Evaluation of factors associated with relapse in telaprevir-based triple therapy for chronic hepatitis C.

BACKGROUND AND RATIONALE: Most patients with chronic hepatitis C show virological response to telaprevir-based triple therapy, and achieve an end-of-treatment response (ETR). However, some patients showing ETR develop virological relapse. This study was carried out to evaluate factors associated with relapse after triple therapy.
MATERIALS AND METHODS: A prospective, multicentric study was conducted in chronic hepatitis C patients who received telaprevir-based triple therapy. We evaluated independent variables such as age, with or without cirrhosis, prior treatment response to interferon (IFN) therapy, IL28B genotype, core amino acid (aa) 70 mutation, drug adherence, white blood cell counts, hemoglobin level, and serum low-density lipoprotein (LDL) cholesterol level. The characteristics of the patients who relapsed after achieving ETR were compared with those who did not.
RESULTS: Among 168 patients, 157 patients achieved ETR (93.5%) and 11 discontinued. Of these 157 patients, relapse occurred in 21 patients (13.4%). Nineteen patients (90.5%) of 21 relapsed patients had the IL28B non-TT genotype (P = 1.79 × 10 -9 ). Multivariate analysis identified core amino acid 70 [P = 0.018, crude odds ratio (OR): 6.927] and the IL28B genotype (P = 3.758 × 10 -5 , crude OR: 39.311) as significantly independent factors that influenced the relapse-related variables. Among the 49 patients with the IL28B non-TT, 18 patients had core aa70 mutation and 31 patients had core aa70 wild-type. In addition, 66.7% (12/18) of those with core aa70 mutation and 22.6% (7/31) of those with core aa70 wild-type developed relapse (P = 0.005). DISCUSSION: Core aa70 mutation and the IL28B non-TT genotype were identified as independent factors that influenced relapse after achievement of ETR for telaprevir-based triple therapy.

Introduction

Recent recommendations suggest the use of an interferon (IFN)-free regimen as the mainstay of hepatitis C virus (HCV) genotype 1 management. However, in some countries and regions, pegylated interferon (PEG-IFN)/ribavirin combined with or without protease inhibitors remains the standard of care for genotype 1b.[1] PEG-IFN/ribavirin therapy in combination with protease inhibitors, such as telaprevir and boceprevir, is a powerful treatment for HCV genotype 1.[234567] The sustained virological response (SVR) rate in genotype 1 chronic hepatitis C treated with PEG-IFN/ribavirin/telaprevir combination therapy is approximately 70-80%.[24567] The three possible outcomes of IFN-based treatment are 1) SVR, 2) relapse, or 3) nonresponse. Very few patients are judged as a nonresponders,[267] therefore the SVR rate in the triple therapy can be judged by the relapse rate after the completion of treatment.

Host, treatment, gender, and genetic factors have been reported to be associated with virological relapse after the completion of dual PEG-IFN/ribavirin combination therapy.[8] Progression of liver fibrosis and lower doses of ribavirin are also reported to be associated with increased relapse rates.[9101112] Most importantly, the IL28B single nucleotide polymorphism (SNP) genotype has a strong impact on treatment response in dual PEG-IFN/ribavirin therapy.[131415] Telaprevir is one part of triple combination treatments. It is possible that the factors associated with relapse in the triple therapy may differ greatly from those in the dual therapy without telaprevir. This retrospective study aimed at identifying baseline and on-treatment factors associated with virological relapse in chronic hepatitis C patients with genotype 1b who received the telaprevir-based triple combination therapy.

Methods

Ethics and Study Design

The study protocol was approved by the institutional Ethics Committee, and this prospective study was initiated after obtaining written informed consent from patients.

Patients, selection criteria

Two hundred and two consecutive (N = 202) patients infected with hepatitis C genotype 1b were recruited to this study between December 2011 and October 2012. Patients were eligible for enrollment if they fulfilled the following criteria: Serum HCV ribonucleic acid (RNA) detectable by real-time polymerase chain reaction (PCR); white blood cell count of more than 2,000/μL; platelet count of more than 50,000/μL; hemoglobin levels of more than 10 g/dL; and alanine aminotransferase (ALT) levels of not more than 300 IU/L before treatment initiation. Asymptomatic carriers with normal ALT levels were included. Exclusions were those positive for hepatitis B surface antigen or antibody to human immunodeficiency virus type 1; with other chronic liver diseases such as alcoholic liver disease, autoimmune hepatitis, and primary biliary cirrhosis; with decompensated cirrhosis of the liver; with liver failure; with a history of gastrointestinal (GI) bleeding; severe renal disorder; abnormal thyroid function; with poorly controlled diabetes or hypertension; on medication with Chinese herbal medicine; with past medical history of interstitial pneumonia; pregnant or with possibility of pregnancy; lactating; with severe depression; with past medical history of allergy to biological preparations such as vaccines; or with past medical history of allergy to IFN, ribavirin, or protease inhibitors.

Treatment protocol

All patients had received combination therapy with PEG-IFNα2b (Peg-Intron; MSD, Tokyo, Japan), ribavirin (Rebetol; MSD, Tokyo, Japan), and telaprevir (Telavic; Mitsubishi Tanabe Pharma, Osaka, Japan) for 12 weeks, followed by 12 weeks of PEG-IFNα2b and ribavirin. The patients received weekly subcutaneous injections of PEG-IFNα2b (1.5 μg/kg/week) and oral administration of ribavirin. The ribavirin dose was adjusted by body weight (600 mg, 800 mg, and 1000 mg per day for <60 kg, 60-80 kg, and >80 kg, respectively) based on the guidelines of the Ministry of Health, Labor and Welfare of Japan. Telaprevir (750 mg or 500 mg) was administered every 8 h. Doses had been appropriately reduced when an adverse event such as anemia or renal insufficiency occurred during the treatment. Treatment was stopped in case of nonvirological response, such as in patients who had serum HCV RNA reductions of less than 3 log IU/mL at 4 weeks or serum HCV RNA detectable at 12 weeks, to avoid unnecessary treatment.

Definition of viral responses

When serum HCV RNA was undetectable at 4 weeks of treatment, patients were considered to have achieved rapid virological response (RVR). Patients who were negative for serum HCV RNA at the completion of treatment were considered to have achieved end-of-treatment response (ETR). Patients who were negative for serum HCV RNA at 24 weeks after treatment completion were considered to have achieved SVR. Patients with ETR in whom viral RNA reappeared after treatment completion were considered to have a relapse. Patients in whom serum HCV RNA levels decreased by 2 log IU/mL or more at 12 weeks of treatment but never became undetectable throughout the duration of treatment and follow-up were considered as partial responder. Patients who did not show serum HCV RNA reductions of less than 2 log IU/mL at 12 weeks of treatment were considered as null responders. Partial and null responders were analyzed as nonresponders.[16]

Laboratory tests

Hematological, liver function, and renal function tests were performed weekly until 24 weeks after the treatment initiation and then monthly until 6 months after the treatment completion. Serum HCV RNA levels were measured by the real-time PCR method (COBAS AmpliPrep; Roche Diagnostics, Tokyo, Japan). Core amino acid 70 and NS5A regions (IFN sensitivity-determining region) of the HCV genome were determined by the direct sequencing method. Core amino acid 70 was defined as wild-type (arginine) or mutant-type (glutamine or histidine). Genomic deoxyribonucleic acid (DNA) was extracted from whole blood using a DNA Isolation kit on a MagNA Pure LC instrument (Roche Diagnostics, Basel, Switzerland). IL28B SNP rs8099917 was determined by the real-time detection PCR using TaqMan SNPs Genotyping Assays on a 7500Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Rs8099917 SNP genotypes were classified into two categories: TT and non-TT (TG or GG). SNPs at rs1127354, which is located in the locus adjacent to the inosine triphosphatase (ITPA) gene, were also genotyped by the real-time detection PCR using TaqMan SNP Genotyping Assay. Rs1127354 SNP genotypes were classified into two categories: CC and non-CC.[1718]

Statistical analysis

Fisher's exact test, chi-square test, and Mann-Whitney U test were performed for comparison of variables between patients with SVR and patients with relapse. Multiple logistic regression analysis was performed to identify significantly independent factors that were associated with relapse. Level of significance was set at P < 0.05. We evaluated independent variables such as age, with or without cirrhosis, prior treatment response to IFN therapy, IL28B genotype, core amino acid 70 mutation, drug adherence, white blood cell counts, hemoglobin level, and serum low-density lipoprotein (LDL) cholesterol level. Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 17.0 (IBM Japan, Tokyo, Japan).

Results

Demographic data

Of the 202 patients enrolled in the study, 168 received telaprevir-based triple combination therapy [Figure 1]. The remaining 34 patients were excluded from the study treatment (10 patients had other liver disease, 4 patient had poorly controlled diabetes, 12 patients had cytopenia, 1 patient had abnormal thyroid function, and 7 patients did not provide written informed consent). Of the 168 patients who were administered the treatment, 157 (93.5%) completed the 24-week treatment course and the remaining 11 (6.5%) prematurely discontinued the treatment; viral breakthrough occurred in 7, nonvirological response to treatment in 3, and severe anemia in 1 [Figure 1]. Thus, 157 patients were finally evaluated.

Figure 1

Flow of patients

The characteristics of the 157 patients are shown in Table 1. The subjects consisted of 74 men and 83 women, with a median age of 60 years (range 18-75 years). There were 108 patients with IL28B TT genotype and 49 with IL28B non-TT genotype, and there were 69 treatment-naïve patients, 62 relapsers, and 26 nonresponders to PEG-IFN/ribavirin therapy (16 partial and 10 null responders). There were 30 patients with advanced fibrosis or cirrhosis (F3 or F4).

Table 1

Baseline clinical and demographic characteristics of the total 157 patients with ETR, 136 patients achieving SVR and 21 patients with relapse

Factor	SVR (n = 136)	Relapse (n = 21)	P value*
Age (years)	59 (18-75)	63 (41-74)	0.045
Gender (Male/Female)	64/72	10/11	1.000
History of interferon therapy (naïve/relapse/partial responder/null responder)	65/56/11/4	4/6/5/6	0.0004
Stage of fibrosis or cirrhosis (F0-F2/F3-4)	106/30	14/7	0.275
BMI (BW/m2)	22.6 (15.9-37.8)	22.5 (15.2-31.9)	0.963
White blood cell count (/µl)	5000 (2000-11100)	4200 (2200-7000)	0.028
Hemoglobin (g/dl)	14.3 (11.3-17.2)	12.9 (11.1-15.9)	0.041
Platelet count (/mm3)×103	172 (70-350)	149 (76-407)	0.787
AST (IU/l)	38 (13-215)	46 (22-139)	0.244
ALT (IU/l)	41 (13-291)	42 (20-201)	0.988
γGTP (U/l)	35 (11-339)	46 (14-296)	0.301
Albumin (g/dl)	4.2 (3.0-4.8)	4.1 (2.8-4.7)	0.509
Total-Bilirubin (mg/dl)	0.8 (0.3-2.0)	0.7 (0.4-2.3)	0.534
LDL-Cholesterol (mg/dl)	101 (45-194)	90 (39-137)	0.020
alpha-fetoprotein (ng/ml)	4.5 (1.4-234.7)	6.0 (2.7-101.5)	0.116
HCV-RNA (LogIU/ml)	6.6 (5.2 -7.8)	6.2 (5.1 -7.6)	0.088
IL28B genotype (rs8099917) (TT/non TT)	106/30	2/19	1.79×10-9
ISDR (0-1/2<)	71/65	11/10	1.000
Core aa70 (wild/mutant)	98/38	8/13	0.005
ITPA (rs1127354) (CC/CA or AA)	112/24	20/1	0.201
RVR/non RVR	116/20	15/6	0.323
Total dose of peg-IFN/body weight/24weeks (µg/kg/week)	1.5 (0.7-2.2)	1.5 (0.7-1.7)	0.817
Total dose of ribavirin/body weight/day (mg/kg/day)	8.0 (3.7-13.1)	7.87 (3.8-12.7)	0.773
Total dose of telaprevir (mg)	126000 (42000-198000)	126000 (29250-207000)	0.062

Eleven patients discontinued therapy due to adverse event, viral breakthrough and poor response (non virological response). Categorical variables are given as number. Continuous variables are given as median (range). *Fisher's exact test and Mann-Whitney U test was performed for comparison of baseline characteristics according to patients with SVR and relapse. BMI: Body mass index, BW: body weight in kg, AST: aspartate aminotransferase, ALT: alanine aminotransferase, γ-GTP: gamma-glutamyltransferase, LDL-cholesterol: low-density lipoprotein cholesterol, IL28B: interleukin 28B, ISDR: interferon sensitivity-determining region, aa: amino acid, ITPA: inosine triphosphatase

Treatment response

Among 168 patients who received the treatment, 157 patients (93.5%) achieved ETR. Among the 157 patients with ETR, 136 patients achieved SVR (86.6%) and 21 patients (13.4%) developed relapse. The comparison between patients with SVR and patients with relapse is shown in Table 1. Among the 136 SVR patients, 106 (77.9%) had the IL28B TT genotype and 30 (22.1%) had the IL28B non-TT genotype. In contrast, among the 21 relapsers, only 2 (9.5%) had the TT genotype and 19 (90.5%) had the non-TT genotype. Univariate analysis showed that age (P = 0.045), prior treatment response (P = 0.0004), white blood cell count (P = 0.028), hemoglobin levels (P = 0.041), LDL cholesterol levels (P = 0.020), the IL28B genotype (P = 1.785 × 10-9), and core amino acid 70 (P = 0.005) were associated with the risk of relapse.

According to prior treatment response, the SVR rates were 94.2% (65/69) in treatment-naïve patients, 90.3% (56/62) in prior relapsers, and 57.7% (15/26) in prior nonresponders. The relapse rates were higher in nonresponders (42.3%, 11/26) than in treatment-naïve patients (5.8%, 4/69, P = 0.0001) and prior relapsers (9.7%, 6/62, P = 0.0009).

The significant factors (age, with or without cirrhosis, prior treatment response to IFN therapy, IL28B genotype, core amino acid 70 mutation, drug adherence, white blood cell counts, hemoglobin level, and serum LDL cholesterol level) were put into a model. The multivariate analysis identified core amino acid 70 [P = 0.018, odds ratio (OR): 6.927, 95% confidence interval (CI): 1.396-34.360] and the IL28B genotype (P = 3.758 × 10-5, OR: 39.311, 95% CI: 6.860-225.270) as significantly independent factors that influenced the relapse-related variables of 157 patients achieving ETR.

The IL28B genotypes were most strongly associated with relapse. Most relapsers had the IL28B non-TT genotype.

Therefore, factors associated with relapse were investigated in the IL28B non-TT genotype patients who achieved ETR.

Of the 157 patients who achieved ETR, 49 had the IL28B non-TT genotype. Of the 49 patients, 30 achieved SVR and 19 showed relapse. The comparison between patients with SVR and patients with relapse is shown in Table 2. Factors associated with relapse were albumin (P = 0.040), LDL cholesterol (P = 0.007), and core amino acid 70 (P = 0.005).

Table 2

Baseline clinical and demographic characteristics and on-treatment factors of 49 patients with the IL28B non TT genotype, 30 patients achieving SVR and 19 patients with relapse

Factor	SVR (n = 30)	Relapse (n = 19)	P value*
Age (years)	57 (18-75)	63 (41-74)	0.134
Prior treatment response (naïve/relapse/partial responder/null responder)	10/11/6/3	3/6/4/6	0.072
Stage of fibrosis or cirrhosis (F0-F2/F3-4)	24/6	12/7	0.319
BMI (BW/m2)	21.3 (17.9-34.3)	22.2 (15.1-31.9)	0.424
White blood cell count (/µl)	4700 (2900-6900)	3900 (2200-7000)	0.361
Hemoglobin (g/dl)	13.4 (11.7-16.8)	13.1 (11.1-15.9)	0.485
Platelet count (/mm3)×103	166 (101-280)	149 (83-270)	0.101
AST (IU/l)	35 (16-205)	46 (22-139)	0.200
ALT (IU/l)	36 (13-291)	37 (20-201)	0.518
γGTP (U/l)	32 (11-339)	46 (14-296)	0.186
Albumin (g/dl)	4.2 (3.8-4.8)	4.1 (2.8-4.7)	0.040
Total-Bilirubin (mg/dl)	0.8 (0.3-1.5)	0.7 (0.4-2.3)	0.066
LDL-Cholesterol (mg/dl)	114 (56-194)	84 (39-137)	0.007
alpha-fetoprotein (ng/ml)	4.5 (1.9-12.4)	6.0 (2.7-101.5)	0.203
HCV-RNA (LogIU/ml)	6.3 (5.1-7.5)	6.1 (5.1-7.6)	0.742
ISDR (0-1/2<)	20/10	11/8	0.559
Core aa70 (wild/mutant)	24/6	7/12	0.005
ITPA (rs1127354) (CC/CA or AA)	25/5	18/1	0.384
RVR (presence/absence)	29/1	14/5	0.086
Total dose of peg-IFN/body weight/24weeks (µg/kg/week)	1.5 (0.8-1.7)	1.5 (1.2-1.9)	0.492
Total dose of ribavirin/body weight/day (mg/kg/day)	7.5 (3.8-11.4)	8.9 (4.1-12.0)	0.242
Total dose of telaprevir (mg)	126000 (73500-207000)	126000 (66000-189000)	0.358

All patients were achieved an ETR after completion of telaprevir/peg-IFN/ribavirin combination therapy. *Fisher's exact test and Mann-Whitney U test was performed for comparison of baseline characteristics according to patients with SVR and relapse. Categorical variables are given as number. Continuous variables are given as median (range). BMI: Body mass index, AST: aspartate aminotransferase, ALT: alanine aminotransferase, γ-GTP: gamma-glutamyltransferase, LDL- cholesterol: low-density lipoprotein cholesterol, ISDR: interferon sensitivity-determining region, aa: amino acid, ITPA: inosine triphosphatase, IL28B: interleukin 28B

Multivariate analysis identified core amino acid 70 (P = 0.021, OR = 5.625, CI = 1.304-24.272) as an independent factor associated with relapse in patients with the IL28B non-TT genotype. Among the non-TT patients, 66.7% (12/18) of those with core 70 mutation and 22.6% (7/31) of those with core 70 wild-type developed relapse (P = 0.005) [Figure 2].

Figure 2

Comparison of relapse rate according to core amino acid 70 substitutions in patients with IL28B non-TT genotype

Next, the patients, divided into treatment-naïve and treatment-experienced groups, were analyzed. Univariate analysis showed that the following factors were significantly associated with the relapse-related variables of 69 treatment-naïve patients: IL28B non-TT (P = 0.019) and HCV RNA (P = 0.036); thus, no influence of core amino acid 70 (identified on analysis in all patients) was noted. On the other hand, univariate analysis of the relapse-related variables of 88 treatment-experienced patients identified liver cirrhosis (P = 0.018), IL28B non-TT (P = 6.34 × 10-7), core amino acid 70 (P = 0.004), white blood cell count (P = 0.022), and LDL cholesterol (P = 0.045) as significant factors, while multivariate analysis identified core amino acid 70 (P = 0.027, OR: 8.479, 95% CI: 1.275-56.388) and IL28B non-TT (P = 0.0007, OR: 81.255, 95% CI: 6.360-1038.055) as significantly independent factors that influenced the relapse-related variables.

Discussion

There have been numerous investigations into factors contributing to relapse after treatment with PEG-IFN/ribavirin dual combination therapy for genotype 1b chronic hepatitis C.[891011121920] In addition, factors contributing to SVR with PEG-IFN/ribavirin/telaprevir combination therapy have been widely investigated.[21222324] However, no studies have focused on relapse after achieving ETR. Here, factors that contribute to relapse after the completion of PEG-IFN/ribavirin/telaprevir combination therapy for chronic hepatitis C were investigated.

Various factors including host, viral, treatment, and genetic factors were reported to influence treatment response and outcome in dual PEG-IFN/ribavirin or triple telaprevir-based combination therapy for chronic hepatitis C genotype 1. Most previous studies focused on the relationship between the treatment and SVR and/or nonresponse. Several studies reported factors associated with virological relapse in the dual combination therapy, such as sex, liver fibrosis, dose of ribavirin, and early viral kinetics.[89122526] It is conceivable that most of the relapsers in previous conventional treatment will achieve SVR with a stronger subsequent treatment. Indeed, approximately 90% of relapsers in the dual therapy without telaprevir achieved SVR with the telaprevir-based triple therapy. Identification of factors associated with relapse in telaprevir-based therapy may make it possible to improve the SVR rate by modifying treatment regimens and algorithm.

ETR could be achieved in most patients treated with PEG-IFN/ribavirin/telaprevir combination.[267] In the present study, the ETR rate was 93.5% (157 of 168 patients). Relapse occurred in 21 (13.4%) of the 157 patients with ETR, resulting in a reduction of the SVR rate (81.0%; 136 of 168 patients) in intention-to-treat analysis.

This study showed that the IL28B SNP genotype was the strongest factor associated with relapse as 19 (90.5%) of the 21 relapsers had the non-TT genotype. The IL28B genotype is one of the most important factors contributing to SVR/nonresponse in dual combination therapy[131415] and telaprevir-based triple combination therapy.[272829] In the present study, all of the 7 patients with viral breakthrough and the 3 patients without virological response had the IL28B non-TT genotype (data not shown). Taken together, this study confirmed that the IL28B SNP genotype is closely associated with virological relapse, as well as SVR, nonresponse, and viral breakthrough.

As shown in the present study, patients with the IL28B non-TT genotype have a risk of relapse after treatment completion. Therefore, this study investigated which factors were significantly associated with relapse in patients with the IL28B non-TT genotype. The multivariate regression analysis identified core amino acid 70 substitutions as a significantly independent factor associated with relapse. In the present study, relapse occurred in 66.7% (12/18) of patients with the IL28B non-TT genotype and core amino acid 70 mutation, despite having achieved ETR. This suggests that core amino acid 70 substitutions are an important factor in predicting the likelihood of posttreatment relapse in the IL28B non-TT genotype patients treated with PEG-IFN/ribavirin/telaprevir combination therapy.

Next, the patients, divided into treatment-naïve and treatment-experienced groups, were analyzed regarding relapse-related variables. As a result, in the treatment-naïve patients, the IL28B genotype and HCV RNA were extracted but not core amino acid 70. On the other hand, in the treatment-experienced patients, core amino acid 70 was identified as a significant factor.

From these results, we speculate as follows: It has been reported that core amino acid 70 is a significant factor correlated with non-SVR and PEG-IFN/ribavirin dual combination therapy. The IL28B non-TT genotype patients in this study were resistant to PEG-IFN/ribavirin combination therapy, and many of them may become non-SVR. The treatment-experienced patients were non-SVR with PEG-IFN/ribavirin combination therapy. Thus, in treatment-resistant patients, such as IL28B non-TT patients and those who failed to achieve SVR with PEG-IFN/ribavirin combination therapy, the core amino acid 70 was a significant, relapse-related factor in the telaprevir-based triple therapy in this study. On the other hand, in the IL28B TT and treatment-naïve patients, core amino acid 70 may have a minor influence on the results of telaprevir-based triple therapy.

There are several limitations to the present study.First, the number of patients studied was low and might be statistically insufficient to draw conclusions. Second, liver fibrosis, an important factor that influences treatment effects, was not thoroughly examined in this study. Thus, relapse rates according to platelet count were analyzed, and no significant differences were observed between patients with SVR and relapse.

In the future, lower relapse rates may be expected with PEG-IFN/ribavirin administration over periods of longer than 24 weeks in chronic hepatitis C patients with the IL28B non-TT genotype and core amino acid 70 mutation. However, there are no reported control studies on this treatment approach, and further research is necessary.

Conclusion

In conclusion, most relapsers after 24 weeks of PEG-IFN/ribavirin/telaprevir combination therapy for HCV genotype 1b had the IL28B non-TT genotype. Furthermore, the relapse rate was significantly higher in the IL28B non-TT genotype patients and treatment-experienced patients with core amino acid 70 mutations than in those with the wild-type virus.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors have no conflicts of interest to declare.