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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

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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

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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

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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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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

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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

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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

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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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