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IL28B polymorphisms predict reduction of HCV RNA from the first day of therapy in chronic hepatitis C
Research Article
IL28B polymorphisms predict reduction of HCV RNA from the first day of therapy in chronic hepatitis C P.-Y. Bochud1,⇑, S. Bibert1, F. Negro2, B. Haagmans3, A. Soulier4,5, C. Ferrari6, G. Missale7, S. Zeuzem7, J.-M. Pawlotsky4,5, S. Schalm3, K. Hellstrand8, A.U. Neumann9, , M. Lagging8, for the DITTO-HCV study group 1
Service of Infectious Diseases, Department of Medicine, University Hospital and University of Lausanne, Switzerland; 2 University Hospital of Geneva, Geneva, Switzerland; 3Erasmus Medical Center, Rotterdam, The Netherlands; 4 National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Hôpital Henri Mondor, Université Paris-Est, Créteil, France; 5 Inserm U955, Créteil, France; 6Azienda Ospedaliera di Parma, Parma, Italy; 7University of Frankfurt, Frankfurt, Germany; 8 Department of Infectious Diseases/Virology, University of Gothenburg, Gothenburg, Sweden; 9Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Background & Aims: Single nucleotide polymorphisms (SNPs) associated with IL28B influence the outcome of peginterferona/ribavirin therapy of chronic hepatitis C virus (HCV) infection. We analyzed the kinetics of HCV RNA during therapy as a function of IL28B SNPs. Methods: IL28B SNPs rs8099917, rs12979860, and rs12980275 were genotyped in 242 HCV treatment-naïve Caucasian patients (67% genotype 1, 28% genotype 2 or 3) receiving peginterferona2a (180 lg weekly) and ribavirin (1000–1200 mg daily) with serial HCV-RNA quantifications. Associations between IL28B polymorphisms and early viral kinetics were assessed, accounting for relevant covariates. Results: In the multivariate analyses for genotype 1 patients, the T allele of rs12979860 (Trs12979860) was an independent risk factor for a less pronounced first phase HCV RNA decline (log10 0.89 IU/ml among T carriers vs. 2.06 among others, adjusted p <0.001) and lower rapid (15% vs. 38%, adjusted p = 0.007) and sustained viral response rates (48% vs. 66%, adjusted p <0.001). In univariate analyses, Trs12979860 was also associated with a reduced second phase decline (p = 0.002), but this association was no longer significant after adjustment for the first phase decline (adjusted p = 0.8). In genotype 2/3 patients, Trs12979860 was associated with a reduced first phase decline (adjusted p = 0.04), but not with a second phase decline. Conclusions: Polymorphisms in IL28B are strongly associated with the first phase viral decline during peginterferon-a/ribavirin therapy of chronic HCV infection, irrespective of HCV genotype.
Keywords: Hepatitis C; IL28 B; Viral kinetics. Received 14 October 2010; received in revised form 13 January 2011; accepted 31 January 2011; available online 25 February 2011 ⇑ Corresponding author. Address: Infectious Diseases Service, Rue du Bugnon 46, 1011 Lausanne CHUV, Switzerland. Tel.: +41 21 314 43 79; fax: +41 21 314 40 60. E-mail address: [email protected] (P.-Y. Bochud). These authors contributed equally to this work.
Ó 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Hepatitis C virus (HCV) infection affects a significant proportion of the world population [1] and patients with chronic HCV infection are at an increased risk of developing cirrhosis, liver failure, and hepatocellular carcinoma [2,3]. The current standard-of-care for chronic HCV infection comprises weekly injections of peginterferon-a (peg-IFN) in combination with daily oral ribavirin [4–6] for 24–48 weeks [7]. Approximately 50% of patients infected with the most common form of HCV (genotype 1) achieve a sustained viral response (SVR) with this treatment. The effectiveness of therapy is mirrored by the kinetics of plasma HCV RNA, which typically follow two main phases: a rapid first phase decline, referring to the viral decline during the initial day(s) after onset of treatment, and a slower second phase decline, which is usually defined as the reduction of HCV RNA levels from the second to the fourth week [8–11]. These phases are assumed to reflect, respectively, the antiviral action of interferon (first phase) and the loss of infected hepatocytes (second phase) [9]. Recently, several genomewide association studies (GWAs) have revealed that single nucleotide polymorphisms (SNPs) within or adjacent to IL28B (19q13), encoding for interferon-k3, predict spontaneous clearance of HCV [12,13] as well as the likelihood of SVR following therapy for chronic hepatitis C [12,14– 16]. Three of these SNPs are highly predictive of SVR in HCV genotype 1 infected patients, i.e. rs12979860 [13,14], rs12980275 [16], and rs8099917 [12,15,16]. The present study was designed to determine the association between IL28B-related SNPs and HCV kinetics during the early phases of therapy, with the aim of identifying predictive markers of treatment outcome and to shed light on functional aspects of IL28B variability.
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY Table 1. Patient’s characteristics.
All n
Genotype 1
Proportion
n 170 119 41.19 25.26
Proportion 1.00 0.70 (10.49) (3.81)
Genotypes 2 and 3 n 72 44 42.57 24.63
Proportion 1.00 0.61 (9.29) (3.28)
0.32 0.68 0.64 0.41 0.51
n Male sex Age, mean (SD) Body mass index, mean (SD) HCV genotype 1 2 3 Baseline ALT (>2*sup.norm) Fibrosis (Ishaak score >2)1 Inflammation (score >5)1 Steatosis2 Low (<30%) Moderate (30-70%) High (>70%) HCV RNA (log10)
242 163 41.60 25.07
1.00 0.67 (10.15) (3.66)
170 23 49 128 76 96
0.70 0.10 0.20 0.53 0.37 0.46
82 51 65
0.48 0.35 0.45
23 49 46 25 31
95 78 35
0.46 0.38 0.17
81 50 16
0.55 0.34 0.11
14 28 19
0.23 0.46 0.31
Baseline, mean (SD) IU/ml 1st phase decline (mean (SD) IU/ml absolute decline on day 1 absolute decline on day 4 maximal decline (day 1 or 4) 2nd phase decline, mean (SD) IU/ml/week Rapid viral response3 Sustained viral response IL28B polymorphisms rs8099917 TT TG GG HWE (exact P) rs12979860 CC CT TT HWE (exact P) rs12980275 AA AG GG HWE (exact P) Diplotypes TCA/TCA TCA/GTG TCA/TTG GTG/TTG TCA/TTA TTG/TTG GTG/GTG Other (carriage freq. <2%)
6.15
(0.76)
6.13
(0.75)
6.18
(0.81)
1.05 1.48 1.55 0.71 89 153
(0.82) (1.09) (1.05) (0.40) 0.39 0.63
0.84 1.11 1.20 0.59 34 90
(0.73) (1.86) (0.83) (0.34) 0.21 0.53
1.55 2.39 2.38 1.02 55 63
(0.78) (1.06) (1.05) (0.34) 0.83 0.88
150 84 8
0.62 0.35 0.03 0.4
97 66 7
0.57 0.39 0.04 0.4
53 18 1
0.74 0.25 0.01 1.0
90 118 34
0.37 0.49 0.14 0.7
44 96 30
0.26 0.56 0.18 0.09
46 22 4
0.64 0.31 0.06 0.5
98 110 34
0.40 0.45 0.14 0.8
52 88 30
0.31 0.52 0.18 0.5
46 22 4
0.64 0.31 0.06 0.5
88 66 40 16 9 9 7 7
0.36 0.27 0.17 0.07 0.04 0.04 0.03 0.03
44 51 35 14 8 8 6 4
0.26 0.30 0.21 0.08 0.05 0.05 0.04 0.02
44 15 5 2 1 1 1 3
0.61 0.21 0.07 0.03 0.01 0.01 0.01 0.04
1
N = 208, 2N = 207; 3N = 229.
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Research Article Table 2. Viral kinetics by IL28B polymorphisms in HCV genotype 1 infected patients.
1st phase decline (log10 IU/ml)
Baseline viral RNA (log10 IU/ml) Polymorphism rs8099917 TT TG GG rs12979860 CC CT TT rs12980275 AA AG GG Diplotypes TCA/TCA TCA/GTG TCA/TTG GTG/TTG TCA/TTA TTG/TTG GTG/GTG Other
p
Mean
SD
<0.001
0.63 0.54 0.38
0.30 0.39 0.36
<0.001
0.72 0.57 0.44
0.28 0.37 0.28
0.80 0.69 0.53
<0.001
0.71 0.57 0.43
0.72 0.61 0.72 0.43 0.33 0.32 0.93 0.60
Ref. <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
0.72 0.59 0.55 0.39 0.63 0.56 0.39 0.35
p
Mean
SD
0.005
1.49 0.82 0.65
0.86 0.57 0.89
0.042
2.06 0.95 0.71
0.72 0.64 0.60
0.71 0.81 0.57
0.08
1.87 0.94 0.77
0.72 0.76 0.65 0.61 0.64 0.49 0.55 1.84
Ref. 0.005 1.0 0.3 0.3 0.6 0.1 NA
2.06 0.87 1.13 0.67 0.83 1.02 0.74 0.13
n
Mean
SD
97 66 7
6.27 5.96 5.87
0.74 0.73 0.51
44 96 30
6.34 6.10 5.96
0.72 0.73 0.79
52 88 30
6.29 6.05 6.11
44 51 35 14 8 8 6 4
6.34 5.91 6.34 6.08 6.02 6.20 5.82 5.66
2nd phase slope (log10 IU/ml/week)
RVR
SVR
p
Prop
p
0.1
0.56 0.52 0.29
0.4
0.002
0.66 0.47 0.53
0.05
0.0023
0.36 0.15 0.11
0.002
0.65 0.47 0.50
0.03
0.05 0.02 0.001 0.5 0.2 0.02 0.04
0.38 0.21 0.09 0.00 0.25 0.38 0.00 0.00
Ref. 0.07 0.006 NA 0.5 1.0 NA NA
0.25 0.66 0.49 0.43 0.64 0.63 0.50 0.33
Ref. 0.1 0.4 0.5 0.2 0.2 0.4 0.8
p
Prop
0.021
0.25 0.16 0.00
0.0023
0.38 0.16 0.11
0.28 0.37 0.28 0.28 0.41 0.32 0.26 0.29 0.24 0.40 0.20
Baseline viral load was available in 170 patients, a few patients may have missing data for the following time points. 1st phase decline is the maximal value observed considering HCV RNA decline from baseline to day 1 or from baseline to day 4. RVR = rapid viral response; SVR = sustained viral response. For convenience, all p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles) which was the most likely model in most situations; when other models were more likely, the p values for those models are indicated by a footnote. 1 p = 0.02 for the additive model (considering a similar effect for each additional copy of the minor allele). 2 p = 0.03 for the additive model (considering a similar effect for each additional copy of the minor allele). 3 p <0.001 for the additive model (considering a similar effect for each additional copy of the minor allele).
Viral kinetics
B
1st phase RNA decline
HCV RNA (log IU/ml)
7 6 5 4 3 CC (n = 44)
CT (n = 96)
5 4 3 2 1 0 -1 -2
TT (n = 30)
CC (n = 44)
p = 0.04
C 1.5 1.0 0.5 0.0 -0.5
CT (n = 53) p = 0.8
CT (n = 96)
TT (n = 30)
p <0.001
2nd phase slope (1st decline ≤1 log)
2.0
CC (n = 2)
Quantification of HCV RNA in plasma was performed on days 0, 1, 4, 7, 8, 15, 22, 29, at end of treatment, and 24 months after completion of treatment, by using the Cobas Amplicor HCV Monitor RT-PCR system, version 2.0 (Roche Diagnostics, Branchburg, NJ). The first phase viral response was calculated using the maximal
982
Baseline viral RNA
TT (n = 20)
D HCV RNA (log IU/ml)
The study was performed among patients enrolled in the Dynamically Individualized Treatment of Hepatitis C Infection and Correlates of Viral/Host Dynamics (DITTO) study, a phase III, open-label, randomized, multi-center trial conducted between 2001 and 2003 in 9 centers from France, Germany, Greece, Israel, Italy, Netherlands, Spain, Sweden, and Switzerland, as described previously [17]. Briefly, the DITTO study enrolled 270 treatment-naïve patients with chronic hepatitis C, defined by a positive test for anti-HCV antibody, an HCV RNA level higher than 1000 IU/ml, and two serum alanine aminotransferase values above the upper limit of normal within 6 months of treatment initiation. Genotyping of HCV was performed using INNO-LiPA HCV II (Innogenetics NV, Ghent, Belgium). All patients were initially treated with a combination of peginterferon a-2a 180 lg weekly (Pegasys, F. Hoffmann-LaRoche, Basel, Switzerland) and ribavirin 1000–1200 mg daily (Copegus, F. Hoffmann-LaRoche). After 6 weeks of treatment, patients were given different regimens depending on their early viral kinetics. There were no major differences in treatment outcome for patients receiving individualized or standard therapy [17]. The study was approved by the local ethical committees, and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Informed consent was obtained from each participant.
HCV RNA (log IU/ml)
Patients
HCV RNA (log IU/ml)
A
Patients and methods
2.0
2nd phase slope (1st decline >1 log)
1.5 1.0 0.5 0.0 -0.5 CC (n = 42)
CT (n = 43)
TT (n = 10)
p = 0.4
FFig. 1. Viral parameters of IL28B rs12979860 genotypes in HCV genotype 1 patients.
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY Table 3. Multivariate analysis of viral kinetics in genotype 1-infected patients.
Baseline viral RNA (log10 IU/ml)
1st phase decline (log10 IU/ml)
2nd phase slope (log10 IU/ml/week)
SVR
RVR
Characteristic
p
p
p
OR (95% CI)
p
OR (95% CI)
p
Male sex Age Body mass index Baseline ALT (>2*sup.norm) Baseline viral RNA (log10 IU/ml) Fibrosis (Ishaak score >2) Inflammation (score >5) Steatosis Low (<30%) Moderate (30-70%) High (>70%) 1st phase decline (>median) IL28B rs12979860
0.1 0.03
0.2 1.0 0.07
0.8 0.3
1.28 (0.46-3.59) 0.97 (0.93-1.02) 0.89 (0.78-1.02)
0.6 0.5 0.1
0.34 (0.18-0.66)
0.001
3.32 (1.31-7.93) 1.01 (0.81-1.05) 0.91 (0.81-1.02) 0.48 (0.21-1.08) 0.22 (0.10-0.45)
0.01 0.7 0.1 0.1 <0.001
Ref. 0.49 (0.20-1.20) 0.83 (0.22-3.12)
0.1 0.8
0.22 (0.09-0.56)
0.0013,4
0.04 0.04
0.081
<0.001
Ref. 0.1 0.7 <0.001 0.8
0.17 (0.06-0.48)
<0.0012
Multivariate models were obtained by backward selection, using a p value <0.15 for removal from the model, with age and sex forced into the model. For convenience, all p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles) which was the most likely model in most situations; when other models were more likely, the p values for those models are indicated by a footnote. RVR = rapid viral response; SVR = sustained viral response. 1 p = 0.04 for the additive model (considering a similar effect for each additional copy of the minor allele). 2 The association between IL28B rs12979860 and RVR was lost when the 1st phase decline (OR = 3.73, 95% CI 1.18–11.8, p = 0.03, not shown) was entered into the model (for IL28B rs12979860, new OR = 0.41, 95% CI 0.12–1.42, adjusted p = 0.16, not shown). 3 The association between IL28B rs12979860 and SVR was lost when the 1st phase decline (OR = 4.08, 95% CI 1.4–11.8, adjusted p = 0.01, not shown) was entered into the model (for IL28B rs12979860, new OR = 0.55, 95% CI 0.17–1.75, adjusted p = 0.3, not shown). 4 The association between IL28B rs12979860 and SVR was lower when RVR (OR = 7.71, 95% CI, adjusted p = 0.004, not shown) was entered into the model (for IL28B rs12979860, new OR = 0.34, 95% CI 0.12–0.93, adjusted p = 0.03, not shown).
HCV RNA decline during the first 4 days following the treatment start (considering the viral load decline from the baseline level to that measured on day 1 and the decline from the baseline level to that measured on day 4; the maximal value was selected in each individual), and the second phase slope computed as previously reported [17]. Rapid viral response (RVR) was defined as having undetectable HCV RNA on day 29, and sustained viral response (SVR) as having undetectable HCV RNA 24 weeks after the completion of therapy (Intention-toTreat analysis). IL28B genotyping Among 270 DITTO patients, 242 Caucasian individuals had available blood samples and were included in the genetic sub-study. DNA from peripheral blood mononuclear cells was isolated using the QIAamp DNA mini kit (Qiagen) and quantified with a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific). DNA samples were genotyped for the IL28B rs8099917, rs12979860, and rs12980275 polymorphisms with TaqMan SNP genotyping assays (Applied Biosystems Inc., Foster City, CA), using the ABI 7500 Fast real time thermocycler, according to manufacturer’s protocols. TaqMan probes and primers were designed and synthesized using Applied Biosystems Inc. software (Supplementary Fig. 2). Automated allele calling was performed using SDS software from Applied Biosystems Inc. Positive and negative controls were used in each genotyping assay.
Statistical analyses Testing for Hardy-Weinberg equilibrium and pairwise linkage disequilibrium (LD) calculations were performed with the genhw and pwld packages in Stata, respectively (version 9.1, StataCorp., College Station, TX). Strong LD was defined by R2 >0.8. Haplotypes were inferred by using the haplo.stats package in R (version 2.8.1). The association of IL28B polymorphisms with continuous (baseline viral load, 1st phase viral decline and 2nd phase slopes) and dichotomic variables (rapid and sustained viral response) was assessed in linear and logistic regression models, respectively. After univariate analyses, multivariate analyses were performed for significant associations. Multivariate models were obtained by backward selection, using a p value <0.15 for removal from the model, with age and gender forced into the model. SNPs were tested using three models assuming one of the following modes of inheritance: dominant (comparing presence of one or two copies of the minor allele vs. none), recessive (comparing presence of two copies of the minor allele versus none or one copy), and additive (none, one or two copies of the minor allele were coded 0, 1 and 2, respectively, assuming greater effect with increased copy number of the minor allele). The dominant model was shown in the tables and the best fitting model, when other than dominant, was indicated by a footnote. For diplotypes, the most frequent diplotype in the study population was considered to be the wild-type diplotype and used as a reference against all other diplotypes in the regression models. Rare diplotypes (e.g. frequency <0.05) were grouped together.
Liver biopsies
Results Liver biopsies were performed in patients in the DITTO study within 12 months prior to enrollment, and only biopsy samples with a length exceeding 1.5 cm and containing more than six portal tracts were evaluated. Hematoxylin–eosin and Sirius Red stains were centrally staged and graded by two independent observers in a blinded fashion according to the Ishak protocol [18]. In addition, steatosis was graded as follows: absent = 0, less than 30% of hepatocytes involved = 1, 30–70% of hepatocytes involved = 2, or more than 70% of hepatocytes involved = 3 [19].
The minor allele frequencies of IL28B SNPs rs8099917, rs12979860, and rs12980275 were 21%, 38%, and 37%, respectively. Since the SNPs rs12979860 and rs12980275 were in strong linkage disequilibrium (R2 = 0.86, Supplementary Table 2), only results associated with rs12979860 are discussed below.
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Research Article Table 4. Viral kinetics of IL28B polymorphisms in HCV genotype 2/3 infected patients.
Baseline viral RNA (log10 IU/ml) Polymorphism rs8099917 TT TG GG rs12979860 CC CT TT rs12980275 AA AG GG Diplotypes TCA/TCA TCA/GTG TCA/TTG Other
1st phase decline (log10 IU/ml)
p
Mean
SD
0.3
2.57 1.90 1.54
0.98 1.11 N.A.
0.1
2.61 2.06 1.62
1.02 0.93 1.40
0.87 0.76 0.49
0.6
2.60 2.07 1.62
0.86 0.77 0.80 0.73
Ref. 0.4 0.8 0.3
2.64 1.92 2.59 1.72
n
Mean
SD
52 18 1
6.23 5.99 6.57
0.85 0.72 ND
45 22 4
6.28 6.00 5.93
0.85 0.78 0.49
45 22 4
6.21 6.14 5.93
43 15 5 8
6.26 6.05 6.17 5.95
2nd phase slope (log10 IU/ml/week)
RVR
p
Prop
0.5
0.88 0.71 1.00
0.1
0.88 0.76 0.67
0.34 0.35 0.23 0.33 0.37 0.32 0.37
p
Mean
SD
0.011
1.03 0.94 1.43
0.34 0.34 N.A.
0.012
1.07 0.90 1.12
0.33 0.36 0.23
1.03 0.93 1.40
0.023
1.06 0.90 1.12
1.03 1.05 0.49 0.94
Ref. 0.02 0.9 0.02
1.08 0.93 0.88 0.91
SVR
p
Prop
p
0.1
0.89 0.83 1.00
0.6
0.2
0.89 0.82 1.00
0.6
0.1
0.90 0.71 0.67
0.051
0.91 0.77 1.00
0.2
Ref. 0.1 0.2 0.2
0.90 0.71 0.80 0.71
Ref. 0.1 0.5 0.2
0.88 0.91 0.80 0.80
0.8 0.7 0.7
Baseline viral load was available in 71 patients, a few patients may have missing data for the following time points. N.A. = not assessable. RVR = rapid viral response; SVR = sustained viral response. For convenience, all p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles) which was the most likely model in most situations; when other models were more likely, the p values for those models are indicated by a footnote. 1 p = 0.01 for the additive model (considering a similar effect for each additional copy of the minor allele). 2 p = 0.009 for the additive model (considering a similar effect for each additional copy of the minor allele). 3 p = 0.01 for the additive model (considering a similar effect for each additional copy of the minor allele).
Consistent with previous data (Supplementary Fig. 1), the linkage disequilibrium between rs12979860 or rs12980275 and rs8099917 was smaller (R2 = 0.41 and R2 = 0.42, respectively). All SNPs were at Hardy-Weinberg equilibrium, but SNPs frequencies diverged across viral genotypes, with the minor alleles being more frequent among patients infected with genotype 1 (24% for rs8099917 and 46% for rs12979860) compared to those infected with genotype 2 or 3 (14% and 21%, respectively, Table 1). Approximately 95% of the study population carried one of the six most common diplotypes, with diplotype TCA/TCA, i.e. carriage of the favorable SNPs T at rs8099917, C at rs12979860 and A at rs12980275 on both copies of chromosome 19, being the most frequent (wild-type, 36%). In patients infected with HCV genotype 1, the minor alleles and rare diplotypes of IL28B SNPs were associated with lower baseline HCV RNA levels (rs8099917 and rs12979860), but had deleterious effects on viral kinetics (rs8099917 and rs12979860) and RVR and SVR rates (rs12979860 only, Table 2). Carriage of the rare T allele of rs12979860 influenced viral kinetics from the first day after the initiation of treatment (Figs. 1 and 2, Supplementary Table 3). This allele had a deleterious impact on the first phase viral decline (mean log10 decline 0.89 vs. 2.06, p <0.001, adjusted p <0.001) and was associated with lower rates of RVR (15% for T carriers vs. 38% for others, p = 0.002, adjusted p = 0.007, adjusted OR = 0.28, 95% CI 0.11– 0.70). This allele was also associated with a slower second phase viral decline (mean 0.54 for T carriers vs. 0.72 for others, p = 0.002), but this association was no longer significant in the multivariate model after adjustment for the first viral decline 984
(p = 0.8, Table 3), or after stratification (p = 0.8 and p = 0.4 for first decline 61 and >1 log IU/ml, Fig. 1C and D, respectively). The T allele was associated with a lower SVR rate (48% vs. 66%, adjusted OR = 0.17, 95% CI 0.06–0.48, adjusted p <0.001), but this association was smaller when RVR was entered into the model (adjusted OR = 0.34, 95% CI 0.12–0.93, adjusted p = 0.03). Among patients who did not achieve RVR, the T allele was still associated with a lower SVR rate (48% vs. 54%, adjusted p = 0.05). Among T allele carriers, the 1st phase decline was associated with SVR (adjusted OR = 4.30, 95% CI 1.64–11.3, adjusted p = 0.003). The T allele was also associated with lower baseline viral load (mean log10 6.06 IU/ml in T carriers vs. 6.34 in others, p = 0.04), although this association was not significant in the multivariate model (adjusted p = 0.08). Carriage of the G allele of rs8099917 influenced baseline viral load (mean log10 HCV RNA 5.87 among G carriers vs. 6.27 among others, p = 0.005, Supplementary Fig. 2), and this association remained significant in the multivariate analysis (p = 0.01). Again, the minor allele G of this SNP had a deleterious effect on the first phase viral decline (maximal decline 0.80 among G carriers vs. 1.49 among others, p <0.001, adjusted p <0.001). It was also associated with a reduced second phase decline (mean 0.52 for G carriers vs. 0.63 for others, p = 0.04), but this association was not significant in a multivariate model, after adjustment for the first phase decline (adjusted p = 0.8) or stratification (p = 0.8 and p = 0.3 for first decline 61 and >1 log IU/ml, respectively). In contrast, rs8099917 G was not significantly associated with lower RVR (15% in G allele carriers vs. 25% in the others, p = 0.1) or SVR rates (49% for G carriers vs. 56% in the others, p. = 0.4), but
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY A
Mean HCV RNA decline (log10 IU/ml)
Mean HCV RNA decline (log10 IU/ml)
rs12979860
B
HCV genotype 1 0 -1 -2 -3 -4 0
5
10
20
15
25
HCV genotypes 2 and 3 0 -1 -2 -3 -4
30
0
10
5
Days on PIFN/RIB treatment CT (n = 96)
CC (n = 44)
TT (n = 4)
30
CT (n = 22)
TT (n = 4)
D Mean HCV RNA decline (log10 IU/ml)
Mean HCV RNA decline (log10 IU/ml)
Diplotypes
C
25
20
rs12979860
rs12979860
CC (n = 44)
15
Days on PIFN/RIB treatment
0 -1 -2 -3 -4 0
5
10
15
20
25
30
0 -1 -2 -3 -4
0
5
Days on PIFN/RIB treatment TCA/TCA (n = 44) GTG/TTG ( n =13) OTHER (n = 10)
TCA/GTG (n = 51) TCA/TTA (n = 8)
10
15
20
25
30
Days on PIFN/RIB treatment
TCA/TTG (n = 35) TTG/TTG (n = 8)
TCA/TCA (n = 42) TCA/TTG (n = 5)
TCA/GTG (n = 15) OTHER (n = 8)
Fig. 2. Viral kinetics of IL28B polymorphisms and HCV genotypes. Numbers are mean absolute RNA decline (Log10 IU/ml) on indicated post-treatment days per IL28B polymorphisms.
this may be secondary to the smaller sample size of rs809991 G carriers as compared to T carriage of rs12979860. In order to assess non-genetic factors influencing RVR, genotype 1 infected patients were stratified according to rs12979860 (Supplementary Table 4). Among rs12979860 CC carriers, those who achieved RVR were younger (p = 0.05), and had a lower BMI (p = 0.04) as well as lower rates of severe steatosis (p = 0.04) than those not achieving RVR, with similar non-significant trends noted for inflammation (p = 0.07), fibrosis (p = 0.08), and gender (p = 0.1). Among rs12979860 CT/TT carriers, the only factor significantly associated with RVR was lower baseline viral load (p <0.001). In patients infected with HCV genotypes 2 or 3, IL28B SNPs (rs8099917, rs12979860) and some IL28B diplotypes were associated with the first phase viral decline, but not with other endpoints (Table 4). Again, the strongest association was observed for the rare allele T of rs12979860. This allele had a deleterious effect on the first phase decline in HCV RNA (mean log10 decline 1.88 in T carriers vs. 2.57 in others, p = 0.01, Supplementary Fig. 2B). As noted in the genotype 1 infected patients, the reduced decline among T carriers was observed already from the first day after the initiation of treatment (p <0.001, Supplementary
Fig. 3D). After adjustment for age and gender, the minor T allele of rs12979860 remained significantly associated with reduced first phase decline (p = 0.02). We examined whether IL28B polymorphisms were associated with baseline AST levels, fibrosis stage, and steatosis grade (Table 5). In genotype 1-infected patients, the percentage of individuals with severe steatosis (>30%) tended to increase in an additive mode according to rs8099917 genotypes (9% for TT, 13% for TG, and 17% for GG carriers), although this association was not significant (p = 0.4). In genotype 2/3 patients, the G allele of rs8099917 was significantly associated with lower ALT levels (72% high ALT for TT vs. 42% in TG and GG carriers, p = 0.02).
Discussion The first phase decline in HCV RNA during combination therapy, i.e. the rapid viral elimination during the first day or days of treatment, is assumed to result from the blocking of the production or release of virions, and thus primarily reflects the antiviral effectiveness of treatment [8,9]. This phase of viral elimination is reportedly dependent upon the fibrosis stage, the severity of
Journal of Hepatology 2011 vol. 55 j 980–988
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Research Article Table 5. Other characteristics of IL28B polymorphisms in HCV infected patients.
Genotype 1 Baseline ALT (>2*sup.norm) Polymorphism rs8099917 TT TG GG rs12979860 CC CT TT rs12980275 AA AG GG Diplotypes TCA/TCA TCA/GTG TCA/TTG Other
Steatosis1 (≥30%)
p
Prop
0.8
0.09 0.13 0.17
0.8
0.08 0.12 0.12
0.48 0.42 0.67 0.50 0.43 0.43 0.57
n
Prop
97 66 7
0.47 0.47 0.71
44 96 30
0.44 0.50 0.60
52 88 30 44 51 35 40
Genotypes 2 and 3 Fibrosis1 (Ishaak score >2)
Baseline ALT (>2*sup.norm)
p
n
Prop
0.4
53 18 1
0.72 0.44 0
0.9
46 22 4
0.72 0.45 0.75
0.6
0.35 0.37 0.27
1.0
46 22 4
Ref. 0.3 0.8 0.6
0.36 0.44 0.78 0.61
Ref. 0.5 0.6 0.4
44 15 5 8
p
Prop
0.4
0.32 0.40 0.33
0.5
0.36 0.36 0.28
1.0
0.09 0.13 0.08
Ref. 0.5 0.5 0.5
0.08 0.15 0.09 0.12
Steatosis2 (≥30%)
p
Prop
0.02
0.33 0.27 0
0.07
0.36 0.28 0
0.70 0.50 0.75 0.70 0.40 0.60 0.75
Fibrosis2 (Ishaak score >2)
p
Prop
p
0.5
0.44 0.33 0
0.4
0.3
0.49 0.22 0.50
0.1
0.2
0.36 0.28 0
0.3
0.46 0.28 0.50
0.3
Ref. 0.04 0.6 0.7
0.35 0.25 0.25 0.25
Ref. 0.5 0.7 0.6
0.49 0.25 0.25 0.38
Ref. 0.2 0.4 0.6
1 N = 147, 2N = 61. All p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles).
steatosis, gamma-glutamyl transpeptidase and insulinemia levels, but independent of baseline viral load or alanine aminotransferase (ALT) levels [20]. Similarly, the first phase viral decline predicts the rate of the slower second phase decline [21], as well as the final treatment outcome [20,22]. In contrast, the second phase decline, which is typically defined as the slower viral elimination from the second through the fourth week of therapy [8,10], is assumed to reflect the death rate of infected cells and immune-mediated clearance of residual infection [11]. This phase of viral decay has been demonstrated to be inversely correlated with baseline viral load and low baseline ALT levels [9]. Thompson et al. have previously been reported that IL28B polymorphisms influence viral decline, although the earliest quantification of HCV RNA occurred 2 weeks after the initiation of therapy [23]. In the present study, HCV RNA assessments were performed on days 0, 1, 4, 7, 8, 15, 22, and 29, allowing for distinct and very detailed evaluations of the first and second phase declines. We observed that IL28B polymorphisms significantly influenced viral load decline from the very first day of antiviral therapy. In addition, they were associated with the first phase of viral decline for all genotypes, but not with the second phase decline, when the latter was stratified for the first phase reduction in HCV RNA. In genotype 1-infected patients, the minor allele of rs12979860 was associated with lower rapid (15% vs. 38%) and sustained (48% vs. 66%) viral response rates, consistent with previous reports, although slightly less pronounced [23], possibly due to demographic differences between study populations. Considering that SNPs investigated are all located in close proximity to three cytokine genes (IL28A, IL28B, and IL29) belonging to the IFN-k (i.e. type III IFN) family, their association with the first phase, but not the second phase decline, was expected under the assumption that the SNPs influence the expression or function of this family of interferons. However, two seemingly counterintuitive observations were made in the present study: (i) that 986
the carriage of the risk alleles, although associated with poorer first phase viral decline, was also associated with lower baseline viral load, corroborating the previously reported findings [14,24], and (ii) that favorable alleles rs12979860 C, rs12980275 A, and rs8099917 T are significantly more common in the setting of HCV genotype 2 or 3 infection than 1 or 4 in a population consisting only of Caucasian patients, confirming the findings reported by McCarthy et al. [24]. Concerning the former finding, it is tempting to hypothesize that minor alleles may be associated with a higher expression of interferon-stimulated genes (ISGs), which would translate into both a lower pretreatment HCV RNA replication level and an impaired antiviral and/or early immune-activating properties of exogenously administered peginterferon-a. This would be in keeping with previous reports showing that transcriptional activation of ISGs [25–28] as well as elevated pre-treatment IP-10 [29] are strongly predictive of reduced therapeutic efficacy of interferon. The latter finding may result from SNP-related differences in the resolution of HCV infection [30], but definite demonstration of this hypothesis is still lacking. Il28B polymorphisms have been associated with liver histological features of chronic HCV infection. In two studies including genotype 1-infected patients, the minor allele of rs12980275 was associated with liver steatosis [31,32]. In the present study, with a smaller sample size, genotype 1-infected patients carrying the minor alleles of rs8099917 had a non-significant trend toward higher rates of severe steatosis. Similarly, in a study of 364 patients with chronic hepatitis C (primarily infected with genotypes 1 or 2), carriage of the minor allele of rs8099917 was associated with reduced liver inflammation and fibrosis [33]. In the present study, these observations regarding fibrosis stage could not be confirmed, although a significant association with lower ALT levels was noted in patients infected with genotype 2 or 3.
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY In conclusion, we propose that polymorphisms in IL28B are highly predictive of response to peginterferon-a and ribavirin combination therapy for HCV genotype 1 infection in analogy with previous reports [12,14–16,24], and that their impact on response is primarily associated with the first phase decline irrespective of HCV genotype. Thus, these findings provide compelling evidence for a major role of SNP polymorphisms in proximity of IL28B in predicting the eradication of HCV infection following therapeutic intervention, although the underlying mechanisms of action remain to be elucidated. The strong association between IL28B polymorphisms and the viral kinetics in the first days of therapy, possibly combined to other baseline features, such as the measurements of IP-10 and/or insulin resistance levels, as well as on-treatment viral kinetics, should allow for improved prediction of response to HCV combination therapy as well as for the development of tailored treatment durations, leading to more cost-effective and side effect sparing strategies.
Authors’ contributions P.Y.B. S.B.
F.N., S.Z.
B.H.
A.S., C.F., G.M., J.M.P., S.S., K.H.
A.U.N.
M.L.
Organised genotyping, performed statistical analyses, and wrote the manuscript Performed single-nucleotide polymorphisms genotyping and critically reviewed the manuscript Initiated and/or developed the DITTO study, performed clinical data and samples collection, contributed significantly in the genetic study design, and critically reviewed the manuscript Performed DNA extraction, edited clinical database, and critically reviewed the manuscript Initiated and/or developed and/or contributed the DITTO study, performed clinical data and samples collection and/or performed HCV RNA quantifications, and critically reviewed the manuscript Initiated and developed the DITTO study, performed part of statistical analyses, and critically reviewed the manuscript Developed and/or contributed to the DITTO study and wrote the manuscript
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support This study was supported by the European Community (QLK22000-00836), The Swedish Society Against Cancer (Cancerfonden), The Swedish Society of Medicine, the Swedish Research Council, The Torsten and Ragnar Söderberg Foundation, the Swedish Foundation for Strategic Research, the VIRGO consor-
tium (BSIK 03012), the Leenaards foundation, the Swiss National Science Foundation (SNF 32003B-127613), Hoffmann La Roche, and The Epicept Corporation. Acknowledgments We thank Tao Cai for calculating haplotypes.
Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jhep.2011.01.050. References [1] WHO. Hepatitis C-global prevalence (update). Weekly Epidemilogical Report 1999;74:425–427. [2] Lagging LM, Westin J, Svensson E, Aires N, Dhillon AP, Lindh M, et al. Progression of fibrosis in untreated patients with hepatitis C virus infection. Liver 2002;22:136–144. [3] Saito I, Miyamura T, Ohbayashi A, Harada H, Katayama T, Kikuchi S, et al. Hepatitis C virus infection is associated with the development of hepatocellular carcinoma. Proc Natl Acad Sci USA 1990;87:6547–6549. [4] Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales Jr FL, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347:975–982. [5] Hadziyannis SJ, Sette Jr H, Morgan TR, Balan V, Diago M, Marcellin P, 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;140:346–355. [6] Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, 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;358:958–965. [7] Lagging M, Wejstal R, Uhnoo I, Gerden B, Fischler B, Friman S, et al. Treatment of hepatitis C virus infection: updated Swedish Consensus recommendations. Scand J Infect Dis 2009;41:389–402. [8] Dahari H, Ribeiro RM, Perelson AS. Triphasic decline of hepatitis C virus RNA during antiviral therapy. Hepatology 2007;46:16–21. [9] Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferonalpha therapy. Science 1998;282:103–107. [10] Zeuzem S, Herrmann E, Lee JH, Fricke J, Neumann AU, Modi M, et al. Viral kinetics in patients with chronic hepatitis C treated with standard or peginterferon alpha2a. Gastroenterology 2001;120:1438–1447. [11] Herrmann E, Lee JH, Marinos G, Modi M, Zeuzem S. Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon. Hepatology 2003;37:1351–1358. [12] Rauch A, Kutalik Z, Descombes P, Cai T, Di Iulio J, Mueller T, et al. Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study. Gastroenterology 2010;138: 1338–1345, 1345 e1331–e1337. [13] Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O’Huigin C, et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 2009;461:798–801. [14] Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009;461:399–401. [15] Suppiah V, Moldovan M, Ahlenstiel G, Berg T, Weltman M, Abate ML, et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet 2009;41:1100–1104. [16] Tanaka Y, Nishida N, Sugiyama M, Kurosaki M, Matsuura K, Sakamoto N, et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009;41:1105–1109. [17] Zeuzem S, Pawlotsky JM, Lukasiewicz E, von Wagner M, Goulis I, Lurie Y, et al. International, multicenter, randomized, controlled study comparing dynamically individualized versus standard treatment in patients with chronic hepatitis C. J Hepatol 2005;43:250–257.
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IL28B polymorphisms predict reduction of HCV RNA from the first day of therapy in chronic hepatitis C P.-Y. Bochud1,⇑, S. Bibert1, F. Negro2, B. Haagmans3, A. Soulier4,5, C. Ferrari6, G. Missale7, S. Zeuzem7, J.-M. Pawlotsky4,5, S. Schalm3, K. Hellstrand8, A.U. Neumann9, , M. Lagging8, for the DITTO-HCV study group 1
Service of Infectious Diseases, Department of Medicine, University Hospital and University of Lausanne, Switzerland; 2 University Hospital of Geneva, Geneva, Switzerland; 3Erasmus Medical Center, Rotterdam, The Netherlands; 4 National Reference Center for Viral Hepatitis B, C and Delta, Department of Virology, Hôpital Henri Mondor, Université Paris-Est, Créteil, France; 5 Inserm U955, Créteil, France; 6Azienda Ospedaliera di Parma, Parma, Italy; 7University of Frankfurt, Frankfurt, Germany; 8 Department of Infectious Diseases/Virology, University of Gothenburg, Gothenburg, Sweden; 9Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Background & Aims: Single nucleotide polymorphisms (SNPs) associated with IL28B influence the outcome of peginterferona/ribavirin therapy of chronic hepatitis C virus (HCV) infection. We analyzed the kinetics of HCV RNA during therapy as a function of IL28B SNPs. Methods: IL28B SNPs rs8099917, rs12979860, and rs12980275 were genotyped in 242 HCV treatment-naïve Caucasian patients (67% genotype 1, 28% genotype 2 or 3) receiving peginterferona2a (180 lg weekly) and ribavirin (1000–1200 mg daily) with serial HCV-RNA quantifications. Associations between IL28B polymorphisms and early viral kinetics were assessed, accounting for relevant covariates. Results: In the multivariate analyses for genotype 1 patients, the T allele of rs12979860 (Trs12979860) was an independent risk factor for a less pronounced first phase HCV RNA decline (log10 0.89 IU/ml among T carriers vs. 2.06 among others, adjusted p <0.001) and lower rapid (15% vs. 38%, adjusted p = 0.007) and sustained viral response rates (48% vs. 66%, adjusted p <0.001). In univariate analyses, Trs12979860 was also associated with a reduced second phase decline (p = 0.002), but this association was no longer significant after adjustment for the first phase decline (adjusted p = 0.8). In genotype 2/3 patients, Trs12979860 was associated with a reduced first phase decline (adjusted p = 0.04), but not with a second phase decline. Conclusions: Polymorphisms in IL28B are strongly associated with the first phase viral decline during peginterferon-a/ribavirin therapy of chronic HCV infection, irrespective of HCV genotype.
Keywords: Hepatitis C; IL28 B; Viral kinetics. Received 14 October 2010; received in revised form 13 January 2011; accepted 31 January 2011; available online 25 February 2011 ⇑ Corresponding author. Address: Infectious Diseases Service, Rue du Bugnon 46, 1011 Lausanne CHUV, Switzerland. Tel.: +41 21 314 43 79; fax: +41 21 314 40 60. E-mail address: [email protected] (P.-Y. Bochud). These authors contributed equally to this work.
Ó 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Introduction Hepatitis C virus (HCV) infection affects a significant proportion of the world population [1] and patients with chronic HCV infection are at an increased risk of developing cirrhosis, liver failure, and hepatocellular carcinoma [2,3]. The current standard-of-care for chronic HCV infection comprises weekly injections of peginterferon-a (peg-IFN) in combination with daily oral ribavirin [4–6] for 24–48 weeks [7]. Approximately 50% of patients infected with the most common form of HCV (genotype 1) achieve a sustained viral response (SVR) with this treatment. The effectiveness of therapy is mirrored by the kinetics of plasma HCV RNA, which typically follow two main phases: a rapid first phase decline, referring to the viral decline during the initial day(s) after onset of treatment, and a slower second phase decline, which is usually defined as the reduction of HCV RNA levels from the second to the fourth week [8–11]. These phases are assumed to reflect, respectively, the antiviral action of interferon (first phase) and the loss of infected hepatocytes (second phase) [9]. Recently, several genomewide association studies (GWAs) have revealed that single nucleotide polymorphisms (SNPs) within or adjacent to IL28B (19q13), encoding for interferon-k3, predict spontaneous clearance of HCV [12,13] as well as the likelihood of SVR following therapy for chronic hepatitis C [12,14– 16]. Three of these SNPs are highly predictive of SVR in HCV genotype 1 infected patients, i.e. rs12979860 [13,14], rs12980275 [16], and rs8099917 [12,15,16]. The present study was designed to determine the association between IL28B-related SNPs and HCV kinetics during the early phases of therapy, with the aim of identifying predictive markers of treatment outcome and to shed light on functional aspects of IL28B variability.
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY Table 1. Patient’s characteristics.
All n
Genotype 1
Proportion
n 170 119 41.19 25.26
Proportion 1.00 0.70 (10.49) (3.81)
Genotypes 2 and 3 n 72 44 42.57 24.63
Proportion 1.00 0.61 (9.29) (3.28)
0.32 0.68 0.64 0.41 0.51
n Male sex Age, mean (SD) Body mass index, mean (SD) HCV genotype 1 2 3 Baseline ALT (>2*sup.norm) Fibrosis (Ishaak score >2)1 Inflammation (score >5)1 Steatosis2 Low (<30%) Moderate (30-70%) High (>70%) HCV RNA (log10)
242 163 41.60 25.07
1.00 0.67 (10.15) (3.66)
170 23 49 128 76 96
0.70 0.10 0.20 0.53 0.37 0.46
82 51 65
0.48 0.35 0.45
23 49 46 25 31
95 78 35
0.46 0.38 0.17
81 50 16
0.55 0.34 0.11
14 28 19
0.23 0.46 0.31
Baseline, mean (SD) IU/ml 1st phase decline (mean (SD) IU/ml absolute decline on day 1 absolute decline on day 4 maximal decline (day 1 or 4) 2nd phase decline, mean (SD) IU/ml/week Rapid viral response3 Sustained viral response IL28B polymorphisms rs8099917 TT TG GG HWE (exact P) rs12979860 CC CT TT HWE (exact P) rs12980275 AA AG GG HWE (exact P) Diplotypes TCA/TCA TCA/GTG TCA/TTG GTG/TTG TCA/TTA TTG/TTG GTG/GTG Other (carriage freq. <2%)
6.15
(0.76)
6.13
(0.75)
6.18
(0.81)
1.05 1.48 1.55 0.71 89 153
(0.82) (1.09) (1.05) (0.40) 0.39 0.63
0.84 1.11 1.20 0.59 34 90
(0.73) (1.86) (0.83) (0.34) 0.21 0.53
1.55 2.39 2.38 1.02 55 63
(0.78) (1.06) (1.05) (0.34) 0.83 0.88
150 84 8
0.62 0.35 0.03 0.4
97 66 7
0.57 0.39 0.04 0.4
53 18 1
0.74 0.25 0.01 1.0
90 118 34
0.37 0.49 0.14 0.7
44 96 30
0.26 0.56 0.18 0.09
46 22 4
0.64 0.31 0.06 0.5
98 110 34
0.40 0.45 0.14 0.8
52 88 30
0.31 0.52 0.18 0.5
46 22 4
0.64 0.31 0.06 0.5
88 66 40 16 9 9 7 7
0.36 0.27 0.17 0.07 0.04 0.04 0.03 0.03
44 51 35 14 8 8 6 4
0.26 0.30 0.21 0.08 0.05 0.05 0.04 0.02
44 15 5 2 1 1 1 3
0.61 0.21 0.07 0.03 0.01 0.01 0.01 0.04
1
N = 208, 2N = 207; 3N = 229.
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Research Article Table 2. Viral kinetics by IL28B polymorphisms in HCV genotype 1 infected patients.
1st phase decline (log10 IU/ml)
Baseline viral RNA (log10 IU/ml) Polymorphism rs8099917 TT TG GG rs12979860 CC CT TT rs12980275 AA AG GG Diplotypes TCA/TCA TCA/GTG TCA/TTG GTG/TTG TCA/TTA TTG/TTG GTG/GTG Other
p
Mean
SD
<0.001
0.63 0.54 0.38
0.30 0.39 0.36
<0.001
0.72 0.57 0.44
0.28 0.37 0.28
0.80 0.69 0.53
<0.001
0.71 0.57 0.43
0.72 0.61 0.72 0.43 0.33 0.32 0.93 0.60
Ref. <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
0.72 0.59 0.55 0.39 0.63 0.56 0.39 0.35
p
Mean
SD
0.005
1.49 0.82 0.65
0.86 0.57 0.89
0.042
2.06 0.95 0.71
0.72 0.64 0.60
0.71 0.81 0.57
0.08
1.87 0.94 0.77
0.72 0.76 0.65 0.61 0.64 0.49 0.55 1.84
Ref. 0.005 1.0 0.3 0.3 0.6 0.1 NA
2.06 0.87 1.13 0.67 0.83 1.02 0.74 0.13
n
Mean
SD
97 66 7
6.27 5.96 5.87
0.74 0.73 0.51
44 96 30
6.34 6.10 5.96
0.72 0.73 0.79
52 88 30
6.29 6.05 6.11
44 51 35 14 8 8 6 4
6.34 5.91 6.34 6.08 6.02 6.20 5.82 5.66
2nd phase slope (log10 IU/ml/week)
RVR
SVR
p
Prop
p
0.1
0.56 0.52 0.29
0.4
0.002
0.66 0.47 0.53
0.05
0.0023
0.36 0.15 0.11
0.002
0.65 0.47 0.50
0.03
0.05 0.02 0.001 0.5 0.2 0.02 0.04
0.38 0.21 0.09 0.00 0.25 0.38 0.00 0.00
Ref. 0.07 0.006 NA 0.5 1.0 NA NA
0.25 0.66 0.49 0.43 0.64 0.63 0.50 0.33
Ref. 0.1 0.4 0.5 0.2 0.2 0.4 0.8
p
Prop
0.021
0.25 0.16 0.00
0.0023
0.38 0.16 0.11
0.28 0.37 0.28 0.28 0.41 0.32 0.26 0.29 0.24 0.40 0.20
Baseline viral load was available in 170 patients, a few patients may have missing data for the following time points. 1st phase decline is the maximal value observed considering HCV RNA decline from baseline to day 1 or from baseline to day 4. RVR = rapid viral response; SVR = sustained viral response. For convenience, all p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles) which was the most likely model in most situations; when other models were more likely, the p values for those models are indicated by a footnote. 1 p = 0.02 for the additive model (considering a similar effect for each additional copy of the minor allele). 2 p = 0.03 for the additive model (considering a similar effect for each additional copy of the minor allele). 3 p <0.001 for the additive model (considering a similar effect for each additional copy of the minor allele).
Viral kinetics
B
1st phase RNA decline
HCV RNA (log IU/ml)
7 6 5 4 3 CC (n = 44)
CT (n = 96)
5 4 3 2 1 0 -1 -2
TT (n = 30)
CC (n = 44)
p = 0.04
C 1.5 1.0 0.5 0.0 -0.5
CT (n = 53) p = 0.8
CT (n = 96)
TT (n = 30)
p <0.001
2nd phase slope (1st decline ≤1 log)
2.0
CC (n = 2)
Quantification of HCV RNA in plasma was performed on days 0, 1, 4, 7, 8, 15, 22, 29, at end of treatment, and 24 months after completion of treatment, by using the Cobas Amplicor HCV Monitor RT-PCR system, version 2.0 (Roche Diagnostics, Branchburg, NJ). The first phase viral response was calculated using the maximal
982
Baseline viral RNA
TT (n = 20)
D HCV RNA (log IU/ml)
The study was performed among patients enrolled in the Dynamically Individualized Treatment of Hepatitis C Infection and Correlates of Viral/Host Dynamics (DITTO) study, a phase III, open-label, randomized, multi-center trial conducted between 2001 and 2003 in 9 centers from France, Germany, Greece, Israel, Italy, Netherlands, Spain, Sweden, and Switzerland, as described previously [17]. Briefly, the DITTO study enrolled 270 treatment-naïve patients with chronic hepatitis C, defined by a positive test for anti-HCV antibody, an HCV RNA level higher than 1000 IU/ml, and two serum alanine aminotransferase values above the upper limit of normal within 6 months of treatment initiation. Genotyping of HCV was performed using INNO-LiPA HCV II (Innogenetics NV, Ghent, Belgium). All patients were initially treated with a combination of peginterferon a-2a 180 lg weekly (Pegasys, F. Hoffmann-LaRoche, Basel, Switzerland) and ribavirin 1000–1200 mg daily (Copegus, F. Hoffmann-LaRoche). After 6 weeks of treatment, patients were given different regimens depending on their early viral kinetics. There were no major differences in treatment outcome for patients receiving individualized or standard therapy [17]. The study was approved by the local ethical committees, and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Informed consent was obtained from each participant.
HCV RNA (log IU/ml)
Patients
HCV RNA (log IU/ml)
A
Patients and methods
2.0
2nd phase slope (1st decline >1 log)
1.5 1.0 0.5 0.0 -0.5 CC (n = 42)
CT (n = 43)
TT (n = 10)
p = 0.4
FFig. 1. Viral parameters of IL28B rs12979860 genotypes in HCV genotype 1 patients.
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY Table 3. Multivariate analysis of viral kinetics in genotype 1-infected patients.
Baseline viral RNA (log10 IU/ml)
1st phase decline (log10 IU/ml)
2nd phase slope (log10 IU/ml/week)
SVR
RVR
Characteristic
p
p
p
OR (95% CI)
p
OR (95% CI)
p
Male sex Age Body mass index Baseline ALT (>2*sup.norm) Baseline viral RNA (log10 IU/ml) Fibrosis (Ishaak score >2) Inflammation (score >5) Steatosis Low (<30%) Moderate (30-70%) High (>70%) 1st phase decline (>median) IL28B rs12979860
0.1 0.03
0.2 1.0 0.07
0.8 0.3
1.28 (0.46-3.59) 0.97 (0.93-1.02) 0.89 (0.78-1.02)
0.6 0.5 0.1
0.34 (0.18-0.66)
0.001
3.32 (1.31-7.93) 1.01 (0.81-1.05) 0.91 (0.81-1.02) 0.48 (0.21-1.08) 0.22 (0.10-0.45)
0.01 0.7 0.1 0.1 <0.001
Ref. 0.49 (0.20-1.20) 0.83 (0.22-3.12)
0.1 0.8
0.22 (0.09-0.56)
0.0013,4
0.04 0.04
0.081
<0.001
Ref. 0.1 0.7 <0.001 0.8
0.17 (0.06-0.48)
<0.0012
Multivariate models were obtained by backward selection, using a p value <0.15 for removal from the model, with age and sex forced into the model. For convenience, all p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles) which was the most likely model in most situations; when other models were more likely, the p values for those models are indicated by a footnote. RVR = rapid viral response; SVR = sustained viral response. 1 p = 0.04 for the additive model (considering a similar effect for each additional copy of the minor allele). 2 The association between IL28B rs12979860 and RVR was lost when the 1st phase decline (OR = 3.73, 95% CI 1.18–11.8, p = 0.03, not shown) was entered into the model (for IL28B rs12979860, new OR = 0.41, 95% CI 0.12–1.42, adjusted p = 0.16, not shown). 3 The association between IL28B rs12979860 and SVR was lost when the 1st phase decline (OR = 4.08, 95% CI 1.4–11.8, adjusted p = 0.01, not shown) was entered into the model (for IL28B rs12979860, new OR = 0.55, 95% CI 0.17–1.75, adjusted p = 0.3, not shown). 4 The association between IL28B rs12979860 and SVR was lower when RVR (OR = 7.71, 95% CI, adjusted p = 0.004, not shown) was entered into the model (for IL28B rs12979860, new OR = 0.34, 95% CI 0.12–0.93, adjusted p = 0.03, not shown).
HCV RNA decline during the first 4 days following the treatment start (considering the viral load decline from the baseline level to that measured on day 1 and the decline from the baseline level to that measured on day 4; the maximal value was selected in each individual), and the second phase slope computed as previously reported [17]. Rapid viral response (RVR) was defined as having undetectable HCV RNA on day 29, and sustained viral response (SVR) as having undetectable HCV RNA 24 weeks after the completion of therapy (Intention-toTreat analysis). IL28B genotyping Among 270 DITTO patients, 242 Caucasian individuals had available blood samples and were included in the genetic sub-study. DNA from peripheral blood mononuclear cells was isolated using the QIAamp DNA mini kit (Qiagen) and quantified with a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific). DNA samples were genotyped for the IL28B rs8099917, rs12979860, and rs12980275 polymorphisms with TaqMan SNP genotyping assays (Applied Biosystems Inc., Foster City, CA), using the ABI 7500 Fast real time thermocycler, according to manufacturer’s protocols. TaqMan probes and primers were designed and synthesized using Applied Biosystems Inc. software (Supplementary Fig. 2). Automated allele calling was performed using SDS software from Applied Biosystems Inc. Positive and negative controls were used in each genotyping assay.
Statistical analyses Testing for Hardy-Weinberg equilibrium and pairwise linkage disequilibrium (LD) calculations were performed with the genhw and pwld packages in Stata, respectively (version 9.1, StataCorp., College Station, TX). Strong LD was defined by R2 >0.8. Haplotypes were inferred by using the haplo.stats package in R (version 2.8.1). The association of IL28B polymorphisms with continuous (baseline viral load, 1st phase viral decline and 2nd phase slopes) and dichotomic variables (rapid and sustained viral response) was assessed in linear and logistic regression models, respectively. After univariate analyses, multivariate analyses were performed for significant associations. Multivariate models were obtained by backward selection, using a p value <0.15 for removal from the model, with age and gender forced into the model. SNPs were tested using three models assuming one of the following modes of inheritance: dominant (comparing presence of one or two copies of the minor allele vs. none), recessive (comparing presence of two copies of the minor allele versus none or one copy), and additive (none, one or two copies of the minor allele were coded 0, 1 and 2, respectively, assuming greater effect with increased copy number of the minor allele). The dominant model was shown in the tables and the best fitting model, when other than dominant, was indicated by a footnote. For diplotypes, the most frequent diplotype in the study population was considered to be the wild-type diplotype and used as a reference against all other diplotypes in the regression models. Rare diplotypes (e.g. frequency <0.05) were grouped together.
Liver biopsies
Results Liver biopsies were performed in patients in the DITTO study within 12 months prior to enrollment, and only biopsy samples with a length exceeding 1.5 cm and containing more than six portal tracts were evaluated. Hematoxylin–eosin and Sirius Red stains were centrally staged and graded by two independent observers in a blinded fashion according to the Ishak protocol [18]. In addition, steatosis was graded as follows: absent = 0, less than 30% of hepatocytes involved = 1, 30–70% of hepatocytes involved = 2, or more than 70% of hepatocytes involved = 3 [19].
The minor allele frequencies of IL28B SNPs rs8099917, rs12979860, and rs12980275 were 21%, 38%, and 37%, respectively. Since the SNPs rs12979860 and rs12980275 were in strong linkage disequilibrium (R2 = 0.86, Supplementary Table 2), only results associated with rs12979860 are discussed below.
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Research Article Table 4. Viral kinetics of IL28B polymorphisms in HCV genotype 2/3 infected patients.
Baseline viral RNA (log10 IU/ml) Polymorphism rs8099917 TT TG GG rs12979860 CC CT TT rs12980275 AA AG GG Diplotypes TCA/TCA TCA/GTG TCA/TTG Other
1st phase decline (log10 IU/ml)
p
Mean
SD
0.3
2.57 1.90 1.54
0.98 1.11 N.A.
0.1
2.61 2.06 1.62
1.02 0.93 1.40
0.87 0.76 0.49
0.6
2.60 2.07 1.62
0.86 0.77 0.80 0.73
Ref. 0.4 0.8 0.3
2.64 1.92 2.59 1.72
n
Mean
SD
52 18 1
6.23 5.99 6.57
0.85 0.72 ND
45 22 4
6.28 6.00 5.93
0.85 0.78 0.49
45 22 4
6.21 6.14 5.93
43 15 5 8
6.26 6.05 6.17 5.95
2nd phase slope (log10 IU/ml/week)
RVR
p
Prop
0.5
0.88 0.71 1.00
0.1
0.88 0.76 0.67
0.34 0.35 0.23 0.33 0.37 0.32 0.37
p
Mean
SD
0.011
1.03 0.94 1.43
0.34 0.34 N.A.
0.012
1.07 0.90 1.12
0.33 0.36 0.23
1.03 0.93 1.40
0.023
1.06 0.90 1.12
1.03 1.05 0.49 0.94
Ref. 0.02 0.9 0.02
1.08 0.93 0.88 0.91
SVR
p
Prop
p
0.1
0.89 0.83 1.00
0.6
0.2
0.89 0.82 1.00
0.6
0.1
0.90 0.71 0.67
0.051
0.91 0.77 1.00
0.2
Ref. 0.1 0.2 0.2
0.90 0.71 0.80 0.71
Ref. 0.1 0.5 0.2
0.88 0.91 0.80 0.80
0.8 0.7 0.7
Baseline viral load was available in 71 patients, a few patients may have missing data for the following time points. N.A. = not assessable. RVR = rapid viral response; SVR = sustained viral response. For convenience, all p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles) which was the most likely model in most situations; when other models were more likely, the p values for those models are indicated by a footnote. 1 p = 0.01 for the additive model (considering a similar effect for each additional copy of the minor allele). 2 p = 0.009 for the additive model (considering a similar effect for each additional copy of the minor allele). 3 p = 0.01 for the additive model (considering a similar effect for each additional copy of the minor allele).
Consistent with previous data (Supplementary Fig. 1), the linkage disequilibrium between rs12979860 or rs12980275 and rs8099917 was smaller (R2 = 0.41 and R2 = 0.42, respectively). All SNPs were at Hardy-Weinberg equilibrium, but SNPs frequencies diverged across viral genotypes, with the minor alleles being more frequent among patients infected with genotype 1 (24% for rs8099917 and 46% for rs12979860) compared to those infected with genotype 2 or 3 (14% and 21%, respectively, Table 1). Approximately 95% of the study population carried one of the six most common diplotypes, with diplotype TCA/TCA, i.e. carriage of the favorable SNPs T at rs8099917, C at rs12979860 and A at rs12980275 on both copies of chromosome 19, being the most frequent (wild-type, 36%). In patients infected with HCV genotype 1, the minor alleles and rare diplotypes of IL28B SNPs were associated with lower baseline HCV RNA levels (rs8099917 and rs12979860), but had deleterious effects on viral kinetics (rs8099917 and rs12979860) and RVR and SVR rates (rs12979860 only, Table 2). Carriage of the rare T allele of rs12979860 influenced viral kinetics from the first day after the initiation of treatment (Figs. 1 and 2, Supplementary Table 3). This allele had a deleterious impact on the first phase viral decline (mean log10 decline 0.89 vs. 2.06, p <0.001, adjusted p <0.001) and was associated with lower rates of RVR (15% for T carriers vs. 38% for others, p = 0.002, adjusted p = 0.007, adjusted OR = 0.28, 95% CI 0.11– 0.70). This allele was also associated with a slower second phase viral decline (mean 0.54 for T carriers vs. 0.72 for others, p = 0.002), but this association was no longer significant in the multivariate model after adjustment for the first viral decline 984
(p = 0.8, Table 3), or after stratification (p = 0.8 and p = 0.4 for first decline 61 and >1 log IU/ml, Fig. 1C and D, respectively). The T allele was associated with a lower SVR rate (48% vs. 66%, adjusted OR = 0.17, 95% CI 0.06–0.48, adjusted p <0.001), but this association was smaller when RVR was entered into the model (adjusted OR = 0.34, 95% CI 0.12–0.93, adjusted p = 0.03). Among patients who did not achieve RVR, the T allele was still associated with a lower SVR rate (48% vs. 54%, adjusted p = 0.05). Among T allele carriers, the 1st phase decline was associated with SVR (adjusted OR = 4.30, 95% CI 1.64–11.3, adjusted p = 0.003). The T allele was also associated with lower baseline viral load (mean log10 6.06 IU/ml in T carriers vs. 6.34 in others, p = 0.04), although this association was not significant in the multivariate model (adjusted p = 0.08). Carriage of the G allele of rs8099917 influenced baseline viral load (mean log10 HCV RNA 5.87 among G carriers vs. 6.27 among others, p = 0.005, Supplementary Fig. 2), and this association remained significant in the multivariate analysis (p = 0.01). Again, the minor allele G of this SNP had a deleterious effect on the first phase viral decline (maximal decline 0.80 among G carriers vs. 1.49 among others, p <0.001, adjusted p <0.001). It was also associated with a reduced second phase decline (mean 0.52 for G carriers vs. 0.63 for others, p = 0.04), but this association was not significant in a multivariate model, after adjustment for the first phase decline (adjusted p = 0.8) or stratification (p = 0.8 and p = 0.3 for first decline 61 and >1 log IU/ml, respectively). In contrast, rs8099917 G was not significantly associated with lower RVR (15% in G allele carriers vs. 25% in the others, p = 0.1) or SVR rates (49% for G carriers vs. 56% in the others, p. = 0.4), but
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY A
Mean HCV RNA decline (log10 IU/ml)
Mean HCV RNA decline (log10 IU/ml)
rs12979860
B
HCV genotype 1 0 -1 -2 -3 -4 0
5
10
20
15
25
HCV genotypes 2 and 3 0 -1 -2 -3 -4
30
0
10
5
Days on PIFN/RIB treatment CT (n = 96)
CC (n = 44)
TT (n = 4)
30
CT (n = 22)
TT (n = 4)
D Mean HCV RNA decline (log10 IU/ml)
Mean HCV RNA decline (log10 IU/ml)
Diplotypes
C
25
20
rs12979860
rs12979860
CC (n = 44)
15
Days on PIFN/RIB treatment
0 -1 -2 -3 -4 0
5
10
15
20
25
30
0 -1 -2 -3 -4
0
5
Days on PIFN/RIB treatment TCA/TCA (n = 44) GTG/TTG ( n =13) OTHER (n = 10)
TCA/GTG (n = 51) TCA/TTA (n = 8)
10
15
20
25
30
Days on PIFN/RIB treatment
TCA/TTG (n = 35) TTG/TTG (n = 8)
TCA/TCA (n = 42) TCA/TTG (n = 5)
TCA/GTG (n = 15) OTHER (n = 8)
Fig. 2. Viral kinetics of IL28B polymorphisms and HCV genotypes. Numbers are mean absolute RNA decline (Log10 IU/ml) on indicated post-treatment days per IL28B polymorphisms.
this may be secondary to the smaller sample size of rs809991 G carriers as compared to T carriage of rs12979860. In order to assess non-genetic factors influencing RVR, genotype 1 infected patients were stratified according to rs12979860 (Supplementary Table 4). Among rs12979860 CC carriers, those who achieved RVR were younger (p = 0.05), and had a lower BMI (p = 0.04) as well as lower rates of severe steatosis (p = 0.04) than those not achieving RVR, with similar non-significant trends noted for inflammation (p = 0.07), fibrosis (p = 0.08), and gender (p = 0.1). Among rs12979860 CT/TT carriers, the only factor significantly associated with RVR was lower baseline viral load (p <0.001). In patients infected with HCV genotypes 2 or 3, IL28B SNPs (rs8099917, rs12979860) and some IL28B diplotypes were associated with the first phase viral decline, but not with other endpoints (Table 4). Again, the strongest association was observed for the rare allele T of rs12979860. This allele had a deleterious effect on the first phase decline in HCV RNA (mean log10 decline 1.88 in T carriers vs. 2.57 in others, p = 0.01, Supplementary Fig. 2B). As noted in the genotype 1 infected patients, the reduced decline among T carriers was observed already from the first day after the initiation of treatment (p <0.001, Supplementary
Fig. 3D). After adjustment for age and gender, the minor T allele of rs12979860 remained significantly associated with reduced first phase decline (p = 0.02). We examined whether IL28B polymorphisms were associated with baseline AST levels, fibrosis stage, and steatosis grade (Table 5). In genotype 1-infected patients, the percentage of individuals with severe steatosis (>30%) tended to increase in an additive mode according to rs8099917 genotypes (9% for TT, 13% for TG, and 17% for GG carriers), although this association was not significant (p = 0.4). In genotype 2/3 patients, the G allele of rs8099917 was significantly associated with lower ALT levels (72% high ALT for TT vs. 42% in TG and GG carriers, p = 0.02).
Discussion The first phase decline in HCV RNA during combination therapy, i.e. the rapid viral elimination during the first day or days of treatment, is assumed to result from the blocking of the production or release of virions, and thus primarily reflects the antiviral effectiveness of treatment [8,9]. This phase of viral elimination is reportedly dependent upon the fibrosis stage, the severity of
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Research Article Table 5. Other characteristics of IL28B polymorphisms in HCV infected patients.
Genotype 1 Baseline ALT (>2*sup.norm) Polymorphism rs8099917 TT TG GG rs12979860 CC CT TT rs12980275 AA AG GG Diplotypes TCA/TCA TCA/GTG TCA/TTG Other
Steatosis1 (≥30%)
p
Prop
0.8
0.09 0.13 0.17
0.8
0.08 0.12 0.12
0.48 0.42 0.67 0.50 0.43 0.43 0.57
n
Prop
97 66 7
0.47 0.47 0.71
44 96 30
0.44 0.50 0.60
52 88 30 44 51 35 40
Genotypes 2 and 3 Fibrosis1 (Ishaak score >2)
Baseline ALT (>2*sup.norm)
p
n
Prop
0.4
53 18 1
0.72 0.44 0
0.9
46 22 4
0.72 0.45 0.75
0.6
0.35 0.37 0.27
1.0
46 22 4
Ref. 0.3 0.8 0.6
0.36 0.44 0.78 0.61
Ref. 0.5 0.6 0.4
44 15 5 8
p
Prop
0.4
0.32 0.40 0.33
0.5
0.36 0.36 0.28
1.0
0.09 0.13 0.08
Ref. 0.5 0.5 0.5
0.08 0.15 0.09 0.12
Steatosis2 (≥30%)
p
Prop
0.02
0.33 0.27 0
0.07
0.36 0.28 0
0.70 0.50 0.75 0.70 0.40 0.60 0.75
Fibrosis2 (Ishaak score >2)
p
Prop
p
0.5
0.44 0.33 0
0.4
0.3
0.49 0.22 0.50
0.1
0.2
0.36 0.28 0
0.3
0.46 0.28 0.50
0.3
Ref. 0.04 0.6 0.7
0.35 0.25 0.25 0.25
Ref. 0.5 0.7 0.6
0.49 0.25 0.25 0.38
Ref. 0.2 0.4 0.6
1 N = 147, 2N = 61. All p values are given for the dominant model (comparing presence of 1 or 2 minor alleles vs. 0 alleles).
steatosis, gamma-glutamyl transpeptidase and insulinemia levels, but independent of baseline viral load or alanine aminotransferase (ALT) levels [20]. Similarly, the first phase viral decline predicts the rate of the slower second phase decline [21], as well as the final treatment outcome [20,22]. In contrast, the second phase decline, which is typically defined as the slower viral elimination from the second through the fourth week of therapy [8,10], is assumed to reflect the death rate of infected cells and immune-mediated clearance of residual infection [11]. This phase of viral decay has been demonstrated to be inversely correlated with baseline viral load and low baseline ALT levels [9]. Thompson et al. have previously been reported that IL28B polymorphisms influence viral decline, although the earliest quantification of HCV RNA occurred 2 weeks after the initiation of therapy [23]. In the present study, HCV RNA assessments were performed on days 0, 1, 4, 7, 8, 15, 22, and 29, allowing for distinct and very detailed evaluations of the first and second phase declines. We observed that IL28B polymorphisms significantly influenced viral load decline from the very first day of antiviral therapy. In addition, they were associated with the first phase of viral decline for all genotypes, but not with the second phase decline, when the latter was stratified for the first phase reduction in HCV RNA. In genotype 1-infected patients, the minor allele of rs12979860 was associated with lower rapid (15% vs. 38%) and sustained (48% vs. 66%) viral response rates, consistent with previous reports, although slightly less pronounced [23], possibly due to demographic differences between study populations. Considering that SNPs investigated are all located in close proximity to three cytokine genes (IL28A, IL28B, and IL29) belonging to the IFN-k (i.e. type III IFN) family, their association with the first phase, but not the second phase decline, was expected under the assumption that the SNPs influence the expression or function of this family of interferons. However, two seemingly counterintuitive observations were made in the present study: (i) that 986
the carriage of the risk alleles, although associated with poorer first phase viral decline, was also associated with lower baseline viral load, corroborating the previously reported findings [14,24], and (ii) that favorable alleles rs12979860 C, rs12980275 A, and rs8099917 T are significantly more common in the setting of HCV genotype 2 or 3 infection than 1 or 4 in a population consisting only of Caucasian patients, confirming the findings reported by McCarthy et al. [24]. Concerning the former finding, it is tempting to hypothesize that minor alleles may be associated with a higher expression of interferon-stimulated genes (ISGs), which would translate into both a lower pretreatment HCV RNA replication level and an impaired antiviral and/or early immune-activating properties of exogenously administered peginterferon-a. This would be in keeping with previous reports showing that transcriptional activation of ISGs [25–28] as well as elevated pre-treatment IP-10 [29] are strongly predictive of reduced therapeutic efficacy of interferon. The latter finding may result from SNP-related differences in the resolution of HCV infection [30], but definite demonstration of this hypothesis is still lacking. Il28B polymorphisms have been associated with liver histological features of chronic HCV infection. In two studies including genotype 1-infected patients, the minor allele of rs12980275 was associated with liver steatosis [31,32]. In the present study, with a smaller sample size, genotype 1-infected patients carrying the minor alleles of rs8099917 had a non-significant trend toward higher rates of severe steatosis. Similarly, in a study of 364 patients with chronic hepatitis C (primarily infected with genotypes 1 or 2), carriage of the minor allele of rs8099917 was associated with reduced liver inflammation and fibrosis [33]. In the present study, these observations regarding fibrosis stage could not be confirmed, although a significant association with lower ALT levels was noted in patients infected with genotype 2 or 3.
Journal of Hepatology 2011 vol. 55 j 980–988
JOURNAL OF HEPATOLOGY In conclusion, we propose that polymorphisms in IL28B are highly predictive of response to peginterferon-a and ribavirin combination therapy for HCV genotype 1 infection in analogy with previous reports [12,14–16,24], and that their impact on response is primarily associated with the first phase decline irrespective of HCV genotype. Thus, these findings provide compelling evidence for a major role of SNP polymorphisms in proximity of IL28B in predicting the eradication of HCV infection following therapeutic intervention, although the underlying mechanisms of action remain to be elucidated. The strong association between IL28B polymorphisms and the viral kinetics in the first days of therapy, possibly combined to other baseline features, such as the measurements of IP-10 and/or insulin resistance levels, as well as on-treatment viral kinetics, should allow for improved prediction of response to HCV combination therapy as well as for the development of tailored treatment durations, leading to more cost-effective and side effect sparing strategies.
Authors’ contributions P.Y.B. S.B.
F.N., S.Z.
B.H.
A.S., C.F., G.M., J.M.P., S.S., K.H.
A.U.N.
M.L.
Organised genotyping, performed statistical analyses, and wrote the manuscript Performed single-nucleotide polymorphisms genotyping and critically reviewed the manuscript Initiated and/or developed the DITTO study, performed clinical data and samples collection, contributed significantly in the genetic study design, and critically reviewed the manuscript Performed DNA extraction, edited clinical database, and critically reviewed the manuscript Initiated and/or developed and/or contributed the DITTO study, performed clinical data and samples collection and/or performed HCV RNA quantifications, and critically reviewed the manuscript Initiated and developed the DITTO study, performed part of statistical analyses, and critically reviewed the manuscript Developed and/or contributed to the DITTO study and wrote the manuscript
Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Financial support This study was supported by the European Community (QLK22000-00836), The Swedish Society Against Cancer (Cancerfonden), The Swedish Society of Medicine, the Swedish Research Council, The Torsten and Ragnar Söderberg Foundation, the Swedish Foundation for Strategic Research, the VIRGO consor-
tium (BSIK 03012), the Leenaards foundation, the Swiss National Science Foundation (SNF 32003B-127613), Hoffmann La Roche, and The Epicept Corporation. Acknowledgments We thank Tao Cai for calculating haplotypes.
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