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)