JWM 11/99 Resistance Collaborative Group Re-Analysis of Studies and Review of Ongoing Prospective Studies John W. Mellors, MD Director, HIV/AIDS Program Chief, Division of Infectious Diseases University of Pittsburgh and Pittsburgh VA

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 HIV Resistance Collaborative Group Clinical Validation Subcommittee John Mellors (Chair, Pitt) Richard D’Aquila (Harvard) Veronica Miller (Germany) Louise Pedneault (GW) Amy Patick (Agouron) Victor DeGruttola (Harvard) Andrew Phillips (UK) Lynn Dix (GW) Dan Holder (Merck) Jeff Murray (FDA)

JWM 11/99 Goals of Clinical Validation Subcommittee Compile and evaluate existing data on clinical validation of resistance tests Review issues relevant to clinical validation of resistance tests –study design –patient populations studied –definitions of drug sensitivity and resistance –definitions of virologic endpoints –methods of analysis (control for covariates)

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 HIV Resistance Collaborative Group Clinical Validation Subcommittee Workshop April 21-22, 1999 Goal: review existing clinical data on relationship between genotype/phenotype and response –studies identified by review of meeting abstract –presentations by lead investigators –questions/clarifications by Subcommittee Studies presented and reviewed –13 retrospective studies –2 prospective, intervention-based studies

JWM 11/99 Clinical Validation Subcommittee Workshop April 21-22, 1999 Impressions: –consistent associations between baseline genotype or phenotype and virological response –highly variable methods of analysis definitions of resistance (mutations/cut-offs) virological endpoints methods of analysis control for key covariates –need for standardized data analysis Action Item: –develop Data Analysis Plan (DAP) for standardized re-analysis of studies

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 Development of Standardized Data Analysis Plan (DAP) Victor DeGruttola (Chair, Harvard) Dan Holder (Merck) Andrew Phillips (Royal Free, UK) Lynn Dix (Glaxo Wellcome)

JWM 11/99 Presentation of Data Analysis Plan by Victor DeGruttola

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 DAP Mutation Table Goal: Standardization of genotype analysis –not intended for patient management Consensus of RCG members (clinical data/opinion) –developed before re-analysis of studies Focus on primary mutations for each drug that would be expected to markedly reduced response to that drug –Not inclusive of all possible mutations that influence susceptibility Used to calculate genotypic sensitivity score and number of mutations present for each drug class

Zidovudine70R; 215Y/F; 41L; 67N; 210W; 219Q StavudineSee MNR-1 and MNR-2 Didanosine74V; 65R; 184V/I Zalcitabine65R; 69D; 74V; 184V/I LamivudineM184V/I AbacavirAny 3 or more of: 184V/I; 65R; 74V; 115F; 41L; 67N; 70R; 210W; 215Y/F; K219Q Multi-Nucleoside Q151M Resistance-1 (MNR-1) Secondary: 62V; 75I; 77L; 116Y Multi-Nucleoside 3 amino acid insert between codons (69Ins) Resistance-2 (MNR-2)2’: 41L; 62V; 67N; 70R; 210W; 215Y/F; 219Q Nucleoside Reverse Transcriptase Inhibitors

Adefovir 65R; 70E; MNR-2 184V causes increased susceptibility Non-nucleoside Reverse Transcriptase Inhibitors Nevirapine 103N; 106A; 108I; 181C/I; Y188C/L/H; G190A/S Delavirdine 103N; 181C; 236L Efavirenz103N; 188L; 190S/E Secondary: 100I; 101E/Q; 108I; 188H; 225H Nucleotide Reverse Transcriptase Inhibitors

Indinavir 32I; 82A/T/F; 84V; 90M Ritonavir32I; 82A/T/F/S; 84V; 90M Saquinavir48V; 82A/T; 84V; 90M Nelfinavir30N; 82F; 84V; 90M Amprenavir32I; 50V; 84V Protease Inhibitors

JWM 11/99 Calculation of Genotypic Sensitivity Score Mutation present for drug received = 0 Mutation not present for drug received = 1 Exceptions: –M184V for Adefovir = 1.5 –AZT mutations for d4T, ddI, ddC = 0.75 Total Score = sum of individual drug scores

JWM 11/99 Calculation of Phenotypic Sensitivity Score Resistance present for drug received = 0 Resistance absent for drug received = 1 Total Score = sum of individual drug scores Resistance defined as either –> 4-fold or > 10-fold decrease in susceptibility (increase in IC50) –separate analyses for each “cut-off”

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 Selection of Studies for Re-Analysis Criteria: –Study completed –Adequate size for multivariate analysis 12 of 15 studies qualified –10 retrospective –2 prospective, intervention based

Study Name Investigator N with GT/PT Treatment Experience Resistance Technology Median Baseline HIV RNA (range) [25 th – 75 th ] Median Baseline CD4 (range) [25 th – 75 th ] ABC Pooled R. Lanier 134 / 84 nRTI exp, PI/NNRTI naïve GT (ABI) PT (Virco) 3.7 (2.6 – 5.8) 417 (11– 1266) ACTG 333 M. Para 46 / 0 nRTI/SQV exp, naïve to other PIs GT (ABI/ clonal seq) ACTG 364 D. Katzenstein 144 / 0 Heavily nRTI exp, naïve to PI/NNRTI GT (Stanford) 4.1 [3.6 – 4.6] 323 [242 – 460] ACTG 372 S. Hammer 96 / 80 Heavily nRTI exp, IDV exp GT (Virco) PT (Virco) CNAA 2007 M. Ait-Khaled 94 / 64 Heavily nRTI/ PI exp, 42% NNRTI exp GT (ABI) PT (Virco) 5.1 ( ) 160 ( ) Description of Re-analyzed Studies

Name Investigator N with GT/PT Treatment Experience Resistance Technology Median Baseline HIV RNA (range) [25 th – 75 th ] Median Baseline CD4 (range) [25 th – 75 th ] Stanford A. Zolopa 54 / 0 Heavily nRTI/PI exp GT (Stanford) BC Centre R. Harrigan 58 / 53 nRTI exp, NNRTI naïve GT (Virco) PT (Virco ) 4.8 (2.7 – 5.8) 160 ( ) Frankfurt V. Miller 0 / 50 Heavily pretreated PT (Virco) Swiss S. Yerly 62 / 0 HAART “failures” GT (ABI) 5.2 (3.1 – 6.4) 113 (4 – 633) GS 408 M. Miller 161 / 0 Heavily pretreated GT (Pharmacia) 4.1* 338* *Mean Values Description of Re-analyzed Studies

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 Composite Data Presentations 8 retrospective, then 2 prospective studies –GS 408 and Swiss studies not included –HIV RNA change modeled rather than failure endpoint Dropout = failures analyses (DAF) Models (unadjusted and adjusted) –HIV RNA –Genotypic Sensitivity Score –Number of Mutations by Drug Class –Phenotypic Sensitivity Score Other analyses/models provided in documents

JWM 11/99 Meta-Analysis from Surrogate Marker Working Group

Baseline HIV-1 RNA (Odds Ratio per 1.0 log 10 Increase) Unadjusted - Retrospective Studies - Dropouts as Failures

Baseline HIV-1 RNA (Odds Ratio per 1.0 log10 Increase) Adjusted for Genotypic Sensitivity Score and New Drug Covariates

Baseline Genotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Unadjusted - Retrospective Studies - Dropouts as Failures

Baseline Genotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA and New Drug Covariates

Baseline # of NRTI Mutations (Odds Ratio per 1 additional) Adjusted for Other Classes in Regimen

Baseline # of NRTI Mutations (Odds Ratio per 1 additional) Adjusted for Baseline HIV RNA, New Drug Covariates and Other Classes

Baseline # of PI Mutations (Odds Ratio per 1 additional) Adjusted for Other Classes in Regimen

Baseline # of PI Mutations (Odds Ratio per 1 additional) Adjusted for Baseline HIV RNA, New Drug Covariates and Other Classes

4-FR Phenotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates

10-FR Phenotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates

JWM 11/99 Re-Analysis of Prospective Studies:VIRADAPT and GART

Post-Meeting Correction Baseline HIV-1 RNA (Odds Ratio per 1.0 log10 Increase) Adjusted for Genotypic Sensitivity Score, New Drug Covariates Prospective Studies - Dropouts as Failures

Baseline Genotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates Prospective Studies – Dropouts as failures

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 Presentations by M. Para, M. Ait-Khaled, V. Miller

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 Ongoing Prospective Trials NameLocation (Sponsor)DesignStatus RESA 2026US (GW/Virco)PT vs SOCClosed VIRA 3001US (GW/Virco)PT vs SOCInterim Analysis CERTUS (Military)PT vs GT vs SOCEnrolled CTCG 575US (ViroLogics)PT vs SOCEnrolling NARVALFR (ARNS)PT vs GT vs SOCEnrolling SEARCHUS (VGI)GT vs SOCEnrolling HAVANNASpain2 x 2 (  GT x  PT)Enrolling ERAUK (MRC/Virco)PT vs GT vs SOCOpening A5076US (ACTG)PT vs GT vs GT/PTIn Development

JWM 11/99 VIRA3001 An Open-Label, Randomized Trial Comparing the Effect on Viral Load of Standard HIV Treatment Practice (Delayed Phenotyping) with Treatment Based on the Antivirogram™ (Immediate Phenotyping) Preliminary Results October, 1999

JWM 11/99 Antivirogram (n = 144) (n = 144) Control (n = 130) Follow-up Wk 2, 4, 8, 12 & 16 Follow-up Screening (Week 5) Baseline (Day 1) No therapy changes permitted VIRA3001 Study Design

JWM 11/99 Patient Population Prior therapy history of  2 NRTIs and 1 PI Plasma HIV-1 RNA  2,000 copies/ml Stable ART for 1 month prior to screening No prior phenotypic testing

JWM 11/99 HIV-1 RNA Response Modified ITT (Observed Data)

JWM 11/99 HIV-1 RNA Response Modified ITT (LOCF)

JWM 11/99 Outline Goals of Clinical Validation Subcommittee (CVSC) Review of CVSC Workshop - April 21-22, 1999 Development of standardized Data Analysis Plan (DAP) Review of Mutation Table used for DAP Description of re-analyzed studies Composite data from re-analyzed studies Presentation of representative studies – ACTG 333, CNAA 2007, Frankfurt cohort Review of prospective studies in progress Summary of key points

JWM 11/99 Summary of Key Points Standardized re-analysis of retrospective studies generally confirms associations between baseline genotype or phenotype and virological response –small datasets  variability (broad CIs) Prospective, intervention-based trials support the clinical value of resistance testing for selection of treatment regimens in experienced patients Data accumulating from ongoing clinical trials of approved and investigational agents will refine the interpretation and improve the predictive value of specific resistance test results

JWM 11/99 MELLORS END

JWM 11/99 BACKUP SLIDES

4-FR PI Phenotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates and Other Classes

10-FR NRTI Phenotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates, Other Classes

10-FR PI Phenotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates, and Other Classes

Number of New Drugs in Regimen (Odds Ratio per Additional New Drug) Retrospective Studies - Dropouts as Failure

Baseline # of NRTI Mutations (Odds Ratio per 1 additional) Adjusted for Baseline HIV RNA, New Drug Covariates and Other Classes Prospective Studies - Dropouts as Failure

Baseline # of PI Mutations (Odds Ratio per 1 additional) Adjusted for Baseline HIV RNA, New Drug Covariates and Other Classes

4-FR NRTI Phenotypic Sensitivity Score (Odds Ratio per 1.0 Unit Increase) Adjusted for Baseline HIV RNA, New Drug Covariates, and Other Classes

Potent New Drug (Odds Ratio for Change from No to Yes) All Studies - Dropouts as Failures

Number of New Drugs in Regimen (Odds Ratio per Additional New Drug) Prospective Studies - Dropouts as Failure

JWM 11/99 Planned Analyses* Virologic response - change in log10 RNA from baseline –ITT Observed & LOCF (Wilcoxon Rank-Sum Test controlled for investigator site) Proportion of subjects with <400 HIV RNA copies/mL plasma –Cochran-Mantel-Haenszel Test controlled for investigator site Immunologic (CD4 cell) response - change from baseline –ITT Observed & LOCF (Wilcoxon Rank-Sum Test controlled for investigator site) Number of virologic endpoints reached –Cochran-Mantel-Haenszel Test controlled for investigator site –Virologic failure defined as failure to achieve  0.5 log10 decrease from baseline in HIV RNA at Week 8 or increase above baseline or >0.5 log10 increase above nadir after Week 8 *Analyses performed on subjects who started therapy at Day 1