Anne Ghys

Evolution of Treatment-Emergent Resistant Variants in Telaprevir Phase 3 Clinical Trials

BACKGROUND Telaprevir (TVR), a hepatitis C virus (HCV) NS3/4A protease inhibitor, has been approved to treat genotype 1 HCV. To understand the clinical impact of TVR-resistant variants, we analyzed samples from patients in phase 3 clinical trials to determine the frequency and retention of TVR-resistant variants in patients who did not achieve sustained virologic response (SVR). METHODS A total of 1797 patients were treated with TVR. Resistant variants (V36A/G/I/L/M, T54A/S, I132V [subtype 1a only], R155G/K/T/M, A156F/N/S/T/V, and D168N) were identified after treatment failure and at visits thereafter, by direct (population) sequencing of the NS3/4A region. Kaplan-Meier analysis was used to determine median time to loss of these variants. RESULTS Resistant variants were observed in 77% (299/388) of patients who did not achieve SVR. Resistance occurred more commonly in subtype 1a (86%; 232/269) than subtype 1b infections (56%; 67/119). After treatment failure, 355 patients had at least 1 follow-up visit (median follow-up period: 9.6 months). Of patients with resistance at time of failure and at least 1 follow-up visit, 60% (153/254) lost resistance. Kaplan-Meier analysis, including all patients with any sequence data after treatment failure, indicated that median time to wild type was 10.6 months (95% confidence interval [CI], 9.47-12.20) in subtype 1a and 0.9 months (95% CI, 0.00-2.07) in subtype 1b infections. CONCLUSIONS After failure to achieve SVR with TVR-based treatment, resistant variants are observed in most patients. However, presumably due to the lower fitness of those variants, they tend to be replaced with wild-type virus over time.

Hepatitis C Viral Evolution in Genotype 1 Treatment-Naïve and Treatment-Experienced Patients Receiving Telaprevir-Based Therapy in Clinical Trials

Background In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR. Methods Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment. Results Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients. Conclusions A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.

Prevalence of the hepatitis C virus NS3 polymorphism Q80K in genotype 1 patients in the European region

Hepatitis C virus (HCV) NS3 polymorphism Q80K is mainly found in patients with HCV genotype (G) 1a, and has been associated with a reduced treatment response to simeprevir with pegylated interferon (P) and ribavirin (R). Prevalence of Q80K among G1 patients may vary geographically. Q80K prevalence in the North-American G1 population in a recent study was 34%. We conducted a post hoc meta-analysis of Q80K polymorphism prevalence among HCV G1-infected patients enrolled in simeprevir and telaprevir Phase II/III studies. Baseline HCV NS3/4A protease sequences were analysed by population sequencing to determine Q80K prevalence. Overall, of 3349 patients from 25 countries in the European region analysed, 35.8%, 63.8% and 0.3% of patients had G1a, G1b and other/unknown HCV G1 subtypes, respectively. Q80K was detected at baseline in 7.5% of HCV G1 patients overall. Examination by subtype showed that 19.8%, 0.5% and 18.2% of patients with G1a, G1b and other/unknown HCV G1 subtypes had the Q80K polymorphism, respectively. Among countries in the European region with sequencing data available for either ⩾20 patients with G1a and/or ⩾40 G1 patients overall, the Q80K prevalence in G1 ranged from 0% in Bulgaria to 18.2% in the UK. Q80K prevalence also varied within G1a across different countries. HCV subtype 1a was correctly determined in 99% of patients by the LiPA v2 assay. A low overall prevalence of Q80K was observed in HCV G1-infected patients in the European region, compared with North America. However, the prevalence varied by country, due to differing ratios of G1a/G1b and differing Q80K prevalence within the G1a populations.

Characterization of telaprevir treatment outcomes and resistance in patients with prior treatment failure: Results from the REALIZE trial

In the Phase 3 REALIZE study, 662 genotype 1 hepatitis C virus (HCV)‐infected patients with prior peginterferon/ribavirin treatment failure (including relapsers, partial, and null responders) were randomized to 12 weeks of telaprevir given immediately (T12/PR48) or following 4 weeks of peginterferon/ribavirin (lead‐in T12/PR48), or 12 weeks of placebo (PR48), combined with a total of 48 weeks of peginterferon alfa‐2a/ribavirin. Sustained virologic response (SVR) rates were 64% (T12/PR48), 66% (lead‐in T12/PR48), and 17% (PR48). This analysis aimed to characterize treatment outcomes and viral variants emerging in telaprevir‐treated patients not achieving SVR. HCV NS3·4A population sequencing was performed at baseline, during treatment, and follow‐up. Telaprevir‐resistant variants were classified into lower‐level (3‐ to 25‐fold 50% inhibitory concentration [IC50] increase: V36A/M, T54A/S, R155I/K/M/T, and A156S) and higher‐level (>25‐fold IC50 increase: V36M+R155K and A156T/V) resistance. Resistant variants were uncommon at baseline. Overall, 18% (52%, 19%, and 1% of prior null and partial responders and relapsers, respectively) of telaprevir‐treated patients had on‐treatment virologic failure, with no significant difference with or without a lead‐in. Virologic failure during the telaprevir‐treatment phase was predominantly associated with higher‐level resistance; virologic failure during the peginterferon/ribavirin‐treatment phase was associated with higher‐ or lower‐level, or wildtype variants, depending on genotype. Relapse occurred in 9% of patients completing assigned treatment and was generally associated with lower‐level resistant variants or wildtype. Resistant variants were no longer detectable by study end (median follow‐up of 11 months) in 58% of non‐SVR patients. Conclusion: In REALIZE, variants emerging in non‐SVR, telaprevir‐treated patients were similar irrespective of the use of a lead‐in and were consistent with those previously reported. In most patients, resistant variants became undetectable over time. (HEPATOLOGY 2012;56:2106–2115)

Telaprevir Activity in Treatment-Naive Patients Infected Hepatitis C Virus Genotype 4: A Randomized Trial

BACKGROUND This partially blinded, randomized, phase 2a C210 study evaluated the antiviral activity of telaprevir-based regimens in treatment-naive patients with chronic hepatitis C virus (HCV) genotype 4 infection. METHODS Twenty-four patients received telaprevir 750 mg every 8 hours for 15 days (T; n = 8), telaprevir in combination with pegylated interferon alfa-2a and ribavirin (Peg-IFN/RBV) for 15 days (TPR; n = 8), or Peg-IFN/RBV plus placebo for 15 days (PR; n = 8), followed by Peg-IFN/RBV for 46 or 48 weeks. The primary objective was to assess the effect of telaprevir on HCV RNA levels. RESULTS HCV RNA levels decreased slightly with T and PR; TPR produced substantial, rapid declines. On day 15, median reductions in the HCV RNA load from baseline were -0.77, -4.32, and -1.58 log10 IU/mL for T, TPR, and PR, respectively, and 0 patients in the T group, 1 in the TPR group, and 0 in the PR group had undetectable HCV RNA. Five of 8 patients who received telaprevir monotherapy had viral breakthrough within 15 days of treatment. Adverse event incidence was similar across treatments and comparable with the incidences from previous clinical trials. One patient (in T group) had a serious adverse event (considered unrelated to telaprevir) that led to treatment discontinuation. CONCLUSIONS Telaprevir with Peg-IFN/RBV had greater activity than Peg-IFN/RBV treatment or telaprevir monotherapy against HCV genotype 4. Telaprevir was generally safe and well tolerated. Further investigation of telaprevir combination therapy in patients with HCV genotype 4 infection is warranted.

Deep-Sequencing Analysis of the Gene Encoding the Hepatitis C Virus Nonstructural 3–4A Protease Confirms a Low Prevalence of Telaprevir-Resistant Variants at Baseline and the End of the REALIZE Study

BACKGROUND Population sequencing (PS) has shown that telaprevir-resistant variants are not typically detectable at baseline (prevalence, ≤5% of patients), and most variants present at the time of treatment failure are no longer detectable at the end of the study. METHODS To gain insight into the evolution of telaprevir-resistant variants, their baseline prevalence and persistence after treatment was investigated using a more sensitive, deep-sequencing (DS) technique in a large number of treatment-experienced patients from the REALIZE study who were infected with hepatitis C virus genotype 1. RESULTS Before treatment initiation, telaprevir-resistant variants (T54A, T54S, or R155K in 1%-2% of the viral population) were detected by DS in a fraction (2%) of patients for whom PS failed to detect resistance; these variants were not necessarily detected at the time of treatment failure. Of 49 patients in whom telaprevir-resistant variants were detected by PS at the time of treatment failure but not at the end of the study, DS revealed the presence of variants (V36A/L/M, T54S, or R155K in 1%-36% of the viral population) in 16 patients (33%) at the end of the study. CONCLUSIONS Similar to PS findings, DS analysis revealed that the frequency of telaprevir-resistant variants before treatment was also low, and variants detected at the time of treatment failure were no longer detectable in the majority of patients during follow-up.

Analysis of genotype 2 and 3 hepatitis C virus variants in patients treated with telaprevir demonstrates a consistent resistance profile across genotypes

Study C209 evaluated the activity of telaprevir in treatment‐naïve patients with genotypes 2 or 3 (G2, G3) hepatitis C virus (HCV) infection. Telaprevir monotherapy showed potent activity against HCV G2, but limited activity against G3. This analysis was performed to characterize HCV viral variants emerging during telaprevir‐based treatment of G2/G3 HCV‐infected patients. Patients were randomized to receive 2 weeks of treatment with telaprevir (telaprevir monotherapy), telaprevir plus peginterferon alfa‐2a and ribavirin (triple therapy), or placebo plus peginterferon alfa‐2a and ribavirin (control), followed by 22–24 weeks of peginterferon/ribavirin alone. Viral breakthrough was defined as an increase >1 log10 in HCV RNA from nadir, or HCV RNA >100 IU/mL in patients previously reaching <25 IU/mL. Twenty‐three patients (47%) had G2 and 26 (53%) had G3 HCV. Viral breakthrough occurred during the initial 2‐week treatment phase in six G2 patients (66.7%; subtypes 2, 2a and 2b) and three G3 patients (37.5%; all subtype 3a), all in the telaprevir monotherapy arm. Four breakthrough patients (three G2, one G3) subsequently achieved sustained virologic response (SVR). In all patients with breakthrough and available sequence data, mutations associated with reduced susceptibility to telaprevir in genotype 1 (G1) HCV were observed. No novel G2/G3‐specific mutations were associated with telaprevir resistance. The telaprevir resistance profile appeared consistent across HCV genotypes 1, 2 and 3. Although viral breakthrough with resistance occurred in patients receiving telaprevir monotherapy, half of these patients achieved an SVR upon addition of peginterferon/ribavirin highlighting the importance of combination therapy.

An OPTIMIZE Study Retrospective Analysis for Management of Telaprevir-Treated Hepatitis C Virus (HCV)-Infected Patients by Use of the Abbott RealTime HCV RNA Assay

ABSTRACT Protease inhibitor (PI)-based response-guided triple therapies for hepatitis C virus (HCV) infection are still widely used. Noncirrhotic treatment-naive and prior relapser patients receiving telaprevir-based treatment are eligible for shorter, 24-week total therapy if HCV RNA is undetectable at both weeks 4 and 12. In this study, the concordance in HCV RNA assessments between the Roche High Pure System/Cobas TaqMan and Abbott RealTime HCV RNA assays and the impacts of different HCV RNA cutoffs on treatment outcome were evaluated. A total of 2,629 samples from 663 HCV genotype 1 patients receiving telaprevir/pegylated interferon/ribavirin in OPTIMIZE were analyzed using the High Pure System and reanalyzed using Abbott RealTime (limits of detection, 15.1 IU/ml versus 8.3 IU/ml; limits of quantification, 25 IU/ml versus 12 IU/ml, respectively). Overall, good concordance was observed between the assays. Using undetectable HCV RNA at week 4, 34% of the patients would be eligible for shorter treatment duration with Abbott RealTime versus 72% with the High Pure System. However, using <12 IU/ml for Abbott RealTime, a similar proportion (74%) would be eligible. Of the patients receiving 24-week total therapy, 87% achieved a sustained virologic response with undetectable HCV RNA by the High Pure System or <12 IU/ml by Abbott RealTime; however, 92% of the patients with undetectable HCV RNA by Abbott RealTime achieved a sustained virologic response. Using undetectable HCV RNA as the cutoff, the more sensitive Abbott RealTime assay would identify fewer patients eligible for shorter treatment than the High Pure System. Our data confirm the <12-IU/ml cutoff, as previously established in other studies of the Abbott RealTime assay, to determine eligibility for shortened PI-based HCV treatment. (The study was registered with ClinicalTrials.gov under registration no. NCT01241760.)

Evolution of Hepatitis C Virus Quasispecies during Repeated Treatment with the NS3/4A Protease Inhibitor Telaprevir

ABSTRACT In treating hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections, the rapid reselection of resistance-associated variants (RAVs) is well known in patients with repeated exposure to the same class of antiviral agents. For chronic hepatitis C patients who have experienced virologic failure with direct-acting antiviral drugs, the potential for the reselection of persistent RAVs is unknown. Nine patients who received 14 days of telaprevir monotherapy were retreated with telaprevir-based triple therapy 4.3 to 5.7 years later. In four patients with virologic failure with both telaprevir-containing regimens, population-based and deep sequencing (454 GS-FLX) of the NS3 protease gene were performed before and at treatment failure (median coverage, 4,651 reads). Using deep sequencing, with a threshold of 1.0% for variant calling, no isolates were found harboring RAVs at the baseline time points. While population-based sequencing uncovered similar resistance patterns (V36M plus R155K for subtype 1a and V36A for subtype 1b) in all four patients after the first and second telaprevir treatments, deep sequencing analysis revealed a median of 7 (range, 4 to 23) nucleotide substitutions on the NS3 backbone of the resistant strains, together with large phylogenetic differences between viral quasispecies, making the survival of resistant isolates highly unlikely. In contrast, in a comparison of the two baseline time points, the median number of nucleotide exchanges in the wild-type isolates was only 3 (range, 2 to 8), reflecting the natural evolution of the NS3 gene. In patients with repeated direct antiviral treatment, a continuous evolution of HCV quasispecies was observed, with no clear evidence of persistence and reselection but strong signs of independent de novo generation of resistance. Antiviral therapy for chronic viral infections, like HIV, hepatitis B virus (HBV), or hepatitis C virus (HCV), faces several challenges. These viruses have evolved survival strategies and proliferate by escaping the hosts immune system. The development of direct-acting antiviral agents is an important achievement in fighting these infections. Viral variants conferring resistance to direct antiviral drugs lead to treatment failure. For HIV/HBV, it is well known that viral variants associated with treatment failure will be archived and reselected rapidly during retreatment with the same drug/class of drugs. We explored the mechanisms and rules of how resistant variants are selected and potentially reselected during repeated direct antiviral therapies in chronically HCV-infected patients. Interestingly, in contrast to HIV and HBV, we could not prove long-term persistence and reselection of resistant variants in HCV patients who failed protease inhibitor-based therapy. This may have important implications for the potential to reuse direct-acting antivirals in patients who failed the initial direct antiviral treatment. (The phase IIIb study described in this paper is registered at ClinicalTrials.gov under registration number NCT01054573.)

Modeling Viral Evolutionary Dynamics after Telaprevir-Based Treatment

For patients infected with hepatitis C virus (HCV), the combination of the direct-acting antiviral agent telaprevir, pegylated-interferon alfa (Peg-IFN), and ribavirin (RBV) significantly increases the chances of sustained virologic response (SVR) over treatment with Peg-IFN and RBV alone. If patients do not achieve SVR with telaprevir-based treatment, their viral population is often significantly enriched with telaprevir-resistant variants at the end of treatment. We sought to quantify the evolutionary dynamics of these post-treatment resistant variant populations. Previous estimates of these dynamics were limited by analyzing only population sequence data (20% sensitivity, qualitative resistance information) from 388 patients enrolled in Phase 3 clinical studies. Here we add clonal sequence analysis (5% sensitivity, quantitative) for a subset of these patients. We developed a computational model which integrates both the qualitative and quantitative sequence data, and which forms a framework for future analyses of drug resistance. The model was qualified by showing that deep-sequence data (1% sensitivity) from a subset of these patients are consistent with model predictions. When determining the median time for viral populations to revert to 20% resistance in these patients, the model predicts 8.3 (95% CI: 7.6, 8.4) months versus 10.7 (9.9, 12.8) months estimated using solely population sequence data for genotype 1a, and 1.0 (0.0, 1.4) months versus 0.9 (0.0, 2.7) months for genotype 1b. For each individual patient, the time to revert to 20% resistance predicted by the model was typically comparable to or faster than that estimated using solely population sequence data. Furthermore, the model predicts a median of 11.0 and 2.1 months after treatment failure for viral populations to revert to 99% wild-type in patients with HCV genotypes 1a or 1b, respectively. Our modeling approach provides a framework for projecting accurate, quantitative assessment of HCV resistance dynamics from a data set consisting of largely qualitative information.