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Basics of HIV Resistance and Testing Jorge Villacian, MD Virco TREAT Asia Annual Network meeting Bangkok, Thailand 11 October 2009 Overview z Review basic concepts on Resistance z Determine how resistance tests work and are interpreted z When should resistance tests be used? z What is the relevance of the Resistance phenomenon and what do we know about resistance to new classes? Overview z Review basic concepts on Resistance z Determine how resistance tests work and are interpreted z When should resistance tests be used? z What is the relevance of the Resistance phenomenon and what do we know about resistance to new classes? What is Resistance? z The reduced susceptibility of a patient’s viral isolate to suppression by an antiviral drug z z A change that improves viral replication in the presence of an inhibitor z The point at which an ARV agent can no longer effectively inhibit viral reproduction HIV-1: Replicative Cycle Wild Type Virus z Non-mutant, drugsusceptible virus z No previous effect from medication z Reference virus Quaisi-Species z Viral isolates are composed of various groups of virus z z z Wild Type (Sensitive) Mutated (Resistant) Mutated (Non-resistant) z Acquired resistance z Selective pressure from current medication Selective Drug Pressure z z z EFV Drug pressure drives selective forces for genetic changes in the viral genome Mutations arising under ART allow virus to escape from the inhibitory effect of the drug Mutations that develop are associated with ARV agents being administered “Minority variants” — <20% of population FTC z TDF ATRIPLA z Increasing amount of resistant mutations are able to develop z No longer minority variants EFV Ongoing replication under selective pressure… FTC z TDF Selective Drug Pressure z Ongoing replication under selective pressure… z Increasing amount of resistant mutations are able to develop z What happens if ARVs are discontinued? EFV FTC TDF Selective Drug Pressure z z z z EFV FTC TDF Selective Drug Pressure In absence of ARV pressure, resistant clones are “overgrown” with Wild Type virus and fade to “undetectable” Resistance is a genetic characteristic, so it is “archived” and can be re-expressed rapidly Absence of resistance on RT does NOT guarantee susceptibility! Clinical history is critical Archived Resistance Overview z Review basic concepts on Resistance z Determine how resistance tests work and are interpreted z When should resistance tests be used? z What is the relevance of the Resistance phenomenon and what do we know about resistance to new classes? Types of Resistance Tests and a Few Examples z Genotype z z Phenotype z z z TruGENER (Siemens) Antivirogram® (Virco) PhenoSense™ (Monogram) Calculated Phenotype: z Virco®TYPE HIV-1 (Virco) HIV Resistance Testing Assays RESISTANCE • The (in)ability of HIV to replicate in the presence of Antiretroviral Drugs • Caused by changes in relevant parts of the virus genome (mutations) Genotyping Assay Calculated Phenotyping Assay Conventional Phenotyping Assay • Indirect measure of the viral susceptibility to Antiretroviral Drugs • Correlative database, frequently updated with GT and PT • Based on sequence (mutations) of relevant parts of the viral genome • PT fold change of virus is calculated from mutations in GT, with interpretation supported by clinical outcomes data base • Direct measure of the ability of the virus to grow in the presence of antiretrovirals • Requires interpretation of sequence information • QUALITATIVE • QUANTITATIVE • Compared to laboratory reference strain • QUANTITATIVE GENOTYPE Genotypic assays step by step PLASMA (> 200 µl) Viral Gag/PR/RT gene isolation total RNA extraction cDNA RT Gag/PR/RT GENES (amplicon) PCR PATIENT TCGATGTAC Interpretation -- Translate into aminoacids -- Compare wild type vs sample -- Identify mutational patterns (rules, expert opinion, etc.) Automatic sequencing Target genome regions P7/P1 P1/P6 RT PRO GAG polymerase INT IN RNaseH 560 1 Affymetrix Bayer Celera ViroLogic Virco 242 300 330 303 400 www.iasusa.org Mutations Selected by nRTIs Abacavir Didanosine Emtricitabine Lamivudine Stavudine Tenofovir Zidovudine Interpreting Resistance from Genotypic Reports z Single mutation can confer resistance z M184 (3TC) D30N (NFV) I50L (ATV) z Pairs of mutations z Multiple or stairwise accumulation of mutations z z LPV/r DRV/r AZT (NAMs) ETR Multi-NRTI mutations: K65R, Q151M Advantages and Disadvantages of Genotype Testing Advantages z z z z Rapid turnaround (1-2 weeks) Less expensive Widely available More sensitive than conventional phenotype for detecting mixtures of resistant and wild-type virus Disadvantages z z z z Indirect measure of resistance Relevance of some mutations unclear Complex mutational patterns may be difficult to interpret No standardized algorithms for interpretation of sequences PHENOTYPE Conventional Phenotype Testing z Measures laboratory susceptibility of an HIV isolate to a given drug z Measures the concentration of drug needed to inhibit the replication of a patient's virus z Degree of resistance is quantified z Compares the fold-change in drug concentration required to inhibit the replication of the patient's virus compared to a representative, wild type, sensitive virus isolate Phenotypic Susceptibility: Relationship Between Drug Concentration and Viral Inhibition Inhibition of Virus Replication (%) 100 Wild-type Resistant Fold Change = 10 Fold resistance 50 IC50 100uM IC50 10uM 0 Wild-type IC50 =10uM Resistant IC50 =100uM Drug Concentration = 10 Numbers need to be interpreted according to cut-points: Biological cut offs # samples BCO = 97.5th percentile Wild‐type distribution 1 28 10 1.7 Fold‐change 100 Advantages and Disadvantages of Conventional Phenotype Testing Advantages z z z z Provides direct and quantitative measure of resistance Often uses clinical cutoffs (CCO) derived from clinical cohorts to define spectrum of resistance Indicates which drugs may have partial activity Can assess interactions among mutations Disadvantages z Clinical cut-offs not defined for some agents z Complex technology with longer turnaround (~ 3 wks) z More expensive than genotyping z Limited laboratories perform testing VIRTUAL PHENOTYPE Correlative and Clinical Outcomes Databases* • Routine clinical testing • Clinical trials • Research collaborations Genotypic data >314,000 Phenotypic data >86,000 Correlative database >53,000 G/Ps Nucleotide sequence (…AAGTC TCCGCAT GCATA…) VirtualPhenotype™-LM engine Calculated fold change values in IC50 Clinical Outcomes Database >21,000 patients or >8,800 Treatment Change Episodes Clinical Cut-Offs *Status July 07 Creating the Phenotype from the Genotype z z Define mutations/pairs which impact the Fold Change for each drug Two factors in Virco G/P database: z z z z The “Weight”: How much this mutation/pair changes the phenotypic Fold Change The “Direction”: Does this mutation/pair lead to more resistance or more drug susceptibility? Each mutation (single and pairs) analyzed for these two factors A total “score” is created from the sum of these factors Æ the calculated “fold change” FC Assessment: Example of Tipranavir PI Mutation Analysis 3I, 10I, 14R, 19I, 24I, 37N, 41K, 46I, 53Y, 54V, 55R, 63P, 64V, 71V, 82T, 84V 10C & 33F 13A & 47V 10F 13V & 15V 10F & 47V 13V & 34Q 10F & 54M 13V & 36I 10F & 58E 13V & 43S 10F & 82C 13V & 71L 10F & 82F 13V & 71V 10F & 82L 13V & 82F 10F & 84A 13V & 84V 10F & 84V 14T 10I & 13A 15V & 43I 10I & 33M 15V & 95F 10I & 82F 16A 10I & 82I 18H 10V 20R & 35D 10V & 34A 20R & 53L 10V & 84V 20R & 70E 10V & 88D 20T & 33F 10Y & 13V 20T & 41K 12S & 69K 20T & 53L Decrease in FC 20T & 73T 33I & 35G 35N 20T & 84V 33I & 36I 35N & 43T 20V 33I & 82A 35N & 70E 22V 33I & 95F 35N & 82F 24F & 60E 33V 36A 24I 33V & 43T 36I & 83D 24I & 33F 33V & 54V 36I & 84V 24I & 50V 34D 36L & 53L 24I & 82T 34D & 58E 36L & 84V 30N 34N & 82A 36V & 82F 30N & 50V 34Q & 36I 36V & 84V 30N & 88D 34Q & 54V 38W 33.1Q 35D 41K 33F 35D & 36I 41K & 54V 33F & 36L 35D & 36V 41T & 70E 33F & 48A 35D & 54M 43I & 55R 33F & 50V 35D & 54V 43Q & 73T 33F & 60E 35D & 84V 43T 33F & 66L 35D & 89V 43T & 82F 33F & 82F 35G 43T & 82T 43T & 85L 45I 45Q 45V 46L 46L & 48M 46L & 50L 46L & 80I 46L & 82L 46L & 84V 47V 47V & 54M 47V & 54V 47V & 69K 47V & 82A 47V & 82I 47V & 84V 48A 48E 48M 48Q 48S 48V 48V & 54A 48V & 54S 48V & 54V 48V & 71V 48V & 82A 48V & 84V 50L 50V 50V & 58E 50V & 70E 50V & 76V 53L 53L & 82F 53L & 82I 54A 54A & 82A 54A & 82F 54A & 84V 54L 54L & 76V 54L & 82A 54L & 82S 54M 54M & 83D 54M & 88D 54S 54S & 74S 54S & 82A 54S & 84V 54T & 82T 54V & 84V 55R 55R & 60E 55R & 95F 58E 58E & 80I 58E & 89V 60E 82L 60E & 71V 82S 60E & 82A 82T 60E & 95V 83D 66V 83D & 84V 69K 84A 71V & 73T 84V 71V & 74E 85V 74A 88D 74P 88G 74P & 82S 88S 74P & 84V 89M 74S 89V & 95L 76V 90M 79S 91P 82C 95F 82C & 95F 95V 82F & 89V 82G 82I & 89V Increase in FC FC Assessment: Example of Tipranavir Mutation Analysis: Defining FoldChange PI Mutations Detected and Evaluated 3I, 10I, 14R, 19I, 24I, 37N, 41K, 46I, 53Y, 54V, 55R, 63P, 64V, 71V, 82T, 84V Mutations 24I 41K 55R 82T 84V 24I & 82T 41K & 54V 54V & 84V Intercept (no mutations) Resistance Weight Factor: weight and direction for mutations which impact TPV Linear Models DB0704 implemented on DEC18, 2007 RWF -0.18198 -0.06075 0.05418 0.36480 0.16181 -0.10545 0.03803 0.20493 -0.06039 Log(FC) = 0.415 FC = 100.415 = 2.6 Types of Cut-Offs z Biological Cut-Offs (BCO) z z Define what is resistant and non-resistant based on how a patient’s virus responds to a drug in vitro Clinical Cut-Offs (CCO) z Define what is resistant and non-resistant based on how a patient’s virus responds to a drug in vivo 34 Example Report Advantages and Disadvantages of Calculated Phenotype Testing Advantages z z z z z z Require less interpretation of complex genotypes Less expensive, quicker than conventional phenotyping Assess impact of interactions between mutations Equivalent virologic outcomes in clinical trials to conventional phenotyping Available from many reference labs Rapid turnaround Disadvantages z z z z Not an actual measured phenotype; a calculated phenotype based on genotypic information Reliability will depend on the accuracy of the genotype Only one source but widely distributed through many labs that perform genotyping Slightly more expensive than genotype alone Overview z Review basic concepts on Resistance z Determine how resistance tests work and are interpreted z When should resistance tests be used? z What is the relevance of the Resistance phenomenon and what do we know about resistance to new classes? When to Use Resistance Testing IAS-USA[1] DHHS[2] European[3] Primary/acute Recommend Recommend Recommend Postexposure prophylaxis -- -- Recommend* Chronic, Rx naive Recommend Recommend Recommend Failure Recommend Recommend Recommend Pregnancy Recommend Recommend Recommend -- Recommend Recommend *Test source patient especially if treated with antiretroviral drugs. Pediatric 1. Hirsch MS, et al. Clin Infect Dis. 2008;47:266-285. 2. November 2008 DHHS Guidelines. Available at: http://www.aidsinfo.nih.gov. Accessed November 10, 2008. 3. EACS Guidelines Version 3. Available at: http://www.eacs.eu/guide/index.htm. Accessed October 24, 2008. Limitations of Resistance Testing z z z High cost compared with other tests routinely used in HIV care Cannot be reliably performed when HIV RNA <500-1,000 copies May not be able to detect minority populations of resistant virus (<20%) z z Especially common after drug discontinuation Resistant strains in viral reservoirs are not detected Which Resistance Test and When? z The utility of phenotypic resistance information increases with treatment experience/mutations complexity Phenotypic Information Genotypic Information Treatment Experience/Mutational Complexity When To Use Resistance Testing Naïve Patients / Starting Therapy z If resistance mutations are not detected, it is still possible that the patient has been infected with a drug-resistant strain z The lack of drug pressure can cause the wildtype strain to dominate and minor resistant species may not be detected When To Use Resistance Testing On Therapy z z z z Virologic failure: assist in selecting active drugs for next regimen Suboptimal viral load reduction Perform while patient is taking ARV agents or immediately after discontinuing therapy (within 4 weeks) All pregnant women prior to initiation of therapy and for those entering pregnancy with detectable viremia while on therapy DHHS Treatment Guidelines, January 29, 2008. Resistance Testing in Clinical Practice z z z Genotype preferred for z Treatment naive: acute or chronic infection z Early virologic failure z Patient no longer receiving therapy Phenotype or combined phenotype/genotype preferred for z High-level resistance to NRTIs or PIs on genotype z Multiple regimen failure with limited treatment options Virtual phenotype also used in settings where phenotypic testing would be preferred z Virtual phenotype is a type of complex interpretation of genotype data, intended to predict phenotypic response Overview z Review basic concepts on Resistance z Determine how resistance tests work and are interpreted z When should resistance tests be used? z What is the relevance of the Resistance phenomenon and what do we know about resistance to new classes? Resistance is associated with worse clinical outcomes z In multivariable analyses, patients with drug resistance mutations to ≥ 2 classes during first 2 years of HAART at significantly higher risk of AIDS progression or death Definition of Resistance Adjusted RH (95% CI) P Value Drug-class resistance mutations ≥1 NRTI mutation 1.52 (1.14-2.03) .004 ≥1 NNRTI mutation 1.95 (1.28-2.95) .002 ≥1 PI mutation (major and minor) 1.50 (1.14-1.97) .004 ≥1 PI mutation (major only) 1.79 (1.28-2.50) .0007 Cumulative drug-class resistance (major and minor PI mutations counted) Virologic failure with no resistance 1.32 (0.57-3.06) .52 Single-class resistance 1.03 (0.65-1.63) .90 Double-class resistance 1.55 (1.15-2.08) .004 1.80 (1.20-2.70) .005 Triple-class resistance Cozzi-Lepri A, et al. AIDS. 2008;22:2187-2198. Summary of Key Conclusions z Emergence of drug resistance within 2 years of initiating HAART in EuroSIDA cohort associated with long-term clinical outcomes z z z 55% increased risk of new AIDS events or death among individuals who developed double-class resistance 80% increased risk of new AIDS events or death among individuals who developed triple-class resistance Mechanism driving association unknown z Possibly due to suboptimal adherence or direct effect of resistance Cozzi-Lepri A, et al. AIDS. 2008;22:2187-2198. Resistance Scoring ● Genotypic susceptibility score = sum of genotypic resistance scores for each drug in regimen ■ Based on rule-based algorithms using predefined drug-resistance mutations ● Phenotypic susceptibility score = sum of phenotypic resistance scores for each drug in regimen ■ Based on FC in susceptibility of test sample relative to control (wild-type) isolate 1.0: susceptible 1: susceptible 0.5: possibly resistant 0: resistant 0: resistant Between 0 and 1: partially susceptible 50 Focus on Number of Active Agents z z z DHHS antiretroviral guidelines: ≥ 2, preferably 3, fully active agents in new regimen Highest rate of virologic suppression in patients receiving investigational drug plus OBR containing ≥ 1 other active agent[1-4] Trend toward greater benefit with 3 vs 2 fully active agents[1-4] z z z z Not statistically significant Must also consider potential drug-drug interactions, adverse events, pill burden, absence of future options Contribution of “partially active” agents (eg, 3TC) difficult to calculate No added benefit from using 4 vs 3 fully active agents 1. Cooper DA, et al. N Engl J Med. 2008;359:355-365. 2. Haubrich R, et al. CROI 2008. Abstract 790. 3. Johnson M, et al. CROI 2008. Abstract 791. 4. Nelson M. CROI 2007. Abstract 104aLB. BENCHMRK-1 & -2: HIV-1 RNA < 50 c/mL at Week 48, Overall and by GSS Subgroup n Total 443 228 GSS: 0 112 65 1 166 92 2 109 47 ≥3 49 21 RAL Patients (%) Placebo 64 34 45 3 67 37 77 62 71 52 0 20 Cooper DA, et al. N Engl J Med. 2008;359:355-365. 40 60 80 100 DUET-1 and -2: HIV-1 RNA < 50 c/mL at Week 48, by Active Agents in OBR 100 Placebo (n = 604) 76 80 0 1 ≥2 No. of Active Agents in OBR by PSS (DRV Considered Active if FC < 40) Haubrich R, et al. CROI 2008. Abstract 790. Johnson M, et al. CROI 2008. Abstract 791. 187/305 51/196 0 121/203 0 26 0/35 20 33 229/300 60 40 61 60 12/36 Patients With HIV-1 RNA < 50 copies/mL at Week 48 (%) ETR (n = 599) BENCHMRK 1 & 2: RAL Resistance at Virologic Failure z z z 492 patients treated with RAL 105 (23%) had virologic failure Genotype available at baseline and after virologic failure for 94 patients z 68% (64/94) had genotypic evidence of RAL resistance z Nearly all (62/64) had mutations at position 143, 148, and/or 155 Cooper DA, et al. N Engl J Med. 2008;359:355-365. Integrase Inhibitor CrossResistance z In RAL study, resistance to ELV with mutations at positions 148 and 155[1] z z Patterns associated with high-level resistance to both RAL and ELV z G140S/Q148H z G140S/Q148R In ELV study, mean decrease in susceptibility[2] z To ELV: > 151-fold (range: 1.02- to 301-fold) z To RAL: > 28-fold (range: 0.78- to > 256-fold) z No significant short-term virologic response to RAL in 2 patients switched from ELV/RTV to RAL following failure[3] z Cross-resistance is a clear issue with first-generation of agents 1. Hazuda DJ, et al. HIV Resistance Workshop 2007. Abstract 8. 2. McColl DJ, et al. HIV Resistance Workshop 2007. Abstract 9. 3. DeJesus E, et al. IAS 2007. Abstract TUPEB032. Phenotypic Biological Cutoff for RAL Host Conclusions Bug z z z z Drug The resistance phenomenon is a relevant factor in choosing antiretroviral treatment regimens Knowledge of the different methods to detect resistance is important in order to correctly interpret their results Resistance has been demonstrated in new drug classes and cross-resistance may be a limiting factor in the future Resistance is one of the multiple factors that influence treatment response