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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