Resistance Interpretation Starting from Basic

1
Resistance InterpretationStarting from Basic

• Iván Meléndez-Rivera, MD, FAAFP, AAHIVMS
• Assistant Professor Family Medicine Department
  Ponce School of Medicine
• Medical Director
• Centro Ararat, Inc. Ponce, P.R.

2
Disclosure of Financial RelationshipsThis
speaker has the following significant financial
relationships with commercial entities to
disclose

• Speaker
• Abbott Pharmaceutical
• Bristol-Myers Squibb
• Boehringer
• Gilead
• GlaxoSmithKline
• Monogram Science
• Roche Pharmaceutical
• Johnson Johnson

• Principal Investigator
• Abbott Pharmaceutical
• Bristol-Myers Squibb
• Roche Pharmaceutical
• GlaxoSmithKline
• Pfizer

This slide set has been peer-reviewed to ensure
that there are no conflicts of interest
represented in the presentation.
3
Why does Resistance Develop?

• HIV can develop resistance quickly because
• HIV reverse transcriptase makes errors in
  matching bases when converting HIV RNA to DNA
• HIV replicates at a rapid rate 1 to 10 billion
  viral particles produced daily
• In an untreated infected host, every possible
  mutation occurs at least once every 24 hours,
  some of which may impart drug resistance.

4
When does resistance occur?

• Resistance occurs when the virus has an
  opportunity to replicate in the presence of
  sub-inhibitory concentrations of drugs.
• Non-adherence to the treatment regimen.
• Pharmacokinetics or drug-drug interactions lead
  to inadequate drug exposures
• The pattern of resistance that is observed can be
  influenced by
• Inherent viral factors
• Host factors (HLA type) can lead to immunologic
  escape mutations

5
What is viral resistance?

• A continuum of reduced susceptibility of HIV to
  the inhibitory effects of drugs

More Susceptible
More Resistant
6
What is wild type virus?

• HIV that has not been exposed to drug therapy
• The predominant sequence of nucleotide bases in a
  heterogeneous mixture of virions
• Most fit form of HIV in the absence of drug

7
Selective Pressures of Therapy
Treatment begins
Drug-susceptible quasispecies Drug-resistant
quasispecies
Selection of resistant quasispecies
Viral load

• Incomplete suppression
• Inadequate potency
• Inadequate drug levels
• Inadequate adherence
• Pre-existing resistance

Time
8
Viral dynamics and drug resistance
t
VIRAL LOAD
Mono and dual therapy
wild type virus(drug susceptible)
mutant virus(drug resistant)
TIME
9
Viral dynamics and drug resistance
t
VIRAL LOAD
HAART
wild type virus(drug susceptible)
mutant virus(drug resistant)
TIME
10
How is resistance measured?

• By phenotype (IC50)
• IC50
• the minimum concentration of a drug needed to
  inhibit the growth of the virus by 50 in vitro
• IC50 is analogous to MIC90
• IC90 is not sufficiently reproducible for routine
  clinical use
• The lower the IC50, the more potent the drug

11
Identifying Mutations

• How do we identify a resistance mutation?
• M 184 M
• 184 is the codon position
• M is the wild type amino acid

12
Identifying Mutations

• How do we identify a resistance mutation?
• M 184 V
• 184 is the codon position
• V is the mutant aa
• M is the wild type amino acid

13
Wild Type and Mutant Virus

• M184M Wild type amino acid at position 184
• M184V Mutant amino acid at position 184
• M184I Mutant amino acid at position 184
• M184M/V Mixture of WT and Mutant amino acids at
  positions 184

14
Atazanavir Mutation

• How do we identify a resistance mutation?
• I50L
• 50 is the codon position L( Leusine) is the
  mutant aa
• I (Iso-leusine)is the wild type amino acid
• Some studies demonstrate increase in
  susceptibility when I50L appears
• Less probability with boosted ATV

15
Mutant Virus

• Reduces susceptibility to some drugs
• Most of the time keeps some drugs available
• Always benefits and risk
• In other word RESISTANCE SHOWS

16
How many mutations are needed for resistance?

• Single point mutations
• M184V (emtricitabine, lamivudine)
• K65R (tenofovir)
• L74V (didanosine)
• K103N (NNRTI)
• Multiple mutations
• TAMs (stavudine, zidovudine)
• Protease (protease inhibitors)

17
Mutations by Numbers
18
Types of Resistance Tests

• Genotype
• HIV gene sequencing of the patients virus to
  detect mutations known to confer drug resistance
• Phenotype
• Measures ability of a recombinant virus derived
  from the patient sample to grow in different
  concentrations of antiretroviral drugs
• Virtual phenotype
• Use of genotype results to predict phenotypic
  susceptibility based originally on database of
  paired genotype and phenotype data or, more
  recently, through scores derived from linear
  regression analysis

Hirsch MS, et al. Clin Infect Dis.
200337113-128.
19
Genotypic and Phenotypic Resistance Tests
20
HIV Resistance Testing Consensus
Routine Use of Resistance Tests Recommended by
U.S. Department of Health and Human Services
(July 2003)
International AIDS Society-USA (CID, July 2003)
EuroGuidelines Group (AIDS, Feb 2001)

• RECOMMENDED USES
• Acute/Recent (lt2 yr) HIV infection
• Incomplete viral suppression
• Treatment failure
• Pregnant women with viremia

21
When to Use Resistance Testing
Especially if exposure to someone receiving
antiretroviral drugs is likely or if prevalence
of drug resistance in untreated patients 5
(European 10).
22
The Use of Drug Resistance Testing
23
Advantages and Disadvantages of Genotype Testing

• Advantages
• Rapid turnaround (1-2 wks)
• Less expensive than phenotyping
• Detection of mutations may precede phenotypic
  resistance
• Widely available
• More sensitive than phenotype for detecting
  mixtures of resistant and wild-type virus

• Disadvantages
• Indirect measure of resistance
• Relevance of some mutations unclear
• Unable to detect minority variants (lt 20 to 25
  of viral sample)
• Complex mutational patterns may be difficult to
  interpret

24
Advantages and Disadvantages of Phenotype Testing

• Advantages
• Provides direct and quantitative measure of
  resistance
• Methodology can be applied to any antiretroviral
  agent, including new drugs, for which genotypic
  correlates of resistance are unclear
• Uses 2 clinical cutoffs (CCO) derived from
  clinical cohorts to define spectrum of resistance
• CCO1 value below which reduced virologic
  response is likely
• CCO2 value above which a virologic response is
  unlikely
• Indicates which drugs have partial activity
• Can assess interactions among mutations
• Accurate with non-B HIV subtypes

25
Advantages and Disadvantages of Phenotype Testing

• Disadvantages
• Susceptibility cutoffs not standardized between
  assays
• Clinical cutoffs not defined for some agents
• May be unable to detect minority variants for
  some mutations (lt 20 to 25 of viral sample)
• Complex technology with longer turnaround ( 3
  wks)
• More expensive than genotyping

26
Advantages and Disadvantages of Virtual
Phenotype Testing

• Advantages
• Similar advantages to genotype (turnaround time,
  cost, sensitivity)
• Defines resistance based on database of in vivo
  responses in treated patients
• Uses 2 clinical cutoffs (CCO) to define spectrum
  of resistance
• CCO1 value below which response expected to be
  comparable to wild type
• CCO2 value above which most virologic response
  would be lost
• Indicates which drugs have partial activity

27
Advantages and Disadvantages of Virtual
Phenotype Testing

• Disadvantages
• Is an estimated phenotype based on the patients
  genotype, not an actual measured phenotype
• Reliability will depend on the accuracy of the
  genotype
• Available only from 1 vendor
• More expensive than genotype alone
• Methodology of linking genotype to phenotypic
  database not intuitively obvioususes a
  proprietary virtual phenotype linear regression

28
HIV drug resistance cutoffs

• Clinical cutoffs
• based on outcome data from clinical trials
• Biologic cutoffs
• based on natural variability of wild-type viruses
  from patients
• Reproducibility cutoffs
• based on assay variability with repeated testing
  of patient samples

Highest
Clinical Relevance
Lowest
29
Defining Clinical Cutoffs Based on Loss of Drug
Effect
0
30
When to Use Which Test?

• Graphic is a gross oversimplification
• Utility of genotypic testing is greatest earlier
  in treatment continuum
• Utility of phenotypic testing increases with
  treatment experience

31
General Limitations of Resistance Testing

• Lack of uniform quality controls across different
  laboratories
• High cost compared with other tests routinely
  done in HIV care
• Cannot be reliably performed with HIV RNA lt
  500-1000 copies/mL
• May not be able to detect minority populations of
  resistant virus if they account for lt 20 of the
  sampleespecially common after drug
  discontinuation
• Resistant strains that are in viral reservoirs
  also not detected

32
Interpreting Resistance Tests A Step-by-Step
Approach

• Collect all prior resistance tests for review
• Make sure current treatment, level of adherence,
  HIV RNA, and CD4 cell count are known
• Take each drug class individually
• Start with NNRTIs
• Next is NRTIs M184V alone vs broader resistance
• Finish with PIs Is resistance present at all? If
  so, is cross-resistance minimal, moderate, or
  high?
• Choose new regimen incorporating resistance data,
  treatment history, and results from clinical
  studies

33
Genotype
34
New dx, ART naive
35
Phenotype
36
PhenoSense Inhibition Curves
Patients virus is sensitive to the drug
Inhibition
FC1
IC50(patient) IC50(control)
Fold Change
37
Reduced Susceptibility Data
FC8.97
Inhibition
38
Phenotypic Susceptibility RelationshipBetween
Drug Concentration and Viral Inhibition
100
Wild-type Resistant
Inhibition of Virus Replication ()
50
Fold resistance
0
Resistant IC50
Wild-type IC50
Drug Concentration
39
Hypersusceptibility
40
Interpreting Phenotypic Data
Probability of response
Zone of Intermediate Response
Fold Change
41
Current Rx ZDV/3TC, NVP HIV RNA 45,000, CD4 350
42
Current Rx ZDV/3TC, ATV CD4 424, HIV RNA 14,000
43
Current Rx ZDV/3TC, ATV CD4 424, HIV RNA 14,000
44
Aug 05 CD4 280 HIV RNA 3500 Current Rx FPV/r,
ABC, 3TC, ENF
45
Genotype or Phenotype or Both?

• Genotype can give early warning that resistance
  is on the horizon can detect mutant populations
  more quickly
• Phenotype directly measures virus vs. drug can
  show degree of resistance
• In treatment experienced patients, both tests
  together can provide the optimal picture of
  resistance

46
Aug 04 CD4 210 HIV RNA 2000 Current Rx FPV/r,
ABC, 3TC, ENF
47
(No Transcript)
48
Replication Capacity
49
Which virus is more fit?RC, Fitness, and
Selective Pressure
WT virus replicates better than mutated virus in
the absence of drug
100
Replicates better than WT virus at high drug
concentrations
Viral Replication
Resistant
Increasing Drug Concentration
0
50
Replication Capacity
The RC value indicates the ability of the
patients virus to replicate relative to the
median of wild-type population of viruses
The median RC value of a wild-type population of
viruses defined as 100
51
Resistance Transmission
52
Persistence of Transmitted Resistant HIV

• Natural history of transmitted resistance in 12
  individuals
• NNRTI only (n 7)
• NNRTI PI (n 2)
• NNRTI NRTI PI (n 1)
• NRTI NNRTI (n 1)
• NRTI PI (n 1)
• Persistence of resistant virus acquired during
  primary infection is in contrast to reversion
  that occurs in those that selected for resistance
  on therapy

Little S, et al. CROI 2004. Abstract 36LB.
53
US surveillance of HIV drug resistance

• Drug resistance in 787 newly diagnosed ART-naïve
  subjects from 89 sites in 6 states (20032004)

Prevalence of resistance by drug class
Bennett D, et al. 12th CROI, Boston 2005, 674
54
Baseline Resistance Increases the Risk of
Virologic Failure

• Prospective, international study of 571 ART-naive
  patients
• 95 (546/571) had baseline resistance testing
• 16 (90/546) showed baseline NRTI and/or NNRTI
  resistance

Borroto-Esoda K, et al. CROI 2004. Abstract 672.
55
Persistence of transmitted resistant virus

• 1. Little SJ, et al. XII Resistance Workshop, Los
  Cabos 2003, 115 2. Ravaux I, et al. 2nd IAS,
  Paris 2003, 822 3. Brenner B, et al. J Virology
  2002 7661753 4. Chan K, et al. AIDS 2003
  171256

• Reversion of MDR is rare even after 1 year

56
Complications due to resistance
57
Discordance due to mixtures
58
Major explanations of discordance

• Incomplete genotypic algorithms (rules)
• Novel mutations
• Improper weighting of mutations in algorithms
  (both over- and under-weighted)
• Non-B subtype resistance patterns
• Immaturity of interpretive algorithms
• Mixtures present
• Suppressive mutations or re-sensitization
  caused by specific mutations (e.g. 184V)

59
M184V Increases Susceptibility to d4T, ZDV, and
TDF
60
REPLICATION CAPACITY M184 vs. M184V
M184
M184V
180
140
100
RC Relative to wild-type virus
60
20
20
RC
197
208
n
Mean RC
52
28
61
Advanced Resistance Test
62
Entry Assay

• Unique assay based on the PhenoSense HIV assay
  technology platform
• Multiple applications
• evaluate inhibitors of virus entry
• fusion inhibitors (gp41)
• co-receptor antagonists (CCR5, CXCR4)
• receptor binding (CD4 - gp120)
• define co-receptor tropism
• measure neutralizing antibody
• elucidate mechanisms of virus entry

63
The New HIV Entry Report
64
(No Transcript)
65
SUMMARY
66
Why is understanding resistance important?
67
Choice of Assay by Patient Category
68
Drug Resistance Testing in Clinical Practice
Summary

• Indications for use of resistance testing have
  greatly expanded
• Genotype preferred
• Treatment naive acute or chronic infection
• Early virologic failure
• Patient no longer on therapy
• Phenotype, virtual phenotype, or combined
  phenotype/genotype preferred
• High-level resistance to NRTIs or PIs on genotype
• Multiple regimen failure with limited treatment
  options
• Optimal selection of next regimen requires
  integration of resistance data, treatment
  history, and results from clinical studies

69
How to get help?

• Clinical Consultation Florida/Caribbean AIDS
  Education Training Center
• Iván Meléndez-Rivera, MD
• drmelendez_at_centroararat.org
• 787-284-5874 (fax)
• 787-284-5884 (office)

70
END

• DIOS LES BENDIGA A TODOS
• Dr. Iván Meléndez Rivera
• 787-284-5884
• drmelendez_at_hotmail.com
• drmelendez_at_centroararat.org