Download Resistance 101: Interpreting and Using the Data

Resistance 101:
Interpreting and Using the Data
Harry Lampiris, MD
Professor of Clinical Medicine, UCSF
Infectious Disease Section, Medical Service
San Francisco VA Medical Center
ACTHIV 2012: A State-of-the-Science Conference for Frontline Health Professionals
Learning Objectives
Upon completion of this presentation, learners
should be better able to:
• State when it is appropriate to perform viral
resistance testing in HIV-infected patients
• Describe the uses and limitations of HIV genotypic
and phenotypic resistance testing
• Describe the utility of HIV tropism assay testing
ACTHIV 2012: A State-of-the-Science Conference for Frontline Health Professionals
Faculty and Planning Committee
Disclosures
Please consult your program book.
Off-Label Disclosure
There will be no off-label/investigational uses
discussed in this presentation.
ACTHIV 2012: A State-of-the-Science Conference for Frontline Health Professionals
Important Resources
• IAS – USA Website http://www.iasusa.org
• Stanford resistance database http://hivdb.stanford.edu
• DHHS Guidelines March, 27, 2012
http://www.aidsinfo.nih.gov/guidelines/
• 2011 Update of Drug Resistance Mutations in HIV
(Topics HIV Med, 2011; 19:156 – 163)
• IAS USA Resistance Testing Guidelines
(Hirsch M et al, Clin Infect Dis 2008, 47:266 – 85)
HIV Drug Resistance Overview
• Resistance is due to mutations in the HIV virus which make it
less susceptible to antiretroviral drugs which contributes to
treatment failure
– Mutations can impair viral fitness (ability of virus to replicate in vitro)
– Mutations can make the virus less damaging to the immune system (in
patients)
• Mutations leading to drug resistance can occur due to drug
selection in a patient or can be acquired (primary drug
resistance)
• In patients receiving ARVs, incomplete virologic suppression
leads to mutations associated with resistance due to
– Partial adherence to regimen
– Sub-optimal dosing of drugs
– Drug interactions
– Incomplete absorption in intestinal tract
Hirsch M, et al. Clin Infect Dis. 2008;47:266-285
HIV Drug Resistance: How It Develops
Treatment begins
Drug-susceptible quasispecies
Viral load
Drug-resistant quasispecies
Selection of resistant
quasispecies
Incomplete suppression
Inadequate potency
Inadequate drug levels
Inadequate adherence
Pre-existing resistance
Time
Hirsch M, JAMA 1998; 279:1984
We can measure resistance to all available
drugs except maraviroc in 2012
NRTIs
NNRTIs
Protease Inhibitors
Entry Inhibitors
Abacavir
Efavirenz
Atazanavir
Enfuvirtide
Didanosine
Nevirapine
Darunavir
Maraviroc
Emtricitabine
Etravirine
Fosamprenavir
Lamivudine
Rilpivirine
Indinavir
Stavudine
Lopinavir
Tenofovir
Nelfinavir
Zidovudine
Ritonavir
Saquinavir
Tipranavir
Integrase Inhibitors
Raltegravir
Resistance Testing: DHHS Guidelines
• Recommended when entering into care (consider repeating when
initiating therapy); genotypic testing is preferred
• Should be done for all pregnant women prior to therapy, or for
those entering pregnancy with a detectable HIV VL on therapy
• Perform when managing suboptimal VL decrease
• In the setting of viral failure, testing should be done while the
patient is on therapy, or within 4 wks of stop
• Recommended to assist in selecting active drugs for pts with viral
failure & VL> 1000; Consider in VL>500
• Phenotypic testing when complex mutations especially with PIs
• Consider genotypic testing for integrase inhibitor resistance when
appropriate
DHHS Guidelines available at http://aidsinfo.nih.gov/guidelines March 27, 2012; page C-12
Prevalence of Transmitted HIV Drug Resistance in US,
2006-2009
• Genotypic analysis of samples from newly diagnosed patients in
CDC National HIV Surveillance System (N = 12,668)
20
All cases with sequences
Cases classified as recent infections
Cases classified as long-standing infections
Cases (%)
16 15.6
12
7.8
8
6.8
4.1
4
0
1 or more
1 class
2 class
3 class
NNRTI
Transmitted Drug Resistance Mutations
Ocfemia MC, et al. CROI 2012. Abstract 730.
NRTI
PI
Methods & Limitations of Resistance Testing
• Genotype:
– Direct sequencing of viral genes RT and PR, less commonly IN and ENV
– Resistance to specific drugs is inferred based on known mutations
– Mutations are detected only if mutant virus is at least 5-20% of virus
population
– Frequently called “population-based sequencing” versus more
sensitive assays (“ultra-deep sequencing”)
• Phenotype
– Grow virus in culture with various amounts of drugs added
– Direct measure of viral resistance
• Resistance tests are most accurate in assessing the sensitivity to drugs
which the patient is currently receiving
DHHS Guidelines available at http://aidsinfo.nih.gov/guidelines March 27, 2012
HIV Genotype
• Involve sequencing of various HIV genes and comparing results to
reference wild type (wt) strain
– RT gene for NRTI and NNRTI resistance
– PI gene for PI resistance
– Integrase Gene for integrase inhibitor resistance (separate test)
• Sometimes a single mutation leads to high level resistance to a
drug; sometimes multiple mutations are required
• Multi-Drug Resistance (MDR) mutations can decrease
susceptibility to many or all drugs in a single class
DHHS Guidelines available at http://aidsinfo.nih.gov/guidelines March 27, 2012
The six mutations every HIV provider
should know
• NRTI
– M184 V
– Thymidine-associated mutations (TAMs)
– K65R and L74V
• NNRTI
– K103N
– Y181C
• PI, IN
– none
DHHS Guidelines available at http://aidsinfo.nih.gov/guidelines March 27, 2012
HIV Phenotype
• Standard phenotypic testing
– Results usually expressed as fold-change in susceptibility
compared to a laboratory control isolate (IC 50 patient
isolate divided by IC50 wild type virus)
– Interpretation of drug activity dependent on methodology
used to define cutoffs (clinical, biological, technical)
• Virtual phenotype testing
– Matches genotypic data against database of virus samples
with paired GT and PT data
– Confidence level based on number of matching genotypes
within the database
Hirsch M, et al. Clin Infect Dis. 2008;47:266-285.
Probability of response
Interpreting Phenotypes
Clinical Cutoffs differ for each drug
Lower clinical cutoff
Response is significantly reduced
Upper clinical cutoff
Response is unlikely
“Zone of
Intermediate
Response”
Fold Change
Perno C, Bertol A. Antiretroviral Resistance in Clinical Practice. 2006. Chapter 7;
Available at: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=antiretro&part=A401.
Using clinical cutoffs to predict
virologic response to tenofovir
Average change in viral load over 24 weeks
80
DAVG24 > 0.5 log (%)
70
60
50
40
30
20
10
0
<1.4
Margot N et al, AIDS 2002; 16:1227-1235
1.4-4.0
Tenofovir fold change
>4.0
Which Resistance Test When?
• Genotype
– preferred due to faster result, lower cost and detection of early
resistance (detecting primary resistance or at time of virologic failure
to first or second ARV regimen)
• Phenotype
– “Addition of phenotypic testing to genotypic testing is generally
preferred for persons with known or suspected complex drug
resistance mutation patterns, particularly to protease inhibitors”
– when > 3 PI or RT mutations found on genotype*
– Usually when getting a phenotype also get genotype simultaneously*
• Virtual Phenotype (not specifically stated in DHHS)
– As a substitute when actual phenotype not available
DHHS Guidelines available at http://aidsinfo.nih.gov/guidelines March 27, 2012; *HL personal practice
Example of combined geno/phenotype report when phenotype not necessary
Example of combined genotype/phenotype report when both are necessary
Example of a Virtual Phenotype Report
Drugs
Fold
Change
Cut-offs
Lower
Upper
Resistance Analysis
NRTI/NtRTI
NRTI/NtRTI mutations: 41L, 67N, 70R, 184V, 219Q, 335D, 369wt/A/I/V, 371wt/V
Zidovudine
4.6
1.5
11.4
Reduced Response
Lamivudine
48.8
1.2
4.6
Minimal Response
Didanosine
1.6
0.9
2.6
Reduced Response
Stavudine
1.0
1.0
2.3
Maximal Response
Abacavir
2.2
0.9
3.5
Reduced Response
Emtricitabine
44.8
Tenofovir DF
1.1
3.1
1.0
Resistant
2.3
Reduced Response
PI
NNRTI
NNRTI mutations: None
Nevirapine
1.3
6.0
Susceptible
Efavirenz
0.9
3.3
Susceptible
Etravirine
1.1
1.6
27.6
Susceptible
PI mutations: 10F, 32I, 43T, 46I, 47V, 63P, 82A, 90M, 93L
Indinavir
24.8
1.0
5.4
Minimal Response
Indinavir/r
24.8
2.3
27.2
Reduced Response
Nelfinavir
20.1
1.2
9.4
Minimal Response
Saquinavir/r
1.2
3.1
22.6
Maximal Response
Fosamprenavir/r
17.4
1.5
19.5
Reduced Response
Lopinavir/r
26.1
6.1
51.2
Reduced Response
Atazanavir/r
6.2
2.5
32.5
Reduced Response
Tipranavir/r
0.8
1.5
7.0
Maximal Response
Darunavir/r
3.2
10.0
106.9
Maximal Response
Major nRTI Mutations
• M184V – diminishes viral fitness by approx. 50%
– High level resistance to 3TC and FTC
– Some resistance to ddI and abacavir
– Restores some activity to AZT/d4T, tenofovir
• K65R
– Broad resistance to all nRTI; but ↑ susc. to AZT
• L74V
– Resistant to abacavir & ddI; ↑ susc.to AZT, TDF
 TAMs - 215, 41, 210, 67, 70, 219:↓ susc. to all nRTI
– Selected by a prior tx history of AZT, d4T
– More resistance w/ 41/210/215 than 67/70/219 path
– 44D, 118I: ↑ nRTI resistance with 41/210/215 path
 TAM pathway and K65R pathway do not occur on the same virus (bidirectional
inhibition)
 MDR mutations (69SSS and Q151M)
 M184V, K65R and L74V have major effects on viral fitness
Hirsch M, et al. Clin Infect Dis. 2008;47:266-285
Question 1
Your patient is failing a tenofovir based regimen and
genotypic resistance testing shows the K65R
mutation. Which of the following statements is true?
1. The patient’s virus will likely have high level resistance to
tenofovir and it should not be included in future regimens
2. The patient’s virus has some degree of resistance to
tenofovir but is likely to have some residual antiviral activity
and may be useful in future treatment regimens
3. The patient’s virus is likely resistant to AZT
4. This mutation does not have a major effect on viral fitness
Resistance Patterns after Failure of
Common Initial NRTI Backbones
AZT + 3TC
D4T + 3TC
AZT + 3TC + ABC
M184V
M184V
M184V
TDF + FTC
M184V
ABC + 3TC
M184V
Gallant J, Top HIV Med 2005; 13:138-42
TAMs
TAMs
TAMs
K65R
L74V or K65R
NNRTI mutations
• K103N
– Most common NNRTI mutation
– High level resistance to efavirenz & nevirapine but not to etravirine
or rilpivirine
• Y181C
– High level resistance to nevirapine & increased risk of efavirenz failure
– Has moderate to severe impact on etravirine cross-resistance
• E138K
– Novel mutation associated with rilpivirine failure; leads to etravirine
cross resistance
• Many other mutations can have effects on susceptibility to NNRTIs
Hirsch M, et al. Clin Infect Dis. 2008;47:266-285
Weighted etravirine mutation score
Mutation
Y181I
Y181V
K101P
L100I
Y181C
M230L
E138A
V106I
G190S
V179Fa
V90I
V179D
K101E
K101H
A98G
V179T
G190A
Vingerhoets J, et al. IHDRW 2008; Abstract 24
Prevalence
in the panel of
4,248 HIV-1
clinical isolates, %
1.5
0.9
2.6
8.4
32.0
1.1
2.5
4.4
3.7
0.7
6.8
2.1
9.9
2.2
9.5
0.6
23.3
Weight factor
3
3
2.5
2.5
2.5
2.5
1.5
1.5
1.5
1.5
1
1
1
1
1
1
1
Response to ETV based upon mutation
score
Weighted mutation score corresponded to response
rates as follows :
• 0-2:
74% (highest response)
• 2.5-3.5:
52% (intermediate response)
• > 4:
38% (reduced response)
Vingerhoets J, et al. IHDRW 2008; Abstract 24; Picchio G, et al. 15th CROI 2008. Abstract 866.
Question 2
Which of the following NNRTIs are likely to be fully
susceptible and a reasonable treatment option in a
patient experience treatment failure with the K103N
mutation?
1.
2.
3.
4.
5.
Efavirenz
Nevirapine
Rilpivirine
Etravirine
3 and 4
Key facts about PI resistance
– Boosted PI’s (LPV/r, FPV/r, SQV/r, ATV/r, DRV/r) usually do not
select for resistance if used as 1st PI
– Multiple PI mutations usually required for high level PI
phenotypic resistance
– In highly PI resistant patients, DRV/r and TPV/r typically have
good activity (must be confirmed by phenotypic resistance
testing)
– If multiple PI mutations, obtain phenotype if possible to
determine appropriate PI to use in salvage
Johnson, et al. Topics HIV Med; Dec 2009.
Darunavir Mutation Score:
V11I, V32I, I33F, I47V, I50V, I54L/M, T74P, L76V, I84V, L89V
• Approved in 1st-line & treatment-experienced pts
• May be used QD in treatment-experienced pts if there
are zero DRV resistance mutations (ODIN Study)
• POWER studies showed patients treated with darunavir
and optimized background meds had VL < 50 c/mL
greater than for comparator PIs
• Response to darunavir was found to be dependent on 11
PI mutations at baseline
Levin J. et al, XVIII IDRW 6/9-13, 2009
Darunavir response by DRV score
Patients (%) with HIV-1 RNA
<50 copies/mL at Week 24
100
80
60
50%
40
22%
20
10%
0
0-2 mutations
3 mutations
≥4 mutations
Number of Darunavir mutations at baseline
De Meyer S, et al. Resistance Workshop 2006. Abstract 73.; Levin J et al, XVIII Intl Drug Resistance Workshop, June 9-13, 2009.
Question 3
A patient experiences treatment failure with a
history of 2 prior PI-based ARV regimens. Genotypic
testing shows one resistance mutation to darunavir
and four other PI mutations. Which is likely to be
true?
1. The virus is likely to be susceptible to atazanavir
2. The virus is likely to be susceptible to fosamprenavir
3. Darunavir/ritonavir may be prescribed 800 mg/100 mg daily
in his next regimen
4. Phenotypic resistance testing may show that the virus is fully
susceptible to darunavir
Steps in HIV-1 Integration
2
In-dependent
processing
of 3′ends
Viral DNA Synthesis
1
Assembly on viral DNA in
a nucleoprotein complex
Nuclear
Entry
Nuclear Membrane
3b Concerted
target DNA
cleavage and
joining
Gap Repair
Mature
Provirus
Hazuda D, et al. 2006. Medicinal Chemistry 30th Annual National Symposium 2006.
3a
Target
DNA
binding
Integrase inhibitor resistance:
the example of raltegravir
• Raltegravir failure is assoc. with integrase mutations in at least 3 genetic
paths defined by at least 2 mutations:
– Major mutations: 148,155, or 143 +/- minor changes
• Most common/most resistant = Q148H plus G140S
• Q148H/K/R or Y143R/H/C associated with high-level phenotypic
resistance (> 100-fold change in IC50)
• N155H associated with low-level phenotypic resistance
(< 50-fold change in IC50)
• Continued raltegravir in the presence of viral failure & resistance is not
recommended
• Cross-resistance from mutations selected by RAL may confer reduced
susceptibility to investigational integrase inhibitors (elvitegravir and
dolutegravir)
Hazuda DJ, et al. HIV Res Workshop 2007. Abst 8. Hatano H, et al JAIDS 2010
BENCHMRK 1 & 2: Evolution From N155 to
Q148 Mutations Over Time
First Genotype
(n = 64)
155
Second Genotype
(n = 51)
148
45%
143
18%
Mixed
92
27%
53%
Hazuda D, et al. ICAAC/IDSA 2008. Abstract 898.
Integrase Assay Determines RAL Susceptibility
• Phenotypic integrase resistance assay now available
– Amplification threshold: VL > 500
– Biological cutoff for RAL is FC > 1.5
– Report does not detail genotypic mutations
• Integrase genotype assays are also available; useful for determining
susceptibility to 2nd generation agents
• Transmitted RAL resistance (N155H +2 minors*) now reported;
consider baseline RAL resistance testing prn
Boyd SD, et al. Antiviral Therapy 2011;16:257-61; Fransen S, et al. 48th Annual CAAC/IDSA 46th Annual
Meeting. Abstract 1214;
Question 4
Which of the following are true about maraviroc?
1. It is only FDA approved for use in treatment-experienced
patients with HIV
2. Maraviroc should only be used in patients with R5 tropic HIV
3. Viral tropism switching (from R5 to D/M) is the most
common cause of treatment failure
4. Maraviroc resistance testing is commercially available
Defining Co-Receptor Tropism
CCR5-tropic (R5) virus
enters CD4+ T cells via
CCR5
HIV that can use either
CCR5 or CXCR4 is
called dual tropic
CXCR4-tropic (X4)
virus enters CD4+ T
cells via CXCR4
Virus populations containing a mixture of
R5-tropic, X4-tropic, and/or dual-tropic HIV are
called mixed tropic
Westby E, van der Ryst E. Antivir Chem Chemother. 2005;16(6):339-354.
Maraviroc
• First HIV CCR5 inhibitor / CCR5 chemokine receptor
antagonist
• Effective against strains that use the CCR5 co-receptor for
cell entry
– Little effect on viruses which use CXCR4 co-receptor, or which
use both
• Activity of maraviroc is tested by co-receptor “tropism”
test, which is not a resistance test
• Usually maraviroc more active in viruses from patients
earlier in the course of HIV infection at higher CD4 counts
Coakley E, et al. Second International Workshop Targeting HIV Entry. 2006. #8.
Representative HIV tropism patterns
MOTIVATE 1, 2, and 1026
Antiretroviral-naive
(n = 1,428)
X4
(0.3%)
Antiretroviral-experienced
(n = 2,560)
DM
(14.7%)
DM
(41.4%)
R5
(85%)
R5
(56%)
X4
(2.6%)
Coakley E, et al. Second International Workshop Targeting HIV Entry. 2006. #8.
Mechanisms of Maraviroc Resistance
– Major: Outgrowth of Dual/Mixed or X4 virus from pre-existing
minority population present at levels too low to be detected by
current tropism assays
• Minor: Virus remains R5, with true resistance
– Mutations in HIV gp120 that allow HIV to bind to CCR5 even
though maraviroc is also bound
– Most mutations are variable changes in the V3 loop
– No consistent signature mutations for MRV resistance
• Genotyping not commercially available
Mori J, et al. HIV Resistance Workshop 2007. Abstract 10.Tsibris AMN, et al.HIV Resistance Workshop 2007, #13; Lewis M, et al. HIV
Resistance Workshop 2007. Abstract 56; Mosley et al.13th CROI; Westby et al. J Virol 2007.
Conclusions
• HIV Drug Resistance can occur prior to initiation of ARV therapy
• Resistance testing can be used to optimize an antiretroviral regimen
– Must use in context of treatment history and results of all prior
resistance tests
– Goal for all HIV infected patients is HIV RNA < 50
• Tropism testing also useful in determining eligibility to receive CCR5
antagonist therapy (maraviroc)
• Factors other than resistance may cause regimen failure
• Resistance testing is reliable and cost-effective but must be
interpreted in context and may require expert advice
• Cannot detect “minority” populations (<5-20%?)
• Thank you for your attention!!
ACTHIV 2012: A State-of-the-Science Conference for Frontline Health Professionals
HIV: The Basics
ACTHIV 2012: A State-of-the-Science Conference for Frontline Health Professionals
Activity Code:
T1223