Pawoltsky diagnostic

69
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 Hospital University of East Paris

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Transcript of Pawoltsky diagnostic

Page 1: 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

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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)