Amino acid substitution in HCV core region and genetic variation near IL28B gene predict viral response to telaprevir with peginterferon and ribavirin

Amino acid substitution in HCV core region and genetic variation near IL28B gene predict viral response to telaprevir with peginterferon and ribavirin

Hepatology - Accepted Preprint (see graphs at bottom of report)

26 Mar 2010

Accepted Articles

These articles have been accepted for publication in Hepatology and are currently being typeset. Readers should note that articles published below have been fully refereed, but have not been through the copy-editing and proof correction process. Wiley-Blackwell and the AASLD cannot be held responsible for errors or consequences arising from the use of information contained in these articles; nor do the views and opinions expressed necessarily reflect those of Wiley-Blackwell or the AASLD.

"The present study is the first to report that the combination of aa substitution of the core region and genetic variation near IL28B gene is very useful as pretreatment predictors of sustained virological response by triple therapy, and further studies based on the larger number of patients are necessary to investigate the present results...... The present study indicated that the use of the combination of aa substitution of the core region and genetic variation near IL28B gene had high sensitivity, specificity, PPV, and NPV for prediction of sustained virological response. .....

........In conclusion, triple therapy with telaprevir, PEG-IFN and ribavirin in Japanese patients infected with HCV-1 and high viral load achieved high sustained virological response rates. Furthermore, the aa substitution pattern of the core region and genetic variation near IL28B gene seem to affect treatment efficacy. Further large-scale prospective studies are necessary to investigate whether the present results relate to the efficacy of the triple therapy, and further understanding of the complex interaction between virus- and host- related factors should facilitate the development of more effective therapeutic regimens....... Efficacy of triple therapy was high in the patients with genotype TT who accomplished sustained virological response (84%), irrespective of substitution of core aa 70....a significantly higher proportion of patients with genotype TT (83.8%) showed sustained virological response than that of patients who showed genotype non-TT (27.6%)....Arg70 has high sensitivity and PPV in predicting sustained virological response.....When one or more of the two predictors were used, the sensitivity, specificity, PPV, and NPV were 94.9, 55.6, 75.5, and 88.2%, respectively. These results indicate that the use of the combination of the above two predictors has high sensitivity, specificity, PPV, and NPV for prediction of sustained virological response......Based on a strong power of substitution of core aa 70 and rs8099917 genotype in predicting sustained virological response (Table 3), it was evaluated how they increase the predictive value when they were combined. The results are schematically depicted in Figures 3, respectively. Together they demonstrate three points: (1) efficacy of triple therapy was high in the patients with genotype TT who accomplished sustained virological response at 83.8%, irrespective of substitution of core aa 70; (2) in the patients having genotype TG and GG, those of Arg70 gained high sustained virological response (50.0%); and (3) sustained virological response (11.8%) were the worst in patients who possessed both of genotype TG and GG, and Gln70(His70)."


Table 3. Multivariate analysis of factors associated with sustained virological response of telaprevir, peginterferon and ribavirin triple therapy in Japanese patients infected with HCV genotype 1.

Only variables that achieved statistical significance (p<0.05) on multivariate logistic regression analysis are shown.

95% CI: 95% confidence interval

Norio Akuta 1 *, Fumitaka Suzuki 1, Miharu Hirakawa 1, Yusuke Kawamura 1, Hiromi Yatsuji 1, Hitomi Sezaki 1, Yoshiyuki Suzuki 1, Tetsuya Hosaka 1, Masahiro Kobayashi 1, Mariko Kobayashi 2, Satoshi Saitoh 1, Yasuji Arase 1, Kenji Ikeda 1, Kazuaki Chayama 3, Yusuke Nakamura 4, Hiromitsu Kumada 1

1Department of Hepatology, Toranomon Hospital, Tokyo, Japan

2Liver Research Laboratory, Toranomon Hospital, Tokyo, Japan

3Department of Medical and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan

4Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan

email: Norio Akuta (akuta-gi@umin.ac.jp)


*Correspondence to Norio Akuta, Department of Hepatology, Toranomon Hospital, 2-2-2 Toranomon, Minato-ku, Tokyo, 105-0001, Japan


Genetic variation near IL28B gene and substitution of amino acid (aa) 70 and 91 in the core region of HCV genotype 1b can predict the response to pegylated interferon (PEG-IFN)/ribavirin combination therapy, but its impact on triple therapy of telaprevir/PEG-IFN/ribavirin is not clear. The aims of this study were to investigate the predictive factors of sustained virological response to 12- or 24-week regimen of triple therapy in 72 of 81 Japanese adults infected with HCV genotype 1. Overall, sustained virological response and end-of-treatment response were achieved by 61% and 89%, respectively. Especially, sustained virological response was achieved by 45% and 67% in 12- and 24-week regimen, respectively. Multivariate analysis identified rs8099917 near IL28B gene (genotype TT) and substitution at aa 70 (Arg70) as significant determinants of sustained virological response. Prediction of response to therapy based on combination of these factors had high sensitivity, specificity, positive and negative predictive values. Efficacy of triple therapy was high in the patients with genotype TT who accomplished sustained virological response (84%), irrespective of substitution of core aa 70. In the patients having genotype non-TT, those of Arg70 gained high sustained virological response (50%), and sustained virological response (12%) were the worst in patients who possessed both of genotype non-TT and Gln70(His70). In conclusions, this study identified genetic variation near IL28B gene and aa substitution of the core region as predictors of sustained virological response to triple therapy of telaprevir/PEG-IFN/ribavirin in Japanese patients infected with HCV genotype 1b.

Hepatitis C virus (HCV) usually causes chronic infection that can result in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC).1,2 At present, treatments based on interferon (IFN), in combination with ribavirin, are mainstay for combating HCV infection. In Japan, HCV genotype 1b (HCV-1b) in high viral loads (>100 KIU/ml) accounts for more than 70% of HCV infections, making it difficult to treat patients with chronic hepatitis C.3 Such background calls for efficient treatments of Japanese patients with chronic HCV infection.

Even with pegylated IFN (PEG-IFN) combined with ribavirin, a sustained virological response lasting over 24 weeks after the withdrawal of treatment is achieved in at most 50% of the patients infected with HCV-1b and high viral loads.4,5 Recently, a new strategy was introduced in the treatment of chronic HCV infection by means of inhibiting protease in the NS3/NS4 of the HCV polyprotein. Of these, telaprevir (VX-950) was selected as a candidate agent for treatment of chronic HCV infection.6 60 Later, it was found that telaprevir, when combined with PEG-IFN and ribavirin, gains a robust antiviral activity.7,8 Specifically, HCV RNA is suppressed below the limits of detection in the blood, in almost all patients infected with HCV-1 during triple therapy of telaprevir with PEG-IFN and ribavirin.9 However, treatment resistant patients who 65 do not achieve sustained virological response by the triple therapy, have been reported.9-11 The underlying mechanism of the response to the treatment is still not clear.

Amino acid (aa) substitutions at position 70 and/or 91 in the HCV core region of patients infected with HCV-1b and high viral loads are pretreatment predictors of poor virological response to PEG-IFN plus ribavirin combination therapy,12-14 and also affect clinical outcome, including hepatocarcinogenesis.15,16 Furthermore, recent report showed that aa substitutions in the core region can be also used before therapy to predict very early dynamics (within 48 hours) after the start of triple therapy of telaprevir with PEG-IFN and ribavirin.17 However, it is not clear at this stage whether aa substitutions in the core region can be used before therapy to predict sustained virological response to triple therapy.


Recent reports showed that genetic variations near IL28B gene (rs8099917, rs12979860) on chromosome 19 as host-related factor, which encodes IFN-λ-3, are pretreatment predictors of virological response to 48-week PEG-IFN plus ribavirin combination therapy in individuals infected with HCV-1,18-21 and also affect clinical outcome, including spontaneous clearance of HCV.22 However, it is not clear at this stage whether genetic variation near IL28B gene can be used before therapy to predict sustained virological response to triple therapy.


The present study included 81 patients with HCV-1b and high viral loads, who received the triple therapy of telaprevir with PEG-IFN plus ribavirin. The aims of the study were to identify the pretreatment factors that could predict sustained virological response, including viral- (aa substitutions in the HCV core and NS5A regions) and host-related factors (genetic variation near IL28B gene).

Results

Virological Response to Therapy.

Sustained virological response was achieved by 44 of 72 (61.1%) patients. 64 of 72 (88.9%) patients were considered end-of-treatment response. According to treatment regimen, sustained virological response were achieved by 45.0% (9 of 20 patients) and 67.3% (35 of 52 patients), in the T12PR12 group and the T12PR24 group, respectively. Of 8 patients, who could not achieve end-of-treatment response, 6 (75.0%) patients resulted in re-elevation of viral loads regardless of HCV-RNA temporary negative, and the other 2 patients (25.0%) did not achieve HCV-RNA negative during treatment.

Especially, in the T12PR24 group, according to the past history of treatment, sustained virological response were achieved by 76.4% (13 of 17 patients), 86.4% (19 of 22 patients), and 23.1% (3 of 13 patients), in treatment-naive, relapsers to previous treatment, and non-responders to previous treatment, respectively.

Sustained Virological Response According to Amino Acid Substitutions in Core, and NS5A Regions.

According to the substitution of core aa 70, a significantly higher proportion of patients with Arg70 substitutions (74.4%) showed sustained virological response than that of patients who showed Gln70(His70) (41.4%) (Figure 1, P=0.007). In contrast, according to the substitution of core aa 91, the sustained virological response rate was not significantly different between Leu91 (65.0%) and Met91 (56.3%) (Figure 1). Likewise, according to the numbers of aa substitutions in ISDR, the sustained virological response rate was not significantly different between wild-type (56.3%) and non wild-type (66.7%) (Figure 1). Thus, sustained virological response was influenced by the substitution of core aa 70.

Sustained Virological Response According to Genetic Variation near IL28B Gene.


According to the genetic variation in rs8099917, sustained virological response was achieved by 83.8% (31 of 37 patients), 29.6% (8 of 27 patients), and 0% (0 of 2 patients), in patients with genotype TT, TG, and GG, respectively. Thus, a significantly higher proportion of patients with genotype TT (83.8%) showed sustained virological response than that of patients who showed genotype non-TT (27.6%) (Figure 2, P<0.001) (Table 2).

According to the genetic variation in rs12979860, sustained virological response was achieved by 83.8% (31 of 37 patients), 34.5% (10 of 29 patients), and 0% (0 of 2 patients), in patients with genotype CC, CT, and TT, respectively. Thus, a significantly higher proportion of patients with genotype CC (83.8%) showed sustained virological response than that of patients who showed genotype non-CC (32.3%) (Figure 2, P<0.001) (Table 2).

Predictive Factors Associated with Sustained Virological Response.

Univariate analysis identified 3 parameters that correlated with sustained virological response significantly: substitution of aa 70 (Arg70; OR 4.12, P=0.007), and genetic variation in rs8099917 (genotype TT; OR 13.6, P<0.001) and rs12979860 (genotype CC; OR 10.8, P<0.001). Two factors were identified by multivariate analysis as independent parameters that significantly influenced sustained virological response [(rs8099917 genotype TT; OR 10.6, P<0.001) and (Arg70; OR 3.69, P=0.040)] (Table 3).

Assessment of Amino Acid Substitutions in Core Region and Genetic Variation near IL28B Gene 270 as Predictors of Sustained Virological Response.


The ability to predict sustained virological response by substitution of core aa 70 and rs8099917 genotype near IL28B gene was evaluated. The sustained virological response rates of patients with a combination of Arg70 or rs8099917 genotype TT were defined as PPV (prediction of sustained virological response). The non-sustained virological response rates of patients with a combination of Gln70(His70) or rs8099917 genotype non-TT were defined as NPV (prediction of non-sustained virological response).

In patients with rs8099917 genotype TT, the sensitivity, specificity, PPV, and NPV for sustained virological response were 79.5, 77.8, 83.8, and 72.4%, respectively. 280 Thus, genotype TT has high sensitivity, specificity, and PPV for prediction of sustained virological response. In patients with Arg70, the sensitivity, specificity, PPV, and NPV were 76.9, 63.0, 75.0, and 65.4%, respectively. Thus, Arg70 has high sensitivity and PPV in predicting sustained virological response. Furthermore, when both predictors were used, the sensitivity, specificity, PPV, and NPV were 61.5, 85.2, 85.7, and 60.5%, 285 respectively. When one or more of the two predictors were used, the sensitivity, specificity, PPV, and NPV were 94.9, 55.6, 75.5, and 88.2%, respectively. These results indicate that the use of the combination of the above two predictors has high sensitivity, specificity, PPV, and NPV for prediction of sustained virological response (Table 4).

Predicting Sustained 290 Virological Response by Amino Acid Substitutions in Core Region in Combination with Genetic Variation near IL28B Gene.


Sustained virological response by core aa 70 in combination with rs8099917 genotype was shown in Figure 3. In patients with rs8099917 genotype TT, sustained virological response was not different between Arg70 (85.7%) and Gln70(His70) (77.8%). In contrast, in patients with rs8099917 genotype TG and GG, a significantly higher proportion of patients with Arg70 (50.0%) showed sustained virological response than that of patients with Gln70(His70) (11.8%) (P=0.038).

Based on a strong power of substitution of core aa 70 and rs8099917 genotype in predicting sustained virological response (Table 3), it was evaluated how they increase the predictive value when they were combined. The results are schematically depicted in Figures 3, respectively. Together they demonstrate three points: (1) efficacy of triple therapy was high in the patients with genotype TT who accomplished sustained virological response at 83.8%, irrespective of substitution of core aa 70; (2) in the patients having genotype TG and GG, those of Arg70 gained high sustained virological response (50.0%); and (3) sustained virological response (11.8%) were the worst in patients who possessed both of genotype TG and GG, and Gln70(His70).

Discussion

Two previous studies (PROVE1 in USA, and PROVE2 in Europe) showed that the T12PR12 and T12PR24 group of telaprevir, PEG-IFN and ribavirin could achieve sustained virological response rates of 35-60% and 61-69%, respectively.10,11 In the present Japanese study, sustained virological response rates were 45% and 67% in the T12PR12 and T12PR24 group, respectively, like two previous studies. There were the differences at the three points between the present study and two previous studies: (1) PEG-IFN in two previous studies was used at a fixed dose of PEG-IFNα-2a, but that of the present study was a body weight-adjusted dose of PEG-IFNα-2b; (2) Body mass index of our patients (median; 23 kg/m2) was much lower than that of the participants of the previous study by McHutchison et al (median; >25 kg/m2); and (3) The present study was performed based on the Japanese patients infected with HCV-1b, except for only one patient of HCV-1a. Especially, in PROVE-1, viral breakthrough rate was higher in HCV-1a subjects compared to HCV-1b, and one of the reasons might be due to the low genetic barrier to the emergence of the R155K variant in HCV-1a.10,27 Further studies of larger number of patients matched for background, including genotype, race, body mass index, treatment regimen, and past history of IFN therapy, are required to investigate the rate of the sustained virological response by triple therapy.

IL28A, IL28B, and IL29 (IFN-λ-2, -3, and -1, respectively) are novel IFNs identified recently.28,29 They are similar to type 1 IFNs in terms of biological activities and mechanism of action, in contrast to their differences in structure and genetics.30 The anti-viral effects of IFN-λ against hepatitis B virus and HCV have been already reported.31 Furthermore, α and λ IFNs act synergistically against HCV.32-34 Recent reports showed that genetic variation near IL28B gene (rs8099917, rs12979860) are pretreatment predictors of virological response to 48-week PEG-IFN plus ribavirin combination therapy in individuals infected with HCV-1,18-21 and also affect clinical outcome, including spontaneous clearance of HCV.22 At AASLD 2009, Thompson et al reported that genetic variation near IL28B gene also affected the viral suppression in the first 2 to 4 weeks of PEG-IFN plus ribavirin, and this phenomenon probably explains much of the differences in treatment response rate.35 The present study is the first to report that genetic variation near IL28B gene significantly also affect sustained virological response by triple therapy. This results should be interpreted with caution since the races other than Japanese populations were not included. Any generalization of the results should await confirmation by studies of patients of other races to explore the relationship between genetic variation near IL28B gene and the response to triple therapy.

The present study indicated that the use of the combination of aa substitution of the core region and genetic variation near IL28B gene had high sensitivity, specificity, PPV, and NPV for prediction of sustained virological response. Efficacy of triple therapy was high in the patients with TT, irrespective of substitution of core aa 70. In the patients having non-TT, those of Arg70 gained high sustained virological response, and sustained virological response was the worst in patients who possessed both of non-TT, and Gln70(His70). Along with a high sustained virological response, combined PEG-IFN and ribavirin accompany severe side effects and entail high costs.

Hence, the patients who do not achieve sustained virological response need to be identified, as early as possible, in order to free them of unnecessary side effects and high costs. The present study is the first to report that the combination of aa substitution of the core region and genetic variation near IL28B gene is very useful as pretreatment predictors of sustained virological response by triple therapy, and further studies based on the larger number of patients are necessary to investigate the present results. Another limitations of the present study were that aa substitutions in areas other than the core region and NS5A-ISDR of the HCV genome, such as the interferon/ribavirin resistance determining region (IRRDR),36 were not examined. Furthermore, HCV mutants with aa conversions for resistance to telaprevir during triple therapy, such as the 156S mutation,37 were also not investigated. In this regard, telaprevir-resistant HCV mutants were reported to be susceptible to IFN in both in vivo and in vitro studies.38,39 Thus, viral factors before and during triple therapy should be investigated in future studies, and identification of these factors should facilitate the development of more effective therapeutic regimens.


In conclusion, triple therapy with telaprevir, PEG-IFN and ribavirin in Japanese patients infected with HCV-1 and high viral load achieved high sustained virological response rates. Furthermore, the aa substitution pattern of the core region and genetic variation near IL28B gene seem to affect treatment efficacy. Further large-scale prospective studies are necessary to investigate whether the present results relate to the efficacy of the triple therapy, and further understanding of the complex interaction between virus- and host- related factors should facilitate the development of more effective therapeutic regimens.

Figure 1. According to the substitution of core aa 70, a significantly higher proportion of patients with Arg70 substitutions showed sustained virological response than that of patients who showed Gln70(His70) (P=0.007). In contrast, according to the substitution of core aa 91, the sustained virological response rate was not significantly different between Leu91 and Met91. Likewise, according to the numbers of aa substitutions in ISDR, the sustained virological response rate was not significantly different between wild-type and non wild-type.
Picture 1.png

Figure 2. According to the genetic variation in rs8099917 or rs12979860 near IL28B gene, a significantly higher proportion of patients with genotype TT or CC showed sustained virological response than that of patients who showed genotype non-TT or non-CC, respectively (P<0.001 or P<0.001, respectively).
Picture 2.png

Figure 3. Predicting sustained virological response by aa substitution in core region in combination with genetic variation near IL28B gene. Efficacy of triple therapy was high in the patients with genotype TT who accomplished sustained virological response at 83.8%, irrespective of substitution of core aa 70. In the patients having genotype TG and GG, those of Arg70 gained high sustained virological response (50.0%), and sustained virological response (11.8%) were the worst in patients who possessed both

of genotype TG and GG, and Gln70(His70).

Picture 3.png