582 Background: Resistance to HER2 antagonism in HER2-positive breast cancer has been associated with activation of the PI3K signaling pathway. PIK3CA mutations in exon 9 and exon 20 have been shown to activate the AKT-mTOR pathway downstream of EGFR-family receptor tyrosine kinases, where most targeted drugs for breast cancer exert their effect. Methods: We measured PIK3CA mutation status in exons 9 (E545K and E542K) and 20 (H1047R) using two different methods [pyrosequencing (Monogram) and an allele-specific, real-time PCR assay (D×S)] and correlated the results with clinical outcomes in a cohort of patients (pts) with trastuzumab-treated MBC. Results: 22/85 pts (26%) with available FFPE tissue had mutations in exon 9 or exon 20. Results obtained by the two methods were 100% concordant. No statistically significant association was seen between the presence of PIK3CA mutations and the levels of HER2, HER3, or p95HER2, as measured by VeraTag™ (HERmark), although there was a trend favoring lower p95 levels in mutants (p=0.07). Including all pts, no differences were observed in either PFS (HR 1.23 for mutant vs. WT, p=0.47) or OS (HR 1.56, p=0.19). In HER2 FISH-positive pts (N=65), no differences were observed in either PFS (HR 1.72, p=0.15) or OS (HR 2.08, p=0.09). PIK3CA mutants fared better in the FISH-negative population (N=20) for PFS (HR 0.34, p=0.03) but not for OS (HR 0.74, p=0.6). Among pts that overexpressed HER2 protein (>13.8 by HERmark, N=59), those with exon 9 mutations, but not those with exon 20 mutations, experienced shorter PFS (HR 8.04, p=0.004 and HR 1.06, p=0.89, respectively) and OS (HR 4.85, p=0.03 and HR 1.48, p=0.45, respectively) when compared to PI3K WT pts. Conclusions: Exon 9 mutations appeared to exert a deleterious effect on outcomes, but exon 20 mutations did not. Pts with PIK3CA mutations, but without HER2 gene amplification, experienced better outcomes on trastuzumab treatment than PIK3CA WT pts, while no association was observed in HER2 amplified pts. These data are hypothesis-generating and require confirmation in larger controlled datasets of trastuzumab-treated MBC pts. Grant support from Komen for the Cure.

Background The CPCRA 064 study examined the effect of structured treatment interruption (STI) of up to 4 months followed by salvage treatment in patients failing therapy with multi-drug resistant HIV. We examined the relationship between the reversion rate of major reverse transcriptase (RT) resistance-associated mutations and change in viral replication capacity (RC). The dataset included 90 patients with RC and genotypic data from virus samples collected at 0 (baseline), 2 and 4 months of STI. Principal Findings Rapid shift towards wild-type RC was observed during the first 2 months of STI. Median RC increased from 47.5% at baseline to 86.0% at 2 months and to 97.5% at 4 months. Between baseline and 2 months of STI, T215F had the fastest rate of reversion (41%) and the reversion of E44D and T69D was associated with the largest changes in RC. Among the most prevalent RT mutations, M184V had the fastest rate of reversion from baseline to 2 months (40%), and its reversion was associated with the largest increase in RC. Most rates of reversion increased between 2 months and 4 months, but the change in RC was more limited as it was already close to 100%. The highest frequency of concurrent reversion was found for L100I and K103N. Mutagenesis tree models showed that M184V, when present, was overall the first mutation to revert among all the RT mutations reported in the study. Conclusion Longitudinal analysis of combined phenotypic and genotypic data during STI showed a large amount of variability in prevalence and reversion rates to wild-type codons among the RT resistance-associated mutations. The rate of reversion of these mutations may depend on the extent of RC increase as well as the co-occurring reversion of other mutations belonging to the same mutational pathway.

Objective: To assess the effect of HIV coreceptor tropism (CRT) on the relative risk of progression to a composite outcome of CD4+ count ≤350 cells per microliter, treatment initiation, or death. Methods: CRT assays were performed after study closure in baseline samples obtained from enrollees in a prospectively monitored cohort of treatment-naive adults with ≥450 CD4+ cells per microliter and ≥1000 HIV-1 RNA copies per milliliter. Results: Dual/mixed (D/M) and R5 CRT were detected in 32 and 282 patients, respectively. The baseline CD4+ count (617 versus 694 cells/μL; P = 0.05) differed in patients with D/M versus R5 CRT. Otherwise, baseline laboratory characteristics were similar. The relative risk of progression to the composite end point was 2.15 (P = 0.002) for D/M versus R5 CRT, 2.07 per 1.0 log10 higher viral load (P < 0.001) and 0.87 per 50 cells per microliter higher CD4+ cell count (P < 0.001). The effect of D/M CRT was also significant in separate analyses of time to initiation of antiretroviral therapy or CD4+ cell count ≤350 cells per microliter. Conclusions: Untreated patients with D/M rather than R5 CRT had a faster rate of disease progression, whether assessed by a composite outcome of time to CD4+ count ≤350 cells per microliter, treatment initiation, or death or by separate analyses of time to CD4+ count ≤350 cells per microliter or treatment initiation.

OBJECTIVES:To evaluate the association between baseline (BL) replication capacity (RC) (RCBL) and immunologic/virologic parameters (at BL and after 48 weeks on therapy) in HIV-1-infected subjects initiating antiretroviral therapy. METHODS:RCBL was determined using a modified Monogram PhenoSense HIV drug susceptibility assay on plasma HIV-1 from 321 treatment-naive subjects from AIDS Clinical Trials Group 384. Univariate and multivariable analyses were performed to determine the association of RCBL with BL and on-therapy virologic and immunologic outcomes. RESULTS:Higher RCBL was associated with lower baseline CD4 (CD4BL) (r = -0.23, P < 0.0001), higher baseline HIV-1 RNA (r = 0.25, P < 0.0001), higher CD4BL activation percent (r = 0.23, P < 0.0001), and lower CD4BL memory count (r = -0.21, P = 0.0002). In a multivariable model, week 48 CD4 increase (DeltaCD448) was associated with lower CD4BL memory count and higher CD4BL-naive percent (P = 0.004, P = 0.015, respectively). The interaction between CD4BL and RCBL was significant (P = 0.018), with a positive association between RCBL and DeltaCD448 in subjects with higher CD4BL and a negative association at lower absCD4BL. CONCLUSIONS:At baseline, higher RC was significantly associated with higher HIV-1 RNA, higher CD4 cell activation, lower CD4 cell count, and lower CD4 memory cell count. These factors may interact, directly or indirectly, to modify the extent to which CD4 recovery occurs in patients starting antiretroviral therapy at different CD4BL counts.

Objective. To evaluate the safety and virological response to lopinavir/ritonavir containing second-line therapy after failing a first line nonnucleoside reverse transcriptase inhibitor (NNRTI) based regimen. Design. Prospective 36 months cohort study of patients switched to zidovudine/stavudine plus didanosine plus lopinavir/ritonavir capsules as second-line regimen. Methodology. Structured interview, medical examination, and laboratory assessment performed every 6 months. Results. We enrolled 40 patients; 1 died and 3 were lost to follow-up. Median CD4+ count at baseline was 108 cell/μL, median log viral load was 4.8 copies/mL. Sixteen (40%) patients had baseline genotypic resistant test, 14 (87%) had lamivudine resistance mutations, and all had NNRTIs resistance mutations. At month 36, 82% of the patients achieved viral suppression (<400 copies/ mL) and the median increase in CD4+ count was 214 cell/μL, (interquartile range: 128-295). Twenty-five patients (62%) experienced at least one adverse event. Conclusions. Our study confirms lopinavir/ ritonavir-based second-line regimen but with a high rate of toxicities.

Background: The extent to which HIV-1 replication capacity (RC) influences the response to therapy remains to be established. Methods: Phenotypic susceptibility and RC of baseline isolates (n = 139) from patients enrolled in the ARGENTA trial were measured and correlated to treatment outcomes over 36 months. Results: RC in baseline isolates correlated with the number of phenotypically active drugs (R = 0.34, P < 0.001). Crude viral RC did not predict treatment outcomes. When viral RC was adjusted by baseline CD4 cell counts, HIV-1 RNA levels, and phenotypic susceptibility to the rescue regimen, it showed significant association with the immunologic outcome (per log10 RC higher, mean difference in 36 months' time-averaged change from baseline CD4 count = −68 cells/μL; P = 0.020). In the subgroup of patients with 3 or more phenotypically active drugs in the salvage regimen (n = 35, median RC = 65%), subjects carrying isolates with RC ≤65% as compared to those with RC >65% had better time-averaged HIV-1 RNA responses (mean: −1.04 vs. −0.32 log10 copies/mL; P = 0.012) and CD4 cell responses (mean: 132 vs. −7 cells/μL; P = 0.006). Among patients with HIV-1 RNA levels persistently >500 copies/mL (n = 61), RC, on a log10 basis, was inversely associated with time-averaged 36-month CD4 cell responses (β = −0.26; P = 0.046). Conclusion: After normalizing for viral susceptibility to the employed regimen or in patient subsets with suboptimal virologic response, higher viral RC may predict worse subsequent treatment outcomes.

Background. Human immunodeficiency virus type 1 (HIV-1) coreceptor tropism, the ability of the virus to enter cells via CCR5 or CXCR4, is a viral characteristic mediated by the envelope gene. The impact of coreceptor tropism on the natural history of HIV-1 infection has not been fully explored.Methods. Coreceptor tropism was measured using a recombinant virus single-cycle assay on plasma specimens obtained at baseline from 126 children and adolescents in the Hemophilia Growth and Development Study cohort who were enrolled from 1989 through 1990 and underwent follow-up through 1997.Results. Detectable CXCR4-using virus at baseline was associated with a lower baseline CD4(+) T cell count and a higher plasma HIV-1 RNA level. In addition, it independently predicted a greater decrease in CD4(+) T cell count over time (P < .001) and was associated with a 3.8-fold increased risk of progression to clinical AIDS.Conclusions. This study demonstrates that coreceptor tropism, as assessed by this single-cycle assay, independently influences the natural history of HIV-1 disease.

Purpose: Non-nucleoside reverse transcriptase inhibitor (NNRTI) hypersusceptibility (HS) improves virologic response to those agents, but phenotypic susceptibility cut-points, and methods to determine the cut-points, have not been completely defined. Method: Phenotypic drug susceptibility (fold change in IC50 [FC]) was determined for 96 randomly selected anti retroviral-experienced, NNRTI-naive patients who received a delavirdine (DLV)-containing regimen in ACTG 359. A weighted. FC score was used to account for other regimen agents. Regression models were used to define baseline DLV HS cut-points using week 4 or week 16 responses. Results: At study entry, DLV HS was present in 36% (35/96) of patients. Models explored HS cut-points from 0.2-1.0 using the week 4 virologic response. Using either a binary or continuous endpoint, DLV HS cut-points between 0.3 and 0.4 were identified. The classification and regression tree (CART) analysis identified baseline DLV FC < 0.44 as a predictor of week 4 response. Conclusions: In relating drug HS to virologic response, several different analytic methods identified a DLV HS FC cut-point of 0.3-0.4. In refining phenotypic cut-points, early virologic responses (not confounded by rebound) may be better metrics than later responses, especially for drugs with low genetic barriers, such as DLV.

Objective:Antiretroviral-treated individuals with drug-resistant HIV experience slower CD4 cell count declines than untreated individuals, independent of degree of viremia. As immune activation independently predicts disease progression, we hypothesized that patients with drug-resistant viremia would have less immune activation than patients with wild-type viremia, independent of plasma HIV RNA levels and that these differences would not be explained by a direct drug effect of protease inhibitors. Methods:Percentages of activated (CD38/HLA-DR) T cells were compared between untreated participants with wild-type viremia and antiretroviral-treated participants with drug-resistant viremia, after adjusting for plasma HIV RNA levels among other factors associated with T cell activation. Changes in T cell activation were also assessed in subjects discontinuing protease inhibitors while continuing other antiretroviral medications. Results:Twenty-one untreated participants with wild-type viremia and 70 antiretroviral-treated participants with drug-resistant viremia were evaluated. Relative to untreated participants, those with drug-resistant viremia had 29% fewer activated CD4 (P = 0.051) and CD8 (P = 0.012) T cells after adjustment for plasma HIV RNA levels among other factors. There was no evidence for an early change in T cell activation among 13 subjects with drug-resistant viremia interrupting protease inhibitors while continuing other antiretroviral medications, but a significant increase in T cell activation with complete or partial emergence of wild-type sequences in protease. Conclusions:Antiretroviral-treated patients with drug-resistant viremia have less T cell activation than untreated patients, independent of plasma HIV RNA level. Decreased ability of drug-resistant variants to cause T cell activation likely contributes to slower CD4 cell count declines among patients with drug-resistant viremia.

Background. Access to antiretroviral therapy and human immunodeficiency virus (HIV) care is increasing in resource-limited settings. We evaluated clinical, behavioral, and demographic risk factors associated with virologic suppression in a public, urban clinic in Kampala, Uganda.Methods. We conducted a cross-sectional, observational study of 137 HIV-infected patients who were receiving antiretroviral therapy at the infectious diseases clinic at Mulago Hospital (Kampala). We measured the prevalence of viral suppression, evaluated risk factors associated with virologic failure, and documented phenotypic resistance patterns and genotypic mutations.Results. A total of 91 (66%) of 137 participants had an undetectable viral load (< 400 copies/mL) after a median duration of 38 weeks (interquartile range, 24-62 weeks) of antiretroviral therapy. Median CD4 cell count was 163 cells/mm(3) (interquartile range, 95-260 cells/mm(3)). The majority of the patients (91%) were treated with nonnucleoside reverse-transcriptase inhibitor-based 3-drug regimens. In multivariate analysis, treatment with the first antiretroviral regimen was associated with viral suppression (odds ratio, 2.6; 95% confidence interval, 1.1-6.1). In contrast, a history of unplanned treatment interruption was associated with virologic treatment failure (odds ratio, 0.2; 95% confidence interval, 0.1-0.6). Of 124 participants treated with nonnucleoside reverse-transcriptase inhibitors, 27 (22%) were documented to have experienced virologic treatment failure. The most common mutation detected was K103N (found in 14 of 27 patients with virologic treatment failure).Conclusions. Although many HIV-infected people treated in Kampala, Uganda, have advanced HIV disease, the majority of patients who received antiretroviral therapy experienced viral suppression and clinical benefit. Because of the frequent use of nonnucleoside reverse-transcriptase inhibitor-based therapy, the majority of resistance was against this drug class. In resource-limited settings, initiation of therapy with a potent, durable regimen, accompanied by stable drug supplies, will optimize the likelihood of viral suppression.

This study examined the prevalence of primary human immunodeficiency type 1 (HIV-1) drug resistance among recently infected youth in the United States. Of the 55 subjects studied, major mutations conferring HIV drug resistance were present in 10 (18%). Eight (15%) had nonnucleoside reverse-transcriptase inhibitor (NNRTI) mutations, with the majority (6) having the K103N mutation; 2 (4%) had nucleoside reverse-transcriptase inhibitor (NRTI) mutations; and 2 (4%) had protease inhibitor (PI) mutations. Phenotypic drug resistance was present in 12 (22%) subjects: 10 (18%) for NNRTIs, 2 (4%) for NRTIs, and 3 (5.5%) for PIs. The prevalence of primary HIV-1 drug resistance, particularly to NNRTIs, in this group of recently infected youth was high.

Although CXCR4-tropic viruses are relatively uncommon among untreated human immunodeficiency virus (HIV)-infected individuals except during advanced immunodeficiency, the prevalence of CXCR4-tropic viruses among treated patients with detectable viremia is unknown. To address this issue, viral coreceptor usage was measured with a single-cycle recombinant-virus phenotypic entry assay in treatment-naive and treated HIV-infected participants with detectable viremia sampled from 2 clinic-based cohorts. Of 182 treated participants, 75 (41%) harbored dual/mixed or X4-tropic viruses, compared with 178 (18%) of the 976 treatment-naive participants (P<.001). This difference remained significant after adjustment for CD4+ T cell count and CCR5 Delta 32 genotype. Enrichment for dual/mixed/X4-tropic viruses among treated participants was largely but incompletely explained by lower pretreatment nadir CD4 + T cell counts. CCR5 inhibitors may thus be best strategically used before salvage therapy and before significant CD4 + T cell depletion.

Objective: To determine the influence of pol replication capacity on the natural history of HIV-1 infection. Design: Pol replication capacity was measured using a recombinant virus single cycle assay on baseline plasma specimens from subjects enrolled in the Hemophilia Growth and Development Study. Setting: Children and adolescents with hemophilia and HIV-1 infection were enrolled at multiple sites across the USA into a natural history study. Participants: The Hemophilia Growth and Development Study enrolled 207 HIV-1-infected hemophiliacs between 6 and 19 years of age in 1989 and 1990. Subjects were followed every 6 months through 1997 with pol replication capacity measurements available from 128 of the subjects. Main outcome measures: A univariate model defined the relationship between pol replication capacity and HIV-1 RNA and CD4 T-cell number. A random effects model assessed the ability of this measure to predict CD4 T-cell decline over time and a Cox proportional hazards model and Kaplan–Meier analyses defined how it predicts clinical progression. Results: Pol replication capacity measures correlated with baseline HIV-1 RNA, R2 = 0.189 (P = 0.03) and CD4 T-cell number, −0.197 (P = 0.03). It also independently predicted CD4 T-cell decline over time and progression to AIDS. Conclusions: This study demonstrates that pol replication capacity independently influences the natural history of HIV-1 infection.

Objectives: To analyse the impact of the M1841/V mutation and individual thymidine-associated mutations (TAM) on nucleoside reverse transcriptase inhibitor (NRTI) phenotypic susceptibility and compare these results with those obtained using commercial and public algorithms.Design: An HIV genotypic/phenotypic database with over 27 000 samples was used to obtain the median fold change (5-95th percentile) in NRTI phenotypic susceptibility for viruses from patients containing individual TAM with or without the M1841 or V mutation and for wild-type patient viruses.Results: The resulting data indicated that in vitro, individual TAM do not have an equivalent impact on NRTI resistance, with some individual TAM having little or no impact on NRTI resistance (e.g. M41L or K219Q/E/H/R). In the presence of the M1841/V mutation, re-sensitization to some drugs, including zidovudine, stavudine and tenofovir was observed despite the presence of a TAM. For didanosine and abacavir, the presence of the M184V mutation and a single TAM did not result in a fold-change increase associated with decreased drug susceptibility. Analysis of public and commercial algorithms revealed a lack of concordance regarding the impact of these mutations, and with the observed phenotypic data.Conclusion: These analyses should assist in the creation of rules for genotypic drug resistance algorithms for a better reflection of the impact of individual TAM and also the impact of M1841/V on resistance. These data provide additional evidence that retaining lamivudine in those treatment regimens in which TAM can be selected may provide some therapeutic benefit by maintaining the M184V mutation. (C) 2004 Lippincott Williams Wilkins.

Purpose of review To review recent clinical data regarding viral fitness and its potential utility in clinical practice. Therapeutic failure of antiretroviral regimens is often due to the development of drug resistance. The viral loads achieved by these drug-resistant variants frequently remain suppressed below pretreatment levels. Furthermore, many patients with virologic failure continue to maintain stable CD4 counts and remain clinically well. One possible explanation for these observations is that the resistant virus that emerges during failure of highly active antiretroviral therapy is less fit than its wild-type counterpart as a result of the requirement to maintain resistance mutations that allow it to replicate in the presence of antiretroviral drugs. Since there are many patients with limited treatment options due to extensive resistance or drug toxicities, or both, there is intense interest in exploring the possibility that viral fitness can be exploited for clinical benefit. This review describes the assays that are used to measure fitness and replication capacity, and investigations of their utility in clinical practice. Recent findings A number of methods have been developed to measure viral fitness. Replication kinetic and competitive culture assays are accurate, but labour and time intensive. The availability of a rapid, single-cycle recombinant assay that measures viral replication capacity presents the possibility that assessments of viral fitness could be incorporated into clinical management. The replication capacity assay appears to correlate with more standard measures of viral fitness, and recently accumulated data in both wild-type and drug resistant viral populations suggest that replication capacity describes an intrinsic characteristic of HIV-1. A number of investigations of the clinical utility of fitness and replication capacity assays have established correlations between these measurements and virologic and immunologic outcomes. Summary Much progress has been made in the past year in our understanding of viral fitness and replication capacity. Assays that measure these viral characteristics have the potential to expand the range of clinical strategies employed against HIV-1. Further investigations are needed to firmly establish the clinical utility of these assays.

Evolution and transmission of multiply drug-resistant human immunodeficiency virus type 1 (HIV-1) may limit therapeutic options as global treatment efforts expand. However, the stability of these mutants in the absence of drug selection pressure is not known. We performed a longitudinal analysis of plasma virus from a person who acquired HIV-1 that contained multiple reverse transcriptase (RT) and protease (PR) mutations. In the absence of therapy, 5 of 12 drug resistance mutations reverted in a stepwise fashion to wild type over the course of 52 weeks. Reversion of the M184V mutation alone did not change viral replicative capacity (RC), but it led to enhanced resistance to zidovudine and tenofovir. However, reversions of a second RT mutation and 3 PR mutations were associated with an increase in viral RC, and this was temporally correlated with a marked decrease in CD4 cell number. This study demonstrates the gradual stepwise back-mutation of certain drug resistance mutations in vivo in the absence of ongoing drug selection pressure. Moreover, it suggests that, despite initially impaired viral fitness, a transmitted HIV-1 isolate with multiple drug resistance mutations can evolve to develop increased RC and significant pathogenicity.