virological assessment of patients candidate to...

Virological assessment of patients candidate to DAA

• Italian male patient • Diagnosis of chronic HCV infection in 1994 • Genotype 1b defined in 1998 • Non-responder to IFN+RBV

• He developed follicular lymphoma

Patients’ characteristics

Day 0 Jan 2014

1w 4w 5w 6w

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LLOQ (12 IU/ml)

HC

V- R

NA

(log

IU/m

l)

Asunaprevir + Daclatasvir + RBV (compassionate use)

Non Responder to SOC

HCV genotype: 1b (performed in 1998) Sex: M Follicular Lymphoma

ALT = 337U/L

Chamaya et al., Hepatology 2012

1w 4w 5w 6w

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LLOQ (12 IU/ml)

HC

V- R

NA

(log

IU/m

l)



Day 0 Jan 2014

Asunaprevir + Daclatasvir + RBV (compassionate use)

ALT = 337U/L

Day 0 Jan 2014

1w 4w 5w 6w

1

2

3

4

5

6

7

LLOQ (12 IU/ml)

HC

V- R

NA

(log

IU/m

l)

Asunaprevir + Daclatasvir + RBV (for compassionate use)



GRT after 5 weeks of therapy A genotypic resistance test was performed in our laboratory…

• Sequencing of NS3 and NS5a for: - genotype re-assessment by phylogenetic analysis - drug resistance detection

By phylogenetic analysis, the sample was classified as genotype 4 subtype d (instead of genotype 1B)

Clade 4d

This result was afterwards confirmed by Abbott RealTime-HCV Genotype II

Day 0 Jan 2014

1w 4w 5w 6w

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LLOQ (12 IU/ml)

HC

V- R

NA

(log

IU/m

l)


GRT after 5 weeks of therapy NS3 Resistance Mutations: Q41Q/R, D168V NS5A Resistance Mutation: L28V, M31M/V, Y93H/Y

GRT at Baseline NS3 Resistance Mutations: none NS5A Resistance Mutation: none

Detection of drug resistance mutations in both NS3 and NS5a at week 5 of therapy, not present at baseline

Protease Inhibitor Resistance

HCV DRUG, Forum for Collaborative HIV Research, April 2014

paritaprevir/r

Boceprevir

Telaprevir

Simeprevir

Faldaprevir

Asunaprevir

Paritaprevir

HCV DRUG, Forum for Collaborative HIV Research, April 2014

NS5a Inhibitor Resistance Daclatasvir

Ledipasvir

Ombitasvir

High world-wide prevalence of NS5A RAVs in NS5a inhibitor naive patients

NS5A population-sequencing analysis was performed in > 3000 patients across 14 countries.

The circulation of NS5A RAVs in some European Countries was estimated with a prevalence ranging from 7% to 17% in GT-1a and from 9% to 13% in GT-1b.

Svarovskaia E.S., EASL 2015

NS5A RAVs change from one type to another over time and appear to be much more persistent

Among 43 GT-1a patients analyzed 48 weeks after failure to 3D +/- RBV: • Most still presented NS5A RAVs (100% in

GT-1a patients conpleting treatment as recommended by guidelines)

Krishnan P et al., EASL 2015

The issue of HCV genotyping…..

Consequences of HCV variability at population level: HCV genotypes

hivforum.org

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

Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide as well as in Europe

Esteban JI et al J Hepatol 2008;48:148-162

3a

1b

1b

1b

2

1a

1a 3a

2

2 1b

1b

1b 1b

1b

1b

1b 1b

1a 3a

1a 3a

1a 3a

1a 3a

1a 3a

1a 3a

1a 3a

4 4

4

1a

2

4 3a

Treatment Failure =

Failure to Cure HCV infection =

There remains hepatocytes in the liver that are infected with wt and/or resistant HCV viruses when treatment is

stopped

A correct determination of HCV-genotype is mandatory prior to treatment initiation

Treatment recommendations for HCV-infected patients with chronic hepatitis C and compensated cirrhosis (including naïve and previous Peg/RBV failures)

EASL Recommendations on Treatment of Hepatitis C 2015, J Hepatol 2015

Treatment recommendations for HCV-infected patients with chronic hepatitis C without cirrhosis

(including naïve and previous Peg/RBV failures)

EASL Recommendations on Treatment of Hepatitis C 2015, J Hepatol 2015

Trugene HCV Genotyping assay Direct sequencing INNO-LiPA HCV 1.0 Reverse hybridization INNO-LiPA HCV 2.0 Reverse hybridization Abbott RealTime HCV Genotype II assay Real time PCR

Target Regions: HCV 5’ UTR, CORE & NS5B region

5’ UTR 3’ UTR

• Several commercial assays are available for determining genotype/subtype

• All assays target the 5’NCR gene for genotypes 1-6, in addition, the 2 assays more used in diagnostics, Abbott and INNO-LiPA-HCV-2.0, target also the NS5B and the core gene, respectively, providing additional information also in subtyping: for genotype 1 (1a/1b, both), and for all genotypes (only Innolipa)

Several commercial assays are available for determining genotype/subtype

Trugene HCV Genotyping assay Direct sequencing INNO-LiPA HCV 1.0 Reverse hybridization INNO-LiPA HCV 2.0 Reverse hybridization Abbott RealTime HCV Genotype II assay Real time PCR

Target Regions: HCV 5’ UTR, CORE & NS5B region

5’ UTR 3’ UTR

• Several commercial assays are available for determining genotype/subtype

• All assays target the 5’NCR gene for genotypes 1-6, in addition, the 2 assays more used in diagnostics, Abbott and INNO-LiPA-HCV-2.0, target also the NS5B and the core gene, respectively, providing additional information also in subtyping: for genotype 1 (1a/1b, both), and for all genotypes (only Innolipa)

Several commercial assays are available for determining genotype/subtype

Even newest commercial-assays may miss a precise determination of HCV-genotype

in 9-10% of cases!

HCV genotyping

NS3 sequencing + phylogenetic analysis Innolipa or Abbott assay

Analysis in 343 patients candidate to DAA therapy

Ceccherini-Silberstein F. et al, Hepatology 2015

HCV genotyping

NS3 sequencing + phylogenetic analysis Innolipa or Abbott assay

Analysis in 343 patients candidate to DAA therapy

HCV-sequencing and commercial-assays were concordant in 91.84% of cases analysed


• The sequencing approach allows to assign HCV genotype/subtype in all patients with a previous result of ‘mixed’ or ‘indeterminate’ HCV-genotype/subtype by commercial assays

We reanalyzed genotype data by phylogenetic analysis of 343 HCV-infected patients candidate to DAA-treatment, who performed a genotypic-resistance-test between 2011 and 2014. To confirm the appropriate genotype allocation, HCV-sequencing was performed by home-made protocols, specific for each genotype, on NS3-protease (95% samples), together with/in alternative to NS5A (9%) and/or NS5B (14%).


Patients (N) Patients (%)

Genotype/subtype confirmed 315 91.84%

Mixed/Indeterminate genotypes 8 2.33%

Genotype 1 with no subtype 6 1.75%

Discordant genotypes 4 1.17%

Genotype 1 with discordant subtype 10 2.91%

4.08%

In addition….

HCV genotyping by

Innolipa/Abbot (year of genotyping) NS3 sequencing Abbott

2013/2014 1a (unknown) 2c -

1b (1993) 2c 2 1b (1994) 4d -

2a/2c (2005) 1b 1b

• 4.1% (14/343) patients showed a discordant genotype or subtype according to direct-sequencing

Innolipa/Abbott Sequencing

1a=5 1g=1 1b=4

1b=4 1a=4 Indeterminate/1a =1 1g=1


HCV genotyping by

Innolipa/Abbot (year of genotyping) NS3 sequencing Abbott

2013/2014 1a (unknown) 2c -

1b (1993) 2c 2 1b (1994) 4d -

2a/2c (2005) 1b 1b

Innolipa/Abbott Sequencing

1a=5 1g=1 1b=4

1b=4 1a=4 Indeterminate/1a =1 1g=1


This suggests the importance to test again HCV genotype when the first version of

commercial assays were used

• 4.1% (14/343) patients showed a discordant genotype or subtype according to direct-sequencing

How to manage this patient according to the new genotype (4d) and drug resistance mutations

in both NS3 and NS5a?

Post-treatment virological issues

Currently, there is no data to firmly support retreatment recommendations, which must be based on indirect evidence (HCV genotype, known resistance

profiles of the administered drugs …) (EASL HCV Clinical Practice Guidelines 2015).

– Clinically meaningful NS5B RAVS have been exceptionally reported with sofosbuvir, and they rapidly disappeared after treatment cessation. Thus, retreatment strategies should include sofosbuvir

– NS3 RAVs can persist for several months (also as minority species)

– NS5A RAVs may persist for YEARS (or even forever?). Check the presence (and type) of NS5A RAVs before starting a second all-oral DAA regimen including an NS5A inhibitor!

Can UDPS provide an added value in the setting of re-treatment with the same drug class?

Day 0 Jan 2014

1w 4w 5w 6w

1

2

3

4

5

6

7

LLOQ (12 IU/ml)

HC

V- R

NA

(log

IU/m

l)


GRT after 5 weeks of therapy NS3 Resistance Mutations: Q41Q/R, D168V NS5A Resistance Mutation: L28V, M31M/V, Y93H/Y

GRT at Baseline NS3 Resistance Mutations: none NS5A Resistance Mutation: none

How to manage this patient?

This patient achieved SVR12 after SOF+PEG+RBV therapy

• Re-treatment issue: - may UDPS provide an added value compared to population sequencing to check for the absence of drug resistance mutations?

• HCV genotype assignment: - May the sequencing approach provide an added value in the setting of mixed/indeterminate results by commercial assays? • Genotypic testing at baseline (not only for Q80K in NS3)

Points to discuss:

virological assessment of patients candidate to...

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