Resistance-associated and fitness-associated substitutions on NS3,

NS5A and NS5B genes in real-life HCV positive patient

failed to 3D therapy

Nadia Marascio, PhD

“Magna Graecia” University, Catanzaro, Italy June 8, 2017

Session 1: Hepatitis C Resistance

NO adverse events were reported

during and until the end of therapy

Adherence was good

HCV RNA viral load was determined by Cobas AmpliPrep/Cobas TaqMan HCV test v2.0

(Roche Diagnostics, Milan, Italy) (quantification range 15 to 100 million IU/mL)

Time (week)

HC

V R

NA

vir

al lo

ad (

IU/m

l)

3D – R therapy (12 weeks) therapy failure

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

start week 4 end of treatment SVR12 follow up

TND

baseline

week 12 after treatment

week 32 after treatment

1. Could have we foreseen failure from basic clinical and virological

features?

2. Was this a relapse or a reinfection?

3. What drugs could we use?

4. Should we treat with drugs currently available or better wait?

Questions

Patient characteristics

• 65 year old man, HCV1b positive

• not co-infected with HIV or HBV

• naïve to all HCV treatments

• AST 36U/ml / ALT 65U/l

• liver stiffness 10.5 kPa (F3 according to Metavir scoring system)

• type 2 diabetes

• No cryoglobulinemia

• risk factors: surgery before 90’s.

April, 2016

(adapted from EASL Recommendations on Treatment of Hepatitis C 2016)

One tablet contains ombitasvir 12.5 mg/ paritaprevir 75 mg/ritonavir 50 mg (2 tablets

once daily). The other tablet contains dasabuvir 250 mg (1 tablet twice daily) (EASL 2016)

3D therapy without ribavirin

Treatment outcome with 3D in HCV1b naive patients

without cirrhosis

(adapted from Hussaini T. 2016)

1. Could have we foreseen failure from basic clinical and virological

features?

2. Was this a relapse or a reinfection?

3. What drugs could we use?

4. Should we treat with drugs currently available or better wait?

Questions

Serum samples

at different time points

Population sequencing (ABI PRISM 3500 genetic analyzer Applied Biosystems)

cut-off for substitutions detection > 15% (clinically significant right now)

Phylogenetic analysis

Neighbour-Joining (NJ) under Tamura–Nei (TN) model, generating 1000 bootstrap replicates (MEGA v.5)

Subtyping determination

Bioafrica Oxford HCV v.2.0 and COMET HCV subtyping tools

Methods workflow

HCV genome

5’ 3’

NS3 c E1 E2 P7 NS2 NS4A NS4B

NS5A NS5B

Figure 1. NS3_NS5A_NS5B concatenate regions NJ tree was reconstructed under TN model with 1000 bootstraps replicates.

Genetic distance is at the bottom.

100

HCV1b

100

100

0.03

High

bootstrap support

1. Could have we foreseen failure from basic clinical and virological

features?

2. Was this a relapse or a reinfection?

3. What drugs could we use?

4. Should we treat with drugs currently available or better wait?

Questions

3D therapy is a fixed-dose tablet comprising NS3/4A protease inhibitor, ritonavir (to

boost paritaprevir exposure), NS5A inhibitor and NS5B polymerase inhibitor with or

without ribavirin.

5’ 3’

NS3 c E1 E2 P7 NS2 NS4A NS4B

paritaprevir target

NS5A NS5B

ombitasvir target

dasabuvir target

HCV genome

Virologic failure in HCV1b positive patients who do not achieve

SVR was related to RASs selection to one or more of 3 DAAs

NS3 paritaprevir

RASs

NS5A ombitasvir

RASs

NS5B dasabuvir

RASs

56H

168A/V/H/E/T/Y

28M/T

31F 93H

316Y

414I/T 556G/R

(Feld J.J. et al., 2014; Zeuzem S. et al. 2014; Pilot-Matias et al. 2015; Krishnan et al. 2015; Kati

et al. 2015; Pawlotsky J.M. 2016; Di Maio V.C. et al, 2016)

Population sequencing (ABI PRISM 3500 genetic analyzer Applied Biosystems)

cut-off for substitutions detection > 15% (clinically significant right now)

Non-synonymous substitutions detection

Alignment of newly generated sequences to HCV1b reference sequences (SeaView v.4).

Interpretation by geno2pheno tool and published data

Methods workflow

HCV genome

5’ 3’

NS3 c E1 E2 P7 NS2 NS4A NS4B

NS5A NS5B

Serum samples

at different time points

NS3 region analysis (1-181 amino acids)

Table 1. NS3 non-synonymous substitutions

* resistance-associated polymorphisms (RAPs) (related to 122R simeprevir RAS)

baseline week 12 (SVR12) week 32 (follow up)

RASs Polymorphisms RASs Polymorphisms RASs Polymorphisms

none

48I

none

48I

none

48I

66G 66G 66G

86Q 86Q 86Q

87A 87A 87A

89S P89 P89

122G 122G 122G

125A 125A 125A

147S 147S 147S

* * *

amino acid change

NS5A region analysis (1-213 amino acids)

Table 2. NS5A non-synonymous substitutions

baseline week 12 (SVR12) week 32 (follow up)

RASs Polymorphisms RASs Polymorphisms RASs Polymorphisms

none

6R

93H

6R

93H

6R

34V 34V 34V

44R 44R 44R

61V 61V 61V

78R 78R 78R

R108 R108 108K

138L 138L 138L

ombitasvir RAS

amino acid change

NS5B region analysis (91- 340 aa and 350-556 aa)

Table 3. NS5B non-synonymous substitutions

sofosbuvir RAS

dasabuvir RAS

*fitness associated substitution

amino acid change

baseline week 12 (SVR12) week 32 (follow up)

RASs Polymorphisms RASs Polymorphisms RASs Polymorphisms

159F

98K

159F

98K

159F

98K

117T 117T 117T

127L 127L 127L

189P 189P 189P

213S 213S 213S

218S 218S 218S

254K 254K 254K

S300 300T 300T

316N 316N 316N

335N 335N 335N

work in progress 556G data not shown 556G data not shown

* * *

Time (week)

HC

V R

NA

vir

al lo

ad

(IU

/ml)

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

start week 4 end of treatment SVR12 follow up

“viral population at baseline”

NS5B159F sofosbuvir RAS +

NS5B316N fitness associated substitution +

NS5B556G dasabuvir RAS (?)

“viral population at relapse”

NS5A93H daclatasvir, elbasvir, ledipasvir and velpatasvir RAS +

NS5B159F sofosbuvir RAS +

NS5B316N fitness associated substitution +

NS5B556G dasabuvir RAS

TND

8 months after the end of treatment

NS5A93H + NS5B159F + NS5B556G RASs

are still present

According to new AIFA n. 500/2017, prescriptive and redeemable

combinations by Italian National Health Service (SSN) for HCV1 failure

with NS5A treatment are (EASL Recommendation 2016 and AISF

Recommendation 2017):

Elbasvir/Grazoprevir + Sofosbuvir + Ribavirin for 24 weeks, HCV1b positive

patient with metavir F3-F4.

Ombitasvir/Paritaprevir/Ritonavir + Dasabuvir + Sofosbuvir + Ribavirin for

24 weeks, HCV1b positive patient with metavir F3-F4.

Daclatasvir + Simeprevir + Sofosbuvir + Ribavirin for 24 weeks, HCV1b

positive patient with metavir F3-F4 (suboptimal according to AISF).

1. Could have we foreseen failure from basic clinical and virological

features?

2. Was this a relapse or a reinfection?

3. What drugs could we use?

4. Should we treat with drugs currently available or better wait?

Questions

In the third quarter of 2017, two pipeline salvage

agents will likely be available (Molino et al, 2017):

Sofosbuvir/Velpatasvir/Voxilaprevir (GS-9857) received FDA breakthrough therapy

designation (BTD) for genotype 1 patients who have failed treatment with an NS5A inhibitor.

Glecaprevir/Pibrentasvir (ABT-493/ABT-530), second-generation NS3/4A and NS5A

inhibitors, received FDA BTD for genotype 1 patients who have failed previous DAA

treatment (pan-genotypic activity and higher genetic barrier to resistance).

It is important to perform resistance testing at failure, as well as at baseline, on three eligible

genes for a personalized IFN-free DAA treatment.

Pre-existing RASs (93H?) could be present below detection limit of Sanger sequencing, a new

significant threshold (from 1% to 15%) using deep-sequencing should be determined.

Amino acid changes co-evolving on HCV genome during IFN-free therapy, especially without

ribavirin, could be related to fitness associated effect promoting further accumulation of RASs.

Conclusions

“Magna Graecia” University

Alfredo Focá

Maria Carla Liberto

Carlo Torti

Nicola Perrotti

Grazia Pavia

Emilia Zicca

Vito Marano

Acknowledgments

“Mater Domini” Hospital

Sebastiano Di Salvo

Massimo De Siena

Francesca Giancotti

Tiziana Gravina

Giorgio Settimo Barreca

Fernanda Fabiani

Thank you all for the kind attention!

Campus map