Abstract
It is often difficult to predict the response to telaprevir-pegylated interferon (PEG-IFN)-ribavirin triple therapy and the appearance of telaprevir-resistant variants. The present study determined the predictive factors of a sustained virological response (SVR) to 12- or 24-week triple therapy (T12PR12 or T12PR24, respectively) in 194 Japanese patients infected with hepatitis C virus genotype 1b (HCV-1b). The study also evaluated whether ultradeep sequencing technology can predict at baseline the emergence of resistant variants after the start of therapy. Analysis of the data of the entire group indicated that an SVR was achieved in 78% of the patients. Multivariate analysis identified IL28B rs8099917 (genotype TT), the substitution of amino acid (aa) 70 (Arg70), response to prior treatment (naive or relapse), PEG-IFN dose (≥1.3 μg/kg of body weight), and treatment regimen (T12PR24) as significant determinants of SVR. Among patients of the T12PR24 group, 92% with genotype TT achieved an SVR, irrespective of a substitution at aa 70. In patients with the non-TT genotype, an SVR was achieved in 76% of those with Arg70, while only 14% of patients with the non-TT genotype, Gln70(His70), and nonresponse to ribavirin combination therapy achieved an SVR. Ultradeep sequencing was conducted for 17 patients who did not achieve an SVR to determine the emergence of resistant variants during therapy. De novo resistant variants were detected in 16 of 17 patients (94%), regardless of the variant frequencies detected at baseline. In conclusion, the results indicate that the response to triple therapy can be predicted by the combination of host, viral, and treatment factors and that it is difficult to predict at baseline the telaprevir-resistant variants that emerge during triple therapy, even with the use of ultradeep sequencing.
INTRODUCTION
New strategies have been introduced recently for the treatment of chronic hepatitis C virus (HCV) infection based on the inhibition of protease in the nonstructural NS3/NS4 proteins of the HCV polyprotein. Of the new agents currently available, telaprevir (VX-950) is used for the treatment of chronic HCV infection (1). Three studies (PROVE1, PROVE2, and a Japanese study) showed that a 24-week regimen of triple therapy (consisting of telaprevir, pegylated interferon [PEG-IFN], and ribavirin) for 12 weeks followed by dual therapy (PEG-IFN and ribavirin) for 12 weeks (also called the T12PR24 regimen) achieved sustained virological response (SVR) (defined as negative HCV RNA lasting >24 weeks after withdrawal of treatment) rates of 61%, 69%, and 73%, respectively, for the three studies, in patients infected with HCV genotype 1 (HCV-1) (2–4). However, a recent study (PROVE3) showed lower SVR rates following the T12PR24 regimen (39%) in HCV-1-infected nonresponders to previous PEG-IFN-ribavirin therapy, and who did not achieve HCV RNA negativity during or at the end of the initial triple therapy (5).
Telaprevir-based therapy is reported to induce resistant variants in HCV (6, 7). Recent reports have described the advantages of ultradeep sequencing technology, including faster processing and large-scale sequencing, in addition to providing a better understanding of the dynamics of variants in HCV quasispecies (8–11). However, it is not clear at this stage whether such technology is useful for the prediction of the emergence of telaprevir-resistant variants during or after the administration of triple therapy.
Based on the above background, there is a need to determine the predictive factors of non-SVR to triple therapy with telaprevir-PEG-IFN-ribavirin before the use of this treatment in order to avoid the appearance of telaprevir-resistant variants. The aim of this study was to determine the predictive factors of SVR to triple therapy and the emergence of telaprevir-resistant variants during such therapy (using ultradeep sequencing technology) in patients infected with HCV genotype 1b.
MATERIALS AND METHODS
Study population.
From May 2008 through April 2013, 332 consecutive patients infected with HCV were selected for triple therapy with telaprevir (MP-424 or Telavic; Mitsubishi Tanabe Pharma, Osaka, Japan), PEG-IFN-α-2b (PEG-Intron; MSD, Tokyo, Japan), and ribavirin (Rebetol; MSD, Tokyo) at the Department of Hepatology, Toranomon Hospital (located in metropolitan Tokyo). Subsequently, 194 of these patients received the triple therapy based on the following inclusion criteria: (i) diagnosis of chronic hepatitis C, (ii) HCV genotype 1b confirmed by sequence analysis, (iii) HCV RNA levels of ≥5.0 log IU/ml determined by the cobas TaqMan HCV test (Roche Diagnostics, Tokyo), (iv) age at study entry of 20 to 65 years, (v) follow-up duration of ≥24 weeks after the completion of triple therapy, (vi) no history of treatment with NS3/NS4A protease inhibitors, (vii) lack of decompensated liver cirrhosis and hepatocellular carcinoma (HCC), (viii) negativity for hepatitis B surface antigen (HBsAg), (ix) no evidence of HIV infection, (x) negative history of autoimmune hepatitis, alcohol liver disease, hemochromatosis, and chronic liver disease other than chronic hepatitis C, (xi) negative history of depression, schizophrenia or suicide attempts, angina pectoris, cardiac insufficiency, myocardial infarction, severe arrhythmia, uncontrolled hypertension, uncontrolled diabetes, chronic renal dysfunction, cerebrovascular disorders, thyroidal dysfunction that is uncontrollable by medical treatment, chronic pulmonary disease, allergy to medication, or anaphylaxis at baseline; pregnant or breastfeeding women or those open to becoming pregnant during the study and men with pregnant partners were excluded. The study protocol was in compliance with the Good Clinical Practice Guidelines and the 1975 Declaration of Helsinki and was approved by the institutional review board of Toranomon Hospital. Each patient received ample information about the goals and potential side effects of the treatment and their right to withdraw from the study. Each provided a signed consent form before participating in the trial.
The efficacy of treatment was evaluated by absence of HCV RNA at 24 weeks after the completion of therapy (i.e., SVR), as measured by the cobas TaqMan HCV test (Roche Diagnostics). Furthermore, failure to achieve an SVR was classified as nonresponse (if HCV RNA was detected during or at the end of treatment) or relapse (reelevation of viral load after the end of treatment, even when HCV RNA was negative at the end of treatment).
Twenty patients (10%) were assigned to a 12-week regimen of triple therapy (the T12PR12 group) and were randomly subdivided into two groups treated with either 1,500 mg/day or 2,250 mg/day of telaprevir to evaluate the treatment efficacy during 12 weeks of treatment. Sixty patients (31%) were allocated to a 24-week regimen of the same triple therapy described above followed by dual therapy of PEG-IFN and ribavirin for another 12 weeks (the T12PR24 group) to evaluate treatment efficacy according to the response to prior treatment. All subjects in the T12PR24 group were treated with telaprevir at 2,250 mg/day. Another group of 114 patients (59%) were treated as described above for the T12PR24 group, except for telaprevir dose, and were subdivided into two groups treated with either 1,500 mg/day or 2,250 mg/day of telaprevir, as prescribed by the attending physician. Table 1 summarizes the profiles and laboratory data of the entire group of 194 patients at the commencement of treatment. They included 117 males and 77 females, aged 23 to 65 years (median, 56 years). At the start of treatment, telaprevir was administered at a median dose of 31.8 mg/kg of body weight (range, 14.5 to 59.2 mg/kg) daily. Especially, 120 patients (62%) were treated with telaprevir at a dose of 2,250 mg/day, while the other 74 patients (38%) were treated with telaprevir at a dose of 1,500 mg/day. PEG-IFN-α-2b was injected subcutaneously at a median dose of 1.5 μg/kg (range, 0.9 to 1.7 μg/kg) once a week. Ribavirin was administered at a median dose of 11.0 mg/kg (range, 4.3 to 15.8 mg/kg) daily. Each drug was discontinued or its dose reduced, as required upon judgment of the attending physician, in response to a fall in hemoglobin level, leukocyte count, neutrophil count, or platelet count, or the appearance of side effects. Triple therapy was discontinued when the leukocyte count decreased to <1,000/mm3, neutrophil count to <500/mm3, or platelet count to <5.0 × 104/mm3, or when hemoglobin decreased to <8.5 g/dl.
Table 1.
Profile and laboratory data at commencement of telaprevir, pegylated interferon, and ribavirin triple therapy in patients infected with HCV genotype 1b
| Study or patient characteristics | Data |
|---|---|
| Demographic data | |
| No. of patients | 194 |
| Sex (no.) | |
| Male | 117 |
| Female | 77 |
| Age (median [range]) (yr) | 56 (21–65) |
| Body mass index (median [range]) (kg/m2) | 22.7 (16.0–36.7) |
| Blood plasma levels (median [range]) | |
| Viremia (log IU/ml) | 6.7 (5.0–7.8) |
| Aspartate aminotransferase (IU/liter) | 36 (15–118) |
| Alanine aminotransferase (IU/liter) | 41 (12–175) |
| Albumin (g/dl) | 3.9 (2.9–4.6) |
| Total bilirubin (mg/dl) | 0.8 (0.2–2.0) |
| Gamma-glutamyl transpeptidase (IU/liter) | 34 (3–240) |
| Creatinine (g/dl) | 0.7 (0.4–1.1) |
| Leukocyte count (cells/mm3) | 4,800 (2,000–8,400) |
| Hemoglobin (g/dl) | 14.4 (12.1–17.4) |
| Platelet count (× 104/mm3) | 17.5 (8.9–33.8) |
| Alpha-fetoprotein (μg/liter) | 4 (2–104) |
| Total cholesterol (mg/dl) | 174 (112–301) |
| High-density lipoprotein cholesterol (mg/dl) | 48 (20–117) |
| Low-density lipoprotein cholesterol (mg/dl) | 97 (41–216) |
| Triglycerides (mg/dl) | 97 (36–336) |
| Uric acid (mg/dl) | 5.7 (2.0–8.6) |
| Fasting plasma glucose (mg/dl) | 93 (64–169) |
| Treatment dose | |
| PEG-IFN-α-2b (median [range]) (μg/kg) | 1.5 (0.9–1.7) |
| Ribavirin (median [range]) (mg/kg) | 11.0 (4.3–15.8) |
| Telaprevir (median [range]) (mg/kg) | 31.8 (14.5–59.2) |
| Telaprevir (no.) | |
| 1,500 mg/day | 74 |
| 2,250 mg/day | 120 |
| Treatment regimen (no.) | |
| T12PR12 group | 20 |
| T12PR24 group | 174 |
| Response to prior treatment (no.) | |
| Treatment naive/relapse to prior treatment | 71 |
| Relapse after prior treatment | 78 |
| No response to prior treatment | 44 |
| IFN monotherapy | 10 |
| IFN-ribavirin dual therapy | 34 |
| Unknown | 1 |
| Amino acid substitutions in HCV genotype 1b (no.) | |
| Core aa 70 | |
| Arginine | 128 |
| Glutamine (histidine) | 65 |
| NDa | 1 |
| Core aa 91 | |
| Leucine | 104 |
| Methionine | 89 |
| ND | 1 |
| ISDR of NS5A | |
| Wild type | 155 |
| Non-wild type | 17 |
| ND | 22 |
| IRRDR of NS5A | |
| ≤5 | 144 |
| ≥6 | 38 |
| ND | 1 |
| V3 of NS5A | |
| ≤2 | 49 |
| ≥3 | 144 |
| ND | 1 |
| IL28B genotype (no.) | |
| rs8099917 genotype | |
| TT | 139 |
| Non-TT | 53 |
| ND | 2 |
| ITPA genotype (no.) | |
| rs112735 genotype | |
| CC | 147 |
| Non-CC | 47 |
| Telaprevir-resistant variants by direct sequencing (no.)b | |
| V36 | 1 |
| T54 | 6 |
| R155 | 0 |
| A156 | 1 |
| V170 | 0 |
ND, not determined.
Telaprevir-resistant variants, detected by direct sequencing, included V36A/C/M/L/G, T54A/S, R155K/T/I/M/G/L/S/Q, A156V/T/S/I/G, and V170A.
Follow-up.
Clinical and laboratory assessments were performed at least once every month before, during, and after treatment. Especially, they were performed every week in the initial 12 weeks of treatment. Adverse effects were monitored clinically by careful interviews and a medical examination at least once every month. Compliance with treatment was evaluated by a questionnaire.
Measurement of HCV RNA.
The antiviral effects of triple therapy on HCV were assessed by measuring blood plasma HCV RNA levels. In this study, HCV RNA levels during treatment were evaluated at least once every month before, during, and after therapy. HCV RNA concentrations were determined using the cobas TaqMan HCV test (Roche Diagnostics). The linear dynamic range of the assay was 1.2 to 7.8 log IU/ml, and undetectable levels were defined as negative samples.
Determination of IL28B and ITPA genotypes.
IL28B rs8099917 and ITPA rs112735 genotypes have been reported as predictors of treatment efficacy and side effects to PEG-IFN-ribavirin dual therapy, and they were genotyped by the Invader assay, TaqMan assay, or direct sequencing, as described previously (12–16).
Detection of amino acid substitutions in core and NS5A regions of HCV-1b.
With the use of HCV-J (accession no. D90208) as a reference (17), the sequence of amino acids (aa) 1 to 191 in the core protein of HCV-1b was determined and compared with the consensus sequence constructed in a previous study to detect substitutions at aa 70 of arginine (Arg70) or glutamine/histidine (Gln70[/His70]) and aa 91 of leucine (Leu91) or methionine (Met91) (18). The sequence of aa 2209 to 2248 in the NS5A of HCV-1b (the interferon sensitivity determining region [ISDR]) reported by Enomoto and coworkers (19) was determined, and the numbers of aa substitutions in the ISDR were defined as wild-type (≤1) or non-wild-type (≥2) compared against HCV-J. Furthermore, the sequence of aa 2334 to 2379 in the NS5A of HCV-1b (IFN-ribavirin resistance-determining region [IRRDR]) reported by El-Shamy and coworkers (20), including the sequence of aa 2356 to 2379 referred to as variable region 3 (V3), was determined and compared with the consensus sequence constructed in a previous study. The numbers of aa substitutions in the IRRDR and V3 were divided into two groups for analysis (numbers of aa substitutions in the IRRDR of ≤5 and ≥6, and those in V3 of ≤2 and ≥3). In the present study, aa substitutions of the core region and NS5A-ISDR-IRRDR-V3 of HCV-1b were analyzed by direct sequencing.
Assessment of telaprevir-resistant variants.
The genome sequence of the N-terminal 609 nucleotides (203 amino acids) in the NS3 region of HCV isolates from the patients was examined. HCV RNA was extracted from 100 μl of serum, and the nucleotide sequences were determined by direct sequencing and deep sequencing. The primers used to amplify the NS3 region were NS3-F1 (5′-ACA CCG CGG CGT GTG GGG ACA T-3′; nucleotides 3295 to 3316) and NS3-AS2 (5′-GCT CTT GCC GCT GCC AGT GGG A-3′; nucleotides 4040 to 4019) as the first (outer) primer pair and NS3-F3 (5′-CAG GGG TGG CGG CTC CTT-3′; nucleotides 3390 to 3407) and NS3-AS2 (sequence above) as the second (inner) primer pair (21). Thirty-five cycles of first and second amplifications were performed as follows: denaturation for 30 s at 95°C, annealing of primers for 1 min at 63°C, extension for 1 min at 72°C, and final extension at 72°C for 7 min. The PCR-amplified DNA was purified after agarose gel electrophoresis and then used for direct sequencing and ultradeep sequencing.
All patients were examined for telaprevir-resistant variants by direct sequencing before the start of triple therapy. Furthermore, patients who did not achieve an SVR were analyzed by ultradeep sequencing, at baseline and at the time of reelevation of viral loads. Telaprevir-resistant variants included V36A/C/M/L/G, T54A/S, R155K/T/I/M/G/L/S/Q, A156V/T/S/I/G, and V170A (22, 23).
Direct sequencing was analyzed by the dye-terminator method. Dideoxynucleotide termination sequencing was performed with the BigDye deoxy terminator v1.1 cycle sequencing kit (Life Technologies, Carlsbad, CA) (21). Sequence data were deposited in GenBank. Ultradeep sequencing was performed using the Ion personal genome machine (PGM) sequencer (Life Technologies). An Ion Torrent adapter-ligated library was prepared using an Ion Xpress Plus fragment library kit (Life Technologies). Briefly, 100 ng of fragmented genomic DNA was ligated to the Ion Torrent adapters P1 and A. The adapter-ligated products were nick translated and PCR amplified for a total of 8 cycles. Subsequently, the library was purified using AMPure beads (Beckman Coulter, Brea, CA), and the concentration was determined using the StepOnePlus real-time PCR (Life Technologies) and Ion Library quantitation kit, according to the instructions provided by the manufacturer. Emulsion PCR was performed using Ion OneTouch (Life Technologies) in conjunction with Ion OneTouch 200 template kit v2 (Life Technologies). Enrichment for templated Ion Sphere particles (ISPs) was performed using the Ion OneTouch enrichment system (Life Technologies), according to the instructions provided by the manufacturer. Templated ISPs were loaded onto an Ion 314 chip and subsequently sequenced using 130 sequencing cycles according to the Ion PGM 200 sequencing kit user guide. Total output read length per run is >10 Mb (0.5 m-TAG, 200-base read) (24). The results were analyzed with the CLC Genomics Workbench software (CLC bio, Aarhus, Denmark) (25).
We also included a control experiment to validate the error rates in ultradeep sequencing of the viral genome. In this study, the amplification products of the second-round PCR were ligated with plasmid and transformed in Escherichia coli in a cloning kit (TA Cloning; Invitrogen, Carlsbad, CA). A plasmid-derived NS3 sequence was used as the template by the control experiment. The fold coverage values evaluated per position for aa 36, aa 54, aa 155, aa 156, and aa 170 in the NS3 region were 359,379×, 473,716×, 106,435×, 105,979×, and 49,058×, respectively. Thus, using the control experiment based on a plasmid encoding an HCV NS3 sequence, amino acid mutations were defined as amino acid substitutions at a frequency of >0.2% of the total coverage. This frequency ruled out putative errors caused by the ultradeep sequence platform used in this study (26).
Statistical analysis.
Nonparametric tests (chi-square test and Fisher's exact probability test) were used to compare the characteristics of the groups. Univariate and multivariate logistic regression analyses were used to determine those factors that significantly contributed to SVRs. The odds ratios (OR) and 95% confidence intervals (CI) were also calculated. All P values of <0.05 by the two-tailed test were considered significant. Variables that achieved statistical significance (P < 0.05) on univariate analysis were entered into multiple logistic regression analysis to identify significant independent predictive factors. The potential pretreatment factors associated with SVR included sex, age, body mass index, levels of viremia, aspartate aminotransferase, alanine aminotransferase, albumin, total bilirubin, gamma-glutamyl transpeptidase (GGT), and creatinine, leukocyte count, hemoglobin level, platelet count, alpha-fetoprotein level, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, uric acid, fasting blood plasma glucose, PEG-IFN dose/kg body weight, ribavirin dose/kg body weight, telaprevir dose/kg body weight, telaprevir dose/day, kind of treatment regimen, response to prior treatment, amino acid substitutions in the core region and NS5A-ISDR-IRRDR, IL28B genotype, ITPA genotype, and telaprevir-resistant variants. Statistical analyses were performed using the SPSS software (SPSS, Inc., Chicago, IL). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were also calculated to determine the reliability of the predictors of response to therapy.
RESULTS
Virological response to therapy.
Analysis of the entire group showed that 78% (151 of 194 patients) achieved an SVR. According to the treatment regimen, an SVR was achieved by 45% (9 of 20 patients) and 82% (142 of 174 patients) of the T12PR12 group and T12PR24 groups, respectively. Taking into consideration the response to prior treatment, in the 173 patients of the T12PR24 group, an SVR was achieved in 89% (54 of 61 patients), 89% (66 of 74 patients), and 55% (21 of 38 patients) of treatment-naive patients, patients who showed relapse after prior treatment, and nonresponders to prior treatment, respectively. Furthermore, SVRs were achieved by 100% (8 of 8 patients) and 43% (13 of 30 patients) of the nonresponders to prior IFN monotherapy and IFN-ribavirin dual therapy, respectively.
Predictors of SVR.
Univariate analysis of the data of the entire group identified seven parameters that correlated significantly with SVR: IL28B rs8099917 (genotype TT) (P < 0.001), substitution of aa 70 (Arg70) (P = 0.001), response to prior treatment (naive or relapse) (P < 0.001), PEG-IFN dose (≥1.3 μg/kg) (P = 0.004), treatment regimen (T12PR24 group) (P = 0.001), platelet count (≥15.0 × 104/mm3) (P = 0.013), and GGT (<50 IU/liter) (P = 0.001). Multivariate analysis that included the above variables identified 5 parameters that independently influenced SVR: IL28B rs8099917 (genotype TT) (OR, 9.52; P < 0.001), substitution of aa 70 (Arg70) (OR, 2.67; P = 0.038), response to prior treatment (naive or relapse) (OR, 3.80; P = 0.007), PEG-IFN dose (≥1.3 μg/kg) (OR, 35.5; P < 0.001), and treatment regimen (T12PR24 group) (OR, 12.8; P < 0.001) (Table 2).
Table 2.
Multivariate analysis of factors associated with sustained virological response to telaprevir, pegylated interferon, and ribavirin triple therapy in patients infected with HCV genotype 1b
| SVR-influencing factor | OR (95% CI)a | P |
|---|---|---|
| IL28B rs8099917 genotype | ||
| Non-TT | 1 | |
| TT | 9.52 (3.36–27.0) | <0.001 |
| Substitution of aa 70 | ||
| Gln70 (His70) | 1 | |
| Arg70 | 2.67 (1.05–6.76) | 0.038 |
| Response to prior treatment | ||
| Nonresponse | 1 | |
| Naive or relapse | 3.80 (1.44–10.1) | 0.007 |
| PEG-IFN-α−2b dose (μg/kg of body weight) | ||
| <1.3 | 1 | |
| ≥1.3 | 35.5 (6.37–198) | <0.001 |
| Treatment regimen | ||
| T12PR12 group | 1 | |
| T12PR24 group | 12.8 (3.44–48.1) | <0.001 |
OR, odds ratio; CI, confidence interval.
Host, viral, and treatment factors for prediction of non-SVR.
Using data of the 172 patients of the T12PR24 group, we evaluated the ability to predict non-SVR by host factor (IL28B rs8099917 genotype), viral factor (substitution of aa 70), and treatment factor (response to prior treatment). With the combination of the rs8099917 non-TT genotype, Gln70 (His70), and nonresponse to ribavirin combination therapy, the sensitivity, specificity, PPV, and NPV for non-SVR were 38% (12 of 32 patients), 99% (138 of 140 patients), 86% (12 of 14 patients), and 87% (138 of 158 patients), respectively. These results indicate that using the combination of the above three predictors has high specificity, PPV, and NPV for the prediction of non-SVR.
The SVR rates using the combination of rs8099917 genotype, substitution of aa 70, and response to prior treatment are shown in Fig. 1. In 126 patients with the rs8099917 TT genotype, the degree of SVR was not significantly different between Arg70 (91% [86 of 95 patients]) and Gln70 (His70) (97% [29 of 30 patients]). In contrast, in 46 patients with the rs8099917 non-TT genotype, a significantly higher proportion of patients with Arg70 (76% [16 of 21 patients]) achieved an SVR than did patients with Gln70 (His70) (32% [8 of 25 patients]) (P = 0.004). Furthermore, in 25 patients with the rs8099917 non-TT genotype and Gln70 (His70), a lower proportion of nonresponders to ribavirin combination therapy (14% [2 of 14 patients]) tended to achieve an SVR than did other patients (55%[6 of 11 patients]) (P = 0.081). These results highlight three properties of triple therapy: (i) a high efficacy of triple therapy was seen in patients with the TT genotype who achieved an SVR at 92%, irrespective of substitution of aa 70, (ii) among patients with the non-TT genotype, 76% of those with Arg70 achieved an SVR, and (iii) only 14% of the patients with the three factors of the rs8099917 non-TT genotype, Gln70 (His70), and nonresponders to ribavirin combination therapy achieved an SVR.
Fig 1.
Prediction of sustained virological response (SVR) by the combination of IL28B rs8099917 genotype, substitution of aa 70, and response to prior treatment. In the T12PR24 group, treatment efficacy was high in patients with the TT genotype who achieved an SVR (92%), irrespective of the substitution of aa 70. In patients with the non-TT genotype, those with Arg70 achieved a high SVR (76%). Patients with the non-TT genotype, Gln70 (His70), and nonresponse to ribavirin combination therapy achieved the lowest frequency of an SVR (14%).
Evolution of telaprevir-resistant variants over time detected by ultradeep sequencing.
Between May 2008 and the end of September 2009, 17 patients (4 treatment-naive patients, 3 who had a relapse after prior ribavirin combination therapy, and 10 nonresponders to ribavirin combination therapy) did not achieve SVR with triple therapy, and they were analyzed for telaprevir-resistant variants by ultradeep sequencing at baseline and at the time of reelevation of viral load.
In 6 of 17 patients (35%), telaprevir-resistant variants were detected at baseline by ultradeep sequencing. In 4 of these 6 patients, a very low frequency of variants at baseline (0.2% of 32,413× coverage for V36A, 0.2% of 27,915× coverage for V36A, 0.2% of 26,230× coverage for T54A, and 0.4% of 29,881× coverage for V170A) were replaced after treatment by de novo high-frequency variants (97.2% of 36,757× coverage for V36C, 27.7% of 5,032× coverage for T54A, 50.2% of 15,487× coverage for A156S, and 99.6% of 14,757× coverage for A156T), respectively. In one of the 6 patients, very high-frequency variants of T54S (99.9% of 33,830× coverage) at baseline persisted during treatment as very high-frequency variants of T54S (99.7% of 26,348× coverage), and de novo very high-frequency variants of R155K (96.1% of 20,630× coverage) also emerged during treatment. In another patient, variants of T54A increased from very low frequency at baseline (0.2% of 53,127× coverage) to high frequency during treatment (99.9% of 45,240× coverage).
In the other 11 of 17 patients (65%), telaprevir-resistant variants were not detected by ultradeep sequencing at baseline, but de novo resistant variants were detected according to treatment (4 patients with V36A/C/M [median 41.5% of median 27,769× coverage], 8 with T54A/S [median 40.2% of median 27,067× coverage], 3 with R155K/Q [median 0.3% of median 17,847× coverage], and 8 with A156S/T [median 2.1% of median 18,150× coverage]).
Thus, in 16 of 17 patients (94%), de novo resistant variants were detected according to treatment. In other words, using ultradeep sequencing, the present study detected the emergence of de novo telaprevir-resistant variants regardless of variant frequencies at baseline, and the emergence of variants after the start of treatment could not be predicted at baseline.
DISCUSSION
Along with resulting in a high SVR, triple therapy is expensive and associated with serious side effects. Furthermore, employing ultradeep sequencing, the present study demonstrated the emergence of de novo telaprevir-resistant variants regardless of variant frequencies at baseline, and that the emergence of variants after the start of triple therapy could not be predicted at baseline. Hence, patients who failed to achieve an SVR with triple therapy need to be identified beforehand to avoid unnecessary side effects, high costs, and the emergence of telaprevir-resistant variants. Host genetic factors (e.g., IL28B genotype), and viral factors (e.g., amino acid substitutions in the core-NS5A region) have often been used as pretreatment predictors of poor virological response to PEG-IFN-ribavirin dual therapy (12, 14, 16, 18, 20) and telaprevir-PEG-IFN-ribavirin triple therapy (27, 28). The present study identified that the treatment efficacy of triple therapy could be predicted by the combination of host (IL28B rs8099917 genotype), viral (substitution of aa 70), and treatment (response to prior treatment, PEG-IFN dose, and T12PR24 regimen) factors. Especially, the use of the combination of rs8099917 non-TT genotype, Gln70 (His70), and nonresponse to ribavirin combination therapy had high specificity, PPV, and NPV for the prediction of non-SVR in the T12PR24 group. Unfortunately, the lowest frequency of an SVR (14%) was in patients who possessed the above three factors (namely, the treatment-resistant group). Previous studies showed that IFN monotherapy reduced the risk of HCC (29–31). Furthermore, analysis of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis (HALT-C) cohort recently showed that long-term PEG-IFN monotherapy reduced the incidence of HCC among patients with cirrhosis who did not achieve an SVR after previous IFN treatment, with or without ribavirin (32). Thus, the present study suggests that the treatment-resistant group should be selected for IFN monotherapy to overcome the problem of telaprevir-resistant variants, and to reduce the risk of hepatocarcinogenesis.
Interestingly, ultradeep sequencing identified telaprevir-resistant variants at baseline in 5 patients (2 patients with V36A [0.2%], 2 with T54A [0.2%], and t1 with V170A [0.4%]) at a very low frequency, but the frequency of resistant variants did not increase over time, except for one patient with T54A in whom it increased from 0.2% at baseline to 99.9% during treatment. This finding may be due to one or more reasons. One reason is probably related to the high susceptibility of telaprevir-resistant variants to IFN. A previous study indicated that mice infected with a resistant strain (A156F [99.9%]) developed only low-level viremia, and the virus was successfully eliminated with IFN therapy (9). Furthermore, this finding probably suggests that a small number of mutant-type viral RNAs may be incomplete or defective, since a large proportion of viral genomes are thought to be defective due to the high replication and mutation rates of the virus (33). Further studies should be performed to evaluate the significance of the presence of low-frequency variants detected by ultradeep sequencing.
A recent study using the human hepatocyte chimeric mouse model and deep sequencing reported that the rapid emergence of de novo telaprevir-resistant HCV quasispecies was induced by mutation of the wild-type strain of HCV in vivo (9). In the present study, ultradeep sequencing did not detect any telaprevir-resistant variants at baseline in 11 patients, although de novo resistant variants emerged in all 11 patients over time. The present clinical results based on patients who did not achieve an SVR provide evidence in support of a de novo emergence of telaprevir resistance that is induced by viral mutation.
The results of the present study should be interpreted with caution, since the study was performed in a small number of Japanese patients infected with HCV-1b. Any generalization of the results should await confirmation by a multicenter randomized trial based on a larger number of patients, including patients of other races and those infected with HCV-1a. Furthermore, the other limitation of the present study is that the existence of very low-frequency telaprevir-resistant variants was not investigated long after the cessation of therapy by ultradeep sequencing. Further large-scale studies using ultradeep sequencing should be performed to investigate the effects of telaprevir-resistant variants on the response to treatment using new drugs, including direct-acting antiviral agents.
In conclusion, this study, which is based on Japanese patients infected with HCV genotype 1b, indicated that the efficacy of triple therapy could be predicted by the combination of host, viral, and treatment factors. However, the present results show that it might be difficult to predict at baseline the emergence of telaprevir-resistant variants during triple therapy, even with the use of ultradeep sequencing. Further large-scale prospective studies are needed to investigate the pretreatment predictors of treatment efficacy and the emergence of telaprevir-resistant variants after triple therapy, and to develop more effective therapeutic regimens in patients infected with HCV genotype 1.
ACKNOWLEDGMENTS
This study was supported in part by a grant-in-aid from the Ministry of Health, Labor, and Welfare, Japan.
Norio Akuta has received honoraria from MSD, K.K., and holds a right for royalty from SRL, Inc. Hiromitsu Kumada has received honoraria from MSD, K.K., Mitsubishi Tanabe Pharma, Dainippon Sumitomo Pharma, and Bristol-Myers Squibb and holds a right for royalty from SRL, Inc. The other authors declare no conflict of interest.
Footnotes
Published ahead of print 19 June 2013
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