The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating and addressing the complexities of heterogeneous nanoparticle subpopulations.
PURPOSE Androgen receptor splice variant 7 (AR-V7) results in a truncated receptor, which leads to ligand-independent constitutive activation that is not inhibited by anti-androgen therapies, including abiraterone or enzalutamide. Given that previous reports suggested that circulating tumor cell (CTC) AR-V7 detection is a poor prognostic indicator for the clinical efficacy of secondary hormone therapies, we conducted a prospective multicenter validation study. PATIENTS AND METHODS PROPHECY ( ClinicalTrials.gov identifier: NCT02269982) is a multicenter, prospective-blinded study of men with high-risk mCRPC starting abiraterone acetate or enzalutamide treatment. The primary objective was to validate the prognostic significance of baseline CTC AR-V7 on the basis of radiographic or clinical progression free-survival (PFS) by using the Johns Hopkins University modified-AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay. Overall survival (OS) and prostate-specific antigen responses were secondary end points. RESULTS We enrolled 118 men with mCRPC who were starting abiraterone or enzalutamide treatment. AR-V7 detection by both the Johns Hopkins and Epic AR-V7 assays was independently associated with shorter PFS (hazard ratio, 1.9 [95% CI, 1.1 to 3.3; P = .032] and 2.4 [95% CI, 1.1 to 5.1; P = .020], respectively) and OS (hazard ratio, 4.2 [95% CI, 2.1 to 8.5] and 3.5 [95% CI, 1.6 to 8.1], respectively) after adjusting for CTC number and clinical prognostic factors. Men with AR-V7–positive mCRPC had fewer confirmed prostate-specific antigen responses (0% to 11%) or soft tissue responses (0% to 6%). The observed percentage agreement between the two AR-V7 assays was 82%. CONCLUSION Detection of AR-V7 in CTCs by two blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, and such men with mCRPC should be offered alternative treatments.
Purpose: Biomarkers aiding treatment optimization in metastatic castration-resistant prostate cancer (mCRPC) are scarce. Presence or absence of androgen receptor (AR) splice variants, AR-V7 and ARv567es, in mCRPC patient circulating tumor cells (CTCs) may be associated with taxane treatment outcomes. Experimental design: A novel digital droplet PCR (ddPCR) assay assessed AR splice variant expression in CTCs from patients receiving docetaxel or cabazitaxel in TAXYNERGY (NCT01718353). Patient outcomes were examined according to AR splice variant expression, including prostate-specific antigen (PSA)50 response and progression-free survival (PFS). Results: Of 54 evaluable patients, 36 (67%) were AR-V7+, 42 (78%) were ARv567es+, 29 (54%) were double positive and 5 (9%) were double negative. PSA50 response rates at any time were numerically higher for AR-V7- vs AR-V7+ (78% vs 58%; p=0.23) and for ARv567es- vs ARv567es+ (92% vs 57%; p=0.04) patients. When AR-V mRNA status was correlated with change in nuclear AR from Cycle 1 Day 1 to Day 8 (n=24), AR-V7+ patients (n=16) had a 0.4% decrease vs a 12.9% and 26.7% decrease in AR-V7-/ARv567es- (n=3) and AR-V7-/ ARv567es+ (n=5) patients, respectively, suggesting a dominant role for AR-V7 over ARv567es. Median PFS was 12.02 vs 8.48 months for AR-V7- vs AR-V7+ (HR=0.38; p=0.01), and 12.71 vs 7.29 months for ARv567es- vs ARv567es+ (HR=0.37; p=0.02). For AR-V7+, AR-V7-/ARv567es+, and AR-V7-/ ARv567es- patients, median PFS was 8.48, 11.17 and 16.62 months, respectively (p=0.0013 for trend). Conclusions: While detection of CTC-specific AR-V7 and ARv567es by ddPCR both influenced taxane outcomes, AR-V7 primarily mediated the prognostic impact. Absence of both variants was associated with the best response and PFS with taxane treatment.
Taxanes are the only chemotherapies used to treat patients with metastatic castration-resistant prostate cancer (CRPC). Despite the initial efficacy of taxanes in treating CRPC, all patients ultimately fail due to the development of drug resistance. In this study, we show that ERG over-expression in in vitro and in vivo models of CRPC is associated with decreased sensitivity to taxanes. ERG affects several parameters of microtubule dynamics and inhibits effective drug-target engagement of docetaxel or cabazitaxel with tubulin. Finally, analysis of a cohort of 34 men with metastatic CRPC treated with docetaxel chemotherapy reveals that ERG-overexpressing prostate cancers have twice the chance of docetaxel resistance than ERG-negative cancers. Our data suggest that ERG plays a role beyond regulating gene expression and functions outside the nucleus to cooperate with tubulin towards taxane in sensitivity. Determining ERG rearrangement status may aid in patient selection for docetaxel or cabazitaxel therapy and/or influence co-targeting approaches.
Drug target identification is a crucial step in development, yet is also among the most complex. To address this, we develop BANDIT, a Bayesian machine-learning approach that integrates multiple data types to predict drug binding targets. Integrating public data, BANDIT benchmarked a ~90% accuracy on 2000+ small molecules. Applied to 14,000+ compounds without known targets, BANDIT generated ~4,000 previously unknown molecule-target predictions. From this set we validate 14 novel microtubule inhibitors, including 3 with activity on resistant cancer cells. We applied BANDIT to ONC201—an anti-cancer compound in clinical development whose target had remained elusive. We identified and validated DRD2 as ONC201’s target, and this information is now being used for precise clinical trial design. Finally, BANDIT identifies connections between different drug classes, elucidating previously unexplained clinical observations and suggesting new drug repositioning opportunities. Overall, BANDIT represents an efficient and accurate platform to accelerate drug discovery and direct clinical application.
Circulating Tumor Cells (CTCs) have emerged as a reliable source of tumor cells, and their concentration has prognostic implications. CTC capture offers real-time access to cancer tissue without the need of an invasive biopsy, while their phenotypic and molecular interrogation can provide insight into the biological changes of the tumor that occur during treatment. The majority of the CTC capture methods are based on EpCAM expression as surface marker of tumor-derived cells. However, EpCAM protein expression levels can be significantly down regulated during cancer progression as consequence of the process of epithelial to mesenchymal transition. In this paper, we describe a novel HER2 (Human Epidermal Receptor 2)-based microfluidic device for the isolation of CTCs from peripheral blood of patients with HER2-expressing solid tumors. We selected HER2 as an alternative to EpCAM, as the receptor is biologically and therapeutically relevant in several solid tumors, like breast cancer (BC), where it is overexpressed in 30% of the patients and expressed in 90%, and gastric cancer (GC), in which HER2 presence is identified in more than 60% of the cases. We tested the performance of various anti HER2 antibodies in a panel of nine different BC cell lines with varying HER2 protein expression levels, using immunoblotting, confocal microscopy, live cells imaging and flow cytometry analyses. The antibody associated with the highest capture efficiency and sensitivity for HER2 expressing cells on the microfluidic device, was the one that performed best in live cells imaging and flow cytometry assays as opposed to the fixed cell analyses, suggesting that recognition of the native conformation of HER2 extracellular epitope on living cells was essential for specificity and sensitivity of CTC capture. Next, we tested the performance of the HER2 microfluidic device using blood from metastatic breast and gastric cancer patients. The HER2 microfluidic device exhibited CTC capture in 9/9 blood samples. Thus, the described HER2-based microfluidic device can be considered as a valid clinically relevant method for CTC capture in HER2 expressing solid cancers.
PurposeThe TAXYNERGY trial (ClinicalTrials.gov identifier: NCT01718353) evaluated clinical benefit from early taxane switch and circulating tumor cell (CTC) biomarkers to interrogate mechanisms of sensitivity or resistance to taxanes in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer. Patients and MethodsPatients were randomly assigned 2:1 to docetaxel or cabazitaxel. Men who did not achieve $ 30% prostate-specific antigen (PSA) decline by cycle 4 (C4) switched taxane. The primary clinical endpoint was confirmed $ 50% PSA decline versus historical control (TAX327). The primary biomarker endpoint was analysis of post-treatment CTCs to confirm the hypothesis that clinical response was associated with taxane drug-target engagement, evidenced by decreased percent androgen receptor nuclear localization (%ARNL) and increased microtubule bundling. ResultsSixty-three patients were randomly assigned to docetaxel (n = 41) or cabazitaxel (n = 22); 44.4% received prior potent androgen receptor-targeted therapy. Overall, 35 patients (55.6%) had confirmed $ 50% PSA responses, exceeding the historical control rate of 45.4% (TAX327). Of 61 treated patients, 33 (54.1%) had $ 30% PSA declines by C4 and did not switch taxane, 15 patients (24.6%) who did not achieve $ 30% PSA declines by C4 switched taxane, and 13 patients (21.3%) discontinued therapy before or at C4. Of patients switching taxane, 46.7% subsequently achieved $ 50% PSA decrease. In 26 CTC-evaluable patients, taxane-induced decrease in %ARNL (cycle 1 day 1 v cycle 1 day 8) was associated with a higher rate of $ 50% PSA decrease at C4 (P = .009).Median composite progression-free survival was 9.1 months (95% CI, 4.9 to 11.7 months); median overall survival was not reached at 14 months. Common grade 3 or 4 adverse events included fatigue (13.1%) and febrile neutropenia (11.5%). ConclusionThe early taxane switch strategy was associated with improved PSA response rates versus TAX327. Taxane-induced shifts in %ARNL may serve as an early biomarker of clinical benefit in patients treated with taxanes.
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