1. Jean-Michel PAWLOTSKY

2. 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

3. 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

4. I HCV Virological Tools

5. HCV Genotype Determination

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

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

8. HCV Genotype Determination
HCV genome

9. HCV Genotype Determination
HCV genome
NS5B

10. HCV Genotype Determination
HCV genome
NS5B
5’ noncoding region

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

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

13. 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)

14. 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)

15. 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)

16. 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)

17. 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)

18. 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)

19. 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)

20. 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)

21. 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)

22. 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

23. New LiPA Assay 5’NC region probes core region probes 1a 1b 2a/b 3a 4a6
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
c-l a b
core region probes

24. 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

25. Detection and Quantification of HCV RNA

26. 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

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

28. 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)

29. 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

30. 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

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

32. Calibration of CAP/CTM HCV AcroMetrix HCV RNA Panel
r = ; p <
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)

33. 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)

34. 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)

35. 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)

36. 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)

37. Abbott Real-Time PCR
m2000RT

38. 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)

39. II Treatment Algorithms

40. HCV Genotype Determination1
Types
2, 3
Types
4, 5, 6

41. HCV GENOTYPE 1

42. liver disease severity
HCV GENOTYPE 1
Assessment of
liver disease severity

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

44. 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 g qd
48 weeks

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

46. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

47. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

48. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

49. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

50. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

51. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

52. HCV GENOTYPE 1 Bad Good prognosis prognosis
Assessment of
liver disease severity
Bad
prognosis
Good
prognosis
Follow-up without treatment
Peginterferon
+ ribavirin 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

53. HCV GENOTYPES 2 or 3

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

55. 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

56. 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

57. HCV GENOTYPES 4, 5 or 6

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

59. 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

60. 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 g qd
48 weeks

61. 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 g qd
48 weeks
HCV RNA detection at the end of
treatment and 24 weeks later

62. 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 g qd
48 weeks
HCV RNA detection at the end of
treatment and 24 weeks later
End-of-treatment virological response
Sustained virological response

63. III Future Challenges

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

65. Shorter Treatment

66. 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)

67. 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: )

68. 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)

69. 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)

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

71. 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: )

72. Longer Treatment

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

74. 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: )

75. Longer Treatment in Genotype 1NS 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: )

76. 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

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

78. 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

79. 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 +++

80. 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

81. 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