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Pawoltsky diagnostic

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  • 1. HCV DiagnosticStrategies & Monitoring 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. IHCV Markers and Kinetics
  • 3. HCV Markers• HCV genotype : • Intrinsic characteristic of the infecting HCV strain.• HCV RNA : • Marker of HCV replication.• Total HCV core Ag : • Surrogate marker of HCV replication.• Anti-HCV antibodies : • Marker of past or present infection.
  • 4. Kinetics of HCV Markers Spontaneous resolution HCV RNA HCV core Ag anti-HCV antibodies ALTInfection Acute hepatitis
  • 5. Kinetics of HCV Markers Persistent infection HCV RNA HCV core Ag anti-HCV antibodies ALTInfection Acute Chronic hepatitis hepatitis
  • 6. IIHCV Virological Tools
  • 7. Classical HCV Virological Tests• SerologicalAssays • ELISA tests for anti-HCVantibodydetection• Molecular tests • HCVgenotypingassays • HCV RNA quantification assays
  • 8. New HCV Virological Tests• New serologicaltests • Quantitativecoreantigendetection (ELISA)• Molecular tests • Real-time PCR assays for HCV RNA level quantification • Ultra-deeppyrosequencing (resistance)
  • 9. Anti-HCVantibodydetection
  • 10. Anti-HCVAntibodyDetection• Based on ELISA• Easy to use, automated• 3rd-generation tests available • ADVIA Centaur (Siemens) • VITROS ECi (Ortho-ClinicalDiagnostics) • AXSYM HCV 3.0 (Abbott) • CobasElecsysModular HCV (Roche) • INNOTEST HCV Ab IV (Innogenetics) • Monolisaanti-HCV Plus version 2 (Bio-Rad)
  • 11. “Combo“ Tests (Ag + Ab)• Based on ELISA• Commerciallyavailable• Reduce the serologicalwindowduring acute infection by 20-30 days• No benefit in the diagnostic setting• No benefit in blood screening in the context of Nucleicacidtesting (NAT)
  • 12. HCV core Ag quantification
  • 13. HCV CoreAntigen Quantification(Bouvier-Alias M. et al., Hepatology 2002;36:211-8)
  • 14. Architect HCV Ag Assay RVR (G1b) SVR (G1b) Core Ag RNA Relapser(G1b) NR (G1a)(Ross M. et al., J ClinMicrobiol 2010;48:1161-8)
  • 15. HCV Core Ag Quantification• HCV core Ag quantification canbeused as a surrogate marker of HCV replication in the monitoring of antiviral therapy• However, HCV core Ag assayslacksensitivitycompared to HCV RNA level quantification by real-time PCR (LLD equivalent to 500-3000 IU/mLaccording to the HCV genotype)
  • 16. HCVGenotypedetermination
  • 17. HCV Genotypes
  • 18. HCV GenotypeDetermination• Molecularmethods: • Direct sequenceanalysis – Home-made : NS5B or E1 regions, – Commercial : 5’ noncodingregion (Trugene HCV 5’NC Genotyping Kit, Bayer HealthCare) or NS5B (Trugene HCV NS5B Genotyping Kit, Bayer HealthCare) • Real-time PCR withgenotype-specificprimers and probes • Reverse hybridization of PCR products: – Line Probe Assay (INNO-LiPA HCV II, Innogenetics)• Serologicalmethods: serotypingassay
  • 19. Versant® HCV Genotype 2.0 Assay (INNO-LiPA)
  • 20. HCV Genotype 1 Subtype Determination Sequence 1st Generation of 2nd Generation of RealTime HCV Analysis of the Line Probe Assay Line Probe Assay Genotype II 5’NCR (LiPA 1.0) (LiPA 2.0) GT 1a 77.6% 70.5% 97.5% 93.2%(n=237) (n=184) (n=167) (n=231) (n=220) GT 1b 90.5% 91.3% 96.2% 88.6%(n=263) (n=238) (n=240) (n=253) (n=233)(Chevaliez et al., PLoS One 2009;4:e820)
  • 21. Interest of Genotype 1 Subtyping in Practice• Peg-IFN and ribavirintherapy: • No practicalinterest for clinicaldecisions• Triple combinationtherapywith Pis: • Modest differencebetween 1a and 1b • Differentresistance profiles • No practicalinterest for clinicaldecisions• IFN-freeregimens • Possibly important
  • 22. HCV RNA Quantification
  • 23. Linear Ranges of Quantification 10 102 103 104 105 106 107 108CobasAmplicorHCV Monitor v2.0SuperQuantLCx HCVRNA AssayVersant HCV RNA3.0 (bDNA)(Chevaliez et al., Gastroenterology 2012;142:1303-13)
  • 24. Linear Ranges of Quantification 10 102 103 104 105 106 107 108CobasAmplicorHCV Monitor v2.0SuperQuantLCx HCVRNA AssayVersant HCV RNA3.0 (bDNA)CTM HCV test v2.0(Roche)CAP/CTM HCVtest, v2.0 (Roche)RealTime™ HCV(Abbott)Artus HCV QS-RGQ (Qiagen)Versant HCV RNA1.0 (kPCR, Siemens)(Chevaliez et al., Gastroenterology 2012;142:1303-13)
  • 25. Real-Time PCR Platforms CAP-CTM96 (ROCHE) kPCR (SIEMENS)m2000SP-m2000RT (ABBOTT)
  • 26. CAP/CTM HCV Assay 8 7 Genotype 1 (n=29) 6 Genotype 2 (n=27) Genotype 3 (n=29) 5 Genotype 4 (n=30) Genotype 5 (n=9) 4 Genotype 6 (n=2) r = 0.889; p < 0.0001 3 3 4 5 6 7 8 HCV RNA level in Versant HCV 3.0 Assay bDNA (Log10 UI/mL)(Chevaliez et al., Hepatology 2007;46:22-31)
  • 27. Underestimation of HCV RNA Levels by CAP/CTM in Genotypes 2 and 4 1.5 1.0 Genotype 1 (n=29) 0.5 Genotype 2 (n=27) Genotype 3 (n=29) 0.0 Genotype 4 (n=30) -0.5 Genotype 5 (n=9) Genotype 6 (n=2) -1.0 -1.5 15% 30%(Chevaliez et al., Hepatology 2007;46:22-31)
  • 28. Lack of HCV RNA Detection by CAP/CTM in Genotype 4 CAP/CTM bDNA 3.0 Real-Time PCR Patient Genotype (Roche) (Siemens) (Abbott) Undetectable A 4h 5.4 log IU/ml 5.0 log IU/ml <12 IU/ml Undetectable B 4l 6.0 log IU/ml 5.7 log IU/ml <12 IU/ml(Chevaliez et al., Hepatology 2008;49:1397-8)
  • 29. Genotype 4 Quantification with CAP/CTM v2.0 (Roche) 8 HCV RNA level in CAP/CTM48 v2.0 Genotype 4a (n=43) Genotype 4c (n=4) 7 Genotype 4d (n=34) Genotype 4e (n=9) (Log10 IU/mL) 6 Genotype 4f (n=9) Genotype 4g (n=2) Genotype 4h (n=5) 5 Genotype 4k (n=4) Genotype 4n (n=1) 4 Genotype 4r (n=8) Genotype 4t (n=3) r = 0.9581; p < 0.0001 3 3 4 5 6 7 8 HCV RNA level in Versant HCV 3.0bDNA Assay (Log10 IU/mL)(Chevaliez et al., J ClinMicrobiol 2013; in press)
  • 30. CAP/CTM v1.0 vs v2.0 (Roche) CAP/CTM96 v1.0 CAP/CTM96 v2.0 m2000SP/m2000RT bDNA 3.0 (Log10 IU/mL) (Log10 IU/mL) (Log10 IU/mL) (Log10 IU/mL) Patient 1 (4h) <1.08 5.8 5.4 5.0 Patient 2 (4l) <1.08 6.3 6.0 5.7 Patient 3 (4) <1.08 6.7 6.5 6,2 Patient 4 (4k) <1.08 5.4 5.7 5.8(Chevaliez et al., J ClinMicrobiol 2013; in press)
  • 31. Abbott Real-Time PCR 8.0 R=0,9658, p<0.0001 7.0 m2000sp (Abbott) HCV genotype 1 (n=43) 6.0 HCV genotype 2 (n=11) HCV genotype 3 (n=19) 5.0 HCV genotype 4 (n=17) HCV genotype 5 (n=5) 4.0 3.0 3.0 4.0 5.0 6.0 7.0 8.0 bDNA 3.0(Chevaliez et al., J Clin Microbiol 2009;47:1726-32)
  • 32. Clinical Achievements of Real-Time PCR Assays• Replace qualitative viral genome detection assays• Accurately quantify a broad range of viral levels observed in clinical practice: • High pretreatment levels • Low levels during antiviral treatment• Efficiently monitors viral kinetics (early assessment of virologic responses to therapy)
  • 33. HCV Resistance Testing
  • 34. Quasispecies Distribution of Viral Populations Major viral populationIntermediate viral populations Minor viral populations
  • 35. Viral SequenceAnalysis Tools Major viral population detected by direct sequencing Intermediate viral populations detected by cloning and sequencing Minor viral populations detected by ultra- sensitive techniques such as ultra-deep sequencing
  • 36. Available NGS Techniques Technology Number of Number of Maximum Sequencing (template single reads nucleotides Manufacturer Type sequence Accuracy device preparation/ per run* per run* length* (bp) NGS chemistry) (x 106) (Gb) High 5500 800 9 75 throughput emPCR/ligation 99.6-99.8% Applied High 5500xl 1600 15 75 Biosystems throughput Ion Torrent emPCR/RTsequ Long reads 6.2 >1 >400 99.97% (ChiP 316) encing High MiSeq 3.4 >1 150 throughput Genome High Solid 320 95 150 Analyzer IIx throughput Illumina capture/reversib 96.7-100% le terminator High HiSeq 1000 1500 300 100 throughput High HiSeq 2000 3000 600 100 throughput GS Junior Long reads 0.1 0.035 400 99% emPCR/pyroseq 454 /Roche uencing GS FLX+ Long reads 1 0.7 1000 97.4-99.9% Single PacBio/Gen- PacBio RS molecule/RTseq Long reads 0.035 0.045 1200 99.99% Probe uencing(Chevaliez S, Rodriguez C & Pawlotsky JM, Gastroenterology 2012;142:1303-13)
  • 37. Available NGS Techniques Technology Number of Number of Maximum Sequencing (template single reads nucleotides Manufacturer Type sequence Accuracy device preparation/ per run* per run* length* (bp) NGS chemistry) (x 106) (Gb) High 5500 800 9 75 throughput emPCR/ligation 99.6-99.8% Applied High 5500xl 1600 15 75 Biosystems throughput Ion Torrent emPCR/RTsequ Long reads 6.2 >1 >400 99.97% (ChiP 316) encing High MiSeq 3.4 >1 150 throughput Genome High Solid 320 95 150 Analyzer IIx throughput Illumina capture/reversib 96.7-100% le terminator High HiSeq 1000 1500 300 100 throughput High HiSeq 2000 3000 600 100 throughput GS Junior Long reads 0.1 0.035 400 99% emPCR/pyroseq 454 /Roche uencing GS FLX+ Long reads 1 0.7 1000 97.4-99.9% Single PacBio/Gen- PacBio RS molecule/RTseq Long reads 0.035 0.045 1200 99.99% Probe uencing(Chevaliez S, Rodriguez C & Pawlotsky JM, Gastroenterology 2012;142:1303-13)
  • 38. Ultra-DeepPyrosequencing Data collection RT Viral genome PCR amplification extraction fromserum emPCRPyrosequenci ng Analysis % of each mutations PyroClass©. PyroMute©, PyroDyn©, PyroLink© are protected under IDDN(Rodriguez C. et al., in revision)
  • 39. Pre-existingHCV ResistantVariants by UDPS Response genotype subtype Patient pegIFN V36 T54 Q80 R155 A156 D168 I170 IL28B HCV RBV TVR V55A A/M A/S R/K K/T/Q S/T/V A/V/T/H A/T Pt-1 CT 1a NR - 90.0% - - 0.1% 0.4% 0.1% 0.5% Pt-2 CT 1a NR - - - - 0.1% 1.1% - 0.2% Pt-3 CT 1b RR - - - - 0.5% 0.5% - 0.2% Pt-4 TT 1b RR - 29.4% - - - 1.3% - 0.1% Pt-5 CT 1a RR - - - - 0.1% 2.9% 0.1% - Pt-6 CT 1b RR 4.2% - - - 0.1% 0.1% 0.1% 0.1% Pt-7 CT 1a SVR - 11.1% - 0.7% - 0.3% - 0.3% Pt-8 CT 1a SVR - - - - 0.1% 0.5% 0.1% - Pt-9 CC 1a SVR - - - - 0.6% 1.8% - - Pt-10 CC 1a SVR - - - - 0.6% - - 0.1% Pt-11 TT 1a RR - - 100.0% 0.1% 6.0% 3.2% 0.1% 0.3% Pt-12 CT 1b SVR - - - - - 0.3% - 0.1% Pt-13 CT 1b SVR - - - - 0.2% 0.2% - 0.8% Pt-14 TT 1b NR - - - - 0.1% 0.2% - 0.1% Pt-15 CT 1b SVR - - - - 0.4% 0.2% 0.1% 0.1% Pt-16 CT 1a SVR - - 1.3% 0.5% 7.8% 0.2% 0.1% 0.1% Pt-17 CT 1a SVR - 47.4% - - 0.1% 0.4% 0.1% 0.1% Pt-18 CT 1b SVR - 20.0% - - 0.1% 0.4% 0.1% 0.1% SVR: sustained virological response; RR: response-relapse; NR: non-response *SNP rs12979860(Chevaliez S., et al., manuscript in preparation)
  • 40. Treatment Failure-PROVE2 H28Q+R155K 100% H28Q+R155K+S54T+Y52C % of variants in the quasispecies 80% H28Q+R155K+S54T+Y52C+V36M+H5 7L+P96H 60% 8 40% HCV RNA(Log10 IU/mL) 6 20% 0% 4 0 2 29 0 57 Days of therapy 85 Viral populations *PyroLink®(Chevaliez S., et al., EASL 2011)
  • 41. Treatment Failure-PROVE2 100% H28Q+R155K % of variants in the quasispecies H28Q+R155K+S54T+Y52C % of mutations in the whole quasispecies 80% H28Q+R155K+S54T+Y52C+V36M+H57 L+P96H V36M+R155K+H57L 60% R155K 40% 8 HCV RNA (Log10 IU/mL) HCV RNA (Log10 IU/mL) 20% 6 0% 0 4 29 57 85 2 182 595 0 Days of treatment Days of therapy 686 *PyroLink® 903 Viral populations(Chevaliez S., et al., EASL 2011)
  • 42. UDPS in HCV Resistance• Novel technologies for the study of HCV resistance, such as ultra- deeppyrosequecing,willbring new insights intoitsmolecularmechanisms andmay have clinical utility in the future
  • 43. IIIPractical Use
  • 44. Standard-of-Care for HCV Genotype non-1 Pegylated IFN- + Ribavirin
  • 45. Standard-of-Care (EU label) Genotype 2,3 Genotype 4, 5, 6 PegIFN+ PegIFN+ ribavirin ribavirin 24 weeks 48 weeks
  • 46. Virological Monitoring PegIFN-ribavirin0 4 8 12 24 36 48 60 72 Weeks of treatment
  • 47. On-Treatment Virologic Responses -2 log LLODBaseline Week 4 Week 8 Week 12
  • 48. On-TreatmentVirologicRespons es -2 log LLODBaseline Week 4 Week 8 Week 12
  • 49. On-TreatmentVirologicRespons es -2 log Delayed VR 72 weeks RVR 24 weeks EVR 48 weeks LLODBaseline Week 4 Week 8 Week 12
  • 50. 24 vs 48 Weeks in Genotype 2/3Patients Without an RVR (N-Core) p=0.19 p=0.14 p=0.02 80 73% 70 61% 63% 60 SVR24 (% of patients) 52% 52% 54% 50 Peg-IFN alfa-2a/RBV 24 weeks 40 Peg-IFN alfa-2a/RBV 48 weeks 30 20 10 49 57 49 51 49 46 95 93 95 81 95 63 0 ITT PP Study (n=188) (n=176) completers (n=153)(Cheinquer et al., AASLD 2012)
  • 51. New Standard-of-Care for HCV Genotype 1 Telaprevir Boceprevir Pegylated + Ribavirin IFN-
  • 52. Virological Monitoring PegIFN-ribavirin0 4 8 12 24 36 48 60 72 Weeks of treatment
  • 53. Virological Monitoring Telaprevir PegIFN-ribavirin0 4 8 12 24 36 48 60 72 Weeks of treatment
  • 54. Response-GuidedTherapy Peg-IFN + Ribavirin + TelaprevirTreatment-naive or responder- relapserPartial responderor null-responder W0 W4 W12 W24 W36 W48 W72
  • 55. Response-GuidedTherapy Peg-IFN + Ribavirin + Telaprevir eRVR: undetectable HCV RNA atweeks 4 and 12 Follow-up: 24 weeksTreatment-naive or responder- TVR + PR PR HCV RNA detectableatweeks 4 and/or 12 but ≤1000 UI/mL relapser PR Follow-up: 24 weeksPartial responderor null-responder W0 W4 W12 W24 W36 W48 W72
  • 56. Response-GuidedTherapy Peg-IFN + Ribavirin + Telaprevir eRVR: undetectable HCV RNA atweeks 4 and 12 Follow-up: 24 weeksTreatment-naive or responder- TVR + PR PR HCV RNA detectableatweeks 4 and/or 12 but ≤1000 UI/mL relapser PR Follow-up: 24 weeksPartial responder TVR + PR PR Follow-up: 24 weeksor null-responder W0 W4 W12 W24 W36 W48 W72
  • 57. FutilityRules Peg-IFN + Ribavirin + Telaprevir• HCV RNA >1000 IU/mLatW4 or W12• HCV RNA detectable (>10-25 IU/mL) atW24
  • 58. FutilityRulewithTelaprevir • Retrospectiveanalysis of ADVANCE, ILLUMINATE and REALIZE data (n=903 treatment-naive and 266 treatmentexperienced) • Likelihood of an SVR  HCV RNA atweek 4 >1000 IU/mL (2.1%): SVR: 0%  HCV RNA atweek 4 =100-1000 IU/mL (2.0%):SVR: 22%  HCV RNA atweek 12 >1000 IU/mL (1.5%): SVR: 0%  HCV RNA atweek 12 =100-1000 IU/mL (1.6%): SVR: 15% • Conclusion: A futilityrule of greaterthan 1000 IU/mLatweek 4 and atweek 12 identifies treatment-naïve or -experienced patients unlikely to achieve an SVR(Jacobson et al., EASL 2012)
  • 59. Futility Rule with Telaprevir HCV RNA Profiles in Patients with HCV RNA >1000 IU/mL at week 4(Jacobson et al., EASL 2012)
  • 60. Virological Monitoring Boceprevir Telaprevir PegIFN-ribavirin0 4 8 12 24 36 48 60 72 Weeks of treatment
  • 61. Response-GuidedTherapy Peg-IFN + Ribavirin + BoceprevirTreatmentnaive patients(excluding F4) Treatment- experienced patients(excludingnull-responders and F4)F4 patients andnull-responders
  • 62. Response-GuidedTherapy Peg-IFN + Ribavirin + Boceprevir W0 W4 W8 W12 W24 W28 W36 W48 Undetectable HCV RNA atweek 8 Boceprevir Boceprevir+ PegIFN/RBVTreatmentnaive PegIFN/ patients RBV Detectable HCV RNA atweek 8(excluding F4) Boceprevir + PegIFN/RBV PegIFN/RBV Treatment- experienced patients(excludingnull-responders and F4)F4 patients andnull-responders
  • 63. Response-GuidedTherapy Peg-IFN + Ribavirin + Boceprevir W0 W4 W8 W12 W24 W28 W36 W48 Undetectable HCV RNA atweek 8 Boceprevir Boceprevir+ PegIFN/RBVTreatmentnaive PegIFN/ patients RBV Detectable HCV RNA atweek 8(excluding F4) Boceprevir + PegIFN/RBV PegIFN/RBV Treatment- W0 W4 W12 W24 W36 W48 experienced Boceprevir patients(excludingnull- PegIFN Boceprevir+ PegIFN/RBV PegIFN/RBV RBVresponders and F4)F4 patients andnull-responders
  • 64. Response-GuidedTherapy Peg-IFN + Ribavirin + Boceprevir W0 W4 W8 W12 W24 W28 W36 W48 Undetectable HCV RNA atweek 8 Boceprevir Boceprevir+ PegIFN/RBVTreatmentnaive PegIFN/ patients RBV Detectable HCV RNA atweek 8(excluding F4) Boceprevir + PegIFN/RBV PegIFN/RBV Treatment- W0 W4 W12 W24 W36 W48 experienced Boceprevir patients(excludingnull- PegIFN Boceprevir+ PegIFN/RBV PegIFN/RBV RBVresponders and F4) W0 W4 W12 W24 W48F4 patients and Boceprevirnull-responders PegIFN RBV Boceprevir + PegIFN/RBV
  • 65. FutilityRules Peg-IFN + Ribavirin + Boceprevir• HCV RNA ≥100 IU/mLatW12• HCV RNA detectable (>10-25 IU/mL) atW24
  • 66. FutilityRuleswithBoceprevir • 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)
  • 67. TreatmentFailures on Triple Combinationwith a DAA • Due to an inadequateresponseto Peg- IFN and ribavirin • Results in uncontrolledoutgrowthofresistantHCV variantsselected by the proteaseinhibitor(Pawlotsky JM. Hepatology 2011;53:1742-51)
  • 68. SVR According to Lead-in (SPRINT-2, non-black) 100 90 82% 82% % of patients with SVR 80 70 60 <1 log HCV RNA 50 decrease 39% 40 ≥1 log HCV RNA 29% decrease 30 20 10 0 BOC/RGT BOC/PR48(Poordad et al., N Engl J Med 2011;364:1185-206)
  • 69. HCV Resistance Testing• Prior to therapy: • There is no indication for resistancetestingatbaseline • All patients shouldbeconsidered as harboringminor viral populations that are resistant to telaprevir and boceprevir• In case of treatmentfailure: • There is no indication for resistancetestingduring and aftertherapy, as the resultwill have no impact on treatmentdecisions • Proteaseinhibitor-resistant viral populations have been enriched in every patient treatedwithtelaprevir or boceprevirwhodid not clear infection• Resistance testingisrequired in clinical trials and global surveillance studies (research setting)

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