1. HIV-1 Drug Resistance Testing: A Practical Discussion Lee T. Bacheler, PhD VP Clinical Virology VircoLab, Inc Durham, NC

2. Topics for today National Guidelines for use of resistance testing Types of resistance testing –genotype –conventional phenotype –virtualPhenotype TM -LM A tour of the vircoTYPE HIV-1 report Some Illustrative examples

3. DHHS Guidelines for the Use of Antiretroviral Agents in HIV-1- Infected Adults and Adolescents - October 10, 2006 Similar recommendations from IAS-USA

4. Genotypic sequence Measured Fold Change (FC) Calculated Fold Change (FC) All Resistance Tests begin by Amplifying the Patient’s Virus Population

5. Genotype Test Determine the genetic composition of patient’s virus Compare the genetic composition of patient’s virus to the genetic composition of wild type virus= identify mutations Use established algorithms to determine which mutations are associated with drug resistance

6. Test Result

7. Analysis of Sequencing Results

8. Sample Genotype Report

9. Conventional Phenotype Test ‘Grow’ patient’s virus in the presence of drug Determine the amount drug required to suppress 50% viral growth = IC 50 Compare the IC 50 of the patient’s virus to the IC 50 of a wild type virus = Fold Change “Clinical” or “Biological” cutoff to define drug resistance Commercially Available Tests: –Antivirogram (Virco) –PhenoSense (Monogram) –Phenoscript (Viralliance)

10. The Antivirogram ® Assay Step by Step PATIENT PLASMA (> 200 µl) total RNAcDNA / gag/PRO/RT GENES (amplicon) extraction RT 2x PCR Creation of deleted molecular clone WT molecular clone (HXB2) gag, PRO, RT Delete gag, PRO & RT genes Deleted molecular clone CD4+ T-cells (MT4) Infectious RECOMBINANT virus Susceptibility assay (2-3 complete viral replication cycles) gag (p7/p1-p1/p6); PRO(1-99); RT (1-400) Gene transfer (nucleofection)

11. ANTIVIROGRAM ® : susceptibility assay Res. WT 3TC 0.00640.032 4 0.80.1620100 µM0 AZT

12. Drug Concentration (µM) 100 50 0 low IC 50 Wild-type isolate high 90 Patient isolate < 10x more Fold Change in IC 50 IC 50 100 µM IC 50 10 µM =10 % Inhibition HIV Replication

13. Sample Conventional Phenotype Report Antivirogram (Virco)

14. HIV-1 Resistance Testing Technologies Phenotyping Assay Direct measure of the ability of the virus to grow in the presence of Antiretroviral Drugs Quantitative Simple interpretation based on (quantitative) fold-change and cut-off values (Biological or Clinical) Less sensitive than genotype for the early detection of mutants Costly and long TAT Genotyping Assay Indirect measure of the virus’ susceptibility to Antiretroviral Drugs based on sequence (mutations) of relevant parts of the viral genome Qualitative Requires sophisticated analysis & interpretation of sequence information Early detection of mutants Affordable and rapid TAT RESISTANCE The ability of HIV to replicate in the presence of Antiretroviral Drugs Caused by changes in relevant parts of the virus genome (mutations)

15. Predicting the Drug Susceptibility Phenotype from the Viral Genotype

16. virco ® NET 5-60 minutes For most US customers, genotyping is done locally (eg LabCorps) The resulting nucleic acid sequence is sent to Belgium for vircoTYPE analysis

17. virco ® TYPE HIV-1 report Repeat for each drug GENOTYPE (nucleotide sequence) Compare to reference sequence Identify all mutations in sample virus Identify all mutations and mutation pairs in sample virus that contribute to resistance to a given drug according to LM For each mutation or mutation pair in the sample virus that contributes to resistance, retrieve the Resistance Weight Factor Add all Resistance Weight Factors to obtain a predicted Fold Change Every 2 months G/P correlative database Resistance Weight Factors (drug specific) LM

18. Virco’s correlative database GenotypesPhenotypes Multiple Linear Regression Analysis Determine which mutations contribute to resistance for each ARV, and by how much (Resistance Weight Factor) [Note: RWF’s are re-calculated after every database update (2-monthly)] Obtaining Resistance Weight Factors

19. Virco’s Databases * Clinical Outcomes Database (>17,000 patients on therapy) Routine clinical testing Clinical trials Research collaborations Genotypic data >285,000 Phenotypic data >83,000 VirtualPhenotype ™ -LM engine >50,000 matched G/P samples Predicted fold change values in IC 50 Nucleotide sequence (…AAGTC TCCGCAT GCATA…) * Status December 06

20. FC Assessment: Example of Tipranavir PI Mutation Analysis 10F & 47V22V & 84V34Q & 84V36L46L & 82T53L54V74S 10F & 82A24I & 82A35D & 36I36L & 58E47V & 54M54A & 55R54V & 70E76V 10F & 84V24I & 82T35D & 54A36L & 83D47V & 54V54A & 84V54V & 74P82A & 84V 10V30N35D & 54V36L & 95F47V & 83D54L54V & 84V82C 10V & 33F30N & 74S35D & 58E38W47V & 84V54L & 82A58E & 84V82L 10V & 88D33F35D & 73T41K48A & 71V54M60E82T 13V & 69K33F & 82A35G & 71V41T48M & 53L54M & 74S60F84V 13V & 71V33I & 36I35N & 84V43T48V54M & 82A69K85V 13V & 82S33M36I & 47V43T & 82T48V & 54V54S71V & 73T88D 13V & 84V33V36I & 54T46L & 53L50L54S & 82T71V & 95F90M 20R33V & 84V36I & 84V46L & 71V50V54V74P & 82A 10F, 13wt/V, 32I, 33F, 41K, 46I, 58E, 63P, 71wt/V, 73S, 77I, 84V, 89V, 90M, 93L

21. FC assessment: Example of Tipranavir Mutation Analysis: Defining Fold-Change Mutations 33F 41K 84V 90M 10F & 84V 13V & 71V 13V & 84V 58E & 84V no mut 10F, 13wt/V, 32I, 33F, 41K, 46I, 58E, 63P, 71wt/V, 73S, 77I, 84V, 89V, 90M, 93L RWF (adjusted for mixtures) 0.217 -0.041 0.149 0.061 -0.174 0.016 0.043 0.079 -0.099 FC = 10 0.252 = 1.8 Log(FC) = 0.252 RWF* 0.217 -0.041 0.149 0.061 -0.174 0.064 0.087 0.079 -0.099 *Resistance Weight Factor: Weight and Direction for mutations which Impact TPV

22. virco ® TYPE HIV-1 v4.1.00 Powered by VirtualPhenotype ™ -LM How to Read and Use the Report

23. Page 1 Summary Report Page 2 Detailed Report Page 3 Definitions and Disclaimers virco ® TYPE HIV-1 V4.1.00

24. 1.Resistance-associated mutations 2.VirtualPhenotype ™ -LM predicted Fold Change in IC 50 3.Cut-Offs (BCO or CCO) 4.Resistance Analysis 5.Reference to Additional Clinical Notes BCO: Biological Cut Offs CCO: Clinical Cut Offs Page 1: Summary Report (1) 1 1 1 2 3 45

25. Page 1: Summary Report (2) Mutations –Mutations that are known to be associated with resistance / treatment response and are listed on the report –Mixtures  4 amino acids: all residues are printed –e.g. 41 wt/L = mixture of Wild Type (Methionine) and Leucine at position 41 in reverse Transcriptase –5 or more amino acids: printed as ‘X’ –e.g. 215X = more than 4 residues detected at position 215 in RT

26. CCO 1 (low) CCO 2 (high) FC valueResistance Analysis BCO Page 1: Summary Report (3) Cut-Off values – 2 Clinical Cut-Offs (CCO1 and CCO2) if available – 1 Biological Cut-Off (BCO), if CCO not available BCO printed in italics

27. Terminology Used in the Resistance Analysis

28. Page 2: Detailed Report (1) Resistance Analysis Details Additional Clinical Notes Key

29. 1. Resistance-associated mutations 2. Graphical representation of the Resistance Continuum 3. Predicted Fold change in IC 50, and 95% confidence limits 4. Cut-Off values 1 1 1 234 Page 2: Detailed Report (2)

30. Page 2: Detailed Report (3) CCO1CCO2 Patient Virus FC BCO

31. Page 2: Detailed Report (1) Resistance Analysis Details Additional Clinical Notes Key

32. CaseConditionDrugText 215 Revertants T215A/C/D/E/G/H/I/L/N/S/V detected AND T215F and/or T215Y not detected AZT d4T Mutations T215A,C,D,E,G,H,I,L,N,S or V in the RT gene can arise as revertants of resistance associated mutations T215Y or F, and may lead to reduced response to AZT and d4T treatment NNRTI Cross- resistance Predicted Resistance to one NNRTI (EFV or NVP) AND susceptibility to the other EFV NVP Cross resistance among EFV and NVP resistant viruses is extensive. Resistance to one of these drugs usually leads to reduced response to all currently approved NNRTIs Additional Clinical Notes (1)

33. CaseConditionDrugText TPV/r CCO (permanent note)TPV CCOs for Aptivus ® are based on treatment responses in highly experienced patients in the RESIST studies. At 8 weeks, the median change in log viral load among patients with baseline TPV FC 5.4 was - 1.9, -0.9, and -0.2. The relevance of these CCOs for patients different from those in RESIST has not been evaluated. DRV/r CCO (permanent note)DRV CCOs for Prezista ® are based on treatment responses in experienced patients in the POWER studies. At 8 weeks, the median change in log viral load among patients with baseline DRV FC 96.9 was -2.3, -1.4, and -0.2. The relevance of these CCOs for patients different from those in POWER has not been evaluated. Additional Clinical Notes (2)

34. CaseConditionDrugText Mixtures A mixture including key resistance- associated mutations is detected AND The predicted FC of the mixed population ≤ CCO2 or BCO Drug Specific The predicted FC for this sample may be reduced by the presence of mixtures in the virus population at key positions associated with resistance. Based on the mutations detected in the viral genotype, a more resistant virus, with a more severe impact on treatment response, could be rapidly selected from this mixed virus population Additional Clinical Notes (3) When resistance associated mutations are present in a mixture (e.g., with a WT residue) –Then subpopulations of virus that harbor these mutations present a risk of clinical resistance, even though the FC for the mixed virus population may be below the cut-off suggesting the drug will be (partially) active When a patient is treated with the drug the resistant subpopulation could be selected rapidly and become the predominant quasispecies, leading to clinical resistance and therapy failure Clinicians are made aware of this effect in an additional clinical note whenever mixtures including patterns of mutations clearly associated with significant resistance are detected

35. Page 3: Definitions and Disclaimers (1)

36. A Resistance Test Result for a Naïve Patient

37. A Resistance Report for a Patient With a Mixed Virus Population In virtualPhenotype – LM 4.1, the contribution of mutations in a mixture is divided across the number of amino acids in the mixture  Most accurate prediction of the measured phenotype

38. A Resistance Report for a Patient With a Mixed Virus Population Standing Definition on pg 3 In virtualPhenotype – LM 4.2, only the contribution of the mutation with the highest level of resistance in a mixture will be taken into account  “Worst Case Scenerio”

39. A Resistance Report for a Patient With Extensive Drug Resistance: What should we do next?

40. What about other classes of Antiretroviral Drugs? Fusion inhibitors (enfuvirtide, T-20) –Resistance develops rapidly if virus replicates presence of T-2O –Resistance testing generally not utilized in patient management Integrase Inhibitors ( –Virologic failure associated with emergence of resistance mutations –Cross resistance between different inhibitors in the class is likely to be important –No currently available commercial assays Tropism (maraviroc) –Not a resistance test per se –Used to Identify patients without detectable CXCR4 tropic virus who could be candidates for use of a CCR5 antagonist –Commercially available assays Trofile™ ( Monogram) SensiTrop™ HIV Co-Receptor Tropism Assay ( Pathway Diagnostics, available through Quest, Mayo Reference Labs)

41. Summary: Use of HIV-1 drug resistance testing is recommended in a variety of settings Three types of resistance tests: genotype, phenotype, and virtual phenotype Tour of the vircoTYPE report Examples –Transmitted drug resistance –Mixed virus populations –Extensive resistance to approved drugs