Asparaginase Pharmacokinetics After Intensive Polyethylene Glycol-Conjugated L-Asparaginase Therapy for Children with Relapsed Acute Lymphoblastic Leukemia

Eligibility Criteria.

Patients with an initial relapse of acute lymphoblastic leukemia at Children’s Hospital and Regional Medical Center (Seattle, WA), Deaconess Hospital (Spokane, WA), and James W. Riley Hospital for Children (Indianapolis, IN) between May 1997 and May 2001 were considered for entry onto a prospective treatment protocol (CHMC 1001). Patients were eligible with any site of initial relapse (including isolated or combined bone marrow relapse and isolated or combined extramedullary relapse at any site) any time after initial remission regardless of prior therapy. Central nervous system (CNS) relapse was defined as ≥5 white blood cells/μl in the spinal fluid and a cytologic evaluation demonstrating leukemic blasts or clinical signs of cranial nerve involvement regardless of cell count. Eligible patients had to be 1–21 years old and have normal cardiac function. Patients with a history of allergy to PEG-ASNase, a history of prior pancreatitis associated with ASNase use, trisomy 21, or mature B-cell leukemia were excluded from the study. Patients with a history of hypersensitivity to native E. coli ASNase were eligible. Informed consent was obtained before study enrollment in accordance with local Institutional Review Boards.

Chemotherapy Treatment Plan.

The details of the chemotherapy regimen, its toxicity, treatment after intensification, and outcome will be reported separately. Briefly, patients received induction and at least one course of intensification. Table 1⇓ summarizes the chemotherapy doses and schedule of the regimen.

PEG-ASNase and Amino Acid Analyses.

Blood samples were collected on days 0, 7, 14, 21, and 28 of induction and days 0, 7, 14, 21, and 28 of intensification. CSF samples were collected on induction days 0, 14, and 28 (and days 7 and 21 for patients with CNS relapse at study entry) and on intensification days 0 and 21 (and on days 7 and 14 for patients without CNS relapse at study entry). Blood and CSF samples were placed immediately in an ice-water bath. No inhibitors of ASNase enzymatic activity were added to the collection tubes. Blood was allowed to coagulate under these conditions. Serum was decanted from coagulated blood after refrigerated centrifugation. Both serum and CSF samples were stored at −70 C. A specimen transmittal form was used to record date and dose of PEG-ASNase and specimen collection time. Stored specimens were shipped to the reference laboratory by overnight mail on dry ice.

PEG-ASNase activity was measured by ammonia produced from Asn with a Nessler reaction. Reacted enzymatic activity solutions were placed in an ELISA plate, and the ELISA plate reader was used to read absorbance, calculate calibration line, and quantify specimens (23 , 24) .

PEG-ASNase antibody was assayed by a modified indirect solid-phase ELISA (23 , 24) . The assay was done with a computer-controlled instrument from Dynatech Laboratories, Inc. Sera from patients who had high-titer antibodies PEG-ASNase was used to create a titration curve. The titers were compared with the pretreatment control serum from the same patient and negative control serum from a healthy volunteer. The assay had excellent linearity, reproducibility, and low detection limits, but the absorbance of control varied between assays. Day-to-day variation was corrected by expression of antibody titers as the ratio of sample over negative control for each assay. A positive antibody titer was defined as a ratio of serum antibody to the average control value of ≥1.5, because this ratio correlated with neutralization or rapid clearance of ASNase in our previous study (24) .

Asn and Gln were assayed by modification of a reported high-performance liquid chromatography method in which amino acids are derivatized with phenylisothiocyanate (25) . The derivatized samples were analyzed using a μC18 column by two-step gradient elution with a flow rate of 0.8 ml/min. The first step was composed of a 25-min linear gradient from 100% buffer A [0.05 M ammonium acetate (pH 6.8)] to 90% A and 10% of buffer B [0.1 M ammonium acetate (pH 6.8) and acetonitrile 50:50 ]. The second step consisted of a 10-min gradient from 90% A + 10% buffer B to 0% A + 100% B. Absorbance was followed at 254 nm. Calibration curves were used to quantify amino acid concentrations. The lowest limit of detection from the linear portion of the calibration lines was 0.01 μm for Asn and 1 μm for Gln (24) .

Statistical Considerations.

The principal goals of this study were to measure PEG-ASNase activity, serum Asn depletion, and CSF Asn depletion during induction and intensification. Because serum and CSF sampling were incomplete from all of the study subjects, comparisons of Asn concentrations were made by two-sided, two sample (rather than matched pair) t test assuming unequal variances using SPSS version 10.0 statistical package. The incidence of PEG-ASNase-associated toxicities, including allergy, pancreatitis, and thrombosis, were reported descriptively.

Clinical Features of Patients.

Table 2⇓ outlines the clinical features of patients at time of original diagnosis and time of relapse. Although 30 patients enrolled on the therapeutic study, serum samples were obtained from 28 patients, and only these patients were analyzed for this report. Seventeen patients had bone marrow relapse (3 combined with CNS and 2 with testicle), and 11 had isolated extramedullary relapse (8 in CNS and 3 in other sites). All of the patients were treated previously with ASNase: 22 with native E. coli ASNase only, 3 with PEG-ASNase only, and 3 with both. All of the patients were either enrolled on Children’s Cancer Group trials or institutional standard therapy based upon Children’s Cancer Group trials. All of the patients received ASNase during at least two initial therapy phases: induction and delayed intensification. Twelve patients received ASNase during three initial therapy phases: induction and two delayed intensification courses. Two patients received ASNase during five initial therapy phases: induction, two interim maintenance courses, and two delayed intensification courses. Four had experienced hypersensitivity reactions to native E. coli ASNase. Details regarding the efficacy and toxicity of the therapeutic regimen will be reported separately. In brief, 86% (24 of 28) patients achieved a remission, 7% (2 of 28) patients died from infection during induction, and 7% (2 of 28) failed to achieve a remission despite receiving induction and intensification. Twenty-five of the initial 28 patients received intensification; reasons for not receiving intensification included death during induction (n = 2) and excessive toxicity during induction (n = 1).

ASNase Toxicity and Modifications of Therapy.

During induction, 2 patients had adverse reactions to PEG-ASNase: CNS venous thrombosis (n = 1) and pancreatitis (n = 1). Seven patients had modifications of PEG-ASNase dosing during induction: 5 did not receive PEG-ASNase on day 23 due to severe infection (n = 4) or death due to infection before day 23 (n = 1), 1 patient did not receive PEG-ASNase on days 9, 16, or 23 due to pancreatitis after the initial dose, and 1 patient received native E. coli ASNase due to commercial unavailability of PEG-ASNase (no serum or CSF samples were obtained from this patient during induction). Twenty-two of the 25 patients who received intensification also received PEG-ASNase as scheduled on day 7 of intensification; PEG-ASNase was omitted due to thrombosis (n = 1) or pancreatitis (n = 1) during induction or commercial unavailability of PEG-ASNase (n = 1). During intensification, 3 additional patients had adverse reactions to PEG-ASNase, including hypersensitivity (n = 2) and pancreatitis (n = 1). The incidence of clinical hypersensitivity to PEG-ASNase was 0% during induction and 8% during intensification. All of the cases of PEG-ASNase-associated toxicity resolved spontaneously and without sequelae.

Pharmacokinetics of PEG-ASNase.

Fig. 1⇓ and Table 3⇓ show the median serum PEG-ASNase activity over time. During induction, median PEG-ASNase activity 5 days after the prior dose was 0.8–1.2 IU/ml. Therapeutic serum PEG-ASNase activity, defined as either > 0.03 IU/ml or > 0.1 IU/ml, was maintained throughout induction at 91–100% of patients at each time point. Only 2 patients did not maintain therapeutic serum PEG-ASNase activity levels (> 0.1 IU/ml) throughout induction. The first patient (treated with native E. coli ASNase during initial therapy without clinical hypersensitivity) did not receive PEG-ASNase on day 23, had no detectable serum PEG-ASNase activity on day 28, and developed hypersensitivity to PEG-ASNase during intensification. This patient did not have PEG-ASNase antibody. The second patient (treated with native E. coli ASNase during initial therapy without clinical hypersensitivity) had undetectable serum PEG-ASNase activity throughout induction, never achieved Asn depletion, had detectable serum PEG-ASNase antibody, and was 1 of only 2 patients with refractory leukemia.

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Fig. 1.

Box and whisker plot of serum PEG-ASNase activity during induction and intensification. Center of box represents median, box encloses 50% of values, and whiskers enclose all values excluding outliers. PEG-ASNase was given on days 2, 9, 16, and 23 induction and day 7 of intensification.

The median interval between induction day 23 PEGASNase dose and the start of intensification was 15 days (range, 7–26 days). Intensification did not start until recovery of blood counts, except in the case of persistent leukemia. Residual therapeutic PEG-ASNase activity (before the scheduled day 7 of intensification dose of PEG-ASNase) persisted in 43% and 30% of patients on days 0 and 7 of intensification, respectively. After a single dose of PEG-ASNase on day 7 of intensification, most patients maintained therapeutic PEG-ASNase activity on days 14 and 21 (87–100% were >0.03 IU/ml, and 80–94% were >0.1 IU/ml). The median PEG-ASNase activity was lower during intensification compared with during induction, reflecting less frequent dosing (Fig. 1)⇓ .

Antibodies to PEG-ASNase.

Table 3⇓ shows the incidence of PEG-ASNase antibody during induction and intensification, defined as a PEG-ASNase antibody ratio ≥1.5. During induction and intensification, immunological reaction to PEG-ASNase was uncommon. Serum PEG-ASNase antibody was only observed in 2 patients during induction and in 1 additional patient during intensification (combined incidence, 11%). The 3 patients who developed PEG-ASNase antibody had received only two courses of ASNase during their initial treatment for acute lymphoblastic leukemia. Neither of the 2 patients with clinical hypersensitivity had a positive PEG-ASNase antibody ratio.

Serum and CSF Asn and Gln Concentrations.

Table 3⇓ and Fig. 2⇓ show the median serum Asn concentration over time. Serum Asn was reduced significantly from day 0 of Induction compared with days 7, 14, 21, and 28 during induction and days 0, 7, 14, 21, and 28 during intensification (P ≤ 0.002 for all comparisons). Serum Asn levels were <3 μm in 47–67% and 47–70% of patients in each time point sampled during induction and intensification, respectively. Table 3⇓ also shows Gln depletion, defined as serum levels <100 μm, was also observed at 42–56% and 27–55% of patients in each time point sampled during induction and intensification, respectively.

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Fig. 2.

Box and whisker plot of serum asparagine concentration during induction and intensification. Center of box represents median, box encloses 50% of values, and whiskers enclose all values excluding outliers.

Table 3⇓ and Fig. 3⇓ show the median CSF Asn concentration over time. Median CSF Asn was progressively lower during induction. The frequency of CSF Asn depletion <3 μm also increased from 60% at day 7 to 75% on days 21 and 28 of induction. CSF Asn depletion <3 μm was maintained in 62–90% of patients at each time point sampled during intensification. Table 3⇓ also shows that CSF Gln did not decrease in any patient.

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Fig. 3.

Box and whisker plot of CSF asparagine concentration during induction and intensification. Center of box represents median, box encloses 50% of values, and whiskers enclose all values excluding outliers.