Jean-Michel PAWLOTSKY

How to Use Virological Tools for the Optimal Management of Chronic Hepatitis C ? Prof. Jean-Michel Pawlotsky, MD, PhD French National Reference Center for Hepatitis B, C and delta, Department of Virology & INSERM U841, Henri Mondor Hospital University of Paris 12 Créteil, France

Virological Markers in HCV Treatment Monitoring HCV genotype Tailor ribavirin dose and treatment duration HCV RNA presence and level Decide to treat Assess virological responses Tailor treatment duration to the actual virological response

I HCV Virological Tools

HCV Genotype Determination

HCV Genotypes (Simmonds P., et al. Hepatology 2005;42:962-73)

HCV Genotype Determination Molecular methods (genotyping): Direct sequence analysis Reverse hybridization: “line probe assay“ (Inno-LiPA) Serological methods (“serotyping“) Competitive ELISA

HCV Genotype Determination HCV genome

HCV Genotype Determination HCV genome NS5B

HCV Genotype Determination HCV genome NS5B 5’ noncoding region

Subtyping in the 5’NC Region 491 patients, HCV genotype 1 (local method) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region 491 patients, HCV genotype 1 (local method) NS5B sequence analysis (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region 491 patients, HCV genotype 1 (local method) NS5B sequence analysis 1a (n=226, 46%) 1b (n=245, 50%) other 1 (n=11, 2%) 1d (n=4) 1e (n=2) 1i (n=1) 1l (n=1) 1nd (n=3) non-1 (n=9, 2%) 2l (n=1) 3a (n=2) 6 (n=6) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region NS5B 1a (n=226) 5’NC region 1a (n=180) 1b (n=39) 1nd (n=7) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region NS5B 1a (n=226) 5’NC region 1a (n=180) 1b (n=39) 1nd (n=7) NS5B 1b (n=245) 5’NC region 1b (n=229) 1a (n=8) 1nd (n=8) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region NS5B 1a (n=226) 5’NC region 1a (n=180) 1b (n=39) 1nd (n=7) 20% NS5B 1b (n=245) 5’NC region 1b (n=229) 1a (n=8) 1nd (n=8) 7% (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region 1b (n=3) 1nd (n=1) 1a (n=2) 1a (n=1) 1b (n=1) NS5B 1d (n=4) 1e (n=2) 1i (n=1) 1l (n=1) 1nd (n=3) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region 100% 5’NC region 1b (n=3) 1nd (n=1) 1a (n=2) 1a (n=1) 1b (n=1) NS5B 1d (n=4) 1e (n=2) 1i (n=1) 1l (n=1) 1nd (n=3) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region 3a (n=2) 1a (n=1) 1b (n=5) NS5B 2l (n=1) 3a (n=2) 6 (n=6) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region ? 5’NC region 1 (n=1) 3a (n=2) 1a (n=1) 1b (n=5) NS5B 2l (n=1) 3a (n=2) 6 (n=6) (Chevaliez S., et al., AASLD 2006)

Subtyping in the 5’NC Region ? 5’NC region 1 (n=1) 3a (n=2) 1a (n=1) 1b (n=5) NS5B 2l (n=1) 3a (n=2) 6 (n=6) ? (Chevaliez S., et al., AASLD 2006)

Current LiPA Assay 5’NC region probes 1a 1b 2a/b 3a 4a 5a 6a AC CC Band positions Marker AC CC 5’NC region probes 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

New LiPA Assay 5’NC region probes core region probes 1a 1b 2a/b 3a 4a 6 5’NC region probes Marker CC AC 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 23. AC 24. 6 c-l 25. 1 a 26. 1 b core region probes

New LiPA Assay Genotype Subtype 1 a, b 2 a, b, c 3 a, b, c, k 4 a, b, c, d, e, f, h 5 a 6 a, c-l

Detection and Quantification of HCV RNA

Ranges of Linear Quantification of HCV RNA Assays (IU/ml) 10 102 103 104 105 106 107 108 Cobas Amplicor HCV Monitor v2.0 Versant HCV RNA 3.0 (bDNA) Untreated hepatitis C

Advantages of Real-Time PCR Quantification Improved sensitivity No carryover contamination Extended dynamic range of quantification Precision and reproducibility

Real-Time PCR Platforms for HCV RNA Roche Molecular Systems: Cobas Taqman® HCV Test Amplification: Cobas Taqman® Extraction: Cobas Ampliprep® (CAP/CTM) Abbott Diagnostic Abbott Real-Time HCV Assay Amplification: m2000RT System Extraction: m2000SP (m2000 Real-Time PCR System)

Ranges of Linear Quantification of HCV RNA Assays (IU/ml) 10 102 103 104 105 106 107 108 Cobas Amplicor HCV Monitor v2.0 Versant HCV RNA 3.0 (bDNA) Untreated hepatitis C

Ranges of Linear Quantification of HCV RNA Assays (IU/ml) 10 102 103 104 105 106 107 108 Cobas Amplicor HCV Monitor v2.0 Versant HCV RNA 3.0 (bDNA) Cobas TaqMan HCV Test (Roche) Abbott Real-Time HCV Assay (Abbott) Untreated hepatitis C

Roche Real-Time PCR Cobas Taqman® Cobas Ampliprep® Cobas Ampliprep®/Cobas Taqman® (CAP/CTM) platform

Calibration of CAP/CTM HCV AcroMetrix HCV RNA Panel r = 0.9981; p < 0.0001 HCV RNA level in CAP/CTM (Log10 IU/ml) 6 5 4 3 2 1 7 8 Expected HCV RNA level (Log10 IU/ml) (Chevaliez et al., Hepatology 2007; in press)

Overestimation of HCV RNA Levels in CAP/CTM vs bDNA 1.5 1.0 0.5 Difference between HCV RNA levels measured in CAP/CTM and in bDNA (in Log10 UI/ml) -0.5 -1.0 -1.5 Genotype 1 Genotype 2 Genotype 3 Genotype 4 Genotype 5 (Chevaliez et al., Hepatology 2007; in press)

Overestimation of HCV RNA Levels in CAP/CTM vs bDNA 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 HCV RNA (Log10 IU/ml) Dilutions undiluted 1/5 1/25 1/125 1/625 1/3125 1/15625 1/78125 HCV genotype 1 HCV genotype 2 (Chevaliez et al., Hepatology 2007; in press)

Underestimation of HCV RNA Levels by CAP/CTM in Genotypes 2 and 4 -1.5 1.0 -1.0 1.5 0.0 0.5 -0.5 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; in press)

Underestimation of HCV RNA Levels by CAP/CTM in Genotypes 2 and 4 -1.5 1.0 -1.0 1.5 0.0 0.5 -0.5 Genotype 1 (n=29) Genotype 2 (n=27) Genotype 3 (n=29) Genotype 4 (n=30) Genotype 5 (n=9) Genotype 6 (n=2) 15% 30% (Chevaliez et al., Hepatology 2007; in press)

Abbott Real-Time PCR m2000RT

Abbott Real-Time PCR R=0,9658, p<0.0001 m2000sp (Abbott) bDNA 3.0 4.0 5.0 6.0 7.0 8.0 bDNA 3.0 m2000sp (Abbott) HCV genotype 1 (n=43) HCV genotype 3 (n=19) HCV genotype 4 (n=17) HCV genotype 5 (n=5) HCV genotype 2 (n=11) R=0,9658, p<0.0001 (Chevaliez et al., in progress)

II Treatment Algorithms

HCV Genotype Determination 1 Types 2, 3 Types 4, 5, 6

HCV GENOTYPE 1

liver disease severity HCV GENOTYPE 1 Assessment of liver disease severity

liver disease severity Follow-up without treatment HCV GENOTYPE 1 Assessment of liver disease severity Good prognosis Follow-up without treatment

liver disease severity Follow-up without treatment HCV GENOTYPE 1 Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 ≥ 2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 ≥ 2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression HCV RNA quantification at week 24

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 ≥ 2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression HCV RNA quantification at week 24 HCV RNA ≥ 50 IU/ml Stop treatment OR continue to slow liver disease progression

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 ≥ 2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression HCV RNA quantification at week 24 HCV RNA <50 IU/ml Continue treatment HCV RNA ≥ 50 IU/ml Stop treatment OR continue to slow liver disease progression

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 ≥ 2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression HCV RNA quantification at week 24 HCV RNA <50 IU/ml Continue treatment HCV RNA ≥ 50 IU/ml Stop treatment OR continue to slow liver disease progression HCV RNA detection at the end of treatment and 24 weeks later

HCV GENOTYPE 1 Bad Good prognosis prognosis Assessment of liver disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA quantification at baseline and at week 12 ≥ 2 log HCV RNA decrease or HCV RNA (-) Continue treatment < 2 log HCV RNA decrease Stop treatment OR continue to slow liver disease progression HCV RNA quantification at week 24 HCV RNA <50 IU/ml Continue treatment HCV RNA ≥ 50 IU/ml Stop treatment OR continue to slow liver disease progression HCV RNA detection at the end of treatment and 24 weeks later End-of-treatment virological response Sustained virological response

HCV GENOTYPES 2 or 3

HCV GENOTYPES 2 or 3 Peginterferon + ribavirin 0.8 g qd 24 weeks

HCV RNA detection at the end of treatment and 24 weeks later HCV GENOTYPES 2 or 3 Peginterferon + ribavirin 0.8 g qd 24 weeks HCV RNA detection at the end of treatment and 24 weeks later

HCV GENOTYPES 2 or 3 Peginterferon + ribavirin 0.8 g qd 24 weeks HCV RNA detection at the end of treatment and 24 weeks later End-of-treatment virological response Sustained virological response

HCV GENOTYPES 4, 5 or 6

serological markers of disease severity HCV GENOTYPES 4, 5 or 6 Liver biopsy or serological markers of disease severity

serological markers of disease severity Follow-up without treatment HCV GENOTYPES 4, 5 or 6 Liver biopsy or serological markers of disease severity Good prognosis Follow-up without treatment

serological markers of disease severity Follow-up without treatment HCV GENOTYPES 4, 5 or 6 Liver biopsy or serological markers of disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks

HCV GENOTYPES 4, 5 or 6 Follow-up without treatment Peginterferon Liver biopsy or serological markers of disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA detection at the end of treatment and 24 weeks later

HCV GENOTYPES 4, 5 or 6 Follow-up without treatment Peginterferon Liver biopsy or serological markers of disease severity Bad prognosis Good prognosis Follow-up without treatment Peginterferon + ribavirin 1.0-1.2 g qd 48 weeks HCV RNA detection at the end of treatment and 24 weeks later End-of-treatment virological response Sustained virological response

III Future Challenges

Future Challenges Better tailor treatment duration: Treat shorter when no more is needed Treat longer when more is needed

Shorter Treatment

ACCELERATE Trial (Genotypes 2 & 3) 100 86% 76% 80 70% 70% 77% 60 66% 60% 60% % SVR 82% 71% 16 weeks 40 65% 65% 24 weeks 20 n=346 n=303 n=333 n=327 Genotype 2 Genotype 3 (Shiffman et al., EASL 2006)

Tailored Duration in Genotypes 2/3 Standard duration group (n = 70) 24 weeks of peginterferon-ribavirin therapy SVR: 76% Variable duration group (n = 213) 12 weeks of treatment if HCV RNA negative* at week 4 24 weeks of treatment if HCV RNA positive* at week 4 SVR: 82% * < 50 IU/ml (Mangia et al., N Engl J Med 2005;352:2609-17)

Shorter Duration in Genotype 1 Patients infected with HCV genotype 1 Baseline HCV RNA level < 600,000 IU/ml PegIFN-2b + ribavirin Treatment duration: 24 weeks Comparison with historical data from the Manns’ trial (Lancet 2001) (Zeuzem et al., J Hepatol 2006;44:97-103)

Shorter Duration in Genotype 1 100 90 80 70 60 24 weeks % Sustained virological response 50 40 48 weeks (historical data from the Mann’s trial) 30 20 10 Week 4 Week 12 Week 24 Time to first negative HCV RNA (< 50 IU/ml) (Zeuzem et al., J Hepatol 2006;44:97-103)

Tailored Duration in Genotype 1 PegIFN-2a + ribavirin: 24-LD: 0.8 g/d of ribavirin, 24 weeks 24-SD: 1.0-1.2 g/d of ribavirin, 24 weeks 48-LD: 0.8 g/d of ribavirin, 48 weeks 48-SD: 1.0-1.2 g/d of ribavirin, 48 weeks (Jensen et al., Hepatology 2006;43:954-960)

Tailored Duration in Genotype 1 100 90 80 70 24-LD 60 24-SD % Sustained virological response 50 48-LD 40 30 48-SD 20 10 HCV RNA < 50 IU/ml at week 4 HCV RNA ≥ 50 IU/ml at week 4 (Jensen et al., Hepatology 2006;43:954-960)

Longer Treatment

Patients with HCV Genotypes 2/3 Older age Male gender High BMI Fibrosis ≥ F3

Longer Treatment in Genotype 1 100 90 80 70 60 % SVR 50 53% 54% 40 30 20 10 48 weeks (n=230) 72 weeks (n=225) (Berg et al., Gastroenterology 2006;130:1086-97)

Longer Treatment in Genotype 1 NS 90 NS 80 70 NS 60 48 weeks % Sustained virological response 50 40 p=0.04 72 weeks 30 20 10 Week 4 Week 12 Week 4 Week 12 HCV RNA < 50 IU/ml HCV RNA ≥ 50 IU/ml) (Berg et al., Gastroenterology 2006;130:1086-97)

Summary The rapid virologic responders may receive shorter therapy, whatever the HCV genotype The slow virologic responders may benefit from longer therapy, whatever the HCV genotype

New Algorithms Genotypes 1, 4 (5 and 6 ?) Genotypes 2 and 3 24 weeks

Ranges of Linear Quantification of HCV RNA Assays (IU/ml) 10 102 103 104 105 106 107 108 Cobas Amplicor HCV Monitor v2.0 Versant HCV RNA 3.0 (bDNA) Cobas TaqMan HCV Test (Roche) Abbott Real-Time HCV Assay (Abbott) Untreated hepatitis C

Urgent Needs Retrospective analyses of major trials (including HCV RNA retesting with more sensitive, real-time PCR-based assays) Identification of the decision thresholds with the best predictive value: Definition of the early virologic response Definition of the slow virologic response Prospective validation in randomized clinical trials +++

Conclusions (1) New virological assays are available The new LiPA assay will improve accuracy of genotype and subtype determination Real-time PCR assays are currently replacing classical methods for HCV RNA quantification: They bring: - nearly full automation - improved sensitivity - broader dynamic ranges of quantification The current commercial assays still need to be improved in order to ensure accuracy

Conclusions (2) The current algorithms use the HCV genotype and HCV RNA level monitoring: To tailor initial therapy To stop therapy in those patients unlikely to achieve a sustained virological response, in order to avoid useless costs and side-effects Future algorithms should now be established to tailor treatment duration to the genotype and the early virologic response, in order to increase cure rates