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Genomics and HCV infection: Progression of fibrosis and treatment response

  • Emilie Estrabaud
    Correspondence
    Corresponding authors. Addresses: Faculté de Medecine Xavier Bichat, INSERM U773, 16 rue Henri Huchard, 75018 Paris, France. Tel.: +33 (0) 157277564; fax: +33 (0) 157277531 (E. Estrabaud), Service d’Hépatologie & INSERM U773, Hôpital Beaujon, 100 Bd du Général Leclerc, 92110 Clichy, France. Tel.: +33 (0) 660957890; fax: +33 (0) 147309440 (T. Asselah).
    Affiliations
    INSERM, UMR773, Team «Viral hepatitis», Centre de Recherche Bichat Beaujon, BP 416, F-75018 Paris, France

    Université Denis Diderot Paris 7, site Bichat, BP 416, F-75018 Paris, France

    Service d’Hépatologie, PMAD Hôpital Beaujon, 100 Bd du Général Leclerc, Clichy la Garenne, 92110 Clichy Cedex, France

    Laboratory of Excellence Labex INFLAMEX, PRES Paris Sorbonne Cité, France
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  • Michel Vidaud
    Affiliations
    Service de Biochimie, Hôpital Beaujon, Clichy, France
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  • Patrick Marcellin
    Affiliations
    INSERM, UMR773, Team «Viral hepatitis», Centre de Recherche Bichat Beaujon, BP 416, F-75018 Paris, France

    Université Denis Diderot Paris 7, site Bichat, BP 416, F-75018 Paris, France

    Service d’Hépatologie, PMAD Hôpital Beaujon, 100 Bd du Général Leclerc, Clichy la Garenne, 92110 Clichy Cedex, France

    Laboratory of Excellence Labex INFLAMEX, PRES Paris Sorbonne Cité, France
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  • Tarik Asselah
    Correspondence
    Corresponding authors. Addresses: Faculté de Medecine Xavier Bichat, INSERM U773, 16 rue Henri Huchard, 75018 Paris, France. Tel.: +33 (0) 157277564; fax: +33 (0) 157277531 (E. Estrabaud), Service d’Hépatologie & INSERM U773, Hôpital Beaujon, 100 Bd du Général Leclerc, 92110 Clichy, France. Tel.: +33 (0) 660957890; fax: +33 (0) 147309440 (T. Asselah).
    Affiliations
    INSERM, UMR773, Team «Viral hepatitis», Centre de Recherche Bichat Beaujon, BP 416, F-75018 Paris, France

    Université Denis Diderot Paris 7, site Bichat, BP 416, F-75018 Paris, France

    Service d’Hépatologie, PMAD Hôpital Beaujon, 100 Bd du Général Leclerc, Clichy la Garenne, 92110 Clichy Cedex, France

    Laboratory of Excellence Labex INFLAMEX, PRES Paris Sorbonne Cité, France
    Search for articles by this author
Open AccessPublished:June 01, 2012DOI:https://doi.org/10.1016/j.jhep.2012.05.016

      Summary

      HCV infection is a global health problem that affects 170 million people worldwide. The severity of the disease varies from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma (HCC). Recently, the standard of care for genotype 1 patients has greatly improved with the addition of protease inhibitors (telaprevir or boceprevir) to pegylated interferon (PegIFN) and ribavirin (RBV). The prediction of fibrosis progression and the response to antiviral treatment are two major issues in the management of patients with chronic hepatitis C. Differential expression of mRNAs was first analyzed for both progression of fibrosis and treatment response. Specific polymorphisms, associated with either fibrosis or viral response, were identified thanks to major improvements in genome scanning technologies. Since 2009, several independent genome wide association studies (GWAS) have reported an association between genetic polymorphisms within the IL-28B promoter and both natural and treatment-induced clearance in genotype 1 infected patients. These different studies showed the strong association and the importance of IL-28B polymorphisms in the treatment response. Combining the different genetic factors could improve their predictive value and help identify patients at a high risk of progression of fibrosis as well as those with a lower chance of responding to treatment. The aim of this review was to discuss the genomic factors (mRNAs, miRNAs, and SNPs) and HCV infection with clinical implications for either progression of fibrosis or treatment response. Recent findings on the IL-28B polymorphism and its application in clinical practice will also be discussed.

      Abbreviations:

      CHC (chronic hepatitis C), CRS (cirrhosis risk score), EVR (early virological response), GWAS (genome wide association study), HCV (hepatitis C virus), HSC (hepatic stellate cells), IFN (Interferon), IL (Interleukin), ISG (interferon stimulated gene), NR (non-response), PegIFN (pegylated-interferon), RVR (rapid virological response), SNP (single nucleotide polymorphism), SVR (sustained virological response), IP10 (inducible protein 10)

      Keywords

      Introduction

      Figure thumbnail fx3
      Identifying patients in whom fibrosis will progress rapidly as well as non-responders is crucial for disease prognosis. Some defined markers such as male gender, age, alcohol, and obesity may play a role in accelerating fibrosis and treatment failure. The current standard of care for HCV genotype 1 patients is triple therapy with the addition of protease inhibitors (boceprevir and telaprevir) to pegylated interferon (PegIFN) plus ribavirin (RBV). For genotype non-1 patients, treatment is still PegIFN and RBV. The prediction of non-response to treatment is mandatory to avoid side effects and reduce costs.
      Since the sequencing of the entire human genome in 2001, major advances have been made in genotyping technologies, in particular, the decrease in the cost of genotyping, the large-scale discovery of single nucleotide polymorphisms (SNPs), the development of massive multiplexed genotyping and the efforts of SNPs consortiums and the HapMap project.
      Since 2009, at least 4 different GWAS have investigated genomic markers associated with a response to PegIFN/RBV in patients with chronic HCV (CHC). The same SNPs located in the promoter region of IL-28B were highly associated with natural and treatment-induced viral clearance [
      • Afdhal N.H.
      • McHutchison J.G.
      • Zeuzem S.
      • Mangia A.
      • Pawlotsky J.M.
      • Murray J.S.
      • et al.
      Hepatitis C pharmacogenetics: state of the art in 2010.
      ].
      The aim of this review was to present and discuss the major genomic markers that have been associated with either fibrosis progression or treatment response in patients with CHC. The combination of these different markers may help identify a strong predictive genetic signature for fibrosis progression and treatment response.

      Genome wide association studies, power and limitations

      Figure thumbnail fx4
      GWAS involves identification of statistical associations between a trait or disease and a genetic polymorphism (SNP), throughout the entire genome.
      Linkage disequilibrium can be used to indirectly determine untyped variations around an SNP. The r2 value, which is commonly used to evaluate linkage disequilibriums, measures the correlation between alleles at a nearby genetic variant. If alleles of 2 SNPs are always found on the same chromosome, there is a perfect correlation and r2 = 1. In practice, this means that typing one of these 2 SNPs will provide complete allele information at the 2 sites. If r2 <1 between the 2 alleles, the sample size will need to be proportionally increased to detect a significant association [
      • Karlsen T.H.
      • Melum E.
      • Franke A.
      The utility of genome-wide association studies in hepatology.
      ].
      So far only SNPs with a frequency of more than 5% of the minor allele can be detected effectively by linkage disequilibrium in GWAS. Thus, the HapMAp project does not provide perfect coverage of GWAS so that even in very large GWAS it is impossible to detect rare low frequency genetic variants. However, the technology will probably improve in the future, thus increasing coverage of low frequency variants.
      Different methods of correcting the p values of associations have been described to counteract associations due to chance because of the many statistical tests. The most commonly used approach consists in the adjustment of the p value according to Bonferroni. The threshold of 0.05 is divided by the number of SNPs analyzed, which leads to an adjusted threshold of 10−6 to 10−8, for current available assays. However, several real associations may be lost by deleting all associations with a p value above this threshold. Furthermore, to improve the statistical relevance of GWAS, it is extremely important to replicate significant associations in at least one independent cohort of individuals (trait and control).
      The GWAS technology has greatly improved in the last decade, however, the detection of rare variants would probably improve this approach even more [
      • Karlsen T.H.
      • Melum E.
      • Franke A.
      The utility of genome-wide association studies in hepatology.
      ].

      Fibrosis

      Liver fibrosis is defined as the excess accumulation of extracellular matrix proteins. Fibrogenesis is a complex dynamic process, mediated by necro-inflammation, and activation of stellate cells [
      • Marcellin P.
      • Asselah T.
      • Boyer N.
      Fibrosis and disease progression in hepatitis C.
      ]. Fibrosis progression determines the prognosis and thus the need for treatment. It is therefore crucial to monitor fibrosis in patients with CHC. Several studies have assessed the identification of mRNAs and miRNAs expression during fibrosis (Table 1) and will be discussed in the first part of the review.
      Table 1Variations in mRNAs and miRNAs expression associated with fibrosis progression. (A) Markers identified by candidate gene strategy; (B) markers identified by scanning approach.
      Figure thumbnail fx5

      Variation of mRNAs expression and fibrosis

      A deregulation of gene expression mainly affecting the IFN αβ and γ pathways (STAT1, STAT2, ISGF3G/IRF9, IFI27, G1P3, G1P2, OAS2, MX1, CXCL9, CXCL10, CXCL11, and Viperin) has been reported in patients with CHC and mild fibrosis [
      • Bieche I.
      • Asselah T.
      • Laurendeau I.
      • Vidaud D.
      • Degot C.
      • Paradis V.
      • et al.
      Molecular profiling of early stage liver fibrosis in patients with chronic hepatitis C virus infection.
      ,
      • Helbig K.J.
      • Lau D.T.
      • Semendric L.
      • Harley H.A.
      • Beard M.R.
      Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector.
      ].
      The transition from mild to moderate fibrosis is crucial in the decision to treat. The expression of 240 liver genes has been compared in 62 patients with mild fibrosis (F1) and moderate fibrosis (F2). Twenty-two genes were upregulated in F2 and mainly involved the cytoskeleton, growth factor cytokines, growth factor receptors, extra-cellular matrix remodeling, and the cell junction [
      • Asselah T.
      • Bieche I.
      • Laurendeau I.
      • Paradis V.
      • Vidaud D.
      • Degott C.
      • et al.
      Liver gene expression signature of mild fibrosis in patients with chronic hepatitis C.
      ].
      Liver steatosis is frequent in patients with CHC [
      • Asselah T.
      • Rubbia-Brandt L.
      • Marcellin P.
      • Negro F.
      Steatosis in chronic hepatitis C: why does it really matter?.
      ]. Three genes involved in the inflammatory pathway (SITPEC, SIGIRR, and TOLLIP) have been described as specifically associated with advanced steatosis in CHC [
      • Chiappini F.
      • Barrier A.
      • Saffroy R.
      • Domart M.C.
      • Dagues N.
      • Azoulay D.
      • et al.
      Exploration of global gene expression in human liver steatosis by high-density oligonucleotide microarray.
      ].
      One study investigated changes in liver mRNAs expression in 13 transplanted patients comparing patients in whom fibrosis progressed with those in whom it did not, before and after transplantation [
      • Smith M.W.
      • Walters K.A.
      • Korth M.J.
      • Fitzgibbon M.
      • Proll S.
      • Thompson J.C.
      • et al.
      Gene expression patterns that correlate with hepatitis C and early progression to fibrosis in liver transplant recipients.
      ]. Fifteen of the 31 upregulated genes encoded for markers of myofibroblasts and myofibroblast-like cells. Liver stress injury and fibrosis development can cause an increase in myofibroblasts due to activation of HSCs and their conversion into the contractile phenotype [
      • Smith M.W.
      • Walters K.A.
      • Korth M.J.
      • Fitzgibbon M.
      • Proll S.
      • Thompson J.C.
      • et al.
      Gene expression patterns that correlate with hepatitis C and early progression to fibrosis in liver transplant recipients.
      ]. It is interesting to note that these data suggest that early fibrosis progression may be associated with a reduction in the pools of quiescent HSCs and with an increase in the number of myofibroblast-like cells.

      Variation in micro-RNAs expression and fibrosis

      Micro-RNAs regulate up to 60% of cellular mRNA expression and stability [
      • Bartel D.P.
      MicroRNAs: genomics, biogenesis, mechanism, and function.
      ,
      • Meister G.
      • Landthaler M.
      • Dorsett Y.
      • Tuschl T.
      Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing.
      ,
      • Meister G.
      • Tuschl T.
      Mechanisms of gene silencing by double-stranded RNA.
      ,
      • Napoli C.
      • Lemieux C.
      • Jorgensen R.
      Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans.
      ].
      In one study, mir-21 expression was found to be correlated to both HCV viral load and fibrosis [
      • Marquez R.T.
      • Bandyopadhyay S.
      • Wendlandt E.B.
      • Keck K.
      • Hoffer B.A.
      • Icardi M.S.
      • et al.
      Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.
      ]. Results are conflicting for mir-122 but it is probably only weakly or not associated with viral load [
      • Marquez R.T.
      • Bandyopadhyay S.
      • Wendlandt E.B.
      • Keck K.
      • Hoffer B.A.
      • Icardi M.S.
      • et al.
      Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.
      ,
      • Morita K.
      • Taketomi A.
      • Shirabe K.
      • Umeda K.
      • Kayashima H.
      • Ninomiya M.
      • et al.
      Clinical significance and potential of hepatic microRNA-122 expression in hepatitis C.
      ]. Two studies have reported a correlation between mir-122 expression, liver damage, and stages of fibrosis [
      • Marquez R.T.
      • Bandyopadhyay S.
      • Wendlandt E.B.
      • Keck K.
      • Hoffer B.A.
      • Icardi M.S.
      • et al.
      Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.
      ,
      • Morita K.
      • Taketomi A.
      • Shirabe K.
      • Umeda K.
      • Kayashima H.
      • Ninomiya M.
      • et al.
      Clinical significance and potential of hepatic microRNA-122 expression in hepatitis C.
      ] while another did not find any association with mir-122 expression in the serum of patients with CHC [
      • Bihrer V.
      • Friedrich-Rust M.
      • Kronenberger B.
      • Forestier N.
      • Haupenthal J.
      • Shi Y.
      • et al.
      Serum miR-122 as a Biomarker of Necroinflammation in Patients With Chronic Hepatitis C Virus Infection.
      ].
      A recent study demonstrated that the different members of the mir-29 family were all downregulated in HCV infected patients. Moreover, freshly isolated HSCs expressed a high rate of mir-29, which was rapidly and markedly reduced after HSC activation [
      • Bandyopadhyay S.
      • Friedman R.C.
      • Marquez R.T.
      • Keck K.
      • Kong B.
      • Icardi M.S.
      • et al.
      Hepatitis C virus infection and hepatic stellate cell activation downregulate miR-29: miR-29 overexpression reduces hepatitis C viral abundance in culture.
      ]. Mir-29 targeted various types of collagen and, interestingly, mir-29 inhibition in mice has been shown to upregulate collagen expression in the liver [
      • van Rooij E.
      • Sutherland L.B.
      • Thatcher J.E.
      • DiMaio J.M.
      • Naseem R.H.
      • Marshall W.S.
      • et al.
      Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis.
      ]. Thus, mir-29 downregulation during HSCs activation might play a role in fibrosis by inducing direct accumulation of collagen in the liver.

      Single nucleotide polymorphisms and fibrosis

      Identification of SNPs that modulate gene expression associated with fibrosis and fibrosis progression

      Several studies have tried to indentify SNPs associated with either fibrosis progression or treatment response (Table 2).
      Table 2Identification of SNPs associated with fibrosis and the response to antivirals in patients with chronic hepatitis C.
      Matrix metalloproteinases (MMPs) play an important role in fibrosis progression. MMP-1, MMP-3, and MMP-9 gene polymorphisms have been shown to influence the transcriptional activity of their respective gene promoters. Interestingly, both MMP-1 2G homozygote and MMP-9 C allele were more frequent in HCV patients with cirrhosis than in those without cirrhosis [
      • Okamoto K.
      • Mimura K.
      • Murawaki Y.
      • Yuasa I.
      Association of functional gene polymorphisms of matrix metalloproteinase (MMP)-1, MMP-3 and MMP-9 with the progression of chronic liver disease.
      ].
      Monocyte chemotactic protein 1 (MCP-1) is upregulated in HSCs during CHC. MCP-1 harbors a functional polymorphism located in its promoter. Interestingly, the 2A homozygote genotype in this specific polymorphism was more frequent in patients with mild fibrosis [
      • Muhlbauer M.
      • Bosserhoff A.K.
      • Hartmann A.
      • Thasler W.E.
      • Weiss T.S.
      • Herfarth H.
      • et al.
      A novel MCP-1 gene polymorphism is associated with hepatic MCP-1 expression and severity of HCV-related liver disease.
      ].
      Decreased vitamin D levels and genetic variations in the vitamin D receptor (VDR) gene have been described as an important modulator of multiple diseases, including hepatic disorders [
      • Valdivielso J.M.
      • Fernandez E.
      Vitamin D receptor polymorphisms and diseases.
      ]. VDR genotyping in 251 patients with chronic hepatitis C showed an association between the haplotype rs1544410 C, rs7975232 A, and rs731236 A with fibrosis progression and cirrhosis. Forty-five percent of the [CCA]-haplotype patients had rapid fibrosis progression and 21.1% had cirrhosis [
      • Baur K.
      • Mertens J.C.
      • Schmitt J.
      • Iwata R.
      • Stieger B.
      • Eloranta J.J.
      • et al.
      Combined effect of 25-OH vitamin D plasma levels and genetic Vitamin D Receptor (NR 1I1) variants on fibrosis progression rate in HCV patients.
      ].

      Cirrhosis risk score

      A gene-centric disease association study of 24, 832 putative functional SNPs was performed to assess the association of SNPs with cirrhosis in a cohort of 433 patients with CHC [
      • Huang H.
      • Shiffman M.L.
      • Cheung R.C.
      • Layden T.J.
      • Friedman S.
      • Abar O.T.
      • et al.
      Identification of two gene variants associated with risk of advanced fibrosis in patients with chronic hepatitis C.
      ]. One SNP located in the DEAD box polypeptide 5 was associated with an increased risk of advanced fibrosis while the second SNP, located in the gene encoding carnitine palmitoyltransferase 1A (CPTA1), was associated with a decreased risk of advanced fibrosis [
      • Huang H.
      • Shiffman M.L.
      • Cheung R.C.
      • Layden T.J.
      • Friedman S.
      • Abar O.T.
      • et al.
      Identification of two gene variants associated with risk of advanced fibrosis in patients with chronic hepatitis C.
      ].
      In a second study, the authors confirmed all significant SNPs, and selected 361 markers to build a signature predicting cirrhosis, called the cirrhosis risk score (CRS) [
      • Huang H.
      • Shiffman M.L.
      • Friedman S.
      • Venkatesh R.
      • Bzowej N.
      • Abar O.T.
      • et al.
      A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C.
      ]. Interestingly, DDX5 and CPT1A were not selected for the final 7 SNPs for the CRS. Possible reasons were (i) lower odds ratios and frequencies in the risk group and (ii) decreased robustness and accuracy of these 2 SNPs in multivariate analysis compared to the 7 selected genes. Of the 7 CRS genes, antizyme-inhibitor-1 (AZIN1) and Toll-like receptor 4 (TLR4) have been shown to play a role in fibrosis. A recent study has reported that the association of AZIN1 SNP with the rapid progression of fibrosis, leads to enhanced generation of a novel alternative splice form from AZIN1 that modifies the fibrogenic potential of HSCs [
      • Paris A.J.
      • Snapir Z.
      • Christopherson C.D.
      • Kwok S.Y.
      • Lee U.E.
      • Ghiassi-Nejad Z.
      • et al.
      A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis.
      ]. All 7 SNPs were associated with the risk of cirrhosis with odds ratios ranging from 1.86 to 3.23. However, the AUC of each SNP was <0.6, showing that predictability was moderate when they were used individually [
      • Huang H.
      • Shiffman M.L.
      • Friedman S.
      • Venkatesh R.
      • Bzowej N.
      • Abar O.T.
      • et al.
      A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C.
      ]. The addition of clinical factors to the group of SNPs or each of them individually did not significantly improve the AUC.
      Two major limitations are associated with the use of CRS to identify patients with rapid progression of fibrosis: (i) CRS cut-off values may only distinguish patients with a very high risk of cirrhosis (ii) CRS was identified in Caucasian patients so it may not be applicable to all ethnic groups [
      • Huang H.
      • Shiffman M.L.
      • Friedman S.
      • Venkatesh R.
      • Bzowej N.
      • Abar O.T.
      • et al.
      A 7 gene signature identifies the risk of developing cirrhosis in patients with chronic hepatitis C.
      ].
      In another study, paired liver biopsies from 271 untreated patients with CHC (F0 = 104, F1 = 101, and F2 = 59) were followed-up for at least 60 months. Mean CRS was significantly higher is patients in whom fibrosis progressed, especially in patients with F0 at the initial biopsy [
      • Marcolongo M.
      • Young B.
      • Dal Pero F.
      • Fattovich G.
      • Peraro L.
      • Guido M.
      • et al.
      A seven-gene signature (cirrhosis risk score) predicts liver fibrosis progression in patients with initially mild chronic hepatitis C.
      ].
      CRS remained the only variable associated with fibrosis progression in multivariate analysis, including gender and alcohol intake [
      • Trepo E.
      • Potthoff A.
      • Pradat P.
      • Bakshi R.
      • Young B.
      • Lagier R.
      • et al.
      Role of a cirrhosis risk score for the early prediction of fibrosis progression in hepatitis C patients with minimal liver disease.
      ].
      Genetic studies have reported an association between advanced steatosis and specific SNPs located in genes encoding microsomal triglyceride transfer protein (MTP G493T) [
      • Petit J.M.
      • Masson D.
      • Minello A.
      • Duvillard L.
      • Galland F.
      • Verges B.
      • et al.
      Lack of association between microsomal triglyceride transfer protein gene polymorphism and liver steatosis in HCV-infected patients.
      ,
      • Zampino R.
      • Ingrosso D.
      • Durante-Mangoni E.
      • Capasso R.
      • Tripodi M.F.
      • Restivo L.
      • et al.
      Microsomal triglyceride transfer protein (MTP) −493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 infected patients.
      ,
      • Mirandola S.
      • Osterreicher C.H.
      • Marcolongo M.
      • Datz C.
      • Aigner E.
      • Schlabrakowski A.
      • et al.
      Microsomal triglyceride transfer protein polymorphism (−493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C.
      ], peroxisome proliferator activated alpha (PPAR L162V) [
      • Verdi H.
      • Koytak E.S.
      • Onder O.
      • Ergul A.A.
      • Cinar K.
      • Idilman R.
      • et al.
      Peroxisome proliferator-activated receptor alpha L162V polymorphism in nonalcoholic steatohepatitis and genotype 1 hepatitis C virus-related liver steatosis.
      ], methylenetetrahydrofolate reductase (MTHFR C677T) [
      • Adinolfi L.E.
      • Ingrosso D.
      • Cesaro G.
      • Cimmino A.
      • D’Anto M.
      • Capasso R.
      • et al.
      Hyperhomocysteinemia and the MTHFR C677T polymorphism promote steatosis and fibrosis in chronic hepatitis C patients.
      ,
      • Borgia G.
      • Gentile I.
      • Fortunato G.
      • Borrelli F.
      • Borelli S.
      • de Caterina M.
      • et al.
      Homocysteine levels and sustained virological response to pegylated-interferon alpha2b plus ribavirin therapy for chronic hepatitis C: a prospective study.
      ], cytokines playing a role in the inflammatory response such as interlekin-10 and -6 (IL-10 and IL-6) [
      • Iuliano A.D.
      • Feingold E.
      • Wahed A.S.
      • Kleiner D.E.
      • Belle S.H.
      • Conjeevaram H.S.
      • et al.
      Host genetics, steatosis and insulin resistance among African Americans and Caucasian Americans with hepatitis C virus genotype-1 infection.
      ], transforming growth factor beta-1 (TGFB1) [
      • Iuliano A.D.
      • Feingold E.
      • Wahed A.S.
      • Kleiner D.E.
      • Belle S.H.
      • Conjeevaram H.S.
      • et al.
      Host genetics, steatosis and insulin resistance among African Americans and Caucasian Americans with hepatitis C virus genotype-1 infection.
      ], tumor necrosis factor (TNF, −238 position) [
      • Sanchez-Munoz D.
      • Romero-Gomez M.
      • Gonzalez-Escribano M.F.
      • Torres B.
      • Castellano-Megias V.M.
      • Gomez-Izquierdo L.
      • et al.
      Tumour necrosis factor alpha polymorphisms are not involved in the development of steatosis in chronic hepatitis C.
      ,
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • Galmozzi E.
      • Rametta R.
      • Dongiovanni P.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      ] and leptin receptor (LEPR) [
      • Iuliano A.D.
      • Feingold E.
      • Wahed A.S.
      • Kleiner D.E.
      • Belle S.H.
      • Conjeevaram H.S.
      • et al.
      Host genetics, steatosis and insulin resistance among African Americans and Caucasian Americans with hepatitis C virus genotype-1 infection.
      ].

      PNPLA3 and fibrosis

      A non-synonymous sequence variation (rs738409 C/G) encoding an isoleucine to methionine substitution in the adiponutrin/patatin-like phospholipase-3 (PNPLA3) has been shown to be strongly associated with increased hepatic fat levels [
      • Romeo S.
      • Kozlitina J.
      • Xing C.
      • Pertsemlidis A.
      • Cox D.
      • Pennacchio L.A.
      • et al.
      Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease.
      ]. This SNP was then shown to be associated with disease severity, fibrosis, and steatosis in non-alcoholic fatty liver disease (NAFLD) [
      • Rotman Y.
      • Koh C.
      • Zmuda J.M.
      • Kleiner D.E.
      • Liang T.J.
      The association of genetic variability in patatin-like phospholipase domain-containing protein 3 (PNPLA3) with histological severity of nonalcoholic fatty liver disease.
      ,
      • Valenti L.
      • Al-Serri A.
      • Daly A.K.
      • Galmozzi E.
      • Rametta R.
      • Dongiovanni P.
      • et al.
      Homozygosity for the patatin-like phospholipase-3/adiponutrin I148M polymorphism influences liver fibrosis in patients with nonalcoholic fatty liver disease.
      ] and in alcoholic liver disease (ALD). Moreover, the same SNP was also associated with elevated liver enzymes in healthy subjects [
      • Kollerits B.
      • Coassin S.
      • Kiechl S.
      • Hunt S.C.
      • Paulweber B.
      • Willeit J.
      • et al.
      A common variant in the adiponutrin gene influences liver enzyme values.
      ]. Interestingly, patients with CHC carrying the rs738409 mutant GG allele had a high risk of steatosis as well as fibrosis and fibrosis progression [
      • Tian C.
      • Stokowski R.P.
      • Kershenobich D.
      • Ballinger D.G.
      • Hinds D.A.
      Variant in PNPLA3 is associated with alcoholic liver disease.
      ,
      • Trepo E.
      • Pradat P.
      • Potthoff A.
      • Momozawa Y.
      • Quertinmont E.
      • Gustot T.
      • et al.
      Impact of patatin-like phospholipase-3 (rs738409 C > G) polymorphism on fibrosis progression and steatosis in chronic hepatitis C.
      ,
      • Stickel F.
      • Buch S.
      • Lau K.
      • Zu Schwabedissen H.M.
      • Berg T.
      • Ridinger M.
      • et al.
      Genetic variation in the PNPLA3 gene is associated with alcoholic liver injury in Caucasians.
      ]. However, there are conflicting results reporting that PNPLA3 rs738409 GG mutant variant may be a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects were negligible in patients with HCV cirrhosis [
      • Nischalke H.D.
      • Berger C.
      • Luda C.
      • Berg T.
      • Muller T.
      • Grunhage F.
      • et al.
      The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis.
      ].

      IL-28B and fibrosis

      The analysis of rs8099917 SNPs during liver fibrosis in chronic hepatitis C showed that the G allele, previously shown to be a risk of treatment failure, was associated with lower activity and less fibrosis with a trend towards a lower rate of fibrosis progression. Interestingly, when patients were stratified according to HCV genotype, the association was more significant in HCV genotype non-1 infected patients [
      • Bochud P.Y.
      • Bibert S.
      • Kutalik Z.
      • Patin E.
      • Guergnon J.
      • Nalpas B.
      • et al.
      IL-28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes.
      ]. However, independent studies have reported that fibrosis progression was not associated with either rs12979860 or with rs8099917 [
      • Asselah T.
      • De Muynck S.
      • Broet P.
      • Masliah-Planchon J.
      • Blanluet M.
      • Bieche I.
      • et al.
      IL-28B polymorphism is associated with treatment response in patients with genotype 4 chronic hepatitis C.
      ,
      • Marabita F.
      • Aghemo A.
      • De Nicola S.
      • Rumi M.G.
      • Cheroni C.
      • Scavelli R.
      • et al.
      Genetic variation in IL-28B gene is not associated with fibrosis progression in patients with chronic hepatitis C and known date of infection.
      ].
      The analysis of a larger cohort showed that PNPLA3 polymorphisms were strongly associated with an increased risk of steatosis in patients with HCV (excluding genotype 3) while the association with IL-28B SNPs was weak [
      • Cai T.
      • Dufour J.F.
      • Muellhaupt B.
      • Gerlach T.
      • Heim M.
      • Moradpour D.
      • et al.
      Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL-28B in hepatitis C virus-associated steatosis.
      ]. Steatosis was found in 22.5% and 39.6% of two independent cohorts of patients carrying the favorable IL-28B genotype vs. 47.6% and 67.4% of IL-28B non-favorable genotypes, respectively [
      • Tillmann H.L.
      • Patel K.
      • Muir A.J.
      • Guy C.D.
      • Li J.H.
      • Lao X.Q.
      • et al.
      Beneficial IL-28B genotype associated with lower frequency of hepatic steatosis in patients with chronic hepatitis C.
      ].
      It has been suggested that levels of LDL cholesterol are significantly higher in CHC subjects carrying the rs12979860 CC genotype compared to CT and TT [
      • Li J.H.
      • Lao X.Q.
      • Tillmann H.L.
      • Rowell J.
      • Patel K.
      • Thompson A.
      • et al.
      Interferon-lambda genotype and low serum low-density lipoprotein cholesterol levels in patients with chronic hepatitis C infection.
      ]. However, triglyceride levels were lower in patients carrying the IL-28B CC genotype. IL-28B CC may be associated with less pronounced lipid metabolism disturbances, as shown by serum lipoprotein levels and hepatic steatosis.
      Altogether, the different studies have suggested that various SNPs, especially, AZIN1 and PNLPA3 are associated with fibrosis progression. Moreover, several extra-cellular-matrix and chemokine mRNAs are probably upregulated in patients with more advanced fibrosis. However, it is important to bear in mind that some studies have investigated fibrosis progression by analyzing paired biopsies while other studies were cross-sectional. Taking into account the duration of infection could provide useful information to improve the evaluation of fibrosis progression in cross-sectional liver biopsies.

      Prediction of treatment response

      Figure thumbnail fx6

      Variation in mRNAs expression and treatment response

      An 8-gene subset accurately predicted treatment response (GIP2/IFI15/ISG15, ATF5, IFIT1, MX1, USP18/UBP43, DUSP1, CEB1, and RPS28) using a number of independent classifier analyses [
      • Chen L.
      • Borozan I.
      • Feld J.
      • Sun J.
      • Tannis L.L.
      • Coltescu C.
      • et al.
      Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection.
      ]. Interestingly, another study evaluated the expression profile of a selection of genes in SVRs and NRs. IFI27 and CXLC9, a two-gene signature, predicted the response to treatment in 79% of the patients with a predictive accuracy of 100% for NRs and 70% for SVRs [
      • Asselah T.
      • Bieche I.
      • Narguet S.
      • Sabbagh A.
      • Laurendeau I.
      • Ripault M.P.
      • et al.
      Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C.
      ]. One study showed that SVR could be predicted, prior to treatment, by analyzing gene expression of signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signaling-1. Interestingly, even after 24 h of treatment, IFN-dependent gene expression can help predict the probability of achieving an SVR [
      • Younossi Z.M.
      • Baranova A.
      • Afendy A.
      • Collantes R.
      • Stepanova M.
      • Manyam G.
      • et al.
      Early gene expression profiles of patients with chronic hepatitis C treated with pegylated interferon-alfa and ribavirin.
      ]. Many of the genes, found to be upregulated in non-responders and responders, encode molecules secreted in the serum (cytokines) [
      • Butera D.
      • Marukian S.
      • Iwamaye A.E.
      • Hembrador E.
      • Chambers T.J.
      • Di Bisceglie A.M.
      • et al.
      Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C.
      ,
      • Ji X.
      • Cheung R.
      • Cooper S.
      • Li Q.
      • Greenberg H.B.
      • He X.S.
      Interferon alfa regulated gene expression in patients initiating interferon treatment for chronic hepatitis C.
      ]. Thus, they are a logical approach to the development of serum markers to predict treatment response.

      Variation of micro-RNAs expression and treatment response

      Since cellular miRNAs regulate many cellular pathways, including the immune response, some miRNAs are probably involved in the modulation of antiviral response to PegIFN/RBV in CHC. IFNβ has been shown to rapidly modulate the expression of numerous miRNAs, and 8 cellular IFNβ-induced miRNAs have sequence-predicted targets within the HCV genome [
      • Pedersen I.M.
      • Cheng G.
      • Wieland S.
      • Volinia S.
      • Croce C.M.
      • Chisari F.V.
      • et al.
      Interferon modulation of cellular microRNAs as an antiviral mechanism.
      ].
      The level of expression of intra-hepatic mir-122 has been shown to be associated with HCV treatment response. Expression of mir-122 was significantly lower in primary non-responders compared to early responders [
      • Sarasin-Filipowicz M.
      • Krol J.
      • Markiewicz I.
      • Heim M.H.
      • Filipowicz W.
      Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy.
      ]. Interestingly, the anti mir-122 (SPC3649 or miravirsen) molecules that inhibit HCV replication in chimpanzees [
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
      • Persson R.
      • Lindow M.
      • Munk M.E.
      • et al.
      Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.
      ] have recently been tested in a randomized double-blind study in treatment naïve patients with genotype 1 [

      Janssen LH, Reesink WH, Zeuzem S, Lawitz E, Rodriguez-Torres M, Chen A, et al. A randomized, double-blind, placebo (PLB) controlled safety and anti-viral proof of concept study of miravirsen (MIR), an oligonucleotide targeting MIR-122, in treatment naive patients with genotype 1 (GT1) chronic HCV infection. AASLD, 2011.

      ].
      However, an independent study investigating the expression profile of 470 cellular miRNAs in 99 CHCs, showed no significant difference in mir-122 expression between responders and non-responders [
      • Murakami Y.
      • Tanaka M.
      • Toyoda H.
      • Hayashi K.
      • Kuroda M.
      • Tajima A.
      • et al.
      Hepatic microRNA expression is associated with the response to interferon treatment of chronic hepatitis C.
      ]. Eight miRNAs (mir-34b, mir-145, mir-143, mir-652, mir-18a, mir-27b, mir-422b, and mir-378) were differentially expressed in responders and non-responders. Moreover, mir-34b and mir-422 were consistently and significantly high and low in non-responders both 12 and 24 weeks after the end of the treatment [
      • Murakami Y.
      • Tanaka M.
      • Toyoda H.
      • Hayashi K.
      • Kuroda M.
      • Tajima A.
      • et al.
      Hepatic microRNA expression is associated with the response to interferon treatment of chronic hepatitis C.
      ].

      Single nucleotide polymorphisms and treatment response

      The main SNPs associated with SVR are summarized in Table 2.

      Killer cell immunoglobulin-like receptors (KIR) activating and inhibiting receptors

      Natural killer (NKs) cells are a subset of lymphocytes that interact directly with virus-infected cells, activate dentritic cells and secrete Th1-type cytokines to increase antiviral cytotoxic T cell response. NK responses are controlled by multiple activating and inhibitory signals such as the KIR receptors. The ligands for these receptors are human leukocyte antigen (HLA) class I. The wide genetic diversity of the KIR family and the HLA class I generate a large number of combinations between KIR and HLA.
      The combination of KIR2DL3 and HLA-C1 was associated with both spontaneous clearance [
      • Khakoo S.I.
      • Thio C.L.
      • Martin M.P.
      • Brooks C.R.
      • Gao X.
      • Astemborski J.
      • et al.
      HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection.
      ] and SVR [
      • Knapp S.
      • Warshow U.
      • Hegazy D.
      • Brackenbury L.
      • Guha I.N.
      • Fowell A.
      • et al.
      Consistent beneficial effects of killer cell immunoglobulin-like receptor 2DL3 and group 1 human leukocyte antigen-C following exposure to hepatitis C virus.
      ,
      • Vidal-Castineira J.R.
      • Lopez-Vazquez A.
      • Diaz-Pena R.
      • Alonso-Arias R.
      • Martinez-Borra J.
      • Perez R.
      • et al.
      Effect of killer immunoglobulin-like receptors in the response to combined treatment in patients with chronic hepatitis C virus infection.
      ] in patients with CHC. This specific combination is supposed to have a weaker inhibitory effect than other combinations leading to a stronger NK cell response.
      KIRs are clonally expressed on NKs in a stochastic manner. Interestingly, Khakoo et al. reported a linear trend between the number of KIR2DL3-HLA-C1 interactions and the ORs resolving the infection. In this model, NK cell activity may be mediated through weak inhibitory KIR2DL3-HLA-C1 interactions.
      HLA class II alleles have been shown to play a role in spontaneous clearance in patients with HCV infection [
      • Azocar J.
      • Clavijo O.P.
      • Yunis E.J.
      MHC class II genes in HCV viral clearance of hepatitis C infected Hispanic patients.
      ]. Interestingly, a new interaction between KIR2DL3 and HLA class II DRB11201 was found to be associated with spontaneous clearance [
      • Romero V.
      • Azocar J.
      • Zuniga J.
      • Clavijo O.P.
      • Terreros D.
      • Gu X.
      • et al.
      Interaction of NK inhibitory receptor genes with HLA-C and MHC class II alleles in Hepatitis C virus infection outcome.
      ].
      Prediction of treatment failure improved from 66% with IL-28B to 80% using both IL-28B and HLA-C2/HLA-C2 genes [
      • Suppiah V.
      • Gaudieri S.
      • Armstrong N.J.
      • O’Connor K.S.
      • Berg T.
      • Weltman M.
      • et al.
      IL-28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European Cohort: a cross-sectional study.
      ]. Moreover, the incorporation of two KIRs in a model including viral load and ethnicity (SVR score) provided complementary results with IL-28B across the CC, CT, and TT genotypes [
      • Golden-Mason L.
      • Bambha K.M.
      • Cheng L.
      • Howell C.D.
      • Taylor M.W.
      • Clark P.J.
      • et al.
      Natural killer inhibitory receptor expression associated with treatment failure and interleukin-28B genotype in patients with chronic hepatitis C.
      ].

      Genetic variants in the cytokine system and treatment response

      STAT3 is mainly activated in the liver by IL-6, a cytokine that has been involved in a variety of cell functions including stimulation of hepatocytes to produce acute-phase proteins. The polymorphism rs1800795 within the IL-6 promoter is associated with lower IL-6 levels compared to patients carrying the G allele. Interestingly, higher response rates were reported in patients carrying the rs1800795 G allele compared to C [
      • Nattermann J.
      • Vogel M.
      • Berg T.
      • Danta M.
      • Axel B.
      • Mayr C.
      • et al.
      Effect of the interleukin-6 C174G gene polymorphism on treatment of acute and chronic hepatitis C in human immunodeficiency virus coinfected patients.
      ]. Yee et al. confirmed the importance of rs1800795 in the viral response and reported that several haplotypes containing rs1800797, rs1800796, rs1800795, and rs2069830 within IL-6, were associated with lower SVR rates [
      • Yee L.J.
      • Im K.
      • Borg B.
      • Yang H.
      • Liang T.J.
      Interleukin-6 haplotypes and the response to therapy of chronic hepatitis C virus infection.
      ]. IL-10 is a T-helper type 2 cytokine that plays a major role in T and B cell regulation. Peripheral blood mononuclear cells (PBMCs) from patients with CHC had increased IL-10 mRNA and protein expression. Moreover IL-10 has been shown to inhibit the production of IFNα by stimulation with viral infections [
      • Payvandi F.
      • Amrute S.
      • Fitzgerald-Bocarsly P.
      Exogenous and endogenous IL-10 regulate IFN-alpha production by peripheral blood mononuclear cells in response to viral stimulation.
      ]. SNPs within the IL-10 promoter at positions −819 and −592 have been associated with different IL-10 levels. Interestingly, a strong relationship was found between IL-10 polymorphisms and response to IFNα treatment [
      • Edwards-Smith C.J.
      • Jonsson J.R.
      • Purdie D.M.
      • Bansal A.
      • Shorthouse C.
      • Powell E.E.
      Interleukin-10 promoter polymorphism predicts initial response of chronic hepatitis C to interferon alfa.
      ]. The frequency of −1082 GG genotypes (high IL-10) was higher in patients who did not eliminate the virus compared to controls. The higher level of IL-10 in these patients was associated with a higher risk of ineffective viral clearance and development of chronic infection in female patients [
      • Paladino N.
      • Fainboim H.
      • Theiler G.
      • Schroder T.
      • Munoz A.E.
      • Flores A.C.
      • et al.
      Gender susceptibility to chronic hepatitis C virus infection associated with interleukin 10 promoter polymorphism.
      ]. Of all the 8 SNPs identified in the entire IFNγ gene, the variant C764G was significantly associated with SVR. Functional analyses showed that the G allele conferred higher promoter activity and a stronger binding affinity for HSF1 [
      • Huang Y.
      • Yang H.
      • Borg B.B.
      • Su X.
      • Rhodes S.L.
      • Yang K.
      • et al.
      A functional SNP of interferon-gamma gene is important for interferon-alpha-induced and spontaneous recovery from hepatitis C virus infection.
      ].

      IL-28B and treatment response

      Figure thumbnail fx7
      Almost 85% of the patients with East Asian ancestry harbor the C allele while only 40% of the patients with African-American ancestry carry genotype CC (Fig. 1) [
      • Ge D.
      • Fellay J.
      • Thompson A.J.
      • Simon J.S.
      • Shianna K.V.
      • Urban T.J.
      • et al.
      Genetic variation in IL-28B predicts hepatitis C treatment-induced viral clearance.
      ]. Thus, rs12979860 genotype diversity could explain the higher SVR rate in Asian patients.
      Figure thumbnail gr1
      Fig. 1Frequency of IL-28B rs12979860 protective allele in worldwide populations. rs12979860 CC is present in up to 80% of patients with Asian ancestry and only 40% of patients with African ancestry. Figure adapted from data reported in Thomas et al.
      [
      • Thomas D.L.
      • Thio C.L.
      • Martin M.P.
      • Qi Y.
      • Ge D.
      • O’Huigin C.
      • et al.
      Genetic variation in IL-28B and spontaneous clearance of hepatitis C virus.
      ]
      .
      Interestingly, the rs12979860 CC genotype was also associated with early improved viral kinetics, a higher rate of rapid virological response, and complete early virological response [
      • Thompson A.J.
      • Muir A.J.
      • Sulkowski M.S.
      • Ge D.
      • Fellay J.
      • Shianna K.V.
      • et al.
      Interleukin-28B polymorphism improves viral kinetics and is the strongest pretreatment predictor of sustained virologic response in genotype 1 hepatitis C virus.
      ]. The association of rs12979860 with viral response has been confirmed in independent cohort studies [
      • Thomas D.L.
      • Thio C.L.
      • Martin M.P.
      • Qi Y.
      • Ge D.
      • O’Huigin C.
      • et al.
      Genetic variation in IL-28B and spontaneous clearance of hepatitis C virus.
      ,
      • McCarthy J.J.
      • Li J.H.
      • Thompson A.
      • Suchindran S.
      • Lao X.Q.
      • Patel K.
      • et al.
      Replicated association between an IL-28B gene variant and a sustained response to pegylated interferon and ribavirin.
      ]. Interestingly, IL-28B genotype was associated with treatment response in patients with CHC but not in those with acute hepatitis C [
      • Nattermann J.
      • Vogel M.
      • Nischalke H.D.
      • Danta M.
      • Mauss S.
      • Stellbrink H.J.
      • et al.
      Genetic variation in IL-28B and treatment-induced clearance of hepatitis C virus in HIV-positive patients with acute and chronic hepatitis C.
      ]. Moreover, jaundice was more common in CC patients (64%) during acute infection than in CT (24%) or TT (6%). However, jaundice was only associated with an increased chance of spontaneous viral clearance in non-CC patients [
      • Tillmann H.L.
      • Thompson A.J.
      • Patel K.
      • Wiese M.
      • Tenckhoff H.
      • Nischalke H.D.
      • et al.
      A polymorphism near IL-28B is associated with spontaneous clearance of acute hepatitis C virus and jaundice.
      ].
      It has been suggested that the influence of rs12979860 genotype is a stronger predictive factor of SVR in patients with HCV-G1 than in HCV non-G1 patients [
      • Montes-Cano M.A.
      • Garcia-Lozano J.R.
      • Abad-Molina C.
      • Romero-Gomez M.
      • Barroso N.
      • Aguilar-Reina J.
      • et al.
      Interleukin-28B genetic variants and hepatitis virus infection by different viral genotypes.
      ].
      HCV-G-2 and G-3 patients carrying the rs12979860 CC allele had a more rapid reduction in plasma HCV RNA, 3 days after beginning of treatment. However, in accordance with previous results [
      • Rauch A.
      • Kutalik Z.
      • Descombes P.
      • Cai T.
      • Di Iulio J.
      • Mueller T.
      • et al.
      Genetic variation in IL-28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.
      ], no significant association was found between rs12979860 and SVR in this cohort (98 genotype 2 and 241 genotype 3 patients). Interestingly, the assessment of IL-28B SNPs in patients with genotype 2 or 3 might help determine the suitable treatment duration [
      • Lindh M.
      • Lagging M.
      • Farkkila M.
      • Langeland N.
      • Morch K.
      • Nilsson S.
      • et al.
      Interleukin 28B gene variation at rs12979860 determines early viral kinetics during treatment in patients carrying genotypes 2 or 3 of hepatitis C virus.
      ].
      In a recent study including 164 HCV genotype 4 patients from different ethnic groups, IL-28B rs12979860 CC was associated with a better treatment response rate (81.8% vs. 46.5% and 29.4% for CC, CT and TT respectively). No significant relationship was found between rs12979860 and the stage of fibrosis [
      • Asselah T.
      • De Muynck S.
      • Broet P.
      • Masliah-Planchon J.
      • Blanluet M.
      • Bieche I.
      • et al.
      IL-28B polymorphism is associated with treatment response in patients with genotype 4 chronic hepatitis C.
      ].
      Interestingly, it has been suggested that IL-28B SNP influences HCV re-infection in patients receiving liver transplants. Rs12979860 CC donors were associated with rapid, complete early, and sustained virological response to PegIFN/RBV, compared to CT and TT [
      • Lange C.M.
      • Moradpour D.
      • Doehring A.
      • Lehr H.A.
      • Mullhaupt B.
      • Bibert S.
      • et al.
      Impact of donor and recipient IL-28B rs12979860 genotypes on hepatitis C virus liver graft reinfection.
      ]. While the rs8099917 favorable genotype was associated with a higher SVR rate in both recipients and donors, recipient rs12979860 status will probably have a weaker impact on viral response [
      • Lange C.M.
      • Moradpour D.
      • Doehring A.
      • Lehr H.A.
      • Mullhaupt B.
      • Bibert S.
      • et al.
      Impact of donor and recipient IL-28B rs12979860 genotypes on hepatitis C virus liver graft reinfection.
      ]. The use of both IL-28B genotype in donors and recipients and HCV core substitution predicted an SVR with 83% sensitivity and 82% specificity [
      • Fukuhara T.
      • Taketomi A.
      • Motomura T.
      • Okano S.
      • Ninomiya A.
      • Abe T.
      • et al.
      Variants in IL-28B in liver recipients and donors correlate with response to peg-interferon and ribavirin therapy for recurrent hepatitis C.
      ].

      Which specific IL-28B SNPs for the prediction of SVR?

      Rs8099917 was identified in part by Rauch et al. and Tanaka because rs12979860 was not investigated, since this SNP is not present on either the Illumina Array or the Affymetrix 6.0 genotyping platform. Prediction of SVR with rs12979860 or rs8099917 will probably not matter in Caucasian patients. However, the frequency of these SNPs in other ethnicities may limit its use as predictive factor in the general population. For example, the use of IL-28B in the decision to treat patients with African ancestry, in which the frequency of the rs 12979860 favorable allele is only 40%, would exclude a large number of patients from being eligible for treatment. Therefore, IL-28B may be useful in combination with other predictive factors to avoid excluding patients based on ethnicity.

      CCR5 and HCV spontaneous viral clearance

      The chemokine receptor CCR5 is activated through the binding of RANTES. CCR5 is also a co-receptor of HIV-1. Interestingly, a common 32-base deletion in the CCR5 gene has been associated with resistance to HIV-1 infection [
      • Dean M.
      • Carrington M.
      • Winkler C.
      • Huttley G.A.
      • Smith M.W.
      • Allikmets R.
      • et al.
      Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study.
      ,
      • Michael N.L.
      • Louie L.G.
      • Rohrbaugh A.L.
      • Schultz K.A.
      • Dayhoff D.E.
      • Wang C.E.
      • et al.
      The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression.
      ,
      • Huang Y.
      • Paxton W.A.
      • Wolinsky S.M.
      • Neumann A.U.
      • Zhang L.
      • He T.
      • et al.
      The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.
      ]. The potential role of CCR5 deletion in resistance to HCV has been studied and suggested to adversely affect outcome of HCV infection [
      • Woitas R.P.
      • Ahlenstiel G.
      • Iwan A.
      • Rockstroh J.K.
      • Brackmann H.H.
      • Kupfer B.
      • et al.
      Frequency of the HIV-protective CC chemokine receptor 5-Delta32/Delta32 genotype is increased in hepatitis C.
      ,
      • Hellier S.
      • Frodsham A.J.
      • Hennig B.J.
      • Klenerman P.
      • Knapp S.
      • Ramaley P.
      • et al.
      Association of genetic variants of the chemokine receptor CCR5 and its ligands, RANTES and MCP-2, with outcome of HCV infection.
      ,
      • Mangia A.
      • Santoro R.
      • D’Agruma L.
      • Andriulli A.
      HCV chronic infection and CCR5-delta32/delta32.
      ,
      • Zhang M.
      • Goedert J.J.
      • O’Brien T.R.
      High frequency of CCR5-delta32 homozygosity in HCV-infected, HIV-1-uninfected hemophiliacs results from resistance to HIV-1.
      ,
      • Promrat K.
      • McDermott D.H.
      • Gonzalez C.M.
      • Kleiner D.E.
      • Koziol D.E.
      • Lessie M.
      • et al.
      Associations of chemokine system polymorphisms with clinical outcomes and treatment responses of chronic hepatitis C.
      ,
      • Goulding C.
      • McManus R.
      • Murphy A.
      • MacDonald G.
      • Barrett S.
      • Crowe J.
      • et al.
      The CCR5-delta32 mutation: impact on disease outcome in individuals with hepatitis C infection from a single source.
      ]. However, the different reports are conflicting.
      A recent study has investigated the association of both CCR5Δ32 and rs12979860 with spontaneous HCV clearance [
      • Nattermann J.
      • Timm J.
      • Nischalke H.D.
      • Olbrich A.
      • Michalk M.
      • Tilmann H.L.
      • et al.
      The predictive value of IL-28B gene polymorphism for spontaneous clearance in a single source outbreak cohort is limited in patients carrying the CCR5Delta32 mutation.
      ]. The authors described an association of response rates with rs12979860 CC only in CCR5 WT homozygous while HCV clearance remained poor in CCR5Δ32 carriers even in CC patients. Because the frequency of the CCR5Δ32 homozygous deletion is rare, it is difficult to study the influence of this mutation, either alone or in combination with IL-28B genotype, on the outcome of HCV infection. RANTES is expressed and secreted by HSCs and induces migration, proliferation, and fibrogenic properties [
      • Schwabe R.F.
      • Bataller R.
      • Brenner D.A.
      Human hepatic stellate cells express CCR5 and RANTES to induce proliferation and migration.
      ]. Different studies have reported the overexpression of CCR5/RANTES in models of liver fibrosis and in patients with CHC [
      • Seki E.
      • De Minicis S.
      • Gwak G.Y.
      • Kluwe J.
      • Inokuchi S.
      • Bursill C.A.
      • et al.
      CCR1 and CCR5 promote hepatic fibrosis in mice.
      ]. Pharmaceutical companies have developed RANTES inhibitors/CCR5 antagonists which have been successfully tested in phase III studies in patients with HIV infection [
      • Kuhmann S.E.
      • Hartley O.
      Targeting chemokine receptors in HIV: a status report.
      ]. Interestingly, the administration of the RANTES inhibitor greatly improved liver fibrosis in mice and accelerated fibrosis regression [
      • Affo S.
      • Bataller R.
      RANTES antagonism: a promising approach to treat chronic liver diseases.
      ].

      Combination of the IL-28B genotype with other factors to predict treatment response

      Host factors

      High ISGs expression before treatment has been associated with a low SVR rate [
      • Chen L.
      • Borozan I.
      • Feld J.
      • Sun J.
      • Tannis L.L.
      • Coltescu C.
      • et al.
      Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection.
      ,
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • Christen V.
      • Terracciano L.
      • Filipowicz W.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ]. Since treatment is based on PegIFN intake, patients who already have high ISG expression may not be as stimulated as patients with low ISGs expression. Analysis of post-treatment biopsies in patients with an RVR revealed that PegIFN did not induce ISGs expression above pretreatment levels [
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • Christen V.
      • Terracciano L.
      • Filipowicz W.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ].
      Both IFNα and λ induce ISGs expression in HCV infected cells, in vitro [
      • Marcello T.
      • Grakoui A.
      • Barba-Spaeth G.
      • Machlin E.S.
      • Kotenko S.V.
      • MacDonald M.R.
      • et al.
      Interferons alpha and lambda inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics.
      ]. However, the kinetics of IFNλ-mediated STAT activation and induction of effector genes were different from those of IFNα, suggesting distinct mechanisms of IFNλ- and IFNα-induced antiviral states.
      Intra-hepatic ISGs expression may be a better predictor of SVR than IL-28B genotype [
      • Dill M.T.
      • Duong F.H.
      • Vogt J.E.
      • Bibert S.
      • Bochud P.Y.
      • Terracciano L.
      • et al.
      Interferon-induced gene expression is a stronger predictor of treatment response than IL-28B genotype in patients with hepatitis C.
      ,
      • Honda M.
      • Sakai A.
      • Yamashita T.
      • Nakamoto Y.
      • Mizukoshi E.
      • Sakai Y.
      • et al.
      Hepatic ISG expression is associated with genetic variation in interleukin 28B and the outcome of IFN therapy for chronic hepatitis C.
      ]. Interestingly, global expression of ISGs is strongly associated with genetic variation of IL-28B [
      • Honda M.
      • Sakai A.
      • Yamashita T.
      • Nakamoto Y.
      • Mizukoshi E.
      • Sakai Y.
      • et al.
      Hepatic ISG expression is associated with genetic variation in interleukin 28B and the outcome of IFN therapy for chronic hepatitis C.
      ]. Multivariate analysis of predictors of response showed that a set of 4 ISGs were better predictors than rs12979860 SNP [
      • Dill M.T.
      • Duong F.H.
      • Vogt J.E.
      • Bibert S.
      • Bochud P.Y.
      • Terracciano L.
      • et al.
      Interferon-induced gene expression is a stronger predictor of treatment response than IL-28B genotype in patients with hepatitis C.
      ]. Despite a small sample size, another study reported an association between rs12979860 SNP and 3 ISGs (ISG15, IFI27, and IFI6) [
      • Urban T.J.
      • Thompson A.J.
      • Bradrick S.S.
      • Fellay J.
      • Schuppan D.
      • Cronin K.D.
      • et al.
      IL-28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis C.
      ]. Moreover, it has been suggested that IL-28B genotype is associated with a specific cell-type modulation of ISGs expression (MxA, PKR, OAS1, and ISG15) in hepatic cells and PBMCs [
      • Abe H.
      • Hayes C.N.
      • Ochi H.
      • Maekawa T.
      • Tsuge M.
      • Miki D.
      • et al.
      IL28 variation affects expression of interferon stimulated genes and peg-interferon and ribavirin therapy.
      ].
      Several studies have shown that elevated IP10 levels may be a prognostic marker of HCV treatment outcome in HCV genotype 1 infection [
      • Butera D.
      • Marukian S.
      • Iwamaye A.E.
      • Hembrador E.
      • Chambers T.J.
      • Di Bisceglie A.M.
      • et al.
      Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C.
      ,
      • Diago M.
      • Castellano G.
      • Garcia-Samaniego J.
      • Perez C.
      • Fernandez I.
      • Romero M.
      • et al.
      Association of pretreatment serum interferon gamma inducible protein 10 levels with sustained virological response to peginterferon plus ribavirin therapy in genotype 1 infected patients with chronic hepatitis C.
      ,
      • Zeremski M.
      • Markatou M.
      • Brown Q.B.
      • Dorante G.
      • Cunningham-Rundles S.
      • Talal A.H.
      Interferon gamma-inducible protein 10: a predictive marker of successful treatment response in hepatitis C virus/HIV-coinfected patients.
      ,
      • Romero A.I.
      • Lagging M.
      • Westin J.
      • Dhillon A.P.
      • Dustin L.B.
      • Pawlotsky J.M.
      • et al.
      Interferon (IFN)-gamma-inducible protein-10: association with histological results, viral kinetics, and outcome during treatment with pegylated IFN-alpha 2a and ribavirin for chronic hepatitis C virus infection.
      ,
      • Narumi S.
      • Tominaga Y.
      • Tamaru M.
      • Shimai S.
      • Okumura H.
      • Nishioji K.
      • et al.
      Expression of IFN-inducible protein-10 in chronic hepatitis.
      ,
      • Lagging M.
      • Romero A.I.
      • Westin J.
      • Norkrans G.
      • Dhillon A.P.
      • Pawlotsky J.M.
      • et al.
      IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection.
      ]. The combination of serum IP-10 and rs12979860 significantly improved the prediction of SVR [
      • Lagging M.
      • Askarieh G.
      • Negro F.
      • Bibert S.
      • Soderholm J.
      • Westin J.
      • et al.
      Response prediction in chronic hepatitis C by assessment of IP-10 and IL-28B-related single nucleotide polymorphisms.
      ,
      • Darling J.M.
      • Aerssens J.
      • Fanning G.
      • McHutchison J.G.
      • Goldstein D.B.
      • Thompson A.J.
      • et al.
      Quantitation of pretreatment serum interferon-gamma-inducible protein-10 improves the predictive value of an IL-28B gene polymorphism for hepatitis C treatment response.
      ,
      • Beinhardt S.
      • Aberle J.H.
      • Strasser M.
      Dulic–Lakovic E, Maieron A, Kreil A, et al., Serum Level of IP-10 Increases Predictive Value of IL-28B Polymorphisms for Spontaneous Clearance of Acute HCV Infectio.
      ]. Serum IP-10 was particularly informative in rs12979860 CT carriers, in whom high serum IP-10 levels resulted in a 64% SVR compared to 24% in patients with low IP-10 [
      • Darling J.M.
      • Aerssens J.
      • Fanning G.
      • McHutchison J.G.
      • Goldstein D.B.
      • Thompson A.J.
      • et al.
      Quantitation of pretreatment serum interferon-gamma-inducible protein-10 improves the predictive value of an IL-28B gene polymorphism for hepatitis C treatment response.
      ]. Serum IP-10 levels below 150 pg/ml significantly predicted a greater reduction of HCV RNA and increased SVR rates in genotype 1 rs12979860 CC patients [
      • Lagging M.
      • Askarieh G.
      • Negro F.
      • Bibert S.
      • Soderholm J.
      • Westin J.
      • et al.
      Response prediction in chronic hepatitis C by assessment of IP-10 and IL-28B-related single nucleotide polymorphisms.
      ].
      It has been suggested that mir-122 expression is associated with treatment outcome [
      • Sarasin-Filipowicz M.
      • Krol J.
      • Markiewicz I.
      • Heim M.H.
      • Filipowicz W.
      Decreased levels of microRNA miR-122 in individuals with hepatitis C responding poorly to interferon therapy.
      ]. IL-28B genotype and mir-122 hepatic expression have been reported to be independently associated with viral response [
      • Dill M.T.
      • Duong F.H.
      • Vogt J.E.
      • Bibert S.
      • Bochud P.Y.
      • Terracciano L.
      • et al.
      Interferon-induced gene expression is a stronger predictor of treatment response than IL-28B genotype in patients with hepatitis C.
      ].
      Vitamin D deficiency is common in patients with chronic liver disease. A recent study showed that vitamin D serum levels improve prediction of an SVR in association with the IL-28B rs12979860 polymorphism [
      • Bitetto D.
      • Fattovich G.
      • Fabris C.
      • Ceriani E.
      • Falleti E.
      • Fornasiere E.
      • et al.
      Complementary role of vitamin D deficiency and the interleukin-28B rs12979860 C/T polymorphism in predicting antiviral response in chronic hepatitis C.
      ]. Vitamin D may be a key regulator of the innate immune response [
      • White J.H.
      Vitamin D signaling, infectious diseases, and regulation of innate immunity.
      ]. However, this study was retrospective. Furthermore, it has not been shown if correcting a vitamin D deficiency before starting treatment may increase SVR rates.

      Viral factors and treatment response

      Two amino acids substitutions at positions 70 and 91 within the core protein and an accumulation of mutations in the interferon sensitivity determining region (ISDR) located in the NS5A coding region have been associated with viral response [
      • Akuta N.
      • Suzuki F.
      • Sezaki H.
      • Suzuki Y.
      • Hosaka T.
      • Someya T.
      • et al.
      Association of amino acid substitution pattern in core protein of hepatitis C virus genotype 1b high viral load and non-virological response to interferon-ribavirin combination therapy.
      ,
      • Enomoto N.
      • Sakuma I.
      • Asahina Y.
      • Kurosaki M.
      • Murakami T.
      • Yamamoto C.
      • et al.
      Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region.
      ].
      Interestingly, two studies [
      • Akuta N.
      • Suzuki F.
      • Hirakawa M.
      • Kawamura Y.
      • Yatsuji H.
      • Sezaki H.
      • et al.
      Amino acid substitution in hepatitis C virus core region and genetic variation near the interleukin 28B gene predict viral response to telaprevir with peginterferon and ribavirin.
      ,
      • Hayes C.N.
      • Kobayashi M.
      • Akuta N.
      • Suzuki F.
      • Kumada H.
      • Abe H.
      • et al.
      HCV substitutions and IL-28B polymorphisms on outcome of peg-interferon plus ribavirin combination therapy.
      ] have shown that IL-28B polymorphisms as well as amino acid 70 substitution in the core protein were independently associated with SVR. The SVR rate in patients receiving telaprevir in combination with PegIFN/RBV, was high in those carrying the rs8099917 TT genotype whatever the core 70 substitutions [
      • Akuta N.
      • Suzuki F.
      • Hirakawa M.
      • Kawamura Y.
      • Yatsuji H.
      • Sezaki H.
      • et al.
      Amino acid substitution in hepatitis C virus core region and genetic variation near the interleukin 28B gene predict viral response to telaprevir with peginterferon and ribavirin.
      ].
      Of all the host and viral factors achieving an RVR is probably the strongest predictor of SVR [
      • Fried M.W.
      • Hadziyannis S.J.
      • Shiffman M.L.
      • Messinger D.
      • Zeuzem S.
      Rapid virological response is the most important predictor of sustained virological response across genotypes in patients with chronic hepatitis C virus infection.
      ]. Early viral kinetics were improved and there was a greater probability of achieving an RVR with the rs12979860 CC genotype compared to CT and TT [
      • Thompson A.J.
      • Muir A.J.
      • Sulkowski M.S.
      • Ge D.
      • Fellay J.
      • Shianna K.V.
      • et al.
      Interleukin-28B polymorphism improves viral kinetics and is the strongest pretreatment predictor of sustained virologic response in genotype 1 hepatitis C virus.
      ,
      • Mangia A.
      • Thompson A.J.
      • Santoro R.
      • Piazzolla V.
      • Copetti M.
      • Minerva N.
      • et al.
      Limited utility of IL-28B in the setting of response-guided treatment with detailed on-treatment virological monitoring.
      ]. RVR was a strong predictor of SVR whatever the IL-28B, suggesting that achieving an RVR may compensate for the negative influence of IL-28B genotype. The CC IL-28B genotype was associated with high SVR rates in non-RVR patients [
      • Thompson A.J.
      • Muir A.J.
      • Sulkowski M.S.
      • Ge D.
      • Fellay J.
      • Shianna K.V.
      • et al.
      Interleukin-28B polymorphism improves viral kinetics and is the strongest pretreatment predictor of sustained virologic response in genotype 1 hepatitis C virus.
      ]. However, in patients with an RVR the rate of SVR was high and there were no associations between IL-28B genotype and SVR [
      • Mangia A.
      • Thompson A.J.
      • Santoro R.
      • Piazzolla V.
      • Copetti M.
      • Minerva N.
      • et al.
      Limited utility of IL-28B in the setting of response-guided treatment with detailed on-treatment virological monitoring.
      ]. It is not clear whether the C allele is associated with a higher SVR rate in HCV genotype 2 or 3 patients achieving an RVR [
      • Mangia A.
      • Thompson A.J.
      • Santoro R.
      • Piazzolla V.
      • Tillmann H.L.
      • Patel K.
      • et al.
      An IL-28B polymorphism determines treatment response of hepatitis C virus genotype 2 or 3 patients who do not achieve a rapid virologic response.
      ,
      • Sarrazin C.
      • Susser S.
      • Doehring A.
      • Lange C.M.
      • Muller T.
      • Schlecker C.
      • et al.
      Importance of IL-28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients.
      ].
      IL-28B polymorphisms may be used in combination with several factors; ISGs, viral genotype and other SNPs before, but also during treatment, to improve prediction of treatment response.

      The activity of IFNλ and IL-28B polymorphisms

      Figure thumbnail fx8
      Although multiple SNPs located in the IL-28B promoter region have been identified and associated with SVR, none of them were causal variants. The results of studies assessing variations of circulating IFNλs in the different IL-28B SNPs are contradictory [
      • Urban T.J.
      • Thompson A.J.
      • Bradrick S.S.
      • Fellay J.
      • Schuppan D.
      • Cronin K.D.
      • et al.
      IL-28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis C.
      ,
      • Hayes C.N.
      • Kobayashi M.
      • Akuta N.
      • Suzuki F.
      • Kumada H.
      • Abe H.
      • et al.
      HCV substitutions and IL-28B polymorphisms on outcome of peg-interferon plus ribavirin combination therapy.
      ,
      • Suppiah V.
      • Moldovan M.
      • Ahlenstiel G.
      • Berg T.
      • Weltman M.
      • Abate M.L.
      • et al.
      IL-28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy.
      ,
      • Tanaka Y.
      • Nishida N.
      • Sugiyama M.
      • Kurosaki M.
      • Matsuura K.
      • Sakamoto N.
      • et al.
      Genome-wide association of IL-28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C.
      ,
      • Langhans B.
      • Kupfer B.
      • Braunschweiger I.
      • Arndt S.
      • Schulte W.
      • Nischalke H.D.
      • et al.
      Interferon-lambda serum levels in hepatitis C.
      ].
      The putative causal SNP rs8103142 (Lys70Arg) within the IL-28B coding region was identified in strong linkage disequilibrium with rs12979860. However, there was no significant difference from the Lys70Arg mutation on either IL-28B activity or expression [
      • Urban T.J.
      • Thompson A.J.
      • Bradrick S.S.
      • Fellay J.
      • Schuppan D.
      • Cronin K.D.
      • et al.
      IL-28B genotype is associated with differential expression of intrahepatic interferon-stimulated genes in patients with chronic hepatitis C.
      ].
      Detailed genotyping was performed on the IL-28B coding region in a cohort of 389 HIV/HCV co-infected patients. Homozygotes for the haplotypes rs4803219, rs28416813, rs8103142, and rs4803217 were strongly linked to the rs12979860 CC genotype (r2 = 0.97). This specific haplotype was especially frequent in individuals with spontaneous clearance compared to those with CHC [
      • di Iulio J.
      • Ciuffi A.
      • Fitzmaurice K.
      • Kelleher D.
      • Rotger M.
      • Fellay J.
      • et al.
      Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance.
      ], suggesting a possible effect of these SNPs on viral clearance.
      Interestingly, IFNλs were highly activated in chimpanzees immediately after HCV infection while IFNαβs were slightly induced [
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV Infection Induces a Unique Hepatic Innate Immune Response Associated with Robust Production of Type III Interferons.
      ].
      However, it is important to remember that while a common variant associated with a disease in a GWAS is generally linked to a causal effect that is nearby, this is not necessary the case. Thus rs12979860 may be in strong linkage disequilibrium with an unidentified causal SNP located in another gene.

      IL-28B and future therapies

      IFNλ-based treatment

      The strong association between IL-28B genotype and treatment response suggests that IFNλ may be an attractive alternative to IFNα. Indeed, IFNλ has a limited effect on hematopoietic cells and the central nervous system, promising antiviral activity in the liver and limited receptor expression.
      Zymogenetics and Bristol-Myers Squibb have developed PegIFNλ1 and are assessing its efficacy in the treatment of HCV. A phase 1b study was published in 2010 [
      • Muir A.J.
      • Shiffman M.L.
      • Zaman A.
      • Yoffe B.
      • de la Torre A.
      • Flamm S.
      • et al.
      Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection.
      ].
      The phase IIb study reported a higher early virological response (EVR) following treatment with PegIFNλ combined with RBV than with PegIFNα, in patients with genotype 1 and 4. Interestingly, at different weekly doses of PegIFNλ (240 to 120 μg), cEVR rates were 55–56.3% compared to 37.9% with 180 μg of PegIFNα in genotype 1 and 4 patients. The tolerability and safety of PegIFNλ were better than with PegIFNα, with less anemia and fewer flu-like symptoms [

      Zeuzem S, Arora S, Bacon B, Box T, Charlton M, Diago M, et al. Pegylated interferon-lambda shows superior viral response with improved safety and tolerability versus PegIFN alpha in HCV patients (G1/2/3/4): emerge phase IIB through week 12. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

      ]. These findings show that PegIFNλ has an antiviral effect against HCV. However, further and longer studies on the effect of PegIFNλ are needed. Interestingly, rs12979860 CC was associated with an increase in SVR in patients treated with PegIFN (Fig. 2).
      Figure thumbnail gr2
      Fig. 2SVR in triple therapy according to IL-28B rs12979860. Telaprevir-based therapy improved both RVR and SVR across the different rs12979860 genotypes. In patients with the unfavorable allele, the addition of telaprevir doubled SVR rates. From 87% to 90% of the CC patients achieved an SVR with telaprevir compared to 64% with PegIFN/RBV
      [

      Jacobson IM, Catlett I, Marcellin P, Bzowej NH, Muir AJ, Adda M, et al. Telaprevir substantially improved SVR rates across all IL-28B genotypes in the advance trial. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

      ]
      . Therefore, patients with all different rs12979860 genotypes benefit from telaprevir triple therapy. Among patients carrying the rs12979860 CC allele, 89% of treatment-naïve, and 82% of patients with a treatment-failure had an early response (HCV RNA undetectable at week 8) with boceprevir in addition to PegIFN/RBV. It is interesting to note that these early responder patients were eligible for shorter treatment
      [

      Poordad F. IL-28B polymorphism predicts virologic response in patients with hepatitis C genotype 1 treated with boceprevir combination therapy. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

      ]
      . Naïve genotype 1 patients with the favorable rs12979860 CC allele do not benefit (SVR) from boceprevir triple therapy compared to PegIFN/RBV.

      IL-28B and triple therapies

      In naïve and previously treated genotype 1 patients, the present standard of care is triple therapy with the protease inhibitor boceprevir or telaprevir combined with PegIFN and RBV [
      • Poordad F.
      • McCone Jr., J.
      • Bacon B.R.
      • Bruno S.
      • Manns M.P.
      • Sulkowski M.S.
      • et al.
      Boceprevir for untreated chronic HCV genotype 1 infection.
      ,
      • Jacobson I.M.
      • McHutchison J.G.
      • Dusheiko G.
      • Di Bisceglie A.M.
      • Reddy K.R.
      • Bzowej N.H.
      • et al.
      Telaprevir for previously untreated chronic hepatitis C virus infection.
      ,
      • Zeuzem S.
      • Andreone P.
      • Pol S.
      • Lawitz E.
      • Diago M.
      • Roberts S.
      • et al.
      Telaprevir for retreatment of HCV infection.
      ,
      • Bacon B.R.
      • Gordon S.C.
      • Lawitz E.
      • Marcellin P.
      • Vierling J.M.
      • Zeuzem S.
      • et al.
      Boceprevir for previously treated chronic HCV genotype 1 infection.
      ]. IL-28B status and response to therapy have been evaluated for telaprevir and boceprevir used in combination with PegIFNα/RBV. In studies of boceprevir triple therapy in both naïve and experienced genotype 1 patients, IL-28B CC genotype was associated with a higher SVR rate [
      • Poordad F.
      • McCone Jr., J.
      • Bacon B.R.
      • Bruno S.
      • Manns M.P.
      • Sulkowski M.S.
      • et al.
      Boceprevir for untreated chronic HCV genotype 1 infection.
      ,
      • Bacon B.R.
      • Gordon S.C.
      • Lawitz E.
      • Marcellin P.
      • Vierling J.M.
      • Zeuzem S.
      • et al.
      Boceprevir for previously treated chronic HCV genotype 1 infection.
      ]. Moreover, the favorable genotype was significantly associated with undetectable HCV RNA after 8 weeks of therapy in both naïve (89% for CC vs. 52% for CT and TT) and experienced patients (89–82% for the CC genotype vs. 52% and 51% for CT and TT) [
      • Poordad F.
      • McCone Jr., J.
      • Bacon B.R.
      • Bruno S.
      • Manns M.P.
      • Sulkowski M.S.
      • et al.
      Boceprevir for untreated chronic HCV genotype 1 infection.
      ,
      • Bacon B.R.
      • Gordon S.C.
      • Lawitz E.
      • Marcellin P.
      • Vierling J.M.
      • Zeuzem S.
      • et al.
      Boceprevir for previously treated chronic HCV genotype 1 infection.
      ]. Therefore, IL-28B may be used to identify patients who are eligible for shorter therapy. In naïve genotype 1 patients treated with boceprevir, the IL-28B polymorphism was an independent predictor of SVR. SVR rates were higher in CC, CT, and TT patients receiving triple therapy. The differences in SVR rates in patients receiving either PegIFN/RBV or triple therapy were more marked in CT and TT patients than in CC patients (40% and 30% in CT and TT vs. 3% in CC).
      Telaprevir-based therapy improved both RVR and SVR across all different rs12979860 genotypes (Fig. 2) [

      Jacobson IM, Catlett I, Marcellin P, Bzowej NH, Muir AJ, Adda M, et al. Telaprevir substantially improved SVR rates across all IL-28B genotypes in the advance trial. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

      ]. Although only Caucasians were genotyped for IL-28B SNP, there was an increase in SVR rates across all IL-28B genotypes in genotype 1 naïve patients treated with telaprevir triple therapy. Patients with the CC genotype were most likely to achieve an RVR and to have shorter treatment duration. The difference in SVR rates between patients receiving triple therapy and PegIFN/RBV was more marked in patients with the T allele than in those with the CC genotype [

      Jacobson IM, Catlett I, Marcellin P, Bzowej NH, Muir AJ, Adda M, et al. Telaprevir substantially improved SVR rates across all IL-28B genotypes in the advance trial. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

      ]. There was no significant difference in SVR rates across the different IL-28B genotypes with telaprevir triple therapy in genotype 1 experienced patients [
      • Pol S.
      • Aerssens J.
      • Zeuzem S.
      Similar SVR rates in IL-28B CC, CT or TT prior relapser partial- or null-responder patients treated with telaprevir/peginterferon/ribavirin: retrospective analysis of the REALIZE study.
      ].
      Multivariate analysis identified rs8099917 genotype TT and substitution at aa 70 (Arg70) as significant determinants of SVR after a 12-week or 24-week regimen of triple therapy in patients with HCV genotype 1 [
      • Akuta N.
      • Suzuki F.
      • Hirakawa M.
      • Kawamura Y.
      • Yatsuji H.
      • Sezaki H.
      • et al.
      Amino acid substitution in hepatitis C virus core region and genetic variation near the interleukin 28B gene predict viral response to telaprevir with peginterferon and ribavirin.
      ]. The efficacy of triple therapy was high in patients with the favorable genotype with an SVR rate of 84%, whatever substitution of core aa 70. However, in patients with the unfavorable genotype, the presence of Arg or Gly at position 70 in the core region was associated with a 50% and 12% SVR respectively [
      • Akuta N.
      • Suzuki F.
      • Hirakawa M.
      • Kawamura Y.
      • Yatsuji H.
      • Sezaki H.
      • et al.
      Amino acid substitution in hepatitis C virus core region and genetic variation near the interleukin 28B gene predict viral response to telaprevir with peginterferon and ribavirin.
      ].
      The INFORM-1 study reported that a dual combination of the nucleoside polymerase inhibitor mericitabine and the HCV protease inhibitor danoprevir produced a rapid and substantial decrease in viral load that was maintained throughout two weeks of treatment [
      • Gane E.J.
      • Roberts S.K.
      • Stedman C.A.
      • Angus P.W.
      • Ritchie B.
      • Elston R.
      • et al.
      Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial.
      ]. Finally, the mean reduction in HCV RNA serum levels with the IFN-free regimen was slightly higher in patients with CC polymorphism than in those with CT and TT [
      • Chu T.W.
      • Kulkarni R.
      • Gane E.J.
      • Roberts S.K.
      • Stedman C.
      • Angus P.W.
      • et al.
      Effect of IL-28B Genotype on Early Viral Kinetics during Interferon-Free Treatment of Patients with Chronic Hepatitis C.
      ].
      Overall, the ability to predict the SVR in patients receiving triple therapy has not been fully evaluated. IL-28B may have limited predictive value in patients with prior failure to PegIFN/RBV. Moreover, in patients with the favorable IL-28B genotype, it may be difficult to not use triple therapy considering the overall increase SVR rates, reported in the different trials, and the possibility of a shorten treatment duration.
      For genotype 4, the current treatment remains the combination of PegIFN/RBV, thus the IL-28B polymorphism may be an important factor associated with response. Further studies are needed to determine whether patients with genotype 4 and good predictors of response, including IL-28B CC, can benefit from shorter therapy.

      Conclusions

      Prediction of both progression of fibrosis and SVR are main issues in chronic hepatitis C. Major advances in genetics during the last decade, allow the identification of specific markers associated with either fibrosis progression or viral response. In particular, SNPs located within genes encoding MMP-1 and MMP-9, MCP1, PNLPA3, and vitamin D receptor have been associated with rapid progression of fibrosis. Moreover, CRS comprising 7 different SNPs was able to discriminate patients with high risk of developing a rapid fibrosis progression from those who will remain at low stages of fibrosis. Since the description of IL-28B SNPs association with both natural and treatment-induced HCV clearance, many studies have investigated the use of these factors for the prediction of viral response. IL-28B has been strongly associated with viral response in all reports, including large cohorts of patients.
      However, for the single patient, the use of IL-28B as a predictive factor of treatment outcome and decision to treat is not sufficient. IL-28B should probably be combined with other markers to increase baseline prediction of treatment outcome.
      During treatment, RVR is the strongest predictor of SVR even compared to IL28 SNPs. For individual patients, RVR may represent global predictors (host and viral) for response. Interestingly, the rs12979860 CC genotype was highly associated with viral response in patients who did not achieve an RVR [
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      ]. Recent studies have suggested that in triple therapy IL-28B genotyping identifies patients who might be eligible for shorter therapy [

      Jacobson IM, Catlett I, Marcellin P, Bzowej NH, Muir AJ, Adda M, et al. Telaprevir substantially improved SVR rates across all IL-28B genotypes in the advance trial. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

      ,

      Poordad F. IL-28B polymorphism predicts virologic response in patients with hepatitis C genotype 1 treated with boceprevir combination therapy. In: 46th Annual Meeting of the European Association for the study of the Liver (EASL); 2011.

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      Conflict of interest

      The Authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Financial support

      EE is supported by a fellowship from the French National Agency for Research on AIDS and Viral Hepatitis (ANRS).

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