- Email: [email protected]
Development and persistence of DAA resistance associated mutations in patients failing HCV treatment
Accepted Manuscript Title: Development and persistence of DAA resistance associated mutations in patients failing HCV treatment Author: Stefania Paolucci Loretta Fiorina Bianca Mariani Viviana Landini Roberto Gulminetti Stefano Novati Renato Maserati Giorgio Barbarini Raffaele Bruno Fausto Baldanti PII: DOI: Reference:
S1386-6532(15)00650-2 http://dx.doi.org/doi:10.1016/j.jcv.2015.08.015 JCV 3421
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
17-4-2015 27-8-2015 28-8-2015
Please cite this article as: Paolucci Stefania, Fiorina Loretta, Mariani Bianca, Landini Viviana, Gulminetti Roberto, Novati Stefano, Maserati Renato, Barbarini Giorgio, Bruno Raffaele, Baldanti Fausto.Development and persistence of DAA resistance associated mutations in patients failing HCV treatment.Journal of Clinical Virology http://dx.doi.org/10.1016/j.jcv.2015.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and persistence of DAA resistance associated mutations in patients failing HCV treatment Stefania Paolucci1, Loretta Fiorina1, Bianca Mariani1, Viviana Landini1, Roberto Gulminetti2, Stefano Novati2, Renato Maserati2, Giorgio Barbarini3, Raffaele Bruno3, Fausto Baldanti1,4*
1
Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico
San Matteo, 27100 Pavia, Italy 2
Institute of Infectious Diseases, University of Pavia, 27100 Pavia, Italy
3
Division of Infectious and Tropical Diseases, Fondazione IRCCS Policlinico San Matteo, 27100
Pavia, Italy 4
Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, 27100
Pavia, Italy
*Corresponding author: F. Baldanti, Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Viale Brambilla 74, 27100 Pavia, Italy Tel:
+39
0382
502283;
Fax:
+39
0382
502599;
E-mail:
[email protected];
[email protected]
Highlights HCV NS3 resistance-associated mutations was investigated in treated patients. Sanger sequencing was performed on HCV virus from inhibitor treatment-patients. Overall 16.4% had a detectable RAV in the NS3 region. Persistence of drug-resistant variants were detected in the patients. Resistance testing during DAA treatment should be taken into consideration.
Abstract
Background. Direct-acting antiviral agents (DAAs) combined with pegylated-interferon (PegIFN) and ribavirin (RBV) are still a standard treatment in patients with genotype 1 HCV infection. However, virologic response could be impaired by baseline or early selection of resistant HCV strains. Objectives. The aim of this study was to determine the onset and persistence of resistanceassociated mutations (RAMs) in the NS3 and NS5B genes of DAA-naïve patients failing treatment. Study design. Direct sequencing of HCV NS3 was performed in 49 DAA-naïve patients with HCV genotype 1 infection. Results. Eight out of 23 patients (34.7%) failed PegIFN/RBV/telaprevir during the 12-weeks of therapy. Treatment failure was associated with the development of RAMs at amino-acids 36,54,80 and 155 of the HCV protease in 6/8 patients (75%). Among patients treated with PegIFN/RBV/boceprevir treatment, 4/18 (22.2%) failed therapy. Of these, 2 (50%) carried virus strains which developed a RAM at amino-acids 54 and 155. Among HCV strains with RAMs, 7 belonged to genotype 1a and 1 to 1b. Finally, in 6/10 (60%) patients, drug-resistant variants could still be detected for up to 3-7 months after stopping therapy. Conclusions. A higher rate (p= 0.49) of treatment failure was observed in patients receiving telaprevir- compared to the boceprevir-based combination. In addition, compared with genotype 1b, genotype 1a was associated with higher rates (p= 0.01) of treatment failure due to virus resistant strains. Resistance testing at baseline and during DAA treatment should be taken into consideration when treating patients with new HCV combination therapies.
Keywords: Hepatitis C virus; HCV genotype 1, drug resistance; protease inhibitors;sequencing. 1. Background
Peginterferon/ribavirin (PegIFN/RBV) has been the standard of care for treating HCV infection worldwide, even though adverse reactions have been observed in at least 10% of patients [1] and a sustained virological response is achieved in only 50% of patients infected with the HCV genotype 1 [2]. Recently, approved drugs inhibiting HCV NS3/4A protease have made a major step towards improving sustained virological response (SVR) rates and decreasing treatment times in genotype-1 infected patients[3]. Direct-acting antiviral agents (DAAs),telaprevir boceprevir and faldaprevir, combined with PegIFN and RBV are first generation antivirals in the treatment of patients with genotype 1 HCV infection. Triple therapiesconsisting ofPegIFN/RBVand telaprevir or boceprevir as well as IFN-free regimens with faldapravir have reported a major improvementin HCV treatment [4-6]. However, viral response during treatment with DAAs might be impaired by baseline or early selection of resistance-associated HCV variants. Sequencing data suggest that resistant variants arise in most patients showing treatment failure [3,7,8].
2. Objectives The aims of this study were to determine the onset and persistence of resistance-associated mutations (RAMs) in the HCV genome of DAA-naïve patients failing DAA/PegIFN/RBV or 2DAAs/RBVtherapy,and to evaluate the role of baseline screening for HCV RAMs before DAA therapy.
3. Study Design 3.1. Patients Forty-nine DAA-naive patients with HCV genotype 1 virus started DAA-based triple therapyduring the period 2013 to 2014. Characteristics of the patients arereportedin Table 1. Twenty-three patients, 11 with genotype 1a and 12 with genotype 1b were treated with PegIFN/RBV/telaprevir;18 patients, 7 with genotype 1a and 11 with genotype 1b, were treated with PegIFN/RBV/boceprevir
and 8 patients, all with genotype 1b, were treated with RBV/faldaprevir/deleobuvir. Of these 49 patients, 36 (73.4%) had been previously treated with PegIFN/RBV. Treatment failure was defined as failure to clear HCV RNA by week 12, HCV RNA >1000UI/ml at week 4 in telaprevir patients, HCV RNA >100UI/ml at week 12 or detectable at week 24 in boceprevir patients or viral load rebound of ≥1 log10 IU/ml from nadir at any time point.The study was approved by the Ethics Committee of the Fondazione IRCCS Policlinico San Matteo (protocol no. 20080009620) and informed consent was obtained prior to enrolment.
3.2. HCV-RNA quantification and genotyping Surplus serum samples fromHCV RNA viral load quantification for each patientwere prospectively evaluated for RAM determination. HCV RNA levels were quantified using the Abbott HCV-RNA assay, (Abbott Park, Illinois, U.S.A.). HCV genotyping wasachieved using the Versant HCV Genotype 2.0 Assay LiPA (Siemens Healthcare Diagnostic Inc., Tarrytown, NY USA). The NS3 region was sequenced to further subtype HCV strains and identify genotypes 1a/1b. Data were analyzed using the Blast program (http://blast.ncbi.nlm.nih.gov). Viral RNA was extracted from serum samples using the automatic Easy Mag extractor (Biomerieux, Lyon, France), and full-length HCV NS3/4A sequences were amplified using a nested RT-PCR[9]. Direct sequencing of HCV NS3 was performed using an automatic sequencer (ABI PRISM 3100 genetic analyzer DNA Sequencer, Applied Biosystems, Foster City, CA, USA) and the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) in all patients at baseline and in non-responder patients. Analysis of NS5B was additionally performed in deleobuvir treated patients[10]. Nucleotide sequences were assembled using the Sequencer 5.0 (Gene Codes Corp., Ann Arbor, MI) software program. Nucleotide sequences were aligned with the confirmed references: M62321 for subtype 1a and D90208 for subtype 1b.
The sequences reported in this study have been submitted to the GenBank database under accession numbers KP898266-KP898336.
3.3. IL28B genotyping IL28 polymorphisms were also determined in all patients (Table 1). Genomic DNA extracted from peripheral blood mononuclear cellswas tested for SNPs within the IL28B locus (C or T rs12979860 and G or T for rs8099917) using the TaqMan allelic discrimination assay[11].
3.4. Statistical analysis An univariate analysis was carried out to compare data between groups by applying the Fisher’s exact test.
4. Results Eight
out
of 23
patients (34.7%)failed to
achieve SVR during the 12-weeks of
PegIFN/RBV/telaprevir therapy (Fig. 1). Treatment failure was associated with the development of RAMs at amino-acids 36, 54, 80 and 155 of the HCV protease in 6/8 patients (75 %) (Table 2), while a smaller proportion of patients (2/8, 25%) discontinued treatment due to adverse events (rash, vomit).Four/18 (22.2%) patients failed to achieve SVR during 12-weeks ofthe PegIFN/RBV/boceprevir combination (Fig. 2). Two out of 4 patients (50%) carried HCV strains with RAMs at amino-acids 54 and 155 of the HCV protease(Table 2) and 2/4(50%) discontinued therapy due to adverse events (rash, vomit, leucopenia). Seven out of 8 patients who failed therapy were PegIFN/RBV experienced, cirrhotic and had CT or TT polymorphism patterns. Patients who discontinued treatment were lost to follow-up. InonepatientfailingPegIFN/RBV/telaprevir,a baseline mutation at position 54 of the HCV protease was foundand baseline mutations at positions 54 and 155 were observedinonepatient initiating PegIFN/RBV/boceprevir (Table 2).On the other hand, a baseline RAM at position 54 of the HCV protease was present in 2/3 patients with
successfulPegIFN/RBV/telaprevir treatment (Table 3). In addition, in 6/8 patients failing treatment due to development of RAMs,a wild-type strain began to re-emerge one month after stopping DAA, anddrug-resistant variants were detectedfor up to 7 months. (Table 2). Compensatory mutations 71A and 153V (Table 2) [12] were observed in only 2 patients.Among patients failing telaprevirand boceprevir–including combinations due to the emergence of RAMs, 7 were infected with genotype 1a virus and one with genotype 1b. IL-28B polymorphism rs12979860 analysis showed unfavorable patterns (CT or TT) in about 90% of patients treated with telaprevir- and boceprevir–including combinations (Table 1). Allpatients on RBV-faldaprevir-deleobuvir were responsive to treatment,although mutations at positions 36 and 55 were detected in 1/8 patients.
5. Discussion Different SVR rates and resistance patterns for approved DAAs were observed with respect to the HCV genotype (1a and 1b). In agreement with other studies [13-15],a higher rate (p= 0.49) of treatment failure was observed in patients receiving the telaprevir- compared to the boceprevirbased combination or RBV/faldaprevir/deleobuvir. In addition, a higher rate (p= 0.01) of resistant variants and worse response to triple therapy was associated with genotype 1a with respect to genotype 1b. Virological failure due to drug-resistant variantswas observed only in a single patient with genotype 1b on boceprevir-based therapy. Response rates in patients with genotype 1b were generally more favorable than those with genotype 1a. The probable explanation for this different outcome might be related to the lower genetic barriers of genotype 1a compared with genotype 1b strains [16]. Although, data from in vitro studies report that most resistance-associated variants have reduced viral fitness [17], in this study, 6/8 patients with virus which developed RAMs while on treatment, maintained drug-resistant variants for up to 7 months after stopping DAA. Recent studies [7,18] show that persistence of drug-resistant variants might be explained by compensatory mutations and/or by the improved replication capacity of double variants such as V36M+R155K.
Here, these double mutations were maintained in four patients after stopping therapy.Avariant observed in two patients at position 54 wasreported to be comparable or increased in replicative fitness [19-20]. Although drug-resistant variants were observed at baseline in 2/8 patients failing therapy, treatment failure on triple therapy was not always predicted by baseline PI resistance. In fact, drugresistant variants were observed also in threetreatment responders.The baseline polymorphism Q80K is common in patients with genotype 1a,but has not been associated with resistance to telaprevir, boceprevir or faldaprevir [21,22,23]; thus, treatment response was not influenced. On the other hand, favorable response might be due to PegIFN/RBV sensitivity, predicted by the CC pattern at IL-28B polymorphism rs12979860. In this study, more than 90% of the patients hadunfavorableCT or TT polymorphisms, and 75.6% were PegIFN/RBV experienced including the7/8 patients carrying drug-resistant variants and failing therapy. However, 65.3% and 77.8 % of patients receivingthe telaprevir and boceprevir combination respectively responded to treatment.This suggests that a potent triple therapy including a PI isable to obtain SVR even underadverse conditions. Although more potent treatment options will soon be available with the introduction of new NS5A and NS5B inhibitors able to overcome the problem of different genotypes, caution should be paid to the presence of major mutations at baseline or theirdevelopment in DAA treatment-experienced patients.
Conflict of interest None. Funding This work was supported by the Ministero della Salute, Fondazione IRCCS Policlinico San Matteo Ricerca Corrente grant 80207. Competing interest None declared
Ethical approval The study was approved by the Ethics Committee of the Fondazione IRCCS Policlinico San Matteo (protocol no. 20080009620) and informed consent was obtained.
Acknowledgements We thank Daniela Sartori for careful preparation of the manuscript and Laurene Kelly for English revision.
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S. Zeuzem, J.F. Dufour, M. Buti,V. Soriano, R.J. Buynak, P. Mantry ,et al., Interferon-free treatment of chronic hepatitis C withfaldaprevir, deleobuvir andribavirin: SOUND-C3, a Phase 2b study,Liver Int.35 (2015) 417-21.
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S. Susser,J. Vermehren , N. Forestier, M.W. Welker, N. Grigorian, C. Füller, et al., Analysis of long-term persistence of resistance mutations within the hepatitis C virus NS3 protease after treatment with telaprevir or boceprevir, J Clin Virol. 52 (2011) 321-7.
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D.L. Wyles, J.A. Gutierrez, Importance of HCV genotype 1 subtypes for drug resistance and response to therapy, J Viral Hepat.21 (2014) 229-40.
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S. Paolucci, L. Fiorina, A. Piralla, R. Gulminetti, S. Novati, G. Barbarini, et al., Naturally occurring mutations to HCV protease inhibitors in treatment-naïve patients, Virol J.(2012)9:245. doi: 10.1186/1743-422X-9-245.
[10] S. Paolucci, L. Fiorina, B. Mariani, R. Gulminetti, S. Novati , G. Barbarini et al., Naturally occurring resistance mutations to inhibitors of HCV NS5A region and NS5B polymerase in DAA treatment-naïve patients, Virol J.(2013)10:355. doi: 10.1186/1743-422X-10-355. [11] L. Fiorina, S. Paolucci, S. Papadimitriou, F. Baldanti, Comparison of three different methods for the evaluation of IL28 and ITPA polymorphisms in patients infected with HCV, J Virol Methods.184 (2012) 103-5. [12] F.X. López-Labrador, A. Moya, F. Gonzàlez-Candelas, Mapping natural polymorphisms of hepatitis C virus NS3/4A protease and antiviral resistance to inhibitors in worldwide isolates.Antivir Therapy, 13 (2008)481-494. [13] I.M. Jacobson, J.G. McHutchison , G. Dusheiko , A.M. Di Bisceglie , K.R. Reddy, N.H. Bzowej, et al.,Telaprevir for previously untreated chronic hepatitis C virus infection. N Engl J Med. 364 (2011) 2405-16. [14] S. De Meyer, I. Dierynck, A. Ghys, M. Beumont, B. Daems, B. Van Baelen et al., Characterization of telaprevir treatment outcomes and resistance in patients with prior
treatment failure: results from the REALIZE trial, Hepatology 56 (2012) 2106-15. doi: 10.1002/hep.25962 [15] F. Poordad, J.P. Bronowicki, S.C. Gordon, S. Zeuzem , I.M. Jacobson, M.S. Sulkowski et al., Factors that predict response of patients with hepatitis C virus infection to boceprevir, Gastroenterology 143 (2012) 608-18. [16] T.L. Kieffer, S. DeMeyer, D.J. Bartels, J.C. Sullivan, E.Z. Zhang, A. Tigges, et al., HepatitisC viral evolution in genotype 1 treatment-naïve and treatment-experienced patients receiving telaprevir-based therapy in clinical trials, APLoS One. 7 (2012) e34372. doi: 10.1371/journal.pone.0034372. [17] T. Shimakami, C. Welsch, D. Yamane, D.R. McGivern , S. Yi M,Zeuzem , S.M. Lemon . Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production
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10.1053/j.gastro.2010.10.056. [18] J.C. Sullivan, S. DeMeyer, D.J. Bartels , I. Dierynck , E.Z. Zhang, J. Spanks, Evolution of treatment-emergent resistant variants intelaprevirphase 3 clinical trials, Clin Infect Dis. 57 (2013) 221-9. [19] T. Shimakami, C. Welsch, D. Yamane, D.R. McGivern, M. Yi, S. Zeuzem, et al., Proteaseinhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus, Gastroenterology 140 (2011) 667-75. [20] X. Tong, S. Bogen, R. Chase, V. Girijavallabhan, Z. F.G. Guo,Njoroge, A. Prongay, et al., Characterization of resistance mutations against HCV ketoamide protease inhibitors, Antiviral Res. 77 (2008) 177-85. [21] D.L. Wyles, Beyond telaprevir and boceprevir: resistance and new agents for hepatitis C virus infection, Top Antivir Med. 20 (2012) 139-45.
[22] L. Lagacé, P.W. White, C. Bousquet, N. Dansereau, F. Dô, M. Llinas-Brunet, et al., In vitro resistance profile of the hepatitis C virus NS3 protease inhibitor BI 201335, Antimicrob Agents Chemother.56 (2012) 569-72. [23] K.L. Berger, L. Lagacé, I. Triki, M. Cartier, M. Marquis, C. Lawetz, et al., Viral Resistance in Hepatitis C Virus Genotype 1-Infected Patients Receiving the NS3 Protease Inhibitor Faldaprevir (BI 201335) in a Phase 1b Multiple-Rising-Dose Study, Antimicrob Agents Chemother. 57 (2013) 4928–4936.
Figure 1. Patients failingPegIFN/RBV-telaprevir treatment during the 12-weeks of triple therapy.
Figure 2. Patients failingPegIFN/RBV-boceprevir treatment during the 12-weeks of triple therapy.
S1386-6532(15)00650-2 http://dx.doi.org/doi:10.1016/j.jcv.2015.08.015 JCV 3421
To appear in:
Journal of Clinical Virology
Received date: Revised date: Accepted date:
17-4-2015 27-8-2015 28-8-2015
Please cite this article as: Paolucci Stefania, Fiorina Loretta, Mariani Bianca, Landini Viviana, Gulminetti Roberto, Novati Stefano, Maserati Renato, Barbarini Giorgio, Bruno Raffaele, Baldanti Fausto.Development and persistence of DAA resistance associated mutations in patients failing HCV treatment.Journal of Clinical Virology http://dx.doi.org/10.1016/j.jcv.2015.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development and persistence of DAA resistance associated mutations in patients failing HCV treatment Stefania Paolucci1, Loretta Fiorina1, Bianca Mariani1, Viviana Landini1, Roberto Gulminetti2, Stefano Novati2, Renato Maserati2, Giorgio Barbarini3, Raffaele Bruno3, Fausto Baldanti1,4*
1
Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico
San Matteo, 27100 Pavia, Italy 2
Institute of Infectious Diseases, University of Pavia, 27100 Pavia, Italy
3
Division of Infectious and Tropical Diseases, Fondazione IRCCS Policlinico San Matteo, 27100
Pavia, Italy 4
Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, 27100
Pavia, Italy
*Corresponding author: F. Baldanti, Department of Clinical, Surgical, Diagnostic and Pediatric Sciences, University of Pavia, Viale Brambilla 74, 27100 Pavia, Italy Tel:
+39
0382
502283;
Fax:
+39
0382
502599;
E-mail:
[email protected];
[email protected]
Highlights HCV NS3 resistance-associated mutations was investigated in treated patients. Sanger sequencing was performed on HCV virus from inhibitor treatment-patients. Overall 16.4% had a detectable RAV in the NS3 region. Persistence of drug-resistant variants were detected in the patients. Resistance testing during DAA treatment should be taken into consideration.
Abstract
Background. Direct-acting antiviral agents (DAAs) combined with pegylated-interferon (PegIFN) and ribavirin (RBV) are still a standard treatment in patients with genotype 1 HCV infection. However, virologic response could be impaired by baseline or early selection of resistant HCV strains. Objectives. The aim of this study was to determine the onset and persistence of resistanceassociated mutations (RAMs) in the NS3 and NS5B genes of DAA-naïve patients failing treatment. Study design. Direct sequencing of HCV NS3 was performed in 49 DAA-naïve patients with HCV genotype 1 infection. Results. Eight out of 23 patients (34.7%) failed PegIFN/RBV/telaprevir during the 12-weeks of therapy. Treatment failure was associated with the development of RAMs at amino-acids 36,54,80 and 155 of the HCV protease in 6/8 patients (75%). Among patients treated with PegIFN/RBV/boceprevir treatment, 4/18 (22.2%) failed therapy. Of these, 2 (50%) carried virus strains which developed a RAM at amino-acids 54 and 155. Among HCV strains with RAMs, 7 belonged to genotype 1a and 1 to 1b. Finally, in 6/10 (60%) patients, drug-resistant variants could still be detected for up to 3-7 months after stopping therapy. Conclusions. A higher rate (p= 0.49) of treatment failure was observed in patients receiving telaprevir- compared to the boceprevir-based combination. In addition, compared with genotype 1b, genotype 1a was associated with higher rates (p= 0.01) of treatment failure due to virus resistant strains. Resistance testing at baseline and during DAA treatment should be taken into consideration when treating patients with new HCV combination therapies.
Keywords: Hepatitis C virus; HCV genotype 1, drug resistance; protease inhibitors;sequencing. 1. Background
Peginterferon/ribavirin (PegIFN/RBV) has been the standard of care for treating HCV infection worldwide, even though adverse reactions have been observed in at least 10% of patients [1] and a sustained virological response is achieved in only 50% of patients infected with the HCV genotype 1 [2]. Recently, approved drugs inhibiting HCV NS3/4A protease have made a major step towards improving sustained virological response (SVR) rates and decreasing treatment times in genotype-1 infected patients[3]. Direct-acting antiviral agents (DAAs),telaprevir boceprevir and faldaprevir, combined with PegIFN and RBV are first generation antivirals in the treatment of patients with genotype 1 HCV infection. Triple therapiesconsisting ofPegIFN/RBVand telaprevir or boceprevir as well as IFN-free regimens with faldapravir have reported a major improvementin HCV treatment [4-6]. However, viral response during treatment with DAAs might be impaired by baseline or early selection of resistance-associated HCV variants. Sequencing data suggest that resistant variants arise in most patients showing treatment failure [3,7,8].
2. Objectives The aims of this study were to determine the onset and persistence of resistance-associated mutations (RAMs) in the HCV genome of DAA-naïve patients failing DAA/PegIFN/RBV or 2DAAs/RBVtherapy,and to evaluate the role of baseline screening for HCV RAMs before DAA therapy.
3. Study Design 3.1. Patients Forty-nine DAA-naive patients with HCV genotype 1 virus started DAA-based triple therapyduring the period 2013 to 2014. Characteristics of the patients arereportedin Table 1. Twenty-three patients, 11 with genotype 1a and 12 with genotype 1b were treated with PegIFN/RBV/telaprevir;18 patients, 7 with genotype 1a and 11 with genotype 1b, were treated with PegIFN/RBV/boceprevir
and 8 patients, all with genotype 1b, were treated with RBV/faldaprevir/deleobuvir. Of these 49 patients, 36 (73.4%) had been previously treated with PegIFN/RBV. Treatment failure was defined as failure to clear HCV RNA by week 12, HCV RNA >1000UI/ml at week 4 in telaprevir patients, HCV RNA >100UI/ml at week 12 or detectable at week 24 in boceprevir patients or viral load rebound of ≥1 log10 IU/ml from nadir at any time point.The study was approved by the Ethics Committee of the Fondazione IRCCS Policlinico San Matteo (protocol no. 20080009620) and informed consent was obtained prior to enrolment.
3.2. HCV-RNA quantification and genotyping Surplus serum samples fromHCV RNA viral load quantification for each patientwere prospectively evaluated for RAM determination. HCV RNA levels were quantified using the Abbott HCV-RNA assay, (Abbott Park, Illinois, U.S.A.). HCV genotyping wasachieved using the Versant HCV Genotype 2.0 Assay LiPA (Siemens Healthcare Diagnostic Inc., Tarrytown, NY USA). The NS3 region was sequenced to further subtype HCV strains and identify genotypes 1a/1b. Data were analyzed using the Blast program (http://blast.ncbi.nlm.nih.gov). Viral RNA was extracted from serum samples using the automatic Easy Mag extractor (Biomerieux, Lyon, France), and full-length HCV NS3/4A sequences were amplified using a nested RT-PCR[9]. Direct sequencing of HCV NS3 was performed using an automatic sequencer (ABI PRISM 3100 genetic analyzer DNA Sequencer, Applied Biosystems, Foster City, CA, USA) and the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) in all patients at baseline and in non-responder patients. Analysis of NS5B was additionally performed in deleobuvir treated patients[10]. Nucleotide sequences were assembled using the Sequencer 5.0 (Gene Codes Corp., Ann Arbor, MI) software program. Nucleotide sequences were aligned with the confirmed references: M62321 for subtype 1a and D90208 for subtype 1b.
The sequences reported in this study have been submitted to the GenBank database under accession numbers KP898266-KP898336.
3.3. IL28B genotyping IL28 polymorphisms were also determined in all patients (Table 1). Genomic DNA extracted from peripheral blood mononuclear cellswas tested for SNPs within the IL28B locus (C or T rs12979860 and G or T for rs8099917) using the TaqMan allelic discrimination assay[11].
3.4. Statistical analysis An univariate analysis was carried out to compare data between groups by applying the Fisher’s exact test.
4. Results Eight
out
of 23
patients (34.7%)failed to
achieve SVR during the 12-weeks of
PegIFN/RBV/telaprevir therapy (Fig. 1). Treatment failure was associated with the development of RAMs at amino-acids 36, 54, 80 and 155 of the HCV protease in 6/8 patients (75 %) (Table 2), while a smaller proportion of patients (2/8, 25%) discontinued treatment due to adverse events (rash, vomit).Four/18 (22.2%) patients failed to achieve SVR during 12-weeks ofthe PegIFN/RBV/boceprevir combination (Fig. 2). Two out of 4 patients (50%) carried HCV strains with RAMs at amino-acids 54 and 155 of the HCV protease(Table 2) and 2/4(50%) discontinued therapy due to adverse events (rash, vomit, leucopenia). Seven out of 8 patients who failed therapy were PegIFN/RBV experienced, cirrhotic and had CT or TT polymorphism patterns. Patients who discontinued treatment were lost to follow-up. InonepatientfailingPegIFN/RBV/telaprevir,a baseline mutation at position 54 of the HCV protease was foundand baseline mutations at positions 54 and 155 were observedinonepatient initiating PegIFN/RBV/boceprevir (Table 2).On the other hand, a baseline RAM at position 54 of the HCV protease was present in 2/3 patients with
successfulPegIFN/RBV/telaprevir treatment (Table 3). In addition, in 6/8 patients failing treatment due to development of RAMs,a wild-type strain began to re-emerge one month after stopping DAA, anddrug-resistant variants were detectedfor up to 7 months. (Table 2). Compensatory mutations 71A and 153V (Table 2) [12] were observed in only 2 patients.Among patients failing telaprevirand boceprevir–including combinations due to the emergence of RAMs, 7 were infected with genotype 1a virus and one with genotype 1b. IL-28B polymorphism rs12979860 analysis showed unfavorable patterns (CT or TT) in about 90% of patients treated with telaprevir- and boceprevir–including combinations (Table 1). Allpatients on RBV-faldaprevir-deleobuvir were responsive to treatment,although mutations at positions 36 and 55 were detected in 1/8 patients.
5. Discussion Different SVR rates and resistance patterns for approved DAAs were observed with respect to the HCV genotype (1a and 1b). In agreement with other studies [13-15],a higher rate (p= 0.49) of treatment failure was observed in patients receiving the telaprevir- compared to the boceprevirbased combination or RBV/faldaprevir/deleobuvir. In addition, a higher rate (p= 0.01) of resistant variants and worse response to triple therapy was associated with genotype 1a with respect to genotype 1b. Virological failure due to drug-resistant variantswas observed only in a single patient with genotype 1b on boceprevir-based therapy. Response rates in patients with genotype 1b were generally more favorable than those with genotype 1a. The probable explanation for this different outcome might be related to the lower genetic barriers of genotype 1a compared with genotype 1b strains [16]. Although, data from in vitro studies report that most resistance-associated variants have reduced viral fitness [17], in this study, 6/8 patients with virus which developed RAMs while on treatment, maintained drug-resistant variants for up to 7 months after stopping DAA. Recent studies [7,18] show that persistence of drug-resistant variants might be explained by compensatory mutations and/or by the improved replication capacity of double variants such as V36M+R155K.
Here, these double mutations were maintained in four patients after stopping therapy.Avariant observed in two patients at position 54 wasreported to be comparable or increased in replicative fitness [19-20]. Although drug-resistant variants were observed at baseline in 2/8 patients failing therapy, treatment failure on triple therapy was not always predicted by baseline PI resistance. In fact, drugresistant variants were observed also in threetreatment responders.The baseline polymorphism Q80K is common in patients with genotype 1a,but has not been associated with resistance to telaprevir, boceprevir or faldaprevir [21,22,23]; thus, treatment response was not influenced. On the other hand, favorable response might be due to PegIFN/RBV sensitivity, predicted by the CC pattern at IL-28B polymorphism rs12979860. In this study, more than 90% of the patients hadunfavorableCT or TT polymorphisms, and 75.6% were PegIFN/RBV experienced including the7/8 patients carrying drug-resistant variants and failing therapy. However, 65.3% and 77.8 % of patients receivingthe telaprevir and boceprevir combination respectively responded to treatment.This suggests that a potent triple therapy including a PI isable to obtain SVR even underadverse conditions. Although more potent treatment options will soon be available with the introduction of new NS5A and NS5B inhibitors able to overcome the problem of different genotypes, caution should be paid to the presence of major mutations at baseline or theirdevelopment in DAA treatment-experienced patients.
Conflict of interest None. Funding This work was supported by the Ministero della Salute, Fondazione IRCCS Policlinico San Matteo Ricerca Corrente grant 80207. Competing interest None declared
Ethical approval The study was approved by the Ethics Committee of the Fondazione IRCCS Policlinico San Matteo (protocol no. 20080009620) and informed consent was obtained.
Acknowledgements We thank Daniela Sartori for careful preparation of the manuscript and Laurene Kelly for English revision.
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Figure 1. Patients failingPegIFN/RBV-telaprevir treatment during the 12-weeks of triple therapy.
Figure 2. Patients failingPegIFN/RBV-boceprevir treatment during the 12-weeks of triple therapy.