
anaemias as treatment may be associated with anaemia. Patients who are depressed or suicidal
should also be excluded (2).
To this point little DNA microarray work done has been done with HCV. However, what work has
been done is interesting and shows promise. In studies comparing Hepatitis B (HBV) with
Hepatitis C, HBV infection was found to express genes predominantly involved in
inflammation. In contrast, with HCV infection anti-inflammatory genes were found to be
expressed (3). In another study HCV associated cirrhosis has been shown to be characterized
by proinflammatory, pro-fibrotic and proapoptotic gene expression profiles (4).
The progression of HCV infection by gene expression analysis of liver biopsies in acutely
infected chimpanzees that develop persistent infection, transient viral clearance, or
sustained clearance has been examined. Transient and sustained viral clearance were uniquely
associated with induction of IFN-gamma-induced genes and other genes involved in antigen
processing and presentation and the adaptive immune response. The study revealed genome-wide
transcriptional changes that reflect the establishment, spread, and control of infection,
and they reveal potentially unique antiviral programs associated with clearance of HCV
infection (5).
In-vitro studies in the human cell line Huh7 has shown that on exposure of the cells to
Interferon 50 genes were up-regulated by at least twofold in control clones, whereas
induction of 9 of the 50 genes was significantly reduced in those Huh7 clones expressing
NS5A. Interferon-alpha activity has been found to be inhibited by the HCV protein NS5A. The
strongest effect of NS5A on Interferon response was observed on the OAS-p69 gene with
reduced expression. In addition, Huh7 cells expressing NS5A showed an up-regulation of
interleukin-8 a proinflammatory cytokine. As a result this study postulated a mechanism for
NS5A mediated interferon resistance (6).
In a recent study microarray gene profiling of peripheral blood mononuclear cells from
hepatitis C patients treated with Interferon-alpha was performed. 88 genes directly relating
to functions of immune cells were up-regulated, including genes involved in antigen
processing and presentation, T-cell activation, lymphocyte trafficking, and effector
functions, suggesting that Interferon-alpha up-regulates multiple genes involving different
aspects of immune responses to enhance immunity against hepatitis C virus (7).
Currently, treatment for HCV still has a significant non-responder rate and is associated
with side effects which limits many individuals from receiving treatment. Already molecular
profiling by DNA microarrays has identified potential genetic targets which may have a role
in the pathogenesis of HCV-associated hepatitis and more importantly possible targets which
have a role in response to Interferon treatment. The work proposed here hopes to build on
this body of knowledge and may lead to improved treatment modalities for HCV.
Methods/Experimental Design:
Approval from the Alfred Hospital Ethics Committee will be obtained and subjects will be
recruited via the Alfred Hospital's Hepatitis Clinics. Informed consent for the study will
be obtained after its purpose is explained verbally and via a plain English statement.
Informed consent will also include consent for liver biopsies. Initially, a pilot study will
be undertaken in which only one subject will be recruited to both the experimental and
control arm of the study. Once the results from this pilot study have been analysed the
study will be expanded to encompass a greater number of individuals.
Liver biopsies will be performed on the following groups.
1. HCV positive patients prior to commencement and at the end of treatment with either
standard or pegylated Interferon plus Ribavirin. This group will be subdivided into
responders and non-responders to treatment. Core liver biopsies under ultra-sound
guidance are already routinely performed on this group prior to the commencement of
treatment. For the purpose of this study non-responders to combination therapy are
those individuals who remain HCV-RNA positive at the end of treatment.
2. Control population consisting of patients undergoing laparotomy for diseases not
involving the liver. These biopsies would be obtained at the time of surgery.
Enough liver tissue (approximately 1-2 mg) will be obtained to isolate messenger RNA (mRNA)
by oligo dT chromatography. Once isolated the mRNA will be reverse transcribed to
complementary DNA (cDNA). This cDNA will then be labeled with a fluorescent reporter
molecule. Hybridization of the labeled liver cDNA will then occur with the DNA microarray.
It is envisaged that the microarray will be commercially obtained and consist of human cDNA.
There is enough DNA on the microarray that two lots of labeled liver cDNA with different
fluorescent markers (i.e. red and green) can be hybridized to the array at the same time
without any interference. The following hybridizations would then be set up.
Reporter molecule 1: HCV +ve group. cDNA Reporter molecule 2: HCV -ve group. cDNA
Hybridization 1 Responder to Rx. Before Rx Control cDNA
Hybridization 2 Responder to Rx. After Rx Control cDNA
Hybridization 3 Non responder to Rx. Before Rx Control cDNA
Hybridization 4 Non responder to Rx. After Rx Control cDNA
Rx = treatment.
The hybridized array will then be scanned and commercially available software packages will
be used to help in the interpretation of the results (8). Genes or groups of genes of
particular interest include:
1. Pro-inflammatory response genes
- Th-1 cytokines such as Gamma Interferon (IFN), Interleukin-2 (IL-2) and Tumour
Necrosis Factor-beta (TNF-ß). Th1 cytokines promote production of opsonizing
antibodies (e.g., IgG1) and induction of cellular cytotoxicity and macrophage
activation. Th1 responses are prominent in the defence against pathogens which
replicate intracellularly.
- Tumour Necrosis Factor (TNF) and TNF receptor family of molecules which are
strongly pro-inflammatory and involved in immune regulation.
- Interleukin-12 (IL-12) which is a key cytokine that protects the host from viral
and microbial infection. It links the innate and acquired arms of the immune
system.
- Interleukin-18 (IL-18) which has been shown to act as a chemoattractant.
2. Genes involved in a pro-fibrotic response.
- Transforming growth factor beta (TGFbeta) which causes fibroblast proliferation.
- Procollagen I and II.
- Platelet-derived growth factor (PDGF) and PDGF beta receptor over-expression have
been linked to development of fibrotic disease as well as cancer and
atherosclerosis.
- Matrix metalloproteinases play a pivotal role in angiogenesis and have a role in
tumorigenesis.
- Fibroblast Growth Factor.
- Vascular Endothelial Growth Factor (VEGF) which is known for its angiogenic
properties.
- Connective tissue growth factor which stimulates collagen production.
In addition, novel genes previously unidentified, with differential expression will be
looked for and will be characterized.
References relevant to this project (from literature search)
1. Chopra, S. Treatment of chronic hepatitis C infection: Recommendations. In: Up To Date,
Rose, BR(Ed), Up To Date, Wellesley. MA, 2002.
2. Bonis, PLA, Chopra, S. Administration of combined interferon alfa-2b and ribavirin in
the treatment of hepatitis C infection. In: Up To Date, Rose, BR(Ed), Up To Date,
Wellesley. MA, 2002.
3. Honda, M, Kaneko, S, Kawai, H, et al. Differential gene expression between chronic
hepatitis B and C hepatic lesions. Gastroenterology 2001; 120: 955-66.
4. Shackel, NA, McGuinness, PH, Abbott, CA, et al. Insights into the pathobiology of
hepatitis C virus-associated cirrhosis: analysis of intrahepatic differential gene
expression. Am J Pathol 2002; 160: 641-54.
5. Su, AI, Pezacki, JP, Wodicka L, et al. Genomic analysis of the host response to
hepatitis C virus infection. Proc Natl Acad Sci 2002;99:15669-74.
6. Girard, S, Shalhoub, P, Lescure, P, et al. An altered cellular response to interferon
and up-regulation of interleukin-8 induced by the hepatitis C viral protein NS5A
uncovered by microarray analysis. Virology 2002; 295: 272-83.
7. Ji, X, Cheung, R, Cooper, S, et al.Interferon alfa regulated gene expression in
patients initiating interferon treatment for chronic hepatitis C. Hepatology
2003;37:610-21.
8. Jenkins, RE, Pennington, SR. arrays for protein expression profiling: towards a viable
alternative to two-dimensional gel electrophoresis. Proteomics 2001; 1: 12-29.
