Virological Tools and Monitoring in the DAA Era

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1 Virological Tools and Monitoring in the DAA Era Prof. Jean-Michel Pawlotsky, MD, PhD National Reference Center for Viral Hepatitis B, C and delta Department of Virology & INSERM U955 Henri Mondor Hospital University of East Paris Créteil, France

2 I Virological Tools

3 HCV genotype determination

4 HCV Genotypes

5 HCV Genotype Determination Molecular methods: Direct sequence analysis Home-made : NS5B or E1 regions, Commercial : 5 noncoding region (Trugene HCV 5 NC Genotyping Kit, Bayer HealthCare) or NS5B (Trugene HCV NS5B Genotyping Kit, Bayer HealthCare) Real-time PCR with genotype-specific primers and probes Reverse hybridization of PCR products: Line Probe Assay (INNO-LiPA HCV II, Innogenetics) Serological methods: serotyping assay

6 Versant HCV Genotype 2.0 Assay (INNO-LiPA)

7 HCV Genotype 1 Subtype Determination Sequence Analysis of the 5 NCR 1 st Generation of Line Probe Assay (LiPA 1.0) 2 nd Generation of Line Probe Assay (LiPA 2.0) RealTime HCV Genotype II GT 1a (n=237) 77.6% (n=184) 70.5% (n=167) 97.5% (n=231) 93.2% (n=220) GT 1b (n=263) 90.5% (n=238) 91.3% (n=240) 96.2% (n=253) 88.6% (n=233) (Chevaliez et al., PLoS One 2009;4:e820)

8 Interest of Genotype 1 Subtyping in Practice Peg-IFN and ribavirin therapy: No practical interest for clinical decisions Triple combination therapy with Pis: Modest difference between 1a and 1b Different resistance profiles No practical interest for clinical decisions IFN-free regimens Possibly important

9 HCV RNA Quantification

10 Linear Ranges of Quantification Cobas Amplicor HCV Monitor v SuperQuant LCx HCV RNA Assay Versant HCV RNA 3.0 (bdna) (Chevaliez et al., Gastroenterology 2012;142: )

11 Linear Ranges of Quantification Cobas Amplicor HCV Monitor v SuperQuant LCx HCV RNA Assay Versant HCV RNA 3.0 (bdna) CTM HCV test v2.0 (Roche) CAP/CTM HCV test, v2.0 (Roche) RealTime HCV (Abbott) Artus HCV QS- RGQ (Qiagen) Versant HCV RNA 1.0 (kpcr, Siemens) (Chevaliez et al., Gastroenterology 2012;142: )

12 Abbott Real-Time PCR 8.0 R=0,9658, p< m2000 sp (Abbott) HCV genotype 1 (n=43) HCV genotype 2 (n=11) HCV genotype 3 (n=19) HCV genotype 4 (n=17) HCV genotype 5 (n=5) bdna 3.0 (Chevaliez et al., J Clin Microbiol 2009;47: )

13 CAP/CTM HCV Assay Genotype 1 (n=29) Genotype 2 (n=27) Genotype 3 (n=29) Genotype 4 (n=30) Genotype 5 (n=9) Genotype 6 (n=2) (Chevaliez et al., Hepatology 2007;46:22-31) 3 r = 0.889; p < HCV RNA level in Versant HCV 3.0 Assay bdna (Log 10 UI/mL)

14 Lack of HCV RNA Detection by CAP/CTM in Genotype 4 Patient Genotype CAP/CTM (Roche) bdna 3.0 (Siemens) Real-Time PCR (Abbott) A 4h Undetectable <12 IU/ml 5.4 log IU/ml 5.0 log IU/ml B 4l Undetectable <12 IU/ml 6.0 log IU/ml 5.7 log IU/ml (Chevaliez et al., Hepatology 2008;49:1397-8)

15 Genotype 4 Quantification with CAP/CTM v2.0 (Roche) HCV RNA level in CAP/CTM48 v2.0 (Log 10 IU/mL) r = ; p < HCV RNA level in Versant HCV 3.0 bdna Assay (Log 10 IU/mL) (Chevaliez et al., J Clin Microbiol 2013;51: ) Genotype 4a (n=43) Genotype 4c (n=4) Genotype 4d (n=34) Genotype 4e (n=9) Genotype 4f (n=9) Genotype 4g (n=2) Genotype 4h (n=5) Genotype 4k (n=4) Genotype 4n (n=1) Genotype 4r (n=8) Genotype 4t (n=3)

16 CAP/CTM v1.0 vs v2.0 (Roche) CAP/CTM96 v1.0 (Log 10 IU/mL) CAP/CTM96 v2.0 (Log 10 IU/mL) m2000 SP /m2000 RT (Log 10 IU/mL) bdna 3.0 (Log 10 IU/mL) Patient 1 (4h) < Patient 2 (4l) < Patient 3 (4) < ,2 Patient 4 (4k) < (Chevaliez et al., J Clin Microbiol 2013;51: )

17 HCV core Ag quantification

18 HCV Core Antigen Quantification (Bouvier-Alias M. et al., Hepatology 2002;36:211-8)

19 Architect HCV Ag Assay RVR (G1b) SVR (G1b) Relapser (G1b) Core Ag RNA NR (G1a) (Ross M. et al., J Clin Microbiol 2010;48:1161-8)

20 HCV Core Ag Quantification HCV core Ag quantification can be used as a surrogate marker of HCV replication in the monitoring of antiviral therapy However, HCV core Ag assays lack sensitivity compared to HCV RNA level quantification by real-time PCR (LLD equivalent to IU/mL according to the HCV genotype)

21 II Practical Use

22 Current Standard-of-Care for HCV Genotype non-1

23 Standard-of-Care for HCV Genotype non-1 Pegylated IFN- + Ribavirin

24 Virological Monitoring PegIFN -ribavirin Weeks of treatment

25 On-Treatment Virologic Responses -2 log LLOD Baseline Week 4 Week 8 Week 12

26 On-Treatment Virologic Responses -2 log LLOD Baseline Week 4 Week 8 Week 12

27 On-Treatment Virologic Responses -2 log LLOD Baseline Week 4 Week 8 Week 12

28 On-Treatment Virologic Responses -2 log RVR 24 weeks LLOD Baseline Week 4 Week 8 Week 12

29 On-Treatment Virologic Responses -2 log RVR 24 weeks EVR 48 weeks LLOD Baseline Week 4 Week 8 Week 12

30 24 vs 48 Weeks in Genotype 2/3 Patients Without an RVR (N-Core) 80 p=0.19 p=0.14 p= % SVR24 (% of patients) % 52% 63% 52% 54% Peg-IFN alfa-2a/rbv 24 weeks Peg-IFN alfa-2a/rbv 48 weeks ITT (n=188) PP (n=176) Study completers (n=153) (Cheinquer et al., AASLD 2012)

31 On-Treatment Virologic Responses -2 log RVR 24 weeks EVR 48 weeks LLOD Baseline Week 4 Week 8 Week 12

32 On-Treatment Virologic Responses -2 log RVR 24 weeks EVR 48 weeks Delayed VR 72 weeks LLOD Baseline Week 4 Week 8 Week 12

33 Current Standard-of-Care for HCV Genotype 1

34 New Standard-of-Care for HCV Genotype 1 Telaprevir Boceprevir Pegylated IFN- + Ribavirin

35 Virological Monitoring Telaprevir PegIFN -ribavirin Weeks of treatment

36 Response-Guided Therapy Peg-IFN + Ribavirin + Telaprevir Treatment-naive or responderrelapser Partial responder or null-responder W0 W4 W12 W24 W36 W48 W72

37 Response-Guided Therapy Peg-IFN + Ribavirin + Telaprevir ervr: undetectable HCV RNA at weeks 4 and 12 Treatment-naive or responderrelapser TVR + PR PR Follow-up: 24 weeks HCV RNA detectable at weeks 4 and/or 12 but 1000 UI/mL PR Follow-up: 24 weeks Partial responder or null-responder W0 W4 W12 W24 W36 W48 W72

38 Response-Guided Therapy Peg-IFN + Ribavirin + Telaprevir ervr: undetectable HCV RNA at weeks 4 and 12 Treatment-naive or responderrelapser TVR + PR PR Follow-up: 24 weeks HCV RNA detectable at weeks 4 and/or 12 but 1000 UI/mL PR Follow-up: 24 weeks Partial responder or null-responder TVR + PR PR Follow-up: 24 weeks W0 W4 W12 W24 W36 W48 W72

39 Futility Rules Peg-IFN + Ribavirin + Telaprevir HCV RNA >1000 IU/mL at W4 or W12 HCV RNA detectable (>10-25 IU/mL) at W24

40 Futility Rule with Telaprevir Retrospective analysis of ADVANCE, ILLUMINATE and REALIZE data (n=903 treatment-naive and 266 treatment experienced) Likelihood of an SVR HCV RNA at week 4 >1000 IU/mL (2.1%): SVR: 0% HCV RNA at week 4 = IU/mL (2.0%): SVR: 22% HCV RNA at week 12 >1000 IU/mL (1.5%): SVR: 0% HCV RNA at week 12 = IU/mL (1.6%): SVR: 15% Conclusion: A futility rule of greater than 1000 IU/mL at week 4 and at week 12 identifies treatment-naïve or -experienced patients unlikely to achieve an SVR (Adda et al., Clin Gastroenterol Hepatol 2013;11:193-5)

41 Futility Rule with Telaprevir HCV RNA Profiles in Patients with HCV RNA >1000 IU/mL at week 4 (Adda et al., Clin Gastroenterol Hepatol 2013;11:193-5)

42 Virological Monitoring Boceprevir Telaprevir PegIFN -ribavirin Weeks of treatment

43 Response-Guided Therapy Peg-IFN + Ribavirin + Boceprevir Treatment naive patients (excluding F4) Treatmentexperienced patients (excluding nullresponders and F4) F4 patients and null-responders

44 Response-Guided Therapy Peg-IFN + Ribavirin + Boceprevir W0 W4 W8 W12 W24 W28 W36 W48 Undetectable HCV RNA at week 8 Treatment naive patients (excluding F4) PegIFN/ RBV Boceprevir Boceprevir + PegIFN/RBV Detectable HCV RNA at week 8 Boceprevir + PegIFN/RBV PegIFN/RBV Treatmentexperienced patients (excluding nullresponders and F4) F4 patients and null-responders

45 Response-Guided Therapy Peg-IFN + Ribavirin + Boceprevir W0 W4 W8 W12 W24 W28 W36 W48 Undetectable HCV RNA at week 8 Treatment naive patients (excluding F4) PegIFN/ RBV Boceprevir Boceprevir + PegIFN/RBV Detectable HCV RNA at week 8 Boceprevir + PegIFN/RBV PegIFN/RBV Treatmentexperienced patients (excluding nullresponders and F4) W0 W4 W12 W24 W36 W48 PegIFN RBV Boceprevir Boceprevir + PegIFN/RBV PegIFN/RBV F4 patients and null-responders

46 Response-Guided Therapy Peg-IFN + Ribavirin + Boceprevir W0 W4 W8 W12 W24 W28 W36 W48 Undetectable HCV RNA at week 8 Treatment naive patients (excluding F4) PegIFN/ RBV Boceprevir Boceprevir + PegIFN/RBV Detectable HCV RNA at week 8 Boceprevir + PegIFN/RBV PegIFN/RBV Treatmentexperienced patients (excluding nullresponders and F4) W0 W4 W12 W24 W36 W48 PegIFN RBV Boceprevir Boceprevir + PegIFN/RBV PegIFN/RBV W0 W4 W12 W24 W48 F4 patients and null-responders PegIFN RBV Boceprevir Boceprevir + PegIFN/RBV

47 Futility Rules Peg-IFN + Ribavirin + Boceprevir HCV RNA 100 IU/mL at W12 HCV RNA detectable (>10-25 IU/mL) at W24

48 Futility Rules with Boceprevir Treatment-naïve and -experienced patients In SPRINT-2 None of the 65 patients with an HCV RNA >100 IU/mL at week 12 achieved an SVR 49 out of 79 patients (62%) with detectable HCV RNA <100 IU/mL at week 12 subsequently became HCV RNA undetectable and 43% achieved an SVR In RESPOND-2 Only 1 patient with an HCV RNA >100 IU/mL at week 12 achieved an SVR 5 out of 6 patients (83%) with detectable HCV RNA <100 IU/mL at week 12 subsequently achieved an SVR (Jacobson et al., Hepatology 2012;56:567-75)

49 Future HCV Therapies

50 Sofosbuvir + Daclatasvir ± RBV Treatment-naive, Genotype % 100% 100% SVR24 rate (%) N=15 LI/SOF + DCV N=14 SOF + DCV N=15 SOF + DCV + RBV (Sulkowski et al., AASLD 2012)

51 ABT-450/r (PI) ± ABT-267 (NS5A) ± ABT-333 (NNI)-AVIATOR Treatment-naϊve Patients 97% 89% 90% 88% 85% Null Responders 89% 93% SVR12 rate (%) N=80 N=41 N=79 N=79 N=79 N=45 N=45 8 weeks 12 weeks 12 weeks ABT-450 ABT-267 ABT-333 RBV ABT-450 ABT-333 RBV ABT-450 ABT-267 RBV ABT-450 ABT-267 ABT-333 ABT-450 ABT-267 ABT-333 RBV ABT-450 ABT-267 RBV ABT-450 ABT-267 ABT-333 RBV (Kowdley et al., AASLD 2012)

52 Conclusions HCV RNA levels must be measured by means of wellvalidated commercial real-time PCR-based assays, with a lower limit of detection of the order of IU/mL Interferon-based strategies, including or not protease inhibitors, are response-guided. Thus, HCV RNA monitoring during therapy is key to tailor treatment duration HCV RNA level monitoring might be less important in the future when all-oral, IFN-free regimens become available, but further studies are needed.

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