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Towards optimal treatment for chronic hepatitis C infection Huub Gelderblom

OMSLAG.indd 1

Towards optimal treatment for chronic

hepatitis C infection

Huub Gelderblom

12-12-2007 8:49:45

Towards optimal treatment for chronic hepatitis C infection

Huub Gelderblom

Towards optimal treatment for chronic hepatitis C infection Thesis, University of Amsterdam, the Netherlands Copyright © 2008 Huub Gelderblom, the Netherlands No part of this thesis may be reproduced, stored or transmitted without prior permission of the author Layout:

Chris Bor, Medical Photography and illustration, Academical Medical Center, The Netherlands Printed by: Buijten& Schipperheijn

Towards optimal treatment for chronic hepatitis C infection

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof.dr D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel der Universiteit op donderdag 17 januari 2008, te 12.00 uur

door Hubrecht Cornelis Gelderblom

geboren te Vlissingen

Promotiecommissie Promotor:

Prof.dr P.L.M. Jansen

Co-promotores:

Dr M.G.H.M. Beld Dr H.W. Reesink

Overige leden:

Prof.dr H.L.A. Janssen Prof.dr P. Klenerman Prof.dr R.A.W. van Lier Prof.dr J. van der Meer Prof.dr T. van der Poll Dr H.L. Zaaijer

Faculteit der Geneeskunde

Table of contents 1.

General Introduction

2.

Clinical performance of the new Roche COBAS® TaqMan HCV Test and High Pure System for extraction, detection and quantitation of HCV RNA in plasma and serum

37

3.

Prediction of virologic response in difficult-to-treat chronic hepatitis C patients during high dose interferon induction therapy

47

4.

Low level HCV viraemia after initial response during antiviral therapy: transcription-mediated amplification predicts treatment failure

65

5.

Early and sustained HCV virological responses to therapy despite suppression of HCV specific T cells

71

6.

High incidence of type 1 diabetes mellitus during or shortly after treatment with pegylated interferon alfa for chronic hepatitis C virus infection

89

7.

Neopterin and ALT as markers of inflammation in chronic hepatitis C patients during administration of the HCV NS3•4A protease inhibitor telaprevir (VX-950) and/or peginterferon alfa 2a

99

8.

Monocyte derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile

109

9.

Summary and Conclusion

121

10. Samenvatting en Conclusie Dankwoord

7

127 135

C h a p t e r

1

General Introduction

General Introduction

Introduction Chronic hepatitis C virus (HCV) infection is a major cause of liver disease [1, 2]. HCV is endemic in most parts of the world, and it is estimated that at least 170 million people are or have been infected with HCV worldwide [3]. HCV is transmitted mainly via the percutaneous route. The majority of infected people develop chronic HCV infection, which frequently results in progressive liver disease. HCV related end-stage liver disease is now the main indication for liver transplantation in the USA and Western Europe. There is no vaccine or immunoglobulin to prevent HCV infection. Reliable in vitro culture systems have only recently become available. Current treatment regimens, based on administration of interferon alfa and ribavirin for 24-48 weeks, result in sustained remission of viremia in ~50% of patients. New classes of antiviral drugs that are expected to improve treatment significantly are currently being tested in phase I and phase II clinical trials.

Epidemiology HCV is an important infection in both developed and developing countries [3, 4] (Figure 1). Compared to other viral infectious diseases such as chronic hepatitis B virus infection (HBV, 350 million people with chronic infection worldwide) and infection with human immunodeficiency virus type 1 (HIV-1, 40 million people infected worldwide), HCV is particular in the sense that it also is quite prevalent in developed countries (estimated seroprevalence 0.5% in the Netherlands, 1.1% in France, 2.2% in Italy and 1.8% in the USA, Table 1). The estimate of 170 million people that have been infected worldwide is based on studies that reported seroprevalence data of antibodies to HCV, but did not assess presence

Figure 1. Worldwide anti-HCV seroprevalence in 2001, source WHO, 2002 [242].



Chapter 1 Table 1. Estimated prevalence of HCV, HIV and HBV infection, number of infected people and mortality worldwide and in 3 regions. region total

No. of people with chronic HCV

Prevalence of chronic HCV

128,000,000

2%

Mortality due No. of people to HCV in 2002 with HIV-AIDS 366,000

40,000,000

Western Europe

7,000,000

1%

17,000

720,000

North America

4,000,000

1.3%

10,000

1,300,000

Sub-Saharan Africa

18,000,000

3%*

45,000

25,000,000

Data from [3, 19, 240], [241]*, WHO and UNAIDS

of HCV RNA [3]. Given the fact that about 75% of those infected do not clear the virus and develop chronic infection, the actual number of people with chronic HCV infection is roughly around 130 million worldwide. The data are from selected populations and countries, and data obtained from population based studies are scarce [3, 4]. Therefore, the true number of HCV infected people may be underestimated. About 4.5 million people worldwide may be coinfected with HCV and HIV-1 [5]. About 30% of HIV-1 infected people in the USA and Europe have been co-infected with HCV, the rate of co-infection differs per region and depends on the main mode of transmission with the highest co-infection rates in hemophiliacs and intravenous drug users (IVDU). The worldwide HCV epidemic is a result of the increased use of syringes, the therapeutic use of blood and blood products and other invasive and/or parenteral medical procedures in the 20th century [4, 6]. Re-use of inadequately sterilized syringes and needles during mass treatment and/or vaccination campaigns has caused the spread of HCV in countries around the world [7-9]. In the 1970s and 1980s, post-transfusion non-A non-B (NANB) hepatitis was the most frequent infectious disease transmitted by blood transfusion, with an incidence of 2-20% for Europe and 10-12% for the USA. An estimated 80-90% of post-transfusion hepatitis cases were classified as NANB [10]. After the discovery of HCV in 1989 [11] and the development of a serologic assay for the detection of HCV-antibodies [12], HCV turned out to be the main cause of NANB hepatitis [13-16]. Blood transfusion prior to the start of donation testing in 1991 appears to account for between 6 and 12% of all infections with HCV in the United Kingdom. Approximately 24,000 recipients were estimated to have been infected with HCV, and by the end of 1995, 9100 of these were estimated to be alive [17].

Transmission / Risk factors The main route of HCV transmission is parenteral via contaminated blood [18]. However, in up to 50% of cases no recognizable risk factor can be identified. Persons at considerable risk for HCV infection include recipients of unscreened blood, blood products and organs (before 1991/1992 in developed countries); and anyone who injects drugs or has ever injected drugs, even if only once [19]. Other (minor) risk factors include haemodialysis, occupational exposure to blood, intranasal cocaine use, sexual promiscuity, beauty treatments, professional pedicure and manicure, ear piercing, incarceration, contact sports, barber shop shaving, sharing of injecting paraphernalia other than syringes or needles among injecting

10

General Introduction

Prevalence of Mortality due to HIV-AIDS HIV in 2005

No. of people with chronic HBV

Prevalence of chronic HBV

Mortality due to HBV in 2002

0.67%

2,800,000

350,000,000

5.8%

563,000

0.1%

18,000

1,400,000-3,500,000

0.2-0.5%

2200-5600

6 months after infection. The spectrum of the disease ranges from minimal chronic hepatitis, to chronic active hepatitis (activity may be intermittent), to progressive hepatitis with piecemeal necrosis, 10-20% of patients will develop cirrhosis after 20-30 years of chronic infection. Progression of the disease and risk of developing cirrhosis is slower in females and those who are young at the time of infection, but faster in persons with high disease activity, alcohol consumption, and

14

General Introduction

Figure 6. Generalized course of spontaneously resolving HCV infection (a) and chronic HCV infection (b). HCV RNA (black squares) is detectable in plasma within 1 to 2 weeks after infection. Plasma alanine aminotransferase (ALT) levels indicative of hepatocellular injury rise 2 to 8 weeks after infection. Symptoms are present in a minority of patients. Anti-HCV becomes detectable after a few weeks. After acute resolving infection anti-HCV persist in most patients. HCV RNA and ALT levels fluctuate considerably during the first 24 weeks. Chronic HCV infection is defined as persistence of HCV RNA in plasma > 24 weeks after infection.

HIV co-infection [100]. In cirrhotics, 1-5% per year develop hepatocellular carcinoma (HCC). Death is caused by decompensated cirrhosis or HCC [1, 2, 85, 87, 101]. End-stage liver disease due to HCV infection is now the major indication for liver transplantation in the western world [102]. The incidence of HCC and the number of HCV associated deaths due to cirrhosis or HCC–now estimated at 10,000 per year–will increase in the USA over the coming decades [19, 103]. Although not all infected persons develop an active hepatitis, and many infected persons seem unaffected by the disease, it is not clear to which extent these people are at risk of progressing to active hepatitis and the late sequelae of HCV infection. Caution should be taken though as symptoms may be aspecific, and many patients suffer from extrahepatic diseases that are HCV related such as cryoglobulinemia, lichen planus, Sjögren’s syndrome, or others [104, 105]. All patients with newly diagnosed chronic HCV should therefore be evaluated thoroughly. It is unknown whether the mode of acquisition affects the course of the disease. Young IV drug users with chronic HCV are more likely to die from IVDU related complications rather than from liver disease due to HCV [106]. About 50% of transfusion recipients die of causes other than HCV in the first decade after transfusion, and about 50% of transfusion recipients are alive >10 years after transfusion [97]. However, survival of transfusion recipients is similar to non-transfused people in those who are alive > 10 years after transfusion [97], and HCV related morbidity is expected in the period > 20 years after infection. This suggests that the number of patients with cirrhosis due to blood transfusion acquired HCV infection will increase in the coming decades [86]. Thus, chronic HCV is a slowly progressive disease and only a minority of all HCV infected persons will die from HCV related liver disease. However, due to size of the epidemic HCV now causes approximately 10,000 deaths per year in the USA (Table 1), and this number may increase in the years to come.

15

Chapter 1

Immunology Most patients exhibit strong HCV-specific CD8+-T-cell and CD4+-T-cell responses during the acute phase of infection and the outcome of acute HCV infection appears to be related to certain HLA class I and II alleles [107, 108], underlying the importance of T cell responses in attaining viral control Patients who spontaneously clear HCV infection maintain this strong and broad HCVspecific CD8+-T-cell and CD4+-T-cell response for years after resolution of the acute infection [109-112]. In patients who develop chronic infection the CD4 +-T-cell and CD8+-T-cell responses are not sustained [113-115]. In chronic HCV infection the HCV-specific CD8+-T-cell and CD4+-T-cell response is generally weak and narrowly focused [109, 110]. Possible reasons for this include T cell exhaustion, and viral mutation and escape [116, 117]. However, T cell responses can be identified in patients with chronic HCV in the absence of viral mutation so that other explanations are also required. Chronic HCV infection is associated with a significant loss of IL-2 secreting HCV specific CD4+-T-cells compared to IFN-γ secreting cells, but in spontaneously resolved infection IL-2 secretion is preserved [118]. There is evidence that HCV infects B cells [77-83], CD4+-T-cells [77, 79, 80], CD8+-T-cells [77-80, 82], monocytes [77-79], and dendritic cells [81-83, 119] in vivo, but these findings are controversial and how HCV subverts the functions of infected cells is not clear. Selective impairments in the function of professional antigen presenting cells known as dendritic cells have been described, and as these cells drive the T-cell response, this may be a cause of T-cell failure in chronic HCV infection [119-122]. There is evidence that HCV interferes with intracellular host defense mechanisms [123]. Some exposed but uninfected antibody negative persons do show cellular immune responses, this suggests that the rate of spontaneous clearance may be higher than the current estimate of 25% [98, 124-128]. Studies on the treatment of acute HCV infection have shown response rates of 90-100% with interferon alfa monotherapy, regardless of the HCV genotype [129]. The phenomenon that HCV is easier to treat when the infection is recent (< 6 months duration) suggests that in chronic infection the immune system has more difficulty to clear the virus. Interferon alfa has direct antiviral effects and immunomodulatory properties, ribavirin prevents virological relapse but by which mechanism is unknown [130]. A number of studies have suggested enhancement of HCV specific T-cell responses during successful antiviral therapy [131, 132], especially in patients with a rapid initial decline in HCV RNA [133, 134].

Detection The diagnosis and management of HCV infection is based on a number of laboratory tests: (1) serologic assays detecting HCV core antigen or specific antibody to HCV (anti-HCV), (2) assays to detect and quantify HCV RNA, (3) assays to determine the HCV genotype [135].

Serologic assays The presence of anti-HCV does not discriminate between resolved or ongoing HCV infection, 75% of anti-HCV positive individuals are also positive for HCV RNA [49, 96]. Anti-HCV may

16

General Introduction

disappear after spontaneously resolved infection [112]. Detection and quantitation of HCV core protein is possible using a HCV core antigen quantitative ELISA, but this assay is less sensitive than HCV RNA assays.

HCV RNA assays Nucleic acid testing (NAT) methods such as RT-PCR and transcription-mediated amplification (TMA) can detect HCV RNA levels as low as 5 IU/mL, and can be used to confirm ongoing HCV infection [136]. Quantitative PCR and branched DNA methods are less sensitive but can be used to monitor HCV RNA load and HCV RNA kinetics during antiviral therapy. The branched DNA (bDNA) method is based on hybridization of HCV specific probes to HCV RNA followed by signal amplification. New quantitative real-time PCR methods with increased sensitivity may bridge the gap between lower limit of quantitation and lower limit of detection, but at present these assays are not always reliable [137-139]. The linear dynamic ranges of the quantitative assays and dynamic ranges and lower limits of detection of the qualitative assays are depicted in Figure 7. Figure 7. Comparison of the linear dynamic ranges of the quantitative assays and dynamic ranges and lower limits of detection of the qualitative assays. HCV RNA levels are displayed as log10 IU/mL. HCV RNA levels are within the gray area in approximately 90% of untreated patients. Samples with HCV RNA above 100,000 IU/mL have to be diluted by a factor 10-100 for use with the Monitor assay. The TaqMan assay is qualitative below 43 IU/mL, the x depicts the lower limit of detection at 15 IU/mL. Both the TaqMan and Abbott are real-time PCR assays that have not been approved nor validated properly.

HCV genotype assays The duration of interferon alfa and ribavirin combination therapy depends on the HCV genotype. Hence, the HCV genotype should be assessed in all patients. Two assays are routinely used for determination of the HCV genotype, one is based on direct sequencing and comparison of the sequence to a number of genotype reference sequences, the other is based on hybridization of PCR amplicons with genotype-specific oligonucleotide probes [135].

Persistence of HCV A number of studies have demonstrated presence of low levels of HCV RNA in PBMCs and/or hepatocytes in some patients after spontaneous or treatment-induced resolution of HCV infection [77, 88, 140-153]. These findings may explain the relapses that have been observed in a small number patients [154-159]. Taken together, these findings suggest that spontaneously resolved acute HCV and treatment induced SVR may lead to a state wherein the virus can persist at very low levels, somehow unable to replicate to high levels. Other

17

Chapter 1

viruses such as CMV, EBV, HBV, Herpes Simplex, measles virus, and woodchuck hepatitis virus are also known to persist at very low levels.

Antibody negative HCV infection and occult HCV infection Cases of antibody negative HCV RNA positive infection have been described [50, 160], and prolonged periods of antibody negative HCV infection occur frequently in IVDU [70, 161, 162]. Castillo et al. [163-168] have recently described occult HCV infection in patients with elevated liver enzymes of unknown etiology, undetectable HCV RNA in plasma, and absence of antiHCV. A number of these patients were positive for HCV RNA by in situ hybridisation and RT-PCR in liver tissue and PBMCs. Apparently, a carrier state of HCV infection can occur.

Therapy Antiviral therapy for chronic HCV infection consists of administration of a modified form of recombinant interferon alfa (a cytokine) and ribavirin (a nucleoside analogue) for 12-48 weeks, leading to a sustained virologic response in ~ 50% of patients [169]. Both drugs were around before HCV was discovered and have been applied successfully as anti-HCV drugs. New classes of antiviral drugs that have been designed specifically for treatment of chronic HCV infection are currently being tested in phase I and phase II clinical trials.

The past Interferon alfa Interferons, discovered in the 1950s, are endogenous proteins with immunomodulatory and antiviral properties [170, 171]. The first study indicating that recombinant interferon alfa-2b might have a beneficial effect in chronic HCV infection (then known as chronic non-A, non-B hepatitis) was published in 1986 [172]. The first randomized clinical trials using interferon alfa-2b were started in 1986 and published just after the discovery of HCV [173-175]. These studies demonstrated normalisation of ALT in 38% and histological improvement in 50% of patients, normalisation of ALT was sustained in 16% of patients; HCV RNA levels could not be assessed at that time. The optimal duration of treatment initially appeared to be 48 weeks [176]. Subsequent trials demonstrated that 24 weeks of treatment was sufficient for patients infected with HCV genotype 2 or 3 [177, 178]. Modified forms of interferon alfa were created by adding a polyethyleneglycol (peg) moiety to the interferon alfa molecule, this resulted in an increased half-life facilitating once weekly administration. Three pivotal trials have demonstrated the superiority of one dose of peginterferon alfa per week compared to standard interferon alfa 3 times a week [179-181]

18

General Introduction

Ribavirin Ribavirin is a nucleoside analogue with broad spectrum antiviral activity that was developed in the early 1970s [182]. The mechanism of action of ribavirin is unknown [130]. Ribavirin monotherapy results in minor decreases in HCV RNA [183, 184], but profound decreases in ALT levels [183-186]. The first study suggesting an effect on HCV was published in 1991 [185]. The first randomized controlled trial comparing ribavirin and interferon alfa plus ribavirin was published by Brillanti et al. in 1994 [187], combination therapy with ribavirin plus interferon alfa did not influence the response during treatment, but reduced the relapse rate after cessation of treatment. These findings were confirmed in 3 pivotal trials that were published in 1998 [177, 178, 188], and combination therapy with interferon alfa and ribavirin became the standard treatment for chronic HCV infection.

Amantadine Amantadine is an old antiviral compound that is registered for treatment of Influenza A infection [189]. In vitro studies in Influenza A infected cells have shown that amantadine prevents virus assembly by blocking a virus specific ion channel [190]. Amantadine entered the HCV field in 1997 when Smith [191] reported a spectacular 18% SVR rate after 6 months of amantadine monotherapy in 22 IFN non-responders. To date these results have not been reproduced, Smith did not even mention SVR in her most recent paper [192]. Brillanti et al. published a small study in 1999 wherein 20 previous non-responders were treated for 24 weeks with interferon alfa plus ribavirin with or without amantadine: in the amantadine group 7 of 10 patients became HCV RNA negative during treatment and 3 of 10 achieved SVR, in the group without amantadine only 1 of 10 patients became HCV RNA negative during therapy and 0 of 10 achieved SVR [193]. The design of Brillanti’s amantadine trial was almost identical to his trial that demonstrated the benefits of ribavirin [187]; therefore, these results were considered very promising and led to dozens of clinical trials investigating the benefits of amantadine. The results from these trials are inconclusive, and a decade after the Smith study the benefits of amantadine are still unclear [194]. However, most of these trials were small, some patients received amantadine sulphate and some amantadine hydrochloride, not all patients were treatment naïve, interferon alfa dosing differed between trials, and patients were not stratified according to HCV genotype in some studies. Taken together, these findings suggest that a small antiviral effect of amantadine is likely, but difficult to prove, especially during combination therapy with the more powerful antivirals interferon alfa and ribavirin. The in vitro evidence is solid, a number of studies have shown that amantadine blocks the function of the HCV P7 ion channel, which is essential for virus assembly [195, 196].

19

Chapter 1

The present Peginterferon alfa and ribavirin combination therapy The current standard antiviral therapy for chronic HCV infection consists of administration of peginterferon alfa and ribavirin for 12-24 weeks (genotype 2 and 3) [179-181, 197], 24-48 weeks (genotype 1) [179-181, 198], or 48 weeks (genotype 4) [179] and leads to a sustained virologic response in approximately 50% (genotype 1 and 4) to 80% (genotype 2 and 3) of patients. The duration of interferon alfa and ribavirin therapy is based on HCV genotype, baseline HCV RNA load [198], and the HCV RNA level after 4 weeks [197, 198], 12 weeks [199, 200], or 24 weeks of antiviral therapy [199]. Interferon alfa and ribavirin based therapy is expensive (~ € 8000 in medication alone for 24 weeks) and causes a wide range of side effects in the majority of patients (Table 2)[51, 201, 202]. Therefore, HCV RNA should be assessed frequently during antiviral therapy in order to (i) stop treatment in patients predicted to experience treatment failure (e.g. in patients without a 2 log10 decline in HCV RNA at week 12) [199, 200, 203], and to (ii) shorten treatment duration in rapid responders (e.g., patients who are HCV RNA negative by PCR at week 4) [197, 198].

Viral kinetics The initial decline in HCV RNA levels during antiviral therapy is bi-phasic [65, 67]. The depth of the decline depends on the dose of interferon alfa [65, 204, 205] and the HCV genotype [206, 207]. The greatest decline is usually seen during the first phase, which lasts for 24 to 48 hours in most patients; this phase represents the direct inhibition of virus production by interferon alfa. During the second phase the decline in HCV RNA level is slower, a possible explanation for this decline is a decrease in the production and release of virus from a decreasing number of infected hepatocytes. Some authors interpret the second phase decline as “immune mediated killing of infected cells” [65], but in fact it is unknown if the second phase decline is immune mediated and if infected hepatocytes die or if the virus is cleared from the cell without cell death [208]. First and second phase HCV RNA kinetics can be calculated according a mathematical model of the bi-phasic decline [65]. The efficacy of antiviral therapy at day 1 or 2 (ε) can be calculated as (HCV RNA baseline – HCV RNA day 1 or 2) / HCV RNA baseline. An efficacy of 0.9 corresponds to a 90% or 1log10 reduction in HCV RNA, an efficacy of 0.99 corresponds to a 99% or 2log10 reduction in HCV RNA. The slope of the second phase decline (∂) can be calculated from HCV RNA levels at early time points, preferably day 2, week 1 and week 2. Some studies have shown that the first and/or second phase of decline in HCV RNA during antiviral therapy correlate with treatment outcome [205, 206, 209-211]; yet some patients with a slow initial decline in HCV RNA (ε < 0.90) during the first 2 days of therapy do achieve SVR [206, 210, 211]. A strong independent predictor of SVR, independent of HCV genotype, is the time needed to achieve a HCV RNA negative status during antiviral therapy [197, 198, 212-216]. Patients who are HCV RNA negative by PCR at week 4 have an increased chance of achieving SVR per se

20

General Introduction Table 2. Side effects of treatment with interferon alfa and ribavirin [51, 169, 202]. Frequency of side effect

Interferon alfa

Ribavirin

>30% (very common)

flu-like symptoms

hemolysis

headache

nausea

fatigue fever rigors myalgia thrombocytopenia induction of autoantibodies 1-30% (common)

anorexia

anemia

erythema at injection site

nasal congestion

insomnia

pruritus

alopecia

diarrhea

lack of motivation

eczema

inability to concentrate irritability, agitation emotional lability depression diarrhea autoimmune disease (thyroiditis, M Sjögren) leucocytopenia taste perversion or =2 infected blood donors. J Infect Dis, 2001. 183(4): p. 666-9.

72. Laskus, T., et al., Exposure of hepatitis C virus (HCV) RNA-positive recipients to HCV RNA-positive blood donors results in rapid predominance of a single donor strain and exclusion and/or suppression of the recipient strain. J Virol, 2001. 75(5): p. 2059-66.

27

Chapter 1 73. Moreau, I., et al., Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland. Virol J, 2006. 3: p. 95. 74.

Noppornpanth, S., et al., Genotyping hepatitis C viruses from Southeast Asia by a novel line probe assay that simultaneously detects core and 5’ untranslated regions. J Clin Microbiol, 2006. 44(11): p. 3969-74.

75.

Kageyama, S., et al., A natural inter-genotypic (2b/1b) recombinant of hepatitis C virus in the Philippines. J Med Virol, 2006. 78(11): p. 1423-8.

76.

Kalinina, O., et al., A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg. J Virol, 2002. 76(8): p. 4034-43.

77.

Torres, B., et al., HCV in serum, peripheral blood mononuclear cells and lymphocyte subpopulations in C-hepatitis patients. Hepatol Res, 2000. 18(2): p. 141-151.

78. Ducoulombier, D., et al., Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes. Hepatology, 2004. 39(3): p. 817-25. 79.

Laskus, T., et al., Hepatitis C virus in lymphoid cells of patients coinfected with human immunodeficiency virus type 1: evidence of active replication in monocytes/macrophages and lymphocytes. J Infect Dis, 2000. 181(2): p. 442-8.

80. Roque Afonso, A.M., et al., Nonrandom distribution of hepatitis C virus quasispecies in plasma and peripheral blood mononuclear cell subsets. J Virol, 1999. 73(11): p. 9213-21. 81.

Goutagny, N., et al., Evidence of viral replication in circulating dendritic cells during hepatitis C virus infection. J Infect Dis, 2003. 187(12): p. 1951-8.

82. Mellor, J., et al., Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells. J Gen Virol, 1998. 79 ( Pt 4): p. 705-14. 83. Boisvert, J., et al., Quantitative analysis of hepatitis C virus in peripheral blood and liver: replication detected only in liver. J Infect Dis, 2001. 184(7): p. 827-35. 84. Blackard, J.T., N. Kemmer, and K.E. Sherman, Extrahepatic replication of HCV: insights into clinical manifestations and biological consequences. Hepatology, 2006. 44(1): p. 15-22. 85. Seeff, L.B., Natural history of chronic hepatitis C. Hepatology, 2002. 36(5 Suppl 1): p. S35-46. 86. Koretz, R.L., et al., Non-A, non-B posttransfusion hepatitis--a decade later. Gastroenterology, 1985. 88(5 Pt 1): p. 1251-4. 87.

Sangiovanni, A., et al., The natural history of compensated cirrhosis due to hepatitis C virus: A 17-year cohort study of 214 patients. Hepatology, 2006. 43(6): p. 1303-10.

88. Wawrzynowicz-Syczewska, M., et al., Natural history of acute symptomatic hepatitis type C. Infection, 2004. 32(3): p. 138-43. 89.

Nomura, H., et al., Short-term interferon-alfa therapy for acute hepatitis C: a randomized controlled trial. Hepatology, 2004. 39(5): p. 1213-9.

90. Gerlach, J.T., et al., Acute hepatitis C: high rate of both spontaneous and treatment-induced viral clearance. Gastroenterology, 2003. 125(1): p. 80-8. 91.

Farci, P., et al., Hepatitis C virus-associated fulminant hepatic failure. N Engl J Med, 1996. 335(9): p. 631-4.

92. Kolk, D.P., et al., Significant closure of the human immunodeficiency virus type 1 and hepatitis C virus preseroconversion detection windows with a transcription-mediated-amplification-driven assay. J Clin Microbiol, 2002. 40(5): p. 1761-6. 93. Jackson, B.R., et al., The cost-effectiveness of NAT for HIV, HCV, and HBV in whole-blood donations. Transfusion, 2003. 43(6): p. 721-9.

28

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General Introduction 138. Gelderblom, H.C., S. Menting, and M.G. Beld, Clinical performance of the new Roche COBAS TaqMan HCV Test and High Pure System for extraction, detection and quantitation of HCV RNA in plasma and serum. Antivir Ther, 2006. 11(1): p. 95-103. 139. Colson, P., A. Motte, and C. Tamalet, Broad differences between the COBAS ampliprep total nucleic acid isolation-COBAS TaqMan 48 hepatitis C virus (HCV) and COBAS HCV monitor v2.0 assays for quantification of serum HCV RNA of non-1 genotypes. J Clin Microbiol, 2006. 44(4): p. 1602-3. 140. Pham, T.N., et al., Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C. J Virol, 2004. 78(11): p. 5867-74. 141. Radkowski, M., et al., Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C. Hepatology, 2005. 41(1): p. 106-14. 142. Radkowski, M., et al., Evidence for viral persistence in patients who test positive for anti-hepatitis C virus antibodies and have normal alanine aminotransferase levels. J Infect Dis, 2005. 191(10): p. 1730-3. 143. Muratori, L., et al., Testing for hepatitis C virus sequences in peripheral blood mononuclear cells of patients with chronic hepatitis C in the absence of serum hepatitis C virus RNA. Liver, 1994. 14(3): p. 124-8. 144. Crovatto, M., et al., Peripheral blood neutrophils from hepatitis C virus-infected patients are replication sites of the virus. Haematologica, 2000. 85(4): p. 356-61. 145. Bare, P., et al., HCV recovery from peripheral blood mononuclear cell culture supernatants derived from HCV-HIV co-infected haemophilic patients with undetectable HCV viraemia. Haemophilia, 2003. 9(5): p. 598-604. 146. Bare, P., et al., Continuous release of hepatitis C virus (HCV) by peripheral blood mononuclear cells and B-lymphoblastoid cell-line cultures derived from HCV-infected patients. J Gen Virol, 2005. 86(Pt 6): p. 1717-27. 147. Dries, V., et al., Detection of hepatitis C virus in paraffin-embedded liver biopsies of patients negative for viral RNA in serum. Hepatology, 1999. 29(1): p. 223-9. 148. Haydon, G.H., et al., Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection. Gut, 1998. 42(4): p. 570-5. 149. Schmidt, W.N., et al., Effect of interferon therapy on hepatitis C virus RNA in whole blood, plasma, and peripheral blood mononuclear cells. Hepatology, 1998. 28(4): p. 1110-6. 150. Schmidt, W.N., et al., Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests. J Infect Dis, 1997. 176(1): p. 27-33. 151. Pham, T.N., et al., Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells. J Gen Virol, 2005. 86(Pt 3): p. 657-66. 152. Castillo, I., et al., Hepatitis C virus replicates in the liver of patients who have a sustained response to antiviral treatment. Clin Infect Dis, 2006. 43(10): p. 1277-83. 153. Carreno, V., et al., Detection of hepatitis C virus (HCV) RNA in the liver of healthy, anti-HCV antibodypositive, serum HCV RNA-negative patients with normal alanine aminotransferase levels. J Infect Dis, 2006. 194(1): p. 53-60. 154. McHutchison, J.G., et al., Hepatic HCV RNA before and after treatment with interferon alone or combined with ribavirin. Hepatology, 2002. 35(3): p. 688-93. 155. Veldt, B.J., et al., Long term clinical outcome of chronic hepatitis C patients with sustained virological response to interferon monotherapy. Gut, 2004. 53(10): p. 1504-8. 156. Lee, W.M., et al., Reemergence of hepatitis C virus after 8.5 years in a patient with hypogammaglobulinemia: evidence for an occult viral reservoir. J Infect Dis, 2005. 192(6): p. 1088-92. 157. Melisko, M.E., R. Fox, and A. Venook, Reactivation of hepatitis C virus after chemotherapy for colon cancer. Clin Oncol (R Coll Radiol), 2004. 16(3): p. 204-5.

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General Introduction 179. Fried, M.W., et al., Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med, 2002. 347(13): p. 975-82. 180. Hadziyannis, S.J., et al., Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med, 2004. 140(5): p. 346-55. 181. Manns, M.P., et al., Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet, 2001. 358(9286): p. 958-65. 182. Sidwell, R.W., et al., Broad-spectrum antiviral activity of Virazole: 1-beta-D-ribofuranosyl-1,2,4triazole-3-carboxamide. Science, 1972. 177(50): p. 705-6. 183. Reichard, O., et al., Hepatitis C viral RNA titers in serum prior to, during, and after oral treatment with ribavirin for chronic hepatitis C. J Med Virol, 1993. 41(2): p. 99-102. 184. Di Bisceglie, A.M., et al., A pilot study of ribavirin therapy for chronic hepatitis C. Hepatology, 1992. 16(3): p. 649-54. 185. Reichard, O., et al., Ribavirin treatment for chronic hepatitis C. Lancet, 1991. 337(8749): p. 1058-61. 186. Dusheiko, G., et al., Ribavirin treatment for patients with chronic hepatitis C: results of a placebocontrolled study. J Hepatol, 1996. 25(5): p. 591-8. 187. Brillanti, S., et al., A pilot study of combination therapy with ribavirin plus interferon alfa for interferon alfa-resistant chronic hepatitis C. Gastroenterology, 1994. 107(3): p. 812-7. 188. Davis, G.L., et al., Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C. International Hepatitis Interventional Therapy Group. N Engl J Med, 1998. 339(21): p. 1493-9. 189. Davies, W.L., et al., Antiviral Activity of 1-Adamantanamine (Amantadine). Science, 1964. 144: p. 862-3. 190. Skehel, J.J., Influenza virus. Amantadine blocks the channel. Nature, 1992. 358(6382): p. 110-1. 191. Smith, J.P., Treatment of chronic hepatitis C with amantadine. Dig Dis Sci, 1997. 42(8): p. 1681-7. 192. Smith, J.P., et al., Amantadine therapy for chronic hepatitis C: a dose escalation study. Am J Gastroenterol, 2004. 99(6): p. 1099-104. 193. Brillanti, S., et al., Pilot study of triple antiviral therapy for chronic hepatitis C in interferon alpha nonresponders. Ital J Gastroenterol Hepatol, 1999. 31(2): p. 130-4. 194. Lim, J.K., et al., Amantadine in treatment of chronic hepatitis C virus infection? J Viral Hepat, 2005. 12(5): p. 445-55. 195. Haqshenas, G., et al., A 2a/1b full-length p7 inter-genotypic chimeric genome of hepatitis C virus is infectious in vitro. Virology, 2006. 196. Griffin, S.D., et al., The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett, 2003. 535(1-3): p. 34-8. 197. Mangia, A., et al., Peginterferon alfa-2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or 3. N Engl J Med, 2005. 352(25): p. 2609-17. 198. Zeuzem, S., et al., Efficacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol, 2006. 44(1): p. 97-103. 199. National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C: 2002--June 10-12, 2002. Hepatology, 2002. 36(5 Suppl 1): p. S3-20. 200. Davis, G.L., et al., Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology, 2003. 38(3): p. 645-52.

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35

C h a p t e r

2

Clinical performance of the new Roche COBAS® TaqMan HCV Test and High Pure System for extraction, detection and quantitation of HCV RNA in plasma and serum Huub C Gelderblom1,2 , Sandra Menting1, Marcel G Beld1

1Section

of Clinical Virology, Department of Medical Microbiology, and 2 AMC Liver Center, Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Antiviral Therapy 2006;11:95–103

Antiviral Therapy 11:95–103

Clinical performance of the new Roche COBAS® TaqMan HCV Test and High Pure System for extraction, detection and quantitation of HCV RNA in plasma and serum Huub C Gelderblom 1,2*, Sandra Menting 1 and Marcel G Beld 1 1

Section of Clinical Virology, Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands 2 AMC Liver Centre, Department of Gastroenterology and Hepatology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands *Corresponding author: Tel: +31 20 566 8748; Fax: +31 20 566 9240; E-mail: [email protected]

Preliminary results of these studies were disclosed at the Therapies for Viral Hepatitis Workshop, Boston, MA, USA, 2–3 November 2004. We evaluated the Roche COBAS® TaqMan HCV Test For Use With The High Pure System (TaqMan HPS; Roche Diagnostics), for the extraction, detection and quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The TaqMan HPS is a real-time PCR assay with a reported linear dynamic range of 3.0×101 to 2.0×108 HCV RNA IU/ml, and a reported lower limit of detection (LLD) of 10 IU/ml. Calculation of the HCV RNA titre is based upon an external standard curve in the presence of an internal control. Intra-assay and inter-assay variation were small in reference panel members with HCV RNA ≥100 IU/ml. Genotype performance and quantitative correlation between the TaqMan

HPS and the bDNA (VERSANT® HCV 3.0 assay; Bayer Diagnostics), assessed in 59 patient samples, were good for HCV genotype 1 but poor for genotypes 2, 3 and 4. For genotypes 2, 3 and 4, values obtained from the TaqMan HPS were in general 0.5 log lower than those from the bDNA. Sensitivity was poor in low viral titre samples of genotypes 1, 2, 3 and 4. The LLD (95%) was estimated at 41 HCV RNA IU/ml for genotype 4. The TaqMan HPS underestimates HCV RNA at all levels in plasma and serum from HCV-infected individuals, and the LLD should be reconsidered. This is clinically relevant because underestimation of HCV RNA levels during therapy may lead physicians into making incorrect treatment decisions.

Introduction Worldwide, more than 170 million individuals have been infected with hepatitis C virus (HCV). Considerable advances have been made in the therapy of HCV during recent years. The current antiviral therapy consists of administration of pegylated interferon alpha and ribavirin for 24 (genotype 2 and 3) or 48 weeks (genotype 1 and 4) and leads to sustained virological response (SVR) in approximately 40% (genotype 1 and 4) to 80% (genotype 2 and 3) of patients. The measurement of HCV RNA levels in plasma has become an important tool for monitoring individuals before and during antiviral therapy. The goal of early HCV RNA quantitation during therapy is to predict a negative treatment outcome as early as possible. Failure to achieve a significant decrease in HCV RNA after 12 weeks of treatment (2 log decrease from baseline HCV RNA) is highly predictive of a negative treatment

outcome [1]. These treatment decisions rely on frequent assessment of HCV RNA, and require assays that are both sensitive in the detection of HCV RNA and accurate in the quantitation of HCV RNA over a wide dynamic range, irrespective of HCV genotype. An ideal test for monitoring patients during treatment and prediction of treatment outcome would be a single quantitative test that matches the sensitivity of a qualitative test while accurately quantitating both low and high viral loads. At present, when HCV RNA drops below the detection limit of the quantitative assays during therapy (for example, 615–600 HCV RNA IU/ml), HCV RNA can only be detected by qualitative assays such as PCR [COBAS® Amplicor HCV Test v2.0; Roche Diagnostics, Branchburg, NJ, USA; lower limit of detection (LLD) 50 IU/ml] or TMA (Transcription-Mediated Amplification; 95

38

HC Gelderblom et al.

Incidence of DM during IFN treatment for HCV

Materials and methods

VERSANT ® HCV qualitative assay; Bayer Diagnostics, Berkeley, CA, USA; LLD 5 IU/ml). The pattern of HCV RNA during this period is uncertain, and the significance of very low viral load during antiviral therapy (PCR-negative but TMA-positive) is unknown [2–4]. The limitations of the current assays require testing of low viral titre clinical samples by two or three assays (quantitative, PCR and TMA) to estimate low viral load, resulting in increased costs and laboratory workload. In our laboratory, we use diagnostic algorithms before, during and after treatment. Based on previous test results (if available, for example, during antiviral therapy), the specimen is first tested by either TMA or bDNA (VERSANT® HCV 3.0 assay; Bayer Diagnostics; linear dynamic range of 615 to 7.7×106 IU/ml). Depending on the TMA or bDNA result, the specimen is then tested by bDNA or TMA, respectively. If the viral load is >5 IU/ml but 5 IU/ml but 615 IU/ml by the TaqMan HPS (Table 2).

8

Log TaqMan IU/ml

7 6

LLD of the TaqMan HPS The LLD was assessed using 25–27 replicates of a dilution panel derived from a single clinical HCV genotype 4 sample (containing 5, 10, 15, 20, 33 and 50 IU/ml). HCV RNA levels of 50 IU/ml, 33 IU/ml and 15 IU/ml were detected with a sensitivity of 100%, 88% and 50%, respectively. The LLD (95%) of HCV RNA was estimated at 41 IU/ml. At the reported LLD of 10 IU/ml (according to Roche), no target was detected in 19 out of 25 replicates. In the majority of positive samples, viral titres were underestimated and reported as HCV RNA below 10 IU/ml by the TaqMan HPS (Table 3).

5 4 3 2

2

3

4

5

6

7

8

Log bDNA IU/ml Genotype 1 (� n=25), genotype 2 (� n=9), genotype 3 (� n=17), genotype 4 (� n=6) and genotype 5 (� n=1). The ideal regression line (y=x) is depicted.

Extraction of HCV RNA that values obtained from the TaqMan HPS were in general 0.2 log higher than those from the bDNA assay (Figure 3B). However, for genotypes 2, 3 and 4, it appeared that values obtained from the TaqMan HPS were in general 0.5 log lower than those from the bDNA assay (Figure 3B).

In addition, for comparison of the HPS with the Boom extraction, and quantitative correlation to the bDNA assay, HCV RNA was extracted and quantitated in 14 clinical samples of genotypes 1 to 4 using both extraction methods and the TaqMan assay. Results obtained by both extraction methods and the TaqMan assay were compared with results obtained by the bDNA assay. The differences in quantitation between the TaqMan assay and bDNA assay were smaller in 11 out of 14 clinical samples when the Boom extraction method was used. Quantitation differences in 10 out of 14 samples were within 0.5 log of the bDNA assay with Boom extraction whereas quantitation differences in 6 out of 14 samples were within 0.5 log of the bDNA assay with the HPS (Figure 4).

Linearity of the TaqMan HPS Linearity was determined in dilution series of 10 clinical samples of genotypes 1, 2 and 3, in a range of 3.0×101 to 1.23×107 IU/ml. Linear regression analysis showed a good correlation for the dilution series of clinical genotype 1 samples (n=6; r2, 0.9940; slope, 0.9520; intercept, 0.2007; data not shown), and clinical genotype 2 and 3 samples (n=4; r2, 0.9932; slope, 0.9340; intercept, 0.3728; data not shown).

Table 1. Correlation coefficients and differences in quantitation for HCV RNA obtained by the TaqMan HPS and bDNA assays per genotype Methods compared

Total

Genotype 1

Genotype 2

Genotype 3

Genotype 4

Genotype 5

TaqMan HPS versus bDNA r2 value Difference in quantitation >0.3 log Difference in quantitation >0.5 log

59 0.8758* 35/59 19/59

25 0.9567* 11/25 2/25

9 0.8923† 9/9 6/9

17 0.8929* 10/17 7/17

6 0.7748‡ 5/6 4/6

2 0/2 0/2

TaqMan HPS, COBAS® TaqMan HCV Test, For Use With The High Pure System (Roche Diagnostics); bDNA, VERSANT® HCV 3.0 assay (Bayer Diagnostics); *P 4.74 log10 IU/mL at day 1 became non-SVR (PV for non-SVR 100%). Some patients who achieved SVR had a low efficacy of antiviral therapy and high HCV RNA load at day 1 (marked 1, 2 and 3 in the middle of the graph), making the combination of efficacy of antiviral therapy and HCV RNA load at day 1 a weak predictor of SVR.

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Interferon induction in chronic hepatitis C

Prediction of treatment outcome using TMA Prediction of SVR All 24 treatment naive patients who achieved SVR became TMA negative (< 5 IU/mL) within 16 weeks (Table 2, Figure 5a). The PV for SVR was 100% in treatment naive patients who were negative by TMA at week 1 or week 2 (Table 2, Figure 5a). All 12 previous non-responders/relapsers who achieved SVR became TMA negative (< 5 IU/ mL) within 8 weeks (Table 2, Figure 5a). The PV for SVR was 83% in previous non-responders/ relapsers who were TMA negative at week 2 (Table 2, Figure 5a). Table 2a. PV for SVR for patients who were HCV RNA negative or positive by TMA during treatment. treatment naive PV of TMA patients TMA negativity for negative/positive SVR (95% CI)

PV of TMA positivity for non-SVR (95% CI)

sensitivity PV for SVR

specificity PV for SVR

day 1

0/53

NA

57% (43-69)

0%

100%

day 2

0/51

NA

55% (41-68)

0%

100%

week 1

3/49

100% (38-100)

57% (43-70)

13%

100%

week 2

6/45

100% (55-100)

60% (45-73)

25%

100%

week 4

12/36

83% (54-96)

64% (48-78)

43%

92%

week 6

18/30

94% (72-100)

77% (59-88)

71%

96%

week 8

27/22

78% (59-90)

86% (66-96)

88%

76%

week 12

33/14

67% (49-80)

86% (59-97)

92%

52%

week 16

30/16

80% (62-91)

100% (77-100)

100%

73%

PV of TMA positivity for non-SVR (95% CI)

sensitivity PV for SVR

specificity PV for SVR

previous PV of TMA non-responders/ negativity for relapsers TMA SVR negative/positive (95% CI) day 1

0/43

NA

74% (60-85)

0%

100%

day 2

0/42

NA

74% (59-85)

0%

100%

week 1

0/44

NA

73% (58-84)

0% 

100%

week 2

6/37

83% (42-98)

81% (66-91)

42%

97%

week 4

11/30

64% (35-85)

83% (66-93)

58%

86%

week 6

15/26

67% (41-85)

92% (75-99)

83%

83%

week 8

17/21

65% (41-83)

100% (82-100)

100%

80%

week 12

21/19

57% (37-75)

100% (80-100)

100%

68%

week 16

18/20

61% (39-80)

100% (81-100)

100%

74%

TMA, transcription-mediated amplification, LLD 5 HCV RNA IU/mL; PV, predictive value; NA, not applicable PV of TMA negativity for SVR was defined as % of TMA negative patients with subsequent SVR PV of TMA positivity for non-SVR was defined as % of TMA positive patients with subsequent non-SVR Sensitivity = % of patients with SVR, TMA negative at this time point Specificity = % of patients with non-SVR, TMA positive at this time point Confidence intervals (95%) of PVs were calculated using the modified Wald method

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Chapter 3

Prediction of non-SVR All 16 treatment naive patients who were TMA positive at week 16 developed non-SVR (Table 2, Figure 5b). The mean time to reach a TMA negative status was significantly shorter in treatment naive patients who achieved SVR compared to those who became TMA negative but thereafter broke through or relapsed (5.9 ± 4.1 weeks vs 9.6 ± 3.4 weeks, respectively, P < 0.01, Mann-Whitney U test). Table 2b. Table for the calculation of PV, sensitivity and specificity depicted in table 2a. SVR

non-SVR

total

TMA negative

a

b

a+b

PV for SVR (a/(a+b)*100)

TMA positive

c

d

c+d

PV for non-SVR (d/(c+d)*100)

total

a+c

b+d

a+b+c+d

sensitivity of TMA specificity of TMA negative and SVR positive and non-SVR (a/(a+c)*100) (d/b+d*100) Figure 5. Prediction of SVR and non-SVR during treatment using TMA.

Figure 5a. PV (predictive value) of TMA negative test results for SVR, defined as the percentage of patients with a TMA negative test result that achieved SVR, during treatment in treatment naive patients and previous nonresponders/relapsers. Figure 5b. PV of TMA positive test results for non-SVR, defined as the percentage of patients with a TMA positive test result that did not achieve SVR, during treatment in treatment naive patients and previous non-responders/relapsers. TMA, Transcription-Mediated Amplification, LLD 5 HCV RNA IU/mL; SVR, sustained virologic response.

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Interferon induction in chronic hepatitis C

All 21 previous non-responders/relapsers who were not TMA negative at week 8 developed non-SVR (Table 2, Figure 5b). The mean time to reach a TMA negative status was significantly shorter in previous non-responders/relapsers with SVR compared to those with eventual breakthrough or relapse (4.3 ± 2.4 weeks vs 9.2 ± 5.6 weeks, respectively, P = 0.03, MannWhitney U test).

Safety Thirteen patients (13%) dropped out during the study, 8 during the first 4 weeks and 5 between week 4 and 24. Five patients were hospitalized, 3 during the first 4 weeks (pancytopenia, gastroenteritis, ketoacidosis secondary to preexisting diabetes mellitus) and 2 between week 4 and 24 (new onset diabetes mellitus, pneumonia), 1 of the 13 dropouts stopped treatment at week 4 due to non-medical reasons. All hospitalized patients recovered. Three patients who stopped prematurely because of asthenia between 24 and 48 weeks of treatment were not considered dropouts, and were analyzed according to their virological response (1 BT, 2 REL). Four patients developed diabetes mellitus during or shortly after treatment. Nine patients already suffered from diabetes mellitus before treatment, 1 patient with type 2 diabetes mellitus became temporarily insulin dependent during antiviral therapy. All patients experienced known side effects associated with interferon or ribavirin. If necessary patients were referred to other specialists (i.e., dermatology (27%), psychiatry (31%), pulmonology (4%), neurology (7%), ophthalmology (12%), ear-nose-and-throat (7%), surgery (2%), and internal medicine (4%)). The dose of interferon alfa was reduced during the first 6 weeks in 9 patients, and at week 8 in 1 patient. The dose of ribavirin was reduced in 28 patients (12 during the first 6 weeks, 27 during week 6 to 48). Dose reduction was not associated with subsequent non-SVR (P = 0.38, Fisher’s exact test). Incidence or recurrence of psychiatric disease during treatment was not associated with subsequent non-SVR (P = 0.37, Fisher’s exact test). Thirteen (42%) of the 31 patients with a history of depression or other psychiatric disease did not have any psychiatric complaints during treatment, and conversely 13 (42%) of the 31 patients with psychiatric complaints during treatment did not have a history of psychiatric disease.

Discussion In this high dose interferon induction study, the SVR rates in treatment naive genotype 1 and 4 patients (44%) and previous non-responders/relapsers (26%) were apparently not higher than described for patients treated with standard of care (peginterferon alfa and ribavirin for 24-48 weeks). However, high dose interferon induction enabled us to predict treatment outcome already within the first weeks of treatment. In fast-responders (≥ 3 log10 HCV RNA decline at week 4 of treatment) a significantly higher proportion of patients (47%) developed SVR compared to slow-responders (25%, < 3 log10 HCV RNA decline at week 4 of

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Chapter 3

treatment). In fast-responders the relapse rate in patients treated for 24 or 48 weeks was not significantly different (27% vs 20% respectively) indicating that a treatment duration of 24 weeks may suffice. In slow-responders none of the previous non-responders/relapsers but 40% of treatment naive patients achieved SVR. In agreement with an earlier study [17], a fast-response (≥ 3 log10 decline in HCV RNA at week 4) was a weak predictor for SVR (PV 47%), but a slow response (< 3 log10 decline in HCV RNA at week 4) was a strong predictor of non-SVR in previous non-responders/relapsers, since all 12 patients did not develop SVR. In a recent study by Zeuzem et al. [6] a 24 week treatment regimen was only possible, without compromising SVR rate, in patients with baseline HCV RNA < 600,000 IU/mL and RVR (HCV RNA negative by PCR at week 4). In our studies, SVR in fast-responders was independent of baseline viral load. Also, of the 19 fast-responders that achieved SVR after a 24 week treatment regimen in our studies, 4 patients would not have met the criteria for RVR, i.e., they were still HCV RNA positive by PCR at week 4, and 8 of these 19 patients were still HCV RNA positive by the more sensitive TMA at week 4. These differences between our results and Zeuzem’s results are probably the result of the high dose interferon induction scheme applied in our studies. In contrast to the results from Layden et al. [24], but in agreement with others [9, 27, 28], the utility of 1st phase viral kinetic parameters for prediction of SVR in individual patients was limited in our study. However, in previous non-responders/relapsers a decline in HCV RNA at day 1 < 0.7 log10 and/or HCV RNA load > 4.74 log10 IU/mL at day 1 had a 100% PV for non-SVR. Thus after one day high dose interferon induction can identify previous nonresponders/relapsers who may benefit from retreatment. This early prediction of non-SVR is in agreement with the results from Layden et al. [24]. The utility of 2nd phase viral kinetics for prediction of SVR or non-SVR was also limited. This is probably related to the rapid initial HCV RNA decline in most patients due to the high dose interferon induction (18 MU/day) applied during the first 2 weeks of treatment. This decline is comparable to the rapid HCV RNA decline observed during administration of HCV protease inhibitors as monotherapy [29] or in combination with peginterferon alfa for 2 weeks [30]. Many patients in our study had HCV RNA levels below the lower limit of detection (615 IU/mL) of the quantitative bDNA assay after just 2 days of treatment. In these patients, in contrast to other studies [9, 24, 27, 28], the 2nd phase decline could not be assessed and therefore the utility of 2nd phase viral kinetics was limited in our study. On the other hand our study demonstrated the value of repeated testing during the first 6 weeks of high dose interferon induction treatment for prediction of response. A negative TMA test within the first 6 weeks of treatment was a strong predictor of SVR. A positive TMA test at week 8 predicted non-SVR in previous non-responders/relapsers and a positive TMA test at week 16 predicted non-SVR in treatment naive patients. The association between a TMA negative status within the first treatment weeks and SVR is in line with the observations of others that with standard treatment [20, 31] or consensus interferon [32] an HCV RNA negativity at week 4 was a strong predictor for SVR. Compared

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Interferon induction in chronic hepatitis C

to TMA, the predictive values of the less sensitive PCR and bDNA tests were lower at all time points in our study. Our results also show that frequent assessment of HCV RNA with TMA changes the classification of “non-response”, “relapse” or “breakthrough” in patients with non-SVR. Some patients classified as TMA non-responders would have been classified as breakthrough or relapse using PCR. Conversely a number of breakthrough patients would have been classified as non-responders if we had not tested frequently with TMA. We identified 10 patients who became HCV RNA negative by TMA, but thereafter showed reappearance of low level viremia at week 16 or 20. This turned out to be an early sign of impending treatment failure. These findings are described in detail in a separate article [33]. As in previous studies performed in our centre [11, 12], high dose interferon was associated with significant, but tolerable, side effects. Before treatment, all patients had been thoroughly informed about the possible adverse events. Furthermore, all patients visited the outpatient clinic at days 0, 1 and 2, and weeks 1, 2, 3, 4, 6, 8 and thereafter every 4 weeks until end of treatment. The 13% dropout rate is comparable to other studies [34], 8 of the 13 dropouts (61%) stopped during the first 4 weeks. Patients were promptly referred to other specialists if side effects occurred. Taken together, high dose interferon and ribavirin combination therapy was well tolerated, but it should be administered in centres that routinely exercise close patient monitoring and experienced multidisciplinary support [34]. In conclusion, high dose interferon induction followed by peginterferon based therapy allows early prediction of SVR and non-SVR in difficult-to-treat chronic hepatitis C patients. In patients with a ≥ 3 log10 HCV RNA decline at week 4 a 24 week treatment duration may suffice, regardless of baseline viral load, but this should be confirmed by other studies. Finally, the development of Specifically Targeted Antiviral Therapy for HCV (STAT-C) in combination with interferon holds great promise for these difficult-to-treat patients [29, 30, 35]. It may be that high dose interferon induction, as in this study, in combination with STAT-C will result in a rapid 1st and a sustained 2nd phase HCV RNA decline, preventing the development or selection of resistance mutations [30, 36] and enabling shorter treatment duration.

Acknowledgements We are grateful to the patients who participated in the studies, and acknowledge Martine Peters, Ruth Culbard and Marja Voskuilen for follow-up of the patients at the outpatient clinic, Sjoerd Rebers and Sandra Menting for HCV RNA assessments, Frits Schöler for assistance with data management, and Ellie Barnes for critical revision of the manuscript. These investigator-initiated studies were funded in part by Schering-Plough and Bayer Diagnostics.

Conflict of interest statement Dr Reesink is a consultant for Schering-Plough, he has received unrestricted grants from Hoffmann-La Roche, Schering-Plough and Vertex Pharmaceuticals. Dr Beld is a consultant for Bayer Diagnostics. ISRCTN59358441 and ISRCTN81536220

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Preliminary results of these studies were disclosed at the American Association for the Study of Liver Diseases (AASLD) Hepatitis Single Topic Conference “Interferon & Ribavirin in Hepatitis C Virus Infection: mechanisms of Response and Non-Response”, Chicago IL, USA, 1-3 March 2007, and at the 17th European Congress of Clinical Microbiology and Infectious Diseases & 25th International Congress of Chemotherapy (ECCMID-ICC), Munich, Germany 31 March - 3 April 2007

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Weegink, C.J., et al., Chronic hepatitis C patients with a post-treatment virological relapse re-treated with an induction dose of 18 MU interferon-alpha in combination with ribavirin and amantadine: a two-arm randomized pilot study. J Viral Hepat, 2003. 10(3): p. 174-82.

12. Sentjens, R.E., et al., Viral kinetics of hepatitis C virus RNA in patients with chronic hepatitis C treated with 18 MU of interferon alpha daily. Eur J Gastroenterol Hepatol, 2002. 14(8): p. 833-40. 13.

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Steindl-Munda, P., et al., Impact of high-dose interferon induction and ribavirin therapy in patients with chronic hepatitis C relapsing after or not responding to interferon monotherapy. Liver Int, 2003. 23(4): p. 269-75.

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Davis, G.L., et al., Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology, 2003. 38(3): p. 645-52.

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Beld, M., et al., Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, assay. J Clin Microbiol, 2002. 40(3): p. 788-93.

22. Lee, S.C., et al., Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcriptionPCR tests for hepatitis C virus RNA: calibration to international units, enhanced genotype reactivity, and performance characteristics. J Clin Microbiol, 2000. 38(11): p. 4171-9. 23. Ross, R.S., et al., Performance characteristics of a transcription-mediated nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA. J Clin Lab Anal, 2001. 15(6): p. 308-13. 24. Layden, J.E., et al., First phase viral kinetic parameters as predictors of treatment response and their influence on the second phase viral decline. J Viral Hepat, 2002. 9(5): p. 340-5. 25. Agresti, A. and B.A. Coull, Approximate is better than “exact” for interval estimation of binomial proportions. American Statistician, 1998. 52(2): p. 119-126. 26. Wai, C.T., et al., A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology, 2003. 38(2): p. 518-26. 27.

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Reesink, H.W., et al., Rapid decline of viral RNA in hepatitis C patients treated with VX-950: a phase Ib, placebo-controlled, randomized study. Gastroenterology, 2006. 131(4): p. 997-1002.

30. Reesink, H.W., et al., Initial results of a 14-day study of the hepatitis C virus inhibitor protease VX-950, in combination with peginterferon-alpha-2a. J Hepatol, 2006. 44(S2): p. S272. 31.

Moucari, R., et al., High predictive value of early viral kinetics in retreatment with peginterferon and ribavirin of chronic hepatitis C patients non-responders to standard combination therapy. J Hepatol, 2006.

32. Cornberg, M., et al., Treatment with daily consensus interferon (CIFN) plus ribavirin in non-responder patients with chronic hepatitis C: a randomized open-label pilot study. J Hepatol, 2006. 44(2): p. 291-301. 33. Gelderblom, H.C., et al., Low level HCV viraemia after initial response during antiviral therapy: transcription-mediated amplification predicts treatment failure. Antivir Ther, 2007. 12(3): p. 423-27. 34. Vrolijk, J.M., et al., High sustained virological response in chronic hepatitis C by combining induction and prolonged maintenance therapy. J Viral Hepat, 2003. 10(3): p. 205-9. 35. McHutchison, J.G., et al., The face of future hepatitis C antiviral drug development: recent biological and virologic advances and their translation to drug development and clinical practice. J Hepatol, 2006. 44(2): p. 411-21.

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C h a p t e r

4

Low level HCV viraemia after initial response during antiviral therapy: transcription-mediated amplification predicts treatment failure Huub C Gelderblom1,2, Henk W Reesink 2, Marcel GHM Beld1, Christine J Weegink 2, Peter LM Jansen2, Marcel G Dijkgraaf 3, Hans L Zaaijer1

1Section of Clinical Virology, Department of Medical Microbiology, 2 AMC Liver Center, Department

of Gastroenterology and Hepatology, and 3Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Antiviral Therapy 2007;12:423–427

Chapter 4

Antiviral Therapy 12:423–427

Short communication Low-level HCV viraemia after initial response during antiviral therapy: transcription-mediated amplification predicts treatment failure Huub C Gelderblom1,2*, Henk W Reesink 2, Marcel GHM Beld1, Christine J Weegink 2 , Peter LM Jansen2, Marcel GW Dijkgraaf 3 and Hans L Zaaijer1 1

Section of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 2 AMC Liver Center, Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands 3 Department of Clinical Epidemiology and Biostatistics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands *Corresponding author: Tel: +31 20 5668748; Fax: +31 20 5669240; E-mail: [email protected] Preliminary results of these studies were disclosed at the 11th International Symposium on Hepatitis C and Related Viruses, Heidelberg, Germany, 3–7 October 2004 (p43), and the 12th International Symposium on Viral Hepatitis and Liver Disease, Paris, France, 1–5 July 2006 (p249).

treat patients. Therapy consisted of amantadine hydrochloride and ribavirin, combined with interferon2b induction during the first 6 weeks and thereafter combined with weekly pegylated interferon-2b. Results: Among the 57 IVR patients, we detected transient or persistent reappearance of low levels of HCV RNA in 10 of the 23 (43%) patients with eventual breakthrough or relapse; but in none of the 34 SVR patients. In 5 of 10 patients reappearing HCV RNA was only detectable by TMA. Conclusion: Reappearance of low levels of HCV RNA in patients with IVR predicts treatment failure.

Background: In chronic hepatitis C patients with an initial virological response (IVR) during antiviral therapy (that is, HCV RNA becomes negative before week 16 of treatment) the significance of reappearing viraemia below the detection limit of PCR is not known. We studied this phenomenon in subsets of patients. Methods: We assessed HCV RNA at weeks 16 and 20 of therapy by PCR and by more sensitive transcription-mediated amplification (TMA) in 23 patients with breakthrough or relapse and in 34 patients with sustained virological response (SVR). All patients participated in a high-dose-interferon induction study for difficult-to-

Introduction and causes a wide range of side effects in the majority of patients. Therefore, the goals of HCV RNA detection and quantification during antiviral therapy are (i) to stop treatment in patients predicted to experience treatment failure [6,7], and (ii) to shorten treatment duration in rapid responders [1,5]. HCV RNA levels below the lower limits of detection (LLD) of quantitative assays (600–615 IU/ml) can be detected by the more sensitive, qualitative PCR (LLD: 50 IU/ml). The consensus rules for decisions on treatment duration at 4 and 24 weeks of treatment are based on qualitative PCR [1,5–7]. However, more sensitive

The current antiviral therapy for chronic hepatitis C virus (HCV) infection consists of administration of pegylated interferon- and ribavirin for 12–24 weeks (genotype 2 and 3) [1–4], 24–28 weeks (genotype 1) [2–5], or 48 weeks (genotype 4) [3] and leads to a sustained virological response (SVR) in ~50% (genotype 1 and 4) to 80% (genotype 2 and 3) of patients. The duration of interferon- and ribavirin therapy is based on HCV genotype, baseline HCV RNA load [5] and the HCV RNA level after 4 weeks [1,5], 12 weeks [6,7] or 24 weeks of antiviral therapy [6]. Interferon-- and ribavirin-based therapy is expensive © 2007 International Medical Press 1359-6535

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detection of HCV RNA is possible using transcriptionmediated amplification (TMA; LLD: 5 IU/ml). The relevance of this 10-fold increased sensitivity of TMA to decisions regarding treatment duration is unknown. Detection of HCV RNA by TMA but not by PCR (PCR-negative, TMA-positive viraemia, HCV RNA
50 but 5 IU/ml) after 12 or 24 weeks of treatment has been observed in very few patients, and was followed by virological non-response in most cases [8–11]. One-hundred and three HCV-infected patients were included in a study on the effects of high-dose-induction treatment for difficult-to-treat patients. In a substudy of this study we determined the significance for treatment outcome of low-level viraemia during antiviral therapy. We performed PCR and TMA at weeks 16 and 20 on samples from patients with an initial response, who later experienced breakthrough, relapse or sustained virological response.

Patients and methods Patients and trial design A trial was designed to study the influence of daily highdose interferon induction on early viral kinetics and treatment outcome in difficult-to-treat hepatitis C patients (that is, patients with any HCV genotype who had not responded to previous interferon treatment, and treatment-naive patients infected with HCV genotype 1 or 4). In total, 103 patients were recruited in the trial, 97 patients, with baseline characteristics as presented in Table 1, received one or more doses of treatment. A flow chart of the trial is depicted in Figure 1. Among the 97 patients who received treatment, 57 patients showed an initial virological response (IVR): HCV RNA was undetectable by TMA (see below) before 16 weeks. Subsequently they experienced breakthrough, relapse or sustained virological response. These 57 IVR patients are the subjects of the analysis presented here: they were

studied for reappearance of low-level HCV RNA at week 16 and 20. The baseline characteristics of the 57 IVR patients are presented in Table 2. All patients were treated with triple therapy consisting of: amantadine hydrochloride 200 mg/day (Symmetrel®; Novartis, Basel, Switzerland) and ribavirin (Rebetol®; Schering-Plough, Kenilworth, NJ, USA) 1,000 or 1,200 mg/day (dependent on body weight) for a total of 24 or 48 weeks, combined with interferon-2b induction (IntronA®; Schering-Plough) during the first 6 weeks, and thereafter combined with weekly pegylated interferon-2b (PegIntron®; Schering-Plough), 1.5 g/kg for a total of 24 or 48 weeks. The interferon induction scheme during the first 6 weeks was as follows: weeks 1 and 2: 18 MU/day in 3 divided doses; weeks 3 and 4: 9 MU/day in 3 divided doses; week 5 and 6: 6 MU/day in 2 divided doses. Patients with a decrease of HCV RNA
3 log at week 4 were treated for 48 weeks, whereas patients with a decrease of HCV RNA
3 log at week 4 were randomized to stop treatment early at 24 weeks or continue to 48 weeks. Treatment was stopped in all patients who were positive for HCV RNA by PCR at week 24. All patients were followed for 24 weeks after completion of therapy. The study was approved by the institutional review board. Written informed consent was obtained from each patient. Data regarding early viral kinetics are not shown: these results will be presented in another publication.

Figure 1. Flow chart of the trial

103 included 6 did not show 97 intention to treat 13 dropped out

Table 1. Baseline characteristics of patients who received one or more doses of treatment Treatmentnaive

Previous treatment failure Total

Patients (intention to treat), n

53

44

97

Male/female, n/n

39/14

35/9

74/23

Mean age (range), years 44.2 (19–67)

46.6 (30–63)

45.3 (19–67)

Genotype, n HCV 1 HCV 2 HCV 3 HCV 4 HCV 5

25 2 7 9 1

66 2 7 21 1

424

41 0 0 12 0

27 non-responders (TMA remains positive during treatment)

57 initial viral responders (TMA-negative before 16 weeks)

34 SVR

23 BT/ REL

BT/REL, breakthrough or relapse; SVR, sustained virological response; TMA, transcription-mediated amplification.

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Table 2. Baseline characteristics of the 57 patients with IVR (i.e. TMA-negative before week 16 of antiviral therapy) Total

Sustained virological response

BT/REL

BT/REL and low-level viraemia at week 16 or 20

Patients, n

57

34

23

10

Male/female, n/n

44/13

26/8

18/5

8/2

Mean age (range), years

43.5 (19–63)

44.1 (25–63)

42.7 (19–50)

39.9 (19–49)

Treatment naive/previous treatment failure, n/n

34/23

23/11

11/12

5/5

Genotype, n HCV 1 HCV 2 HCV 3 HCV 4 HCV 5

39 2 7 8 1

21 1 5 6 1

18 1 2 2 0

9 0 0 1 0

Decrease of HCV RNA
3 log10 at week 4, n (%)

48 (84)

27 (79)

21 (91)

9 (90)

BT/REL, breakthrough or relapse; HCV, hepatitis C virus; IVR, initial virological response; TMA, transcription-meditated amplification.

Specimen collection and HCV RNA assessment

HCV genotype.

Blood samples were obtained in EDTA-containing Vacutainer tubes (Becton Dickinson, Alphen aan den Rijn, the Netherlands); the samples were centrifuged and plasma samples were frozen at -80°C within 24 h of collection. We collected and tested samples during antiviral therapy at standard time points at day 0, weeks 4 and 12, at end-of-treatment (week 24 or 48), and at end-of-follow-up (week 48 or 72) by TMA, PCR and bDNA (see below). To determine the presence of low-level viraemia, we tested additional samples drawn at weeks 16 and 20. The presence and level of HCV RNA was determined and categorized as follows:

HCV genotypes were determined using the TruGene® HCV genotyping assay (Bayer Diagnostics).

HCV RNA levels
615 IU/ml. HCV RNA levels were determined quantitatively using the bDNA VERSANT® HCV 3.0 assay (Bayer Diagnostics, Berkeley, CA, USA). The dynamic range of this assay is 615–7.7106 IU/ml HCV RNA [12]. HCV RNA
50 and
615 IU/ml. The HCV RNA level was determined to be between 50 and 615 IU/ml if the sample tested negative in the bDNA assay, but positive in the qualitative PCR (COBAS® Amplicor HCV Test v2.0, Roche Molecular Systems, Branchburg, NJ, USA; LLD: 50 IU/ml HCV RNA) [13]. HCV RNA
5 and
50 IU/ml. The HCV RNA level was determined to be between 5 and 50 IU/ml if the sample tested negative in the PCR assay, but positive in the TMA (VERSANT® HCV qualitative assay, Bayer Diagnostics; LLD: 5 IU/ml) [14]. HCV RNA
5 IU/ml. The HCV RNA level was determined to be
5 IU/ml when the TMA tested negative. Antiviral Therapy 12:3

Statistical analysis. Statistical analysis was performed using SPSS version 12.0.2 for Windows (SPSS Inc., Chicago, IL, USA). The association between the presence of HCV RNA at weeks 16 or 20 on the one hand and treatment failure on the other was determined using the McNemar test with a two-tailed P-value. Associations were assessed for each week separately (presence of HCV RNA at week 16 or at week 20) as well as for both time points in a row (presence of HCV RNA at weeks 16 and/or 20). The level of significance was reset to 0.025 to correct for multiple comparisons.

Results We studied 57 patients with IVR (that is, TMA-negative before week 16 of antiviral therapy). Subsequently 9/57 patients showed a breakthrough during therapy, 14/57 patients relapsed after cessation of therapy, and 34/57 patients achieved an SVR. Sensitive testing for presence of HCV RNA by TMA at weeks 16 and 20 revealed that the TMA assay remained negative in all 34 patients who achieved an SVR, but 10 of the 23 patients with eventual breakthrough or relapse showed transient or persistent reappearance of low levels of HCV RNA. In eight patients HCV RNA reappeared at week 16, in two patients at week 20 (Table 3). The association between reappearance of HCV RNA at week 16 or 20 and subsequent treatment failure was significant (week 16: P
0.001; week 20: P
0.001; week 16 and/or 20: P=0.002). In five of these 10 patients the reappearing HCV RNA was also

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TMA+ TMA+ PCRTMA+ PCR+ TMATotal

Total

Week 16

Week 20

14* 9 5 92 106

8 5 3 45 53

6 4 2 47 53

Figure 2. Three patterns of low-level viraemia observed in patients

A

8 7

Log HCV RNA, IU/ml

Table 3. TMA and PCR results at weeks 16 and 20 for the 57 patients with IVR

*Four patients were hepatitis C virus (HCV)-positive by transcription-mediated amplification (TMA+) at both time points; samples from eight patients were available at only one of the two time points. IVR, initial virological response; PCR+, HCV-positive by PCR; PCR-, HCV-negative by PCR; TMA-, HCV-negative by TMA.

Antiviral therapy

6 5 4 bDNA 3 PCR TMA

2 1 0 -4 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Time, weeks

B

8

6 5 4 bDNA 3 PCR TMA

2 1 0 -4 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Time, weeks

C

8 7

426

Log HCV RNA, IU/ml

Discussion In patients with an IVR, we detected transient or persistent reappearance of low-level viraemia after 16 and 20 weeks of antiviral therapy in 10/23 patients with eventual breakthrough or relapse, but not in 34 patients who achieved SVR. The reappearing HCV RNA was detected with TMA, and in some cases also with PCR. Low-level viraemia preceded breakthrough or relapse by 4–32 weeks. Thus, sensitive detection of reappearing HCV RNA enables early prediction of treatment failure, with a high predictive value. Persistence of HCV RNA detectable by TMA but not by PCR during treatment in patients with eventual breakthrough, relapse or non-response has been described previously [8–10]. In three studies, these patients were studied at weeks 12 [15] and 24 [9,10,15] only, during 48 week treatment regimens. Our results confirm the significance of reappearance of HCV RNA detected only by TMA [8]. Two important differences between these studies and our study are that we sampled more frequently during treatment and we included patients with SVR in our analysis, thus enabling the calculation of the predictive value of reappearing viraemia by TMA for failure of treatment.

Antiviral therapy

7

Log HCV RNA, IU/ml

detectable by PCR (Table 3). There was no correlation between the decrease in HCV RNA at week 4 and subsequent breakthrough, relapse or SVR. The predictive value of HCV RNA detection by TMA at week 16 or 20 for treatment failure was 100%. The predictive value of a negative TMA test result at week 16 and 20 for SVR was 72%. We categorized the low-level viraemia of the 10 patients into three patterns (Figure 2): (i) breakthrough, but with detection by TMA 4 weeks earlier than by PCR in 3/10 patients (Figure 2A); (ii) transient low-level viraemia preceding breakthrough by 16–30 weeks in 3/10 patients (Figure 2B); and (iii) transient low-level viraemia preceding relapse by 4–32 weeks in 4/10 patients (Figure 2C).

Antiviral therapy

6 5 4 bDNA 3 PCR 2 TMA

1 0 -4 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72

Time, weeks HCV, hepatitis C virus; TMA, transcription-mediated amplification.

Our analysis revealed the presence of HCV RNA between the standard sampling points at week 12 and week 24. The fact that reappearance of viraemia during treatment seems limited to patients with eventual breakthrough or relapse makes it a clinically relevant phenomenon. In such patients treatment could be stopped earlier. By contrast, results from the HALT-C trial have shown that 6/27 patients with reappearance of HCV detectable by TMA at week 24 did achieve SVR [16]. However, (i) the HALT-C trial deals with a different

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patient population (previous non-responders with advanced liver disease), (ii) patients were treated with a different treatment regime, and (iii) patients were not evaluated at week 16. The reappearance of low-level HCV viraemia we observed might be related to the therapeutic regimen that was applied to the patients. We treated our patients with high doses of interferon- during the first 6 weeks of treatment (6 MU/day in two divided doses during weeks 5 and 6) and then switched to standard, lower, dose pegylated interferon-. This may have provoked a viral rebound after the first 6 weeks. The rate of breakthrough/relapse after initial response in our study was 40% (52% in previous non-responders and 32% in naive patients). This is slightly higher than the 25% breakthrough/relapse rate after initial response in naive patients during standard treatment [7]. However, the reappearance of HCV RNA at week 16 or 20 in 43% of patients with eventual breakthrough or relapse suggests that transient or persistent reappearance of low-level viraemia after 16 and 20 weeks of antiviral therapy is also likely to occur during standard treatment. To verify this, samples taken between weeks 16 and 24 from patients treated with the standard of care (pegylated interferon/ribavirin) should be (re)analysed by TMA. Our results indicate that reappearance of low levels of HCV RNA is a reliable predictor for treatment failure in patients with an IVR during therapy.

3. 4.

5.

hepatitis C: a randomised trial. Lancet 2001; 358:958–965. Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347:975–982. Hadziyannis SJ, Sette H Jr, Morgan TR, et al. Peginterferonalpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann Intern Med 2004; 140:346–355. Zeuzem S, Buti M, Ferenci P, et al. Efficacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol 2006; 44:97–103.

National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C: 2002 – June 10-12, 2002. Hepatology 2002; 36:S3–20. 7. Davis GL, Wong JB, McHutchison JG, Manns MP, Harvey J, Albrecht J. Early virologic response to treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C. Hepatology 2003; 38:645–652. 8. Desombere I, Van Vlierberghe H, Couvent S, Clinckspoor F, Leroux-Roels G. Comparison of qualitative (COBAS AMPLICOR HCV 2.0 versus VERSANT HCV RNA) and quantitative (COBAS AMPLICOR HCV monitor 2.0 versus VERSANT HCV RNA 3.0) assays for hepatitis C virus (HCV) RNA detection and quantification: impact on diagnosis and treatment of HCV infections. J Clin Microbiol 2005; 43:2590–2597. 9. Germer JJ, Zein NN, Metwally MA, et al. Comparison of the VERSANT HCV RNA qualitative assay (transcriptionmediated amplification) and the COBAS AMPLICOR hepatitis C virus test, version 2.0, in patients undergoing interferon-ribavirin therapy. Diagn Microbiol Infect Dis 2003; 47:615–618. 10. Mihm U, Hofmann WP, Kronenberger B, Wagner M, Zeuzem S, Sarrazin C. Highly sensitive hepatitis C virus RNA detection assays for decision of treatment (dis)continuation in patients with chronic hepatitis C. J Hepatol 2005; 42:605–606. 11. Weegink CJ, Sentjens RE, Beld MG, Dijkgraaf MG, Acknowledgements Reesink HW. Chronic hepatitis C patients with a posttreatment virological relapse re-treated with an induction dose of 18 MU interferon-alpha in combination with We are grateful to Sjoerd Rebers and Sandra Menting, ribavirin and amantadine: a two-arm randomized pilot who performed the HCV RNA tests, to Frits Schöler study. J Viral Hepat 2003; 10:174–182. for assistance with data management, and to Ed Hull, 12. Beld M, Sentjens R, Rebers S, et al. Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitaDanny Haddad and Paul O’Grady for critical revision tion of hepatitis C virus RNA in plasma and serum: of the manuscript. conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, assay. Potential conflict of interest: Dr Reesink is a consultant J Clin Microbiol 2002; 40:788–793. for Schering-Plough and has received grants from 13. Lee SC, Antony A, Lee N, et al. Improved version 2.0 qualHoffmann-La Roche and Schering-Plough. Dr Beld is a itative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: calibraconsultant for Bayer Diagnostics. tion to international units, enhanced genotype reactivity, Funded in part by: Schering-Plough and Bayer and performance characteristics. J Clin Microbiol 2000; 38:4171–4179. Diagnostics. Schering-Plough and Bayer Diagnostics 14. Ross RS, Viazov SO, Hoffmann S, Roggendorf M. were not involved in analysis of the data or reporting Performance characteristics of a transcription-mediated of the results. nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA. J Clin Lab Anal 2001; 15:308–313. 15. McHutchison JG, Blatt LM, Ponnudurai R, Goodarzi K, References Russell J, Conrad A. Ultracentrifugation and concentration of a large volume of serum for HCV RNA during treat1. Mangia A, Santoro R, Minerva N, et al. Peginterferon alfament may predict sustained and relapse response in chronic 2b and ribavirin for 12 vs. 24 weeks in HCV genotype 2 or HCV infection. J Med Virol 1999; 57:351–355. 3. N Engl J Med 2005; 352: 2609–2617. 16. Morishima C, Morgan TR, Everhart JE, et al. HCV RNA 2. Manns MP, McHutchison JG, Gordon SC, et al. detection by TMA during the hepatitis C antiviral longPeginterferon alfa-2b plus ribavirin compared with interterm treatment against cirrhosis (Halt-C) trial. Hepatology feron alfa-2b plus ribavirin for initial treatment of chronic 2006; 44:360–367. Accepted for publication 15 November 2006

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Early and sustained HCV virological responses to therapy despite suppression of HCV specific T cells Eleanor Barnes4*, Huub C Gelderblom1,2*, Isla Humphreys4, Nasser Semmo4, Henk W Reesink1, Marcel GHM Beld 2 , René AW van Lier3, Paul Klenerman4 *These authors contributed equally to the work.

1 AMC

Liver Center, Department of Gastroenterology and Hepatology, 2Section of Clinical Virology, Department of Medical Microbiology, and 3Department of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 4Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, UK

Submitted

Chapter 5

Abstract Background/Aims: The role of the adaptive immune response in sustaining hepatitis C viral control during therapy with interferon-α and ribavirin is unclear. We examined the effect of high dose interferon-α induction therapy on T-cell responses and addressed the hypothesis that a sustained virological response (SVR) after treatment is associated with a restoration of IL-2 and IFN-γ secreting HCV specific T-cells. Methods: 31 treatment-naïve chronic HCV patients were treated with amantadine and ribavirin, combined with 6 weeks of high dose IFN-alpha2b induction therapy followed by weekly PEG-IFN-alpha-2b, for 24 or 48 weeks. Using IFN-γ and IL-2 ELISpots, we analysed the pattern of cytokine secretion by structural and non-structural HCV, CMV and influenza specific T-cells before, during and following therapy. CD4+ and CD8+ T-cell subsets were distinguished using CFSE proliferation assays. Viral kinetics were assessed using bDNA, PCR and TMA assays. Results: 15/31 patients achieved SVR. HCV specific T-cell responses that secreted predominantly IFN-γ and correlated with ALT (r2=0.45, p=0.001) were found in 10/15 SVR and 11/16 non-SVR patients before treatment. There was a striking loss of IFN-γ and IL-2 HCV specific T-cells during therapy seen predominantly in the SVR group, which recovered following cessation of therapy. Suppression of CMV and influenza T-cell responses in addition to total lymphocyte counts were also observed. Conclusions: High dose IFN-α induction therapy leads to a profound decline in IL-2 and IFN-γ secreting HCV specific T-cells. This data indicates that restoration of T-cell responses is not causally linked to early or SVR to therapy.

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SVR despite HCV specific T-cell decline

Introduction HCV is currently a worldwide epidemic. The development of antiviral therapies against HCV and understanding the mechanism of action of currently available successful therapies will be crucial in controlling this epidemic. Combination therapy with PEG-α–IFN and ribavirin, currently the best available treatment, leads to long term resolution of infection in 50-80% of individuals depending on viral genotype [1]. The mechanism of action is not completely understood, but both drugs have wide ranging effects on the immune system [2, 3]. During acute HCV infection, robust Tcellular immune responses can be detected in the majority of individuals [4, 5]. These are maintained in those that spontaneously resolve infection [6]. However, once persistent HCV is established HCV specific cellular immune responses are generally weak and narrowly focused [7]. It is plausible then that combination therapy leads to long-term viral control through enhancement of HCV specific T-cell responses. In support of this theory a number of studies have shown some enhancement of HCV specific T-cells during therapy. However, it remains controversial whether this is associated with an SVR to therapy [8-11]. Additionally, whether the re-emergence of HCV specific T-cell responses during therapy is simply a consequence rather than a cause of a falling viral load is currently unclear. Furthermore, studies of T-cell responses during successful treatment of acute HCV showed either a decline or no enhancement in T-cell responses despite the very high efficacy of therapy at this stage of infection. In one patient in this study subsequent treatment with OKT3 antibody led to the abolishment of detectable responses against HCV without viral recrudescence, suggesting that T-cell immunosurveillance may not be required to establish a SVR [12]. The quality as well as the magnitude of the HCV specific T-cell response both in the context of antiviral therapy and in the spontaneous resolution of HCV infection may also be critically important in determining a successful outcome. We have previously shown that persistent HCV viremia is associated with a significant loss of IL-2 secreting HCV specific T-cells compared to IFN-γ secreting T-cells, whilst in spontaneously resolved HCV infection IL-2 secretion is preserved [13]. Similarly, CD4 + T-cells in human immunodeficiency viremic patients that fail to respond to therapy have been shown to produce exclusively IFN-γ, whilst in aviremic patients the production of both IL-2 and IFN-γ is preserved [14]. Others have suggested the emergence of Th1 type responses during combination therapy are related to a beneficial outcome [8]. The rate of viral decline may also impact on the emergence of T-cell responses during therapy. During the first few weeks of combination therapy two phases of viral decline occur. Mathematical modelling has proposed that the first more rapid decline in viral load is due to direct antiviral effects of therapy, whilst the second phase is due to immune pressure [15]. There is some experimental data to show that a rapid fall in viral load during therapy is more likely to be associated with restoration of T-cell responses than a more gentle decline [16]. In these studies causality is not established.

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In this study we explore these issues using high dose IFN-α induction therapy. High dose IFN-α induction therapy has been shown to increase the likelihood of an early virological response [17, 18]. However data regarding the SVR rate is conflicting [17-20]. We focus on HCV specific CD4+ T-cell responses detectable with IFN-γ and IL-2 ELISpot as we find reproducible responses using this approach [9, 13]. We address two specific hypotheses. Firstly, that a rapid fall in viral load is causally linked with restoration of HCV-specific responses and a SVR to therapy. Secondly, that a SVR to treatment is associated with the restoration of IL-2 secreting HCV-specific T-cell responses as has been shown in the spontaneous resolution of HCV infection [13] and in the treatment of HIV infection [14].

Methods Patients The patients in this study were part of a larger clinical study of 100 HCV patients (previous non-responders to conventional interferon and ribavirin, and treatment naïve patients), designed to assess the efficacy of the treatment regime described in detail below (currently in submission). Of these, 31 treatment naïve patients (21 males, 10 females) with persistent HCV genotype-1 infection were included in this immunological study (Table 1). In all patients, HCV RNA was detectable by RT-PCR in the serum on at least two consecutive occasions 6 months apart (Roche v2.0 Amplicor assay, Roche Diagnostics Ltd, Branchburg, NJ, USA) before treatment. All patients were HCV genotype-1 (TruGene® HCV genotyping assay, Bayer Diagnostics, Berkeley CA, USA) and negative for HIV and HBV antibodies. 5 patients were cirrhotic (4 biopsy proven and one on clinical grounds). All patients were treated with triple therapy consisting of: amantadine hydrochloride 200 mg/d (Symmetrel®; Novartis, Basel, Switzerland) and ribavirin (Rebetol®; Schering-Plough, Kenilworth, NJ, USA) 1000 or 1200 mg/d (based on body weight) for a total of 24 or 48 weeks, combined with interferon alfa-2b induction (IntronA®; Schering-Plough) during the first 6 weeks, and thereafter combined with weekly pegylated interferon alfa-2b (Pegintron®; Schering-Plough), 1.5 µg/kg for a total of 24 or 48 weeks. The interferon induction scheme during the first 6 weeks was as follows: week 1 and 2: 18 MU/d in 3 divided doses; week 3 and 4: 9 MU/d in 3 divided doses; week 5 and 6: 6 MU/d in 2 divided doses. Patients with a decrease of HCV RNA < 3 log at week 4 were treated for 48 weeks. Patients with a decrease of HCV RNA ≥ 3 log at week 4 were randomized at week 24 to stop treatment at 24 weeks or continue to 48 weeks. Treatment was stopped in all patients with HCV RNA > 615 IU/ml at week 24. All patients were followed for 24 weeks after completion of therapy. The study was approved by the institutional review board.

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Samples Peripheral blood mononuclear cells (PBMC) were isolated from blood by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway) and frozen immediately before treatment (week 0), 12 and 24 and 48 weeks after the start of treatment, and 24 weeks after completion of treatment (follow up-FU). Analysis of viral load, alanine transaminase (ALT) and full blood counts were performed at week 0, 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 48 and then weeks 12 and 24 after treatment.

Viral kinetics Viral kinetics were assessed using the following three assays; quantitative bDNA (VERSANT ® HCV 3.0 assay; Bayer Diagnostics; linear dynamic range 6.15 x 102-7.7 x 106 IU/ml) , qualitative PCR (COBAS® Amplicor HCV Test v2.0; Roche diagnostics; Lower limit of detection (LLD) 50 IU/ml, and qualitative TMA (Transcription-Mediated Amplification; VERSANT ® HCV qualitative assay; Bayer Diagnostics; LLD 5 IU/ml)

Clinical definitions Clinical definitions are based on the highly sensitive TMA assay. SVR was defined as loss of HCV RNA from the serum that was maintained for 24 weeks after therapy. Non-responders had detectable HCV RNA levels in serum throughout treatment. Breakthrough was defined as initial loss of HCV RNA from the serum followed by recurrence of HCV RNA whilst on therapy. Relapse was defined as absence of viremia during therapy followed by detectable viremia within 24 weeks of completion of therapy. For the purpose of analyses non-SVR is defined as non-response, breakthrough or relapse.

ELISpot assays Thawed PBMC were tested by IFN-γ (MABTECH, Stockholm, Sweden) and IL-2 (BD Biosciences, Oxford, UK) ELISpot assays as previously described [9]. PBMC from the same patient derived at multiple time-points were assessed concurrently. Briefly, PBMCs (0.2 million/well following exclusion of dead cells with tryphan blue) were plated in duplicate in anti–IFN-γ or anti-IL-2 precoated 96-well ELISpot plates. Antigens used included; HCV core-derived peptides spanning amino acids (AA) 1-191 in a single pool, NS5a derived peptides AA 2201-2410 in a second pool (each peptide 20AA in length overlapping by 10 AA, 10µg/ml final concentration, corresponded to HCV isolate H77 genotype 1a), recombinant proteins NS3, NS4, and NS5 (Chiron, Emeryville, CA, USA) (final concentration of 1 µg/ml) in a single pool as previously described [9, 13], CMV lysate (final concentration 0.05µg/ml; Virusys, North Berwick, ME, USA), medium alone as negative control, phytohemagglutinin (PHA; 4µg/ml) as a positive control and influenza vaccine (enzira split virion inactivated, Chiron). Plates were developed after 18-20 hours incubation and analyzed for spot forming units (SFU) using an ELISpot plate reader (AID Reader System, Strassberg, Germany, ELISpot 3.1 SR program).

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Chapter 5

A positive response was recorded where the probability of a spot appearing in the stimulated well was significantly different (p< 0.05) from the probability of a spot appearing in the negative control well (Excel BINOMDIST) [9, 13]. The frequency of T-cells was calculated by subtracting the average SFC in negative control duplicate wells from the average SFC in stimulated duplicate wells and expressed per106 PBMC.

CFSE based proliferation assays. PBMC (1 x 107 /ml) in PBS were incubated at 37°C for 7 min with 0.5µM CFSE (Molecular Probes, Eugene, Oregon, USA). PBMC were washed with PBS containing 10% pooled human serum and then PBS only. Cells were resuspended at 2 x 106/ml in RPMI containing 10% human serum. Stained cells (1 x 106/well, 1ml) were cultured in 48-well plates with either medium alone, PHA, core peptide pools 1-4 (10µg/ml final concentration), CMV lysate (optimized final concentration 0.05µg/ml; Virusys) or influenza vaccine (enzira split virion inactivated). After six days cells were washed and stained at 4°C with: anti-human CD4-APC, CD8-PE and Viaprobe (7-AAD) (BD Pharmingen, Oxford, UK). Flow cytometric analysis was performed on a FACSCalibur and analysed using FlowJo (Treestar, Ashland, Oregon, USA). T-cell proliferation was determined by gating on the lineage-positive CFSElow and CFSEhigh subset. A stimulation index (SI) was derived by dividing the CD4+ proliferative frequency (%) in presence of antigen by the CD4+ proliferative frequency without antigen using the following formula: CD4+ proliferative frequency(%)=Number of CD4+ CFSElow cells/(Number of CD4+ CFSElow cells + number of CD4+ CFSEhigh cells) x100. A SI > 2 was considered positive. The CD4+ proliferative frequency (%) for a positive response was achieved by subtracting the proliferative frequency with antigen from the proliferative frequency without antigen.

T-cell FACS analysis 100,000 viable PBMC (following staining with tryphan blue) were incubated for 20’ at 4°C with CD3-PE, CD4-APC and CD8-FITC and aquired/analysed as above.

Statistical analysis Significance of the proportion of viraemic patients was determined using the Fishers’ exact test. Comparisons between SVR and non-SVR patient cohorts pre-treatment were made using the non-parametric Mann-Whitney U test. Correlations were determined using the Pearson correlation coefficient. T-cell responses at each time point were compared with baseline (pre-treatment) values using the paired non-parametric Wilcoxon ranked sign test. P values 3 log Treatment (years) pre-Rx (IU/ml) week 4 week 12 decrease in duration (U/l) Pre-Rx viral load at (weeks) week 4 19

F

354

42

311

49

322

42

F

324

67

F

325

51

M

330

47

F

336

47

341

46

346

46

M

314

49

F

316

35

M

145

319

49

M

243

326

48

M

94

327

50

M

67

339

47

M

65

305

55

M

415

313

41

M

315

40

317

35

32

Response to treatment

50

no

178000

+

–

yes

Breakthrough

M

208

yes

1380000

–

+

yes

24

Breakthrough

M

494

no

381000

+

+

no

48

Non-responder

55

no

2550000

+

+

no

24

Non-responder

96

yes

1290000

+

+

no

24

Non-responder

217

yes

5850000

+

+

no

48

Non-responder

34

no

1760000

+

+

no

24

Non-responder

M

72

no

313000

+

+

no

24

Non-responder

M

81

nd

5400000

+

+

no

24

Non-responder

43

nd

1060000

+

+

no

48

Non-responder

194

no

1350000

+

–

yes

24

Relapse

nd

368000

+

–

yes

48

Relapse

no

1390000

+

+

yes

48

Relapse

yes

140000

+

–

yes

48

Relapse

no

854000

+

–

yes

24

Relapse

no

1420000

+

–

yes

48

Relapse

no

3270000

+

–

yes

48

SVR

62

no

3100000

+

–

yes

24

SVR

F

31

no

296000

+

–

no

48

SVR

M

170

no

2920000

–

–

yes

48

SVR

318

32

F

79

nd

132000

–

–

yes

24

SVR

320

50

M

205

no

1590000

–

–

yes

24

SVR

323

47

M

51

no

2910000

–

–

yes

24

SVR

329

40

F

37

no

2850000

+

+

no

48

SVR

333

32

F

82

no

196000

–

–

yes

24

SVR

334

55

F

49

no

161000

+

–

no

48

SVR

335

39

M

263

no

915000

+

–

yes

48

SVR

340

34

M

58

no

1880000

+

–

yes

24

SVR

343

49

M

77

yes

239000

+

+

no

48

SVR

347

48

M

121

nd

119000

–

–

yes

24

SVR

350

50

M

76

no

32200

–

–

yes

24

SVR

HCV specific T-cell responses, ALT and viral load before therapy in relation to outcome Before therapy HCV core specific T-cell responses could be detected in 21/31 patients; 10/15 patients with a subsequent SVR and 11/16 patients with a non-SVR to therapy. The mean viral load, ALT and HCV core specific responses as detected by IFN-γ ELISpot were lower in patients who achieved a SVR (viral load IU/ml: 5.791 ± 0.1723 vs 5.990 ± 0.1181, ALT IU/L 118.4

78

SVR despite HCV specific T-cell decline Figure 1b. T-cell proliferation

T-cell proliferation was assessed following 6 days of stimulation with medium alone, HCV core pooled antigens, CMV lysate and PHA following incubation with CFSE. FACS analysis of two representative patients (322 and 324) is shown following gating on the viaprobe negative T-cell subset. The % in the upper left quadrants = number of CD4+CFSElow/ total CD4+ cells x100.

± 27.25 vs 134.9 ± 29.44, core responses SFU/106 PBMC 85.07 ± 31.58 vs 187.9 ± 94.09), although this did not reach statistical significance. There was no significant association between the magnitude of the core specific response and the viral loads before treatment. However, there was a significant correlation between the IFN-γ core specific HCV response and both ALT (Pearson correlation r2=0.45, p=0.001)(Figure 1a) and AST levels (Pearson correlation r2=0.42, p=0.001) before treatment. Figure 2a: HCV core specific T-cell responses by IFN- ELISpot in all patients at each time point.

A single data-point at each time point represents total HCV core specific T-cell responses (using peptides spanning the entire core region) in a single patient assessed by IFN- ELISpot. Values at each time point are compared with baseline (Wilcoxon signed rank test). Bars = mean.

79

Chapter 5 Figure 2b: HCV core specific T-cell responses by IFN- ELISpot in relation to viral loads and treatment outcome.

HCV core specific T-cell responses in patients with non-SVR (A) and SVR (B). A single data-point at each time point represents total HCV core specific T-cell responses (using peptides spanning the entire core region) in a single patient assessed by IFN- ELISpot. Values at each time point are compared with baseline (Wilcoxon signed rank test). Bars = mean. HCV viral loads before during and after therapy in patients with a Non-SVR (C) and SVR (D) to therapy. The threshold for the bDNA, PCR and TMA assays are shown using dotted lines.

In 13 patients (5 SVR and 8 non-SVR) with HCV core specific T-cell responses that were detected by IFN-γ ELISpot, sufficient PBMC were available pre-treatment for further analysis using CFSE assays. Using this assay 9/13 patients showed CD4+ T-cell proliferation to HCV core antigens. In 2 of these patients proliferative responses were also observed for the CD8+ T-cell subset, consistent with previous studies [13] showing T-cell responses to core were

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SVR despite HCV specific T-cell decline

largely CD4+. Generally proliferative responses to HCV core antigens were very weak (mean CD4+CFSE low/total CD4+ =1.15%) compared to proliferative responses that were observed using CMV (mean CD4+CFSE low/total CD4+ =42.96%) and influenza (mean CD4+CFSE low/total CD4+=30.58%) antigens consistent with a previous study [21]. Two representative patients are shown (Figure 1b). The magnitude of the HCV proliferative response pretreatment did not differ between patients with a SVR and a non-SVR to treatment (data not shown).

Effect of therapy on HCV core IFN-γ responses HCV core specific responses were assessed in all patients by IFN-γ ELISpot before treatment, weeks 12, 24 and 48 weeks during treatment, and 24 weeks after the completion of treatment. Overall there was a significant decline in IFN-γ T-cell responses at weeks 12 (week 0 vs week 12 p=0.007) and 24 (week 0 vs week 24 p=0.009) compared to pre-treatment, that recovered after therapy (Figure 2a). Further subgroup analysis of patients with a SVR and a non-SVR to therapy showed that the decline in T-cell responses was statistically significant only in patients with a SVR to therapy (p= 0.02 week 0 vs. 12, p= 0.019 week 0 vs. 24, Figures 2bA and 2bB). The viral kinetics during therapy for these subgroups are shown (Figures 2bC and 2bD). The viral kinetics and HCV specific T-cell responses are detailed in 4 representative patients; 311, 341, 335 and 333 (Figure 3). Figure 3: Kinetics of HCV core specific T-cell responses and viral loads in individual patients.

HCV core specific T-cell responses (using peptides spanning the entire core region) are assessed by IFN- ELISpot before during and after treatment. HCV viral loads (determined by bDNA, PCR and TMA assays) are plotted. The threshold for the bDNA, PCR and TMA assays are shown using dotted lines.

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Figure 4: HCV core specific T-cell responses before, during and after treatment by IL-2 ELISpot. A single data-point at each time point represents total HCV core specific T-cell responses (using peptides spanning the entire core region) in a single patient assessed by IL-2 ELISpot. Bars = mean. Values at each time point were not significantly different compared with baseline (Wilcoxon signed rank test). Figure 5: CMV specific T-cell responses before, during and after treatment using IFN- ELISpot. A single data-point at each time point represents total CMV specific T-cell responses (using CMV lysate) in a single patient assessed by IFN- ELISpot. Values at each time point are compared with baseline (Wilcoxon signed rank test). Only patients found to be CMV positive by ELISpot before treatment were included in this analysis. Bars = mean. Figure 6: Influenza specific T-cell responses to before, during and after treatment using IFN- ELISpot. A single data-point at each time point represents total influenza specific T-cell responses in a single patient assessed by IFN- ELISpot. Values at each time point are compared with baseline (Wilcoxon signed rank test). Bars = mean.

Effect of therapy on HCV core IL-2 responses The effect of therapy on HCV core specific T-cell responses was assessed using IL-2 ELISpot. IL-2 secretion could be detected before therapy in 8/31 patients. The mean magnitude of IL-2 production by HCV core specific T-cells was significantly weaker than IFN-γ production (35.72±14.38 vs. 138.1±50.97 SFU/106PBMC) (p=0.030) before treatment and remained lower at all time points during therapy compared to IFN-γ secretion. Similar to the decline in IFNγ response, a decrease in IL-2 production was seen during therapy, which recovered after the cessation of therapy) (Figure 4). This was true for both SVR and non-SVR groups (data not shown). The mean number of IL-2 secreting HCV core specific T-cells was higher at the follow up time point in both the SVR and the non-SVR group compared to pre-treatment. However, this did not reach statistical significance (data not shown).

Effect of therapy on HCV non-structural specific T-cell responses IFN-γ and IL-2 production was then assessed by ELISpot following stimulation with HCV proteins NS3-5. Responses were detected in fewer patients (5/31; 3 patients with a SVR and 2 with a non-SVR) and were significantly weaker (IFN-γ mean 42.4±8.9 SFU/106PBMC) pre-

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SVR despite HCV specific T-cell decline

treatment than those observed using core peptides (IFN-γ mean 138.1±50.97 SFU/106PBMC, consistent with our previous studies [13]). Reponses to NS3-5 did not increase during therapy, and were lower at all time points during therapy compared to pre-treatment, though this did not reach statistical significance. There was no significant increase in HCV NS3-5 IL-2 secretion during therapy. In a subset of 6 patients we assessed T-cell responses to a pool of overlapping peptides spanning NS5a before treatment and at week 12. No T-cell responses to NS5a were observed at either time point.

Effect of therapy on CMV and Influenza specific T-cell responses To determine if the suppression of T-cell responses observed during therapy was specific to HCV we assessed the magnitude of CMV specific T-cell responses during therapy. CMV responses could be detected in 14/31 patients by IFN-γ ELISpot before treatment (mean magnitude 461.4±46.31 SFU/106PBMC) There was a marked decline in the magnitude of the CMV specific T-cell response at 12 weeks (mean 178.2±49.86 SFU/106PBMC p=0.003) and 24 weeks p=0.037 (mean 236.0±80.0 SFU/106PBMC) into therapy compared to baseline. These recovered again after the cessation of therapy (mean 307.5±60.02 SFU/10 6PBMC p=n/s) (Figure 5). As both CMV and HCV are persistent viral infections and as IFN-α has broad anti-viral effects it was unclear if therapy was exerting suppressive effects directly on T-cells or indirectly through suppression of antigen. We therefore examined the effect of therapy on influenza specific T-cells, where there is no circulating antigen, in a subgroup of 6 patients (Figure 6). In these six patients a significant fall in the influenza specific T-cell response was observed between the pre-treatment and 12 week time point (p=0.03).

Effect of therapy on global T-cell populations As HCV, CMV and Influenza specific T-cell responses fell during therapy and recovered following therapy we assessed the impact of therapy on global T-cell populations at week 0 and week 12 by FACS analysis. We selected 6 patients where HCV specific T-cell responses were detectable at week 0 but undetectable at week 12 by IFN-γ ELISpot (Figure 7a). FACS analysis of PBMC showed a fall in the CD4+ and CD8+ T-cell subsets within the total PBMC population of approximately 50% in both T-cell subsets (Figure 7b and 7c, representative patient Figure 7d). Analysing the blood lymphocyte counts in all patients at week 0 and week 12 we observed the mean in the SVR group was 5.8 109/ml pre-treatment and this declined in 13/15 patients by week 12 to a mean count of 3.3 109/ml. Similarly in the nonSVR group the mean count before treatment was 6.4 109/ml declining in 14/15 patients (data not available in 1 patient at 12 week time point) to a mean count of 3.3 109/ml (Figure 7e). Thus although 200,000 cells were used in each ELISpot well, the T-cell fraction within each well would, at least in some cases, be approximately 50% less at week 12 compared to pretreatment. However, this observation cannot explain the complete loss of HCV specific T-cell responsiveness observed in many patients during the first 12 weeks of therapy.

83

Chapter 5 Figure 7: Total T-cell populations

Total T-cell populations in 6 patients with detectable HCV core specific T-cell responses by IFN- ELISpot pretreatment (week 0) and and undetectable responses at week 12 (A). CD4+CD3+ (B) and CD8+CD3+ (C) T-cell subsets at week 0 and 12 in each patient. Bars=mean. The dot/plot of PBMC from patient 355 highlighting % CD3+CD4+ and CD3+CD4+/total PBMC at week 0 and 12 (D). Blood lymphocyte counts in non-SVR and SVR patients at week 0 and 12(E).

Discussion A number of studies have shown a successful outcome with combination therapy is associated with enhanced cellular immune responses [8, 10]. However, others have failed to demonstrate such a clear association [9, 11] and a causal relationship between enhanced T-cell responses and viral load decline has not been demonstrated. In this study then we adopted a treatment regime that used high dose IFN-α in the early weeks of therapy, followed by conventional doses of PEG-IFN and ribavirin with the expectation that this may increase SVR rates, either through direct anti-viral effects or through enhancement of HCV-specific T-cell responses. It was therefore surprising that the main observation in this study was that there was a profound decline in HCV specific Tcell responses during the first 12 weeks of therapy, that was maintained at 24 weeks and that was more pronounced in those patients that had a subsequent SVR to therapy. Whilst a decrease in T-cell responses has been observed during resolving primary HCV infection in

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SVR despite HCV specific T-cell decline

parallel with a declining HCV viral load [12], this has not been previously described in the context of HCV antiviral therapy. It is tempting to speculate that high dose IFN-α leads to a rapid decline in viral load and that as antigen is rapidly lost, antigen-dependent T-cell responses wane in the absence of ongoing antigenic stimulation. Mathematical modelling predicts that a very rapid loss of antigen below a critical threshold during therapy may lead to a failure of T-cell antigen recognition [22]. The finding that a decline in T-cell response at 12 weeks is most evident in those that achieve a SVR is significant. Of 15 patients who developed a SVR only 2/15 had detectable viraemia using the highly sensitive TMA assay at 12 weeks, and 0/15 patients at 24 weeks whereas viraemia was observed in 9/16 patients at 12 weeks and 10/16 at 24 weeks who failed to make a SVR, suggesting that indeed T-cell responses (whose decline is greatest in SVR patients at week 12 and 24) parallel a decline in HCV viral load. However, this does not explain the observation that in many individuals T-cell responses recover once therapy has stopped whilst HCV viral loads remain undetectable (Figure 4 representative patients 335 and 333). In this study, there was a decline in blood lymphocyte counts between baseline and 12 weeks in almost all patients. Furthermore, we observed not only a decline in HCV specific T-cells but also in CMV and influenza specific T-cell responses during therapy. The decline in influenza T-cell responses is particularly informative as any effect on T-cell responses must be independent of viral load, since influenza virus does not persist. FACS analysis of PBMC at week 0 and 12 weeks into therapy confirmed that there was a decline in the CD4+ and CD8+ T-cell subsets. Taken together, this data suggests that the observed decline in T-cell responses is due, at least in part to a direct effect of therapy on T-cells through direct suppression of their generation (IFN-α is known to exert suppressive effects on bone marrow), increased apoptosis or more speculatively through Tcell redistribution away from the peripheral blood compartment. We analysed HCV specific T-cell responses using antigen pools. The first of these, a core peptide pool generates a reproducible response in a number of previously studied cohorts [9, 13, 23]. This approach is analogous to the use of overlapping peptide pools for CMV pp65 or HIV gag in other antiviral studies. It is not a comprehensive analysis but provides a target population of T-cells to track. To add breadth we also used an NS3-5 whole antigen pool as has been used extensively elsewhere [9, 13]. Finally to provide data on peptide specific responses against a non-structural protein we also analysed an NS5a peptide pool. Importantly, no enhancement of these responses was observed during treatment. Whilst we showed no association between the magnitude of the HCV specific T-cell responses before therapy and the subsequent response to therapy we did observe a highly significant correlation between liver inflammation as assessed by both ALT, AST and the HCV specific T-cell response. Whilst it is widely assumed that liver inflammation in HCV is due to an influx of HCV specific T-cells this is the first time that this association has been clearly made. In defining a successful immune response simple quantification of cellular responses is likely to represent an over-simplistic approach. The phenotype and cytokine profile of viral specific T-cells clearly differs between different viral infections. Furthermore we have previously shown that the functional status of HCV specific T-cells is important in HCV control [13].

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In individuals that resolve HCV, CD4+ HCV specific T-cells maintain IL-2 secretory capacity compared to IFN-γ secretion, whilst in persistent infection IL-2 secreting CD4+ T-cells are rarely observed. Such populations have a relatively low proliferative capacity as assessed using CFSE assays [24]. Furthermore in HIV infection it has been shown that in the presence of high viral loads HIV specific CD4+ T-cell populations do exist, but lack proliferative capacity and secrete IFN-γ but are unable to secrete IL-2 in response to antigenic stimulation. Once HIV antigen loads decline through drug therapy IL-2 secretion and proliferative capacity is restored [14]. In this study then we addressed the hypothesis that successful combination therapy is associated with the restoration of IL-2 secreting cellular immune responses. We observed that IL-2 secretion by HCV specific T-cells was significantly weaker than IFN-γ secretion before treatment and declined further during treatment in both the SVR and nonSVR groups. Following therapy IL-2 secretion was restored to pre-treatment levels in those with a SVR but was not enhanced above baseline. Overall, in this study we demonstrate that rather than enhancing the T-cell response, as might have been predicted, high dose α–interferon induction therapy is associated with a profound loss of IL-2 and γ-IFN secreting HCV specific T-cells. This effect is probably mediated both through loss of antigenic stimulation of HCV specific T-cells and also through direct immunosuppressive effects of high dose IFN-α therapy. Although the magnitude of this effect is of most significance in the setting of high dose IFN-α, the significance of these observations is much more general. Firstly, the very rapid virological response seen can readily occur in the absence of immune restoration-indeed in the context of loss of HCV specific T-cells. This strongly implies that this phase of viral dynamics is independent of Tcell responses. Secondly, long term antiviral effects are also obtained whilst T-cell responses are suppressed. Again, this supports an argument that any change in T-cell responses seen during conventional doses of IFN-α therapy are not causally related to clinical outcome. The challenge remains to develop new efficacious antiviral therapies for HCV. It is probable that HCV protease and polymerase inhibitors will be used in clinical practice in the next few years. These specific antiviral therapies will shed further light on the relationship between HCV viral loads and T-cell responses during therapy. Additionally, the fact that Tcell responses induced during current antiviral therapies appear not to influence virological outcomes does not mean that further direct manipulation of such responses would not confer some further benefit. In the meantime further evaluating the host pathways which contribute to successful outcomes for antiviral therapy remains an important objective.

References 1.

Fried, M.W., et al., Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med, 2002. 347(13): p. 975-82.

2.

Barnes, E., et al., Impact of alpha interferon and ribavirin on the function of maturing dendritic cells. Antimicrob Agents Chemother, 2004. 48(9): p. 3382-9.

3.

Brinkmann, V., et al., Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells. J Exp Med, 1993. 178(5): p. 1655-63.

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Cox, A.L., et al., Cellular immune selection with hepatitis C virus persistence in humans. J Exp Med, 2005. 201(11): p. 1741-52.

5.

Lechner, F., et al., CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained. Eur J Immunol, 2000. 30(9): p. 2479-87.

6.

Takaki, A., et al., Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C. Nat Med, 2000. 6(5): p. 578-82.

7.

Lauer, G.M., et al., High resolution analysis of cellular immune responses in resolved and persistent hepatitis C virus infection. Gastroenterology, 2004. 127(3): p. 924-36.

8.

Kamal, S.M., et al., Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper 1 responses in patients with chronic hepatitis C. Gastroenterology, 2002. 123(4): p. 1070-83.

9.

Barnes, E., et al., The Dynamics of T-Lymphocyte Responses During Combination THerapy for Chronic Hepatitis C Virus Infection. Hepatology, 2002. 36: p. 743-754.

10.

Cramp, M.E., et al., Hepatitis C virus-specific T-cell reactivity during interferon and ribavirin treatment in chronic hepatitis C. Gastroenterology, 2000. 118(2): p. 346-55.

11.

Aberle, J.H., et al., CD4+ T Cell Responses in Patients with Chronic Hepatitis C Undergoing Peginterferon/ Ribavirin Therapy Correlate with Faster, but Not Sustained, Viral Clearance. J Infect Dis, 2007. 195(9): p. 1315-9.

12. Lauer, G.M., et al., Full-breadth analysis of CD8+ T-cell responses in acute hepatitis C virus infection and early therapy. J Virol, 2005. 79(20): p. 12979-88. 13.

Semmo, N., et al., Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection. Hepatology, 2005. 41(5): p. 1019-28.

14.

Younes, S.A., et al., HIV-1 viremia prevents the establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed with proliferative capacity. J Exp Med, 2003. 198(12): p. 1909-22.

15.

Neumann, A., et al., HCV Viral dynamics in vivo and the efficacy of Interferon alpha therapy. Science, 1998. 282: p. 103-7.

16.

Tang, K.H., et al., Relationship between early HCV kinetics and T-cell reactivity in chronic hepatitis C genotype 1 during peginterferon and ribavirin therapy. J Hepatol, 2005. 43(5): p. 776-82.

17.

Steindl-Munda, P., et al., Impact of high-dose interferon induction and ribavirin therapy in patients with chronic hepatitis C relapsing after or not responding to interferon monotherapy. Liver Int, 2003. 23(4): p. 269-75.

18.

Rosen, H.R., et al., Early hepatitis C viral kinetics correlate with long-term outcome in patients receiving high dose induction followed by combination interferon and ribavirin therapy. J Hepatol, 2002. 37(1): p. 124-30.

19.

Yasui, K., et al., Dynamics of hepatitis C viremia following interferon-alpha administration. J Infect Dis, 1998. 177(6): p. 1475-9.

20. Ferenci, P., et al., Combination of interferon induction therapy and ribavirin in chronic hepatitis C. Hepatology, 2001. 34(5): p. 1006-11. 21.

Semmo, N., et al., Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection. J Viral Hepat, 2007. 14(7): p. 492-502.

22. Komarova, N.L., et al., Boosting immunity by antiviral drug therapy: a simple relationship among timing, efficacy, and success. Proc Natl Acad Sci U S A, 2003. 100(4): p. 1855-60. 23. Semmo, N., et al., T-cell responses and previous exposure to hepatitis C virus in indeterminate blood donors. Lancet, 2005. 365(9456): p. 327-9. 24. Penna, A., et al., Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection. Hepatology, 2007. 45(3): p. 588-601.

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Acknowledgements E.B. is an M.R.C.(U.K.) clinician scientist, I.H. is funded by the M.R.C, (U.K.), P.K. is a Senior Wellcome clinician scientist. We would like to thank Ester Remmerswaal and Nelly Baylon for technical assistance. We would also like to thank the patients for their participation in this study.

Conflict of interest statement: E.B., H.C.G., I.H., N.S., R.A.W.VL., P.K: none to declare H.W.R. is a consultant for Schering-Plough, he has received unrestricted grants from Hoffmann-La Roche, Schering-Plough and Vertex Pharmaceuticals. M.G.H.M.B. is a consultant for Bayer Diagnostics.

Grant support This study was supported by the Medical Research Council (UK), Wellcome trust, ScheringPlough and Bayer Diagnostics

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C h a p t e r

6

High incidence of type 1 diabetes mellitus during or shortly after treatment with pegylated interferon alfa for chronic hepatitis C virus infection Tim CMA Schreuder1*, Huub C Gelderblom1*, Christine J Weegink1, Dörte Hamann3, Henk W Reesink1, J Hans DeVries2 , Joost BL Hoekstra2 , Peter LM Jansen1 *These authors contributed equally to this study 1 AMC

Liver Centre, Department of Gastroenterology and Hepatology, and 2Department of Internal Medicine, Academic Medical Centre, University of Amsterdam, 3Department of Autoimmune Diseases, Sanquin Diagnostics at CLB, Amsterdam

Online Early, Liver International DOI:10.1111/j.1478-3231.2007.01610.x

Chapter 6 Liver International ISSN 1478-3223

CLINICAL STUDIES

High incidence of type1diabetes mellitus during or shortly after treatment with pegylated interferon a for chronic hepatitis C virus infection ¨ Tim C. M. A. Schreuder1,�, Huub C. Gelderblom1,�, Christine J. Weegink1, Dorte Hamann2, Henk W. Reesink1, J. Hans DeVries3, Joost B. L. Hoekstra3 and Peter L. M. Jansen1 1 Department of Gastroenterology and Hepatology, AMC Liver Centre, University of Amsterdam, Amsterdam, the Netherlands 2 Department of Autoimmune Diseases, Sanquin Diagnostics at CLB, Amsterdam, the Netherlands 3 Department of Internal Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

Keywords autoimmune – diabetes mellitus – hepatitis C – interferon a

Correspondence T. C. M. A. Schreuder, Department of Gastroenterology and Hepatology, AMC Liver Centre, Room C2-331, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands Tel: 131 20 5668748 Fax: 131 20 5669240 e-mail: [email protected] Received 29 April 2007 accepted 9 September 2007 DOI:10.1111/j.1478-3231.2007.01610.x

Abstract Background: Development of diabetes mellitus (DM) during or shortly after treatment with interferon a (IFN-a) in patients with chronic hepatitis C virus (HCV) infection has been reported sporadically. We prospectively screened for DM during and after IFN-a therapy for chronic HCV infection. Methods: Blood glucose levels of patients with chronic HCV infection were routinely assessed at all outpatient visits during and after treatment with pegylated-IFN-a (Peg-IFN-a) and ribavirin (Riba). Results: Between December 2002 and October 2005, 189 non-diabetic patients were treated with Peg-IFN-a/Riba, of whom five developed type 1 DM (2.6%), three type 2 DM (1.6%) and one an indeterminate type of DM. Classical symptoms of DM were present in three patients who developed DM shortly after cessation of Peg-IFN-a/Riba. In the other patients, symptoms of DM were either indistinguishable from side effects caused by Peg-IFN-a/Riba or absent. Conclusion: Our study showed a high incidence of type 1 DM during PegIFN-a/Riba therapy for chronic HCV infection. Symptoms of DM may be absent or mistaken for Peg-IFN-a/Riba-associated side effects. To diagnose DM without delay, we propose routine assessment of blood glucose at all outpatient visits during and after Peg-IFN-a/Riba treatment in chronic HCV patients.

Interferon a (IFN-a) and ribavirin (Riba)-based therapy for chronic hepatitis C virus (HCV) infection is effective in 40–80% of patients but causes a wide range of side effects, such as influenza-like symptoms, depression, insomnia, headache, fatigue, fever and anaemia (1). IFN-a treatment for chronic HCV infection is associated with the development of autoimmune disorders such as autoimmune thyroiditis, hypothyroidism, hyperthyroidism and Sj¨ogren’s syndrome in 2.5–20% of patients (1, 2). Type 1 diabetes mellitus (DM) can also occur during or after IFN-a treatment for chronic HCV infection. Several cases have been published (3–10), but the exact incidence of DM during or after treatment with IFN-a is unknown. An Italian retrospective study among 11 241 patients with chronic HCV infection treated with IFN-a revealed

only 10 new diagnoses of DM (0.08%) after at least 16 weeks of treatment (11). Development of type 1 DM during treatment with IFN-a for chronic hepatitis B infection, hairy cell leukaemia, Kaposi’s sarcoma and renal cell carcinoma has also been reported (3), but the majority of cases occurred in patients with chronic HCV infection (3). The prevalence of type 2 DM was found to be significantly higher in a cohort of IFN-a-naı¨ve chronic HCV patients (14.5%) compared with patients suffering from other chronic liver diseases (7.3%) and the general population (7.8%); the difference was even more pronounced in patients with advanced histological disease (12, 13). Recent studies have shown that insulin resistance is common in non-diabetic chronic HCV patients, and that insulin resistance decreases during treatment with IFN-a (14–18). We screened for DM during and after treatment with pegylated-IFN-a (Peg-IFN-a) and Riba for

�These authors contributed equally to this study.

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Determination of autoantibodies

chronic HCV infection. This started after we observed two index cases of DM during Peg-IFN-a/Riba treatment in our hospital in December 2002. In the first patient, DM-related symptoms, commencing after 16 weeks of antiviral treatment, had been attributed to Peg-IFN-a/Riba; DM was diagnosed because symptoms were persistent after cessation of Peg-IFN-a/Riba treatment. The second patient was presented at the emergency room after 11 weeks of Peg-IFN-a/Riba therapy because of severe debilitation, where DM was diagnosed (blood glucose 54 mmol/L). Because of the atypical clinical picture of DM in these two patients, we amended our treatment protocols, and blood glucose levels of patients with HCV were routinely measured in all patients at all outpatient visits before, during and after treatment with Peg-IFN-a/Riba as of December 2002.

In patients who developed DM, we retrospectively determined autoantibodies to glutamic acid decarboxylase (anti-GAD65), protein tyrosine phosphatase (anti-IA2) and pancreatic islet cells [islet cell antibodies (ICA)] in stored plasma samples obtained before and after development of DM. Detection of anti-GAD65 and anti-IA2 was performed by a fluid phase assay using [35]S-labelled recombinant human GAD65 and IA2 as tracers, and protein A sepharose beads for precipitation. Anti-GAD65 and anti-IA2 values were determined according to the WHO 97/550 standard (19). ICA was determined by indirect immunofluorescence on monkey pancreas tissue (INOVA Diagnostics, San Diego, CA, USA). AntiGAD65 and anti-IA2 test results were noted in international units (IU) per millilitre and converted to negative, indeterminate, weakly positive or positive. The following cut-off values (in IU/mL) were used: for anti-GAD65, 0–29 = negative, 30–42 = indeterminate, 43–80 = weakly positive and 4 80 = positive; for antiIA2, 0–23 = negative, 24–46 = indeterminate, 47–68 = weakly positive and 4 68 = positive. Seroconversion was defined as a transition from a negative or an indeterminate state to a (weakly) positive state. Autoantibodies directed to thyroid peroxidase (antiTPO) and antinuclear antibodies (ANF) were measured using standard immunoassays before antiviral therapy. In patients who developed DM with seroconversion, anti-TPO was measured again. Thyroid function (thyroid-stimulating hormone and free T4) was tested before treatment and every 3 months during treatment in all patients according to standard recommendations.

Methods Treatment protocols Patients were treated with either (i) standard therapy consisting of Peg-IFN-a2b 1.5 mg/kg/week (PegIntrons, Schering-Plough Corporation, Kenilworth, NJ, USA) or Peg-IFN-a2a 180 mg/week (Pegasyss, Roche, Basel, Switzerland) combined with Riba (Rebetols, ScheringPlough or Copeguss, Roche) 1000–1200 mg daily for a total of 24 or 48 weeks according to HCV genotype (n = 107) or (ii) in a clinical trial with triple therapy consisting of amantadine hydrochloride (Symmetrels, Novartis Pharma, Arnhem, the Netherlands) 200 mg daily and Riba 1000–1200 mg daily for a total of 24 or 48 weeks, combined with high-dose IFN-a induction (IntronAs, Schering-Plough) during the first 6 weeks (week 1–2: 18 MU daily, week 3–4: 9 MU daily, week 5–6: 6 MU daily), thereafter combined with Peg-IFNa2b 1.5 mg/kg/wk for a total of 24 or 48 weeks (n = 100). In the group receiving standard therapy (n = 107), 17 patients had been treated with IFN-a previously, 90 were treatment naı¨ve, 34 were female and 73 male; 95 were treated with PegIntron and Rebetol, 1 with IntronA and Rebetol, and 11 with Pegasys and Copegus. In the group receiving triple therapy (n = 100), 46 patients had been treated with IFN-a previously, 54 were treatment naı¨ve, 23 were female and 77 male.

Human leucocyte antigen typing In those who developed DM, the human leucocyte antigen (HLA) haplotype was analysed using PCR with sequence-specific primers (SSP, GenoVision, West Chester, PA, USA) and sequence-specific probes (SSO, Dynal, Carlsbad, CA, USA) on stored or fresh peripheral blood mononuclear cells. C-peptide reserve In those who developed DM, C-peptide levels were measured before and after glucagon stimulation to assess the degree of pancreatic islet cell destruction (20). Briefly, an intravenous cannula was placed in the forearm of the patient in a fasting state; blood samples for assessment of C-peptide and glucose levels were

Assessment of blood glucose levels Random blood glucose levels of patients with HCV were routinely measured at all outpatient visits before, during and after treatment with Peg-IFN-a/Riba as of December 2002.

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labelled as type 1 or 2; this patient declined assessment of C-peptide reserve. Five of 98 patients (5.1%) developed DM during (n = 4) or after (n = 1) standard treatment, and four of 91 patients (4.4%) during (n = 2) or after (n = 2) triple treatment. Patient characteristics are summarized in Table 1. Classical symptoms of DM were indistinguishable from the side effects of antiviral therapy with PegIFN-a/Riba in these patients. Three patients presented with typical symptoms (e.g. thirst and polyuria), but they developed DM 4 weeks after cessation of treatment, when Peg-IFN-a/Riba-related side effects had disappeared. All nine patients were initially treated with insulin. After cessation of antiviral therapy, seven patients remained insulin dependent, whereas two patients without detectable antibodies (Table 2, patients 1 and 2) switched to oral antidiabetic drugs. One of these two patients was even able to stop oral antidiabetic drugs after losing weight. Peg-IFN-a/Riba treatment was stopped prematurely when DM was diagnosed in two of nine patients. Three patients achieved a sustained virological response, defined as undetectable HCV RNA 24 weeks after cessation of antiviral therapy.

obtained 15 and 0 min before administration of 1 mg glucagon intravenously (i.v.) and 6 min afterwards. Results Patients Between December 2002 and October 2005, 207 patients with chronic HCV infection were treated. One hundred and seven patients received standard antiviral therapy and 100 received triple therapy. Eighteen patients already suffered from DM before therapy and were therefore excluded from this analysis. One hundred and eighty-nine patients were analysed, including the two index patients and including 24 patients who were either already on treatment or during the 24-week follow-up period to determine the virological response, when the study started in December 2002. In total, nine of 189 patients (4.8%) developed DM defined as two random glucose samples above 11.1 mmol/L, according to the ADA classification (21). All 189 patients had normal glucose levels at baseline. Type 1 DM was defined as the presence of associated autoantibodies with or without susceptible HLA constitution; type 2 DM was defined as the absence of associated autoantibodies and no susceptible HLA constitution. Five patients (2.6%) developed type 1 DM, and three patients (1.6%) developed type 2 DM. One patient without autoantibodies but with a susceptible HLA-constitution DM could not be

b-cell autoantibodies As depicted in Table 2, two of the nine patients were positive for anti-GAD65 or ICA before treatment. Three of the nine patients seroconverted for antiGAD65, anti-IA2 or ICA during treatment.

Table 1. Patient characteristics Patient

Sex

Age (years)

BMI (kg/m2)

DM in family

HCV genotype

Previous IFN-a

1 2 9 3 4 6 7 8 5

M M M M M M F M M

61 50 43 54 46 25 44 46 54

25 30 33 25 23 22 24 22 25

Yes No No Yes No Yes Yes Yes No

2 1b 1 3 1a 4 3a 3a 1

� � � � 1 � � � �

Cirrhosis

Antiviral treatment

Onset of DM

Virologic outcome

1 1 � 1 � � � � 1

Standard Triple Standard Standard Triple Triple Standard Standard Triple

Week 16� Week 11 Week 12 4 weeks after ET Week 11 4 weeks after ET Week 22 Week 14 4 weeks after ET

NR NRw NR NR NR SVR SVR SVRw NR

�DM was diagnosed after cessation of Peg-IFN-a/Riba, but based on symptoms, the onset of DM was estimated after 16 weeks of antiviral treatment. wTherapy was stopped after the development of DM. Patients were treated for 24–48 weeks with either (i) standard therapy consisting of Peg-IFN-a/Riba or (ii) in a clinical trial with triple therapy consisting of amantadine hydrochloride and Peg-IFN-a/Riba, with high-dose IFN-a induction instead of Peg-IFN-a during the first 6 weeks. BMI, body mass index, calculated as weight in kilograms divided by square height in metres. DM in family, first degree family members with type 1 or 2 DM. ET, end of treatment (24–48 weeks). NR, non-response, defined as persistence of HCV RNA during and after antiviral treatment. SVR, sustained virologic response, defined as undetectable HCV RNA 24 weeks after cessation of antiviral treatment. DM, diabetes mellitus; HCV, hepatitis C virus; Peg-IFN-a, pegylated-interferon-a; Riba, ribavirin.

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Table 2. Type 1 diabetes mellitus-associated autoantibodies before antiviral therapy and after development of diabetes mellitus, human leucocyte antigen haplotype, C-peptide reserve, diabetes mellitus classification and non-b-cell autoantibodies in all patients who developed diabetes mellitus anti-GAD65

Positive for anti-GAD65, anti-IA2 or ICA before Patient Before After Before After Before After treatment

Seroconversion for anti-GAD65, anti-IA2 or ICA Risk during treatment HLA�

C-peptide DM Antireservew type TPO ANF

1 2 9 3 4 6 7 8 5

No No No No No Yes Yes Yes No

Yes ND ND ND ND No No ND ND

� � � � 1 � � 11 �

� � � � 11 11 11 11 �

anti-IA2

� � � � � � � � �

ICA

� � � � � � � 1 �

� � � � � � � 1 �

� � � � � 1 � 11 �

No No No No Yes No No Yes No

� � ND DR3 � DR3, DQ2 DR3, DR4, DQ2 ND DR4, DQ8

2 2 2 1 1 1 1 1 ?

� � � � � � � � �

� � � � � � 1 � �

�HLA haplotype was analysed using stored or fresh PBMCs. Two patients (8 and 9) declined extra sampling of blood for these tests. wC-peptide reserve was assessed in three patients, four patients refused the test, two patients who had developed hepatocellular carcinoma were not contacted. Autoantibodies to thyroid peroxidase (anti-TPO) and antinuclear antibodies (ANF) were measured in stored plasma samples using standard immunoassays before antiviral therapy. Cut-off values for autoantibodies: Anti-GAD65 (IU/mL) 0–29 negative 29–42 indeterminate 42–80 weakly positive 4 80 positive Anti-IA2 (IU/mL) 0–23 negative 24–46 indeterminate 47–68 weakly positive 4 68 positive. 11, positive;1, weakly positive; � , indeterminate; � , negative; seroconversion was defined as transition to a 1 or 11 state; DM, diabetes mellitus; HLA, human leucocyte antigen; ICA, islet cell antibodies; ND, not determined; PBMCs, peripheral blood mononuclear cells.

a DM-associated HLA haplotype also seroconverted for anti-GAD65 (Table 2). Two patients declined additional blood sampling for assessment of HLA haplotype.

Figure 1 shows glucose levels and increasing titres of anti-GAD65 before, during and after antiviral treatment in patient 4 (Tables 1 and 2), who developed type 1 DM after 11 weeks of triple antiviral treatment. Non-b-cell antibodies

C-peptide reserve

One patient (patient 7, Table 2) developed thyroiditis, followed by hypothyroidism, without the presence of anti-TPO, shortly after the development of type 1 DM; this patient was positive for ANF before treatment. The other two seroconverters remained anti-TPO negative (data not shown).

In three patients, we assessed the C-peptide reserve, four patients refused the test and two patients, who had developed hepatocellular carcinoma, were not contacted (Table 2). Two patients with positive b-cell autoantibodies had low fasting C-peptide levels (o 100 pmol/L) that did not increase after stimulation with 1 mg of glucagon i.v., suggesting complete destruction of pancreatic b-cells (Table 2). One patient without b-cell autoantibodies had a fasting C-peptide level of 1060 pmol/L that increased to 1700 pmol/L 6 min after stimulation with 1 mg of glucagon i.v., indicating adequate function of pancreatic b-cells (Table 2).

Human leucocyte antigen typing The human leucocyte antigen haplotype was determined in seven of the nine patients. Four patients had an HLA haplotype associated with an increased susceptibility (DR3, DR4, DQ8 and DQ2) for the development of type 1 DM. Two of these four patients with

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Fig. 1. Glucose levels and autoantibodies to glutamic acid decarboxylase (anti-GAD65) in patient 4 who developed type 1 diabetes mellitus during antiviral therapy. Glucose (mmol/L, red line) and anti-GAD65 (E/mL, grey columns). Glucose levels were assessed prospectively, autoantibodies were assessed retrospectively in stored plasma samples. Anti-GAD65 was weakly positive at baseline (�46 E/mL), increased to positive (��84 E/mL) before hyperglycaemia occurred at 11 weeks of treatment, and continued to increase thereafter (���112 E/mL week 11, ����335 E/mL week 28). Treatment with insulin was initiated immediately after the peak in blood glucose level at week 11. The broken horizontal line depicts the upper limit of normal (ULN) for non-fasting blood glucose levels (11.1 mmol/L).

cases and one possible case of de novo type 1 DM during or after treatment of chronic HCV infection with Peg-IFN-a/Riba. In our study, only two of the nine patients who developed DM tested positive for type 1 DM-associated autoantibodies before IFN-a was started. These findings agree with those of Fabris et al. (3), who analysed the presence of b-cell autoantibodies in a number of studies: anti-GAD65, ICA or anti-IA2 was present in 3% of patients with chronic HCV infection (12 of 440) before treatment; two of 440 patients (0.45%) developed type 1 DM during antiviral therapy, and both tested positive for ICA before treatment (i.e. two of the 12 patients with anti-GAD65, ICA or anti-IA2 before treatment). In contrast, pretreatment autoantibodies associated with the development of type 1 DM – assessed in 26 of the 31 reported cases so far – were positive in 50% of patients who developed type 1 DM (3, 5, 9). Taken together, both the positive and the negative predictive value of b-cell autoantibodies seem too low to identify patients at a high risk or to effectively rule out the possibility of developing IFN-a-associated type 1 DM. The destruction of pancreatic b-cells in type 1 DM may be mediated by IFN-a (22, 23). Enhanced

Ethnic origin of the type 1 diabetes mellitus patients Two of the five patients who developed type 1 DM were of Dutch origin, one patient was born in Portugal, one patient was born in Egypt and one patient was born in Surinam. Sex One of 50 females (2%) vs eight of 119 (6.7%) males developed DM (P = 0.45, Fisher’s exact test). Discussion In this prospective cohort study, we found the incidence of DM, especially type 1, during or shortly after treatment with Peg-IFN-a/Riba for chronic HCV infection to be higher than reported previously. Most patients who developed DM were identified through routine assessment of blood glucose levels, as DMrelated complaints were absent or mistaken for PegIFN-a/Riba-related side effects. So far, 31 cases of de novo type 1 DM during or after treatment of chronic HCV infection with IFN-a, IFNa/Riba or Peg-IFN-a/Riba have been described (3, 5–10). Through screening, we identified five certain Liver International (2007) c 2007 Blackwell Munksgaard 2007 The Authors. Journal compilation �

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Whether pegylation of IFN-a has an effect is uncertain. Three of the 31 previously described patients and all five patients in our study who developed type 1 DM during IFN-a treatment were treated with PegIFN-a (5, 6). Our study has several limitations: (i) we only assessed type 1 DM-associated autoantibodies in patients who developed DM and (ii) we did not use a control group such as chronic hepatitis B patients undergoing IFN-a treatment, or untreated chronic HCV patients. However, our study, prompted by two index patients who developed DM during treatment for chronic HCV infection, was based on a clinical – ad hoc – adjustment of two treatment protocols to monitor development of DM. Interestingly, the two index cases in our study (patients 1 and 2), who both required insulin at the time of diagnosis, had developed type 2 DM. Five of the seven patients who were subsequently identified had developed type 1 DM. Given the low positive and negative predictive values of type 1 DM-associated autoantibodies (�50%) (3), and the similar clinical presentation of both types of DM, we chose to monitor glucose levels rather than rely on autoantibodies. No recommendations have been made in HCV treatment guidelines on glucose assessment during therapy (33). Considering (i) the high incidence of types 1 and 2 DM in our study, (ii) the atypical clinical picture of DM during Peg-IFN-a/Riba therapy, (iii) the availability of an easy method to identify patients who develop DM and (iv) the importance of the treatment of diabetes, we suggest that routine glucose assessment at all outpatient visits for all HCV patients before, during and shortly after PegIFN-a/Riba treatment should be incorporated into the treatment guidelines, similar to the existing recommendation to screen for autoimmune thyroid disease. In conclusion, this is the first prospective study on the development of DM in chronic HCV patients during treatment with Peg-IFN-a/Riba. We have shown that the incidence of DM, especially type 1, during treatment with Peg-IFN-a/Riba for chronic HCV infection is markedly higher than reported previously. DM-related complaints are frequently absent or mistaken for Peg-IFN-a/Riba-related side effects. Routine assessment of random blood glucose is an easy method to identify patients who develop DM during Peg-IFN-a/Riba treatment. These results support routine assessment of blood glucose levels at all outpatient visits before, during and in the first month after Peg-IFN-a/Riba treatment to detect DM without delay.

expression of IFN-a by pancreatic islet cells in transgenic mice induces inflammation (22, 24), autoreactive T cells (22, 25) and precedes DM (22, 24, 26). Enhanced expression of IFN-a by pancreatic islet cells has also been demonstrated in patients with type 1 DM (27, 28). Riba and amantadine are not associated with the development of DM. Certain HLA haplotypes (DR3, DQ2, DR4 and DQ8) are associated with increased susceptibility to type 1 DM (29). In the analysis by Fabris et al. (3), HLA haplotype was determined in 13 patients with chronic HCV infection who developed type 1 DM during IFN-a or IFN-a/Riba therapy; all 13 patients had an HLA haplotype associated with increased susceptibility to type 1 DM. In our study, four of the nine patients had a type 1 DM-associated HLA haplotype, of whom three patients also tested positive for one or more autoantibodies (Table 2). We classified one patient as indeterminate owing to the absence of autoantibodies. Taken together, these results suggest that certain HLA haplotypes may predispose to the development of type 1 DM during Peg-IFN-a/Riba therapy in chronic HCV patients. In the patients who seroconverted, the absence of concomitant anti-TPO antibodies argues against induction of a polyglandular autoimmune syndrome by IFN-a. In addition, we described three cases of de novo type 2 DM during treatment of chronic HCV infection with Peg-IFN-a/Riba. The prevalence of type 2 DM is high among chronic HCV patients, especially in patients with advanced fibrosis or cirrhosis (12, 30). The pathogenesis is unclear, but insulin resistance and the development of DM may be mediated by pro-inflammatory cytokines such as IL-6 and TNF-a (31, 32). Four of the nine patients who developed DM were treated with triple antiviral therapy with high doses of IFN-a during the first 6 weeks (Table 1, patients 2, 4, 5, 6); one of these patients had been treated with IFN-a previously. The incidence of DM was similar in the standard treatment group (five of 98 patients, 5.1%) compared with the triple treatment group (four of 91 patients, 4.4%). The prevalence of DM before treatment with triple therapy was higher in previous IFN-a non-responders (six of 46 patients) than in treatmentnaı¨ve patients (three of 54) but this was not significant (P = 0.29, Fisher’s exact test). The prevalence of DM before treatment with standard therapy was similar in previous IFN-a non-responders (two of 17 patients) compared with treatment-naı¨ve patients (seven of 90, P = 0.63, Fisher’s exact test). Taken together, these findings suggest that the development of DM is not associated with higher doses of IFN-a or multiple courses of treatment.

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Acknowledgements We are grateful to Marcel Beld (Section of Clinical Virology, Department of Medical Microbiology, Academic Medical Centre, Amsterdam, the Netherlands) for providing access to stored plasma samples. Preliminary results of these studies were presented at the 41st annual meeting of the European Association for the Study of the Liver, Vienna, Austria, 26–30 April 2006 and at the 42nd annual meeting of the European Association for the Study of Diabetes, Copenhagen/Malm¨o, 14–17 September 2006. Conflict of interest statement: Dr H. W. Reesink is a consultant for Schering-Plough and has received grants from Hoffmann-La Roche and Schering-Plough. The other authors declare that they have no conflict of interest.

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Neopterin and ALT as markers of inflammation in chronic hepatitis C patients during administration of the HCV NS3•4A protease inhibitor telaprevir (VX-950) and/or peginterferon alfa 2a Huub C Gelderblom,1 Stefan Zeuzem, 3 Christine J Weegink,1 Nicole Forestier, 3 Lindsay McNair,4 Susan Purdy,4 Marcel GW Dijkgraaf,2 Peter LM Jansen,1 Henk W Reesink1

1 AMC

Liver Center, Department of Gastroenterology and Hepatology, and 2Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 3Saarland University Hospital, Homburg/Saar, Germany, 4Vertex Pharmaceuticals Incorporated, Cambridge, MA, USA

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Chapter 7

Abstract Background Neopterin is a marker of monocyte/macrophage activity. ALT is a marker of hepatocyte injury. We determined changes in neopterin and ALT levels, as markers of inflammation, in 2 ancillary studies during two phase 1b trials of Hepatitis C virus (HCV) NS3•4A protease inhibitor telaprevir (VX-950), with or without peginterferon alfa 2a (Peg-IFN). Methods 54 chronic hepatitis C patients (genotype 1) received placebo or telaprevir, with or without Peg-IFN, for 14 days in 2 multiple-dose studies. Results During administration of telaprevir, every patient demonstrated a >2-log decrease in HCV RNA. Mean neopterin and ALT levels decreased in all 4 telaprevir alone groups. In contrast, mean neopterin levels increased and ALT levels decreased in the Peg-IFN plus telaprevir and Peg-IFN plus placebo groups. Conclusion These data suggest that treatment of chronic hepatitis C patients with an HCV NS3•4A protease inhibitor ameliorates inflammation. The increase in neopterin levels and decrease in ALT levels during administration of Peg-IFN with or without telaprevir is in accordance with earlier observations that IFN reduces hepatocyte injury but increases monocyte/macrophage activity. The IFN-mediated immunomodulatory effects appear to remain intact when IFN is combined with telaprevir.

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Introduction Neopterin (6-d-erythro-trihydroxypropylpteridine) is a pteridine derivative that is produced during the metabolism of guanosine triphosphate (GTP), and is a marker of inflammation. Neopterin is produced primarily by monocytes and macrophages upon activation by interferon (IFN) gamma or IFN alfa [1]. Neopterin levels are elevated in hepatitis C virus (HCV) infection [2-5], hepatitis A infection [6], hepatitis B infection [6], HIV-1 infection [7, 8], severe acute respiratory distress syndrome (SARS) [9], and a variety of other infectious and inflammatory diseases [7]. In advanced HIV-1 infection, neopterin levels are elevated and independently predict disease progression [8]. Neopterin levels decline during highly active antiretroviral therapy (HAART), but levels remain elevated compared with healthy controls [10-13]. Neopterin levels decline during treatment of SARS [9], or as disease activity naturally subsides in hepatitis A and B [6]. Administration of IFN alfa, IFN gamma, or TNF alfa induces an increase in neopterin in healthy volunteers [14-16]. The current treatment of choice for patients with chronic hepatitis C genotype 1 infection consists of administration of pegylated IFN alfa (PegIFN alfa) in combination with the nucleoside analog ribavirin for 48 weeks. In chronic hepatitis C patients, treatment with IFN alfa based regimens induces an increase in neopterin levels, irrespective of treatment outcome [5, 17-19]. Alanine aminotransferase (ALT) is an enzyme that is present in hepatocytes. ALT is released from damaged hepatocytes into the blood after hepatocellular injury or death. The HCV NS3•4A serine protease mediates proteolysis of the HCV polyprotein at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B junctions and is essential for HCV replication [20]. Telaprevir (VX-950) is a highly selective peptidomimetic inhibitor of the HCV NS3•4A serine protease that is currently under development for the treatment of chronic HCV infection. We determined changes in neopterin as a marker of monocyte/macrophage activity, and ALT as a marker of hepatocyte injury, in 2 ancillary studies during two phase 1b trials of telaprevir with or without Peg-IFN alfa 2a, in chronic hepatitis C patients.

Methods These were 2 ancillary studies to 2 placebo controlled phase 1b trials investigating the safety, pharmacokinetics and HCV RNA kinetics during administration of telaprevir (VX-950, Vertex Pharmaceuticals Incorporated, Cambridge, MA, USA), a specific inhibitor of HCV genotype 1 NS3•4A protease with or without PegIFN alfa 2a, in chronic hepatitis C patients [21, 22]. Clinical and demographic characteristics of the patients are summarized in table 1. Briefly, patients ranged in age from 21 to 64 years, 34 were men and 20 were women. All patients

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were infected with HCV genotype 1, with HCV RNA levels ≥ 100,000 IU/mL. All patients had compensated liver disease, and were HBsAg and anti-HIV negative. Telaprevir or placebo was administered during 14 days in both studies. In study VX04-950101 (hereafter referred to as the 101 study), patients were allocated to: 450 mg telaprevir q8h (n=10); 750 mg telaprevir q8h (n=8); 1250 mg q12h (n=10); or telaprevir matched placebo (n=6). In study VX05-950-103 (hereafter referred to as the 103 study), patients were allocated to: 750 mg telaprevir q8h (n=8); 750 mg telaprevir q8h plus 180 µg Peg-IFN alfa 2a (PEGASYS®, Hoffman-La Roche, Basel, Switzerland) on days 1 and 8 (n=8); telaprevir matched placebo plus 180 µg Peg-IFN alfa 2a on days 1 and 8 (n=4). Telaprevir dosing started at day 2 in both studies. The first telaprevir dose in the 103 study was a 1250 mg loading dose. Serum neopterin levels were measured by a quantitative competitive ELISA (ELItest® Neopterin, Brahms, Hennigsdorf, Germany), according to the manufacturer’s instructions, at pretreatment, day 8 and day 15. The expected plasma level of neopterin in healthy individuals is between 3.1 and 7.7 nmol/L. The minimum level of detection is 2 nmol/L. ALT levels were assessed by a routine technique at frequent intervals during the study. HCV RNA was assessed at frequent intervals during the study by real-time PCR (COBAS® TaqMan HCV Test with HPS extraction; linear dynamic range 3.0 x 101 to 2.0 x 108 HCV RNA IU/mL; lower limit of detection 10 HCV RNA IU/mL; Roche Diagnostics, Branchburg, NJ, USA), according to the manufacturer’s instructions. Statistical analysis was performed using SPSS version 12.0.2 for Windows (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 4.0b for Macintosh (GraphPad Software, San Diego, CA, USA). Values are given as means ± SD. We used the Wilcoxon signed rank test to compare neopterin, log HCV RNA and ALT levels before and during treatment. A p-value