Pawoltsky diagnostic
description
Transcript of Pawoltsky diagnostic
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
I
HCV Markers and Kinetics
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.
Kinetics of HCV MarkersSpontaneous resolution
Infection Acutehepatitis
anti-HCVantibodies
ALT
HCV RNA
HCV core Ag
Chronic hepatitis
anti-HCVantibodies
Kinetics of HCV MarkersPersistent infection
ALT
HCV RNA
HCV core Ag
Infection Acutehepatitis
II
HCV Virological Tools
Classical HCV Virological Tests
• Serological Assays• ELISA tests for anti-HCV antibody detection
• Molecular tests• HCV genotyping assays• HCV RNA quantification assays
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)
Anti-HCV antibody detection
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)
“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)
HCV core Ag quantification
HCV Core Antigen Quantification
(Bouvier-Alias M. et al., Hepatology 2002;36:211-8)
Architect HCV Ag AssayRVR (G1b) SVR (G1b)
Relapser (G1b) NR (G1a)
Core AgRNA
(Ross M. et al., J Clin Microbiol 2010;48:1161-8)
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)
HCV Genotype determination
HCV Genotypes
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
Versant® HCV Genotype 2.0 Assay (INNO-LiPA)
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)
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
HCV RNA Quantification
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)
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)
Real-Time PCR PlatformsCAP-CTM96 (ROCHE)
kPCR (SIEMENS)
m2000SP-m2000RT (ABBOTT)
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)
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%
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)
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)
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)
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)
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)
HCV Resistance Testing
Quasispecies Distribution of Viral Populations
Major viralpopulation
Intermediate viral populations
Minor viral populations
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
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)
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)
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)
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
(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
(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
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
III
Practical Use
RibavirinPegylated IFN- +
Standard-of-Care for HCV Genotype non-1
Standard-of-Care (EU label)
Genotype 2,3 Genotype 4, 5, 6
PegIFN + ribavirin24 weeks
PegIFN + ribavirin48 weeks
Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
On-Treatment Virologic Responses
LLOD
Baseline Week 4 Week 8 Week 12
-2 log
On-Treatment Virologic Responses
LLOD
Baseline Week 4 Week 8 Week 12
-2 log
On-Treatment Virologic Responses
LLOD
Baseline Week 4 Week 8 Week 12
-2 log
RVR24 weeks
Delayed VR72 weeks
EVR48 weeks
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)
TelaprevirBoceprevir
RibavirinPegylated IFN-
+
New Standard-of-Care for HCV Genotype 1
Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
Telaprevir
Response-Guided TherapyPeg-IFN + Ribavirin + Telaprevir
W4 W24 W48 W72W36W12W0
Treatment-naive or responder-
relapser
Partial responder or null-responder
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
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
• HCV RNA >1000 IU/mL at W4 or W12
• HCV RNA detectable (>10-25 IU/mL)at W24
Futility RulesPeg-IFN + Ribavirin + Telaprevir
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)
Futility Rule with Telaprevir
(Jacobson et al., EASL 2012)
HCV RNA Profiles in Patients with HCV RNA >1000 IU/mL at week 4
Virological Monitoring
Weeks of treatment
360 2412 48 6084 72
PegIFN-ribavirin
Telaprevir
Boceprevir
Treatment-experienced
patients (excluding null-responders and
F4)
F4 patients and null-responders
Treatment naive patients
(excluding F4)
Response-Guided TherapyPeg-IFN + Ribavirin + Boceprevir
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
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
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
• HCV RNA ≥100 IU/mL at W12
• HCV RNA detectable (>10-25 IU/mL) at W24
Futility RulesPeg-IFN + Ribavirin + Boceprevir
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)
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)
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)
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)