Research Article
NS5A resistance-associated substitutions in patients with genotype 1 hepatitis C virus: Prevalence and effect on treatment outcome

https://doi.org/10.1016/j.jhep.2017.01.007Get rights and content

Background & Aims

The efficacy of NS5A inhibitors for the treatment of patients chronically infected with hepatitis C virus (HCV) can be affected by the presence of NS5A resistance-associated substitutions (RASs). We analyzed data from 35 phase I, II, and III studies in 22 countries to determine the pretreatment prevalence of various NS5A RASs, and their effect on outcomes of treatment with ledipasvir-sofosbuvir in patients with genotype 1 HCV.

Methods

NS5A gene deep sequencing analysis was performed on samples from 5397 patients in Gilead clinical trials. The effect of baseline RASs on sustained virologic response (SVR) rates was assessed in the 1765 patients treated with regimens containing ledipasvir-sofosbuvir.

Results

Using a 15% cut-off, pretreatment NS5A and ledipasvir-specific RASs were detected in 13% and 8% of genotype 1a patients, respectively, and in 18% and 16% of patients with genotype 1b. Among genotype 1a treatment-naïve patients, SVR rates were 91% (42/46) vs. 99% (539/546) for those with and without ledipasvir-specific RASs, respectively. Among treatment-experienced genotype 1a patients, SVR rates were 76% (22/29) vs. 97% (409/420) for those with and without ledipasvir-specific RASs, respectively. Among treatment-naïve genotype 1b patients, SVR rates were 99% for both those with and without ledipasvir-specific RASs (71/72 vs. 331/334), and among treatment-experienced genotype 1b patients, SVR rates were 89% (41/46) vs. 98% (267/272) for those with and without ledipasvir-specific RASs, respectively.

Conclusions

Pretreatment ledipasvir-specific RASs that were present in 8–16% of patients have an impact on treatment outcome in some patient groups, particularly treatment-experienced patients with genotype 1a HCV.

Lay summary

The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection received treatments that included ledipasvir-sofosbuvir to determine how prevalent NS5A RASs are in patients at baseline, and found that ledipasvir-specific RASs were present in 8–16% of patients prior to treatment and had a negative impact on treatment outcome in subset of patient groups, particularly treatment-experienced patients with genotype 1a HCV.

Introduction

Due to high rates of viral replication and an error prone hepatitis C virus (HCV) RNA polymerase, tremendous variability of HCV has been observed within infected patients (quasispecies). These single mutations that do not abolish viral replication, are thought to be pre-existing [1] and as a result, NS5A resistance-associated substitutions (RASs) are observed at baseline in patients infected with chronic HCV. Deep sequencing enables detection of HCV substitutions, point deletions, or insertions within the quasispecies down to a frequency of 1%. However, commercially available assays based on standard population HCV sequencing or not cross-validated next generation, also called deep sequencing, report variants with a frequency of ⩾15% of the quasispecies.

The prevalence of baseline NS5A RASs has been reported to be 6% to 16% using population sequencing (cut-off 15–25%) or deep sequencing (cut-off 1%) [2], [3], [4]. Interestingly, the prevalence and type of baseline NS5A RASs may vary by geographic regions. For example, the prevalence of the NS5A M28V in genotype 1a-infected patients was shown to be higher in the United States than in Europe, 7% vs. 0%, respectively [5]. Furthermore, the prevalence of genotype 3 NS5A Y93H varied between 0% and 17% in different geographic regions [6]. A comparison of baseline prevalence of RASs in Japanese and Western patients showed that the prevalence of Q80L and S122G in NS3, and L28M, R30Q and Y93H in NS5A was significantly higher in Japanese patients than the Western counterparts [7].

Many currently approved interferon (IFN)-free regimens for the treatment of chronic hepatitis C (HCV) include an inhibitor of HCV NS5A. To date, there are five NS5A inhibitors approved for treatment of chronic HCV infection; ledipasvir (LDV), daclatasvir, and velpatasvir (which are all administered with the NS5B inhibitor sofosbuvir [SOF]), and ombitasvir (in a fixed-dose combination with the protease inhibitor paritaprevir, the nonnucleoside NS5B polymerase inhibitor dasabuvir, and ritonavir, a potent inhibitor of CYP3A4 enzymes), and elbasvir (in a fixed-dose combination with the protease inhibitor grazoprevir) [8], [9], [10], [11], [12]. The presence of baseline NS5A RASs may impact the treatment outcome of some NS5A inhibitor containing HCV regimens due to the intrinsic qualities of the NS5A inhibitor, drug pharmacology, or effects of the other compounds within the treatment regimen. However, depending on how NS5A RASs are defined and included in resistance analysis, as well as what level of variant detection is utilized, different results may be obtained. To date, three definitions of NS5A variants that are associated with resistance have been used most commonly; polymorphisms at RAS positions (RAPs), class RASs, and drug-specific RASs. Polymorphisms at RAS positions are defined as any change from reference sequence for a specific genotype at positions associated with NS5A inhibitor resistance. NS5A class RASs are substitutions that have been shown to emerge on treatment or confer a significant reduction in susceptibility in vitro (e.g., >2.5-fold change in EC50) to any approved or investigational NS5A inhibitor. Drug-specific RASs refer to substitutions that have been shown to emerge on the specific drug treatment or confer significantly reduced susceptibility in vitro to the specific NS5A inhibitor. In addition, drug-specific RASs can be categorized into groups with different levels of reduced susceptibility to the drug.

To enable comparisons of resistance analyses between clinical trials, standardization of RAS definitions and sensitivity cut-offs are needed. In several studies, population sequences were used for resistance analysis (cut-off for variant detection 15–25%) and NS5A polymorphisms at RAS positions were defined as RAPs. In these studies, the presence of baseline NS5A polymorphisms at RAS positions had shown no significant impact on treatment outcome [5], [13]. Further study is needed to understand the role of RASs present at frequencies below 15% and whether substitutions without an in vitro susceptibility change to the NS5A inhibitor may dilute a clinical signal by RASs that do confer reduced susceptibility to a specific NS5A inhibitor.

Here, we characterize the prevalence of baseline NS5A RASs in 5397 NS5A inhibitor-naïve HCV patients infected with genotype 1a or 1b according to geographic regions. Moreover, we assessed the effect of baseline NS5A RASs, defined as NS5A RAPs, NS5A class RASs or LDV-specific RASs using 1% and 15% sensitivity of substitution detection cut-offs, on treatment outcome among 1765 patients treated with currently recommended regimens containing LDV-SOF. A previous analysis using a portion of the same dataset has recently been published [14]. That analysis concerned the prevalence and effect on treatment of NS3, NS5A, and NS5B RASs, and included data on patients who had been treated with regimens/durations that have not been incorporated into label recommendations or treatment guidelines. The current study covers only NS5A RASs and includes data only from patients who received guideline-recommended regimens.

Section snippets

Sequencing analysis

Deep sequencing of baseline plasma samples was performed in 5397 patients from 22 countries across the HCV Gilead clinical development program from 2010 to 2015. The list of clinical trials and identification numbers are included in the supplementary materials (Supplementary Table 1). The HCV NS5A coding regions were amplified by DDL Diagnostic Laboratory (Rijswijk, Netherlands) using proprietary amplification primers and standard reverse transcription polymerase chain reaction (RT-PCR)

Patient baseline characteristics

Demographic and baseline clinical characteristics of the 5393 NS5A inhibitor-naïve patients included in the NS5A baseline prevalence RAS analysis are provided in Table 2. The majority of patients were treatment-naïve (56%) and male (64%), with HCV genotype 1a (65%) and non-CC interleukin (IL) 28B alleles (73%). Approximately one-third (32%) of patients had cirrhosis.

Prevalence and type of pretreatment RASs across geographic regions

Baseline prevalence of NS5A polymorphisms at RAS positions, NS5A class RASs, LDV RASs and the specific Y93H NS5A variant was

Discussion

Current NS5A inhibitors show overlapping but distinct resistance profiles with RASs described at the NS5A amino acid positions 24, 28, 30, 32, 31, 38, 58, 92, and 93. There are advantages and disadvantages with each of the three main approaches to defining NS5A RASs. The advantage of using the NS5A RAPs definition is that it provides a uniform list of variants for all NS5A inhibitors. It does not require extensive phenotypic testing of all variants with several NS5A inhibitors and provides

Financial support

This study was sponsored by Gilead Sciences, Inc. who was also involved with the study design, as well as the analysis and interpretation of data.

Conflict of interest

Stefan Zeuzem: Consultant: Abbvie, BMS, Gilead, Janssen, and Merck; Speaker: Abbvie, BMS, Gilead, Janssen, and Merck. Masashi Mizokami: Research: Asahi Kasei Pharma Corporation, Mitsubishi Tanabe Pharma Corp, G&G Science Co, SRL Technical Services; Speaker: Abbott, Astellas, Bristol-Myers Squibb, Chugai Pharmaceutical, Daiichi Sankyo, Dainippon Sumitomo, Eisai, G&G, GlaxoSmithKline, Minophagen, Merck Sharp & Dohme, Otsuka Pharmaceutical, Taisho Toyama Pharmaceutical, Takeda Pharmaceutical, The

Authors’ contributions

Conception/Design: Stefan Zeuzem, Ross Martin, Evguenia Svarovskaia, Hadas Dvory-Sobol, Brian Doehle, Charlotte Hedskog, Michael D. Miller, Hongmei Mo; Data Acquisition/Analysis: Ross Martin, Evguenia Svarovskaia, Hadas Dvory-Sobol, Brian Doehle, Charlotte Hedskog, Michael D. Miller, Hongmei Mo; Data Interpretation: Stefan Zeuzem, Masashi Mizokami, Stephen Pianko, Alessandra Mangia, Kwang-Hyub Han, Diana M. Brainard, Steven Knox, John G. McHutchison, Wan-Long Chuang, Ira Jacobson, Gregory J.

Acknowledgement

We extend our thanks to the patients, their families, and all participating investigators. This study was sponsored by Gilead Sciences. Editorial assistance was provided by David McNeel and Sandra Chen of Gilead Sciences.

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