After completion of the dose escalation portion of our phase I trial of ABT-751 on the 7-day and 21-day schedules, the study was expanded to include a pilot study of patients with neuroblastoma treated at 200 mg/m2
/day on the 7-day schedule. Study design, eligibility criteria, treatment regimen, definitions of dose-limiting toxicity and monitoring parameters have been reported previously [8
] The clinical trial and all study procedures were approved by the institutional review board at all participating institutions, and all patients and their legal guardians provided informed consent and assent according to institutional guidelines.
Eligibility criteria unique to the neuroblastoma pilot study included a lower threshold for hematologic function [absolute neutrophil count (ANC) >250/mcL, platelet count > 25,000/mcL] and alanine aminotransferase (ALT) ≤5 × the upper limit of normal. Due to clinical benefit observed in patients with neuroblastoma on the phase 1 dose escalation portion of the trial, patients enrolled on the pilot study were not required to have measurable or evaluable disease, but could be enrolled with no evidence of disease (NED). Patients with other solid tumors enrolled on the dose escalation portion of the trial, were required to have measurable or evaluable disease. Hematologic dose-limiting toxicity (H-DLT) was defined as failure to recover an ANC ≥1000/mcL for patients with an ANC ≥1000/mcL at enrollment or to an ANC ≥250/mcL for those with an ANC 250–1000/mcL at enrollment by day 28 of the treatment cycle. Failure to recover the platelet count to ≥50,000/mcL for patients with a platelet count ≥50,000/mcL at enrollment or to≥25,000/mcL for those enrolled with a platelet count 25,000–50,000/mcL by day 28 of the treatment cycle was also considered H-DLT.
Clinical Response Criteria and Survival Analysis
Response was assessed with the CTEP Response Evaluation Criteria in Solid Tumors (RECIST) in those with measurable disease. In patients with neuroblastoma that is evaluable only by MIBG scans, complete response was complete resolution of all lesions, partial response was decrease in the number of sites of disease compared to baseline, progressive disease was any new site of disease, and stable disease was no change in number or size of MIBG positive lesions. Central review of pre-treatment and on study disease evaluations was performed for all patients considered to have disease response by their treating physicians.
For event-free survival analysis, events were defined as discontinuation of protocol therapy for tumor progression, toxicity or withdrawal of consent; whereas the progression-free survival analysis only included tumor progression as an event. Patients were followed until death or date of last documented follow up visit. Event-free survival, progression-free survival, and overall survival (OS) from the date of the first dose of ABT-751 were estimated using the Kaplan-Meier method, with a two-tailed log rank test used to compare EFS in patients with neuroblastoma to patients who were enrolled on the phase I trial of ABT-751 with other solid tumors.
Research evaluation for patients with neuroblastoma in the pilot study included serial assessments of heath related quality of life using the PedsQL Generic Core Scale version 4.0. This 23 item self-administered questionnaire has been validated in children with cancer.[12
] Prior to each cycle, the patient’s performance status was assigned by a health care provider using the Lansky Scale for children 10 years of age or the Karnofsky scale for children >10 years of age.
Peripheral Blood Mononuclear Cells (PBMC)
Whole blood (6 mL) was obtained from patients prior to, 5 hours and 24 hours after the first dose and then prior to the dose of ABT-751 on day 5 of cycle 1. Peripheral blood mononuclear cells (PBMCs) were harvested by density centrifugation using Ficoll Hypaque. PBMCs were washed in PBS and stored at −70°C until analysis. Analysis of tubulin polymerization was performed using a polyclonal anti-detyrosinated α-tubulin antibody (Chemicon International, Temecula, CA) and monoclonal anti-acetylatedα-tubulin antibody (Sigma-Aldrich, St. Louis, MO) as described below.
Pediatric Tumor Cells Lines
Neuroblastoma (SK-N-AS, SK-N-DZ), rhabdomyosarcoma (RD), Ewing sarcoma (TC-71), medulloblastoma (HTB-186 Daoy) and osteosarcoma (HOS) pediatric tumor cell lines were obtained from American Type Culture Collection (ATCC, Manassas, Virginia). A second Ewing sarcoma line (LD) was established at the NCI from a patient with a scapular lesion. The neuroblastoma cell line, KCNR, was obtained from C.P. Reynolds. Cell lines were maintained in 1640 RPMI media with 10% fetal bovine serum (FBS) or 15% FBS for the KCNR line at 37°C/5% CO2 for optimal cell growth.
Vincristine (VCR), 1 mg/ml solution (Vincasar PFS, Sicor Pharmaceuticals, Inc, Irvine, California), was diluted in sterile H2O to a 100 mcM solution. ABT-751, which was supplied as a powdered compound by Abbott Laboratories (Abbott Park, Illinois), was dissolved in 1.25 % DMSO/sterile H2O to a final concentration of 135 mcM. Combretastatin, from a 10 mM DMSO solution, was diluted in sterile H2O to a 10 mcM solution. Aliquots of stock drug solutions were stored at −20°C. Prior to each assay, stock drug solutions were further diluted in sterile H2O to concentrations ranging from 0.1 nM to 1000 nM for VCR, from 0.1 nM to 100 mcM for ABT-751 and from 0.1 nM to 1000 nM for combretastatin.
Sulforhodamine B (SRB) Cytotoxicity Assay
Cells, in 1640 RPMI media with FBS, were plated in triplicate onto 96 well tissue culture plates in numbers determined optimal for confluent monolayer growth (5,000 cells/well for HOS, HTB-186 Daoy; 10,000 cells/well for TC-71, RD, SK-N-AS, SK-N-DZ, LD; 30,000 cells/well for KCNR), with an automated, multichannel pipette system (SerialMate, Matrix Technologies, Hudson, New Hampshire). Cells were incubated for 24 hours at 37°C/5% CO2
then exposed to vehicle control (1.25% DMSO/H2
O), VCR (0.1–1000 nM), ABT-751 (0.1 nM–100 mcM), and in 4 cell lines (SK-N-AS, KCNR, RD, TC-71) combretastatin (0.1–1000 nM) for 72 hours. Cells were fixed with trichloroacetic acid (final concentration 10%) at 4°C, washed, then dried at room temperature, stained with SRB in 1% acetic acid and dye was then solubilized with Tris base. Optical density measurements were performed at 540 and 405 nm dual wavelengths in a Bio-Tek EL 340 UV plate reader (Bio-Tek Instruments, Inc., Winooski, Vermont). [13
ACEA RT-CES Assay
The ACEA RT-CES device (ACEA Biosciences, San Diego, California) utilizes microelectrodes imbedded in the well base of a multi-well tissue culture plate to continuously measure changes in electrical impedance of plated cells as a determinant of cell surface area. The unit of measure, Cell Index (CI), relates the impedance of a media blank at time zero to impedance following cell plating and drug exposure. [17
Each cell line was initially plated in duplicate onto 16 well ACEA E-plates (ACEA Biosciences, San Diego, California) at concentrations ranging from 2,500–40,000 cells/well, to characterize growth curves in drug-free media and determine optimal cell number for confluent monolayer growth. Number of cells plated, drug concentrations and exposure were performed as described for the SRB assay. Hourly measurements of electrical impedance were performed for 96 hours (time of cell plating through the end of the 72 hour drug exposure).
Dose Response Curves and IC50 Determination
Endpoint cytotoxicity results from the SRB assay were compared to the continuous data obtained from the ACEA RT-CES assay following the 72 hour drug exposure. Growth curves initially performed with the ACEA system confirmed all tumor lines remained in the log phase of cell growth 72 hours after plating; therefore, this time point was evaluated. Cell survival for the SRB and ACEA assays was calculated as a percentage of vehicle control.
IC50 concentrations and the slope (h) of the sigmoidal dose-response curves for VCR and ABT-751 in each of the 8 pediatric tumor cell lines were estimated by fitting the mathematical model below to the combined drug concentration-survival data from all experiments using MLAB (Civilized Software Inc, Silver Spring, Maryland).
Where C(d) represents the percent survival at concentration d, ME is the maximum effect, IC50 is the concentration that produces half of the maximum effect and h is the slope of the dose response curve.
Tubulin Polymerization Assay
Acetylation and detyrosination are reversible post-translational modifications made to α-tubulin. Polymerized tubulin has been shown to be the preferred substrate for these enzymatic reactions as compared to tubulin heterodimers, [21
] and smaller amounts of acetylated and detyrosinated α-tubulin have been found in dynamic microtubules. Therefore, these modifications represent markers of microtubule stability. [23
Cells in 1640 RPMI media with FBS were plated onto 100 mm diameter tissue culture dishes (500,000 cells for HOS, HTB-186 Daoy; 1,000,000 cells for TC-71, RD, SK-N-AS, SK-N-DZ, LD; 3,000,000 cells for KCNR), incubated for 24 hours at 37°C/5% CO2 then exposed to vehicle control (1.25% DMSO/H2O), VCR, ABT-751, and in selected cell lines (SK-N-AS, KCNR, RD, TC-71) combretastatin for 72 hours at the IC10, IC50 and IC90 concentrations as estimated from dose response curves for each cell line. Scraped cells were pelleted and then resuspended in lysis buffer (0.1 M PIPES, 1 mcM EGTA, 1 mM MgSO4, 30% glycerol, 5% DMSO, 0.125% IGEPAL, Roche Diagnostics complete mini protease inhibitor; 1 tablet/10 mL, aprotinin; 9,500 units/mL, GTP; 5 mM), vortexed, centrifuged and supernatant samples were standardized for protein concentration with the Bradford Reagent assay and BSA standards by measuring single reference wavelength absorbance (Hewlett Packard 8452A Diode Array Spectrophotometer).
Samples containing 15 mcg total protein were separated by SDS-PAGE gel electrophoresis, transferred to PVDF membrane and then exposed to a polyclonal anti-detyrosinated α-tubulin antibody and monoclonal anti-acetylated α-tubulin antibody, which are antibodies specific for post-translational modifications made to α-tubulin. A monoclonal anti-tyrosinated α-tubulin antibody (Sigma-Aldrich, St. Louis, MO) was used as a measure of dynamic tubulin and a monoclonal anti-α tubulin antibody (Sigma-Aldrich, St. Louis, MO) as a measured control of total tubulin.
Following the protocol described above for cell plating and drug exposure, four cell lines (SK-N-AS, KCNR, RD, TC-71) were lysed with buffer containing 5 mcM paclitaxel (Sigma-Aldrich, St. Louis, MO), immediately vortexed and centrifuged. Pellet samples were resuspended in lysis buffer and supernatant samples were standardized for total protein concentration. Samples of 3 mcg total protein were separated by gel electrophoresis, transferred to a PVDF membrane and exposed to the anti-acetylated and anti-tyrosinated α-tubulin antibodies followed by GAPDH and C-21 monoclonal antibodies as a gel loading control. Membranes were developed by a chemiluminescent technique (Supersignal Femto West Kit, Pierce Biotechnology, Inc, Rockford, IL). [25
SK-N-AS, KCNR, RD, and TC-71 cell lines were plated onto 12 mm diameter glass cover slips, incubated for 24 hours and then exposed to vehicle control (1.25% DMSO/H2
O), VCR, ABT-751, or combretastatin for 72 hours at the IC10
concentrations as estimated from the dose response curves specific for each cell line. Cells were fixed with cold methanol (−20°C), blocked with 5% bovine serum albumin in PBS then exposed to primary antibodies; anti-detyrosinated and anti-acetylatedα-tubulin antibodies specific for post-translationally modified tubulin, anti-tyrosinatedα-tubulin antibody as a measure of dynamic tubulin and anti-α-tubulin as a control for total tubulin, followed by fluorescein-5-isothiocyanate (FITC) labeled secondary antibodies. Cover slips were mounted onto glass slides using Fluor-Gel (Electron Microscopy Sciences). Intracellular microtubule structure was observed on an Axiovert 100M fluorescent microscope (Carl Zeiss, Inc., Oberkochen, Germany) equipped with a PlanApochromat 100x/1.4 oil objective and confocal images were generated on a Zeiss LSM510. [28