What do we know about RAVs today

Francesca Ceccherini-SilbersteinUniversità degli Studi di Roma “Tor Vergata”

Cattedra di Virologia

Disclosures: Advisory/Consulting/Conferences/Grants: Gilead, BMS, AbbVie, Roche Diagnostics, MSD, BMS, Janssen, Abbott Molecular, ViiV

RASsResistance associatedsubstitutions

RAVsResistance associated variants

Pawlotsky JM, Gastroenterology 2016 and EASL 2016

1991 20010

20

40

60

80

100

8-12

SV

R (%

)

15-20

38-43

25-30

50-60

1995 1998

Standard interferon (6 mos)[1]

Standardinterferon

(12-18 mos)[2,3]

Interferon/ribavirin

(6-12 mos)[3,4] PegIFNmonotherapy(6-12 mos)[5,6]

PegIFN/ribavirin(6-12 mos)[6,7]

2011-2013

70-80

PI + PegIFN/RBV(6-12 mos)[8-18]

1. Carithers RL Jr., et al. Hepatology. 1997;26(3 suppl 1):83S-88S. 2. Zeuzem S, et al. N Engl J Med. 2000;343:1666-1672. 3. Poynard T, et al. Lancet. 1998;352:1426-1432. 4. McHutchison JG, et al. N Engl J Med. 1998;339:1485-1492. 5. Lindsay KL, et al. Hepatology. 2001;34:395-403. 6. FriedMW, et al. N Engl J Med. 2002;347:975-982. 7. Manns MP, et al. Lancet. 2001;358:958-965. 8. Poordad F, et al. N Engl J Med. 2011;364:1195-1206. 9. Jacobson IM, et al. N Engl J Med. 2011;364:2405-2416. 10. Sherman KE, et al. N Engl J Med. 2011;365:1014-1024. 11. Jacobson IM, et al.64th Annual Meeting of the American Association for the Study of Liver Diseases, 1-5 November 2013, Washington, DC. 12. Zeuzem S, et al. Gastroeneterology 2014;146:430-41. 13. Lawitz E, et al. Gastroenterology 2013;144:S-151. 14. Jensen D, et al. 64th Annual Meeting of the AmericanAssociation for the Study of Liver Diseases, 1-5 November 2013, Washington, DC. 15. Jacobson I, et al. 64th Annual Meeting of the American Association for the Study of Liver Diseases, 1-5 November 2013, Washington, DC. 16. Marcellin P, et al. Gastroenterology 2013;145:790-800e3. 17.Bronowicki JP, et al. Antiviral Ther 2013;18:885-93. 18. Manns MP, et al. Hepatology 2012;56:884-93. 19. Hezode C, et al. Hepatology 2012;56:553A- 4A. 20. Dore G, et al. J Hepatol 2013;58:S570-1. 21. Lawitz E, et al. Lancet Infect Dis 2013;13:401-8. 22. Kowdley KV, et al. Lancet2013;381:2100-7. 23. Lawitz E, et al. 64th Annual Meeting of the American Association for the Study of Liver Diseases. Washington, DC, 1-5 November 2013. 24. Lawitz E, et al. N Engl J Med 2013;368:1878-87. 25. Jacobson IM, et al. N Engl J Med 2013;368:1867-77. 26. Zeuzem S, et al. NEngl J Med 2014;370:1993-2001. 27. Osinusi A, et al. JAMA 2013;310:804-11. 28. Jacobson IM, et al. 64th Annual Meeting of the American Association for the Study of Liver Diseases. Washington, DC, 1-5 November 2013. 29. Sulkowski MS, et al. N Engl J Med 2014;370:211-21. 30. Zeuzem S,et al. N Engl J Med 2014;370:1889-9. 31. Afdhal N, et al. N Engl J Med 2014;370:1483-9. 32. Feld JJ, et al. N Engl J Med 2014;370:1594-603. 33. Zeuzem S, et al. N Engl J Med 2014;370:1604-14. 34. Ferenci P, et al. N Engl J Med 2014;370:1983-9. 35. Poordad F, et al. N Engl J Med2014;370:1973-82. 36. Lawitz E, et al. 64th Annual Meeting of the American Association for the Study of Liver Diseases, Washington, DC, 1-5 November 2013. 37. Gane EJ, et al. Gastroenterology 2014;146:736-43e1.

IFN-free DAA +/- RBV(3-6 mos) [24-37]

2012-2014

83-9689-100

1 DAA +PegIFN/RBV

(3-6-12 mos) [19-24]

1989HCV

identified

1992HCV

Blood Test

2005HCV in-vitro

Culture

1999HCV

Replicon

1974Mystery

virus

Changes in standard of care for HCV, and improvements in numbers of sustained virological responses

hivforum.org

31%–33% nucleotide difference among the 7 known HCVgenotypes and 20%–25% among the nearly 67 HCV subtypes(Smith et al., 2014).

HCV genetic variability is higher than HIV’s and HBV’s

0% 25%Amino acid variability:

47% amino acids of HCV PROTEASE NS3 are conserved

among all HCV-genotypes

55% amino acids of HCVPOLYMERASE NS5B are conserved

among all HCV-genotypes

0% 25%Amino acid variability:

46% amino acids of HCVNS5A are conserved

among all HCV-genotypes

Cento et al., PLoS ONE 2012 Love et al., J Vir 2009 Di Maio et al., AAC 2014

Today only few DAAs are pangenotypic…….

What about today in the era of new DAAs?Still genotype important predictor for response?

(Grazoprevir) Daclatasvir Sofosbusvir

FROM CURRENT STATUS TO OPTIMIZATION OF HCV TREATMENT: RECOMMENDATIONS FROM AN EXPERT PANEL

Craxi` A*, Perno CF*, Vigano` M, Ceccherini-Silberstein F, Petta S, and The AdHoc (Advancing Hepatitis C for the Optimization of Cure) Working Party: AlbertiA, Aghemo A, Andreone P, Andreoni M, Bonora S, Brunetto MR, Bruno S, Caporaso N, Chirianni A, Ciancio A, Degasperi E, Di PerriG, Fagiuoli S, Gaeta GB, Ferrari C, Pellicelli A, Puoti M, Raimondo G, Taliani G, Villa E , Zignego AL Submitted 2016

HCV genotype dictates the choice of anti-HCV drugs

and can modulate the duration of treatment in infected patients

with chronic hepatitis C

HIV versus HCVBaseline resistance testing

• HIV treatment failure expected at

First documentation of a transmission of an HCV DAA resistant variant from a DAA treated patient to his sexual HIV-infected partner

Patient A, a man chronically infected with HCV genotype 1a and co-infected with HIV- 1, treated with pegIFN/RBV plus telaprevir in July2012 with HCV breakthrough. HCV NS3 protease sequences beforetreatment with telaprevir did not have anymajor substitution associated with NS3 PIs.

Patient B, a man also HIV-1 infected and sexual partner of patient A, diagnosed of acute HCV co-infection in January 2011, with HCV genotype 1a. This patient refused therapy with pegIFN/RBV during the acute phase of HCV infection.In April 2012 he entered a clinical HCV trial and was treated for 24 weeks with pegIFN/RBV plus Daclatasvir, with undetectable HCV RNA at week 24 and 36 after the end of treatment. However, at week 48 after stopping therapy, presented elevated transaminase and detectable HCV RNA, suggesting a HCV re-infection. The patient denied any known riskfor HCV infection except unprotected sexualintercourse with his partner (patient A).After 12 weeks, patient B tested negative for HCV infection.

108 and 28 individualNS3 protease clonesfrom patient A and B early samples, respectively, weresequenced

Franco et al Gastroenterology May 2014

V36M substitution detectedin 9/20 clones in patient A (20 weeks off therapy) and in all late sequencesfrom patient B.

Virological issues in the DAAs EraAfter treatment failure: useful / recommended the

resistance test?

[…] Currently, there is no data to firmly supportretreatment recommendations, which must be based onindirect evidence (HCV genotype, known resistanceprofiles of the administered drugs, number of drugs used,use of ribavirin, treatment duration). Whether assessing thesequence of the target HCV genes (HCV resistance testing)prior to retreatment is helpful to make a decision remainsunknown, as well as which therapeutic decision should bemade based on this result.

For patients with cirrhosis or other patients who requireretreatment urgently, testing for RAVs that conferdecreased susceptibility to NS3 protease inhibitors (eg,Q80K) and to NS5A inhibitors should be performed usingcommercially available assays prior to selecting the nextHCV treatment regimen.

Update of December 11, 2015

Role of RAVs after treatment failure to DAAs

FROM CURRENT STATUS TO OPTIMIZATION OF HCV TREATMENT: RECOMMENDATIONS FROM AN EXPERT PANEL Craxi` A, et al Subitted 2016

Statements• Baseline resistance testing for RAVs before first course with DAAs has limited clinical utility, unless if recommended by specific drug labels.• In case of baseline presence of NS5A RAVs or Q80K, the search of other baseline NS3 andNS5a RAVs might be useful to personalize therapy, and should encourage the use of at least2 active DAAs without resistance and/or longer treatment duration and/or the addition ofRbv.• Resistance testing after treatment failure in all 3 genes (NS3, NS5A, NS5B [for the twodifferent classes of nucleoside and non- nucleoside inhibitors] independently of the failure regimen) is strongly recommended in order to optimize retreatment strategy*.• HCV sequencing can be based on Sanger population method and should also confirm theprevious genotype and subtype assignment.• According to resistance results, current re-treatment strategies for patients failing a firstcourse with DAAs should include at least 2 active drug classes, with a preferential use ofone drug with high genetic barrier to resistance, and with extended treatment durationsand addition of Rbv, otherwise waiting for better future options.• If a deferred treatment has been considered, and in case of presence of RAVs at failure, inorder to assist the therapeutic choice when starting a re-treatment, HCV sequencing shouldbe repeated (only in the gene with previous RAVs) and should be better based on deepsequencing.* NOTE: It would be desirable to preserve a sample before starting treatment with DAA, because in case of failure and presence of RAVs, the study of the baseline sample may help to distinguish if the resistance occurred during failure or it was already present as natural resistance before treatment. This information may help to set to the best the next regimen.

Hepatitis C Virus Resistance to Direct-Acting Antiviral Drugs in Interferon-Free Regimens

Pawlotsky JM: Gastroenterology 2016

Should we be worried about baseline RAVs?SVR rates to NS3 protease inhibitorplus NS5A inhibitor combinationregimens in HCV genotype 1infected patients according to thepresence of baseline RAVs.

SVR rates for NS3 protease inhibitorplus nucleos(t)ide NS5B inhibitorcombination regimens in HCVgenotype 1 infected patientsaccording to the presence of baselineRAVs.

SVR rates NS5A inhibitor plusnucleos(t)ide NS5B inhibitor DAAcombination regimens in HCVgenotype 1 infected patientsaccording to the presence ofbaseline RAVs.

Sarrazin C et al., J Hepatol 2015

Rong L et al., Sci Transl Med 2010

It has been predicted that every nucleoside of the 3.2 kb HBV genome or the 10 kb HIV and HCV genomes theoretically can be substituted every day within a given infected patient

Mutations occur frequently during the HCV replication

Smith et al., 2014

Overall prevalence of Q80K in G1 across different regions

6%

19%

All G1 G1a

Europe

34%

48%

All G1 G1a

North America

3%9%

All G1 G1a

South America

13.7% of patients (274/2007) all HCV G1 29.5% (269/911) of those with HCV GT1a and 0.5% (5/1096) of those with HCV GT1b

Update from Sorbo MC et al., ICAR 2015

Sarrazin C et al., Antivir Res 2015

HCV GT-1a(N=267)

HCV GT-1b(N=352)

16.1%

1.1%

Jacobson et al. Presented at AASLD 2013Lenz et al. Presented at AASLD 2013

Q80K: 34.7% in subtype 1a HCV German treatment-naïve pts (N=170)

Dietz et al., PLoSOne 2016

Italy our data

The prevalence of pre-treatment NS5A RAVs in GT-1 is differentacross different countries, ranging from 6% to 25%, and differentaccording to subtype…..

The analysis of >3000 GT-1 NS5A sequences form 14 countries showed a high prevalence of baseline Y93H mutation (associated with resistance to daclatasvir 100 fold) in GT -1 b infected patients, ranging from 7% to 15%.

Svarovskaia E.S., EASL 2015

The Italian experience: the prevalence of patients with at least 1 natural NS5A RAV

is different according to genotype and subtype

Cento V, 14th European Meeting on HIV & Hepatitis - 2016

M28V: 3.7%L31M: 4.3%Y93HC: 1000)

23/26 86/91 32/34 164/169 17/20 113/113 14/14 44/44

12 Weeks 12 Weeks 24 Weeks 24 WeeksSarrazin et al., EASL 2015

Impact of baseline NS5A RAVs can be reduced/eliminated by changing the regimen (longer duration of treatment, inclusion of

RBV) in patients with compensated cirrhosis treated with Ledipasvir/Sofosbuvir

• 18% (94/511) cirrhotic patients had BL RAVs

Chart1

LDV/SOFLDV/SOF

LDV/SOF+RBVLDV/SOF+RBV

LDV/SOFLDV/SOF

LDV/SOF+RBVLDV/SOF+RBV

With RAVs

No RAVs

SVR12 (%)

88

95

94

97

85

100

100

100

Sheet1

With RAVsNo RAVs

LDV/SOF8895

LDV/SOF+RBV9497

LDV/SOF85100

LDV/SOF+RBV100100

LDV/SOF ± RBV: SVR12 in GT 1 Patients With Cirrhosis ± Baseline NS5A RAVs

Sarrazin et al., EASL 2015

Different impact according to HCV-1 subtype……

… Even in GT 1 and GT 4 patients with advanced liver disease or post liver transplant (SOLAR-1 and 2)

Charlton M et al., EASL 2016

Impact of baseline NS5A RAVs can be reduced/eliminated by longer duration of treatment in patients

treated with Ledipasvir/Sofosbuvir

GT1 a (N=376) & GT1b (N=211) GT 4 (N=35)

Baseline NS5B RAVs did not impact SVR12 rates

At time of relapse, NS5A RAVs were detected in most (91%) patients, whereas NS5B RAVs were uncommon (16%; no S282T, L159F and V321A; E237G was detected in 3 patients)

Baseline resistance polymorphisms

• NS5A: -28, -30, -31, or -93 polymorphisms detected in 22 of 112 patients• 82% (18/22) SVR in patients with NS5A polymorphisms

• 10/14 (71%) in cirrhosis cohort; 8/8 in post-transplant cohort

• 90% (81/90) SVR12 in patients without NS5A polymorphisms• 39/45 (87%) in cirrhosis cohort; 42/45 in post-transplant cohort

• No NS5B-S282 variants detected at baseline or failure

Daclatasvir + Sofosbuvir + Ribavirin Combination for HCV Patients with Advanced Cirrhosis or Post-transplant Recurrence:

ALLY-1 Phase 3 Study

Poordad F et al EASL 2015

HCV resistance to the daclatasvir/sofosbuvir combination across different HCV genotypes in the real life

Impact of preexisting HCV NS5A and/or NS5B resistance-associated variants (RAVs) on treatment outcomes in a cohort of 177 patients infected with HCV genotypes (GT) 1a (N=44), 1b (N=63), 3 (N=29) and 4 (N=41) receiving SOF plus DCV without ribavirin for 12 weeks.

44% of patients were cirrhotic.

Prior to treatment, NS5A RAVs were detected at a frequency of 9% in GT1a-, 32% in GT1b-, 21% in GT3-and 10% in GT4-infected patients. The most frequent RAVs were Y93H in GT1b (11%) and GT3 (3.5%). No NS5B S282T, L320F or V321A variants were detected, while L159F was found in12% of GT1b patients at baseline Fourati S et al., CROI 2016

Although the number of patients with baseline variants other than Q80K was small, there was no difference in SVR24 rates among GT1a-infected patients with any of these variants at baseline as compared to

patients with the reference amino acid residue at the corresponding position

Observed data SVR24 ratea in the presence of baseline variants

Krishnan P et al., AAC 2015

Zeuzem S et al., Ann Intern Med. 2015

For HCV ……….already more than 100 RASs…

HCV DRAG, Forum for Collaborative HIV Research, Lontok et al Hepatology 2015

1a – red 1b – blue 2 – brown 3a – green 4 – orange

Detection10%

Carefully…..Different WT codons in different HCV genotypes /subtypes…..

Sorbo MC et al manuscript in preparation RAS from Lontok et al 2015

1a – red 1b – blue 2 – brown 3a – green 4 – orangeHCV-1b1: D90208; HCV-1b2: AJ238799*; HCV-4a1:Y11604; HCV-4a2:NC_009825*

Carefully…..Different WT codons in different HCV genotypes /subtypes…..

Sorbo MC et al manuscript in preparation

1a – red 1b – blue 2 – brown 3a – green 4 – orange

RAS from Lontok et al 2015

HCV-1b1: D90208; HCV-1b2: AJ238799*; HCV-4a1:Y11604; HCV-4a2:NC_009825*

Not all RASs are Equal…..Fold-change in EC50 1a 1b

Position M28T Q30R L31M/V Y93H/N L31V Y93H/N

Ledipasvir 20x >100x >100x/>100x>1,000x/>10,000 20X >100x/?

Ombitasvir >1000x >100x10,000x/

>10,000x 100x

Daclatasvir >100x >1000x >100x/>1000x>1,000x/>10,000x 100x>10x >1,000x/

>1,000x 100x/-->100x

Velpatasvir

Huang et al., CROI 2016

Open and filled symbols depict viruses with or without mixtures, respectively

Beware of HCV-genotype for NS5A resistance ...

Nakamoto S., WJG 2014

SVR rates were reduced in GT-3 patients with natural NS5A

RAVs treated with grazoprevir, MK-3682 (NS5B), and MK-8408 (NS5A inhibitor) for 8

weeks

Gane EJ, AASLD 2015

ASTRAL-3: phase 3 studyof SOF + VEL for 12 weeks

in GT 3 patients

SVR12 84% (21/25) in patients

with Y93H

Mangia A, AASLD 2015

SVR rates were reduced in GT-3 patients with natural NS5A

RAVs treated with grazoprevir, MK-3682 (NS5B), and MK-8408 (NS5A inhibitor) for 8

weeks

Gane EJ, AASLD 2015

ASTRAL-3: phase 3 studyof SOF + VEL for 12 weeks

in GT 3 patients

SVR12 84% (21/25) in patients

with Y93H

Mangia A, AASLD 2015

Better results if longer duration of treatment and/or inclusion of RBV?

Kwo PY, EASL 2016

SVR rates were NOT reduced in GT-3 non-cirrhotic patients

with natural NS5A RAVstreated with ABT-493 + ABT-530

co-administered for 8 weeks

Muir AJ, EASL 2016

100 % SVR in GT-3 cirrhotic treatment-naïve patients treated with ABT-493 + ABT-530 with/without RBV for 12 weeks

GT1a 24 (20.0)GT1b 44 (36.7)GT2c 6 (5.0)GT3a 24 (20.0)

GT4 (a-d-n-r) 22 (18.3)

0102030405060708090

100

Suboptimal/not recommended (N=70) Recommended (N=50)

Relapse (N=97) Breakthrough (N=14) Non responder (N=9)

75.7%

12.9% 11.4%

88.0%

10.0%2.0%

In a real life italian setting a total of 120 patients whofailed a DAA-regimen were analyzed (cirrhotics 74%)

Di Maio VC et al., EASL 2016Di Maio VC et al., 14th European Meeting on HIV & Hepatitis - 2016

Prev

alen

ce o

f pat

ient

s inc

lude

din

the

anal

ysis

(%)

• SOF+RBV (N=46, HCV-1,3,4)• SMV+DCV+/-RBV (N=8)• SOF+PegIFN+RBV (N=6)• 3D+/-RBV (N=1 HCV-2, N=2 HCV-3)• Other (N=7)

• SMV+SOF+/-RBV (N=33)• 3D+/-RBV (N=4, HCV-1a,1b)• DCV+SOF+/-RBV (N=4)• LDV+SOF+/-RBV (N=4)• SOF+RBV (N=5, HCV-2)

Day 0 4w 8w

1

2

3

4

5

6

7

Relapser to SOCHCV genotype: 1a Sex: M CirrhosisAge: 53ID_1497HC

V-RN

A (lo

g IU

/ml)

LLOQ (12 IU/ml)

Paritaprevir/ritonavir+Ombitasvir+Dasabuvir+RBV

On September 2015 the HCV genotype determination wasrepeated with a commercial assay:HCV genotype 3a

A NS3, NS5A and NS5B genotypic resistance test was performed in our laboratory…

A Clinical case of a patient with cirrhosis starting in August 2015 3D+RBV

Day 0 4w 8w

1

2

3

4

5

6

7

HCV-

RNA

(log

IU/m

l)

LLOQ (12 IU/ml)

GRT 8 weeksBy phylogenetic analysis a HCV- 3a infection was confirmedNS3 Resistance Mutations: NoneNS5A Resistance Mutations: Y93HNS5B Resistance Mutations: None

Relapser to SOCHCV genotype: 3a Sex: M CirrhosisAge: 53ID_1497

Paritaprevir/ritonavir+Ombitasvir+Dasabuvir+RBV

A Clinical case of a patient with cirrhosis starting in August 2015 3D+RBV

Day 0 4w 8w

1

2

3

4

5

6

7

HCV-

RNA

(log

IU/m

l)

LLOQ (12 IU/ml)

GRT 8 weeksBy phylogenetic analysis a HCV- 3a infection was confirmedNS3 Resistance Mutations: NoneNS5A Resistance Mutations: Y93HNS5B Resistance Mutations: None

SOF Interruption

DCV Start

In vitro daclatasvir resistance profile

0

2000

4000

6000

8000

M28

TQ

30H

Q30

RL3

1ML3

1VY9

3CL3

1VY9

3HL3

1M-Y

93H

F28S

L31M

C92R

Y93H

A30K

L31F

Y93H

R30G

R30H

R30S

L30H

Y93H

Y93R

Fold

resi

stan

ceGT 1a1 GT 45GT 23GT 1b2 GT 34

Natural RAS Y93H:2% in 93 GT-3 patientsCento V, 14th European Meeting on HIV & Hepatitis - 2016

Relapser to SOCHCV genotype: 3a Sex: M CirrhosisAge: 53ID_1497

Paritaprevir/ritonavir+Ombitasvir+Dasabuvir+RBV

A Clinical case of a patient with cirrhosis starting in August 2015 3D+RBV

Documento di indirizzo dell’Associazione Italiana per lo Studio del Fegatoper l’uso razionale di antivirali diretti di seconda generazione nelle categorie di pazienti

affetti da epatite C cronica ammesse alla rimborsabilità in Italia

Aggiornamento del 15 Dicembre 2015

EUROPEAN RAVS DATABASE: FREQUENCY AND CHARACTERISTICS OF RAVS IN TREATMENT-NAÏVE AND DAA-EXPERIENCED PATIENTS

Susser & Dietz et al., EASL 2016

Do DAA resistance mutations “disappear” following

discontinuation of therapy?

In the majority of patients PR RAVs disappear….

Lentz O, et al. EASL 2014

Simeprevir

Krishnan P et al. EASL 2015

Paritaprevir/r

Post-treatment 24 Weeks

Post-treatment 48 Weeks

NS3/4A (any) 31/67 (46%) 5/57 (9%)NS5A (any) 68/70 (97%) 49/51 (96%)NS5B (non-nuc) 33/44 (75%) 20/35 (57%)

Persistence of NS5a Resistance Associated Variants Following Ombitasvir/Paritaprevir/r +

Dasabuvir Treatment• Pooled patients with virologic failure from all clinical trials

(n=2510)• 67 patients with HCV genotype 1a • 7 patients with HCV genotype 1b (no long-term follow-up

reported)

Krishnan et al., Abstract #O057, EASL 2015

The absence (by Sanger sequencing) or low prevalence of RAVs does not always means low amount of circulating

resistant virus

WT R155 (65.5%)

WT= wild-type

PT 13PT 11

WT R155 (96.3%)

R155K (35.5%)

R155K (3.7%)

Mutational Load of R155K: 77,966 IU/ml

Mutational Load of R155K: 594,022 IU/ml

HCV-RNA : 1,671,926 IU/ml HCV-RNA: 2,090,903 IU/ml

Mutational Load of R155K: 44,167 IU/ml

WT R155 (96.0%)

R155K (4.0%)

HCV-RNA: 1,104,189 IU/ml

At 100 weeks of therapy interruption At 62 weeks of therapy interruption

PT 211

Three HCV-1a infected patients, all failing TVR-containing regimen with RAVs, still showed the R155K after 60-100 weeks of therapy interruption.

Di Maio VC, EASL 2015

SVR12 Rates With Retreatment of G1 SMV/SOF Failures: Real-World Database

Simeprevir + sofosbuvir failures (n=27)

No RAVs: 18% NS3/NS5B: 52%/15% NS3 plus:

• NS5A: 4%• NS5B: 7%

Retreatment regimens Ledipasvir/sofosbuvir + RBV

12/24 weeks (n=10/13) Ombitasvir/paritaprevir/r +

dasabuvir + RBV 12/24 weeks (n=3/1)

0

20

40

60

80

100

SVR

12 (%

)

100% 100%

12 Weeks(n=10)

Interim SVR12 Rates (ITT)

93%

24 Weeks(n=13)

12 Weeks(n=3)

LDV/SOF + RBV OMB/PTV/r + DAS + RBV

Vermehren J, et al. EASL 2016

SVR12 Rates With Retreatment of Prior SOF/LDV or DCV DAA Failures:

Real-World Study RAVs identified in sofosbuvir plus

daclatasvir or ledipasvir failures (n=22) No RAVs: 5% NS5A: 77% NS5A plus either NS3 (5%), NS5B

(9%) or NS3 + NS5B (5%) Retreatment regimens

Simeprevir + sofosbuvir + RBV 12/24 weeks (n=6/11)

Ombitasvir/paritaprevir/r + dasabuvir + RBV 12/24 weeks (n=4/1)

0

20

40

60

80

100

SVR

12 (%

)

100%

75%

12 Weeks(n=6)

Interim SVR12 Rates (ITT)100%

24 Weeks(n=11)

12 Weeks(n=4)

SIM + SOF + RBV OMB/PTV/r + DAS + RBV

Vermehren J, et al. EASL 2016

Lawitz E. et al., EASL 2015Hadas Dvory-Sobol IWDR Berlin, June 2014

Presence of NS5A RAVs is Associated with Retreatment Failure

SOF/LDV

12 240

N=41 SVR12

weeks

Failed SOF/LDV 8-12 wks (SVR>94%)Regimen SVR: 70%Cirrhosis: 46% BL NS5A RAVs: 73%

Lawitz E. et al., EASL 2015Hadas Dvory-Sobol IWDR Berlin, June 2014

Prior to re-treatno NS5B RAVs(S282T, L159F, V321A)At second VF 4 of 12 (33%) patients had NS5B RAVs:S282T (n=2)L159F (n=1)S282T + L159F (n=1)

Presence of NS5A RAVs is Associated with Retreatment Failure

SOF/LDV

12 240

N=41 SVR12

weeks

Failed SOF/LDV 8-12 wks (SVR>94%)Regimen SVR: 70%Cirrhosis: 46% BL NS5A RAVs: 73%

Retreatment of LDV/SOF Failuresin absence of cirrhosis

Wilson E. et al Clin Infect Dis 2016

SOF/LDV12 240

N=34* SVR12

weeks

2 non-VF excludedBL Y93H/N: 8/32

* Failed: 4wks LDV/SOF + 9451

+/- 9669 (SVR=20-40%)Cirrhosis: 0% (97% F0-2) BL NS5A RAVs: 85%

SVR: 97%

Thirty-two of 34 enrolled participants completed therapy

97 100 96

0

20

40

60

80

100

Combined

31/32 5/526/27

SV

R12

(%)

No RAVs

RAVs

1 Relapse GT1b:BL NS5A: L31M + Y93H >1000x FC in LDV EC50At Failure: L31M + Y93H + NS5B S282T (17.8%)

Hezode C et al EASL 2016

Poordad F et al., AASLD 2015

QUARTZ-I: Retreatment of HCV Genotype 1 DAA-failures with Ombitasvir/Paritaprevir/r, Dasabuvir, and Sofosbuvir

17/22 patients had at BL >1 RAV in 1 of the 3 DAA targets;7 patients had RAVs (other than NS3 Q80K) in 2 targets:2 patients had RAVs in all 3 targets.

• 22 DAA-treated patients 14/20 1a failed 3D

regimen No LDV/SOF

failures

Single treatment failure had no RAVs detected

Retreatment may require «unconventional» approaches with multiple DAAsC-SWIFT retreatment Part B

HCV GT1-infected patients who failed 4, 6, or 8 weeks of EBR/GZR+ SOF in Part A were offered retreatment with EBR/GZR + SOF +RBV for 12 weeks

Lawitz E., AASLD 2015, Poster #LB-12

SOF/VEL + RBV for 24 weeks may be an effective retreatment strategy for patients who

have failed NS5A-based HCV treatment

• 11/13 GT 3 patients with RAVs had Y93H; 9 (82%) achieved SVR12• 5 patients had 2 NS5A RAVs; all 5 achieved SVR12• 3 patients had NS3 RAVs; all 3 achieved SVR12

GT 2 (n=13*)GT 1 (n=34) GT 3 (n=16)

100% SVR 100% SVR100% SVR

38%No RAVs

5/13

62%RAVs8/13

82%No RAVs

28/34

18%RAVs6/34

19%No RAVs

3/16

81%RAVs13/16

100% SVR

77% SVR

10/133/38/85/56/6

96% SVR

27/28

Gane EJ et al., EASL 2016

Considerations in DAA Treatment Failure

Was initial therapy sub-optimal?Drug comboDurationRBV use

Indications of other problems on treatment?Adherence? Significant drug interactions?

Are there other baseline host/disease factors that may have contributed?Cirrhosis, especially decompensation IL28B, age, treatment experience, baseline RASs

By Courtesy of N Terrault 2016

The role of NS5B resistance test for patients who failed a Sofosbuvir containing regimen is not yet defined

Svarovskaia et al, JID 2015

n=901* with deep sequencing

n =12,012 in SOF or LDV/SOF clinical studiesn=1025 with virologic failure

1% 282T SOF virologic failures Gane et al AASLD 2015

Zhdanov K., APASL 2015

Buti M et al J Hepatology 2015

DAA target Regimen HCV-geno/subtypeBaseline RASs Failure RASs

NS3 NS5A NS5B NS3 NS5A NS5B

NS3+NS5B

SMV+SOF 1b V55A L159F+C316NSMV+SOF 1b Q80Q/R L159F+C316N+S556GSMV+SOF 1b D168D/V L31M+Y93H L159F+C316NSMV+SOF 1b Q80Q/R Y93H/Y L159F+C316N

SMV+SOF+RBV 1b D168D/V L159F+C316NNS5A+NS5B LDV+SOF+RBV 1b Y93H L159F+C316N

NS5B

SOF+RBV 1b L159F+C316N L159F+C316NSOF+RBV 1b L159F+C316N L159F+C316NSOF+RBV 1b R30H+Y93H L159F+C316N+S556GSOF+RBV 1b L159FSOF+RBV 3a L159L/FSOF+RBV 3a L159FSOF+RBV 3a L159F

SOF+PegINF+RBV 1b L159F+C316NSOF+PegINF+RBV 1b L159F+C316N+S556S/GSOF+PegINF+RBV 1b L159F+C316N+S556GSOF+PegINF+RBV 1b L159F+C316N

DAA, Direct Acting Antivirals; RASs, Resistance Associated Substitutions

3/20 (15%) HCV-3a infected patients who failed a Sofosbuvircontaining regimen showed L159F RAS at virological failure

Baseline resistance test not available

Di Maio VC et al., EASL 2016Di Maio VC et al., 14th European Meeting on HIV & Hepatitis - 2016

Sex: MALE Country of Origin: ITALYAge: 41 year

Liver status: OLT in 2007HCV genotype: 4d

Outcome to previous pegIFN+RBV treatment: Null responder

The oral combination of daclatasvir + simeprevir was chosen as a new treatment in 2014.

Clinical case

Day 0 Day1 Day2 1w 2w 4w 6w 7w 2w

1

2

3

4

5

6

7

Null Responder to SOCHCV genotype: 4d Age: 41 Sex: MID_451 OLT in 2007

HCV-

RNA

(log

IU/m

l) Daclatasvir+Simeprevir Interruption

LLOQ (15 IU/ml)

GRT 2 weeks after therapy interruptionNS3 Resistance Mutations: A156G, D168ENS5A Resistance Mutations: L28V, R30S

Day 0 Day1 Day2 1w 2w 4w 6w 7w 2w

1

2

3

4

5

6

7

Null Responder to SOCHCV genotype: 4d Age: 41 Sex: MID_451 OLT in 2007

HCV-

RNA

(log

IU/m

l) Daclatasvir+Simeprevir Interruption

GRT Day 0NS3 Resistance Mutations: D168ENS5A Resistance Mutations: R30S

LLOQ (15 IU/ml)

GRT 2 weeks after therapy interruptionNS3 Resistance Mutations: A156G, D168ENS5A Resistance Mutations: L28V, R30S

1w 2w 3w 4w 5w 7w 9w 10w 12w 14w 19w 4w

1

2

3

4

5

6

7

0d 1d 2d

HCV-

RNA

(log

IU/m

l)

LLOQ (15 IU/ml)

Sofosbuvir+Ribavirin

HCV genotype: 4d Age: 41 Sex: MID_451 OLT in 2007

GRT 4 weeks after therapy interruptionNS3 Resistance Mutations: D168ENS5A Resistance Mutation: R30SNS5B Resistance Mutations: None

Days Weeks

Breakthrough to DCV+SMV

Day 0 2w 4w 6w 8w 12w 16w 20w 24w 4w 11w

1

2

3

4

5

6

7

HCV-

RNA

(log

IU/m

l) Ledipasvir+Sofosbuvir+Ribavirin

LLOQ (15 IU/ml)

after EOT

GRT 11 weeks after therapy interruptionNS3 Resistance Mutations: D168ENS5A Resistance Mutations: R30SNS5B Resistance Mutations: None

HCV genotype: 4d Age: 41 Sex: MID_451 Breakthrough to SOF+RBV

Day 0 2w 4w 6w 8w 12w 16w 20w 24w 4w 11w

1

2

3

4

5

6

7

HCV-

RNA

(log

IU/m

l) Ledipasvir+Sofosbuvir+Ribavirin

LLOQ (15 IU/ml)

after EOT

GRT 11 weeks after SOF+LDV+RBV interruptionNS3 Resistance Mutations: D168ENS5A Resistance Mutations: R30SNS5B Resistance Mutations: None

GRT Day 0 (2014)NS53 Resistance Mutations: D168ENS5A Resistance Mutations: R30S

HCV genotype: 4d Age: 41 Sex: M

Second OLT in 2016 Breakthrough to SOF+RBV

GRT 2 weeks after DCV+SMV interruptionNS3 Resistance Mutations: A156G, D168ENS5A Resistance Mutations: L28V, R30S

Breakthrough to DCV+SMVNull Responder to SOC

Relapse to SOF+LDV+RBV

Treatment should be individualized

From Sarrazin C et al., J Hepatol 2015

Both in terms of treatment duration,number of effective drugsand screening after SVR

DAA failure

Genotypic resistance testingNS3+NS5A+NS5B

No NS5A RAVs

SOF/LDV + RBVSOF + DCV + RBV

24 weeksNo Q80K

SOF + SIM + RBV 24 weeks

NS5A RAVs(Q30, L31, H58, Y93)

SOF + SIM + RBV 24 weeks

(even if Q80K)

NS5A and NS3 RAVs

(R155, A156, D168)

Desperationtime

3D + SOFSOF + SIM + DCV + RBVSOF +LDV/DCV + RBV

SOF + pegIFN + RBV12/24 weeks

Investigational triple regimens

Modified by Wyles D, AASLD 2015

Retreatment may require «unconventional» approaches with multiple DAAs

Conclusions• Although systematic HCV resistance testing prior to start DAA

treatment is NOT recommended (exception: NS3-Q80K, soon NS5A-test for elbasvir), it is indeed currently considered (helpful to store a sample in difficult patients).

• SVR rates are high with new IFN-free regimens (in both mono and co-infected HIV populations). However, failures can occur, particularly when treatment is suboptimal.

• At failure: the resistance test should be performed in all 3 genes NS3 + NS5A + NS5B.

• Retreatment options exist, today particularly based on sofosbuvir in combination with one or two other DAAs (better with RBV and longer duration).

• However, the frequent finding at failure of major RAVs involving ≥2 DAAs-targets may advocate also for unconventional, resistance-based regimens.

Thanks for your attention

What do we know about RAVs todayRASs�Resistance associated substitutionsDianummer 3HCV genetic variability is higher than HIV’s and HBV’sDianummer 5HCV genotype dictates the choice of anti-HCV drugs �and can modulate the duration of treatment in infected patients with chronic hepatitis C��HIV versus HCV�Baseline resistance testingDianummer 8Virological issues in the DAAs EraDianummer 10Dianummer 11Dianummer 12Dianummer 13Should we be worried about baseline RAVs?Dianummer 15Overall prevalence of Q80K in G1 across different regionsThe prevalence of pre-treatment NS5A RAVs in GT-1 is different across different countries, ranging from 6% to 25%, and different according to subtype…..�Dianummer 18Impact of baseline NS5A RAVs can be reduced/eliminated by changing the regimen (longer duration of treatment, inclusion of RBV) in patients with compensated cirrhosis �treated with Ledipasvir/SofosbuvirLDV/SOF ± RBV: SVR12 in GT 1 Patients With Cirrhosis ± Baseline NS5A RAVs… Even in GT 1 and GT 4 patients with advanced liver disease or post liver transplant (SOLAR-1 and 2)Dianummer 22Dianummer 23Dianummer 24Dianummer 25Dianummer 26Dianummer 27Dianummer 28Dianummer 29Not all RASs are Equal…..�Dianummer 31Beware of HCV-genotype for NS5A resistance ...SVR rates were reduced in GT-3 patients with natural NS5A RAVs treated with grazoprevir, MK-3682 (NS5B), and MK-8408 (NS5A inhibitor) for 8 weeksSVR rates were reduced in GT-3 patients with natural NS5A RAVs treated with grazoprevir, MK-3682 (NS5B), and MK-8408 (NS5A inhibitor) for 8 weeksSVR rates were NOT reduced in GT-3 non-cirrhotic patients �with natural NS5A RAVs �treated with ABT-493 + ABT-530 co-administered for 8 weeks �Dianummer 36Dianummer 37Dianummer 38Dianummer 39Dianummer 40Dianummer 41Dianummer 42In the majority of patients PR RAVs disappear….Persistence of NS5a Resistance Associated Variants Following Ombitasvir/Paritaprevir/r + Dasabuvir TreatmentDianummer 46SVR12 Rates With Retreatment of G1 �SMV/SOF Failures: Real-World DatabaseSVR12 Rates With Retreatment �of Prior SOF/LDV or DCV DAA Failures: �Real-World StudyDianummer 49Dianummer 50Retreatment of LDV/SOF Failures�in absence of cirrhosisDianummer 53Dianummer 54Retreatment may require «unconventional» approaches with multiple DAAs�C-SWIFT retreatment Part BSOF/VEL + RBV for 24 weeks may be an effective retreatment strategy for patients who have failed NS5A-based HCV treatmentConsiderations in DAA Treatment FailureThe role of NS5B resistance test for patients who failed a Sofosbuvir containing regimen �is not yet definedDianummer 59Clinical case �Dianummer 61Dianummer 62Dianummer 63Dianummer 64Dianummer 65Treatment should be individualizedRetreatment may require «unconventional» approaches with multiple DAAs�ConclusionsDianummer 69