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Background Neurofilament light (NFL) chain concentrations, reflecting axonal damage, are seen in several polyneuropathies but have not been studied in human immunodeficiency virus (HIV) distal sensory polyneuropathy (DSP). We evaluated NFL in cerebrospinal fluid (CSF) and plasma in relation to DSP in people with HIV (PWH) from 2 independent cohorts and in people without HIV (PWoH). Methods Cohort 1 consisted of PWH from the CHARTER Study. Cohort 2 consisted of PWH and PWoH from the HIV Neurobehavioral Research Center (HNRC). We evaluated DSP signs and symptoms in both cohorts. Immunoassays measured NFL in CSF for all and for plasma as well in Cohort 2. Results Cohort 1 consisted of 111 PWH, mean ± SD age 56.8 ± 8.32 years, 15.3% female, 38.7% Black, 49.6% White, current CD4+ T-cells (median, interquartile range [IQR]) 532/µL (295, 785), 83.5% with plasma HIV RNA ≤50 copies/mL. Cohort 2 consisted of 233 PWH of similar demographics to PWH in Cohort 1 but also 51 PWoH, together age 58.4 ± 6.68 years, 41.2% female, 18.0% Black, Hispanic, non-Hispanic White 52.0%, 6.00% White. In both cohorts of PWH, CSF and plasma NFL were significantly higher in both PWH with DSP signs. Findings were similar, albeit not significant, for PWoH. The observed relationships were not explained by confounds. Conclusions Both plasma and CSF NFL were elevated in PWH and PWoH with DSP. The convergence of our findings with others demonstrates that NFL is a reliable biomarker reflecting peripheral nerve injury. Biomarkers such as NFL might provide, validate, and optimize clinical trials of neuroregenerative strategies in HIV DSP.

Introduction: Iatrogenic pseudoaneurysm (PSA) in hemodialysis vascular access puncture site is the most common complication in a patient with ERSD, which needs urgent surgical intervention. This research explained the patients’ characteristics with pseudoaneurysm in vascular access that underwent surgical intervention immediately. Methods: Retrospectively, it reported cases of pseudoaneurysm that underwent immediate surgical intervention in an operating theatre in the emergency department of RS Dr. Soetomo Surabaya. Result: Twenty-one patients, whose age range from 26-to 64 years old (mean 46.57 + 12.14 years), have undergone immediate surgical and vascular intervention in an operating theater in the emergency department of RS Dr. Soetomo from 2017 until March 2021. The site of pseudoaneurysm varies from cubiti (90.4%), with signs and symptoms of impending rupture (57.1%), rupture (28.6%), and infected pseudoaneurysm (14.3%). Surgical management such as arterial repair was conducted in 90.4% of cases, and two patients (9.6%) unfortunately underwent takedown AVF. It was found from all 21 cases that nine cases didn’t have vascular access as recommended, leading to repetitive puncture in the same puncture site. Conclusion: Iatrogenic Pseudoaneurysm in ESRD patients on regular hemodialysis is the most common complication that has been treated. The medical staff’s knowledge about recognition and prevention is a crucial factor in controlling this complication

Purpose: P95HER2 (p95) is a truncated form of the HER2, which lacks the trastuzumab-binding site and contains a hyperactive kinase domain. Previously, an optimal clinical cutoff of p95 expression for progression-free survival (PFS) and overall survival (OS) was defined using a quantitative VeraTag assay (Monogram Biosciences) in a training set of trastuzumab-treated metastatic breast cancer (MBC) patients.Experimental Design: In the current study, the predictive value of the p95 VeraTag assay cutoff established in the training set was retrospectively validated for PFS and OS in an independent series of 240 trastuzumab-treated MBC patients from multiple institutions.Results: In the subset of 190 tumors assessed as HER2-total (H2T)-positive using the quantitative HERmark assay (Monogram Biosciences), p95 VeraTag values above the predefined cutoff correlated with shorter PFS (HR = 1.43; P = 0.039) and shorter OS (HR = 1.94; P = 0.0055) where both outcomes were stratified by hormone receptor status and tumor grade. High p95 expression correlated with shorter PFS (HR = 2.41; P = 0.0003) and OS (HR = 2.57; P = 0.0025) in the hormone receptor-positive subgroup of patients (N = 78), but not in the hormone receptor-negative group. In contrast with the quantitative p95 VeraTag measurements, p95 immunohistochemical expression using the same antibody was not significantly correlated with outcomes.Conclusions: The consistency in the p95 VeraTag cutoff across different cohorts of patients with MBC treated with trastuzumab justifies additional studies using blinded analyses in larger series of patients. Clin Cancer Res; 20(10); 2805–13. ©2014 AACR.

PDF file - 1729KB, Supplemental Figure S2. Kaplan-Meier plots for p95 IHC categories within the HER2 overexpressed subset (H2T>13.8 RF/mm2) with p95 IHC 0 in green, 1+ in red, 2+ in black and 3+ in blue. A, Progression free survival. B, Overall survival. C, Progression free survival in the hormone receptor positive subgroup.

PDF file - 474KB, Supplemental Figure S1. Expanded p95 IHC images from Figure 4A.

The trajectories of acquired immunity to severe acute respiratory syndrome coronavirus 2 infection are not fully understood. We present a detailed longitudinal cohort study of UK healthcare workers prior to vaccination, presenting April-June 2020 with asymptomatic or symptomatic infection. Here we show a highly variable range of responses, some of which (T cell interferon-gamma ELISpot, N-specific antibody) wane over time, while others (spike-specific antibody, B cell memory ELISpot) are stable. We use integrative analysis and a machine-learning approach (SIMON - Sequential Iterative Modeling OverNight) to explore this heterogeneity. We identify a subgroup of participants with higher antibody responses and interferon-gamma ELISpot T cell responses, and a robust trajectory for longer term immunity associates with higher levels of neutralising antibodies against the infecting (Victoria) strain and also against variants B.1.1.7 (alpha) and B.1.351 (beta). These variable trajectories following early priming may define subsequent protection from severe disease from novel variants.

We evaluated whether biomarkers of age-related neuronal injury and amyloid metabolism are associated with neurocognitive impairment (NCI) in people with and without HIV (PWH, PWoH). This was a cross-sectional study of virally suppressed PWH and PWoH. NCI was assessed using a validated test battery; global deficit scores (GDS) quantified overall performance. Biomarkers in cerebrospinal fluid (CSF) were quantified by immunoassay: neurofilament light (NFL), total Tau (tTau), phosphorylated Tau 181 (pTau181), amyloid beta (Aβ)42, and Aβ40. Factor analysis was used to reduce biomarker dimensionality. Participants were 256 virally suppressed PWH and 42 PWoH, 20.2% female, 17.1% Black, 7.1% Hispanic, 60.2% non-Hispanic White, and 15.6% other race/ethnicities, mean (SD) age 56.7 (6.45) years. Among PWH, the best regression model for CSF showed that higher tTau (β = 0.723, p = 3.79e-5) together with lower pTau181 (β = −0.510, p = 0.0236) best-predicted poor neurocognitive performance. In univariable analysis, only higher tTau was significantly correlated with poor neurocognitive performance (tTau r = 0.214, p = 0.0006; pTau181 r = 0.00248, p = 0.969). Among PWoH, no CSF biomarkers were significantly associated with worse NCI. Predicted residual error sum of squares (PRESS) analysis showed no evidence of overfitting. Poorer neurocognitive performance in aging PWH was associated with higher CSF tTau, a marker of age-related neuronal injury, but not with biomarkers of amyloid metabolism. The findings suggest that HIV might interact with age-related neurodegeneration to contribute to cognitive decline in PWH.

BackgroundResistance-associated substitutions (RASs) to HCV direct-acting antivirals (DAAs) can contribute to virologic failure and limit retreatment options. People who inject drugs (PWID) are at highest risk for transmission of resistant virus. We report on RASs at baseline and after virologic failure in DAA-naive and protease inhibitor-experienced PWID.MethodsWe sequenced the NS3/4A, NS5A, and NS5B regions from 150 PWID with genotype 1 (GT1) viruses; 128 (85.3%) GT1a, 22 (14.7%) GT1b.ResultsAmong the 139 (92.7%) DAA-naive PWID, 85 of 139 (61.2%) had baseline RASs—67 of 139 (48.2%) in NS3 (predominantly Q80K/L); 25 of 139 (18.0%) in NS5A; and 8 of 139 (5.8%) in NS5B. Of the 11 protease inhibitor-experienced participants, 9 had baseline NS3 RASs (V36L N = 1, Q80K N = 9) and 4 had baseline NS5A RASs (M28V N = 2, H58P N = 1, A92T N = 1). Among the 11 participants who had posttreatment samples with detectable virus (7 treatment failures, 1 late relapse, 3 reinfections), 1 sofosbuvir/ledipasvir failure had a baseline H58P. Two sofosbuvir/ledipasvir-treated participants developed new NS5A mutations (Q30H, Y93H, L31M/V). Otherwise, no RASs were detected.ConclusionsOur results demonstrate RAS prevalence among DAA-naive PWID is comparable to that in the general population. Only 2 of 150 (1.3%) in our longitudinal cohort developed treatment-emergent RASs. Concern for transmission of resistant virus may therefore be minimal.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is normally controlled by effective host immunity including innate, humoral and cellular responses. However, the trajectories and correlates of acquired immunity, and the capacity of memory responses months after infection to neutralise variants of concern - which has important public health implications - is not fully understood. To address this, we studied a cohort of 78 UK healthcare workers who presented in April to June 2020 with symptomatic PCR-confirmed infection or who tested positive during an asymptomatic screening programme and tracked virus-specific B and T cell responses longitudinally at 5-6 time points each over 6 months, prior to vaccination. We observed a highly variable range of responses, some of which - T cell interferon-gamma (IFN-γ) ELISpot, N-specific antibody waned over time across the cohort, while others (spike-specific antibody, B cell memory ELISpot) were stable. In such cohorts, antiviral antibody has been linked to protection against re-infection. We used integrative analysis and a machine-learning approach (SIMON - Sequential Iterative Modeling Over Night) to explore this heterogeneity and to identify predictors of sustained immune responses. Hierarchical clustering defined a group of high and low antibody responders, which showed stability over time regardless of clinical presentation. These antibody responses correlated with IFN-γ ELISpot measures of T cell immunity and represent a subgroup of patients with a robust trajectory for longer term immunity. Importantly, this immune-phenotype associates with higher levels of neutralising antibodies not only against the infecting (Victoria) strain but also against variants B.1.1.7 (alpha) and B.1.351 (beta). Overall memory responses to SARS-CoV-2 show distinct trajectories following early priming, that may define subsequent protection against infection and severe disease from novel variants.* These authors contributed equally.# These authors jointly supervised this work and contributed equally.Corresponding authors: Eleanor Barnes, email: ellie.barnes@ndm.ox.ac.ukand Adriana Tomic, email: 19info@adrianatomic.com

Purpose: Expression of p95HER2 (p95), a truncated form of the HER2 receptor, which lacks the trastuzumab binding site but retains kinase activity, has been reported as a prognostic biomarker for poor outcomes in patients with trastuzumab-treated HER2-positive metastatic breast cancer. The impact of p95 expression on trastuzumab treatment efficacy in early HER2-positive breast cancer is less clear. In the current study, p95 was tested as a predictive marker of trastuzumab treatment benefit in the HER2-positive subset of the FinHer adjuvant phase III trial. Experimental Design: In the FinHer trial, 232 patients with HER2-positive early breast cancer were randomized to receive chemotherapy plus 9 weeks of trastuzumab or no trastuzumab treatment. Quantitative p95 protein expression was measured in formalin-fixed paraffin-embedded samples using the p95 VeraTag assay (Monogram Biosciences), specific for the M611 form of p95. Quantitative HER2 protein expression was measured using the HERmark assay (Monogram Biosciences). Distant disease-free survival (DDFS) was used as the primary outcome measure. Results: In the arm receiving chemotherapy only, increasing log10(p95) correlated with shorter DDFS (HR, 2.0; P = 0.02). In the arm receiving chemotherapy plus trastuzumab (N = 95), increasing log10(p95) was not correlated with a shorter DDFS. In a combined analysis of both treatment arms, high breast tumor p95 content was significantly correlated with trastuzumab treatment benefit in multivariate models (interaction P = 0.01). Conclusions: A high p95HER2/HER2 ratio identified patients with metastatic breast cancer with poor outcomes on trastuzumab-based therapies. Further investigation of the p95HER2/HER2 ratio as a potential prognostic or predictive biomarker for HER2-targeted therapy is warranted. Clin Cancer Res; 24(13); 3046–52. ©2018 AACR.

Background. Grape peel and seed extract (Vitis vinifera), that has resveratrol, is one of many antioxidants that can pass through blood brain barrier and can induce release neurotrophic factor that contribute in ERK 1/2 pathway mechanism in post stroke. Objective . To prove that grape peel and seed extract can regenerate neuron in brain functional. Methods. True experimental design with five groups in this research. The five groups are negative control, positive control, grape peel and seed extract 50mg/KgBW, 100mg/KgBW, and 200mg/KgBW. rats are given grape peel and seed extract in variable dose to know how extract’s effect in neuron repairment. The repairment is monitored from ladder rung walking test score. Results. Range average score ladder rung walking test post stroke dan post treatment group N, K, Ra, Rb, dan Rc, were 0 ± 0, 0.001028933 ± 0.011664445, 0.123214286 ± 0.019834983, 0.064744427 ± 0.024296721, 0.03781401 ± 0.006888803. Statistical test used Annova significantly p;0,001.Dose 50mg/KgBW is effective in repairing neuron. Conclusion . Grape’s leather and Seed extract 50 mg/kgBW can improve neuron regeneration on animal model.

Background: Expression of p95HER2 (p95), a truncated form of the HER2 receptor that lacks the trastuzumab binding site but retains kinase activity, has been reported as a prognostic biomarker for poor outcome in trastuzumab-treated HER2-positive metastatic breast cancer (MBC). However, the ability of p95 to predict trastuzumab benefit has not been demonstrated due to the difficulty in obtaining the appropriate control group, namely HER2+ MBC patients not treated with trastuzumab. In the current study, the predictive value of p95 expression was tested in a cohort comprised of HER2-positive MBC patients treated before the availability of trastuzumab and trastuzumab-treated HER2-positive MBC patients. Methods: The current cohort was derived from 206 HER2-positive MBC patients in the Munich Cancer Registry with a median follow up of 64 months. Cases were divided between those that received trastuzumab (n=115) and those that were treated before the availability of trastuzumab (n=91). Quantitative p95 protein expression was measured in formalin-fixed paraffin-embedded samples using the p95 VeraTag® assay (Monogram Biosciences), which is specific for the active M611 form of p95. Quantitative total HER2 protein expression was measured using the HERmark® assay (Monogram Biosciences). p95 and HERmark cutoffs were pre-specified (Duchnowska, Clin Cancer Res, 20:2805, 2014 and Huang, Am J Clin Pathol, 134:303, 2010). Analyses with p95 were restricted to samples with confirmed HER2 overexpression by HERmark. All hazard ratios (HR) were stratified by estrogen receptor status and grade. Results: Consistent with previous training (Sperinde, Clin Cancer Res, 16:4226, 2010) and validation (Duchnowska, Clin Cancer Res, 20:2805, 2014) datasets, subjects treated with trastuzumab experienced a shorter time to progression (TTP) when p95 expression levels were above the cutoff versus below the cutoff (HR = 3.8, p = 0.019). However, only a trend was observed between p95 expression levels and overall survival (HR = 2.2, p = 0.20), possibly due to a lower frequency of events and relatively small sample size. The predictive value of p95 was assessed by determining the benefit of adding trastuzumab to chemotherapy treatment in subsets below and above the p95 cutoff. As expected, patients with p95 below the cutoff experienced significant benefit in TTP from adding trastuzumab (HR = 0.13, p Conclusions: In this dataset, quantitative p95 expression was predictive of trastuzumab treatment benefit in MBC. Patients with high p95 expression may be particularly good candidates for dual HER2 blockade, as reported in the NeoALTTO trial (Scaltriti, Clin Cancer Res, 21:569, 2015), or other additional therapies. Citation Format: Sperinde J, Bachmeier B, Weidler JM, Lie Y, Chenna A, Winslow J, Engel J, Schubert-Fritschle G, Sommerhoff C, Petropoulos C, Bates M, Huang W, Nerlich A. Quantitative p95HER2 protein expression is predictive of trastuzumab response in HER2-positive metastatic breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-07-09.

Background: Expression of p95HER2 (p95), a truncated form of the HER2 receptor that lacks the trastuzumab binding site but retains kinase activity, appears to be a prognostic biomarker for poor trastuzumab treatment outcome in HER2-positive metastatic breast cancer. The impact of p95 expression on trastuzumab treatment efficacy in early HER2-positive breast cancer is less clear. In the current study, p95 expression levels were measured in HER2-positive patients from the phase III FinHer adjuvant trastuzumab trial and correlated with treatment outcome. Methods: In the FinHer phase III trial, 232 HER2-positive early breast cancer patients were randomized to receive 9-weeks of trastuzumab treatment versus no trastuzumab treatment (control). Quantitative p95 protein expression was measured in formalin-fixed paraffin-embedded samples using the p95 VeraTag® assay (Monogram Biosciences), specific for the M611 form of p95. Quantitative HER2 protein expression was measured using the HERmark® assay (Monogram Biosciences). Time to distant recurrence (TDR) was used as the primary outcome measure. Results: Sufficient tissue was available to measure p95 in 192 HER2-positive patients randomized to receive chemotherapy vs chemotherapy plus trastuzumab. The chemotherapy only (n=97) and chemotherapy plus trastuzumab (n=95) arms were first analyzed separately. In the chemotherapy only arm, increasing log(p95) correlated with shorter TDR (HR = 2.0; p = 0.02) when stratified by hormone receptor status, nodal status and chemotherapy regimen. In the chemotherapy plus trastuzumab arm, increasing log(p95) was not correlated with a shorter TDR (HR = 0.58; p = 0.19). Log(HER2) was not significantly correlated with TDR in either arm. In a combined analysis of both treatment arms, log(p95) was significantly correlated with trastuzumab treatment outcome in a multivariate model that included hormone receptor status, nodal status, chemotherapy regimen, log(p95) and treatment arm. Subset analyses of hormone receptor positive and negative groups indicated that the interaction of p95 expression with trastuzumab treatment was largely driven by the hormone receptor negative subset. Conclusions: In the FinHer phase III adjuvant breast cancer trial, HER2-positive patients with elevated breast tumor p95HER2 expression experienced poor outcomes when treated with chemotherapy alone, whereas patients with elevated p95 expression experienced the most benefit when trastuzumab was added to chemotherapy. The different influence between hormone receptor subsets of p95 expression on trastuzumab response resembles the effect of HER2 expression on trastuzumab response in the NSABP B-31 trial (JNCI 105:1782, 2013). Citation Format: Jeff Sperinde, Weidong Huang, Aki Vehtari, Ahmed Chenna, Pirkko-Liisa Kellokumpu-Lehtinen, John Winslow, Petri Bono, Yolanda Lie, Jodi Weidler, Heikki Joensuu. Quantitative p95HER2 and HER2 correlations with outcome in the FinHer trial [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-03.

Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how “minor” polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3′ processing indicates the importance of the 3′ processing step and highlights a therapeutic angle that needs to be better exploited.

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice. A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica.

Purpose: P95HER2 (p95) is a truncated form of the HER2, which lacks the trastuzumab-binding site and contains a hyperactive kinase domain. Previously, an optimal clinical cutoff of p95 expression for progression-free survival (PFS) and overall survival (OS) was defined using a quantitative VeraTag assay (Monogram Biosciences) in a training set of trastuzumab-treated metastatic breast cancer (MBC) patients. Experimental Design: In the current study, the predictive value of the p95 VeraTag assay cutoff established in the training set was retrospectively validated for PFS and OS in an independent series of 240 trastuzumab-treated MBC patients from multiple institutions. Results: In the subset of 190 tumors assessed as HER2-total (H2T)-positive using the quantitative HERmark assay (Monogram Biosciences), p95 VeraTag values above the predefined cutoff correlated with shorter PFS (HR = 1.43; P = 0.039) and shorter OS (HR = 1.94; P = 0.0055) where both outcomes were stratified by hormone receptor status and tumor grade. High p95 expression correlated with shorter PFS (HR = 2.41; P = 0.0003) and OS (HR = 2.57; P = 0.0025) in the hormone receptor-positive subgroup of patients (N = 78), but not in the hormone receptor-negative group. In contrast with the quantitative p95 VeraTag measurements, p95 immunohistochemical expression using the same antibody was not significantly correlated with outcomes. Conclusions: The consistency in the p95 VeraTag cutoff across different cohorts of patients with MBC treated with trastuzumab justifies additional studies using blinded analyses in larger series of patients. Clin Cancer Res; 20(10); 2805–13. ©2014 AACR.

Background: Patients with HER2-positive breast cancer are at high risk for brain metastases. A large number of HER2-positive tumors also express p95HER2 (p95), a truncated form of HER2 that lacks the trastuzumab binding site but retains kinase activity. Although p95 expression in primary breast tumors is well studied, the prevalence and significance of p95 expression in brain metastases is unknown. In the current study we examined expression of p95 in brain metastases and in matched primary breast tumors. Methods: Seventy-five pairs of formalin-fixed paraffin-embedded samples from matched primary breast cancers and brain metastases were assayed for quantitative p95 protein expression using the p95 VeraTag® assay (Clin Cancer Res, 16:4226, 2010) specific for the M611 form of p95. Sufficient material to obtain p95 data in both primary and matched brain metastasis samples was available in 52 cases. In the remaining 23 cases, a p95 measurement was obtained in either the primary or brain metastasis sample. Estrogen (ER) and progesterone (PR) receptor status were scored using immunohistochemistry. Hormone receptor positivity was defined as either ER or PR positive. Quantitative HER2 protein expression was measured using the HERmark® assay. Both the p95 VeraTag assay and the HERmark assay measure tumor-averaged protein expression in units of relative fluorescence per mm2 tumor (RF/mm2). Measurements of p95 > 2.8 RF/mm2 and HER2 > 17.8 RF/mm2 were considered as positive results. Results: There was a net increase in p95 expression in brain metastases relative to the matched primary tumor with a median increase of 1.5-fold (p = 0.001, range 0.2-fold to 35-fold). The increase in p95 expression was only weakly correlated with the increase in quantitative HER2 expression (R2 = 0.18; p = 0.0018). Cases with HERmark-positive tumors were more likely to have the largest (≥ 5-fold) increase in p95 expression compared to those with lower HER2 expression (odds ratio = 6.3; p = 0.018). Changes in p95 levels from primary to brain metastasis were unrelated to hormone receptor status (p = 0.59). P95 positivity in the primary tumor correlated with time from breast cancer diagnosis to first progression (HR = 2.2; p = 0.012) when stratified by hormone receptor status and tumor grade. Although there was a trend towards correlation of p95 positivity in the brain metastasis with time from diagnosis to brain metastasis (HR = 1.7; p = 0.058, stratified as above), p95 positivity did not correlate with overall survival from the time of brain metastasis diagnosis (HR = 1.3; p = 0.42, stratified as above). Conclusions: This is the first study of quantitative p95 expression in matched primary tumors and brain metastases. Brain metastases of breast cancer show significant increases in p95 protein expression compared to matched primary tumors. These data provide a rationale for future correlative studies on p95 levels in brain metastases. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-11-07.

Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF).SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48.Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed.Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance data are unavailable.

Background: HER3 is thought to play a prominent role in resistance to HER2-directed breast cancer therapies. Recent data suggest that HER3 levels also influence HER2-normal breast tumor biology. HER3 and PI3K signaling are linked in that in HER3 signaling activates PI3K and inhibition of PI3K activity can upregulate HER3 expression. Here, we measured quantitative HER3 protein expression levels and PIK3CA mutation status in matched tissues from the primary tumor and site of metastasis to assess correlations with time to recurrence. Methods: 44 pairs (8 HER2+ by HERmark®) of matched tissues from the primary tumor and the site of metastasis were evaluated for HER3 protein expression using a sensitive, quantitative assay for HER3 protein expression in FFPE tissue sections (VeraTag®). Matched samples were also evaluated for quantitative HER2 expression (HERmark) and for PIK3CA mutations at exon 9 (E542K and E545K) and exon 20 (H1047R). Results: HER3 protein expression at the metastatic site was largely independent of HER3 levels at the primary site (Spearman p = 0.50) in contrast to HER2 expression (Spearman p = 0.0004). HER3 expression in the primary tumor correlated with time to recurrence (TTR) (HR = 2.0 per 2-fold increase in HER3; p < 0.0001). Conversely, HER3 expression measured at the site of metastasis was not correlated with TTR (p = 0.55). Estrogen receptor negative tumors were less likely to have PIK3CA mutations (p = 0.023). In cases of primary tumors with PIK3CA mutations, no reversions to wild-type PIK3CA were observed in the metastatic sites. In metastatic tumors, mutations detected in the primary tumor as well as new mutations were observed. A gain of an exon 9 mutation at the metastatic site correlated with shorter TTR (HR = 2.5; p = 0.043). Excluding the 8 samples that were HER2+ by HERmark, longer TTR was observed for patients with PIK3CA mutations in the primary tumor (HR = 0.47; p = 0.042), which is consistent with previous reports. Interestingly, the longer TTR for those with PIK3CA mutations appeared to be dependent on quantitative HER3 protein level (interaction p = 0.065). Conclusions: HER3 protein expression in matched primary and metastatic breast cancer tissues were unrelated. This may indicate that HER3 protein is influenced by the different tumor microenvironments of the primary and metastatic sites. PIK3CA mutations were either maintained or acquired at metastatic sites. Both low HER3 protein expression and the presence of PIK3CA mutations in the primary tumor but not the metastatic tumor were associated with longer TTR. These observations suggest that HER3 protein expression may be an important prognostic factor for breast cancer progression. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-16.

Background: Accurate determination of HER2 status is critical in determining appropriate therapy for breast cancer patients. The HERmark® assay is a novel method to quantitatively measure HER2 total protein expression (H2T) in breast cancer. In this study, we compared HERmark H2T with central laboratory HER2 retesting and local (site reported) HER2 testing of formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. The quantitative total HER2 measurements (H2T) by HERmark and results of local HER2 tests were correlated with tumor pathohistological characteristics and overall survival of breast cancer patients. Methods: 232 FFPE breast cancer tissues were provided by 11 CBS study sites for HER2 testing by the HERmark assay and central laboratory IHC re-testing performed in blinded fashion. Local HER2 immunohistochemistry and/or fluorescence in situ hybridization (FISH) results and valid HERmark H2T and central HER2 IHC results were obtained in 192 cases for analysis. Results: H2T showed a significant correlation with central HER2 IHC staining intensity (P < 0.0001). The concordance rates of positive and negative HERmark status (excluding equivocal) with those of local HER2 status determined by the CBS sites, and with those of central HER2 IHC status were 84% (Kappa = 0.68) and 96% (Kappa = 0.91), respectively. Higher H2T levels significantly correlated with higher tumor grade (p = 0.007) and negative ER/PR status (p = 0.002). Twenty-six (14%) cases showed discordant (conversion of negative and positive) results between local HER2 status and HERmark status. Of the discordant cases, HERmark significantly agreed with H-score of central HER2 IHC retesting (p = 0.014), as compared with local HER2 status. The concordant negative group (local HER2 negative/H2T low) demonstrated better overall survival (OS) (HR = 0.198, p = 0.0001), compared to that of concordant positive group (local HER2 positive/H2T high). The concordant negative group also showed better OS than that of discordant local HER2 negative/H2T high group (HR = 0.065, p = 0.0003), but showed no significant difference in OS as compared to that of discordant local HER2 positive/H2T low group (HR = 1.774, p = 0.499).). In 24 cases (13%) considered to be “triple negative” by local HER2, ER and PR testing, HERmark re-classified 4 cases (17%) as HER2 positive. Conclusions: H2T by HERmark yields a continuum of quantitative HER2 protein measurements that shows an excellent correlation with central HER2 IHC retesting and confirms the known correlations between HER2 expression with tumor grade and ER/PR status. OS results of concordant HER2 positive or negative groups (between local HER2 testing and HERmark H2T) confirmed that HER2 positive patients (excluding adjuvant trastuzumab therapy) have worse OS than patients with HER2 negative disease. However, in the HERmark and local HER2 discordant groups, OS appeared to track better with H2T by HERmark and not with the local HER2 status. Novel quantitative HER2 measurements may identify patients with false (+) and (−) HER2 status by local HER2 testing and may provide added clinical value to routine “real world” HER2 testing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-06.

Background: Expression of p95HER2 (p95), a truncated form of HER2 also known as p110 or M611-CTF, is a possible trastuzumab resistance mechanism and has been associated with poor prognosis in trastuzumab-treated HER2-positive metastatic breast cancer (MBC). Previously we reported on optimal clinical cutoffs for quantitative p95 (Clin Cancer Res, 16:4226, 2010) and quantitative HER2 protein expression (H2T) by HERmark® (Cancer, 116:5168, 2010) that defined patient subsets with different progression-free survival (PFS). These cutoffs were confirmed in an independent trastuzumab-treated MBC cohort (ASCO 2011, #586). Here, using individual patient data, we performed an analysis on the combined data set of 243 cases from the discovery and validation cohorts to derive optimal cutoffs for quantitative p95 and H2T. Methods: Both quantitative H2T (HERmark, Monogram Biosciences) and p95 assays employed the VeraTag® method to quantify protein expression in formalin-fixed, paraffin-embedded tumor samples from two cohorts of 101 and 142 cases of trastuzumab-treated MBC with 7.4 and 9.2 months median PFS, respectively. All analyses were stratified by hormone receptor status, tumor grade (3 vs. 1+2) and cohort. H2T measurements were compared to pre-specified cutoffs for HERmark negative (H2T17.8 RF/mm2), derived from the 95th percentile of centrally determined HER2-negatives, respectively, within a reference database of 1,090 breast cancer patient samples. Results: Patients classified as HERmark-positive had longer PFS than those classified as HERmark-negative (HR = 0.52; p = 0.0006; medians 10.0 and 5.9 months). The previously determined optimal H2T cutoff of 13.8 RF/mm2 in the center of the HERmark-equivocal zone, gave a similar result (HR = 0.54; p = 0.0005). This was close to the optimal cutoff of 12.75 RF/mm2 (HR = 0.48; p < 0.0001, unadjusted) for the combined data set. The PFS for the small group of patients in the HERmark-equivocal zone (n = 20) was more similar to the HERmark-negatives (equivocal vs. negative: HR=0.98; p = 0.9) than the HERmark-positives (positive vs. equivocal: HR=0.57; p = 0.057). The pre-specified p95 cutoff at 2.8 RF/mm2 separated the 174 HERmark-positive cases into two groups of longer (p95 Conclusions: HERmark positive and negative categories, defined by analytical comparison with centrally determined HER2 status, were confirmed to have significantly different PFS in trastuzumab-treated MBC patients. The optimal H2T clinical cutoff for this combined analysis was centered in the HERmark analytical equivocal zone. An optimal p95 clinical cutoff of 2.7 RF/mm2 derived from this combined analysis was nearly identical to the previously established cutoff of 2.8 RF/mm2. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-31.

Emergence of HIV resistance is a concerning consequence of global scale-up of antiretroviral therapy (ART). To date, there is no published information about HIV resistance from the Dominican Republic. The study's aim was to determine the prevalence of transmitted drug resistance (TDR) to reverse transcriptase and protease inhibitors in a sample of chronically HIV-1-infected patients in one clinic in Santo Domingo. The data are presented in the context of a review of the TDR literature from Latin America and the Caribbean. Genotype testing was successfully performed on 103 treatment-naive adults planning to initiate antiretroviral therapy; the World Health Organization (WHO) list of surveillance drug resistance mutations (SDRM) was used to determine the presence of TDR mutations. WHO SDRM were identified in eight patients (7.8%); none had received sdNVP. There were no significant differences in epidemiologic or clinical variables between those with or without WHO SDRM. The prevalence of WHO SDRM was 1.0% and 6.8% for nucleoside reverse transcriptase inhibitors and nonnucleoside reverse transcriptase inhibitors, respectively. No WHO SDRMs for protease inhibitors were identified. Among 12 studies of TDR in the region with a sample size of at least 100 subjects, the reported prevalence of SDRM ranged from 2.8% to 8.1%. The most commonly identified SDRM was K103N. This information adds to our understanding of the epidemiology of TDR in the region and the possible role such mutations could play in undermining first-line treatment. Ongoing surveillance is clearly needed to better understand the TDR phenomenon in the Caribbean.

Objectives The aim of the study was to determine the prognostic value of HIV replication capacity (RC) for subsequent antiretroviral (ARV) treatment response in ARV-experienced patients. Methods RC and phenotypic resistance testing were performed at baseline and week 12 on plasma samples from patients randomized to undergo a 12-week ARV drug-free period (ARDFP) or initiate immediate salvage therapy (no-ARDFP group) in the Options in Management with Antiretrovirals (OPTIMA) trial. Dichotomous and incremental phenotypic susceptibility scores (dPSSs and iPSSs, respectively) were calculated. The predictive value of RC and PSS for ARV therapy response and/or ARDFP was evaluated using multivariate regression analysis and Pearson correlations. Results In 146 no-ARDFP subjects, baseline RC (50.8%) did not change at week 12 and was not correlated with CD4 cell count or viral load changes at week 12 (P = 0.33 and P = 0.79, respectively) or at week 24 (P = 0.96 and P = 0.14, respectively). dPSS predicted virological but not CD4 cell count response to ARV therapy at weeks 12, 24 and 48 (P = 0.002, P

Background. Patients with HER2−positive breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Within HER2−positive breast cancer tumors, it is possible to resolve a ∼1.5-log range of HER2 protein expression using a novel quantitative HER2 assay (HERmark®). We investigated the correlation between quantitative HER2 protein expression in primary breast cancers and the time to brain metastases (TTBM) in HER2−positive advanced breast cancer patients treated with trastuzumab. Methods. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER2−positive metastatic breast cancer, defined as 3+ categorical staining by immunohistochemistry (IHC). HER-2/neu gene copy number was subsequently quantified as HER2/CEP17 ratio by central laboratory fluorescence in situ hybridization (FISH). HER2 protein was quantified as total HER2 protein expression (H2T) by the HERmark assay in formalin-fixed, paraffin-embedded primary tumor samples. HER2 variables were correlated with clinical features and TTBM measured from the initiation of trastuzumab-containing therapy. Results. H2T level (continuous variable) was correlated with shorter TTBM (HR=2.3; p=0.013), whereas HER2 gene amplification by FISH (p=0.28) and continuous HER2/CEP17 ratio (p=0.25) had no significant prognostic impact. The correlation between continuous H2T level and TTBM was confirmed in a multivariate analysis (HR=3.2; p=0.021). Controlling for the competing risk of death from causes other than brain metastases, continuous H2T remained a strong correlate of TTBM (HR=2.7; p=0.0009). In the subset of patients that was centrally-determined HER2 positive by FISH (117 patients), above-median H2T level was significantly associated with shorter TTBM (HR=2.4; p=0.005), whereas this was not true for median FISH/CEP17 ratio (p=0.4). In a multivariate analysis of this subset, continuous H2T (p=0.021) and a time dependent covariate capturing time to non-brain metastases (p=0.0044) were prognostic for TTBM, whereas FISH/CEP17, ER, PgR and grade were not. Conclusions. These data reveal a strong relationship between quantitative HER2 protein expression levels and the risk of brain relapse in HER2−positive advanced breast cancer patients. Consequently, quantitative assessment of HER2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-05.

Background: Accurate assessment of the HER2 status is critical in determining appropriate therapy for patients with invasive breast cancer. ASCO/CAP HER2 testing guidelines caution that up to 20% of routine HER2 testing by IHC/FISH may be unreliable (Wolff et al. JCO 2007;25:118). The HERmark assay is a novel quantitative HER2 protein measurement for determining HER2 status in breast cancer. Central HER2 testing showed high concordance (96-98%) with HERmark for positive and negative categories when equivocal subsets were excluded (Huang et al. Am J Clin Pathol 2010;134:303; Joensuu et al, 2008 SABCS, abstract 2071). In this study, we examined concordance between HERmark and routine HER2 testing by IHC and FISH from “real world” formalin-fixed, paraffin-embedded (FFPE) breast cancers submitted commercially for HERmark testing. Methods: 717 HERmark results on FFPE breast cancers tested from 2008 to 2010 and corresponding HER2 IHC/FISH results were reviewed. The IHC and FISH results, per pathology reports submitted at the time of HERmark testing, were compared to HERmark categorical (negative, equivocal, positive) results. Results: 590 (419) samples had IHC (FISH) and HERmark results available. Of these cases, 92% (94%) were either negative or equivocal by IHC (FISH). The HERmark testing reported 33% HERmark negative, 33% HERmark equivocal and 34% HERmark positive. Comparisons of HER2 status by IHC and FISH vs. HERmark are detailed in Table 1. 19% of cases classified as IHC 0 or 1+ were positive by HERmark; 35% classified as FISH negative were HERmark positive. Of the 158 triple negative cases by IHC/FISH (ER, PR and HER2 negative, data not shown), 38 (24%) were reclassified as HER2 positive by HERmark. Conclusions: Higher than expected discordance between HER2 IHC/FISH and the HERmark assay was observed in this study compared with central HER2 testing of prior study cohorts. Selection bias may have impacted these results as ≥ 92% of cases submitted for HERmark testing were either equivocal or negative by routine HER2 IHC/FISH. Central HER2 FISH retesting of these samples will be performed and compared with HERmark results to further evaluate the discordance observed in this analysis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-12.

Background: Accurate assessment of the HER2 status is essential for identifying patients who may benefit from HER2 targeted therapy. The current methods, immunohistochemistry (IHC) and in situ hybridization (ISH), determine HER2 status semi-quantitatively as positive (+), equivocal (+/−) and negative (−) with predefined cutoff values. Recent studies have suggested that current HER2 cutoffs may not be optimal for all clinical settings of HER2 targeted therapy. In a small subset of adjuvant NCCTG N9831 patients confirmed as HER2−normal by round-robin review of HER2 testing, trastuzumab benefit was observed (Perez et al, SABCS 2010). Quantification of HER2 as continuous variable may enable a more accurate optimization of HER2 cutoffs for various HER2 targeted therapies. In this study, we measured continuous HER2 protein expression by the HERmark™ assay and continuous mRNA expression by quantitative real time polymerase chain reaction (qPCR), and compared these results with central IHC and central chromogenic in situ hybridization (CISH) results of FinHer. Methods: Total HER2 protein expression (H2T) was quantified using the HERmark assay as previously described (Huang et al. Am J Clin Pathol 2010;134:303). HER2 mRNA expression (H2N) was measured by qPCR as previously published (Noske et al. Br Cancer Res Treat 2011;126:109). The results of H2T and H2N as continuous variables and as predefined categories were compared with central CISH results from FinHer (Joensuu et al, N Engl J Med 2006;354), and central IHC retesting. Results: H2T in 899 evaluable samples described a continuum of 0.4 to 721.2 (relative HERmark unit); while H2N in 915 evaluable samples showed a continuum of 31.4 to 42.8 (delta-Ct). Significant correlation between H2T and H2N as continuous variable was found (R2= 0.56, P< .0001). Paired method comparison was performed for samples with valid results in any two of the four testing methods. Overall concordance of H2T and H2N with predefined categories (+, +/−, -) was 81%, and concordance of (+) and (−) subsets was 95% when (+/−) cases (H2T 11%; H2N 6%) were excluded. Overall concordance of central IHC and H2T categories (+, +/−, -) was 75%, and concordance of (+) and (−) subsets was 96% when (+/−) cases (IHC 16%; H2T 11%) were excluded. Overall concordance of IHC and H2N categories (+, +/−, -) was 84%, and concordance of (+) and (−) subsets was 99% when (+/−) cases (IHC 16%; H2N 6%) were excluded. Concordance of central CISH (+, -) with H2T and H2N categories (+, -) was 89% and 91%, respectively, when (+/−) cases were excluded from H2T (13%) and H2N (8%), respectively. Conclusions: All four methods identified HER2−positive breast cancers. The discordance rate between the methods tested was approximately 10 to 20% despite careful delineation of cancerous tissue in the sample and analysis of adjacent tumor sections. No combination of assays could be identified with concordance rate >95% when the equivocal subsets were included in comparisons. Exclusion of the equivocal subsets (about 10% of samples) yielded high concordance rates of approximately 95% or higher. H2T and H2N showed comparable continuous distribution patterns and significant concordance with standard HER2 status by central IHC and CISH. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-01.

Article approval pending, Drug resistance poses a significant challenge for the successful application of highly active antiretroviral therapy (HAART) globally. Furthermore, emergence of HIV-1 isolates that preferentially use CXCR4 as a coreceptor for cell entry, either as a consequence of natural viral evolution or HAART use, may compromise the efficacy of CCR5 antagonists as alternative antiviral therapy.

Percentages of activated T cells correlate with HIV-1 disease progression, but the underlying mechanisms are not fully understood. We hypothesized that HLA-DR + CD38 + (DR + 38 + ) CD4 + T cells produce the majority of HIV-1 due to elevated expression of CCR5 and CXCR4. In phytohemagglutinin (PHA)-stimulated CD8-depleted peripheral blood mononuclear cells (PBMC) infected with HIV-1 green fluorescent protein (GFP) reporter viruses, DR − 38 + T cells constituted the majority of CCR5 (R5)-tropic (median, 62%) and CXCR4 (X4)-tropic HIV-1-producing cells (median, 61%), although cell surface CCR5 and CXCR4 were not elevated in this subset of cells. In lymph nodes from untreated individuals infected with R5-tropic HIV-1, percentages of CCR5 + cells were elevated in DR + 38 + CD4 + T cells (median, 36.4%) compared to other CD4 + T-cell subsets (median values of 5.7% for DR − 38 − cells, 19.4% for DR + 38 − cells, and 7.6% for DR − 38 + cells; n = 18; P < 0.001). In sorted CD8 − lymph node T cells, median HIV-1 RNA copies/10 5 cells was higher for DR + 38 + cells (1.8 × 10 6 ) than for DR − 38 − (0.007 × 10 6 ), DR − 38 + (0.064 × 10 6 ), and DR + 38 − (0.18 × 10 6 ) subsets ( n = 8; P < 0.001 for all). After adjusting for percentages of subsets, a median of 87% of viral RNA was harbored by DR + 38 + cells. Percentages of CCR5 + CD4 + T cells and concentrations of CCR5 molecules among subsets predicted HIV-1 RNA levels among CD8 − DR/38 subsets ( P < 0.001 for both). Median HIV-1 DNA copies/10 5 cells was higher in DR + 38 + cells (5,360) than in the DR − 38 − (906), DR − 38 + (814), and DR + 38 − (1,984) subsets ( n = 7; P ≤ 0.031). Thus, DR + 38 + CD4 + T cells in lymph nodes have elevated CCR5 expression, are highly susceptible to infection with R5-tropic virus, and produce the majority of R5-tropic HIV-1. PBMC assays failed to recapitulate in vivo findings, suggesting limited utility. Strategies to reduce numbers of DR + 38 + CD4 + T cells may substantially inhibit HIV-1 replication.

The HIV gp41 N-trimer pocket region is an ideal viral target because it is extracellular, highly conserved, and essential for viral entry. Here, we report on the design of a pocket-specific d -peptide, PIE12-trimer, that is extraordinarily elusive to resistance and characterize its inhibitory and structural properties. d -Peptides (peptides composed of d -amino acids) are promising therapeutic agents due to their insensitivity to protease degradation. PIE12-trimer was designed using structure-guided mirror-image phage display and linker optimization and is the first d -peptide HIV entry inhibitor with the breadth and potency required for clinical use. PIE12-trimer has an ultrahigh affinity for the gp41 pocket, providing it with a reserve of binding energy (resistance capacitor) that yields a dramatically improved resistance profile compared to those of other fusion inhibitors. These results demonstrate that the gp41 pocket is an ideal drug target and establish PIE12-trimer as a leading anti-HIV antiviral candidate.

To assess the effect of HIV coreceptor tropism (CRT) on the relative risk of progression to a composite outcome of CD4 countor =350 cells per microliter, treatment initiation, or death.CRT assays were performed after study closure in baseline samples obtained from enrollees in a prospectively monitored cohort of treatment-naive adults withor =450 CD4 cells per microliter andor =1000 HIV-1 RNA copies per milliliter.Dual/mixed (D/M) and R5 CRT were detected in 32 and 282 patients, respectively. The baseline CD4 count (617 versus 694 cells/microL; 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 (P0.001) and 0.87 per 50 cells per microliter higher CD4 cell count (P0.001). The effect of D/M CRT was also significant in separate analyses of time to initiation of antiretroviral therapy or CD4 cell countor =350 cells per microliter.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 countor =350 cells per microliter, treatment initiation, or death or by separate analyses of time to CD4 countor =350 cells per microliter or treatment initiation.

Background: Frequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration. Design: Cross-sectional analysis. Methods: Subjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq™, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models. Results: Between April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002). Conclusion: Despite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI.

Introduction Accurate assessment of HER2 status is critical in determining appropriate therapy for breast cancer patients but the best HER2 testing methodology has yet to be defined. In this study, we compared quantitative HER2 expression by the HERmark™ Breast Cancer Assay (HERmark) with routine HER2 testing by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and correlated HER2 results with overall survival (OS) of breast cancer patients in a multicenter Collaborative Biomarker Study (CBS). Methods Two hundred and thirty-two formalin-fixed, paraffin-embedded breast cancer tissues and local laboratory HER2 testing results were provided by 11 CBS sites. HERmark assay and central laboratory HER2 IHC retesting were retrospectively performed in a blinded fashion. HER2 results by all testing methods were obtained in 192 cases. Results HERmark yielded a continuum of total HER2 expression (H2T) ranging from 0.3 to 403 RF/mm2 (approximately 3 logs). The distribution of H2T levels correlated significantly (P

Breast cancer is the second most likely malignancy to metastasize to the brain, with up to 30% of patients showing signs of metastases at autopsy.1,2 This occurrence is particularly common in some subsets of advanced breast cancer, including tumors positive for human epidermal growth factor receptor 2 (HER2) and triple-negative (hormone receptor [HR]–negative and HER2-negative) tumors.3–8 Paradoxically, more effective treatment of advanced breast cancer allows more time for the development of brain metastasis (BM), and the relative incidence of BM is increasing.6,7 In HER2-positive advanced breast cancer, approximately half of the patients treated with trastuzumab, a recombinant humanized anti-HER2 monoclonal antibody (mAb), will relapse in the brain.6,7 With the emergence of investigational targeted therapies to treat or delay BM, it is increasingly important to understand the relationship between biomarker expression in the primary and metastatic lesions in the brain.8 Anti-HER2 therapies seem to effectively delay the development of BM,8–11 yet little is known about HER2 expression in BM and its relation to HER2 expression in primary tumors. Recent data suggest that quantitating HER2-total (H2T) levels (measured by HER mark and other methods) in HER2-positive breast cancers may be important in predicting response to HER2-directed therapies, at least in the neoadjuvant setting.12–14 Further, elevated expression of p95HER2 (p95) has been found to be a resistance marker for trastuzumab in advanced HER2-positive breast cancer15–18 and to play a role in the development of metastasis.19,20 Thus far, p95 has only been measured in primary breast cancer samples. In the current study, including breast cancers with various phenotypes, we compared quantitative p95 and HER2 expression in primary tumors and in matched BM and assessed clinical implications of these alterations.

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.

The HIV protease inhibitor P-1946 is a member of a novel family of l-Lysine derivatives. The compound is a specific HIV-1 protease inhibitor that has potent and selective in vitro antiviral activity (EC50 152 nM) against a range of isolates resistant to commercially available protease inhibitors. The presence of at least four primary and four secondary drug resistance mutations is required to achieve greater than four-fold resistance to P-1946. P-1946's favorable resistance profile makes it a good lead for the development of new agents active against existing PI-resistant virus in treatment-experienced patient.

Replication capacity and transmission of human immunodeficiency virus (HIV)-1 in antiretroviral drug-naive Malawian women who had subtype C infection were investigated. Infant children of these women received either 1 dose of nevirapine or 1 dose of nevirapine plus 1 week of daily doses of zidovudine. PhenoSense HIV was used to determine replication capacity in 49 women whose infants were infected with HIV-1 and in 47 women whose infants were uninfected by 6-8 weeks of age. Mean replication capacity was higher in transmitters than in nontransmitters (P=.01). In a multivariate model, higher replication capacity was associated with transmission (odds ratio, 1.45 for each 10% increase in replication capacity [95% confidence interval, 1.11-1.90]; P = .0063), after adjustment for maternal HIV-1 load and other factors.

We have investigated a potential transmission chain of HIV-1 with drug resistance-associated mutations between three individuals over a period of 5 years by use of cloning and sequencing of viral genes, and phenotypic characterization. Viruses containing reverse transcriptase drug resistance-associated mutations were transmitted sequentially between three homosexual men (A, B, and C), and persisted in one individual for at least 4 years, despite intermittent therapy and reduced viral replicative capacity compared with wild-type strains. Clonal analysis of the envelope gene from semen and blood virus showed that the virus transmitted to patient C was more closely related to virus from the semen than the blood of patient B. Our data suggest that HIV variants with drug resistance-associated mutations can persist following primary infection, despite intervening antiretroviral therapy, and subsequently sexually transmitted. We provide "proof of principle" that such mutations can therefore become "fixed" within the circulating virus pool.

SUMMARY Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germ-line B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design., In Brief Understanding the molecular basis of HIV Env-specific broadly neutralizing antibodies (bnAbs) development is key for vaccine design. Landais et al. find that glycan heterogeneity played a role in the activation of V2 apex PCT64 bnAbs precursor and that viral evolution was similar to CAP256, another donor with V2 apex bnAbs.

Although combination antiretroviral therapy has resulted in a considerable improvement in the treatment of human immunodeficiency virus (HIV) type 1 (HIV-1) infection, the emergence of resistant virus is a significant obstacle to the effective management of HIV infection and AIDS. We have developed a novel phenotypic drug susceptibility assay that may be useful in guiding therapy and improving long-term suppression of HIV replication. Susceptibility to protease (PR) and reverse transcriptase (RT) inhibitors is measured by using resistance test vectors (RTVs) that contain a luciferase indicator gene and PR and RT sequences derived from HIV-1 in patient plasma. Cells are transfected with RTV DNA, resulting in the production of virus particles that are used to infect target cells. Since RTVs are replication defective, luciferase activity is measured following a single round of replication. The assay has been automated to increase throughput and is completed in 8 to 10 days. Test results may be useful in facilitating the selection of optimal treatment regimens for patients who have failed prior therapy or drug-naive patients infected with drug-resistant virus. In addition, the assay can be used to evaluate candidate drugs and assist in the development of new drugs that are active against resistant strains of HIV-1.