Sue Chow

Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations

Previous studies of intracellular expression of phospho‐epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho‐epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho‐Thr 202/Tyr 204‐p44/42 extracellular‐regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho‐epitopes in leukocyte populations in whole blood.

Potential Use of Cetrimonium Bromide as an Apoptosis-Promoting Anticancer Agent for Head and Neck Cancer

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with γ radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H+-ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G1 cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models.

Benzethonium Chloride: A Novel Anticancer Agent Identified by Using a Cell-Based Small-Molecule Screen

Purpose: This study aims to identify a novel therapeutic agent for head and neck cancer and to evaluate its antitumor efficacy. Experimental Design: A cell-based and phenotype-driven high-throughput screening of ∼2,400 biologically active or clinically used compounds was done using a tetrazolium-based assay on FaDu (hypopharyngeal squamous cancer) and NIH 3T3 (untransformed mouse embryonic fibroblast) cells, with secondary screening done on C666-1 (nasopharyngeal cancer) and GM05757 (primary normal human fibroblast) lines. The “hit” compound was assayed for efficacy in combination with standard therapeutics on a panel of human cancer cell lines. Furthermore, its mode of action (using transmission electron microscopy and flow cytometry) and its in vivo efficacy (using xenograft models) were evaluated. Results: Benzethonium chloride was identified as a novel cancer-specific compound. For benzethonium (48-hour incubation), the dose required to reduce cell viability by 50% was 3.8 μmol/L in FaDu, 42.2 μmol/L in NIH 3T3, 5.3 μmol/L in C666-1, and 17.0 μmol/L in GM05757. In vitro, this compound did not interfere with the effects of cisplatin, 5-fluorouracil, or γ-irradiation. Benzethonium chloride induced apoptosis and activated caspases after 12 hours. Loss of mitochondrial membrane potential (ΔΨM) preceded cytosolic Ca2+ increase and cell death. In vivo, benzethonium chloride ablated the tumor-forming ability of FaDu cells, delayed the growth of xenograft tumors, and combined additively with local tumor radiation therapy. Evaluation of benzethonium chloride on the National Cancer Institute/NIH Developmental Therapeutics Program 60 human cancer cell lines revealed broad-range antitumor activity. Conclusions: This high-throughput screening identified a novel antimicrobial compound with significant broad-spectrum anticancer activity.

Pharmacodynamic Monitoring of Molecular-Targeted Agents in the Peripheral Blood of Leukemia Patients Using Flow Cytometry

The introduction of specific, molecular-targeted drugs is radically changing cancer treatment. Pharmacodynamics, which measures drug effects on the host, is key during early-phase clinical trials of novel agents to determine the relations between drug dose and target inhibition as well as measure the downstream effects of target inhibition on the cancer. In this article, we describe the application of flow cytometry to the pharmacodynamic monitoring of molecular-targeted agents in leukemia patients. The methods are based on current clinical flow-cytometry applications, with the addition of phosphospecific antibodies to measure the activation states of intracellular signaling elements and the introduction of techniques that maintain drug–target equilibrium during sample preparation. Using this approach, we successfully showed dose-dependent inhibition of c-Kit during a phase I clinical trial treating acute leukemia patients with the novel agent sorafenib. Further refinements identify considerable interpatient variation in signaling activity within leukemic blast populations, suggesting that an individualized approach to treatment based on flow cytometric monitoring might be advantageous. Improvements in sample turnaround offer the potential to introduce real-time pharmacodynamic monitoring during early-phase clinical trials.

A phase I study of elesclomol sodium in patients with acute myeloid leukemia.

Elesclomol is a novel, injectable, small molecule chemotherapeutic agent that has demonstrated preclinical efficacy in AML.[1,2] Mechanistically, elesclomol binds copper (Cu) in the form of Cu in the serum and enables the reduction reaction of Cu (II) to Cu (I) once inside the malignant cell. This redox reaction disrupts mitochondrial respiration and elevates the level of reactive oxygen species (ROS) beyond sustainable levels.[3] Ultimately, elesclomol perturbs cellular energy production and metabolism and triggers the mitochondrial apoptosis pathway in cancer cells resulting in cell death.[4] We and others have previously shown that AML cells and stem cells are uniquely dependent on mitochondrial metabolism and function.[5–7] Thus, elesclomol-mediated mitochondrial disruption could be efficacious for a subset of AML patients. Due to the mechanism of action of elesclomol, it is most effective under oxygen conditions where mitochondrial respiration is active. In contrast, under hypoxic conditions, where the energy production of a cell shifts to glycolysis in the cytoplasm, elesclomol’s anti-cancer activity is diminished.[8,9] This shift in cellular respiration is often associated with high lactate dehydrogenase (LDH) levels in the cell, and may be reflected by high levels in the serum as well.[10] In support of this hypothesis, preliminary findings in a phase 3 metastatic melanoma study showed a differential response to treatment with elesclomol based on baseline levels of LDH. Improved progression-free survival was achieved in patients with normal LDH. In contrast, patients with high LDH levels had no difference in progression-free survival, and in fact, had decreased overall survival.[11] We therefore conducted a phase I dose escalation study assessing the safety and efficacy of elesclomol in patients with relapsed or refractory AML. A total of nine patients with relapsed and refractory AML with LDH 0.8 times the upper limit of normal (ULN) were enrolled in the study from February 2011 to September 2013. Baseline characteristics are shown in Table 1. Patients at least 18 years of age with adequate performance status and renal and hepatic function were eligible to participate in this study. Patients were removed from study treatment if they developed unacceptable toxicity or adverse events, progressive disease, or a sustained increase in LDH 1.2 times the ULN (see Supplementary materials and methods for more details). The Ethics Review Board at the University Health Network as well as Health Canada approved this study before it commenced. All patients provided written consent and written authorization permitting the use of protected health information prior to the initiation of any study related procedures. The study is registered at clinicaltrials.gov (NCT01280786). A sodium salt formulation of elesclomol sodium was used in this study at a starting dose of 200 mg/m via a 60 min infusion once weekly (days 1, 8, 15, and 22) based on prior studies.[11–13] Dose-limiting toxicity (DLT) was defined as grade 3 or 4 non-hematologic toxicity possibly related to elesclomol occurring during the first four-week cycle of therapy. The primary study objective was to characterize the safety and tolerability of elesclomol administered to subjects with AML. The secondary objectives were to determine the optimal phase II dose and schedule for elesclomol in subjects with AML and evaluate response to the drug by pharmacodynamic assessment. Efficacy of elesclomol was evaluated based on bone marrow aspirate at cycle 1, day 29. Responses were recorded as defined by Cheson et al.[14] In the event of disease progression during cycle 1, study drug dosing was stopped and the bone marrow aspirate was not performed. Of the nine patients enrolled in the trial, six received one complete cycle of elesclomol. Two of the nine

Abstract A106: A phase 1 study of ENMD‐2076 in patients with relapsed or refractory acute myeloid leukemia (AML)

ENMD‐2076 is a novel, orally‐active molecule that inhibits Aurora A kinase as well as multiple receptor tyrosine kinases that drive tumor vascularization, including VEGFR2 (KDR), PDGFR and FGFR. A phase 1 study was conducted to determine the maximum tolerated dose (MTD) and toxicities of ENMD‐2076 in patients with refractory hematological malignancies. Fifteen patients with AML (cohorts of 6 patients per dose level) have been treated with 225 (n=7), 325 (n=2), and 375 (n=6) mg of ENMD‐2076 administered orally once daily. Median age was 76 years (range, 60 to 82 years). Median ECOG status was 1 (range, 0 to 2). Fourteen patients had received prior therapy (median, 2 regimens; range, 0 to 4 regimens). A total of 17 cycles have been administered to date, with a median of 1 cycle (range, 0 to 3 cycles); 2 patients (14%) have received 3 or more cycles of therapy. The most common ENMD‐2076‐related adverse events were grade ≤ 2 and consisted of dizziness, petechiae, hypertension, nausea, fatigue, diarrhea, and reflux. Dose‐limiting toxicity consisted of grade 3 fatigue in 2 patients at the 375 mg/day dose level. Therefore, the dose was decreased to 325 mg/day. Following a drug holiday, both patients who had experienced the grade 3 fatigue were restarted at the 325 mg/day dose level. No patient experienced grade 4 toxicities or death from ENMD‐2076. Of the 13 evaluable patients, 1 patient achieved a morphologic leukemic free state (MLFS) with platelet transfusion independence. One patient achieved a HI‐P. Two other patients had a 12% and 14% reduction in marrow blast count, respectively. At the time of analysis, 3 patients discontinued therapy due to disease progression. Peripheral blood and/or bone marrow were obtained at baseline for ex vivo drug sensitivity testing, and on Days 8 and 29 of cycle 1 for pharmacodynamic (PD) monitoring, using a whole blood flow cytometry protocol to measure ENMD‐2076 effects on cell signalling pathways. This assay uses combined labelling for P‐ERK, P‐Akt, P‐STAT5, and P‐S6 as the readout, and tests the effects of acute stimulation with the ligands SCF and FL in the presence or absence of pathway inhibitors, including ENMD‐2076. Pre‐incubation with drug concentrations in the range 0.5 – 2 µM suppressed growth factor stimulation in the blast cells of all patients tested to date. Decreases in the ability to stimulate ERK,Akt, STAT5, and S6 were seen in the Day 8 and 29 samples, including a striking inhibition of cell signalling in one patient who achieved a MLFS. Assays are also in progress to monitor specific effects on Aurora kinase and the cell cycle in these patient samples. In conclusion, single agent ENMD‐2076 has activity in a heavily pretreated group of AML patients that may correlate with inhibition of ERK,Akt, STAT5, and S6 activity. Enrollment, as well as PK and PD monitoring of this study, is ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A106.

Abstract 4647: Tie2-expressing monocytes (TEMs) as potential biomarkers of angiopoietin-Tie2 (Ang/Tie2) directed therapies: correlative analysis of a phase I study of AMG386 + temsirolimus (T).

Background: The Ang/Tie2 pathway plays a critical role in tumor angiogenesis, and several agents targeting this axis are in development. Tie2 is expressed by monocytes (M), and TEMs may be angiogenic mediators. Thymidine phosphorylase (TP), an angiogenic enzyme, is increased in TEMs upon Tie2 stimulation. We used flow cytometry to evaluate TEMs in a phase I study of AMG386 (Ang1/2 peptibody) + T in patients (pts) with solid tumors. Methods: Peripheral blood was collected from healthy volunteers and pts with advanced cancer (n=10 each) for assay development. For phase I study pts (n=5 to date), blood was collected on Cycle 1 Day 1 (D1), D3 and D8. Extracellular Tie2 staining was performed after hypotonic red blood cell lysis. TP staining followed formaldehyde fixation and Triton X-100 permeabilization. M and lymphocytes (L) were gated with CD45 and CD33. Median fluorescence intensities (MFI) were measured and normalized by calculating a M/L ratio. Tumor response was assessed every 2 cycles. Results: Tie2 staining was present on all M (absent in other leukocytes) in healthy controls and cancer pts, with no discrete Tie2+/- populations. Tie2 and TP M/L were similar between these 2 groups. Tie2, TP and response data from the first 5 pts receiving AMG386 + T are presented. Preliminary results reveal an association between change in TP D1 to D3 and tumor response. TP M/L decreased in all pts with tumor shrinkage (mean -18%), but increased (+6%) in the pt with tumor growth. No consistent association between Tie2 M/L and response was observed. Conclusions: Measurement of Tie2 and TP in circulating M is feasible. No discrete Tie2+ M population is identifiable. Tie2 and TP staining are similar in cancer pts and healthy volunteers. Preliminary analyses reveal an association between an initial reduction in TP and tumor shrinkage in pts receiving AMG386+T. Enrolment continues and additional data will be presented. Citation Format: David W. Cescon, Philippe L. Bedard, Sue Chow, Helen Chen, Albiruni Razak, Monika Wizemann, Lillian L. Siu, David Hedley. Tie2-expressing monocytes (TEMs) as potential biomarkers of angiopoietin-Tie2 (Ang/Tie2) directed therapies: correlative analysis of a phase I study of AMG386 + temsirolimus (T). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2013-4647