1. HIV Resistance Testing Clinical Implications Cyril K. Goshima, M.D. Director, AIDS Education Project June, 2009

2. I am a? 1. Physician 2. Nurse 3. Pharmacist 4. Dentist 5. Student 6. Patient 7. Other

3. Resistance Testing will tell us what meds will work for a patient? 1. True 2. False

4. Resistance Testing is recommended before starting HAART. 1. True 2. False

5. Resistance Testing can be done on patients with viral load less than 500. 1. True 2. False

6. The K103N mutation is an NNRTI Mutation 1. True 2. False

7. M184V is a common PI Mutation 1. True 2. False

8. When to Use Resistance Testing DHHS Guidelines –Recommend testing: acute infection, suboptimal virologic suppression after treatment is initiated, treatment failure, prior to the initiation of therapy. –Consider: chronic infection < 2 yrs. –Which test is not recommended

9. Resistance Testing Genotypic Resistance Testing Phenotypic Resistance Testing Combined Geno/Pheno “Virtual Phenotype” Testing Trofile (HIV Tropism Assay) PhenoSense Entry (FI Resistance Testing) Integrase Resistance Testing Replication Capacity

10. Requirements for Resistance Testing Viral Load must be greater than 500 for genotype & phenotype resistance testing, integrase resistance testing Viral load must be greater than 1000 for Trofile and PhenoSense Entry

11. Genotypic Resistance Testing Detects mutations in the HIV genome associated with resistance to specific drugs. Advantages –Adequate turn-around time (1-2 wks) –Less expensive –Detect mutations that may precede phenotypic resistance –Widely available –More sensitive in detecting mixtures of resistant and wild type viruses

12. Genotypic Resistance Testing Disadvantages –Indirect measure of resistance –Relevance of some mutations unclear –Unable to detect minority variants (<20 – 25% of viral sample) –Complex patterns may be difficult to interpret –Genotypic correlates of resistance not well defined for non-B subtypes.

13. Phenotypic Resistance Testing Measures the patient’s HIV isolates ability to replicate in the presence of varying concentration of specific drugs. Advantages –Direct and quantitative measure of resistance –Method can be applied to any agent incl. new where genotypic correlates are unclear –Can assess interactions among mutations –Accurate with non-B HIV subtypes.

14. Phenotypic Resistance Testing Disadvantages –Susceptibility cut-offs not standard between assays –Clinical cut-offs not defined for some drugs –Unable to detect minority species –Complex technology –More expensive –Longer turn-around time.

15. Other Tests Geno/Phenotype Resistance Testing –e.g. Phenosense GT from Monogram –Both tests are performed –The discordance is reported “Virtual” Phenotype –Genotyping is performed and the phenotype is determined by looking at all the matched pairs of genotype with phenotype in a data set to give the best estimate

16. Other Tests Fusion Inhibitor Resistance Testing –Resistance to Enfuvirtide Replication Capacity –How weak is your patient’s virus? Chemokine Receptor Identification –CCR5, CXCR4, or mixed virus present Integrase Inhibitor Resistance Testing

17. How We Identify a Mutation How do we identify a resistance mutation? “M” is the “wild type” amino acid “184” is the codon position M 184 M

18. How We Identify a Mutation How do we identify a resistance mutation? “M” is the “wild type” amino acid “184” is the codon position “V” is the mutant amino acid M 184 V

19. How We Identify a Mixture “M” is the “wild type” amino acid “184” is the codon position “M/V” is the mixture of wild type & mutant amino acid M 184 M/V

20. Definitions for Phenotypic Resistance Testing IC50 = Concentration of drug required to inhibit replication by 50% Fold Change = IC50 pt./IC50 reference Cut Off = Fold change or concentration below which the virus is considered susceptible, above which non-susceptible Biological Cut Off = Fold change based on variations in clinical samples from treatment naïve individuals.

21. Definitions for Phenotypic Resistance Testing Clinical Cut Off = Fold change based on virologic response to ARV in Clinical Trials Replication Capacity: The ability of a pt’s virus to replicate in the absence of drug

22. NRTIs

23. NRTI Mutations Single point mutation can result in high level resistance e.g. M184V (3TC, FTC), K65R (TDF) TAMS pattern of mutations e.g. codons 41, 67, 70, 210, 215, 219 (AZT, D4T) 2 other patterns that are selected for by AZT/DDI & DDI/D4T –Q151M:resist. all NRTI except TDF –T69insertion + 1 or more TAMS @ 41, 210, 215: resist. all NRTI

24. Common Mutations: NRTIs TAMS = thymidine analog mutations (aka ZDV mutations): M41L, D67N, K70R, L210W, T215F/Y, K219E/Q NAMS = nucleoside analog mutations: TAMS plus E44A/D, A62V*, K65R, T69D, T69ins, L74I/V, V75A/I*/M/S/T, V77L*, Y115F, F116Y*, V118I, Q151M, M184I/V *Secondary mutations seen with Q151M

25. NRTI Signature Mutations *TAMS=Thymidine analog mutations. PositionDrug 74, 65, TAMS ddI (41, 67, 70, 210, 215, 219),* 333 ZDV 184 (TAMS, 44, 65,118) 3TC/FTC 75, TAMS d4T 184 + TAMs or 65, 74, 115 ABC 65; 41, 210, 215Y TDF

26. NNRTIs

27. Common Mutations: NNRTIs Delavirdine (DLV) –L100I, K103N, V106M, Y181C, I; Y188L, G190E/Q – P236L(rare), Y318F Efavirenz (EFV) –L100I, K103N, V106M, Y181C, I; Y188L, G190A, S, E, Q…; P225H Nevirapine (NVP) –L100I, K103N, V106A, M; Y181C, I; Y188C, L, H; G190A, E, S, Q…,F227L

28. NNRTI Multi-Drug Resistance Class Resistance –L100I, K101E or P, K103N or S, V106A or M, Y188C, H, or L, M230L Resistance to one NNRTI usually confers cross resistance to all other agents (exceptions: 181 and EFV, 190A/S and DLV) Continued viral replication in the presence of NNRTI results in accumulation of additional resistance mutations –May impact clinical utility of future NNRTIs

29. NNRTI Novel Mutations Those exhibiting a > 10 fold change: –K103R and V179D (in combination) –K101P

30. NNRTI: Etravirine K103N NNRTI mutation is not associated with resistance to Etravirine Multiple Resistance Associated Mutations (RAMS) Scoring of the number of RAMS determines resistance to Etravirine Similar to Protease Inhibitor Scoring

31. PIs

32. PI Resistance Cross resistance is common PI mutations are uncommon in boosted PI regimens Multiclass experienced pts. may have been exposed to unboosted regimens The number of primary PI mutations may predict the response to therapy e.g. TPV score 0-3 good, 4-7 intermediate, >8 poor or Kaletra

33. PI Common Mutations LopinavirV32I, M46I/V, I47A/V, G48V, I50V, I54A/L/M/S/T/V, V82A/F/S/T, I84V, L90M (need several of these) FosAmprenavirV32I, M46I/V,I47A/V, I50V, I54A/L/M/S/T, I84A/C/V, V82F, L33F, M46I/L (in certain combinations)? NelfinavirD30N, V82A/F/S/T*, N88D/S/T, I84A/C/V, L90M SaquinavirG48V, V82A/F/S/T*, I84A/C/V, L90M* IndinavirM46I/L, V82A/F/S/T*, N88S/T, I84A/C/V, L90M* RitonavirM46I/L, V82A/F/S/T*, N88S/T, I84A/C/V, L90M* Tipranavir10V, 13V, 20M/R/V, 33F, 35G, 36I, 43T, 46L, 47V, 54A/M/V, 58E, 69K, 74P, 82L/T, 83D, and 84V DarunavirV11I, V32I, L33F, I47V, I50V, I54M/L, G73S, L76V, I84V, L89V AtazanavirNeeds 5 or more from list. Following exposure to ATV: I50L

34. Hypersusceptibility

35. NRTI Increased Susceptibility MutationSelected By…Increases Susceptibility K65RTDF, ABC, ddIZDV L74I or VddI, ABCZDV, TDF L100INNRTIZDV, TDF Y181C, I, or VNVP, DLVZDV, TDF M184V3TC, FTC, ABC, ddIZDV, TDF, d4T

36. NNRTI Increased Susceptibility MutationSelected by…Increases Susceptibility G190A, C, S, T, or V EFV, NVPDLV P225HEFV, NVP?DLV F227LEFV, NVPDLV L100INNRTIZDV, TDF Y181C, I, or VNVP, DLVZDV, TDF

37. PI Increased Susceptibility MutationSelected by… Increases Susceptibility N88SNFV, IDV, ATV APV I50LATVRTV, LPV, SQV, other PIs?

38. PI Hypersusceptibility Mutation I50V, selected by LPVr and APV, increased susceptibility to ATV, TPV.

39. Integrase and Entry Inhibitor Resistance Resistance has been seen against the Entry Inhibitors and Integrase Inhibitors There are resistance tests that can be ordered For CCR5 it may just be a repeat of the Trophile test to determine change to mixed or dual tropic viruses

40. Case Discussion Patient CB, 42 y/o, homosexual male Current Regimen (05/31/06): CBV/TDF/EFV Past Drugs: CBV/ IDV, CBV/NFV CD4/VL –Date: 09/08/05 349/8,810 –Date: 03/07/06 192/10,300 –Date: 06/02/06 186/9,400 –Date: 09/18/06 92/6,610 –Date: 10/17/06 /12,000

45. Case Discussion NRTI –M184V present (3TC/FTC resist, TDF hs) –Multiple TAMs –No K65R (TDF sens despite 41 & 215 mut) NNRTI –No significant mutations PI –4 TPV assoc mut (intermediate response) –DRV sens

46. Case Discussion Was the CBV/TDF/EFV regimen a reasonable one? There has been no response to this therapy after 3 mos. What should you do? Any suggestions on a possible new regimen?

47. Discordance Inaccurate genotype interpretation algorithm that does not account for novel or previously unknown mutation effect Mixtures of wild type and resistant strains. Phenotype underestimates resistance Variability in phenotypic susceptibility with specific mutations Believe the genotype. Genotypic change may precede phenotypic resistance.

48. Clinical Implications Is there evidence for sequencing of NRTIs? Should the initial regimen be a boosted PI or a NNRTI? Is 3TC = FTC as far as resistance is concerned?

49. Clinical Implications Try to use at least 2 new potent agents to switch from a failing regimen. The longer a failing regimen is continued, the more mutations accumulate. If there is no new agent, better to cont. the same regimen unless compelled to do otherwise. Resistance is relative. 3TC cont. to have virological effect despite M184V mutation. Boosted PIs may have more of a response than an unboosted PI evidenced by a lower fold change.

50. Clinical Implications NRTI –TAMs can prevent K65R mutation. K65R is associated with multiple NRTI resistance and TDF resistance. ? Add ZDV to failing regimen –Continue 3TC or FTC despite a M184V mutation (hypersusc. ZDV, TDF, D4T; RC) NNRTI –DC NNRTI as soon as mutations develop. There is no virological or RC advantage.

51. Clinical Implications PI –Never use an unboosted PI. Antiretroviral susceptibility is on a continuum. Using drugs with the most activity (lower fold change) is a reasonable choice.

52. Clinical Implications In initial therapy, a boosted PI regimen may have an advantage over a NNRTI regimen because of fewer HIV mutations. ( J. Bartlett, et al, JAIDS, 4(3): 323-331; Swiss HIV Cohort Study, oral abstract 72, XV International HIV Drug Resistance Workshop) Possible explanations maybe lower genetic barrier and pharmacokinetics with missed doses.

53. Clinical Implications Replication Capacity –Lower RC with certain NRTI (3TC) and PI (NFV). –No change in RC with NNRTI

54. Resistance Testing will tell us what meds will work for a patient? 1. True 2. False

55. Resistance Testing is recommended before starting HAART. 1. True 2. False

56. Resistance Testing can be done on patients with viral load less than 500. 1. True 2. False

57. The K103N mutation is an NNRTI Mutation 1. True 2. False

58. M184V is a common PI Mutation 1. True 2. False

59. Acknowledgements Monogram Bioscience, Sharon Martens, MN, ARNP/FNP Dr. Joel Gallant, MD, MPH from Clinical Care Options HIV LLC, “Use and Interpretation of Resistance Tests in Multi- Class Experienced Patients,” September 2, 2005.

60. Thank You Questions?