pawoltsky diagnostic
DESCRIPTION
TRANSCRIPT
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HCV Diagnostic Strategies & Monitoring
Prof. Jean-Michel Pawlotsky, MD, PhD
National Reference Center for Viral Hepatitis B, C and delta
Department of Virology & INSERM U955
Henri Mondor HospitalUniversity of East Paris
Créteil, France
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I
HCV Markers and Kinetics
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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.
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Kinetics of HCV MarkersSpontaneous resolution
Infection Acutehepatitis
anti-HCVantibodies
ALT
HCV RNA
HCV core Ag
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Chronic hepatitis
anti-HCVantibodies
Kinetics of HCV MarkersPersistent infection
ALT
HCV RNA
HCV core Ag
Infection Acutehepatitis
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II
HCV Virological Tools
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Classical HCV Virological Tests
• Serological Assays• ELISA tests for anti-HCV antibody detection
• Molecular tests• HCV genotyping assays• HCV RNA quantification assays
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New HCV Virological Tests
• New serological tests• Quantitative core antigen detection (ELISA)
• Molecular tests• Real-time PCR assays for HCV RNA level
quantification• Ultra-deep pyrosequencing (resistance)
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Anti-HCV antibody detection
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Anti-HCV Antibody Detection• Based on ELISA
• Easy to use, automated
• 3rd-generation tests available• ADVIA Centaur (Siemens)• VITROS ECi (Ortho-Clinical Diagnostics)• AXSYM HCV 3.0 (Abbott)• Cobas Elecsys Modular HCV (Roche)• INNOTEST HCV Ab IV (Innogenetics)• Monolisa anti-HCV Plus version 2 (Bio-Rad)
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“Combo“ Tests (Ag + Ab)• Based on ELISA
• Commercially available
• Reduce the serological window during acute infection by 20-30 days
• No benefit in the diagnostic setting
• No benefit in blood screening in the context of Nucleic acid testing (NAT)
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HCV core Ag quantification
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HCV Core Antigen Quantification
(Bouvier-Alias M. et al., Hepatology 2002;36:211-8)
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Architect HCV Ag AssayRVR (G1b) SVR (G1b)
Relapser (G1b) NR (G1a)
Core AgRNA
(Ross M. et al., J Clin Microbiol 2010;48:1161-8)
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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 500-3000 IU/mL according to the HCV genotype)
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HCV Genotype determination
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HCV Genotypes
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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
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Versant® HCV Genotype 2.0 Assay (INNO-LiPA)
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HCV Genotype 1 Subtype Determination
1st Generation of Line Probe Assay
(LiPA 1.0)
2nd Generation of Line Probe Assay
(LiPA 2.0)
RealTime HCV Genotype II
Sequence Analysis of the
5’NCR
GT 1a(n=237)
GT 1b(n=263)
77.6%(n=184)
90.5%(n=238)
70.5%(n=167)
91.3%(n=240)
97.5%(n=231)
96.2%(n=253)
93.2%(n=220)
88.6%(n=233)
(Chevaliez et al., PLoS One 2009;4:e820)
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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
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HCV RNA Quantification
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Linear Ranges of Quantification10 102 103 104 107 108
Cobas AmplicorHCV Monitor v2.0
SuperQuant
LCx HCV RNA Assay
Versant HCV RNA3.0 (bDNA)
105 106
(Chevaliez et al., Gastroenterology 2012;142:1303-13)
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Linear Ranges of Quantification10 102 103 104 107 108
Cobas AmplicorHCV Monitor v2.0
SuperQuant
LCx HCV RNA Assay
Versant HCV RNA3.0 (bDNA)
CTM HCV test v2.0 (Roche)
RealTime™ HCV(Abbott)
Artus HCV QS-RGQ (Qiagen)
CAP/CTM HCV test, v2.0 (Roche)
Versant HCV RNA1.0 (kPCR, Siemens)
105 106
(Chevaliez et al., Gastroenterology 2012;142:1303-13)
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Real-Time PCR PlatformsCAP-CTM96 (ROCHE)
kPCR (SIEMENS)
m2000SP-m2000RT (ABBOTT)
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CAP/CTM HCV Assay
HCV RNA level in Versant HCV 3.0 Assay bDNA(Log10 UI/mL)
Genotype 1 (n=29)
Genotype 2 (n=27)
Genotype 3 (n=29) Genotype 4 (n=30)
Genotype 5 (n=9)
Genotype 6 (n=2)
r = 0.889; p < 0.0001
8
7
6
5
4
3876543
(Chevaliez et al., Hepatology 2007;46:22-31)
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Genotype 1 (n=29)
Genotype 2 (n=27)
Genotype 3 (n=29)
Genotype 4 (n=30)
Genotype 5 (n=9)
Genotype 6 (n=2)
-1.5
1.0
-1.0
1.5
0.0
0.5
-0.5
Underestimation of HCV RNA Levels by CAP/CTM in Genotypes 2 and 4
(Chevaliez et al., Hepatology 2007;46:22-31)
15% 30%
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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)
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r = 0.9581; p < 0.0001
3 4 5 6 7 83
4
5
6
7
8H
CV
RN
A le
vel i
n C
AP/
CTM
48 v
2.0
(Log
10 IU
/mL)
HCV RNA level in Versant HCV 3.0 bDNA Assay(Log10 IU/mL)
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)
Genotype 4 Quantification with CAP/CTM v2.0 (Roche)
(Chevaliez et al., J Clin Microbiol 2013; in press)
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CAP/CTM v1.0 vs v2.0 (Roche)
CAP/CTM96 v1.0(Log10 IU/mL)
CAP/CTM96 v2.0(Log10 IU/mL)
m2000SP/m2000RT
(Log10 IU/mL)bDNA 3.0
(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 Clin Microbiol 2013; in press)
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Abbott Real-Time PCR
3.0 4.0 5.0 6.0 7.0 8.0
bDNA 3.0
m20
00sp
(Abb
ott)
3.0
4.0
5.0
6.0
7.0
8.0
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., J Clin Microbiol 2009;47:1726-32)
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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)
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HCV Resistance Testing
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Quasispecies Distribution of Viral Populations
Major viralpopulation
Intermediate viral populations
Minor viral populations
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Viral Sequence Analysis ToolsMajor 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
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Available NGS TechniquesManufacturer Sequencing
device
Technology(template
preparation/NGS chemistry)
TypeNumber of
single reads per run*
(x 106)
Number of nucleotides
per run*
(Gb)
Maximum sequence
length* (bp)Accuracy
Applied Biosystems
5500emPCR/ligation
High throughput 800 9 75
99.6-99.8%5500xl High
throughput 1600 15 75
Ion Torrent (ChiP 316)
emPCR/RTsequencing Long reads 6.2 >1 >400 99.97%
Illumina
MiSeq
Solid capture/reversible terminator
High throughput 3.4 >1 150
96.7-100%
Genome Analyzer IIx
High throughput 320 95 150
HiSeq 1000 High throughput 1500 300 100
HiSeq 2000 High throughput 3000 600 100
454 /RocheGS Junior
emPCR/pyrosequencing
Long reads 0.1 0.035 400 99%
GS FLX+ Long reads 1 0.7 1000 97.4-99.9%
PacBio/Gen-Probe PacBio RS
Single molecule/RTseq
uencingLong reads 0.035 0.045 1200 99.99%
(Chevaliez S, Rodriguez C & Pawlotsky JM, Gastroenterology 2012;142:1303-13)
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Available NGS TechniquesManufacturer Sequencing
device
Technology(template
preparation/NGS chemistry)
TypeNumber of
single reads per run*
(x 106)
Number of nucleotides
per run*
(Gb)
Maximum sequence
length* (bp)Accuracy
Applied Biosystems
5500emPCR/ligation
High throughput 800 9 75
99.6-99.8%5500xl High
throughput 1600 15 75
Ion Torrent (ChiP 316)
emPCR/RTsequencing Long reads 6.2 >1 >400 99.97%
Illumina
MiSeq
Solid capture/reversible terminator
High throughput 3.4 >1 150
96.7-100%
Genome Analyzer IIx
High throughput 320 95 150
HiSeq 1000 High throughput 1500 300 100
HiSeq 2000 High throughput 3000 600 100
454 /RocheGS Junior
emPCR/pyrosequencing
Long reads 0.1 0.035 400 99%
GS FLX+ Long reads 1 0.7 1000 97.4-99.9%
PacBio/Gen-Probe PacBio RS
Single molecule/RTseq
uencingLong reads 0.035 0.045 1200 99.99%
(Chevaliez S, Rodriguez C & Pawlotsky JM, Gastroenterology 2012;142:1303-13)
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Ultra-Deep Pyrosequencing
Viral genome
extraction from serum
RT PCR amplification
emPCR Pyrosequencing
PyroMute® PyroDyn®
% of each mutations
Analysis
Data collection
PyroClass®
PyroClass©. PyroMute©, PyroDyn©, PyroLink© are protected under IDDN
Modeling of population growth
Sequence quality filters
PyroLink®
Linkages
Classificationof generated sequences
(Rodriguez C. et al., in revision)
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Pre-existing HCV Resistant Variants by UDPS
PatientIL28B
genotype
HCV subtype
pegIFN
RBV
TVR
Response
V36A/M
T54A/S V55A Q80
R/KR155
K/T/QA156S/T/V
D168A/V/T/H
I170A/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%
*SNP rs12979860
(Chevaliez S., et al., manuscript in preparation)
SVR: sustained virological response; RR: response-relapse; NR: non-response
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(Chevaliez S., et al., EASL 2011)
0
57
0%
20%
40%
60%
80%
100%
H28Q+R155K
H28Q+R155K+S54T+Y52C
H28Q+R155K+S54T+Y52C+V36M+H57L+P96H
0
2
4
6
8
Days of therapy
% o
f var
iant
s in
the
quas
ispe
cies
*PyroLink®
TVR + PegIFN
HCV
RNA(
Log 10
IU/m
L)
Viral populations
Treatment Failure-PROVE2
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(Chevaliez S., et al., EASL 2011)HC
V RN
A (L
og10
IU/m
L)
0
2
4
6
8
Days of treatment
% o
f mut
ation
s in
the
who
le q
uasi
spec
ies
*PyroLink®
0
29
57
85
182
595
686
903
0%
20%
40%
60%
80%
100% H28Q+R155K
H28Q+R155K+S54T+Y52C
H28Q+R155K+S54T+Y52C+V36M+H57L+P96H
V36M+R155K+H57L
R155K
% o
f var
iant
s in
the
quas
ispe
cies
Days of therapy
HCV
RNA
(Log
10 IU
/mL)
Viral populations
Treatment Failure-PROVE2
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UDPS in HCV Resistance
• Novel technologies for the study of HCV resistance, such as ultra-deep pyrosequecing, will bring new insights into its molecular mechanisms and may have clinical utility in the future
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III
Practical Use
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RibavirinPegylated IFN- +
Standard-of-Care for HCV Genotype non-1
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Standard-of-Care (EU label)
Genotype 2,3 Genotype 4, 5, 6
PegIFN + ribavirin24 weeks
PegIFN + ribavirin48 weeks
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Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
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On-Treatment Virologic Responses
LLOD
Baseline Week 4 Week 8 Week 12
-2 log
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On-Treatment Virologic Responses
LLOD
Baseline Week 4 Week 8 Week 12
-2 log
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On-Treatment Virologic Responses
LLOD
Baseline Week 4 Week 8 Week 12
-2 log
RVR24 weeks
Delayed VR72 weeks
EVR48 weeks
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24 vs 48 Weeks in Genotype 2/3 Patients Without an RVR (N-Core)
80
70
60
50
40
30
20
10
0ITT
(n=188)PP
(n=176)Study
completers (n=153)
Peg-IFN alfa-2a/RBV 24 weeks
Peg-IFN alfa-2a/RBV 48 weeks
SVR
24 (%
of p
atie
nts)
4995
5793
4995
5181
4995
4663
52%
61%
52%
63%
54%
73%p=0.19 p=0.14 p=0.02
(Cheinquer et al., AASLD 2012)
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TelaprevirBoceprevir
RibavirinPegylated IFN-
+
New Standard-of-Care for HCV Genotype 1
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Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
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Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
Telaprevir
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Response-Guided TherapyPeg-IFN + Ribavirin + Telaprevir
W4 W24 W48 W72W36W12W0
Treatment-naive or responder-
relapser
Partial responder or null-responder
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Response-Guided TherapyPeg-IFN + Ribavirin + Telaprevir
W4 W24 W48
Follow-up: 24 weeks
Follow-up: 24 weeks
TVR + PR
PR
eRVR: undetectable HCV RNA at weeks 4 and 12
HCV RNA detectable at weeks 4 and/or 12 but ≤1000 UI/mL
W72
PR
W36W12W0
Treatment-naive or responder-
relapser
Partial responder or null-responder
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Response-Guided TherapyPeg-IFN + Ribavirin + Telaprevir
W4 W24 W48
Follow-up: 24 weeks
Follow-up: 24 weeks
TVR + PR
PR
eRVR: undetectable HCV RNA at weeks 4 and 12
HCV RNA detectable at weeks 4 and/or 12 but ≤1000 UI/mL
W72
PR
W36W12
Follow-up: 24 weeksPRTVR + PR
W0
Treatment-naive or responder-
relapser
Partial responder or null-responder
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• HCV RNA >1000 IU/mL at W4 or W12
• HCV RNA detectable (>10-25 IU/mL)at W24
Futility RulesPeg-IFN + Ribavirin + Telaprevir
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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 =100-1000 IU/mL (2.0%): SVR: 22%
HCV RNA at week 12 >1000 IU/mL (1.5%): SVR: 0% HCV RNA at week 12 =100-1000 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
(Jacobson et al., EASL 2012)
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Futility Rule with Telaprevir
(Jacobson et al., EASL 2012)
HCV RNA Profiles in Patients with HCV RNA >1000 IU/mL at week 4
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Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
Telaprevir
Boceprevir
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Treatment-experienced
patients (excluding null-responders and
F4)
F4 patients and null-responders
Treatment naive patients
(excluding F4)
Response-Guided TherapyPeg-IFN + Ribavirin + Boceprevir
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W0 W4 W8 W12 W24 W28 W36 W48
BoceprevirUndetectable HCV RNA at week 8
PegIFN/RBV
PegIFN/RBVDetectable HCV RNA at week 8
Treatment-experienced
patients (excluding null-responders and
F4)
F4 patients and null-responders
Treatment naive patients
(excluding F4)
Boceprevir + PegIFN/RBV
Boceprevir + PegIFN/RBV
Response-Guided TherapyPeg-IFN + Ribavirin + Boceprevir
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W0 W4 W8 W12 W24 W28 W36 W48
BoceprevirUndetectable HCV RNA at week 8
PegIFN/RBV
PegIFN/RBVDetectable HCV RNA at week 8
BoceprevirW4W0 W12 W24 W36 W48
Boceprevir + PegIFN/RBVPegIFNRBV PegIFN/RBV
Treatment-experienced
patients (excluding null-responders and
F4)
F4 patients and null-responders
Treatment naive patients
(excluding F4)
Boceprevir + PegIFN/RBV
Boceprevir + PegIFN/RBV
Response-Guided TherapyPeg-IFN + Ribavirin + Boceprevir
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W0 W4 W8 W12 W24 W28 W36 W48
BoceprevirUndetectable HCV RNA at week 8
PegIFN/RBV
PegIFN/RBVDetectable HCV RNA at week 8
BoceprevirW4W0 W12 W24 W36 W48
Boceprevir + PegIFN/RBVPegIFNRBV PegIFN/RBV
W0 W4 W12 W24 W48
Boceprevir + PegIFN/RBVPegIFNRBV
Boceprevir
Treatment-experienced
patients (excluding null-responders and
F4)
F4 patients and null-responders
Treatment naive patients
(excluding F4)
Boceprevir + PegIFN/RBV
Boceprevir + PegIFN/RBV
Response-Guided TherapyPeg-IFN + Ribavirin + Boceprevir
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• HCV RNA ≥100 IU/mL at W12
• HCV RNA detectable (>10-25 IU/mL) at W24
Futility RulesPeg-IFN + Ribavirin + Boceprevir
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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)
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Treatment Failures on Triple Combination with a DAA
• Due to an inadequate response to Peg-IFN and ribavirin
• Results in uncontrolled outgrowth of resistant HCV variants selected by the protease inhibitor
(Pawlotsky JM. Hepatology 2011;53:1742-51)
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SVR According to Lead-in (SPRINT-2, non-black)
29%
39%
82%
% o
f pat
ient
s w
ith S
VR
0
10
20
30
40
50
60
70
80
90
100
BOC/RGT BOC/PR48
82%
<1 log HCV RNA decrease
≥1 log HCV RNAdecrease
(Poordad et al., N Engl J Med 2011;364:1185-206)
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HCV Resistance Testing• Prior to therapy:
• There is no indication for resistance testing at baseline• All patients should be considered as harboring minor viral
populations that are resistant to telaprevir and boceprevir
• In case of treatment failure:• There is no indication for resistance testing during and after
therapy, as the result will have no impact on treatment decisions• Protease inhibitor-resistant viral populations have been enriched
in every patient treated with telaprevir or boceprevir who did not clear infection
• Resistance testing is required in clinical trials and global surveillance studies (research setting)