Evaluation of cutpoints for phenotypic hypersusceptibility to efavirenz (2003)

Phenotypic assays measure the susceptibility of HIV-1 to antiretroviral drugs, and are used to guide treatment regimen choices. Analyses of baseline predictors of virological failure support the use of a 0.4-fold cutpoint for defining increased susceptibility (hypersusceptibility) to efavirenz in the PhenoSense HIV assay. The enhancement of the virological activity of efavirenz, associated with resistance to reverse transcriptase inhibitors, provides an opportunity to consider mutational and drug interactions in optimizing treatment. High-throughput phenotypic assays, which measure the inhibition of reverse transcriptase and protease activity by increasing concentrations of drug, provide accurate and reproducible measures of drug susceptibility to HIV-1. With one of the commercial phenotypic assays, the ViroLogic PhenoSense assay (ViroLogic, Inc., South San Francisco, CA, USA), increased susceptibility or hypersusceptibility to each class of antiretroviral drugs has been observed. Increased susceptibility to non-nucleoside reverse transcriptase inhibitor (NNRTI) drugs is more common in nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients and is most closely associated with resistance to zidovudine. Site-directed mutagenesis studies have shown that combinations of the classic thymidine analog mutations, such as M41L, D67N, T215Y, and K219R, or other NRTI-associated mutations, such as M184V and T69 insertions, are associated with NNRTI hypersusceptibility [1]. In the ACTG 364 study cohort of highly zidovudine-experienced subjects, nearly 40% demonstrated hypersusceptibility associated with D67N, L210W and T215Y/F [2,3]. In three clinical trials, CCTG 575 [4], ACTG 364 [3,5] and ACTG 398 [6] and one observational study [7], hypersusceptibility to NNRTI drugs has been associated with improved virological outcome, and with immunological outcome in one study [4]. The original definition of NNRTI hypersusceptibility, a less than 0.4-fold change in susceptibility relative to a wild-type reference virus, was based on the assay variability and the variability of drug susceptibility among wild-type clinical isolates [1]. With a view to refining the definition to a more clinically relevant threshold for efavirenz hypersusceptibility, we examined multiple potential cutpoints in the ACTG 364 trial database and related these to the virological outcome of treatment over 144 weeks. Two different approaches were used to evaluate thresholds for efavirenz hypersusceptibility. In the first approach, a separate co-variate for baseline efavirenz hypersusceptibility was examined in models that included other predictors of treatment outcome. Cox proportional hazards models evaluated the relationship between baseline efavirenz hypersusceptibility and the risk of on-treatment virological failure, defined as confirmed HIV-1 RNA 2000 copies/ml or greater at week 16 or later [3]. Table 1 displays the hazard ratios for efavirenz hypersusceptibility, using various efavirenz hypersusceptibility definitions, in separate multivariate Cox models that also adjust for baseline log10 HIV-1 RNA and susceptibility to drugs in the treatment regimen. The phenotypic susceptibility score (PSS) was used as a summary measure of the susceptibility of baseline virus to the assigned treatment regimen. The PSS of baseline viruses was determined for each study subject by summing the levels of drug susceptibility to each drug in the treatment regimen [3]. In the PSS calculation, each drug in the regimen could receive a maximum score of 1 for complete sensitivity, and a minimum score of 0 for complete resistance, with a continuous range between 0 and 1 for ‘partly sensitive’ viruses. Although many hypersusceptibility cutpoints were independently associated with treatment failure hazard ratios of less than 1, the best estimate for the cutpoint was 0.4-fold (based on maximizing the partial likelihood of the Cox model). Whereas an efavirenz hypersusceptibility cutpoint of 0.3-fold was associated with an even lower risk of treatment failure (Table 1), the P value observed at this cutpoint was greater than 0.05 as a result of the smaller patient numbers. The a priori choice of 0.4 as a cutpoint, as used in three clinical trials [3,4,6], is thus supported by these additional analyses.Table 1: Hazard ratios for treatment failure associated with various efavirenz hypersusceptibility thresholds among efavirenz-treated subjects, adjusted for baseline log10 HIV-1 RNA and phenotypic susceptibility score.The second approach to evaluate the efavirenz hypersusceptibility threshold utilized models that incorporated efavirenz hypersusceptibility into the baseline PSS. In these models, the PSS could be increased by a maximum of 0.5 units for efavirenz hypersusceptibility, such that an efavirenz score between 1.0 and 1.5 was given to viruses that showed hypersusceptibility to efavirenz. For example, when evaluating the efavirenz hypersusceptibility definition of less than 0.4-fold, the PSS contribution for efavirenz was calculated as for efavirenz drug susceptibility values less than 0.4-fold, 1 for efavirenz drug susceptibility values between 0.4- and 2.5-fold, and between 0 and 1 for susceptibility values above 2.5-fold [3]. In multivariate Cox models using this approach, the threshold of 0.4-fold was again shown to be the maximum likelihood estimate, and yielded the smallest hazard ratio (hazard ratio 0.45, P = 0.003, for composite PSS incorporating efavirenz hypersusceptibility). In summary, this re-analysis of ACTG 364 outcome data supports the use of a 0.4-fold cutpoint for defining efavirenz hypersusceptibility in the PhenoSense HIV drug susceptibility assay. The increase in virological efficacy of efavirenz in three clinical trials (ACTG 398, ACTG 364 and CCTG 575) among NRTI-experienced subjects with efavirenz hypersusceptibility, compared with those without evidence for efavirenz hypersusceptibility (all with a cutpoint of 0.4) provides new challenges and opportunities. A challenge is how to use these observations in the sequencing of antiretroviral treatments to sustain a long-term virological response. Treatment regimens that contain efavirenz in combination with other active drugs may have enhanced potency in salvage therapy for hypersusceptible viruses. Among these patients, it may be advantageous to continue NRTI treatment to sustain the resistance mutations associated with efavirenz hypersusceptibility. The enhancement of the virological activity of efavirenz, associated with resistance to NRTI, provides an opportunity to consider mutational and drug interactions in optimizing treatment.