Patients older than 12 months and younger than 22 years with measurable or evaluable tumors refractory to therapy were eligible. Histologic verification of malignancy was required except for patients with intrinsic brainstem glioma. Eligible diagnoses included patients with recurrent or refractory solid tumor (parts A, D); leukemia with more than 25% blasts in the bone marrow (part B); neuroblastoma, medulloblastoma, CNS PNET or ATRT (part C). Other eligibility criteria included: Lansky or Karnofsky score ≥ 60; body-surface area of ≥ 0.5 m2; recovery from the acute toxic effects of prior therapy; ≥ 3 months since total-body irradiation, craniospinal or hemi-pelvic radiation and ≥ 2 months since a stem-cell transplant; adequate bone marrow function for patients with solid tumors (peripheral absolute neutrophil count ≥ 1,000/μL, platelets ≥ 100,000/μL [transfusion independent], hemoglobin ≥ 8.0 g/dL), for patients with leukemia (part B) platelets ≥ 20,000/μL, hemoglobin ≥ 8.0 g/dL; adequate renal function (age-adjusted normal serum creatinine or a glomular filtration rate ≥ 70 mL/min/1.73 m2), adequate liver function (total bilirubin ≤ 1.5× institutional upper limit of normal for age, ALT ≤ 5× institutional upper limit of normal for age and albumin ≥ 2 g/dL). Patients who were to receive 13cRA (part C) had to have ≤ grade 1 skin toxicity, serum triglycerides lower than 300 mg/dL, a negative urine dipstick for protein, or lower than 1,000 mg/24 hour urine collection, and no gross hematuria. Patients were excluded if they had received valproic acid in the previous 2 weeks, were on enzyme-inducing anticonvulsants, or other noncytotoxic anticancer agents, were pregnant or lactating or had uncontrolled infections. Patients with solid tumor with bone marrow involvement and patients with active CNS leukemia were excluded. Patients with CNS malignancies receiving dexamethasone had to be on a stable or decreasing dose for ≥ 7 days before study enrollment.
The institutional review boards of participating institutions approved the protocol. Informed consent and assent, as appropriate, were obtained according to local institutional guidelines.
Drug Administration and Study Design
Vorinostat was supplied by the Cancer Therapy Evaluation Program (National Cancer Institute, Bethesda, MD) as a white, opaque gelatin capsule, containing 100 mg of vorinostat. A dosing nomogram was used to minimize interpatient dosing variability. For part D, a suspension was prepared locally by the investigational pharmacists by mixing 20 mL of OraPlus (Humco, Texarcana, TX) with the contents of twenty 100 mg vorinostat capsules in a 4 ounce glass bottle. After shaking for up to 3 minutes to disperse, an additional 20 mL of OraSweet (Paddock Lab, Minneapolis, MN) was added. The container was again shaken to disperse, resulting in a final concentration of 50 mg/mL. The suspension was stored at room temperature and based on manufacturer's recommendation was stable for a maximum of 2 weeks. Vorinostat was administered orally each day, preferably with food.
The starting vorinostat dose for part A (patients with recurrent or refractory solid tumor) was 180 mg/m2
/d (approximately 80% of the adult recommended dose of 400 mg daily) with dose escalations in 30% increments. Once the MTD for vorinostat was defined, vorinostat was administered to six patients with recurrent or refractory leukemia to assess its tolerability at the solid tumor MTD (part B). In part C, additional cohorts of patients with recurrent neuroblastoma, medulloblastoma, PNET, or ATRT were enrolled to determine the MTD of vorinostat administered in combination with standard neuroblastoma dosing of 13cRA (80 mg/m2
/dose twice per day for 14 days).22
The starting dose for vorinostat in part C was 180 mg/m2
/dose daily. In part D, patients with recurrent or refractory solid tumor received vorinostat as a 50- mg/mL suspension at the part A MTD in order to characterize the pharmacokinetics of the suspension compared with the capsule.
In the absence of disease progression, and if laboratory parameters as defined in the eligibility section were met, each 28-day course was repeated without interruption for up to 12 courses. In parts A and C, a minimum of three evaluable patients were treated at each dose level. If one of three patients at a given dose level experienced a DLT, up to three more were accrued at the same dose level. If ≥ two patients experienced DLT, then the MTD was exceeded and three more patients were treated at the next lower dose level. The MTD was defined as the dose level at which at most one patient experienced DLT with at least two of three to six patients experiencing a DLT at the next higher level. If observed DLTs were different classes of adverse effects, then expansion of the cohort to 12 patients was considered if the following conditions were met: one of the DLTs did not appear to be dose related; the toxicity was readily reversible; the study chair, statistician, Children's Oncology Group Developmental Therapeutics Chair, and investigational new drug sponsor all agreed that cohort expansion was acceptable. If fewer than one third of patients in the expanded cohort experienced DLT at the dose, further dose escalation could proceed.
In part B, six patients with recurrent or refractory leukemia received vorinostat at the solid tumor MTD to assess its tolerability.
To study the pharmacokinetics of an oral suspension formulation in part D, 12 patients with recurrent or refractory solid tumors (six patients < 12 years, six > 12 years) were enrolled. The suspension was administered on day 1 of course 1 at the solid tumor MTD; after day 1, patients could continue their course using either capsules or suspension.
Toxicities were graded according to the Common Terminology Criteria for Adverse Events version 3.0. Hematologic DLT was defined as grade 4 neutropenia or thrombocytopenia (children with leukemia were not considered evaluable for hematologic toxicity). Nonhematologic DLT was defined as grade 3 or 4 nonhematologicatoxicity with the specific exclusion of: grade 3 nausea and vomiting of fewer than 5 days duration responsive to antiemetic therapy, grade 3 transaminase elevations that met eligibility criteria within 7 days of interruption and did not recur on rechallenge with study drug, grade 3 fever or infection fewer than 5 days, any grade 2 nonhematologic toxicity that persisted for ≥ 7 days and was considered sufficiently medically significant or sufficiently intolerable by patients that it required treatment interruption, grade 2 allergic reactions that necessitated discontinuation of study drug, or any adverse event requiring interruption of study drug for longer than 7 days or which recurred on drug rechallenge. Before opening part D of the study, the protocol was amended to exclude grade 3 hypokalemia, hypophosphatemia, hypocalcemia, and hypomagnesemia responsive to oral supplementation from the definition of DLT.
Pretreatment evaluations included a history, physical examination and CBC, electrolytes, renal and liver function tests, serum protein and albumin, triglycerides (part C only). CBCs were obtained twice weekly during the first course and weekly thereafter. History, physical examinations, and laboratory studies were obtained weekly in course 1 and before each subsequent course. Disease evaluations were obtained at baseline, at the end of course 1 and after every other course. Tumor response was reported using the Response Evaluation Criteria in Solid Tumors (RECIST).23
Participation in pharmacokinetic studies in parts A, B, and C was voluntary24
whereas all subjects enrolled in part D had to agree to participate in pharmacokinetics studies before enrollment. Blood samples (2 mL) were collected in heparinized tubes before the vorinostat dose, and at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 (± 2) hours after the first dose. The plasma concentrations of vorinostat and its metabolite, 4-anilio-4-oxobutanoic, acid were determined using a previously described validated liquid chromatography, tandem mass spectrometry method.25
The lower limit of quantitation was 2 ng/mL for vorinostat and 10 ng/mL for 4-anilio-4-oxobutanoic acid. The within-day and between-day precision (coefficient of variation) values and accuracy values for both analytes met standard assay validation criteria.26
Pharmacokinetic parameters for vorinostat and 4-anilio-4-oxobutanoic acid were calculated using noncompartmental methods. For each patient, the maximum concentration and time to maximum concentration were the observed values. The area under the plasma concentration-time curve (AUC0→t where t was the last measured time point) was calculated by the trapezoidal rule.
PBMC protein lysates were isolated as described previously27
from patients' whole blood drawn before, and at 1, 6, and 24 hours (± 2 hours) after the first vorinostat. Ten to 25 micrograms of each lysate was analyzed by Western blotting using a purified rabbit polyclonal antiacetyl-H3 antibody (Upstate Biotechnology, Lake Placid, NY) and chemiluminescence. The level of acetyl-H3 in each sample was determined relative to the expression of the housekeeping gene HPRT
(Abcam, Cambridge, MA).