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Presentación de PowerPoint - Fundació LLuita contra la SIDA

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Presentación de PowerPoint - Fundació LLuita contra la SIDA
Actualització en tropisme
Roger Paredes
IrsiCaixa i Lluita contra la SIDA
Hospital Germans Trias i Pujol
Predicció tropisme
CD4
Binding
Co-receptor
Binding
gp41
gp120
V3 loop
CD4
Cell
Membrane
CCR5/CXCR4
(R5/X4)
Adapted from Doms et al. Genes Dev. 2000;14:2677-2688.
Virus-Cell
Fusion
Predicció tropisme
Dual
tropic
X4 (SI)
CD4
CCR5
CXCR4
T-cell lines
R5 (NSI)
Primary lymphocytes
CD4 Naïve
CD4 memory
Monocyte/macrophages
Predicció tropisme
Koot M, et al: Prognostic Value of
HIV-1 Syncytium-Inducing
Phenotype for Rate of CD4+ Cell
Depletion and Progression to AIDS.
Annals Int Med 1993
Predicció tropisme
Conclusions MERIT Trial
1.
Twice-daily maraviroc was not
noninferior to efavirenz at <50
copies/mL in the primary analysis.
2.
However, 15% of patients would
have been ineligible for inclusion
by a more sensitive screening
assay.
3.
Their retrospective exclusion
resulted in similar response rates in
both arms
Cooper D et al. JID 2010
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
Predicció tropisme
Subject
18
Subject
07
Wee
k
Wee
k
Subject
19
Wee
k
Tsibris et al. PLoS One 2009
Subject
47
Wee
k
Predicció tropisme
Problemes clínics
• Tests fenotípics
– ESTA  Car, lent, no útil a CV < 1000 copies/mL
– MT2  Difícil de recuperar virus infecciosos, Restringit a laboratoris
especialitzats
• Tests genotípics
– X4 preexisteixen a virtualment tots els pacients  origen de la majoria de
fracassos a antagonistes del CCR5  Males eines diagnòstiques generen
incertesa al clínic  Mercat potencial dels antagonistes CCR5 reduït
– No validats
– Seqüenciació Poblacional  Poc sensible  Se’ns escapen X4
– 454 (deep sequencing)  En desenvolupament  Detecta X4 a tothom 
Punt de tall?
– Plasma vs. PBMC  Són equivalents pel diagnòstic?
Temes tractats
• Tests fenotípics
– ESTA a PBMCs
• Tests genotípics
– Tropisme a DNA episomal
– Milloria de la predicció fenotípica a partir del genotip
– Tests genotípics de tropisme més exactes
– Tropisme genotípic i resposta a maraviroc
Temes tractats
• Tests fenotípics
– ESTA a PBMCs
• Tests genotípics
– Tropisme a DNA episomal
– Milloria de la predicció fenotípica a partir del genotip
– Tests genotípics de tropisme més exactes
– Tropisme genotípic i resposta a maraviroc
Toma J et al -. Determining HIV-1 Coreceptor Tropism Using PBMC
Proviral DNA Derived from Aviremic Blood Samples. Poster 541
• Tropism was successfully determined in all 20 PBMC samples
– 10/10 viremic  PBMC and plasma tropism concordant (8 R5, 2 DM).
– 10/ WITH low or undetectable viral load 6 R5 and 4 DM.
• Extensive homology of plasma and PBMC Env sequences.
• No phylogenetic distinctions between plasma and PBMC
samples from the 5-paired samples.
• Tropism of clones derived from plasma and PBMC were
concordant:
– one DM had solely of dual-tropic variants
– the other DM virus population was comprised predominantly of R5
variants with a minor subpopulation of dual-tropic variants
– 3 R5 virus populations were comprised of R5 variants
Temes tractats
• Tests fenotípics
– ESTA a PBMCs
• Tests genotípics
– Tropisme a DNA episomal
– Milloria de la predicció fenotípica a partir del genotip
– Tests genotípics de tropisme més exactes
– Tropisme genotípic i resposta a maraviroc
Babic D et al. - Predicting HIV-1 Co-receptor usage and Response to
CCR5 Inhibitor Therapy through Episomal cDNA. Poster 540
PATIENTS AND METHODS:
14 patients from ACTG 5211 study who failed vicriviroc therapy
 5 with emergence of DM virus
PBMCs collected from study entry through week 48
Co-receptor usage determined based on phenotypes conferred by episomal fulllength env genes or V3 genotypes
Babic D et al. - Predicting HIV-1 Co-receptor usage and Response to
CCR5 Inhibitor Therapy through Episomal cDNA. Poster 540
Episomal env sequences were amplified in 81% (74/91) of PBMC samples. 
Mostly in subjects with high VL
No. of patients
(n=14)
Phenotype/Genotype
(episomal cDNA)
Trofile assay
(plasma RNA)
2
DM
DM
8
R5
R5
1
X4 at baseline
X4 at week 8
1
DM at week 2
DM at week 48
1
R5
DM
1
DM
R5
Babic D et al. - Predicting HIV-1 Co-receptor usage and Response to
CCR5 Inhibitor Therapy through Episomal cDNA. Poster 540
CONCLUSIONS
1.
The Trofile assay and phenotypic assay based on episomal env sequences
were largely concordant.
2.
In two patients, episomal sequence analysis predicted the emergence of
X4 or dual-tropic virus in plasma viral RNA.
3.
These results support the use of episomal cDNA as an additional approach
to predict the virologic response to entry inhibitors.
4.
However, subjects were essentially viremic through the study follow-up.
The efficiency of episomal DNA recovery in aviremic subjects might be
lower than 80%
Temes tractats
• Tests fenotípics
– ESTA a PBMCs
• Tests genotípics
– Tropisme a DNA episomal
– Milloria de la predicció fenotípica a partir del genotip
– Tests genotípics de tropisme més exactes
– Tropisme genotípic i resposta a maraviroc
Victoria Sánchez et al. A highly sensitive and specific model for predicting
HIV-1 tropism in treatment-experienced patients combining
interpretation of V3 loop sequences and clinical parameters. Poster 543
MOTIVATE 2
EAP maraviroc
Clinical cohort
4 patients screened
15 patients screened
157 patients screened
4 patients enrolled
4 samples collected
1 patient excluded
16 patients excluded
14 patients enrolled
14 samples collected
159 patients enrolled
170 phenotype / genotype samples
141 patients enrolled
152 samples collected
Victoria Sánchez et al. A highly sensitive and specific model for predicting
HIV-1 tropism in treatment-experienced patients combining
interpretation of V3 loop sequences and clinical parameters. Poster 543
V3 sequence
Patients
characteristics
Clinical
parameters
Binary multivariate regression analyses
Treatment-experienced patients (Nagelkerke R square: 0.937)
Geno2pheno clinical model 15% prediction
Geno2pheno clonal model 1% prediction
Net charge prediction
Nadir CD4
Exposure >3 and >4 ART classes
AIDS development
Victoria Sánchez et al. A highly sensitive and specific model for predicting
HIV-1 tropism in treatment-experienced patients combining
interpretation of V3 loop sequences and clinical parameters. Poster 543
V3 sequence
Patients
characteristics
Clinical
parameters
Binary multivariate regression analyses
Treatment-experienced patients (Nagelkerke R square: 0.937)
Geno2pheno clinical model 15% prediction
Geno2pheno clonal model 1% prediction
Net charge prediction
FPR score geno2pheno clinical
Years since HIV diagnosis
Nadir CD4
Log HIV-1 viral load
Exposure >3 and >4 ART classes
AIDS development
Victoria Sánchez et al. A highly sensitive and specific model for predicting
HIV-1 tropism
in treatment-experienced
patients combining
Predictive
modelling
of the co-receptor
usage
interpretation of V3 loop sequences and clinical parameters. Poster 543
Results (6):
P= - Log10 (1 / 1+e-y)
y = a1x1+a2x2+a3x3+a4x4+a5x5+a6x6
• a1 - a6: Regression coefficients of the variables in the model
1,0
(a1: -12; a2: -8; a3: -14; a4: +0.055; a5: -0.087 and a6: +3.6)
0,8
Sensitivity
• x1 - x6: Variables included in the equation :
X1: geno2pheno 15% FPR clinical model prediction
X2: V3 net charge prediction
X3: Exposure to > 3 antiretroviral classes
X4: FPR score of geno2pheno-clinical model
X5: Years since HIV infection diagnosis
X6: log HIV-1 RNA viral load
0,6
0,4
0,2
0,0
0,0
0,2
0,4
0,6
1 - Specificity
AUC: 0.966 (CI 95% 0.930-1.000, p<0.001)
Cut-off ≥ 5.75: CXCR4 (S: 96.6%, E: 92.3%)
0,8
1,0
Temes tractats
• Tests fenotípics
– ESTA a PBMCs
• Tests genotípics
– Tropisme a DNA episomal
– Milloria de la predicció fenotípica a partir del genotip
– Tests genotípics de tropisme més exactes
– Tropisme genotípic i resposta a maraviroc
Pou C et al. High Resolution Tropism Kinetics by Quantitative Deep Sequencing in
HIV-1 Infected Subjects Initiating Suppressive First-Line ART. Poster 544
Study design and per-subject tropism prediction
* FPR 10%
** FPR 10%, cut-off X4: 1%
Pou C et al. High Resolution Tropism Kinetics by Quantitative Deep Sequencing in
HIV-1 Infected Subjects Initiating Suppressive First-Line ART. Poster 544
ROC curves for different G2P FPRs
X4 prevalence in different compartments
% subjects with predicted X4
100
90
80
Significance relative to ESTA:
* P<0.05
** P<0.01
*** P<0.001
Plasma
PBMC T1
PBMC T2
70
* *
60
50
***
40
30
**
20
*
10
0
ESTA
PS
QDS
MT2
Phylogeny reconstruction for one subject
Test characteristics
PLASMA
PBMC
ESTA
PS*
QDS**
PS*
QDS**
MT2
Sensitivity
-
36.4
90
36.3
90.9
45.5
Specificity
-
94.4
82.4
100
55.6
100
PPV
-
80
75
100
55.6
100
NPV
-
70.8
93.3
73.1
90.9
76
* FPR 10%
** FPR 10%, cut-off X4: 1%
Pou C et al. High Resolution Tropism Kinetics by Quantitative Deep Sequencing in
HIV-1 Infected Subjects Initiating Suppressive First-Line ART. Poster 544
C O N C L U S I O N S
1.
V3-loop QDS is the most accurate tropism test, followed by V3-loop PS.
2.
The most reliable settings to explore the ability of V3-loop QDS to predict clinical outcomes are:
• Using a G2P[coreceptor] FPR ≤10% to define “predicted CXCR4 coreceptor usage”.
• Defining an “X4” result as the presence of predicted X4 viruses in at least 1% of the virus population.
3.
In the absence of CCR5 antagonist pressure, X4 viruses from plasma and PBMCs tend to cluster together
and there are no clear evidences of viral evolution over time. These findings suggest that either plasma or
PBMCs, and current or stored samples, could be used in most subjects for tropism testing.
4.
However, discrepancies in X4 detection between plasma and PBMCs indicate that each tropism assay
should be specifically validated in clinical trials assessing outcomes of CCR5 antagonist therapy.
Temes tractats
• Tests fenotípics
– ESTA a PBMCs
• Tests genotípics
– Tropisme a DNA episomal
– Milloria de la predicció fenotípica a partir del genotip
– Tests genotípics de tropisme més exactes
– Tropisme genotípic i resposta a maraviroc
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
•
To retrospectively evaluate virological response to maraviroc in treatment-naïve individuals
where viral tropism was predicted using population-based sequencing of the HIV-1 V3 loop
Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC)
Randomization
1:1:1
Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC)
Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC)
Original Trofile Screening
(6 weeks)
48 wk
96 wk
Primary
analysis
Patient eligibility criteria:
• ≥ 16 years of age
• Treatment naive
• R5 HIV-1 infection
0
• HIV-1 RNA ≥ 2,000 copies/mL
• No evidence of resistance to EFV, ZDV, or 3TC
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
Saag M, et al. 4th IAS 2007; Sydney, Australia. Abstract WESS104.
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
• Plasma samples from the MERIT study (N=705) were amplified in triplicate
using RT-PCR and sequenced using an ABI 3730
• Automated base-calling was performed using custom ReCall software
without manual review
• Tropism prediction using “geno2Pheno” co-receptor algorithm (g2P) with
5.75% FPR blinded to ESTA results and clinical outcome
• Outcome measures include: change in plasma viral load (pVL), percent
undetectable (<50 copies/mL) and time to tropism change
• Trofile “X4” and “D/M” results were combined into a single “non-R5”
category
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
-1
Concordant R5 (N=283)
-2
Concordant nonR5 (N=9)
ESTA nonR5, Geno R5 (N=39)
-3
ESTA R5, Geno nonR5 (N=20)
-4
Log Viral Load Change
0
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
0
8
16
24
32
40
Maraviroc BID; LOCF
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
48
Week
60
72
84
96
Concordant R5 (N=283)
0.6
Concordant nonR5 (N=9)
0.4
ESTA nonR5, Geno R5 (N=39)
ESTA R5, Geno nonR5 (N=20)
0.0
0.2
Percent copies <50
0.8
1.0
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
0
8
16
24
32
40
Maraviroc BID; Missing=Failure
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
48
Week
60
72
84
96
0.8
Genotype R5
(n= 322)
0.2
0.4
0.6
Genotype nonR5
(n= 29)
0.0
Percent with R5 by Original Trofile
1.0
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
0
8
16
24
Maraviroc BID
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
32
40
48
Week
60
72
84
96
0.8
ESTA R5
Genotype R5
0.2
0.4
0.6
Genotype nonR5
ESTA nonR5
0.0
Percent with R5 by Original Trofile
1.0
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
0
8
16
24
32
40
48
Week
Maraviroc BID
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
60
72
84
96
McGovern RA et al. Population-based Sequencing of the V3-loop is Comparable
to the Enhanced Sensitivity Trofile Assay (ESTA) in Predicting Virologic Response
to Maraviroc of Treatment-naïve Patients in the MERIT Trial. ORAL 92
CONCLUSIONS
• In MERIT patients re-screened as R5 by population-based V3 sequence
analysis, MVC BID was non-inferior to EFV, confirming previous results with
ESTA
• Performance of population-based sequencing is similar to that of Enhanced
Sensitivity Trofile assay where results were discordant
• Patients randomized to either MVC BID and QD called R5 by populationbased sequencing saw greater virological success than those called non-R5
• These results were also obtained with “deep” sequence analysis of V3
(see Abstract M-268)
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
Swenson et al. Large-Scale Application of “Deep” Sequencing
Using 454 Technology to HIV Tropism Screening. Poster 545
•
Screening plasma samples from the MVC arms of trials in treatment-naïve “TN” (MERIT) and
treatment-experienced patients “TE” (MOTIVATE-1 & -2 and A4001029) were examined. The
EFV arm was also examined for comparison.
Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC)
Randomization
1:1:1
Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC)
Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC)
Original Trofile Screening
(6 weeks)
ESTA vs 454
0
48 wk
96 wk
Primary
analysis
Patient eligibility criteria:
• ≥ 16 years of age
• HIV-1 RNA ≥ 2,000 copies/mL
• Treatment naive
• No evidence of resistance to EFV, ZDV, or 3TC
• R5 HIV-1 infection
Maraviroc no está aprobado en España
Saag M, et al. 4th IAS 2007; Sydney, Australia. Abstract
para el tratamiento de pacientes naïve
WESS104.
Swenson et al. Large-Scale Application of “Deep” Sequencing
Using 454 Technology to HIV Tropism Screening. Poster 545
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
Swenson et al. Large-Scale Application of “Deep” Sequencing
Using 454 Technology to HIV Tropism Screening. Poster 545
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
Swenson et al. Large-Scale Application of “Deep” Sequencing
Using 454 Technology to HIV Tropism Screening. Poster 545
Conclusions
“Deep” sequence analysis of the HIV V3-loop gives improved
prediction of virological response to MVC relative to the original
Trofile assay, and similar to the currently-available Trofile assay
(ESTA).
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
Alerta!
El reanàlisi amb ESTA i
454 es va fer a les
mostres d’screening
La seqüenciació poblacional
d’V3 es va fer en pacients
que van entrar a l’estudi, és a
dir, després de l’screening
amb Trofile
Efavirenz (EFV 600 mg QD) + Combivir™ (ZDV+3TC)
Randomization
1:1:1
Maraviroc (MVC 300 mg BID) + Combivir (ZDV+3TC)
Maraviroc (MVC 300 mg QD) + Combivir (ZDV+3TC)
Original Trofile Screening
(6 weeks)
0
48 wk
Primary
analysis
Maraviroc no está aprobado en España
para el tratamiento de pacientes naïve
96 wk
Conclusions
• Tests fenotípics
– Possible realitzar-los a PBMCs  De moment DNA total >> episomal
• Tests genotípics
– La seqüenciació poblacional d’V3 + screening previ amb Trofile és
equiparable a ESTA
– 454 és equiparable a ESTA a mostres pre-screening  454 és la millor
– 454 és l’eina genotípica més exacta però
• no disponible a molts laboratoris
• En desenvolupament tècnic / bioinformàtic
– El clínic pot sentir-se força tranquil fent genotip convencional (useu FPR
altes (>20%))
– Ambdós possibles a PBMCs o plasma a mostres actuals o guardades
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