13th European Meeting on HIV & Hepatitis Treatment Strategies & Antiviral Drug Resistance, Barcelona 3-5 June 2015

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1 13th European Meeting on HIV & Hepatitis Treatment Strategies & Antiviral Drug Resistance, Barcelona 3-5 June 2015 Session 6: Drug Resistance to HCV DAA's Drug Resistance to Protease Inhibitors Francesca Ceccherini-Silberstein Università degli Studi di Roma Tor Vergata Dipartimento di Medicina Sperimentale e Chirurgia Cattedra di Virologia Barcelona 4 June 2015

2 Disclosures F. Ceccherini-Silberstein has received funds for attending symposia, speaking, organizing educational activities, grantresearch support, consultancy and advisory board from AbbVie, Merck Sharp & Dohme, Gilead, Janssen Cilag, Roche Diagnostics, Bristol MyersSquibb, and ViiV.

3 New Approved DAAs Sofosbuvir Simeprevir Daclatasvir Harvoni Nucleotide analogue 400 mg qd All genotypes High barrier January mg / 12.5 mg / 50 mg qd Paritaprevir/ ombitasvir/ ritonavir (Viekirax) Protease inhibitor 150 mg qd Genotypes 1 and 4 Low barrier May 2014 NS3 protease inhibitor + NS5A inhibitor + ritonavir + HCV-1 and HCV-4 Low barrier NS5A inhibitor 30 or 60 mg qd All genotypes Low barrier September 2014 Dasabuvir (Exviera) Nucleotide analogue + NS5A inhibitor 400 mg sofosbuvir + 90 mg of ledipasvir qd HCV-1 and HCV-4 High barrier September mg bd NS5B polymerase inhibitor

4 The importance of genotype.

5 The 7 HCV genotypes vary in their geographical distribution and in their level of genetic diversity A high degree of genetic variability among the endemic HCV strains have been acquired during long-term evolution Strains that exhibit lower genetic distance and are widely spread likely have a shorter history and a higher transmission rate Jackowiak P. Genetics and Evolution, 2014

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7 Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide as well as in Europe 3a 1a 1b 3a 4 3a 2 1a 1b 1a 1b 3a 1b 1a 3a 1a 1b 3a 1a 1b 3a 2 1a 1b 3a 1b 1a 4 1a 3a 1b b 3a 4 1a 1b Esteban JI et al J Hepatol 2008

8 30% 28,2 Proportion of HCV genotypes in HCV-RNA+ patients N= % 20% 23,9 17,7 37.5% HCV-1 30% HCV % HCV-4 15% 10% 10,8 6,8 5% 5,1 2,8 1,8 1,7 1,4 0% 3a n=354 1a n=300 not tested n=222 1,462 PLHIV-HCV patients 1b n=136 4 n=85 Distribution of HCV genotypes (G) was: G1a, 439 (30%); G1b, 178 (12%); G2, 35 (2%); G3a, 339 (23%); G4, 147 (10%). 92% were currently receiving ART. M Shanyinde et al ICAR 2015 other n=64 1 n=35 3 n=23 4c/4d n=21 2a/2c n=17

9 HCV genotype was the most important baseline predictor for response to Peg-IFN + Ribavirin Combination Therapy HCV genotypes 2 and 3 SVR = % HCV-2= 80-95% HCV-3 Low viremia = 75-80% HCV-3 High viremia = 60-70% HCV genotype 1 HCV genotypes 4 and 6 SVR = % HCV-1 Low viremia = 50% HCV-1 High viremia = 30-35% SVR = %

10 What about today or tomorrow in the era of new DAAs? Still genotype important predictor for response? Today only few DAAs are pangenotypic. (Grazoprevir) Daclatasvir Sofosbusvir 47% amino acids of HCV PROTEASE NS3 are conserved among all HCV-genotypes 46% amino acids of HCV NS5A are conserved among all HCV-genotypes 55% amino acids of HCV POLYMERASE NS5B are conserved among all HCV-genotypes Amino acid variability: 0% <1% 1-5% 5-10% 10-25% >25% Amino acid variability: 0% <1% 1-5% 5-10% 10-25% >25% Cento et al., PLoS ONE 2012 Love et al., J Vir 2009 Di Maio et al., AAC 2014

11 HIV-1 protease conservation 68% 45% Ceccherini-Silberstein et al., AIDS 2004

12 Comparative Activity of PIs Against HCV NS3 Genotype 1-6 Proteases from Patient Isolates from Genotypes 1-6 in Cell-Based SEAP assay IC 50 (nm) Gt1a Gt1b Gt2a #1 Gt2a #2 Gt2b #1 Gt2b #2 Gt2b #3 Gt2b #4 Gt2b #5 Gt3a #1 Gt3a #2 G3a #3 Gt3a #4 Gt4a #1 Gt4a #2 Gt 5a #3 Gt5a #4 Gt 6a telaprevir TMC-435 boceprevir MK-5172 Graham D et al. AASLD 2011 Poster # 370 NS3 Genotype

13 Association between HCV genotype and prevalence of liver steatosis

14 OMV/PTV/RTV + DSV + RBV treatment in cirrhotic GT1a patients requires 24 weeks instead of 12 TURQUOISE-II, phase 3 trial, combination of coformulated paritaprevir ombitasvir and dasabuvir + RBV for 12/24 weeks in previously untreated and previously treated adults with chronic HCV genotype 1 infection and compensated cirrhosis. Poordad F. et al., NEJM 2014

15 Treatment recommendations for HCV-monoinfected or HCV/HIV coinfected patients with chronic hepatitis C with compensated (Child-Pugh A) cirrhosis, including treatment-naïve patients and patients who failed on a treatment based on PegIFN-a and ribavirin (RBV). EASL HCV clinical guidelines 2015

16 ID_476 HCV genotype: 1b Age: 55 Sex: M Naive Cirrhotic Paritaprevir/ritonavir+Ombitasvir+ Dasabuvir+RBV IU/ml GRT Day 0 NS3 Resistance mutations: None NS5A Resistance mutations: None NS5B Resistance Mutations: None HCV- RNA (log IU/ml) IU/ml 193 IU/ml 45 IU/ml 1 LLOQ (12 IU/ml) 30 IU/ml No TND!!! <12 IU/ml Day 0 4w 5w 6w 7w 9w 11w 13w 15w 19w

17 The importance of resistance.

18 Mutations occur frequently during the replication of HCV

19 CONSEQUENCES OF HCV VARIABILITY AT PATIENT S LEVEL: QUASISPECIES POPULATION It has been predicted that every nucleoside of the 3.2 kb HBV genome or the 10 kb HIV and HCV genomes theoretically can be substituted every day within a given infected patient Rong L et al., Sci Transl Med 2010 HCV resistant variants are naturally produced during the HCV life cycle M.D. Schneider, C. Sarrazin Antiviral Research 2014

20 Different distribution of TVR RAVs in TVR treated patients according to subtype Kieffer, PLoS ONE 2012

21 Different emergency of resistance mutations in GT1a and GT1b also in BOC-failing patients Ogert RA et al., AASLD 2011 (poster n 927)

22 Slow HCV-RNA decay and early resistance predict the risk of failure to TVR/BOC treatment N=5 T54S N=2 V36L (1.5%) (3.8%) N=1 V55A (0.8%) N=13 Q80K (10.0%) N=1 V36M (2.1%) N=1 T54S+R155K (2.1%) N=1 T54S (2.1%) N=1 T54A (2.1%) N=2 Q80K (4.3%) N=1 A156V (2.1%) N=3 Q80K (16.7%) N=109 wild-type (83.8%) Baseline resistance test (N=130, TVR & BOC) Baseline RAVs in 21 patients (16.2%) N=40 wild-type (85.1%) Resistance test 48h (N=47, TVR & BOC) 48h new RAVs in 4 patients (8.5%) N=10 wild-type (55.6%) Resistance test 3-17 days (N=18, TVR & BOC) 3-17 days new RAVs in 4 patients (22.2%) N=2 A156T (14.3%) N=1 V36L+Q80K 5.6%) N=1 V36M+R155K (5.6%) N=1 R155T+A156V (5.6%) Characteristics Crude OR 95% C.I. 95% C.I. P-value P-value Adjusted OR Lower Upper Lower Upper HCV-RNA >100 IU/ml at week < At least one baseline/early resistance mutation HCV genotype Cirrhosis Unfavourable IL-28b genotype (TT or CT) Previous null responder to SOC OR, odds ratio; CI, confidence interval; SOC, standard of care Ceccherini-Silberstein et al CROI 2014 Cento V. et al., DLD 2014

23 Simeprevir Bolded type under the amino acid position number represents the key mutations that are clearly associated with virologic failure and result in a resistant phenotype. IAS USA 2013 IAS list 2013

24 Protease Inhibitor Resistance paritaprevir/r HCV DRAG, Forum for Collaborative HIV Research, April 2014

25 Beware of HCV-genotype for daclatasvir resistance... Nakamoto S., WJG 2014

26 Useful a HCV sequencing test before starting treatment?

27 Currently, all international guidelines list several new treatment options with similar high efficacy for initial treatment and retreatment of prior non-responders. The new DAAs may be used with or without ribavirin for 12 or 24 weeks, depending on factors including HCV genotype, subtype (1a or 1b), presence of liver cirrhosis, and prior treatment history..

28 Virologic failure during simeprevir treatment was more common in patients with genotype 1a with Q80K No differences between 1b and 1a without Q80K SMV + P/R P/R SVR12 (%) /147 36/74 GT 1a 105/117 29/56 GT 1b Differences in SVR12 by Subgroup (95% CIs) GT 1a/other HCV - With baseline Q80K vs Pbo - Without baseline Q80K vs Pbo GT 1b HCV Favors Placebo Favors SMV 28.2 ( ) 4.7 (-14.6 to 24.1) 40.3 ( ) 42.1 ( ) SMV (n) Pbo (n) Jacobson I, et al. EASL 2013

29 Overall prevalence of Q80K in G1 across different regions 13.7% of patients (274/2007) all HCV G1 29.5% (269/911) of those with HCV GT1a and 0.5% (5/1096) of those with HCV GT1b North America 48% Europe Italy our data : all G1 9% 34% 6% All G1 19% G1a Patients with Q80K (%) 25,0 20,0 15,0 10,0 5,0 0,0 20/89 1/136 HCV GT- HCV GT- 1a (N=89) 1b (N=136) All G1 G1a South America Overall 30% 14% 3% 9% All G1 G1a All G1 G1a Includes 15 patients with non-1a/b genotype. Jacobson et al. Presented at AASLD 2013 Lenz et al. Presented at AASLD 2013

30 Different clusters of HCV genotypes 1 Figure 1. Radial dendrograms of the 109 HCV NS3 sequences obtained together with reference sequences for subtypes 1a, 1b and 1c (outgroup). Vicenti el al JAC 2012

31 Different frequency of natural resistance mutations in GT1a NS3 by clade n=293 De Luca et al IWDR 2013 Overall prevalence of Q80K in QUEST-2 Simeprevir study was 10.2% (EASL 2013) Prevalence of Q80K in GT1a infected patients was 24.2%.

32 McCloskey RM et al., J Infect Dis 2014

33 The majority (96%) of HCV infections carrying Q80K descended from a single lineage in which a Q80K substitution occurred around the 1940's in the United States, which implies that this polymorphism is likely highly transmissible McCloskey RM et al., J Infect Dis 2014

34 SMV + SOF (12 weeks) SVR12 rates according to Q80K presence at baseline in G1a non-cirrhotic patients OPTIMIST (92.8,100) 96 (88.7,100) 97 (92.5,100) 80 SVR12 (%) /116 44/46 68/70 G1a G1a with Q80K G1a without Q80K Q80K did not impact SMV + SOF efficacy when administered for 12 weeks to G1a non-cirrhotic treatment-naïve and -experienced patients Asselah T, EASL 2015 SYMPOSIUM Kwo P, et al. EASL Poster LP14

35 SMV + SOF (12 weeks) SVR12 rates according to Q80K presence at baseline in G1a cirrhotic patients: OPTIMIST (74.0,92.6) 74 (57.2,89.8) 92 (82.2,100) SVR12 (%) /72 25/34 35/38 G1a G1a with Q80K G1a without Q80K Eligible patients were years with chronic HCV G1 infection; HCV RNA > IU/mL at screening, platelets >50 000/mm 3, albumin >3 g/dl and presence of cirrhosis determined by any of: FibroScan with kpa >12.5 within 6 months of screening; FibroTest score of >0.75 and AST to platelet ratio >2 at screening; liver biopsy documenting cirrhosis Treatment-experienced = IFN-intolerant, prior relapsers, prior nonresponders, other Brackets = 95% CI; AST = aspartate aminotransferase Asselah T, EASL 2015 SYMPOSIUM Lawitz E, et al. EASL Poster LP04

36 SMV + SOF (12 weeks) SVR12 rates according to Q80K presence at baseline in G1a cirrhotic patients 82 cirrhotic CPT A pts with chronic HCV G1a SVR12 (%) /58 22/23 32/35 Overall G1a with Q80K G1a without Q80K Discrepant results were reported regarding the impact of Q80K in cirrhotic patients Randomised, prospective, open-label trial that was investigator-initiated. Patients were assigned randomly to receive either SMV + SOF for 12 weeks or SOF + PR for 12 weeks. Randomisation was stratified according to previous PR treatment (non- or null responder). SVR12 rate in the SOF + PR arm was 75% CPT = Child-Pugh Turcotte score Asselah T, EASL 2015 SYMPOSIUM Pearlman B, et al. Gastroenterology 2015

37 Natural RAVs were detected in 55/286 (19.2%) NS3 sequences and in 30/96 (31.3%) NS5A sequences from PI-naïve patients with an available baseline resistance test DAA naïve pts were analysed for NS3 (1a=121, 1b=154, 3=6, 4=5) and NS5a (1a=42, 1b=42, 3=7, 4=5). Prevalence of NS3 RAVs was more frequent in GT-1a (28.3%) compared to genotype 1b (7.8%). Genotype, n Genotype, n NS3 RAVs 1a 1b 3a NS5A RAVs 1a 1b (N=121) (N=154) (N=6) (N=42) (N=42) V36L M28V 1 V55A/I 2/3 Q30H 1 T54S 3 3 Q30R 1 Q80L/R 5/1 6/0 L31M 1 4 Q80K 19 1 Q54H 17 S122A 1 Q54Y 1 R155K 1 Y93H 1 3 D168E 2 1 3a (N=7) Sorbo MC et al., ICAR 2015

38 Natural RAVs were detected in 55/286 (19.2%) NS3 sequences and in 30/96 (31.3%) NS5A sequences from PI-naïve patients with an available baseline resistance test DAA naïve pts were analysed for NS3 (1a=121, 1b=154, 3=6, 4=5) and NS5a (1a=42, 1b=42, 3=7, 4=5). Prevalence of NS3 RAVs was more frequent in GT-1a (28.3%) compared to genotype 1b (7.8%). Genotype, n Genotype, n NS3 RAVs 1a 1b 3a NS5A RAVs 1a 1b (N=121) (N=154) (N=6) (N=42) (N=42) V36L M28V 1 V55A/I 2/3 Q30H 1 T54S 3 3 Q30R 1 Q80L/R 5/1 6/0 L31M 1 4 Q80K 19 1 Q54H 17 S122A 1 Q54Y 1 R155K 1 Y93H 1 3 D168E 2 1 3a (N=7) Sorbo MC et al., ICAR 2015

39 Comparative study of RAVs prevalence in NS3 and NS5A genes in different HCV sub-genotypes, in liver and plasma specimens in patients with HCC / transplanted and not 13 HCC/LT pts infected by HCV GT1a [N=4], 1b [N=5], 3a [N=3], and 4d [N=1] underwent LT due to HCC, in RAVs Patient Genotype Compartment NS3-Protease NS5A TT none Q54H Pt.2 1b NT none Q54H, Y93H/P/Y/S PL none Q54H TT T54T/S Y93H/Y Pt.22 1b NT none Y93H/Y PL none none TT S122A none Pt.1 3a NT S122A none PL S122A none PL 49w S122A none Table 1. Prevalence of NS3 and NS5A RAVs found in different compartments of HCC/transplanted pts: tumorous tissue (TT), normal tissue (NT), plasma (PL), 49 week post-lt plasma (PL 49w). RAVs were not present in any compartments of the other 10 remaining pts included in the current study (neither in NT/TT /PL for 5 pts nor in PL for additional 5 pts). Interestingly, the NS3 RAV S122A found in 1/13 HCC/LT (1, GT3) was detected in only 1 control-pt GT1b: in HCC pts 7.7% (1/13) vs 0.3% in controls (1/286), p= 0.08 Fisher test. Sorbo MC et al., ICAR 2015

40 Clinical case Sex: MALE Age: 41 year Country of Origin: ITALY Liver status: OLT in 2007, today decompensated cirrhosis (ascites) HCV genotype: 4d Outcome to previous pegifn+rbv treatment: Null responder

41 New treatment strategies are available for GT-4 The main limitations of these clinical trials on HCV-G4 are their small sample size and the relatively mild stage of liver diseases Asselah T, J Hepatol 2015

42 Treatment selection in patients before and after OLT based on currently available evidence The oral combination of daclatasvir + simeprevir was chosen for this patient. Ferenci, Nat Rev 2015

43 Would you have tested this patient for Q80K presence? Analyzing 77 GT-4 NS3-sequences from Los-Alamos DB, the Q80K prevalence is 6.5% (5/77). Sarrazin C et al., Antivir Res 2015

44 ID_451 HCV genotype: 4d Age: 41 Sex: M Null Responder to SOC OLT in Daclatasvir+Simeprevir Interruption GRT 2 weeks after therapy interruption NS3 Resistance Mutations: A156G, D168E NS5A Resistance Mutation: L28V, T58P HCV- RNA (log IU/ml) LLOQ (12 IU/ml) Day 0 Day1 Day2 1w 2w 4w 6w 7w 2w

45 Generation of multiple-resistant viruses Bolded type under the amino acid position number represents the key mutations that are clearly associated with virologic failure and result in a resistant phenotype. IAS USA 2013 IAS list 2013

46 ID_451 HCV genotype: 4d Age: 41 Sex: M Null Responder to SOC OLT in Daclatasvir+Simeprevir GRT Day 0 NS3 Resistance Mutations: D168E NS5A Resistance Mutation: T58P Interruption GRT 2 weeks after therapy interruption NS53 Resistance Mutations: A156G, D168E NS5A Resistance Mutation: L28V, T58P HCV- RNA (log IU/ml) LLOQ (12 IU/ml) Day 0 Day1 Day2 1w 2w 4w 6w 7w 2w

47 Useful a genotypic HCV resistance test after failure?

48 In the era of DAA.. Treatment Failure = Failure to Cure HCV infection = There remains hepatocytes in the liver that are infected with wt and/or resistant HCV viruses when treatment is stopped

49 Virological failures to all-oral, IFN-free, regimens are likely to involve multiple RAVs on multiple drug targets Pooled prevalence of NS3 RAVs at failure: 76% Pooled prevalence of NS5A RAVs at failure: 68% Pooled prevalence of NS5B RAVs at failure: 55% Krishnan P et al., EASL 2015

50 The rate of decline is specific to each drug resistant mutation and it is related to the replication capacity Dynamics of Resistant HCV Variants Post-Treatment (TVR) Sullivan et al., EASL 2011, Sullivan et al CID 2013 % resistant viral variants detected 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 Dynamics of Resistant HCV Variants Post-Treatment (BOC) All T54S R155K V36M Genotype 1a Time after treatment failure (years) 2 % Variant viral résistant détecté 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Genotype 1b All T54S T54A Time after treatment failure (years) Barnard et al., Virology 2013

51 Long-term follow-up data on a cohort of 314 BOCfailing patients indicated that NS3 RAVs reverted to wild-type in 73% of cases within 3 years post-therapy From a different perspective, these data indicate that 27% of patients still presented dominant resistant (and presumably fit) variants after 3 years since boceprevir discontinuation. Furthermore, the remaining 73% can still harbor minority residual resistant strains, since reversion was evaluated by population sequencing, able of detecting variants at a frequency >20%. Howe, Antivir Res 2014

52 An HCV-1a infected patient who failed Telaprevir triple therapy with V36M+R155K still showed R155K at 100 weeks after therapy interruption ID_13 HCV genotype: 1a Age: 44 Sex: M Null responder to SOC IL-28: CT HCV- RNA (log UI/ml) TVR + pegifn+rbv pegifn+rbv Interruption GRT Day 0 NS3-protease (bulk+udps): None GRT Day 2 NS3-protease bulk: None By UDPS: V36A 2.5%,R155K 4.1% Mutational Load: V36A=183 IU/ml R155K=301 IU/ml GRT 18 weeks NS3-protease (bulk+udps): V36M 100%, R155K 100% GRT 72 weeks after therapy interruption NS3-protease bulk: None By UDPS V36M 1.3%, R155K 32.8% Mutational Load: V36M: IU/ml R155K: IU/ml GRT 100 weeks after therapy interruption NS3-protease bulk: None By UDPS R155K 3.7% Mutational Load: R155K:77966 IU/ml 2 LLOQ (12 IU/ml) 1 TVR INTERRUPTION PegIFN+RBV INTERRUPTION Day 0 Day 2 2 w 4 w 8 w 12 w 16 w 18 w 6w 12w 20w 25w 34 w 45 w 72 w 100 w Follow-up on treatment Follow-up after treatment

53 The low prevalence of minority RAVs does not always means low amount of circulating resistant virus Three HCV-1a infected patients, all failing TVR-containing regimen with RAVs, still showed the R155K after weeks of therapy interruption. At 100 weeks of therapy interruption At 62 weeks of therapy interruption HCV-RNA : 1,671,926 IU/ml R155K (35.5%) HCV-RNA: 2,090,903 IU/ml R155K (3.7%) HCV-RNA: 1,104,189 IU/ml R155K (4.0%) WT R155 (65.5%) WT R155 (96.3%) WT R155 (96.0%) WT= wild-type PT 11 Mutational Load of R155K: 594,022 IU/ml PT 13 Mutational Load of R155K: 77,966 IU/ml PT 211 Mutational Load of R155K: 44,167 IU/ml Di Maio VC, EASL 2015

54 First documentation of a transmission of an HCV DAA resistant variant from a DAA treated patient to his sexual HIV-infected partner Patient A, a man chronically infected with HCV genotype 1a and co-infected with HIV- 1, treated with pegifn/rbv plus telaprevir in July 2012 with HCV breakthrough. HCV NS3 protease sequences before treatment with telaprevir did not have any major substitution associated with NS3 PIs. Patient B, a man also HIV-1 infected and sexual partner of patient A, diagnosed of acute HCV coinfection in January 2011, with HCV genotype 1a. This patient refused therapy with pegifn/rbv during the acute phase of HCV infection. In April 2012 he entered a clinical HCV trial and was treated for 24 weeks with pegifn/rbv plus Daclatasvir, with undetectable HCV RNA at week 24 and 36 after the end of treatment. However, at week 48 after stopping therapy, presented elevated transaminase and detectable HCV RNA, suggesting a HCV re-infection. The patient denied any known risk for HCV infection except unprotected sexual intercourse with his partner (patient A). After 12 weeks, patient B tested negative for HCV infection. V36M substitution detected in 9/20 clones in patient A (20 weeks off therapy) and in all late sequences from patient B. 108 and 28 individual NS3 protease clones from patient A and B early samples, respectively, were sequenced Franco et al Gastroenterology May 2014

55 Risk of Late Relapse or Re-Infection with Hepatitis C After Sustained Virological Response: Meta-Analysis of 66 Studies in 11,071 Patients 5-yr recurrence rate post SVR, % Five-Year Rate (95%CI) of Recurrence Post-SVR, by Risk Group Low Risk 43 studies N = 9,419 Avg. FU = 4.1±2.1y 1.1% (95%CI %) High Risk (IDUs/prisoners) 16 studies N = 819 Avg. FU = 2.9±1.6y 13.2% (95%CI %) HIV/HCV Co-Infected 7 studies N = 833 Avg. FU = 3.1±1.2 years 21.7% (95%CI %) Low risk High risk HIV/HCV co-infected Hill A, et al. 22nd CROI; Seattle, WA; February 23-26, Abst. 654.

56 When treating patients with chronic viral hepatitis C with direct antiviral agents we should always remember: The complexity: virus, host, clinical aspects, previous treatment outcome, DAA The virus is very variable: Conclusions At population level: different genotypes and subtypes, different response to PegIFN/RBV and to new DAA! Different PI, NS5A and NS5B resistance development and prevalence (1a vs 1b subtype, genotypes). At patient level: quasispecies, minor variants, pre-existing resistance.

57 Conclusions The performance of a baseline sequencing of HCV can provide at the same time two important virological information for clinical management of patients with chronic HCV infection: 1) a correct genotype/subtype assignment based on sequence analysis (often incomplete, or even wrong, with old diagnostic methods). 2) detection of variants that are potential non responders to therapy (natural resistance or previous failure resistance). In GT-1a patients: test for Q80K in patients eligible for simeprevir treatment. What about NS5A natural resistance Y93H and other mutations? What about NS5B L159F polymorphism in GT-1b patients for sofosbuvir? Potential role for the duration of therapy? HCV-RNA decay at early time points (i.e. 8? 24-48h? week 2) may help in predicting treatment outcome: HCV-RNA >100 IU/ml at week 2 may identify patients at risk of failure in clinical practice. Attention to early resistance development/selection!