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© 1999 American Society for Clinical Oncology Cerebrospinal Fluid Asparagine Concentrations After Escherichia coli Asparaginase in Children With Acute Lymphoblastic LeukemiaFrom the Departments of Pharmaceutical Sciences, Hematology-Oncology, and Biostatistics and Epidemiology, St Jude Children's Research Hospital, and Colleges of Pharmacy and Medicine, University of Tennessee, Memphis, TN. Address reprint requests to Mary V. Relling, PharmD, Pharmaceutical Department, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105; email mary.relling{at}stjude.org
PURPOSE: The CNS is an important sanctuary site in childhood acute lymphoblastic leukemia (ALL). CSF asparagine concentration reflects asparaginase systemic pharmacodynamics. We evaluated the time course of CSF asparagine depletion in children with ALL during and after a course of Escherichia coli asparaginase. PATIENTS AND METHODS: Thirty-one children (24 newly diagnosed and seven at relapse) received E coli asparaginase 10,000 IU/m2 intramuscularly three times weekly for six and nine doses, respectively, as part of multiagent induction chemotherapy. CSF asparagine levels were measured before, during, and after asparaginase dosing. RESULTS: The percentage of patients with undetectable (< 0.04 µmol/L) CSF asparagine was 3.2% (one of 31 patients) at baseline, 73.9% (17 of 23) during asparaginase therapy, and 56.3% (nine of 16) 1 to 5 days, 43.8% (seven of 16) 6 to 10 days, 20.0% (two of 10) 11 to 30 days and 0% (zero of 21) more than 30 days after asparaginase therapy. The proportion of patients with depleted CSF asparagine was higher during asparaginase therapy than at baseline (P < .001), 11 to 30 days (P = .003), and more than 30 days after asparaginase therapy (P < .001). Median CSF asparagine concentrations were 4.42 µmol/L before, less than 0.04 µmol/L during, and less than 0.04 µmol/L at 1 to 5 days, 1.63 µmol/L at 6 to 10 days, 1.70 µmol/L at 11 to 30 days, and 5.70 µmol/L at more than 30 days after asparaginase therapy, respectively. CSF depletion was more common in patients with low baseline CSF asparagine concentrations (P = .003). CONCLUSION: CSF asparagine concentrations are depleted by conventional doses of E coli asparaginase in the majority of patients, but they rebound once asparaginase therapy is completed.
ASPARAGINASE IS an effective chemotherapeutic agent in childhood acute lymphoblastic leukemia (ALL) and is a component of most multiagent remission induction regimens for both newly diagnosed and relapsed ALL.1 Asparaginase hydrolyzes asparagine to aspartic acid and ammonia in the extracellular space. Most normal cells are able to synthesize asparagine, whereas some leukemic cells have decreased asparagine synthetase and thus require exogenous asparagine.2 Asparagine-dependent protein synthesis is halted with subsequent inhibition of nucleic acid synthesis. Leukemic cell cytotoxicity occurs in vitro when asparagine is depleted for at least 4 days.3 There are different preparations of asparaginase from various natural sources with different pharmacologic properties: Escherichia coli, Erwinia chrysanthemi, and E coli asparaginase covalently bound to polyethylene glycol. The elimination half-life after Erwinia asparaginase (0.65 ± 0.13 days) is significantly shorter than that for an E coli preparation (1.24 ± 0.17 days), but both native preparations have shorter half-lives than polyethylene glycol asparaginase (5.73 ± 3.24 days).4 Moreover, markedly different pharmacokinetic properties were observed with two different manufacturer's formulations of E coli asparaginase.5 CSF asparagine depletion has been proposed as an informative pharmacodynamic indicator of asparaginase effect. Asparagine depletion in plasma has also been reported after asparaginase therapy,5-11 but such measurements are complicated by the possibility of ex vivo hydrolysis of asparagine by circulating plasma asparaginase. Because asparaginase does not penetrate into the CNS, asparagine in CSF is protected from such ex vivo hydrolysis. Pilot data indicated detection of CSF asparagine was related to plasma asparaginase levels.12 Depletion of CSF asparagine after Erwinia asparaginase therapy has been demonstrated,13 and asparagine levels in CSF correlated with those in carefully processed plasma.6 Additionally, the CNS is an important therapeutic target in childhood ALL, and thus CSF asparagine depletion may play a role in the treatment or prevention of meningeal leukemia. The course of CSF asparagine depletion over time after standard doses of three-times-weekly intramuscular E coli asparaginase (Elspar; Merck & Co, Inc, West Point, PA), the most common formulation used in the United States, has not been well characterized. We determined the time course of asparagine concentrations in the CSF of children with newly diagnosed and relapsed ALL during and after a course of E coli asparaginase therapy.
Patient Eligibility and Treatment Schedules Serial CSF samples were obtained from 24 children with newly diagnosed ALL and seven with relapsed ALL who were enrolled onto the St Jude Children's Research Hospital protocols Total XIIIB and R15, respectively. Of the seven patients with relapsed ALL, five had hematologic relapse and two had combined hematologic and testicular relapse. During remission induction therapy in the Total XIIIB protocol, all children received E coli asparaginase 10,000 IU/m2 intramuscularly on days 2, 4, 6, 8, 10, and 12; prednisone 40 mg/m2/d orally on days 1 to 28; vincristine 1.5 mg/m2 intravenously on days 1, 8, 15, and 22; daunorubicin 25 mg/m2 intravenously on days 1 and 8; and etoposide 300 mg/m2 plus cytarabine 300 mg/m2 intravenously on days 22, 25, and 29. Patients enrolled onto the R15 protocol received asparaginase at the same dose on days 8, 10, 12, 15, 17, 19, 22, 24, and 26; oral etoposide 50 mg/m2 daily on days 1 to 22; dexamethasone 8 mg/m2/d orally on days 8 to 35; and vincristine 1.5 mg/m2 intravenously on days 8, 15, 22, and 29. Serum albumin was measured on day 29. Informed consent was obtained from patients' parents according to institutional review board guidelines.
Sample Collection
Determination of CSF Asparagine Concentrations
Statistical Analysis
Patient characteristics are listed in Table 1. All patients achieved complete remission after induction (Total XIIIB) or reinduction (R15) therapy, except one patient with leukemia in relapse who died during reinduction. Otherwise, all scheduled doses of asparaginase were administered without any modifications; the only exception was one newly diagnosed patient who had an allergic reaction to the second E coli asparaginase dose (generalized erythema), and thus completed the remainder of asparaginase treatment with the Erwinia preparation.
Figure 1 shows the proportion of patients who had levels of asparagine less than the detection limit (ie, < 0.04 µmol/L). The proportion of newly diagnosed ALL patients with CSF asparagine depletion did not differ significantly over time from that of relapsed patients (P = .195). Thus, all patients were pooled for analysis. The proportion of patients with undetectable CSF asparagine levels was significantly greater during asparaginase therapy than at baseline (P < .001), at 11 to 30 days (P = .003), and more than 30 days after the last dose of asparaginase (P < .001). Once asparaginase therapy was completed, the proportion of patients with undetectable levels consistently decreased. Five of 10 patients with CNS status 1 (no identifiable blasts in CSF), 14 of 18 patients with CNS status 2 (WBC count < 5/µL with identifiable blasts in CSF), and two of three patients with CNS status 3 (WBC count
Figure 2 depicts the distribution of CSF asparagine levels with respect to asparaginase dosing. The median CSF asparagine concentration before therapy was 4.42 µmol/L (normal range, 3.7 to 8.3 µmol/L).14 This declined to less than 0.04 µmol/L during and 1 to 5 days after asparaginase therapy. Subsequently, median CSF asparagine concentrations increased to 1.63 µmol/L at 6 to 10 days, 1.70 µmol/L at 11 to 30 days, and 5.70 µmol/L at 30 days or more after the last dose of asparaginase. The distribution of CSF asparagine concentrations during asparaginase treatment differed significantly from those at baseline and from those measured more than 30 days after asparaginase (both P < .001).
None of the patients in this study developed clinical pancreatitis, severe hepatic dysfunction, or thrombotic events. Three patients developed transient hyperglycemia during asparaginase treatment that required insulin therapy. All three had CSF asparagine concentrations less than the limit of detection, two during asparaginase therapy and the other within 10 days after asparaginase therapy. Although the numbers are small, there was no difference in the frequency of CSF asparagine depletion among patients who required insulin therapy and those who did not (P = .53). CNS status, WBC count, and CSF WBC count at diagnosis as well as serum albumin at day 29 were not significantly different between patients who developed undetectable CSF asparagine levels and those whose CSF asparagine concentrations remained greater than 0.04 µmol/L. Only the baseline CSF asparagine concentration was significantly associated with whether CSF asparagine became depleted at some point after baseline; the median baseline CSF asparagine concentration was 3.9 µmol/L (range, < 0.04 to 22.2 µmol/L) for patients who had CSF asparagine depletion and 6.6 µmol/L (range, 3.1 to 13.5 µmol/L) for those who never had levels less than 0.04 µmol/L (P = .003). Sixty-three percent of patients on Total XIIIB and 86% of patients on R15 experienced CSF asparagine depletion at some point after baseline. One patient with newly diagnosed ALL had an allergic reaction to E coli asparaginase while on Total XIIIB and had undetectable CSF asparagine concentrations during and 1 to 5 days after the last asparaginase dose, although this patient had a low CSF asparagine level at baseline (0.65 µmol/L). Four patients with relapsed ALL had previous allergic reactions to E coli asparaginase—local pruritis (n = 1), generalized pruritis and hives (n = 2), and local pain and swelling in addition to generalized pain (n = 1)—occurring a median of 2.9 years (range, 2.6 to 8.8 years) before relapse. All four patients received the E coli preparation on the R15 protocol with diphenhydramine premedication; none experienced allergic reactions while receiving R15 therapy and all completed asparaginase therapy as directed. All four patients had undetectable CSF asparagine concentrations: two during asparaginase therapy and two during and 6 to 10 days after the last asparaginase dose. The median baseline CSF asparagine level for these four patients was 3.23 µmol/L (range, 0.98 to 5.94 µmol/L). As expected, levels of glutamine in the CSF were within the normal range at the time of diagnosis or relapse (350 to 680 µmol/L) and did not change significantly throughout the treatment course (P = .5).
This is the first kinetic analysis of CSF asparagine concentrations during conventional remission induction treatment with three-times-weekly intramuscular E coli asparaginase (Elspar), the most commonly used preparation in the United States, in patients with newly diagnosed or relapsed ALL. Because the cytotoxic effect of asparaginase is related to depletion of asparagine,3 estimates of the extent and duration of asparagine depletion in vivo may be important pharmacodynamic surrogates of asparaginase effect. Using E coli asparaginase, we found that CSF asparagine levels were depleted in the majority of patients during and 1 to 5 days after asparaginase therapy was completed. The proportion of patients with asparagine depletion we observed (74%) during and for the first 5 days after E coli asparaginase is similar to that achieved with the Erwinia preparation (70% of patients).13 The similarity is somewhat surprising because E coli asparaginase has a longer half-life and maintains active asparaginase plasma levels for a longer period of time than does Erwinia asparaginase at the same doses.4 In fact, we administered the E coli compound at a slightly more intense schedule (three times a week for either six or nine doses) than the study using Erwinia asparaginase (every 3 days for a total of eight doses). However, it should be noted that the assay results across laboratories may not be directly comparable because our baseline CSF asparagine levels were lower than those reported by some investigators,13 although comparable to others.14 Boos et al5 detected a more prolonged asparagine depletion in plasma with E coli asparaginase compared with that from Erwinia, although they used a different E coli preparation than we did. We did find that 20% of patients had CSF asparagine levels less than 0.04 µmol/L at 11 to 30 days after the last dose of E coli asparaginase compared with 5.6% of patients studied 19 days after Erwinia asparaginase,13 which suggests that there may be a more pronounced depletion of CSF asparagine at later time points after three-times-weekly E coli asparaginase therapy than every-third-day Erwinia therapy. Nonetheless, a controlled comparison of CSF depletion after different preparations would be helpful. Baseline CSF asparagine concentrations were lower among patients with depleted levels some point after baseline than in those who never developed CSF asparagine levels less than 0.04 µmol/L. More than 75% of patients who had an undetectable CSF asparagine concentration at some point after baseline had a baseline level less than 5 µmol/L, whereas only 10% of patients who never achieved complete CSF asparagine depletion had a baseline CSF less than this level. Analysis of CSF asparagine concentrations before a course of asparaginase may be of clinical value in identifying a subset of patients who are refractory to the pharmacologic effect of conventional asparaginase dosing. Interestingly, one of the 31 patients had an undetectable baseline CSF asparagine level, which has not been previously observed in children with ALL.13 Although the reason for this is unclear, undetectable CSF asparagine concentrations have also been reported in children with bacterial meningitis.15 Our patient had CNS status 1 (absence of blasts in the CSF) and undetectable CSF asparagine up to 30 days after asparaginase treatment.
It has been suggested that there may be loss of pharmacologic effect with repeated applications of either Erwinia or E coli preparations.5-7 For example, depletion of plasma asparagine was reported to be more common6 or more pronounced6,7 when assessed after Erwinia asparaginase given during induction (first exposure) compared with the level of depletion during reinduction (second exposure).7 Another study showed a difference in the potency (eg, decrease of plasma asparagine level) of E coli asparaginase from two different manufacturers.5 Both Asparaginase medac (Medac GmbH, Hamburg, Germany; produced by Kyowa Hakko, Kogyo, Japan) and Crasnitin (Bayer AG, Leverkusen, Germany) depleted asparagine plasma levels ( CSF measurements were also made in a single patient on the R15 protocol whose data were not included in these analyses because of treatment with only Erwinia asparaginase. The patient had a history of prior allergy to E coli asparaginase, manifested as local pain, erythema, and swelling, approximately 2.5 years before diagnosis of relapse. The patient subsequently received the Erwinia preparation to complete initial remission reinduction therapy. While on R15, the patient received a complete course of Erwinia asparaginase (nine doses), as directed by the protocol, without experiencing any reaction. CSF asparagine was never depleted, even during asparaginase therapy, with an asparagine concentration of 1.51 µmol/L, compared with a median of less than 0.04 µmol/L in the other seven patients with relapsed ALL at the same time point. Moreover, this patient had a higher CSF asparagine level more than 30 days after the last dose of asparaginase (36.40 µmol/L) than anyone we studied after E coli asparaginase therapy. In vitro studies have indicated that a minimum of 4 days of asparagine depletion is necessary for optimal leukemic cell kill.3 One might then argue that in the majority of patients, asparagine depletion for a 19-day period is sufficient for efficacy. However, clinical data obtained in a prior study of E coli asparaginase suggested an improved response rate for ALL treated with 12,000 IU/m2 compared with lower doses given three times weekly,16 with 63% responding at 12,000 IU/m2 compared with 54% at 6,000 IU/m2 three times weekly. Thus, it is possible that efficacy could be enhanced by intensifying asparaginase dosing further beyond the 10,000 IU/m2 three times weekly that we have used, although the majority of patients exhibited prolonged asparagine depletion. Consistent with this notion is that Erwinia asparaginase, with its shorter half-life, was associated with inferior event-free survival compared with E coli asparaginase treatment.17 It is unclear whether the effects of treatment with asparaginase (ie, asparagine depletion) will have any prognostic significance. The dosage and schedule for the administration of asparaginase in induction therapy can be easily monitored by measuring asparagine concentrations in the CSF, and baseline CSF asparagine levels may predict future CSF asparagine depletion. Further studies are needed to assess whether asparagine depletion is associated with event-free survival, which would provide direction for asparaginase treatment scheduling and dosing.
Supported by the National Institutes of Health, Bethesda, MD, Cancer Center Support grant no. CA-20180, and grants no. CA-21765 and CA-51001; a Center of Excellence Grant from the State of Tennessee; a grant from Rhône-Poulenc Rorer Pharmaceuticals, Inc, Collegeville, PA; American Society of Health-System Pharmacists Foundation Fellowship in Immunology/Immunotherapy; and American Lebanese Syrian Associated Charities. We thank our nurses, Sheri Ring, Margaret Edwards, Terri Kuehner, and Lisa Walters, and Amy Atkinson, Jean Cai, Ken Cox, Ya Qin Chu, Krystal Effinger, Sherree Johns, Natasha Lenchik, and Yi Su for their excellent technical assistance.
1. Jones B, Holland JF, Glidewell O, et al: Optimal use of L-asparaginase (NSC-109229) in acute lymphocytic leukemia. Med Pediatr Oncol 3:387-400, 1977[Medline] 2. Capizzi RL, Bertino JR, Skeel RT, et al: L-Asparaginase: Clinical, biochemical, pharmacological, and immunological studies. Ann Intern Med 74:893-901, 1971
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Asselin BL, Ryan D, Frantz CN, et al: In vitro and in vivo killing of acute lymphoblastic leukemia cells by L-asparaginase. Cancer Res 49:4363-4368, 1989
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Asselin BL, Whitin JC, Coppola DJ, et al: Comparative pharmacokinetic studies of three asparaginase preparations. J Clin Oncol 11:1780-1786, 1993 5. Boos J, Werber G, Ahlke E, et al: Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations. Eur J Cancer 32A:1544-1550, 1996
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Gentili D, Conter V, Rizzari C, et al: L-Asparagine depletion in plasma and cerebro-spinal fluid of children with acute lymphoblastic leukemia during subsequent exposures to Erwinia L-asparaginase. Ann Oncol 7:725-730, 1996
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Boos J, Nowak-Göttl U, Jürgens H et al: Loss of activity of Erwinia asparaginase on repeat applications. J Clin Oncol 13:2474-2475, 1995 (letter)
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Miller HK, Salser JS, Balis ME: Amino acid levels following L-asparagine amidohydrolase (EC.3.5.1.1) therapy. Cancer Res 29:183-187, 1969 9. Ahlke E, Nowak-Göttl U, Schulze-Westhoff P, et al: Dose reduction of asparaginase under pharmacokinetic and pharmacodynamic control during induction therapy in children with acute lymphoblastic leukemia. Br J Haematol 96:675-681, 1997[Medline] 10. Gentili D, Zucchetti M, Conter V, et al: Determination of L-asparagine in biological samples in the presence of L-asparaginase. J Chromatogr B Biomed Sci Appl 657:47-52, 1994
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Asselin BL, Lorenson MY, Whitin JC, et al: Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor. Cancer Res 51:6568-6573, 1991 12. Riccardi R, Holcenberg JS, Glaubiger DL, et al: L-Asparaginase pharmacokinetics and asparagine levels in cerebrospinal fluid of rhesus monkeys and humans. Cancer Res 41:4554-4558, 1981[Medline]
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Dibenedetto SP, Di Cataldo A, Ragusa R, et al: Levels of L-asparagine in CSF after intramuscular administration of asparaginase from Erwinia in children with acute lymphoblastic leukemia. J Clin Oncol 13:339-344, 1995
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Cooney DA, Capizzi RL, Handschumacher RE: Evaluation of L-asparagine metabolism in animals and man. Cancer Res 30:929-935, 1970 15. Heiblim DI, Evans HE, Glass L, et al: Amino acid concentrations in cerebrospinal fluid. Arch Neurol 35:765-768, 1978[Medline]
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Ertel IJ, Nesbit ME, Hammond D, et al: Effective dose of L-asparaginase for induction of remission in previously treated children with acute lymphocytic leukemia: A report from Childrens Cancer Study Group. Cancer Res 39:3893-3896, 1979 17. Conter V, Schrappe M, Aricó M, et al: Role of cranial radiotherapy for childhood T-cell acute lymphoblastic leukemia with high WBC count and good response to prednisone: Associazione Italiana Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster groups. J Clin Oncol 15:2786-2791, 1997[Abstract] Submitted November 9, 1998; accepted January 14, 1999. This article has been cited by other articles:
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test and Fisher's exact test were used to determine whether CNS status at diagnosis and/or the requirement of insulin during asparaginase therapy differed between patients whose CSF asparagine concentrations did and did not become depleted at any time after baseline, respectively. Bonferroni's correction was applied to comparisons that involved CSF asparagine concentrations during asparaginase treatment against other time periods; therefore, such comparisons were considered significant at a type I error rate of alpha = 0.05/5 = 0.01. All other comparisons were considered significant at a type I error rate of alpha = 0.05. All P values reported are two-sided, and all statistical analyses were conducted using SAS Release 6.12 (SAS Institute, Inc, Cary, NC) and StatXact Version 3.0.2 (Cytel Software Corp, Cambridge, MA). 
5/µL with identifiable blasts in CSF) had CSF asparagine levels lower than the detection limit (P = .387). 
). Lower and upper ends of boxes represent the 25% and 75% quartiles, respectively.
0.5 µmol/L) in 99.4% and 100% of samples, respectively, during initial induction treatment; however, during reinduction treatment, 100% of Asparaginase medac samples but only 66.6% of Crasnitin samples exhibited complete asparagine depletion. In contrast, we observed no significant decrease in the proportion of patients with asparagine depletion in CSF after E coli asparaginase when studied at relapse (six of seven; 86%) compared with newly diagnosed patients (15 of 24; 63%). However, patients on our relapse protocol did receive more doses of E coli asparaginase (nine doses) than did newly diagnosed patients (six doses), which could have accounted for this finding. All four patients with both relapsed ALL and a previous allergic reaction to E coli asparaginase had undetectable asparagine levels during treatment; however, all four patients received retrieval therapy at least 2.5 years after initial asparaginase treatment. Moreover, three of these four patients had baseline CSF asparagine levels less than 5 µmol/L, thereby predisposing them to depletion. 
