HCV Research and News: 01/15/11

January 15, 2011

The impact of human immunodeficiency virus on viral hepatitis

Future hepatitis C virus treatment: interferon-sparing combinations

Figure 1. Targets for direct-acting antivirals against the hepatic C virus (HCV).

The DAAs closest to being marketed are the protease inhibitors, boceprevir and telaprevir. Phase 3 global registration studies of both will be completed this year and these protease inhibitors are expected to be the first DAAs to gain regulatory approval as add-on therapy to current SOC PEG-IFN plus ribavirin. The benefits in terms of efficacy will be significant – 48 weeks of boceprevir plus SOC increased the SVR rates in treatment-naïve GT1 patients from 38 to 66%, while 12 weeks of telaprevir plus 24 weeks SOC increased the SVR rates from 43 to 75% (15, 16). Triple therapy may also offer hope in treatment-experienced patients, especially previous responder relapsers and partial responders (17– 19). However, both have specific toxicities (notably anaemia and dysgeusia with boceprevir and anaemia and rash with telaprevir), which increased the rate of treatment withdrawal in the DAA combination arms.

Although triple therapy (addition of either telaprevir or boceprevir to PEG-IFN plus ribavirin) is likely to become the new SOC in late 2011, this will not be suitable for patients either intolerant of or with contraindications to IFN or ribavirin, including patients with decompensated cirrhosis or following solid organ transplantation. Also, the efficacy of this triple therapy will probably be reduced in treatment-experienced patients, especially those who were non-responders to a previous treatment with SOC. Moreover, although telaprevir has similar antiviral activity against HCV GT2, this agent has no effect in patients with HCV GT3 infection (20, 21). All current protease inhibitors and most non-nucleoside polymerase inhibitors in development are active primarily against HCV GT1. PEG-IFN plus ribavirin will remain the SOC for non-GT1 HCV until nucleoside polymerase inhibitors and cyclophyllin inhibitors enter clinical practice.

Finally, the 30–40% of GT1 patients who do not respond to this new triple therapy will have developed resistance to protease inhibitors, which will limit future treatment options.

Therefore, although the addition of a single DAA to PEG-IFN and ribavirin may improve cure and shorten the treatment duration of SOC, this approach will not meet the needs of many difficult-to-treat patient groups.

Towards an all oral regimen

The successful development of an all-oral, IFN-free regimen of multiple DAAs should address this current extensive unmet medical need and could potentially become the standard of care for all patients with chronic HCV infection. The rationale for this approach is based on the current human immunodeficiency virus (HIV) treatment paradigm, in which a combination of different DAA agents, which target different steps of viral replication, has been shown to increase viral suppression as well as delay or prevent the emergence of antiviral resistance. There is a rapidly increasing list of potential candidates for such a combination including NS3 helicase inhibitors, NS3/4A protease inhibitors, cyclophyllin B inhibitors, NS5A inhibitors, NS5B nucleoside inhibitors and NS5B non-nucleoside inhibitors (see Table 1). The primary criteria for a DAA combination should be to increase antiviral efficacy without increasing toxicity. The combination of DAAs should exhibit in vivo at least additive and preferably synergistic antiviral efficacy [i.e. rather than interference as observed with telbivudine plus lamivudine in patients with hepatitis B virus (HBV) infection]. The combination should lack cross resistance and should prevent virological breakthrough from the emergence of resistance mutants to either or both DAAs. Finally, the combination should lack direct drug interactions and overlapping toxicities.

The first in vitro study of combination DAAs was performed in the replicon model (22). The addition of RG7128, a nucleoside polymerase inhibitor, to RG7227, a protease inhibitor, provided additive viral suppression and completely prevented the development of phenotypic resistance to the protease inhibitor. Similar in vitro effects have been demonstrated for other DAA combinations, including a protease inhibitor plus a non-nucleoside polymerase inhibitor and also the combination of two nucleoside polymerase inhibitors (23, 24). The in vivo efficacy of combining DAAs was first reported in chimpanzees treated with a combination of Merck NS3/4A protease inhibitor MK-7009 and non-nucleoside inhibitor MK-608 for 7 days, whereby both had rapid and sustained viral suppression and one animal eradicated the HCV infection (25). The first study of combination DAAs in patients was the proof-of-concept INFORM-1 study completed last year (26). In this randomised, placebo-controlled double-blind trial, 87 patients with HCV GT1 infection were randomised to receive up to 13 days of either oral combination therapy with RG7128, a nucleoside polymerase inhibitor, and RG7227/danaprevir, an NS3/4A protease inhibitor or with matched placebos. Both agents had already been administered to patients for 12 weeks in combination with SOC. Direct drug interactions between RG7128 and danaprevir were considered very unlikely, because of the different mechanisms of action and routes of elimination and the lack of overlapping toxicities identified in any of the preclinical or human clinical studies. This combination achieved profound antiviral suppression, greater than the additive effects of either treatment alone. The median reduction in HCV RNA from baseline was 5 logs, falling below the level of detection in 88% in the cohort who received the highest dose of both RG7128 (1000 mg b.i.d.) and danaprevir (900 mmg b.i.d.). No evidence of the emergence of resistance to either compound was observed during this study. This combination was well tolerated, with no serious adverse events, treatment-related dose modifications, discontinuations or study withdrawals. An important observation was that antiviral efficacy was similar in treatment-naïve and treatment-experienced patients including non-responders. Because the total duration of therapy was only 13 days, all patients rolled over into SOC. The rates of RVR, EVR and ETR were markedly increased by the 2 weeks of pretreatment. In the final cohort of patients who received the highest dose of RG7227 and RG7128, 100% achieved ETR after 24 weeks SOC. Although SVR results are still pending, the benefit of pretreatment with combination DAA on subsequent responses to SOC suggests that the strategy of combination of DAA lead-in before starting SOC could be an alternative strategy to IFN-free DAA therapy.

However, the primary goal of combination DAA therapy in HCV infection will be to provide a safe and effective substitute for IFN regimens in all treatment-naïve and -experienced patients.

Although the new treatment paradigm for HCV is based on HIV, the goals are very different. In HIV infection, a cure is not achievable because it is impossible to eradicate infection from lymphocytes and macrophage reservoirs and from nuclear integration. Therefore, lifelong combination DAA therapy is needed to maintain viral suppression and prevent disease progression. In HCV infection, however, replication is entirely cytoplasmic and limited to hepatocytes. Therefore, viral eradication should be possible with short-course combination DAA.

The duration of combination DAA necessary to eradicate HCV infection is unknown. The early viral kinetic profile of single DAA therapy demonstrates a rapid Phase 1 decline in serum HCV RNA levels of 3–4 log in the initial 36 h, attributed to the clearance of free virions from the circulation. The addition of a second DAA appears to increase this initial slope, suggesting at least additive effects of both agents. This is followed by Second Phase decline in serum HCV RNA of 1-1.2 log/week, attributed to the loss of infected hepatocytes (Fig. 2). This rate of viral decline continues until the infection is eradicated, unless DAA-resistant variants emerge. Therefore, based on the estimated total body viral burden of 1011 virions, between 8 and 12 weeks of DAA therapy should be sufficient to eradicate HCV infection in most patients. The addition of a second DAA targeting a different step of HCV replication and lacking cross resistance should both increase the slope of the Phase 1 decline as well as prevent virological breakthrough during Phase 2.

Figure 2. Early viral kinetic profile of combined direct-acting antivirals' therapy. HCV, hepatic C virus.

A finite duration of combination DAA therapy without IFN assumes that viral suppression alone will eradicate HCV, which seems reasonable in the absence of evidence of either viral latency or extrahepatic reservoirs of replication (as seen in HBV). Another important factor for the maintenance of end-of-treatment response may be the indirect effect of combination DAA therapy on host immune responses. In chronic HCV infection, the HCV NS3 protease may directly impair host IFN responses through the inhibition of phosphorylation of IFN regulatory factor-3 (27). Administration of the NS3/4A protease inhibitor should restore this immune responsiveness. Chronic HCV infection is also associated with high levels of IP-1(CXCL-10), a chemokine involved in lymphocyte chemotaxis, reflecting endogenous IFN activation levels. High levels of IP-10 during SOC are correlated with the risk of post-treatment relapse (28). In the INFORM study, the rapid and profound viral suppression achieved with 13 days of combination RG7128 and danaprevir (5 logs within 14 days) correlated with normalisation of IP-10 levels (29). Furthermore, it has been shown that in non-responders, some IFN-stimulated genes were highly expressed; thus, preactivation of the IFN system in patients appears to limit the effect of IFN antiviral therapy (30). Current and future studies will determine whether the IL28 genotype influences sustained response rates in an IFN-free DAA regimen.

Future studies of combination DAA therapy in chronic HCV should include the evaluation of HCV-specific and non-specific immune responses to determine whether immune reconstitution does occur and whether this is a prerequisite for the prevention of relapse after treatment withdrawal.

First studies with all-oral treatments

The shift from triple therapy (single DAA plus SOC) to IFN-free combination DAA studies has been impeded by the reluctance of regulatory authorities to approve the combination of two experimental compounds still in early-phase clinical development. However, such studies should be reasonable as long as safety data are available for each candidate DAA for the duration of the proposed treatment. The rapid emergence of resistant variants during monotherapy with either NS3/4a or NS5b non-nucleoside inhibitors has restricted the duration of DAA monotherapy studies to 3–5 days; thus, longer duration safety data must be obtained from studies of DAA in combination with SOC. An additional requirement before embarking on combination DAA studies in patients should be data from preclinical and human clinical studies for each candidate DAA, confirming lack of cross-resistance, lack of overlapping toxicities and a low likelihood of any drug–drug interactions, which could affect antiviral activity, bioavailability or clearance. INFORM-1 fulfilled all of these requirements but was performed in Australia and New Zealand, because the conservative regulatory environment of the FDA and EMEA did not allow this study to be performed in either Europe or the US at that time. The success of this proof-of-concept and widespread enthusiasm in the HCV field over these results will push the regulatory authorities to modify their previously conservative position.

Following the INFORM proof-of-concept study, seven Phase 2 studies of combination DAA studies are already entering Phase 2 clinical trials in patients with treatment-naïve HCV infection (see Table 2), with many more planned. All studies include an NS3/4a protease inhibitor with or without ritonavir boosting, combined with an agent targeting the HCV polymerase complex – either a non-nucleoside NS5b (n=4), nucleoside NS5b (n=1) or an NS5a inhibitor (n=2). The potential advantage of the nucleoside polymerase inhibitor is the relatively high genetic barrier to resistance and the low prevalence of pre-existing nucleoside polymerase resistance. In a recent survey, no treatment-naïve GT 1 patients had detectable signature S282T mutations, while the prevalence of mutations conferring resistance to either NS3/4a inhibitors or non-nucleoside polymerase inhibitors could be detected in more than 8% of treatment-naïve patients (31). However, it is not clear whether these pre-existing mutations will affect the response to combination DAA therapy. Most of these mutations are associated with decreased replicative fitness and none confer cross-resistance to either NS3/4a inhibitors or non-nucleoside polymerase inhibitors. Unlike INFORM-1, the aim of these studies is curative, with combination DAAs administered for 12–24 weeks. Most study designs incorporate response-guided therapy, with the 2-week rather than the 4-week RVR adopted as a predictor of efficacy (and shortened treatment duration). Although IFN-free, most studies have retained ribavirin as a third oral agent (based on observations of higher relapse rates in the ribavirin-sparing treatment arms in the telaprevir plus SOC studies) (32). However, the impact of ribavirin on the efficacy and tolerability of combination DAA therapy needs to be established.

Summary

The addition of a protease inhibitor to PEG-IFN plus ribavirin will increase the cure rate in both treatment-naïve and treatment-experienced patients and is likely to become the new SOC. However, there will still be a large ‘unmet need’, including patients unable to or unwilling to receive IFN or ribavirin therapy and previous non-responders to SOC. The rationale for combining different DAAs is to increase viral suppression and prevent or delay the emergence of antiviral resistance. The ultimate goal is to develop a short-duration, IFN-free oral combination, with excellent tolerability and efficacy in both treatment-naïve and treatment-experienced patients.

Conflicts of interest

Edward Gane is a member of local or international Advisory Boards and Invited Speaker for GSK, Roche, Pharmasset, Abbott, Novartis and Merck.

References

1 Lavanchy D. The global burden of hepatitis C. Liver Int 2009; 29 (Suppl. 1): 74–81.

2 CDC Compressed Mortality File. Available at http://www.wonder.cdc.gov/mortsql.html/ (accessed 1 June 2008).

3 National Health and Nutrition Examination Surveys 1988–2006. Available at http://www.cdc.gov/nchs/about/major/nhanes (accessed 1 June 2008).

4 Armstrong G, Wasley A, Simard E, et al. Prevalence of HCV infection in the United States. Ann Int Med 2006; 144: 705–14.

5 Wise M, Bialek S, Bell B, et al. Changing trends in HCV-related mortality in USA. Hepatology 2008; 47: 1128–35.

6 Davila J, Morgan R, Shaib Y, et al. HCV and increasing incidence of hepatocellular carcinoma: a population-based study. Gastroenterology 2004; 127: 1372–80.

7 Davis G, Albright J, Cook S. Projecting future complications of chronic hepatitis C in United States. Gastroenterology 2010; 139: 331–8.

8 Manns MP, McHutchison JG, Gordon SC, 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: 958–65.

9 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: 975–82.

10 Ge D, Fellay J, Thompson A, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009; 461: 399–401.

11 Suppiah V, Moldovan M, Ahlenstiel G, et al. IL28B is associated with response to chronic hepatitis C interferonalpha and ribavirin therapy. Nat Genet 2009; 41: 1100–4.

12 Tanaka Y, Nishida N, Sugiyama M, et al. Genome-wide association of IL28B with response to pegylated interferonalpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009; 41: 1105–9.

13 Thompson A, Muir A, Sulkowski M, et al. Interleukin-28B polymorphism improves viral kinetics and is the strongest pretreatment predictor of sustained virologic response in genotype 1 hepatitis C virus. Gastroenterology 2010; 139: 120–29.

14 Fellay J, Thompson AJ, Ge D, et al. ITPA gene variants protect against anaemia in patients treated for chronic hepatitis C. Nature 2010; 464: 405–8.

15 Jacobson I, McHutchison J, Dusheiko G, et al. Telaprevir in combination with peginterferon and ribavirin in genotype 1 HCV treatment-naïve patients: final results of phase 3 ADVANCE study. Hepatology 2010; 52: 427A.

16 Poordad F, McCone J, Bacon B, et al. Boceprevir (BOC) combined with Peginterferon alfa-2b/ribavirin (P/R) for Treatment-naïve patients with hepatitis C Virus (HCV) genotype (G) 1: SPRINT-2 final Results. Hepatology 2010; 52: 402A.

17 McHutchison JG, Manns MP, Muir AJ, et al. Telaprevir for previously treated chronic HCV infection. N Engl J Med 2010; 362: 1292–303.

18 Berg T, McHutchison N. Adda, SVR with telaprevir, peginterferon alfa-2a and ribavirin in HCV patients with well-characterized prior null response, partial response, viral breakthrough or relapse after PR: rollover study 107. J Hepatol 2010; 52: S2.

19 Bruce R, Bacon B, Gordon S, et al. HCV RESPOND-2 final results: high sustained virologic response among Genotype 1 previous non-responders and relapsers to peginterferon/ribavirin when re-treated with Boceprevir plus pegintron (peginterferon alfa- 2b)/ribavirin. Hepatology 2010; 52: 430A.

20 Asselah T, Benhamou Y, Marcellin P. Protease and polymerase inhibitors for the treatment of hepatitis C. Liver Int 2009; 29 (Suppl. 1): 57–67.

21 Foster G, Hézode C, Bronowicki JP, et al. Activity of telaprevir alone or in combination with peginterferon alfa-2a and ribavirin in treatment-naïve, genotype 2 and 3, hepatitis C patients: Final Results of Study C209. J Hepatol 2010; 52: S27.

22 Tan H, Rajyaguru S, Wu T, et al. Combination of NS3/4a protease inhibitor ITMN-191 (R7227) with the active moiety of the NS5b inhibitors R1626 or R7128 enhances viral clearance and reduces emergence of drug-resistant variants. Hepatology 2008; 48: 1153A.

23 Koev G, Dekhtyar T, Han L, et al. Antiviral interactions of an HCV polymerase inhibitor with an HCV protease inhibitor or interferon in vitro. Antiviral Res 2007; 73: 78–83.

24 Zennou V, Lam A, Keilman M, et al. Combination of two complementary mucleotide analogues PSI7977 and PSI938 effectively clears wild type and NS5b-S282T HCV replicons. J Hepatol 2010; 52: S400.

25 Olsen DB, Carroll SS, Handt L, et al. HCV antiviral activity andresistance analysis in chronically infected chimpanzees treated with NS3/4A protease and NS513 polymerase inhibitors. J Hepatol 2007; 46: S298.

26 Gane E, Roberts S, Stedman C, et al. Interferon-free oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and protease inhibitor (danoprevir/RG7227) for the treatment of chronic hepatitis C genotype 1 infection: results of the INFORM-1 trial. Lancet 2010; 376: 1467–75.

27 Foy E, Li K, Wang C, et al. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 2003; 300: 1145–8.

28 Lagging M, Romero AI, Westin J, et al. IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection. Hepatology 2006; 44: 1617–25.

29 Shulman N, Smith P, Ipe D, et al. Reduction of IP10, a biomarker of endogenous IFN, during therapy with two direct-acting antiviral agents (DAA-combo) in INFORM−1 suggests that reducing HCV RNA with IFN−free therapy may enhance response to Interferons. 16th International Symposium on HCV and Related Viruses, October 3–7 2009, Nice, France.

30 Asselah T, Bieche I, Narguet S, et al. Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C. Gut 2008; 57: 516–24.

31 Kuntzen T, Timm J, Berical A, et al. Naturally occurring dominant resistant mutations to HCV protease and polymerase inhibitors in treatment-naive patients. Hepatology 2008; 48: 1769–78.

32 Hézode C, Forestier N, Dusheiko G, et al. Telaprevir and peginterferon with or without ribavirin for chronic HCV infection. N Engl J Med 2009; 360: 183–50.

Source

The role of genetic markers in hepatitis C virus therapy: a major step for individualized care

Liver International
Special Issue: Proceedings of the 4th Paris Hepatitis Conference. The publication of this supplement was supported by an unrestricted educational grant from F. Hoffmann-Laroche Ltd.
Volume 31, Issue Supplement s1, pages 29–35, January 2011

John G. McHutchison

Article first published online: 4 JAN 2011
DOI: 10.1111/j.1478-3231.2010.02389.x
© 2011 John Wiley & Sons A/S

Author Information
Gilead Sciences, Foster City, CA, USA
* Correspondence: Correspondence John G. McHutchison, MD, 333 Lakeside Blvd, Foster City, CA, USA Tel: +1 650-522-5302 Fax: +1 650-522-1975 e-mail: john.mchutchison@gilead.com

Keywords
Genetics; HCV; IL28B;treatment response

Abstract

It has been understood for some time that the treatment outcome of hepatitis C virus (HCV) infection is influenced by host genetic factors. Three independent genome-wide association studies have recently identified that a genetic variation in the IL28B gene [interferon-λ3 (IFN-λ3)] determines the outcome of IFN-α-based therapy in patients with genotype 1 chronic hepatitis C infection. This genetic polymorphism is also strongly associated with a higher likelihood of spontaneous clearance following acute hepatitis C infection. These results confirm the importance of specific host genetic markers in predicting outcome and treatment response. They also provide the framework and potential for a clinically relevant and meaningful pharmacogenomic approach to personalizing anti-HCV treatment.

Host genetics have long been suspected to play a role in determining response to interferon-α (IFN-α)-based therapy for chronic hepatitis C virus (HCV). The current standard of care (SOC) therapy is pegylated IFN (PEG-IFN) and ribavirin (RBV) combination therapy. The sustained virological response (SVR) rate is approximately 40–45% in patients with genotype 1 HCV (1–4). Predictors of treatment response are poorly defined for any individual patient and do not allow personalization of therapy; both host (e.g. gender, age and liver fibrosis) and viral factors (e.g. genotype and viral levels) are recognized to be important, but do not adequately explain the variation in response that is observed. African-American ancestry is strongly associated with a poor response, and the SVR rate is half that observed in Caucasians (5, 6). These ethnic differences cannot be explained by the severity of disease, compliance, viral kinetics or more basic immunological or other parameters. Hispanic ethnicity has also been associated recently with lower response rates compared with Caucasians (7). In contrast, Asian populations appear to have the highest response rates (8, 9). Taken together, these ethnic or racial differences in the treatment response rates strongly suggest a genetic basis.

Technology that can screen the entire human genome for variants associated with human diseases has recently become widely available. Although they are not yet ready for large-scale clinical use, these tools could potentially define polymorphic sites that might translate into clinical diagnostics, as well as provide an insight into systems biology. Three published genome-wide association studies (GWAS) have been published recently that link a genetic variation in the IL28B gene region to response to PEG-INF-α plus RBV in patients with genotype 1 chronic HCV (10–12). Here, we explain these technologies and principles and summarize the publicly available data as well as discuss future directions and potential applications.

Genetic association studies

A case/control genetic association analysis involves a comparison of the frequency of a particular allele(s)/genotype(s) in a sample of affected patients compared with a control group of unaffected individuals. The existence of stretches of linkage disequilibrium (LD) in the human genome and the wealth of data that have been generated by the HapMap Project (http://www.hapmap.org) make it possible to assess common genetic variation (i.e. variants that are present in at least 5% of a given population) on a genome-wide scale using ‘tag’ SNPs. GWAS are therefore a hypothesis-free method for systematically testing the association between all common variants in the human genome and any polymorphic trait, notably clinical phenotypes (disease, drug response, drug toxicity and others). An important note about the use of tag SNPs is that GWAS are only able to identify a region within the genome that is associated with a phenotype. They do not usually identify the polymorphism itself that is directly responsible for the effect (the causal or the functional variant), although this may occur by chance. Follow-up of a discovery involves fine sequencing of the associated region, as well as more basic biological studies. As GWAS involve hundreds of thousands of association tests, the threshold for statistical significance must be stringent and correction for multiple testing must be performed (e.g. Bonferroni correction, Table 1). This requires large, well-characterized cohorts to have enough power to detect real associations. Even then, unless the P-value of the association is clearly beyond the genome-wide significance, replication of the study in an independent cohort should be undertaken. For further discussion, readers are directed to a recent overview of the interpretation of GWAS (13).

Before genome-wide approaches became available, most studies used a candidate gene approach, in which polymorphisms affecting plausible biological pathways are chosen to be tested for association with a particular phenotype. A limited number of SNPs are selected, minimizing cost. The threshold for statistical significance is lower, although correction for multiple testing should still be performed. Unfortunately, such statistical vigilance has not always been the case. Another limitation of these targeted studies is the difficulty of correcting for population stratification: invalid associations can be created by systematic differences due to shared ancestry between subgroups of study subjects. Panels of ancestry markers, or informative markers extracted from genome-wide genotyping data, can be used to address this issue. Consequently, many candidate gene findings have not been replicated in follow-up studies, and this is also true for candidate gene studies of HCV treatment response (14–26). All have been limited by a small sample size and borderline significance in the setting of multiple testing. Results have been inconsistent and reproducibility limited.

Genome-wide analyses of genotype 1 hepatitis C virus treatment response

Genetic variation in the IL28B gene region has recently been shown to be strongly associated with viral clearance following treatment with PEG-IFN and RBV in patients with chronic genotype 1 HCV infection.

Ge and colleagues: the first genome-wide association analysis to investigate genetic predictors of treatment response used patients from the IDEAL study, a large randomized-controlled trial that confirmed the similar efficacy of the two commercially available PEG-IFN preparations (PEG-IFN α-2b vs PEG-IFN α-2a) in North American patients with chronic genotype 1 HCV infection (1). One thousand six hundred and four patients consented to genetic testing. An additional 67 patients were enrolled from a second randomized clinical trial that compared the efficacy of PEG-IFN-2b/RBV in Caucasians vs African Americans (5). This study therefore had the advantage of an extremely well-characterized patient phenotype, defined within the context of clinical trials. Genome-wide analysis was performed using the Illumina Human 10-quad BeadChip (610 000 SNPs on chip/565 759 passed QC and were used for the association analysis). The primary analysis for treatment outcome compared SVR with true biological non-response in 1137 patients, in three independent ethnic groups – Caucasians, African Americans and Hispanics, defined by genetic ancestry (336 patients who did not attain SVR were excluded on the basis of <80% compliance to PEG-IFN or RBV; a further 198 were excluded for technical reasons).

Seven SNPs on chromosome 19 in the region of the IL28B gene (coding for IFN-λ3) met the threshold for Bonferroni-corrected significance in the GWAS, adjusting for other clinical factors known to affect the treatment response, including baseline HCV RNA level, hepatic fibrosis stage and steatosis grade, age, body mass index, baseline ALT level, blood sugar levels and weight-adjusted RBV dose (1). The P-value for association of the top discovery SNP, rs12979860, located 3 kb upstream of the IL28B gene, was 1.06 × 10−25 in Caucasians (P=1.37 × 10−28, combined across the three ethnic populations). The six other SNPs displayed different degrees of LD with rs12979860 (Table 1), and their effects were largely explained by rs12979860. Another two SNPs, not present on the genome-wide chip, were shown to be highly associated with rs12979860 by fine sequencing studies. These were both putative functional variants, rs28416813 lying in the promoter region 37 base pairs upstream of the IL28B start codon and rs8103142 a non-synonymous polymorphism in exon two (Lys70Arg). Given the high degree of correlation, it was not possible to resolve which of these three polymorphisms might be solely responsible for the association signal. A validation cohort was not required, given the strength of the association signal (exceeding genome-wide significance by a factor of 10−20).

In this compliant cohort, the IL28B polymorphism was associated with a two-fold increase in the SVR rate in all ethnic groups. In a multivariable logistic regression model, ‘IL28B-type’ was a stronger independent predictor of SVR than viral load (±600 000 IU/ml), hepatic fibrosis stage or ethnicity [adjusted odds ratio (OR) for SVR in Caucasians=7.3 (5.1–10.4)]. Furthermore, the favourable CC genotype was more common in Caucasians than African Americans, and it was estimated that this difference in genotype frequency was responsible for approximately half of the recognized discrepancy in the treatment response rates between the two populations. Random sampling of a healthy population then identified the rs12979860 CC genotype to be most common in Asian patients, suggesting that this genetic variant may also contribute to the high response rates that have been reported in this group (8). Finally, the frequency of the C allele in the study population was found be lower than that of an ethnically matched population of unknown HCV status, suggesting that the genetic variation in the IL28B gene region may be relevant to the natural clearance of HCV. This last point was subsequently confirmed in a separate study that examined the association between rs12979860 and the spontaneous clearance of HCV (28).

Tanaka and colleagues: a second GWAS, conducted in a Japanese population, also identified an important role for the IL28B gene region in HCV treatment response (11). A two-stage design was used with a GWAS discovery stage in 154 patients, using Affymetrix SNP 6.0 genome-wide SNP typing array (900 000 SNPs/621 220 SNPs passed QC), followed by a subsequent validation stage in 172 patients. The association analysis used null virological response as a phenotype (NVR, defined as a <2log10 IU/ml reduction in serum HCV RNA by week 12 of therapy and detectable HCV RNA at week 24) rather than SVR. Adherence >80% during the first 12 weeks was required for inclusion. Two SNPs in the IL28B gene region showed strong associations in the GWAS phase I, rs8099917 and rs12980275 (P=3.11 × 10−15 and P=1.93 × 10−13 respectively). These two SNPs were validated in the replication phase [combined P=2.68 × 10−32, OR 27.1; (14.6–50.3) and P=2.84 × 10−27, OR 17.7 (10.0–31.3) respectively]. Six other SNPs were associated beyond the genome-wide threshold, found either on the basis of LD and haplotype structure (HapMap release 23a, Japanese ancestry), or by fine resequencing studies. All were in the IL28B gene region, in strong LD, and it was concluded that the association signal was driven by one of the identified SNPs, although again it was not possible to conclude which, if any, was the causal variant. Logistic regression modelling confirmed a strong independent role for SNP rs8099917 in predicting NVR. In a secondary analysis, Tanaka and colleagues also observed that SNPs rs8099917 and rs12980275 were significantly associated with SVR vs no SVR [unadjusted OR 12.1 (6.5–22.4), P=1.18 × 10−18 and 8.8 (5.1–15.4), P=1.17 × 10−16 respectively]. This study therefore confirmed that genetic variation in the IL28B gene region was associated with viral clearance, as well as null response (an expected finding, given the recognized role of NVR in strongly predicting overall non-response).

Suppiah and colleagues: a third GWAS was conducted in genotype 1 HCV-infected patients of European ancestry (Australian/western Europe) (12). The investigators tested for polymorphisms associated with SVR. Adherence to therapy was not defined. A two-stage approach was adopted. The initial GWAS was performed in an Australian cohort (n=293) using the Illumina Infinium HumanHapMap300 or the CNV 370-Quad genotyping BeadChip (311 159 SNPs used for the association analysis). One hundred and seventy-two SNPs were taken forward to the second stage, based on a P-value for association <10−5, or P<10−3 and immune-regulatory/antiviral function. The replication cohort included patients from the UK, Germany, Italy and Australia. The polymorphism rs8099917 exceeded the threshold for genome-wide significance in the GWAS stage and replication phases [OR 1.98 (1.57–2.52), P=9.25 × 10−9]. Seven other SNPs in the IL28B gene region were noted to be associated with SVR in a subsequent haplotype analysis using tagging SNPs identified within the distinct IL28B haplotype block (Table 1). Although only one of these seven SNPs reached genome-wide significance, it was notable that six of the seven were common to the Ge/Tanaka papers (Table 1). Several other SNPs were described as being of interest in this study, on the basis of suggestive but not genome-wide significance (P<10−4)±biological plausibility. These included SNPs related to the genes IL21R and CASP1. However, given the absence of these association signals in the other two studies, it is reasonable to conclude that they probably represent false-positive associations.

Summary

Three independent original GWAS have now identified variants within the IL28B gene region that are strongly associated with response to PEG-IFN plus RBV combination therapy in patients chronically infected with genotype 1 HCV. Furthermore, a strong association of rs12979860 with both an early virological response and an SVR in IFN-naïve patients treated with PEG-IFN and RBV was also reported. In particular, the association of rs12979860 with virological response appeared to be stronger in the naïve patients' population compared with prior non-responders (27).

There are now more than 10 independent validations of the importance of this genetic variant related to HCV treatment response in different populations of chronically infected individuals (other HCV genotypes, HIV coinfected patients and in the setting of transplantation, among others). Another study has also shown that the SNP rs12979860 is strongly associated with the spontaneous clearance of HCV. Most probably, studies have detected the same genetic signal. The fact that multiple SNPs have been identified, with differing effect sizes, almost certainly reflects the differences between the studies with respect to ethnicity, phenotype definition and SNP genotyping platform, and also suggests the existence of a strong LD in the region. The causal variant(s) have yet to be defined.

Genome-wide analysis of spontaneous clearance of hepatitis C virus

Data supporting a role for IL28B genetic variations in spontaneous clearance of HCV followed rapidly after the treatment response discovery. Thomas and colleagues used a candidate gene approach to investigate the potential role of rs12979860 variation in determining the natural clearance rate following acute HCV, in cohorts of individuals who spontaneously cleared the virus (n=388) or had persistent infection (n=620) (28). Patients with the CT/TT genotypes at rs12979860 were three times less likely to spontaneously clear HCV [clearance rate: CT/TT=28% vs CC=53% vs OR 0.33 (0.25–0.45), P=10−13, overall cohort]. Similar effects were found in individuals of European or African American ethnicity. Nineteen per cent of the cohort was co-infected with HIV, and 10% were HBsAg-positive; neither infection altered the effect of this locus on the outcome of an acute HCV infection. These data were extended by a second group that performed a GWAS for spontaneous clearance in a Swiss/German cohort (chronic hepatitis C, n=1015; spontaneous clearance, n=347; n=448 were co-infected with HIV). They confirmed that variants of the IL28B gene region are the only common genetic variants associated with the spontaneous clearance of HCV infection, using a genome-wide approach [top association SNP=rs8099917, OR 2.31 (1.74–3.06), P=6.7 × 10−9] (29).

Mechanism

The mechanism by which genetic variation in the IL28B gene region influences the treatment response is unknown. None of the identified variants has an obvious effect on gene function (e.g. non-sense mutation). But even without a dramatic impact on protein production or structure, polymorphisms may exert a functional influence through gene expression, mRNA splicing, protein stability/half-life or, for cytokines like IFNs, receptor binding and activation. The data available to date are limited to studies of gene expression, with conflicting results. Ge and colleagues did not identify any association between the polymorphism rs12980275 and IL28B RNA expression in peripheral blood mononuclear cells from 80 HCV-negative individuals. In contrast, Suppiah and colleagues observed a weak association between rs8099917 and IL28B/IL28A expression in 49 healthy volunteers, where a higher expression was noted in the responder genotype (P=0.044); this effect was largely driven by lower expression in three patients homozygous for the non-responder genotype. Tanaka and colleagues were the only group to examine expression in HCV-infected patients. In a small cohort (n=20), higher expression levels of IL28B mRNA in patients with the responder genotype were observed. Further studies are required. Finally, although the association signals in all three studies were mainly due to IL28B gene variants, an effect from the nearby IL28A gene cannot be excluded.

We have to recall that in non-responders, some IFN-stimulated genes were highly expressed before treatment; thus, pre-activation of the IFN system in patients appears to limit the effect of IFN antiviral therapy (30, 31). Further ongoing studies evaluating the inter-relationships between IL28B polymorphisms and intrahepatic gene expression (in particular, the IFN pathway and immune response) may also shed some additional light.

Nevertheless, the finding that polymorphisms in genes of the IFN-λ family, also known as type III IFNs, are important for both spontaneous and treatment-related clearance of genotype 1 HCV has significant biological plausibility. Three members of the type III IFN family have been described, IFN-λ1/2/3 (IL29, IL28A, IL28B). IFN-λs appear to be ubiquitously expressed in response to the same viral stimuli that induce type 1 IFN (TLR ligands, double-stranded RNA) (34, 35). The IFN-LR is made up of two subunits – IFN-λ receptor 1 (IFN-LR1=IL28RA) and interleukin 10 receptor 2 (IL10R2=IL10RB); all three members of the IFN-λ signal via this receptor. The IFN-LR is believed to share a common downstream signalling pathway with the type 1 IFN receptor, activating the complex IFN-stimulated gene factor 3 (ISGF3=STAT-1/2·IRF-9), which binds to the IFN-stimulated response element to induce the transcription of IFN-stimulated genes. The major distinction recognized between type 1 and type III IFN appears to be the tissue distribution of the receptor. Whereas the type 1 IFN receptor is expressed by all cells, the type III IFN receptor (specifically, the IFN-LR chain) is more restricted. It is expressed on epithelial cells, including hepatocytes, but not on haemopoietic cells that therefore have an impaired response to IFN-λ. IFN-λs have been shown to inhibit HCV in cell culture models: it was less potent than IFN-α, but also at least additive with IFN-α. More recently, a subcutaneous preparation of IFN-λ1 has shown antiviral activity in HCV patients. Importantly, it remains unclear how IFN-α and IFN-λ interact in vivo, and the relevance of genetic variation in the IL28B gene region to IFN-λ1 therapy is not known.

Clinical applicability

Genotyping of one of the IL28B polymorphisms has great potential as a clinical diagnostic tool to aid in assessing the probability of response to current therapy. To test for a single polymorphism is not technically difficult or time consuming (the PCR-based assay uses the same technology as the current test used for haemochromatosis). A licensed assay has been available in the USA since July 2010. The SNP rs12979860 appears to be the most suitable simple genetic predictor of treatment outcome, because it has the strongest association signal in the largest cohort studied to date, thus explaining the lower observed response rates in AA patients. Knowledge of host IL28B type could help in the clinical decision-making process. The use of this genetic predictor might allow genotype 1 HCV-infected patients to be divided into 2 groups in the future: (i) those with the good response genotype, who might be managed in the same way as patients with genotypes 2/3 HCV, and (ii) those with the less favourable genetic response genotypes, in whom the decision about starting therapy must be evaluated in light of the expected availability of direct antivirals in the near future.

Future studies

A number of important clinical and more translational questions now must be addressed. These include whether IL28B type can be used to personalize the duration of therapy with SOC. That is, will rs12979860 CC ‘good responder’ patients only require 24 weeks of PEG-IFN and RBV therapy? Will direct antivirals overcome or attenuate the IL28B-type effect? It is likely that they will, at least to some degree, with fixed-duration regimens. However, perhaps IL28B type will allow a shortened duration of therapy, minimizing toxicity and drug resistance. Is IL28B type relevant to non-genotype 1 HCV infection and treatment outcomes? And is this relevant for those with HIV/HCV co-infection or for the rapidity and severity of post-transplant HCV recurrence [which might also depend on IL28B type of both the donor and the recipient liver as reported recently (40)]? What is the future for IFN-λ as a therapeutic, and will the IL28B type be relevant? And how will the geographical differences in the favourable IL28B allele frequency relate to treatment response, as well as to newer therapeutic regimens and durations? Is this a marker of IFN response above and beyond HCV infection, and if so, do other diseases with IFN-based regimens (such as hepatitis B, some malignancies, multiple sclerosis and others) interact differentially with IL28B types? And finally, the functional underpinnings of this genetic marker should, once unravelled, provide greater biological insight into treatment response in this disease and potential novel therapeutic approaches.

Should IL28B typing be included in hepatitis C virus clinical trials?

Because of the effect of the IL28B on treatment outcomes and early viral kinetics (Thompson A. J., personal communication), it seems very relevant that this factor, like HCV genotype, race, viral load and ethnicity, be incorporated into clinical trial designs. This is particularly relevant for new small-molecule studies, where an imbalance in IL28B type could be associated with significant numbers of patients, with the favourable IL28B type being randomized unequally into different arms of a particular study. This is especially true in small proof-of-concept studies (with less than 100 patients) and to a lesser degree in studies with approximately 250 subjects (Fig. 1). If this factor is not taken into account in trials of this size, there is an approximately 25–50% chance of a mismatch in terms of the proportion of patients with a favourable IL28B type randomized into each arm of any trial. Because of these discrepancies, the results of these trials could be incorrectly interpreted as favouring one treatment arm or dose of a drug, while in fact it is due to this factor alone.

What important clinical trials should now be performed?

It seems clear that many opportunities now exist to segment our HCV patients into groups of patients who are highly and less likely to respond to PEG-IFN and RBV therapy. Being able to determine the IL28B type before treatment makes it possible to position and perform certain trials:

1 A trial comparing PEG-IFN and RBV for 24 and 48 weeks in IL28B C/C favourable patients. Similar to genotype 2- and 3-infected patients, who only require 24 weeks of therapy, IL28B favourable patients might also only require a shorter duration of therapy. This is particularly relevant to East Asia, where the higher prevalence of the favourable C/C alleles and higher response rates might make a shorter duration of therapy possible for most patients.

2 A trial comparing PEG-IFN and RBV for 48 and 72 weeks (or longer) in IL28B unfavourable patients.

3 Evaluation of shorter triple therapy direct antiviral combination regimens (12 and 24 weeks) in IL28B C/C patients.

Conclusions

In conclusion, the identification of the genetic variation in the IL28B gene region as a predictor of genotype 1 chronic HCV treatment outcome is an exciting discovery. It sheds new light on virus–host interaction, and appears to have immediate clinical use as a diagnostic tool, providing potentially helpful information to both patients and clinicians considering therapy with PEG-IFN plus RBV. It is therefore a step towards a personalized approach to anti-HCV therapy. However, the treatment paradigm for genotype 1 HCV is about to change with the introduction of direct antivirals. The role of the IL28B variation in this setting as well as determining the duration of therapy required in these favourably genetically determined individuals with both current two-drug and future three-drug regimens requires semi-urgent investigation.

Conflicts of interest

John McHutchison is an employee of Gilead. He has received funding for research and acted as a consultant and advisor for Schering Plough and Merck. He is co-inventor of patents relating to the IL 28B and ITPA discoveres.

References

1 McHutchison JG, Lawitz EJ, Shiffman ML, et al. Peginterferon alfa-2b or alfa-2a with ribavirin for treatment of hepatitis C infection. N Engl J Med 2009; 361: 580–93.

2 Manns MP, McHutchison JG, Gordon SC, 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: 958–65.

3 Fried MW. Side effects of therapy of hepatitis C and their management. Hepatology 2002; 36: S237–44.

4 Hadziyannis SJ, Sette H Jr, Morgan TR, 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: 346–55.

5 Muir AJ, Bornstein JD, Killenberg PG. Peginterferon alfa-2b and ribavirin for the treatment of chronic hepatitis C in blacks and non-Hispanic whites. N Engl J Med 2004; 350: 2265–71.

6 Conjeevaram HS, Fried MW, Jeffers LJ, et al. Peginterferon and ribavirin treatment in African American and Caucasian American patients with hepatitis C genotype 1. Gastroenterology 2006; 131: 470–7.

7 Rodriguez-Torres M, Jeffers LJ, Sheikh MY, et al. Peginterferon alfa-2a and ribavirin in Latino and non-Latino whites with hepatitis C. N Engl J Med 2009; 360: 257–67.

8 Liu CH, Liu CJ, Lin CL, et al. Pegylated interferon-alpha-2a plus ribavirin for treatment-naive Asian patients with hepatitis C virus genotype 1 infection: a multicenter, randomized controlled trial. Clin Infect Dis 2008; 47: 1260–9.

9 Dev AT, McCaw R, Sundararajan V, Bowden S, Sievert W. Southeast Asian patients with chronic hepatitis C: the impact of novel genotypes and race on treatment outcome. Hepatology 2002; 36: 1259–65.

10 Ge D, Fellay J, Thompson AJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009; 461: 399–401.

11 Tanaka Y, Nishida N, Sugiyama M, et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009; 41: 1105–9.

12 Suppiah V, Moldovan M, Ahlenstiel G, et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet 2009; 41: 1100–4.

13 Pearson TA, Manolio TA. How to interpret a genome-wide association study. JAMA 2008; 299: 1335–44.

14 Morgan TR, Lambrecht RW, Bonkovsky HL, et al. DNA polymorphisms and response to treatment in patients with chronic hepatitis C: results from the HALT-C trial. J Hepatol 2008; 49: 548–56.

15 Welzel TM, Morgan TR, Bonkovsky HL, et al. Variants in interferon-alpha pathway genes and response to pegylated interferon-alpha2a plus ribavirin for treatment of chronic hepatitis C virus infection in the hepatitis C antiviral long-term treatment against cirrhosis trial. Hepatology 2009; 49: 1847–58.

16 Airoldi A, Zavaglia C, Silini E, et al. Lack of a strong association between HLA class II, tumour necrosis factor and transporter associated with antigen processing gene polymorphisms and virological response to alpha-interferon treatment in patients with chronic hepatitis C. Eur J Immunogenet 2004; 31: 259–65.

17 Constantini PK, Wawrzynowicz-Syczewska M, Clare M, et al. Interleukin-1, interleukin-10 and tumour necrosis factor-alpha gene polymorphisms in hepatitis C virus infection: an investigation of the relationships with spontaneous viral clearance and response to alpha-interferon therapy. Liver 2002; 22: 404–12.

18 Dai CY, Chuang WL, Chang WY, et al. Tumor necrosis factor-alpha promoter polymorphism at position −308 predicts response to combination therapy in hepatitis C virus infection. J Infect Dis 2006; 193: 98–101.

19 Schiemann U, Glas J, Torok P, et al. Response to combination therapy with interferon alfa-2a and ribavirin in chronic hepatitis C according to a TNF-alpha promoter polymorphism. Digestion 2003; 68: 1–4.

20 Vidigal PG, Germer JJ, Zein NN. Polymorphisms in the interleukin-10, tumor necrosis factor-alpha, and transforming growth factor-beta1 genes in chronic hepatitis C patients treated with interferon and ribavirin. J Hepatol 2002; 36: 271–7.

21 Yee LJ, Tang J, Gibson AW, et al. Interleukin 10 polymorphisms as predictors of sustained response in antiviral therapy for chronic hepatitis C infection. Hepatology 2001; 33: 708–12.

22 Yu ML, Dai CY, Chiu CC, et al. Tumor necrosis factor alpha promoter polymorphisms at position −308 in Taiwanese chronic hepatitis C patients treated with interferon-alpha. Antiviral Res 2003; 59: 35–40.

23 Su X, Yee LJ, Im K, et al. Association of single nucleotide polymorphisms in interferon signaling pathway genes and interferon-stimulated genes with the response to interferon therapy for chronic hepatitis C. J Hepatol 2008; 49: 184–91.

24 Hijikata M, Ohta Y, Mishiro S. Identification of a single nucleotide polymorphism in the MxA gene promoter (G/T at nt −88) correlated with the response of hepatitis C patients to interferon. Intervirology 2000; 43: 124–7.

25 Suzuki F, Arase Y, Suzuki Y, et al. Single nucleotide polymorphism of the MxA gene promoter influences the response to interferon monotherapy in patients with hepatitis C viral infection. J Viral Hepat 2004; 11: 271–6.

26 Bonkovsky HL, Naishadham D, Lambrecht RW, et al. Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C. Gastroenterology 2006; 131: 1440–51.

27 Asselah T, Essioux L, Marcellin P, et al. Differential association of a chromosome 19 single nucleotide polymorphism (SNP RS12979869) to outcome (early and sustained virological response (EVR/SVR)) in naïve vs retreated HCV patients treated with IFN-based therapies. Hepatology 2010; 52.

28 Thomas DL, Thio CL, Martin MP, et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 2009; 461: 798–801.

29 Rauch A, Kutalik Z, Descombes P, et al. Genetic variation in IL28B is associated with chronic Hepatitis C and treatment failure – a genome-wide association study. Gastroenterology 2010; 138: 1338–45.

30 Younossi ZM, Baranova A, Afendy A, et al. Early gene expression profiles of patients with chronic hepatitis C treated with pegylated interferon-alfa and ribavirin. Hepatology 2009; 49: 763–74.

31 Asselah T, Bieche I, Narguet S, et al. Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C. Gut 2008; 57: 516–24.

32 Ank N, West H, Paludan SR. IFN-lambda: novel antiviral cytokines. J Interferon Cytokine Res 2006; 26: 373–9.

33 Uze G, Monneron D. IL-28 and IL-29: newcomers to the interferon family. Biochimie 2007; 89: 729–34.

Source