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11/28/2011
1
BHIVA AUTUMN CONFERENCE 2011Including CHIVA Parallel Sessions
17–18 November 2011, Queen Elizabeth II Conference Centre, London
Dr Erasmus SmitBirmingham Heartlands Hospital
BHIVA AUTUMN CONFERENCE 2011Including CHIVA Parallel Sessions
17–18 November 2011, Queen Elizabeth II Conference Centre, London
Dr Erasmus SmitBirmingham Heartlands Hospital
COMPETING INTEREST OF FINANCIAL VALUE > £1,000:
Speaker Name Statement
Erasmus Smit None
Date 3 November 2011
11/28/2011
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The importance of ultraThe importance of ultra--deep sequencing in deep sequencing in
clinical practiceclinical practiceDr Erasmus Smit
Consultant VirologistHPA, West Midlands Public Health Laboratory
Heart of England Foundation Trust, Birmingham
3
OverviewOverview
� Basic Concepts:
◦ Concept 1: Where do drug resistance minority variants
(MV) come from?
◦ Concept 2: Sensitivity issues and disappearance of
mutations
◦ Concept 3: Different ultra sensitive assays
◦ Concept 4: Cut-offs, repeatability and assay errors
� Clinical Importance of DR Minority Variants for:◦ NNRTIs
◦ 2nd generation NNRTIs, PIs, INI and CCR5 antagonists
◦ Treatment experienced
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Concept 1: Where do drug resistance Concept 1: Where do drug resistance minority variants (MV) come from?minority variants (MV) come from?
� Transmitted Drug Resistance (TDR)+ disappearance
(archived)
� Natural low level unselected mutations (de novo
production)
� Drug pressure selected clones (archived)
� Evolution of minority CXCR4 tropic variants over time
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Rate of TDR mutation lossRate of TDR mutation loss
Jain. JID 2011:203
*
*HR for replacement (95% CI)
=77.5 (14.7–408.2), P<0.001
6Kaplan-Meier plot of cumulative probability of mutation replacement in 75 ART-naïve pts from two seroconverter cohorts
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TDR and MVTDR and MV
A Buckton. Antiviral Therapy 2011
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•Most published studies used AS-PCR
•Studies looked at fold increase of common mutations in Tx naive pts
•Found at least 2-3 fold increase in mutation detection
•Sys review & met analysis of 13 studies
•Studies are small and someshow huge fold increase
Viral load
Time
Drug-susceptible quasispeciesDrug-resistant quasispecies
Treatment begins
Pt stop Tx
5
50
500
5000
50000
500000
5000000
Pt fails Tx
Concept 2: Sensitivity issues and Concept 2: Sensitivity issues and disappearance of mutationsdisappearance of mutations
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9
X
VirologicFailure
XX XX
X
X
XX
X X
X
X
cART
ReboundViraemia
Survivalof Fittest
PCR
XXXXXXXXXXXX
XXXXXX
SingleMutation
MultipleMutations
Standard Genotypic test
PCR
PCR
PCR
Concept 3: Current standardConcept 3: Current standard resistance testresistance test
Standard Genotypic test
X= drug resistance mutation
10Concept 3: Different ultra sensitive assaysConcept 3: Different ultra sensitive assays
X
X
X
ReboundViraemia X
XX
XX
XXXX
XX
P C R
X
Ultra deep sequencing
Allele specific PCR
Cycle number
Delta ct
Mutant template
Wild type template
PCR
PCR
P C RP C RP C R P C R
Flu
ore
scence
SingleMutation
MultipleMutations
11/28/2011
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11Concept 3: Different ultra sensitive assaysConcept 3: Different ultra sensitive assays
X
X
X
ReboundViraemia
X
Allele specific PCR
Cycle number
Delta ct
Mutant template
Wild type template
PCR
Flu
ore
scence
� Allele specific PCR (AS-PCR)◦ Selective amplification of a resistance
codon◦ Very sensitive (0.01 to 0.1%)◦ Amplifies only one mutation at a time◦ Limited by genetic variation in the
region complementary to the primers →variability
◦ Subtype issues◦ Quick but impractical for clinical use
SingleMutation
MultipleMutations
12Concept 3: Different ultra sensitive assaysConcept 3: Different ultra sensitive assays
X
X
X
ReboundViraemia X
XX
XX
XXXX
XX
P C R
Ultra deep sequencingPCR
P C RP C RP C R P C R
� Ultra-deep Sequencing (UDS) or second generation sequencing◦ Clonally amplified fragments akin to single
genome sequencing x 1000◦ High throughput ◦ Ability to detect archived / minority variants at
a 0.1 - 1% level◦ Current limits are: costs, high viral load,
processing huge amount of data, performance issues between different labs with no EQA programmes and clinical interpretation
SingleMutation
MultipleMutations
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Which technology is the best?Which technology is the best?
Ultra deep sequencing
� Sensitive (0.1- 1%)
� Higher viral loads (1000-10,000 copies/ml)
� Quantitative
� Clonal sequencing and info on mutation linkage
� Expensive & capital outlay
� Labour intensive
� Automation can reduce cost for use in clinical practice
� Needs good bioinformatics software
Allele specific PCR
� Very sensitive (0.01 – 0.1%)� Lower viral loads (≤1000
copies/ml)� Quantitative with calibration
standards � Needs to optimised for each
subtype� Detects one mutation at a time� Less likely to make it into clinical
practice� Needs good quality control and
standardization� >>Expensive than normal PCR� << Expensive than standard
genotypic resistance test
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Concept 4: CutConcept 4: Cut--offs, repeatability offs, repeatability and assay errorsand assay errors
Tests
� RT-PCR in tests also makes mistakes (UDS)
� Inaccuracy of very low % MV (AS-PCR)
� Lower limit of detection for AS-PCRs differ in studies
� Need for lab external quality assessments and validation
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Studies
� Usually small
� Unable to define clinically relevant
MV cut-offs
� AS-PCR studies only assessed the
effect of selected mutations
� Few UDS studies explored
mutation linkage
� Not all studies showed or looked
for enhancement of MV clones
following VF
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OverviewOverview
� Basic Concepts:
◦ Concept 1: Where do drug resistance minority variants
(MV) come from?
◦ Concept 2: Sensitivity issues and disappearance of
mutations
◦ Concept 3: Different ultra sensitive assays
◦ Concept 4: Cut-offs, repeatability and assay errors
� Clinical Importance of DR Minority Variants for:◦ NNRTIs
◦ 2nd generation NNRTIs, PIs, INI and CCR5 antagonists
◦ Treatment experienced
15
NNRTIsNNRTIs
LI, JAMA. 2011;305(13):1327-1335
� A systematic review and pooled analysis1
� 10 studies (n=985); DR MV (n=187) was associated ↑ risk of VF (HR = 2.3 [1.7-3.3])
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LI, JAMA. 2011;305(13):1327-1335
Figure 3. Effect of MV and Antiretroviral Therapy Adherence on Virologic Failure
17
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Fig. 2. A threshold quantity of K103N at baseline that is predictive of virologic failure
(2000 copies/ml)
Goodman AIDS 2011
Retrospective AS-PCR analysis of GS-01-934 (EFV+TDV/FTC vs EFV +AZT/3TC)
Mutational load = % DR MV x sample viral load
5/6 individuals failed their EFV based therapy with a predicted odds ratio of 47 (95% CI 5.2–429.2)
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22ndnd gen NNRTIs / PIs / INI / CCR5 antagonistsgen NNRTIs / PIs / INI / CCR5 antagonists
� No ↑ risk of VF with ETV1 or LRV2 when baseline DR MV present
� No ↑ risk of VF with 1st-line PI-based therapy3,4,5
� TDR INI mutations are uncommon – no baseline resistance tests recommended
� UDS for INI will become important once TDR mutations becomes more prevalent
� INI mutations disappear quickly – potential UDS added value6
� Re-analysis of Merit data using deep V3 loop sequencing showed similar sensitivity as ESTA7
191) Geretti 51st ICAAC 2011 2) Craig 6th IAS 2011 3) Garcia-Lerma J Virol 2003 4) Peuchant AIDS 20085) Simen JID 2009 6) Ferns AIDS 2009 7) Swenson CID 2011
Swenson CROI 200920
Pfizer 1029 = MVC vs placebo in Tx experienced with D/M or X4 tropic virus + OBT
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Treatment ExperiencedTreatment Experienced
1)Roquebert AIDS 2006 2) Svarovskaia JAIDS 2007 3) Le PLos One 2009 4) Varghese JAIDS 2009
5) Codoňer PLos 2011
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� No study conclusively demonstrated clinical usefulness of
detecting MV in 2nd line or salvage Rx
� An association between MV and VF has been suggested1,2
� DR MV correlate with historical cART use which is useful in
complex pts3
� NNRTI experienced pts - UDS often detected additional
major NNRTI mutations 4,5
� Adequately powered studies are need to define clinical
relevant cut-offs and explore potential of mutation linkage
Effective use of UDS Effective use of UDS
� Past history of strategic / chaotic treatment interruptions
� Pts with high risk of X4 tropic MV e.g. low CD4 counts
� Pts with unknown past cART history
� Highly treatment experienced patients with ltd options
� In all children after 2nd regimen failure
� Patients in whom superinfection is suspected
� When TDR mutations are present on standard resistance
test – expect other DR MV
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Drug Class Added Value Level of evidence
1st gen NNRTIs – naive (EFV, NVP)
Added value IIa
2nd gen NNRTI - naive(ETV, RPV, LRV)
No added value but under powered
IIb
PI/r - naive No added value but under powered
IIb
2nd gen NNRTI -experience
Potential added value, needs to be proven
III
INI - naive Potential added value in future when TDR becomes prevalent
III
INI - experienced Potential added value, needs to be proven
III
Geno2pheno Tropism Added value IIa
Added value of UDS (currently)Added value of UDS (currently)
Conclusion Conclusion --11
� Current resistance tests are inadequate to detect MV or
archived virus
� US resistance assays increase detection of TDR MV by
at least 2–3-fold
� DR MV increase the risk of VF on EFV/NVP by 2-3 fold
� AS-PCR is a good research tool (0.01% MV sensitivity)
� UDS provides added value for low genetic barrier drugs
& ↑ sensitivity of tropism assay
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Conclusion Conclusion –– 22 (clinical cut(clinical cut--offs)offs)
� Without clinical cut-offs the PPV is low i.e. more Rx
success than Rx failures in those with low % MV →
↑use of more expensive drugs
� Clinical cut-offs have not yet been established and all
results will have to interpreted with caution
� Clinical cut-offs will probably differ for each mutation
and be dependent on viral load and other drugs in
regimen
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