NGS in clinical Italian practice: impact of minor quasispecies on - - PowerPoint PPT Presentation

NGS in clinical Italian practice: impact of minor quasispecies on antiretroviral drug resistance and viral tropism

AREVIR-GenaFor-Meeting Bonn 3th May 2012

• Dr. Daniele Armenia

HIV RNA Level Time Treatment begins Before starting Incomplete suppression Virological Failure Sensitive variant Minority resistant variant

Why use NGS in management of HIV-1 infection?

HIV RNA Level Time Treatment begins Before starting Incomplete suppression Virological Failure Sensitive variant Minority resistant variant

Why use NGS in management of HIV-1 infection?

HIV RNA Level Time Treatment begins Before starting Incomplete suppression Virological Failure Sensitive variant Minority resistant variant

Why use NGS in management of HIV-1 infection?

Involved in resistance development of:

• Low-mean genetic barrier

ARVs such as NNRTI or INI

• CCR5-Antagonists

(pre-existing minority X4 virus)

HIV RNA Level Time Treatment begins Before starting Incomplete suppression Virological Failure Sensitive variant Minority resistant variant

Why use NGS in management of HIV-1 infection?

Involved in resistance development of:

• Low-mean genetic barrier

ARVs such as NNRTI or INI

• CCR5-Antagonist

(pre-existing minority X4 virus)

NGS allows to detect hidden minority resistance variants and theoretically to prevent their selection during treatment

Overview

1. To define new genetic markers of HIV that can predict the presence

• f transmitted RTIs drug resistant minority species.

2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.

Overview

1. To define new genetic markers of HIV that can predict the presence

• f transmitted RTIs drug resistant minority species.

2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.

Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche,

• n plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without

primary resistance detected by GRT.

In patients with L210M and T69S an higher number of minority drug resistance strains has been detected and these strains are present with a frequency higher than 1%

Alteri et al, JAC 2011

In patients with L210M and T69S an higher number of minority drug resistance strains has been detected and these strains are present with a frequency higher than 1%

This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance

Overview

1. To define new genetic markers of HIV that can predict the presence

• f transmitted RTIs drug resistant minority species.

2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.

The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1–infected patients, who started a salvage raltegravir-containing regimen, were investigated. Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure

At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected

Any statistically association neither with virologic response at 24-weeks nor with the development of resistant variants at failure was observed

Armenia et al, JID 2011

UDPS Dynamics of Integrase Mutations During Raltegravir Treatment

Armenia et al, JID 2011

Armenia et al, JID 2011

Armenia et al, JID 2011

Resistance to raltegravir in integrase strand transfer inhibitor–naive patients remains today a rare event. Pathways of resistance at failure were not predicted by baseline mutations.

Overview

1. To define new genetic markers of HIV that can predict the presence

• f transmitted RTIs drug resistant minority species.

2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.

V Svicher, V Cento, G Rozera, I Abbate, MM Santoro, D Armenia, L Fabeni, G Palamara, A Latini, G Rizzardini, V Micheli, AR Buonomini, MP Trotta, A Antinori, M Andreoni, MR Capobianchi, F Ceccherini-Silberstein, CF Perno Manuscript submitted

The Genotypic False Positive Rate Determined by Population V3-Sequencing Predicts the Burden of X4 Minority Quasispecies

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

FPR of V3 sequences detected by UDPS

FPR at population V3 sequencing

0-2 (N=5) 5-10 (N=4) 10-20 (N=6) 20-60 (N=16) >60 (N=12) 2-5 (N=3)

5.75% Cut-off

R5 species X4 species Bulk V3 species

Prevalence >20% Prevalence 10-20% Prevalence <10%

V3 quasispecies composition at UDPS according to the false positive rate determined by population V3 sequencing

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

FPR of V3 sequences detected by UDPS

FPR at population V3 sequencing

0-2 (N=5) 5-10 (N=4) 10-20 (N=6) 20-60 (N=16) >60 (N=12) 2-5 (N=3)

5.75% Cut-off

R5 species X4 species Bulk V3 species

No X4 variants are detected by both UDPS and ESTA in all patients with FPR >60

Prevalence >20% Prevalence 10-20% Prevalence <10%

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

FPR of V3 sequences detected by UDPS

FPR at population V3 sequencing

0-2 (N=5) 5-10 (N=4) 10-20 (N=6) 20-60 (N=16) >60 (N=12) 2-5 (N=3)

5.75% Cut-off

R5 species X4 species Bulk V3 species

Prevalence >20% Prevalence 10-20% Prevalence <10%

The majority of patients with FPR ranging from 20 to 60 at population V3 sequencing harbour R5-species.

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

FPR of V3 sequences detected by UDPS

FPR at population V3 sequencing

0-2 (N=5) 5-10 (N=4) 10-20 (N=6) 20-60 (N=16) >60 (N=12) 2-5 (N=3)

5.75% Cut-off

R5 species X4 species Bulk V3 species

Prevalence >20% Prevalence 10-20% Prevalence <10%

X4 species at a level >10% are present in all patients with FPR<5, and reach 90% of the entire population in patients with FPR<2

Overview

1. To define new genetic markers of HIV that can predict the presence

• f transmitted RTIs drug resistant minority species.

2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on virological response to MVC in clinical practice.

Geno2pheno [coreceptor]

Analysis pipeline

15 Patients Starting Maraviroc R5-infected (Esta) Plasma samples Baseline 454-junior and Sanger genotyping Sff output Geno2pheno [454] HIV-RNA monitoring to evaluate virological failure* + Fasta X4 tropism: >2% of species with FPR ≤3.75% X4 tropism: FPR ≤ 10%

*Virological failure: 2 consecutive HIV-1 RNA measurments >50copies/mL after at least 6 months of MVC pressure

Overall patients 15 Age, Median (IQR) 41(37-46) Male % 66.7 Subtype (N) B (14) BF (1) R5 Tropism (%) By phenotype (Esta) By genotyping (geno2pheno FPR set at 10%) 100 86.6 Median (IQR) viral load at baseline (log10 copies/ml) 4.9 (4.5-4.3) Median (IQR) CD4 cell count at baseline (cells/µl) 146 (120-172) Median (IQR) GSS (rega 8.02) 2(2-3) Median (IQR) Number of experienced regimen 12(7-15) Novel drugs co-admistered for the first time with MVC (%) DRV ETR RAL T20 13.3 6.7 33.3 6.7

Patient characteristics

Patient ID FPR pop

20 40 60 80

FPR values per UDPS species detected

3.75% cut-off

Variant (UDPS) prevalence (%)

≥80 60-80 40-60 20-40 10 20-40 <20 30 50 70 90 7323 569 77 6971 449 4879 621 3488 8782 434 2350 179 9019 380 199

1.7 4 17 17 26.9 35.9 42.5 45.7 48.7 49 51.3 57 62.8 65.4 87.8

2331 4917 4685 4234 2960 2776 4277 4278 6903 2507 2833 3298 5154 3393 4111

Tot Reads

FPR at population sequencing

V3 quasispecies composition at UDPS according to the false positive rate determined by population V3 sequencing

At high FPR at population sequencing no X4 species were detected by UDPS

Patient ID FPR pop

20 40 60 80

FPR values per UDPS species detected

3.75% cut-off

Variant (UDPS) prevalence (%)

≥80 60-80 40-60 30-40 10 20-40 <20 30 50 70 90 7323 569 77 6971 449 4879 621 3488 8782 434 2350 179 9019 380 199 1.7 4 17 17 26.9 35.9 42.5 45.7 48.7 49 51.3 57 62.8 65.4 87.8 2331 4917 4685 4234 2960 2776 4277 4278 6903 2507 2833 3298 5154 3393 4111

Tot Reads

FPR at population sequencing

7323 569 77 6971 449 4879 621 3488 8782 434 2350 179 9019 380 199 1.7 4 17 17 26.9 35.9 42.5 45.7 48.7 49 51.3 57 62.8 65.4 87.8 2331 4917 4685 4234 2960 2776 4277 4278 6903 2507 2833 3298 5154 3393 4111

FPR at population sequencing ranging around from 20 to 60%, X4 species were occasionally detected by UDPS

Patient ID FPR pop

20 40 60 80

FPR values per UDPS species detected

3.75% cut-off

Variant (UDPS) prevalence (%)

≥80 60-80 40-60 20-40 10 20-40 <20 30 50 70 90

Tot Reads

FPR at population sequencing

As previously observed, if FPR at population sequencing is less than 5%, X4 species were ever detected by UDPS

Patient ID FPR pop

20 40 60 80

FPR values per UDPS species detected

3.75% cut-off

Variant (UDPS) prevalence (%)

≥80 60-80 40-60 20-40 10 20-40 <20 30 50 70 90 7323 569 77 6971 449 4879 621 3488 8782 434 2350 179 9019 380 199 1.7 4 17 17 26.9 35.9 42.5 45.7 48.7 49 51.3 57 62.8 65.4 87.8 2331 4917 4685 4234 2960 2776 4277 4278 6903 2507 2833 3298 5154 3393 4111

Tot Reads

FPR at population sequencing

HIV-RNA (log10 copies/ml) CD4 cell count (cell/mm3

X4 Tropic N=4 R5 Tropic N=11

100 200 300 400 500 600 1.0 2.0 3.0 4.0 5.0 6.0

4/15 (26%) patients carried >2% of X4 species (FPR≤3.75) in viral

• population. At baseline these X4-infected patients showed a trend
• f lower CD4 cell count than R5 infected patients

20 40 60 80 100

7323 569 77 6971 449 4879 621 3488 8782 434 2350 179 9019 380 199 13.9 13.5 17 2.7

% X4 species detected by UDPS

Patient ID

The rate of failure to maraviroc containing regimen is higher in patients carrying X4-tropic viruses in comparison with R5- infected patients.

Patient ID Genotypic sensitive score (Rega 8.02) FPR at population sequencing Percentage

• f X4

variant Virologic Failure* Tropism 6971 2 17.0 17.0 Yes

X4

569 2.5 4.0 15.6 No 7323 2 1.7 13.9 Yes 434 0.5 49 2.7 Yes 8782 3 48.7 0.2 No

R5

449 1.5 26.9 0.0 Yes 2350 1.5 51.3 0.0 Yes 179 2 57.0 0.0 Yes 621 3 42.5 0.0 Yes 77 3.5 17.0 0.0 No 199 87.8 0.0 No 380 1 65.4 0.0 No 3488 2.5 45.7 0.0 No 4879 1.5 35.9 0.0 No 9019 1.5 62.8 0.0 No 3/4 (75%) X4-infected patients failed MVC regimen. Only 1 patient with GGS>2 responded 4/11 (36.4%) R5-infected patients failed MVC regimen. The majority

• f

these failing patients had GGS≤2

* VF: 2 consecutive viremia value >50 copies/ml after at least 6 months of MVC treatment

Conclusions

• The proportion of patients with X4 variants increases by decreasing the FPR

by population V3-sequencing.

• X4 species at a level >10% are present in all patients with FPR<5.
• No X4 variants are detected by both UDPS and ESTA in all patients with FPR

>60

• The rate of virological failure to Maraviroc was higher in X4-infected

patients (carrying >2% of X4 species with FPR≤3.75) in comparison to those were R5-infected.

• The baseline Genotypic Sensitive Score, together with the tropism

determination, might have a relevant role on tailoring Maraviroc containing regimens to avoid the virological failure.

Virco BVBA I Vandenbroucke K Van Baelen H Van Marck L Van Wesenbeeck L Stuyver San Martino Hospital, Genoa

• B. Bruzzone
• A. Di Biagio

San Gallicano Hospital, Rome

• G. Palamara
• M. Giuliani
• A. Antinori

MR Capobianchi G Rozera I Abbate A Brusselles

• P. Narciso
• C. Gori
• E. Girardi
• R. d’Arrigo
• F. Forbici

M.P. Trotta

• A. Ammassari
• R. Bellagamba
• M. Zaccarelli
• G. Liuzzi
• V. Tozzi
• P. Sette
• N. Petrosillo
• F. Antonucci
• E. Boumis
• E. Nicastri
• U. Visco
• P. De Longis
• G. D’Offizi
• G. Ippolito

And the Resistance Study Group

INMI “L. Spallanzani” Rome

• G. Rizzardini
• V. Micheli
• A. Capetti
• P. Meraviglia
• M. Moroni
• L. Sacco University Hospital

Milan

• The Patients

ACKNOWLEDGEMENTS

University of Rome “Tor Vergata” F Ceccherini-Silberstein C.F. Perno M.M. Santoro

• V. Svicher
• L. Fabeni
• V. Cento
• C. Alteri
• M. Pollicita
• F. Scopelliti
• D. Di Pinto
• M. Romani
• A. Bertoli
• S. Dimonte
• R. Salpini
• F. Stazi
• M. Andreoni
• L. Dori
• L. Sarmati

AR Buonomini University of Turin

• G. Di Perri
• V. Ghisetti

Infectious Diseases Unit Florence

• S. Lo Caputo
• F. Mazzotta