Persisting long-term benefit of genotype-guided treatment for HIV-infected patients failing HAART. The Viradapt study: week 48 follow-up

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1 Antiviral Therapy 5: Persisting long-term benefit of genotype-guided treatment for HIV-infected patients failing HAART. The Viradapt study: week 48 follow-up P Clevenbergh 1 *, J Durant 1, P Halfon 2, P del Giudice 1, V Mondain 1, N Montagne 3, JM Schapiro 4, CAB Boucher 5 and P Dellamonica 1 1 Department of Infectious Diseases, Nice University Hospital, France 2 Virology Department, Alphabio, Marseille, France 3 Bonnet Hospital, Fréjus, France 4 National Haemophilia Centre, Tel Hashomer, Israel 5 Virology Department, University Hospital, Utrecht, The Netherlands *Corresponding author: Tel: ; Fax: ; clevenbergh.ph@easynet.fr Objective: We report the 12 months follow-up of the patients who participated in the Viradapt study. Methods: A total of 108 HIV-infected patients failing antiretroviral (ARV) therapy (HIV RNA > copies/ml, therapy >6 months with nucleoside reverse transcriptase inhibitors, >3 months with protease inhibitors (PIs) were randomized into two arms: standard of care in the control arm, and treatment according to the resistance mutations in the protease and reverse transcriptase genes in the study arm. After the first 6 months of the randomized study, open-label, genotype-guided treatment was offered in both arms. A multivariate analysis was performed to assess the predictive factors of treatment success (HIV RNA <200 copies/ml). Results: The two arms were comparable in terms of risk factors, age, sex, previous treatments, CD4 cell count and log 10 HIV-1 RNA at baseline. At week 24, an interim combined analysis showed a statistically significant difference in the drop in viral load at months 3 and 6 (P=0.015, repeated measures analysis of variance) in favour of the genotype group. Patients in both arms were then offered open-label genotyping. Genotype analysis was performed every 3 months, and treatment changes could accordingly be made. As some of the patients in the control arm had already progressed to months 9 or 12, only 69% (30/43) of these patients received genotypeguided treatment changes. In the genotype arm, the mean drop in HIV RNA of 1.15 log 10 copies/ml, obtained at month 6, persisted at months 9 and 12 (1.15 log 10 copies/ml ±0.17). In the control arm, an additional drop in HIV RNA to 0.98 log 10 ±0.22 copies/ml was observed by month 12. In control patients receiving open-label genotype, the percentage of patients with HIV-1 RNA levels below detection limit (200 copies/ml) rose from 14% at month 6 to 30.5% at month 12. This percentage in the study arm remained stable at 31.3% and 30% at months 9 and 12, respectively. Genotype-guided therapy, primary protease mutations and PI plasma concentrations were significantly correlated with virological success. Conclusion: In this heavily pretreated patient population, genotype-guided therapy resulted in a sustained reduction in HIV RNA of greater than one log 10 throughout a 1 year follow-up period. Performance of genotype-guided therapy may have contributed to the additional viral load reduction seen in patients in the control group who received open-label genotyping after the 6 month point. Multivariate analysis showed that the presence of primary protease mutations, performance of genotypeguided treatment changes and PI plasma concentrations independently affected virological response. Introduction Although combination therapy has significantly decreased HIV-associated morbidity and mortality [1], a large number of patients are not able to achieve or maintain complete viral suppression with their current antiretroviral (ARV) drug regimen [2]. Salvage therapy is often of poor efficacy [3], due to the high prevalence of drug-resistant virus in previously treated patients and the broad cross-resistance that is observed between ARV drugs [4]. Salvage patients given drugs to which their virus was phenotypically or genotypically resistant did worse than patients receiving a regimen containing more than one drug to which their virus was still sensitive [5 13]. The short-term benefits for patient management with the assistance of drug-resis International Medical Press /00/$

2 P Clevenbergh et al. tance testing in the selection of a alternative ARV treatment has been shown in two prospective pilot studies, GART [14] and Viradapt [15]. In the Viradapt study, patients managed with the use of genotype-resistance testing fared better at month 6 than patients managed with standard of care with regard to the absolute drop in HIV RNA and the proportion of patients with an HIV RNA below level of quantification [15]. The GART study showed a statistically significant advantage for the genotype group at month 3 in terms of both virological parameters [14]. The long-term benefit of genotype resistance testing and the efficacy of repeated genotyping of patients failing therapy have not yet been evaluated. Here, we report the 12 months of follow-up to the Viradapt study. After 6 months the interim analysis showed a statistically significant advantage for patients in the genotype arm, both arms received open-label genotyping if viral load failed to demonstrate a decline of at least 0.5 log 10 from baseline. Trial design Patients From March 1997 until March 1998, consecutive HIVinfected patients failing antiretroviral therapy (HIV RNA > copies/ml, therapy >6 months with nucleoside reverse transcriptase inhibitors, >3 months with PIs) who consented to participate were randomized into two arms. Standard of care consisted of a therapeutic best guess on the part of the clinician for the development of a salvage regimen in the control arm, and a change in therapy guided by the resistanceassociated mutations in protease and reverse transcriptase (RT) genes from genotyping in the study arm. Forty-three patients were randomized to the control group and 65 to the genotypic group. The demographic data on these patients has already been reported [15]. Imbalance in the number of patients between the two arms is due to the fact that a secondary randomization was initially planned for the genotype arm. The genotype study arm was initially designed to also assess the utility of a treatment modification based on the appearance of mutations during treatment before the appearance of a rise in viral load. However, as the lower limit of sensitivity for the utilized genotyping techniques is approximately 1000 copies/ml, virus below the limit of detection could not be amplified for sequencing. This second randomization was abandoned and the patients were kept in their initial arm. Study design The first 6 month period was a randomized study in which the study arm received genotype-guided treatment and the control arm received standard of care. An interim analysis was performed when all patients had reached at least the 6 months follow-up end-point. The results of this analysis have previously been published and led to the study being changed to an open-label format due to the superior results seen in the genotype arm [15]. During the second 6 month period, both arms received genotype-guided treatment changes in an open-label fashion. Genotyping was performed every 3 months. For patients in the control arm who had already progressed beyond 6 months, genotype-guided treatment change was performed only at month 9 and for those who had already reached the 1 year study end-point, no genotype-guided treatment change could be performed. Virological studies Complete sequencing of the major part of the RT gene and the entire protease gene was performed on plasma HIV RNA. HIV RNA was extracted from patient plasma samples, and then amplified using RT PCR methodologies. Owing to the lack of registered kits at the time the study was initiated, multiple procedures were utilized for determining the genetic sequences during the course of the trial. From the beginning of the trial until January 1998, several home-brew PCR extraction and DNA sequencing technologies developed by ACT Gene Laboratory (Paris, France) were used following standard laboratory procedures. Qiagen- or Boehringer Mannheim-based RNA extractions were used, followed by RT PCR and nested-pcr using pol gene oligonucleotides producing two separate fragments: a 800 bp reverse transcriptase amplicon and a 350 bp protease amplicon. These amplicons were then sequenced using ALF Express and ABI 337 (Foster City, Calif., USA) sequencers, using either single- or double-stranded data. From February 1998 until July 1998, the pre-release version of the TRUGENE HIV-1 kit was used for the RNA extraction and RT PCR in a single step, while DNA sequencing was performed on DNA sequencers. From August 1998 until study completion, the Visible Genetics TRUGENE HIV-1 kit (version 1.0) was used. Plasma RNA extraction (500 µl) was performed after centrifugation. The pellet was treated using RNAzol (Biotecs Laboratories, Houston, Tex., USA), followed by chloroform, isopropanol and ethanol steps. Extracted RNA was stored at 80 C until use. The viral RNA was retrotranscribed in cdna and subsequently amplified by a single tube RT PCR, using the TRUGENE HIV-1 kit High Resolution Genotyping Genekit (Visible Genetics Inc., Toronto, Ont., Canada), producing a 1.3 kb amplicon covering the entire protease gene and the majority of the reverse transcriptase gene of HIV-1. Bidirectional DNA sequencing of the amplification International Medical Press

3 Genotype-guided treatment for HIV-infected patients failing HAART Figure 1. Mean changes in plasma HIV-RNA from baseline throughout 12 months in control and genotype arms HIV RNA (log10 copies/ml) Control Randomized study 3 Genotypic 6 Time (months) products was performed using CLIP technology (Visible Genetics). Each sequencing reaction was loaded on a MicroGene Clipper (Visible Genetics), an automated DNA sequencer. For each sample sequenced, the resulting assays were base called using GeneObjects software (Visible Genetics), aligned and assembled together using the GeneLibrarian software (Visible Genetics). The resulting sequence for each sample was then compared to a database containing known drug resistance mutations, to detect which mutations were present in the patient s HIV-1 viral RNA. HIV RNA was quantified using the Roche Amplicor HIV assay (Roche Molecular Systems, Alameda, Calif., USA). Treatment Decisions regarding treatment changes were guided by correlating specific mutations to decreased susceptibility to a specific drug. The interpretation of resistance-associated mutations is a rapidly evolving field and the resistance/mutation table [15] has evolved over the duration of study as the knowledge about genotypic resistance patterns has increased. Three separate resistance/mutations tables were used along the study period [15 17]. New drugs (nevirapine, efavirenz and abacavir) became available during the study period and were included in the allowed therapeutic choices. Use of newly available drugs was equally distributed in both arms. Statistical analysis For the first 6 months of the randomized study, analyses were performed on an intent-to-treat basis. All patients who had observations after randomization were included in the analysis. The primary end-point of 9 Open study 12 the study was the variation of HIV-RNA from baseline to months 3 and 6 (log 10 -transformed). Secondary efficacy parameter was the proportion of patients with plasma HIV RNA below the limit of detection (200 copies/ml). Differences in the proportion of patients in the two groups were analysed using a χ 2 test. Mean changes of HIV RNA from baseline were compared by analysis of variance and two-tailed t-tests. For the 6 12 month open-label follow-up study, the primary end-point was the evolution of HIV RNA from baseline to months 9 and 12. The secondary endpoint was the proportion of patients with HIV RNA below the level of detection at 200 copies/ml. All data collected were included in the analysis. Data from patients who died or were lost to follow-up were censored and not included in the analysis. Statistics were performed in an on-treatment fashion for this part of the study. The correlation between quantitative variables (HIV-RNA, PI plasma concentrations and PI primary mutations) was evaluated by linear regression. Presence or absence of primary protease gene mutations (including 30, 46, 48, 82, 90) at baseline, were correlated with reduction in viral load at 3 and 6 months. The χ 2 test or the Fisher s Exact test was used for nominal variables. Multivariate analysis (logistical regression) was performed to determine the independent and predictive values of virological response (HIV RNA below the level of detection at 200 copies/ml at months 3 and 6). All statistical tests reported are twotailed and all confidence intervals are at 95%. A P-value <0.05 was considered statistically significant. Data were analysed using the Statview version 5.0 software. Results A total of 108 patients were included in the Viradapt study. In the genotype group, 60/65 (92%) of patients completed month 9 and 56/65 (86%) patients reached month 12 of follow-up. In the control group, 39/43 (90%) patients completed month 9 and 36/43 (84%) patients reached month 12 of follow-up. In the control arm, a genotype-guided treatment change was performed one or two times in 30/43 (69%) patients during the entire open-label period, in 18/43 patients (42%) at month 6 and in 23/43 patients (53%) at month 9. In 48 patients in the genotype group, a third or a fourth genotype-guided therapy change was performed during the last 6 months of follow-up: 31/65 patients (48%) at month 6 and 32/65 patients (49%) at month 9. Nine of 43 patients (21%) in the control arm and 15/65 (23%) in the genotype arm benefited from two consecutive genotype-guided therapy changes during the second 6 months. In the genotype arm, the reduction in viral load was Antiviral Therapy 5:1 67

4 P Clevenbergh et al. Figure 2. Percentage of patients with plasma HIV RNA below the limit of detection (200 copies/ml) in control and genotype arms Figure 3. Correlation of baseline primary protease mutations and randomization arm with changes in HIV RNA Randomized Study Open Study Control Genotypic 0 Time (months) Control 0,00 14,00 14,00 12,5 30,5 Genotypic 0,00 29,20 32,30 31,3 30,4 Proportion of patients <200 copies/ml 35 HIV RNA (log10 copies/ml) Time (months) Control wild-type Control mutant Genotypic wild-type Genotypic mutant maintained throughout the 12 months of study with a mean drop in HIV RNA of 1.15 log 10 ±0.17 copies/ml at month 12. In control arm, at the completion of the randomized study, viral load decreased by only 0.67 log 10 copies/ml. During the second 6 months with open label genotyping, there was an additional decrease in viral load to 0.98 log 10 ±0.22 copies/ml (Figure 1). In the genotype arm, the percentage of patients with an HIV RNA below level of detection remained stable round 30% throughout the 12 months of follow-up. In the control arm, the proportion of patients with an HIV RNA below the level of detection rose from 14% at month 6 to 30.5% at month 12 (Figure 2). The multivariate analysis showed that genotype-guided therapy [odds ratio, 2.24 (95% confidence interval ); P=0.025], primary protease mutations (odds ratio, 2.47 [95% confidence interval ]; P=0.014) and PI plasma concentration [odds ratio, 2.37 (95% confidence interval ); P=0.017] were all independent predictors of success. Discussion In the genotype-guided treatment group the benefit of using resistance testing persisted for 1 year. With intensive use of genotyping, 30/65 (46%) of the study patients reached an HIV RNA below the level of quantification at least once during the study period opposed to 11/43 (26%) patients in the control arm. However, despite repeated genotype-guided treatment changes in the study arm, the proportion of patients with HIV RNA levels below detection did not increase further after month 6. This could be due to the selection of patients with broadly resistant HIV strains and/or to the lack of active drug(s) against resistant viruses. Incomplete patient adherence to drug regimens could also have blunted the effectiveness of repeated genotype-guided treatment changes. During the second 6 months of the study, open-label genotyping was performed on all study participants. During this phase, there was an additional drop in HIV RNA levels in the control arm. Since this phase was not controlled, no conclusions can be drawn regarding the cause or causes of the additional reduction in HIV RNA. This could be due to the availability of more potent drugs like abacavir or non-nucleoside reverse transcriptase inhibitors [18,19]. This could also be due to the increased skill of the physicians in caring for salvage. Use of genotyping could also have played a role in this additional drop in HIV RNA. Empirical salvage therapies using more than three drugs combinations have been shown to be more powerful than the standard three-drug regimen [20], and the number of patients given a four- or five-drug regimen increased during the study period. In the former control patients the percentage of patients with an HIV RNA level below the limit of quantification rose at month 12. This delayed increase in the percentage of patients with undetectable HIV RNA is possibly due to the fact that most patients in the former control group received open-label genotype-guided treatment changes only at month 9. Many retrospective studies [6 13] have established a link between baseline resistance profiles and the changes in HIV RNA levels in drug-experienced patients put on salvage therapy. A multivariate analysis to determine predictive factors affecting HIV RNA changes was performed. The presence of primary International Medical Press

5 Genotype-guided treatment for HIV-infected patients failing HAART protease mutations, performance of genotype-guided treatment and PI plasma concentrations all independently affected virological response. Regarding the effect of primary protease mutations on HIV RNA changes, the greatest reduction was seen for patients who did not have primary protease mutations and received genotype-guided treatment. A decrease in viral load of 1.5 log 10 was seen in these patients. The poorest response was seen in those in whom primary protease mutations were present and received standard of care. Intermediate results were seen in the groups in whom protease mutations were absent and received standard of care, or patients in whom primary protease mutations where present and received genotype-guided treatment changes (Figure 3). Even with the use of genotype-guided therapy changes, 70% of the patients in the Viradapt study did not achieve complete viral suppression. Suboptimal plasma concentration of PIs has also been shown to be an independent predictive factor of failure and may have been a contributing factor in this low suppression rate [21]. A different analysis using the standardized plan of the Resistance Collaborative Group (DAP data analysis plan) [22] demonstrated that the predictive factors of success (low baseline viral load, higher number of new drugs, higher genotype sensitivity score, lesser resistance mutations to reverse transcriptase or protease inhibitors) were the same as have previously been described in the literature [3,23 27]. In conclusion, in this heavily pretreated-patient population, genotype-guided therapy changes resulted in a sustained reduction in HIV RNA levels of greater than 1 log 10 throughout the 1-year follow-up period. Performance of genotype-guided therapy changes may have contributed to the additional viral load reduction seen in control patients after the introduction of openlabel genotyping to all study participants at 6 months. Multivariate analyses showed that the presence of primary protease mutations, performance of genotypeguided treatment and PI plasma levels independently affected the virological response. Acknowledgements Co-investigators: P Puglièse, V Rahelinirina, I Perbost, C Pradier, L Bentz, H Etesse (Infectious Diseases Department, Nice, France), P Simonet (Hôpital Les Broussailles, Cannes, France), E Counillon (Bonnet Hospital, Frejus, France). Study nurses: M Massard, J Charlier, G Valentini, C Rascle). Sponsors and collaborative assistance: Produits Roche France E Dohin; ACT Gene and Visible Genetics Europe C Sayada, Ch Holtzer; Data Management M Andriamanamihaja, R Boulme; Genotypic Analysis A Beyou, C Domingo. References 1. Palella FJ, Delaney KM, Moorman AC, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. New England Journal of Medicine 1998; 338: Perrin L & Telenti A. HIV Treatment failure: testing for HIV resistance in clinical practice. Science 1998; 280: Deeks SG, Hecht FM, Swanson M, et al. HIV RNA and CD4 cell count response to protease inhibitor therapy in an urban AIDS clinic: response to both initial and salvage therapy. AIDS 1999; 13:F35 F Palmer S, Shafer RW & Merigan TC. Highly drug-resistant HIV-1 clinical isolates are cross-resistant to many antiretroviral compounds in current clinical development. AIDS 1999; 13: Patick AK, Zhang M, Hertogs K, et al. Correlation of virological response with genotype and phenotype of plasma HIV-1 variants in patients treated with nelfinavir in the US expanded access program. Antiviral Therapy 1998; 3 (Suppl. 1): Zolopa AR, Shafer RW, Warford A, et al. Predictors of antiviral response to saquinavir/ritonavir therapy in a clinical cohort who have failed prior protease inhibitors: a comparison of clinical characteristics, antiretroviral drug history and HIV genotype. Antiviral Therapy 1998; 3 (Suppl. 1): Lanier R, Danehower S, Daluge S, et al. Genotypic and phenotypic correlates of response to abacavir (ABC, 1592). Antiviral Therapy 1998; 3 (Suppl. 1): Harrigan PR, Montaner JS, Hogg RS, et al. Baseline resistance profile predicts response to ritonavir/saquinavir therapy in a community setting. Antiviral Therapy 1998; 3 (Suppl. 1): Walmsley S, Walach C, Moses A, Becker M & Harrigan PR. Can baseline genotype predict response to salvage therapy with nelfinavir. 6th Conference on Retroviruses and Opportunistic Infections. Chicago, February 1999; Abstract Harrigan PR, Raboud J, Hertogs K, et al. Drug resistance and short term virological response in patients prescribed multidrug rescue therapy. Antiviral Therapy 1999; 4 (Suppl. 1): Hammer S, Demeter L, degruttola V, et al., for the ACTG 372 Study Team. Relationship of phenotypic and genotypic resistance profiles to virological outcome in a trial of abacavir, nelfinavir, efavirenz and adefovir dipivoxil in patients with virological failure receiving indinavir (ACTG 372). Antiviral Therapy 1999; 4 (Suppl. 1): Katzenstein D, Bosch R, Shafer R, et al. Virological response to nelfinavir, efavirenz, or both in patients with > 4 years of previous nucleoside RT inhibitors in ACTG 364. Antiviral Therapy 1999; 4 (Suppl. 1): Lorenzi P, Opravil M, Hirschel B, et al., for the Swiss HIV Cohort Study Impact of drug resistance mutations on virologic response to salvage therapy. AIDS 1999; 13:F17 F Baxter JB, Mayers DL, Wentworth DN, et al., for the CPCRA 046. Final results of CPCRA 046: a pilot study of antiretroviral management based on plasma genotypic antiretroviral resistance testing (GART) in patients failing antiretroviral therapy. Antiviral Therapy 1999; 4 (Suppl. 1): Durant J, Clevenbergh P, Halfon P, et al. Drug-resistance genotyping in HIV-1 therapy: the Viradapt randomised controlled trial. Lancet 1999; 353: Sande MA, Gilbert DN & Moellering RC. In The Sanford Guide to HIV/AIDS Therapy, 6 th Edition, Antimicrobial Therapy, Inc. 17. Hirsch M S, Conway B, D Aquila RT, Johnson VA, Brun- Vézinet F, Clotet B, Demeter L M, Hammer SM, Jacobsen DM, Kuritzkes DR, Loveday C, Mellors JW, Vella S & Antiviral Therapy 5:1 69

6 P Clevenbergh et al. Richman DD. Antiretroviral Drug resistance testing in adults with HIV infection. Consensus Statement. Journal of the American Medical Association 1998; 279: Foster RH & Faulds D. Abacavir. Drugs 1998; 55: De Clercq E The role of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy of HIV-1 infection. Antiviral Research 1998; 38: Duvivier C; Katlama C, Mouroux M, et al. GigaHAART: a rescue therapy in HIV infected patients with multiple failures to HAART. 7 th Conference on Clinical Aspects and treatment of HIV infection. October 1999, Lisbon; LB Durant J, Clevenbergh P, Garraffo R, et al. Relevance of protease inhibitor plasma levels in patients treated with genotypic adapted therapy: pharmacological data from the Viradapt study. AIDS 2000 (in press). 22. FDA meeting on HIV resistance testing. Gaithersburg, Md., USA, 2 3 November Fätkenheuer G, Theisen A, Rockstroh J, et al. Virological treatment failure of protease inhibitor therapy in an unselected cohort of HIV-Infected patients. AIDS 1997; 11:F113 F Staszewski S, Miller V, Sabin C, et al. Virological response to protease inhibitor therapy in an HIV clinic cohort. AIDS 1999; 13: Mocroft A, Gill MJ, Davidson W & Phillips A Predictors of a viral response and subsequent virological treatment failure in patients with HIV starting a protease inhibitor. AIDS 1998; 12: Casado JL, Perez-Elias MJ, Antela A, et al. Predictors of long-term response to protease inhibitor therapy in a cohort of HIV-infected patients. AIDS 1998; 12:F131 F Wit FWNM, van Leeuwen R, Weverling GJ, et al. Outcome and predictors of failure of highly active antiretroviral therapy: one-year follow-up of a cohort of Human Immunodeficiency Virus type 1-infected persons. Journal of Infectious Diseases 1999; 179: Received 1 December 1999; accepted 29 March International Medical Press