Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Therapy

Pharmacokinetic, pharmacodynamic and intracellular effects of PEG-asparaginase in newly diagnosed childhood acute lymphoblastic leukemia: results from a single agent window study

Leukemia volume 22, pages 1665–1679 (2008)Cite this article

Abstract

L-asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is thought to result from depletion of asparagine in serum and cells. We investigated the clinical response in vivo of 1000 IU/m2 pegylated (PEG)-asparaginase and its pharmacokinetic, pharmacodynamic and intracellular effects in children with newly diagnosed ALL before start of combination chemotherapy. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common/pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (P=0.01) and BCRABL-positive ALL (P=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-asparaginase (P=0.02) and both were prognostic factors for long-term event-free survival (hazard ratio 6.4, P=0.004; hazard ratio 3.7, P=0.01). After administration of one in vivo dose of PEG-asparaginase no changes in apoptotic parameters or in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. One additional dose of PEG-asparaginase upfront ALL treatment did not lead to other severe toxicities.

Download PDF

Introduction

L-asparaginase is an effective drug to treat newly diagnosed acute lymphoblastic leukemia (ALL).1 Prolonged L-asparaginase intensification significantly improved the outcome of ALL patients as was demonstrated in the Dana–Farber Cancer Institute ALL Consortium Protocol 91-01.2 Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL.3, 4, 5, 6, 7, 8 Also a poor early in vivo response to L-asparaginase as a single drug has been linked to an unfavorable outcome in pediatric ALL.9

The administration of L-asparaginase results in the deamination of asparagine into aspartic acid leading to a rapid and complete depletion of serum asparagine, which ultimately affects the intracellular asparagine levels.10, 11 L-asparaginase also has 3–4% glutaminase activity leading to serum glutamine depletion.12 L-asparaginase enzymatic activity should be >100 IU/l to sufficiently diminish the asparagine serum levels required to induce leukemic cell kill.13 Amino-acid deficiency impairs protein synthesis and leads to apoptosis and cell death.14

In vitro studies demonstrated that cellular deprivation of asparagine and glutamine leads to increased levels of asparagine synthetase.15 This enzyme opposes the action of L-asparaginase and, thereby can rescue cells from the effect of L-asparaginase.16 Human leukemia cell line studies suggested that only L-asparaginase-resistant cells upregulate the activity of asparagine synthetase.15 However, we recently demonstrated that upregulation of asparagine synthetase mRNA occurs in both sensitive and in resistant cases within 24 h of in vivo exposure to L-asparaginase.17 These data imply that mechanisms other than increased expression levels of asparagine synthetase contribute to cellular L-asparaginase resistance.

Recently, gene expression profiling studies by microarray analysis revealed that leukemic cells of L-asparaginase-resistant ALL patients express higher levels of genes involved in protein synthesis than L-asparaginase-sensitive cells.18 A deficiency in amino acids finally induces apoptosis in malignant cells. L-asparaginase activates caspase 3 and inactivates poly-ADP-ribose-polymerase (PARP) in patients leukemic cells and resistance to L-asparaginase is linked to an impaired capacity of cells to trigger the apoptotic pathway.19 Hypothetically, an altered intracellular amino-acid composition might rescue cells from the effects of L-asparaginase. It is yet unknown whether the amino-acid metabolism of L-asparaginase differs between resistant and sensitive leukemic cells of patients.

In the present study we investigated pharmacokinetic and pharmacodynamic as well as intracellular effects of one in vivo dose of pegylated-L-asparaginase (PEG-asparaginase) in children with newly diagnosed ALL. In vivo response to PEG-asparaginase was monitored by analyzing the decrease in leukemic cells during a therapeutic window of 5 days before start of combination chemotherapy. In vivo response to PEG-asparaginase was compared with the baseline as well as PEG-asparaginase-induced changes in serum and intracellular amino-acid levels and with parameters of apoptosis. In vitro resistance to L-asparaginase was determined and compared to the mentioned factors that might influence drug resistance. In addition, the influence of PEG-asparaginase given upfront regular antileukemic treatment was evaluated on the incidence of allergic reactions during treatment and changes in hemostasis.

Methods

Patients and treatment

In close collaboration between our institution and the Dutch Childhood Oncology Group (DCOG), a window study with PEG-asparaginase upfront to the ALL-9 treatment schedule was initiated in July 2000. The ALL-9 protocol was based on the ALL-6 treatment strategy of the DCOG.20 Children with newly diagnosed ALL and presenting white blood count (WBC)>10 × 109 per liter were eligible for the window study with PEG-asparaginase. Patients received a single dose of 1000 IU/m2 PEG-asparaginase in an 1-h infusion 5 days before starting combination chemotherapy.21 PEG-asparaginase (Oncaspar), kindly provided by Medac (GmbH, Hamburg, Germany), was used because of its lower immunogenicity than native (unpegylated) L-asparaginase,22 which was important as native E. coli L-asparaginase (Paronal, four doses of 6000 IU/m2 on days 29, 33, 36 and 40) was used as part of the regular combination chemotherapy given after the investigational window. After the PEG-asparaginase window, patients were divided into non-high-risk and high-risk criteria. Non-high-risk ALL was defined as WBC<50 × 109 per liter, no mediastinal mass, absence of t(9;22), t(4;11) or other mixed lineage leukemia (MLL) rearrangements, no T-cell phenotype and no central nervous system or testicular involvement. All other leukemias were defined as high risk. All children received the combination induction therapy of dexamethasone, vincristine and intrathecal therapy during the first 4 weeks, and additional daunorubicin only in case of high-risk ALL.

The window study with PEG-asparaginase was approved by the local ethical committee and by the institutional research board of the DCOG. The patient and/or the parents/guardians gave informed consent for this study.

The immunophenotyping and cytogenetic characterizations were performed at the central reference laboratory of the DCOG and at laboratories of the participating centers. B-lineage ALL (CD19+, HLA-DR+) was classified into the following differentiation stages: proB-ALL cells were CD10−, cytoplasmic μ chain− (cμ−) and surface immunoglobulin− (sIg−); c-ALL cells were CD10+/cμ−/sIg−; pre-B cells were CD10+or−/cμ+/sIg−. B-ALL cells characterized by CD10−/cμ−/sIg+ were excluded from the study. Cytogenetic analyses were performed by regular karyotyping and fluorescence in situ hybridization.23

Patient samples

Bone marrow and peripheral blood samples were obtained at initial diagnosis of ALL before the administration of PEG-asparaginase. Serum and leukemic cells from peripheral blood were collected 1 h after the end of the infusion with PEG-asparaginase and thereafter daily during 5 consecutive days till the start of combination chemotherapy at day 0. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep (density 1.077 g/ml; Lucron bioproducts, Gennep, the Netherlands) as described before.17 Contaminating nonleukemic cells were removed by immunomagnetic beads as described earlier.24 All samples contained over 90% of leukemic cells, as determined morphologically on May-Grünwald-Giemsa (Merck, Darmstadt, Germany) stained cytospins.

Clinical response

In correspondence with the definition of the window response to prednison in ALL25 the clinical window response to PEG-asparaginase on day 0 (5 days after the PEG-asparaginase infusion) was defined as good when the number of leukemic cells was <1 × 109 per liter of peripheral blood, as intermediate when leukemic cells were 1–10 × 109 per liter, and as poor when leukemic cells were >10 × 109 per liter.

In vitro cytotoxicity

In vitro L-asparaginase cytotoxicity in leukemic cells taken at initial diagnosis (untreated) was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were exposed to six different concentrations of L-asparaginase (Paronal; Christiaens BV, Breda, the Netherlands) ranging from 0.0032 to 10 IU/ml in duplicate for 4 days. Control cells were cultured without L-asparaginase. After 4 days of incubation at 37 °C in humidified air containing 5% CO2, the MTT assay was performed. Drug sensitivity was assessed by the LC50, the drug concentration lethal to 50% of the cells. Evaluable assay results were obtained when a minimum of 70% of leukemic cells was present in the control wells after 4 days of incubation and when the control OD was ⩾0.050.8

In vitro sensitivity toward L-asparaginase was defined as LC50 ⩽0.033 IU/ml, in vitro resistance toward L-asparaginase was defined as LC50 ⩾0.912 IU/ml, and intermediate sensitive was defined as LC50 0.033–0.912 IU/ml.8

As LC50 values were highly correlated between Paronal and L-asparaginase Medac (Rs 0.93, P<0.002) and between Paronal and PEG-asparaginase (Oncaspar) (Rs 0.86, P<0.002) all consecutive MTT assays were performed using Paronal.3, 8

Apoptotic features

Determination of Annexin V, DIOC6, cleaved-caspase-3 and cleaved PARP were performed by fluorescence-activated cell sorting analysis as described earlier.19

For ex vivo exposure to L-asparaginase, cells were incubated with culture medium as control or incubated with 10 IU/ml of L-asparaginase (Paronal) in culture medium. A total of 5 × 106 cells were harvested after 18, 24, 30 and 44 h of incubation with L-asparaginase and apoptotic markers were analyzed.

L-asparaginase activity and amino-acid levels in serum

Serum levels of L-asparaginase and asparagine, glutamine, aspartic acid and glutamic acid were determined in the laboratory of Prof Dr J Boos (University Children's Hospital Muenster, Department of Pediatric Hematology/Oncology, Germany). Blood samples were taken daily from day −5 to 0, and twice a week from day 0 to 28. L-asparaginase activity was analyzed as described before.26 After collection serum was isolated by centrifugation and stored immediately at −80 °C; serum levels of asparagine, aspartic acid, glutamine and glutamic acid were performed using high-performance liquid chromatography.27 The lower limit of detection was 0.2 μM for all amino acids; glutamine levels higher than 250 μM were not further analyzed and reported as >250 μM. The serum levels of asparagine and the L-asparaginase activity in a part of the patients included in the present study have been published before.28

Intracellular amino-acid levels

Intracellular levels of all essential and nonessential amino acids were measured by liquid column chromatography on a Biochrome 20 amino-acid analyzer with ninhydrin detection (Biochrome, Cambridge, UK). Blood samples were taken daily from day –5 at diagnosis to day 0. At each time point 5 × 106 leukemic cells were lyzed in 100 μl of lysis buffer (150 ml NaCl, 30 mM Tris (pH 7.6), 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulphonylfluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptine) for 15 min on ice. Cleared supernatants were stored at −80 °C until the time of analysis. The intracellular amounts of amino acids were expressed as μmol of amino acid per mg of protein. The lower limit of detection was 7 μmol (coefficient of variation 10%). Intracellular protein content was measured using the bicinchoninic acid assay (Interchim Omnilabo, Rockford, Illinois, USA29).

For ex vivo exposure to L-asparaginase, cells were incubated with culture medium as control or incubated with 0.1 or 10 IU/ml of L-asparaginase (Paronal) in culture medium. A total of 5 × 106 cells were harvested after 1, 3, 6, 24 and 30 h, and if possible after 48 and 72 h of incubation with L-asparaginase and amino-acid levels were analyzed.

Clinical toxicity

On the basis of clinical manifestations of diabetes mellitus, pancreatitis and hyperlipidemia, neurotoxicity and stroke laboratory parameters were monitored. Changes in coagulation and fibrinolysis were monitored throughout induction therapy. On days −5 and 0 and during induction on days 29, 33, 36, 40 and 43, peripheral blood samples were collected from the infusion line just before each Paronal infusion. All coagulation assays were performed with commercially available reagents and methods as described in detail previously.30 Reference values were applied from the literature.31, 32, 33

Statistics

Differences between multiple groups were calculated using the Kruskal–Wallis test. The Mann–Whitney test was used when two groups were compared. P=0.05 (two sided) was used as level of significance.

Median follow-up time of patients at risk of an event was 4.1 years (range 1.9–5.5 years). Event-free survival (EFS) defined as relapse-free survival was calculated according to Kaplan–Meier (±s.e.). Multiple regression was conducted using Cox proportional hazards regression models to assess prognostic factors for EFS.

Results

Patients

Between July 2000 and July 2004, 57 children with newly diagnosed ALL were enrolled in the PEG-asparaginase-window study (Table 1). As only patients with initial WBC>10 × 109 per liter were eligible for this study, more than half of these children were high-risk patients according to ALL-9 criteria.

Table 1 Patient characteristics of PEG-asparaginase window study
Full size table

Clinical response

Administration of PEG-asparaginase at day −5 resulted in a steadily drop in the number of leukemic cells in the peripheral blood over 5 consecutive days. The median leukemic cell count was reduced 192-fold from 34.5 × 109 per liter at day −5 to 0.18 × 109 per liter at day 0 (Figure 1). Out of 57, 35 (61%) children were defined as good responders to PEG-asparaginase, 16 (28%) were intermediate responders and 6 (11%) children were poor responders.

Figure 1
figure 1

Clinical response to one dose of 1000 IU/m2 PEG-asparaginase administered intravenously at day −5, that is 5 days upfront regular chemotherapy. A good clinical response to PEG-asparaginase was defined as <1 × 109/l leukemic cells in the peripheral blood at day 0, an intermediate response was defined as 1–10 × 109/l, and a poor response as >10 × 109/l leukemic cells in the peripheral blood at day 0. The data of individual patients are given in gray, medians with ranges (25th and 75th percentiles) are drawn in black. PEG, pegylated.

Full size image

The good prognostic genotypes (hyperdiploid and TELAML1/t(12;21)-positive ALL) were associated with a good clinical window response, whereas the poor prognostic genotype BCRABL/t(9;22) was associated with a poor clinical window response (P=0.04) (Table 2). The 17 T-ALL patients had a significant poorer response to PEG-asparaginase compared to the 36 common/pre B cases (P=0.01), BCRABL patients were not included.

Table 2 Correlation between clinical response to one dose of PEG-asparaginase and immunophenotype and genotype in 57 children with newly diagnosed ALL
Full size table

In vitro cytotoxicity of L-asparaginase was measured in 41 leukemic cell samples. Children with a poor or intermediate in vivo response to PEG-asparaginase treatment had a median LC50 value of 1.0 IU/ml compared to 0.04 IU/ml for children with a good clinical response. So poor and intermediate responders were in vitro 25-fold more resistant to L-asparaginase (P=0.02) than children with a good in vivo response (Figure 2). In concordance with earlier studies,34 T-ALL cells were significantly more in vitro resistant to L-asparaginase than precursor B-ALL cells (LC50 median 1.22 IU/ml versus 0.10 IU/ml; P<0.001). Only one 8-year-old girl with T-ALL and an initial WBC of 132 × 109 per liter had a poor clinical response (WBC 25 × 109 per liter at day 0, absolute leukemic cell count 19.3 × 109 per liter), whereas her blasts were in vitro sensitive to L-asparaginase. She still is in continuous complete remission.

Figure 2
figure 2

Comparison between in vivo and in vitro response to L-asparaginase. In 41 children in vitro toxicity of L-asparaginase (LC50 in IU/ml) was measured and related to the in vivo response to 1000 IU/m2 PEG-Asparaginase given upfront regular chemotherapy. Good responders (leukemic cells at day 0 <1 × 109/l) were significantly more sensitive to L-Asparaginase than intermediate responders (leukemic cells 1–10 × 109/l at day 0) (P=0.03) and intermediate and poor responders together (leukemic cells >1 × 109/l at day 0) (P=0.02). The sensitivity to L-Asparaginase did not differ between the intermediate and poor responders.

Full size image

Apoptosis

Analysis of in vivo induced apoptosis by PEG-asparaginase

We analyzed 25 patients for different apoptotic parameters over time (Figure 3). At diagnosis samples from 13 patients were available, immediately after the PEG-asparaginase infusion samples from 21 patients, and on the consecutive days from 10 to 24 patients. The median percentage of ALL cells showing phosphatidylserine externalization was 10.5% before starting treatment (day −5). These values did not significantly differ over time (Figure 3a). The median percent of cells with changes in mitochondrial transmembrane potential was small at all time points: before PEG-asparaginase exposure 5.4% cells were DIOC6 positive, this did not change over time (P=NS) (Figure 3b). The median percentage of cells with cleaved (and hence activated) caspase 3 at diagnosis was 2.7% and remained low (P=NS) (Figure 3c). The median percent of cells with PARP inactivation at diagnosis was 2.9% and remained low (P=NS) (Figure 3d).

Figure 3
figure 3

In vivo induced apoptosis registered before and during a five day period after the administration of 1000 IU/m2 PEG-asparaginase upfront regular chemotherapy. Percent of cells with PS externalization (a), with Δψm depolarization (b), caspase 3 activation (c) and PARP inactivation (d) are shown. The effect of in vitro exposure to 10 IU/ml L-asparaginase in time is given in (e) for two patients. PARP, Poly-ADP-ribose-polymerase; PEG, pegylated; PS, phosphatidylserine.

Full size image

These data show that the drop in WBC count and number of leukemic cells (Figure 1) seen after PEG-asparaginase administration was not simultaneously associated with an increase in in vivo apoptotic markers. None of the apoptotic markers was linked to immunophenotype or genotype.

Analysis of in vitro induced apoptosis by L-asparaginase

In contrast to data obtained after in vivo exposure, significant changes in apoptotic parameters were detected after in vitro exposure to L-asparaginase (Figure 3e). This is in correspondance with earlier studies showing that activation of these apoptotic markers can be measured after in vitro exposure to L-asparaginase.19

L-asparaginase activity and levels of amino acids in serum

The median serum level of asparagine before treatment was 38.7 μM (p25th–75th: 28.1–44.9 μM). In all patients serum asparagine levels decreased below the limit of detection of 0.2 μM after the administration of PEG-asparaginase (P<0.001) (Figures 4a and b). With the exception of two patients these asparagine levels remained below 0.2 μM until day 21, so for a total of at least 26 days. The L-asparaginase activity in the serum was >100 IU/l for a total of at least 15 days (Figure 4a). The level of serum aspartic acid (Figure 4b) increased after 1 h of PEG-asparaginase infusion from 7.1 μM (p25th–75th: 4.1–14.3 μM) to 22.1 μM (p25th–75th: 15.5–47.3 μM) (P<0.001), followed by a decrease to median 11.7 (p25th–75th: 7.3–21.3 μM) and 15.5 μM (p25th–75th: 9.6–23.5 μM) at days −3 and 0. Compared to the level measured at diagnosis before the PEG-asparaginase infusion, the serum aspartic acid levels were still increased at both time points (P=0.02 at day –3) and (P=0.05 at day 0).

Figure 4
figure 4

(a) The effect of 1000 IU/m2 PEG-asparaginase administered i.v. 5 days upfront regular chemotherapy on serum asparagine levels in time. The levels of PEG-Asparaginase (left Y-axis in IU/ml) are given in black, the levels of asparagine (right Y-axis in μM) in gray; 0.0001 μM is the detection limit of asparagine, the upper dotted line is the reference L-asparaginase activity level of 100 IU/ml. (b) Serum amino-acid levels of asparagine, aspartic acid, glutamine and glutamic acid were measured in 24 children with newly diagnosed ALL before and after in vivo administration of one dose of 1000 IU/m2 PEG-Asparaginase. Significant changes between the levels at diagnosis and 1 h after the administration of PEG-asparaginase are indicated by P<0.001. i.v., intravenously; PEG, pegylated; ALL, acute lymphoblastic leukemia.

Full size image

Most glutamine levels stayed beyond the upper limit of detection (>250 μM) after PEG-asparaginase treatment (Figure 4b). However, glutamic acid levels significantly increased from median 87.6 μM (p25th–75th: 57.8–124 μM) before treatment to 310 μM (p25th–75th: 178–396 μM) 1 h after therapy (P<0.001) and to 239 μM (p25th–75th: 125–306 μM) at day–3 (P<0.001) and 159 μM (p25th–75th: 127–300 μM) at day 0 (P<0.001) (Figure 4b).

The serum peak levels of PEG-asparaginase did not differ between clinical response groups. No correlation with age, immunophenotype or WBC was observed.

Intracellular amino-acids levels

In vivo PEG-asparaginase exposure

Intracellular levels of 20 different amino acids were measured in leukemic cells of 19 children with newly diagnosed ALL and in peripheral blood cells of 9 healthy control children without bone marrow disease. The protein concentration of ALL cells was median 176 μg/ml (25th–75th percentile: 146–226 μg/ml) (N=9), which significantly differed from the protein content of mononuclear peripheral blood cells of normal controls (447 μg/ml; 25th–75th percentile: 347–568 μg/ml) (P<0.0001). The amino-acid levels were therefore expressed as μmol/mg of cellular protein.

The intracellular levels for 16 out of 20 amino acids did not differ between the ALL cells at diagnosis and normal controls (Table 3). Aspartic acid (P<0.05), glutamic acid (P<0.05) and cystathionine (P<0.01) were twofold higher in ALL compared to normal controls, whereas taurine levels were 5.4-fold lower in leukemic cells (P<0.001). PEG-asparaginase did not affect the intracellular amino-acid levels over time (Table 3), as is also shown in Figure 5a for asparagine, aspartic acid, glutamine and glutamic acid. The intracellular amino-acid levels at diagnosis and the in vivo response to PEG-asparaginase did not correlate (Figure 6; Table 4). Results were borderline significant for leucine, but not significant if adjusted for multiple testing. One remarkable difference demonstrated valine, which was two times higher in nonresponding patients.

Table 3 Effect of one single dose of PEG-asparaginase on intracellular amino-acid levels in newly diagnosed ALL
Full size table
Figure 5
figure 5

The effect of L-asparaginase on intracellular amino-acid levels of asparagine, aspartic acid, glutamine and glutamic acid in leukemic cells of children with newly diagnosed ALL. (a) In vivo effect of one dose of 1000 IU/m2 PEG-asparaginase measured in leukemic cells of 19 children with ALL compared to the levels found in peripheral blood cells of nine healthy control cases. Data are given as μmol/mg of intracellular protein. (b) The effect of in vitro exposure to L-asparaginase on intracellular amino-acid levels of asparagine, aspartic acid, glutamine and glutamic acid of leukemic cells of six patients that were in vitro exposed to 10 IU/ml L-asparaginase. Asparagine levels (P<0.05) and glutamine levels (P<0.001) decreased significantly before and after incubation with L-asparaginase. ALL, acute lymphoblastic leukemia; PEG, pegylated.

Full size image
Figure 6
figure 6

Intracellular amino-acid levels (in μmol/mg of intracellular protein) of asparagine, aspartic acid, glutamine and glutamic acid at diagnosis of 19 children with ALL, related to their clinical response to one dose of 1000 IU/m2 PEG-asparaginase 5 days later, as defined in figure 1. ALL, acute lymphoblastic leukemia; PEG, pegylated.

Full size image
Table 4 Intracellular amino-acid levels at diagnosis related to in vitro and in vivo sensitivity
Full size table

As amino-acid levels did not change over time, the relation between clinical response and amino-acid levels over time was not further evaluated.

In correspondence with the in vivo data, the levels of intracellular amino acids did not significantly differ between in vitro L-asparaginase-resistant and -sensitive patients (Table 4).

In vitro L-asparaginase exposure

In contrast to the lack of in vivo intracellular depletion of asparagine and glutamine (Figure 5a), significant intracellular depletion was detected after in vitro exposure to L-asparaginase. Leukemic blasts of six patients were in vitro exposed to 0.1 IU/ml L-asparaginase, 10 IU/ml L-asparaginase and to culture medium only as control, and analyzed before exposure and during 72 h after exposure. Significant intracellular asparagine depletion was observed with 0.1 IU/ml and 10 IU/ml of L-asparaginase at all time points tested (P<0.05) (Figure 5b). Only after exposure to 10 IU/ml of L-asparaginase, intracellular glutamine levels declined significantly within 3 h (P<0.001), whereas no significant rise in glutamic acid could be observed (Figure 5b).

Clinical toxicity

No clinical toxicity related to PEG-asparaginase was seen during the window phase. At day 29 of the induction phase in which patients received twice-weekly Paronal, one patient out of 57 patients experienced a period of diabetes mellitus. Another child was diagnosed with transient hyperlipidemia. No clinical signs of pancreatitis, severe neurotoxicity or thrombotic events were observed.

Allergic reactions

Four infusions with Paronal were planned during induction on day 29, 33, 36 and 40, and only the 30 high-risk patients were scheduled to receive nine additional Paronal infusions during intensification after 3 months.

No child demonstrated an allergic reaction to the first Paronal infusion on day 28. One out of 57 children had a grade 3 allergic reaction on the second infusion. Treatment was switched to Oncaspar and could not completely be finished. So 56/57 patients received the total of four Paronal doses during induction.

Of 30 high-risk patients, 1 relapsed within 2 months after diagnosis and changed protocol before the intensification phase. Of the remaining 29, 15 (52%) children demonstrated an allergic reaction. Moreover, 9 of these 15 could complete the intensification therapy by switching to Erwinase or to Oncaspar. The allergy rate of 52% was not higher than the 81% allergy rate in a group of 16 high-risk patients treated with the same ALL-9-HR protocol but without the upfront PEG-asparaginase window.

Changes in hemostasis

At diagnosis data point to enhanced thrombin generation. Consumption coagulopathy was monitored in 17 patients (Table 5).

Table 5 Coagulation parameters of 57 patients before the window with PEG-asparaginase at day –5, before starting ALL-9 induction therapy (day 0) and during four Paronal infusions
Full size table

On day 0, 5 days after the administration of 1000 IU/m2 PEG-asparaginase many coagulation proteins demonstrated a significant decrease. The decrease in coagulation factors on day 0 was not related to the clinical response to PEG-asparaginase.

The changes in coagulation parameters before Paronal administration at day 28 and at the end of induction on day 43 (after four doses of Paronal) demonstrated the same pattern as for patients who did not receive PEG-asparaginase window treatment, as published recently.35

Outcome and prognostic factors

Survival analysis of in vivo response to PEG-asparaginase showed that clinically good window responders had a more favorable outcome than clinically poor window responders (3-year EFS±s.e.: 91±6% for good, 81±10% for intermediate and 33±19% for poor responders (median follow-up 4.1 years; Figure 7a). The hazard ratio for the good and intermediate responders compared to the poor responders was 6.4 (95% CI 1.81–22.86) (Table 6). The prognosis of good and intermediate responders was significantly higher than for the poor responder group (P=0.004, univariate analysis). The P-value of the trend analysis for all three groups was 0.014.

Figure 7
figure 7

(a) Relationship between event free-survival (EFS) and clinical response to the PEG-asparaginase window: 35 good responders, 16 intermediate and six poor responders. The prognosis differed between good and intermediate responders compared to poor responders (P=0.004). (b) Relationship between EFS and in vitro sensitivity to L-asparaginase. Fifteen children were sensitive, 11 intermediate sensitive and 15 were resistant to L-asparaginase. The prognosis differed between the sensitive and intermediate sensitive group compared to the group resistant to L-asparaginase (P<0.01). PEG, pegylated.

Full size image
Table 6 Univariate analyses not stratified for treatment arm
Full size table

Patients who were in vitro sensitive or intermediate sensitive to L-asparaginase had a 3-year EFS of 93±7 and 100%, which is significantly different from the EFS of 53±13% for the in vitro resistant patients (Figure 7b). The hazard ratio for the good and intermediate-sensitive patients compared to the resistant patients was 3.7 (95% CI 1.3–10.7; P=0.014) (Table 6). The P-value of the trend analysis for all three groups was 0.16.

Of the other variables including age, sex, WBC, immunophenotype and genotype, only WBC (>or <50 × 109 per liter) at diagnosis was significantly related to outcome in the Cox univariate analysis (P=0.02) (Table 6).

In a multivariate analysis, including clinical response, in vitro L-asparaginase resistance, age, white blood cell count and immunophenotype, only the clinical response was an independent risk factor (P=0.042) (χ2-trend 6.602, P=0.0013).

Discussion

In the present study we investigated the effect of a therapeutic window with one single dose of PEG-asparaginase before start of combination chemotherapy on several pharmacodynamic and pharmacokinetic parameters, and on clinical response in pediatric ALL.

Clinical response

Monotherapy with 1000 IU/m2 caused a gradual decrease of the leukemic cell burden in most patients within 5 days of treatment (Figure 1). Children with unfavorable characteristics like T-ALL or BCRABL/t(9;22)-positive ALL were in vivo more resistant to PEG-asparaginase than children with a more favorable immunophenotype and genotype. This is in concordance with the fact that T-ALL cells were found to be in vitro more resistant to L-asparaginase.34 For BCRABL/t(9;22), the in vivo resistance to L-asparaginase is a new finding. In the present study we also showed that in vitro resistance to L-asparaginase is related to the clinical response to PEG-asparaginase as single drug.

Parameters of apoptosis

Apoptotic markers at diagnosis did not predict the clinical response. We, like others36 were not able to demonstrate a change in the percentage of in vivo apoptotic cells after administration of PEG-asparaginase. If however, ALL cells were in vitro exposed to L-asparaginase, a significant increase in time of the apoptotic markers was found, confirming previous findings.19 Together with the fact that there was a rapid decrease in leukemic cells upon in vivo PEG-asparaginase exposure, these findings indicate that apparently no apoptotic cells remain in the circulation in vivo. Most likely, these apoptotic cells are rapidly being removed by phagocytosis.37

L-asparaginase activity in serum and intracellular amino acids

One dose of 1000 IU/m2 PEG-asparaginase immediately resulted in complete serum asparagine depletion with a concomitant rise in serum aspartic acid and glutamic acid. Our data are in correspondence with previous data observed in a larger group of patients treated with L-asparaginase and confirm that PEG-asparaginase will yield its pharmacodynamic effects for 2–4 weeks.38 However, in vivo no changes were observed in the levels of intracellular amino acids (including asparagine, aspartic acid, glutamine and glutamic acid) directly after PEG-asparaginase administration nor on successive days thereafter, except for valine. So far, no explanation can be given for the high level of valine in the nonresponding group. In contrast, in vitro exposure to L-asparaginase of ALL cells significantly changed the intracellular asparagine and glutamine levels that reflected the changes observed in serum suggesting that the surviving leukemic cells are capable of keeping the intracellular amino-acid pools in balance, whereas those cells that are not capable of doing so die and are immediately removed from the blood circulation. Therefore the in vivo process of intracellular amino-acid depletion in leukemic cells cannot be measured which is in correspondence with the finding that no in vivo effect on apoptosis parameters could be measured. These data underline the fact that only in vitro studies have been published that show a relationship between amino-acid depletion and apoptosis,39, 40 whereas literature on in vivo measurements is lacking.

Iwamoto et al.41 recently described the protective effect of mesenchymal cells in L-asparaginase cytotoxicity. They postulated that the interaction between ALL cells and the microenvironment in which these cells reside protected the leukemic cells from asparagine depletion by a high expression of asparagine synthetase in these mesenchymal cells. Surviving leukemic cells may therefore be rescued with asparagine produced by mesenchymal cells. There was no correlation between the upregulation of asparagine synthetase mRNA and the amount of asparagine in the leukemic cells (data not shown). At present protection of ALL cells from L-asparaginase toxicity by mesenchymal cells is part of an ongoing debate.

Another possible explanation for the discrepancy of cellular asparagine levels in vivo and in vitro comes from the Italian group of Bussolati.42, 43 In vitro L-asparaginase rapidly hydrolyzes asparagine and subsequently glutamine if asparagine is depleted.44 As the main amino group for in vivo asparagine synthesis is provided by glutamine, depletion of glutamine impacts endogenous asparagine synthesis. However, glutamine is abundantly present in vivo, and hence a donor for the amino group needed for asparagine synthesis is not lacking. This theory might also explain our finding that asparagine remains detectable in leukemic blasts after in vivo exposure to PEG-asparaginase. Not only Boos et al.44 but also Fine et al.45 emphasized the fact that leukemic cell lines and primary samples from leukemic patients are different from each other and cell line data cannot be extrapolated to primary patients’ samples that easily.

At diagnosis the intracellular levels of glutamic acid and cystathionine were higher and taurine levels were lower in leukemic cells compared to normal peripheral blood mononuclear cells. Proliferating leukemic cells may produce more glutamic acid due to a fast turnover of glutamine to provide nitrogen and carbon needed for the synthesis of purines and pyrimidines energy metabolism.46 It is known that malignant cells are more dependent on an exogenous source of asparagine and glutamine than normal cells.47

The high levels of cystathionine and low levels of taurine may be linked to each other. Leukemic cells were shown to have consistently lower taurine levels compared to normal lymphocytes and granulocytes.48 A low activity of γ-cystathionase in leukemic cells might result in an abnormal methionine–cysteine pathway leading to higher levels of its starting product cystathionine and lower levels of the end product taurine.

Chakrabarti et al.49 also tried to explain differences in L-asparaginase resistance by differences in intracellular amino-acid metabolism. They pointed to a hypothesis of Ryan and Keefer, that depletion of asparagine might result in decreased glycine and serine concentrations in L-asparaginase-sensitive but not in L-asparaginase-resistant tumor cells.50, 51 Asparagine is required for the transamination of glyoxylate resulting in glycine and serine depletion, which could have severe consequences for purine biosynthesis.49 However, we did not find evidence to support these hypotheses as we found no difference in the serine or glycine content between in vivo good and poor responders to L-asparaginase nor between in vitro L-asparaginase-sensitive and -resistant leukemic cells.

It has also been suggested that L-asparaginase treatment caused reduced incorporation of valine into proteins in L-asparaginase-sensitive cells but not in L-asparaginase-resistant tumor cells.52 In our group valine levels before L-asparaginase treatment were two times lower in in vivo good/intermediate responders compared to poor responders. However, no difference in the level of valine between in vitro L-asparaginase-resistant and -sensitive ALL cells were observed in the present study.

Clinical toxicity, allergic reactions and changes in hemostasis

There was no acute toxicity of one dose of PEG-asparaginase in this investigational window. One extra dose of PEG-asparaginase did not lead to higher incidence of allergic reactions during induction treatment and also not during intensification therapy 3 months later. This is of importance as Silverman et al. observed that L-asparaginase tolerance is an important prognostic factor in ALL.2

The single dose of PEG-asparaginase in this study induced a fall in more than half of the coagulation proteins. We demonstrated that the serum asparagine depletion due to one PEG-asparaginase infusion of 1000 IU/m2 lasted about 4 weeks. This did not result in a persistent inhibition of coagulation protein synthesis. At day 29, so almost 5 weeks after PEG-asparaginase administration, all coagulation parameters had recovered to normal or even increased levels. The levels of all coagulation parameters were in the same range as published recently in children treated with the same protocol without the PEG-asparaginase window.35 Disturbances in hemostasis due to amino-acid depletion are probably a local effect, that is, in the liver only.53, 54

Outcome and prognostic factors

Both the clinical response to PEG-asparaginase window and the in vitro sensitivity to L-asparaginase are predictive for outcome. The prognostic relevance of the in vitro sensitivity has been previously shown.5, 6, 7, 8 In the present study we also show that the in vivo response to a window with L-asparaginase predicts outcome, which is in concordance with the data of Asselin et al.9 These results suggest that children with ALL with a poor clinical response to PEG-asparaginase might benefit from a more intensive antileukemic therapy.

Conclusion

The clinical response to one dose of 1000 IU/m2 PEG-asparaginase intravenously as an investigational window is an independent prognostic marker related to outcome. Children with the favorable common/pre B ALL especially those with TELAML1 positivity and hyperdiploidy show a good clinical response to PEG-asparaginase and children with the prognostic unfavorable factors T-cell immunophenotype or BCRABL/t(9;22) have a relatively poor response to PEG-asparaginase. The in vivo response to PEG-asparaginase correlates well with in vitro sensitivity to L-asparaginase. Intracellular changes in apoptotic features and amino acids in ALL cells cannot be monitored in vivo; this may be explained by the fact that apoptotic cells are immediately removed from the circulation by phagocytosis. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-asparaginase. Intracellular amino-acid concentrations at diagnosis are not related to PEG-asparaginase response. One additional dose of PEG-asparaginase proved to be safe in children with ALL because it did not cause a higher incidence of allergic reactions to L-asparaginase further on in treatment and it caused no severe toxicities.

References

  1. Capizzi RL, Bertino JR, Skeel RT, Creasey WA, Zanes R, Olayon C et al. L-asparaginase: clinical, biochemical, pharmacological, and immunological studies. Ann Intern Med 1971; 74: 893–901.

    CAS  Article  PubMed  Google Scholar 

  2. Silverman LB, Gelber RD, Dalton VK, Asselin BL, Barr RD, Clavell LA et al. Improved outcome for children with acute lymphoblastic leukemia: results of Dana–Farber Consortium Protocol 91-01. Blood 2001; 97: 1211–1218.

    CAS  Article  PubMed  Google Scholar 

  3. Pieters R, Loonen AH, Huismans DR, Broekema GJ, Dirven MW, Heyenbrok MW et al. In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions. Blood 1990; 76: 2327–2336.

    CAS  PubMed  Google Scholar 

  4. Yamada S, Hongo T, Okada S, Watanabe C, Fujii Y, Ohzeki T . Clinical relevance of in vitro chemoresistance in childhood acute myeloid leukemia. Leukemia 2001; 15: 1892–1897.

    CAS  Article  PubMed  Google Scholar 

  5. Janka-Schaub GE, Harms DO, den Boer ML, Veerman AJ, Pieters R . [In vitro drug resistance as independent prognostic factor in the study COALL-O5-92 Treatment of childhood acute lymphoblastic leukemia; two-tiered classification of treatments based on accepted risk criteria and drug sensitivity profiles in study COALL-06-97]. Klin Padiatr 1999; 211: 233–238.

    CAS  Article  PubMed  Google Scholar 

  6. Pieters R, Huismans DR, Loonen AH, Hahlen K, van der Does-van den Berg A, van Wering ER et al. Relation of cellular drug resistance to long-term clinical outcome in childhood acute lymphoblastic leukaemia. Lancet 1991; 338: 399–403.

    CAS  Article  PubMed  Google Scholar 

  7. Kaspers GJ, Veerman AJ, Pieters R, Van Zantwijk CH, Smets LA, Van Wering ER et al. In vitro cellular drug resistance and prognosis in newly diagnosed childhood acute lymphoblastic leukemia. Blood 1997; 90: 2723–2729.

    CAS  PubMed  Google Scholar 

  8. Den Boer ML, Harms DO, Pieters R, Kazemier KM, Gobel U, Korholz D et al. Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia. J Clin Oncol 2003; 21: 3262–3268.

    CAS  Article  PubMed  Google Scholar 

  9. Asselin BL, Kreissman S, Coppola DJ, Bernal SD, Leavitt PR, Gelber RD et al. Prognostic significance of early response to a single dose of asparaginase in childhood acute lymphoblastic leukemia. J Pediatr Hematol Oncol 1999; 21: 6–12.

    CAS  Article  PubMed  Google Scholar 

  10. Ollenschlager G, Roth E, Linkesch W, Jansen S, Simmel A, Modder B . Asparaginase-induced derangements of glutamine metabolism: the pathogenetic basis for some drug-related side-effects. Eur J Clin Invest 1988; 18: 512–516.

    CAS  Article  PubMed  Google Scholar 

  11. Broome JD . -asparaginase: discovery and development as a tumor-inhibitory agent. Cancer Treat Rep 1981; 65 (Suppl 4): 111–114.

    CAS  PubMed  Google Scholar 

  12. Miller HK, Salser JS, Balis ME . Amino acid levels following L-asparagine amidohydrolase (EC.3.5.1.1) therapy. Cancer Res 1969; 29: 183–187.

    CAS  PubMed  Google Scholar 

  13. Muller HJ, Boos J . Use of L-asparaginase in childhood ALL. Crit Rev Oncol Hematol 1998; 28: 97–113.

    CAS  Article  PubMed  Google Scholar 

  14. Bussolati O, Belletti S, Uggeri J, Gatti R, Orlandini G, Dall’Asta V et al. Characterization of apoptotic phenomena induced by treatment with L-asparaginase in NIH3T3 cells. Exp Cell Res 1995; 220: 283–291.

    CAS  Article  PubMed  Google Scholar 

  15. Andrulis IL, Argonza R, Cairney AE . Molecular and genetic characterization of human cell lines resistant to L-asparaginase and albizziin. Somat Cell Mol Genet 1990; 16: 59–65.

    CAS  Article  PubMed  Google Scholar 

  16. Jousse C, Averous J, Bruhat A, Carraro V, Mordier S, Fafournoux P . Amino acids as regulators of gene expression: molecular mechanisms. Biochem Biophys Res Commun 2004; 313: 447–452.

    CAS  Article  PubMed  Google Scholar 

  17. Appel IM, den Boer ML, Meijerink JP, Veerman AJ, Reniers NC, Pieters R . Up-regulation of asparagine synthetase expression is not linked to the clinical response L-asparaginase in pediatric acute lymphoblastic leukemia. Blood 2006; 107: 4244–4249.

    CAS  Article  PubMed  Google Scholar 

  18. Holleman A, Cheok MH, den Boer ML, Yang W, Veerman AJ, Kazemier KM et al. Gene-expression patterns in drug-resistant acute lymphoblastic leukemia cells and response to treatment. N Engl J Med 2004; 351: 533–542.

    CAS  Article  PubMed  Google Scholar 

  19. Holleman A, den Boer ML, Kazemier KM, Janka-Schaub GE, Pieters R . Resistance to different classes of drugs is associated with impaired apoptosis in childhood acute lymphoblastic leukemia. Blood 2003; 102: 4541–4546.

    CAS  Article  PubMed  Google Scholar 

  20. Veerman AJ, Hahlen K, Kamps WA, Van Leeuwen EF, De Vaan GA, Solbu G et al. High cure rate with a moderately intensive treatment regimen in non-high-risk childhood acute lymphoblastic leukemia. Results of protocol ALL VI from the Dutch Childhood Leukemia Study Group. J Clin Oncol 1996; 14: 911–918.

    CAS  Article  PubMed  Google Scholar 

  21. Muller HJ, Loning L, Horn A, Schwabe D, Gunkel M, Schrappe M et al. Pegylated asparaginase (Oncaspar) in children with ALL: drug monitoring in reinduction according to the ALL/NHL-BFM 95 protocols. Br J Haematol 2000; 110: 379–384.

    CAS  Article  PubMed  Google Scholar 

  22. Yoshimoto T, Nishimura H, Saito Y, Sakurai K, Kamisaki Y, Wada H et al. Characterization of polyethylene glycol-modified L-asparaginase from Escherichia coli and its application to therapy of leukemia. Jpn J Cancer Res 1986; 77: 1264–1270.

    CAS  PubMed  Google Scholar 

  23. Slater RM, Smeets DF, Hagemeijer A, De Jong B, Beverstock CG, Geraedts JP et al. Update of the cytogenetic study of childhood non-high-risk acute lymphocytic leukemia at diagnosis in protocol VI of the Dutch Childhood Leukemia Study Group. Haematol Blood Transfus 1990; 33: 169–173.

    CAS  PubMed  Google Scholar 

  24. Kaspers GJ, Veerman AJ, Pieters R, Broekema GJ, Huismans DR, Kazemier KM et al. Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay. Br J Cancer 1994; 70: 1047–1052.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Schrappe M, Reiter A, Zimmermann M, Harbott J, Ludwig WD, Henze G et al. Long-term results of four consecutive trials in childhood ALL performed by the ALL-BFM study group from 1981 to 1995. Berlin-Frankfurt-Munster. Leukemia 2000; 14: 2205–2222.

    CAS  Article  PubMed  Google Scholar 

  26. Lanvers C, Vieira Pinheiro JP, Hempel G, Wuerthwein G, Boos J . Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum. Anal Biochem 2002; 309: 117–126.

    CAS  Article  PubMed  Google Scholar 

  27. Lenda K, Svenneby G . Rapid high-performance liquid chromatographic determination of amino acids in synaptosomal extracts. J Chromatogr 1980; 198: 516–519.

    CAS  Article  PubMed  Google Scholar 

  28. Appel IM, Pinheiro JP, den Boer ML, Lanvers C, Reniers NC, Boos J et al. Lack of asparagine depletion in the cerebrospinal fluid after one intravenous dose of PEG-asparaginase: a window study at initial diagnosis of childhood ALL. Leukemia 2003; 17: 2254–2256.

    CAS  Article  PubMed  Google Scholar 

  29. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150: 76–85.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Appel IM, Hop WC, Pieters R . Changes in hypercoagulability by asparaginase: a randomized study between two asparaginases. Blood Coagul Fibrinolysis 2006; 17: 139–146.

    CAS  Article  PubMed  Google Scholar 

  31. Monagle P, Barnes C, Ignjatovic V, Furmedge J, Newall F, Chan A et al. Developmental haemostasis. Impact for clinical haemostasis laboratories. Thromb Haemost 2006; 95: 362–372.

    CAS  Article  PubMed  Google Scholar 

  32. Flanders MM, Phansalkar AR, Crist RA, Roberts WL, Rodgers GM . Pediatric reference intervals for uncommon bleeding and thrombotic disorders. J Pediatr 2006; 149: 275–277.

    Article  PubMed  Google Scholar 

  33. Ries M, Klinge J, Rauch R . Age-related reference values for activation markers of the coagulation and fibrinolytic systems in children. Thromb Res 1997; 85: 341–344.

    CAS  Article  PubMed  Google Scholar 

  34. Pieters R, den Boer ML, Durian M, Janka G, Schmiegelow K, Kaspers GJ et al. Relation between age, immunophenotype and in vitro drug resistance in 395 children with acute lymphoblastic leukemia—implications for treatment of infants. Leukemia 1998; 12: 1344–1348.

    CAS  Article  Google Scholar 

  35. Appel IM, Hop WCJ, van Kessel-Bakvis C, Stigter R, Pieters R . Age-related changes by asparaginase on coagulation and fibrinolysis in childhood acute lymphoblastic leukemia. Thromb Haemost August 2008.

  36. Stahnke K, Fulda S, Friesen C, Strauss G, Debatin KM . Activation of apoptosis pathways in peripheral blood lymphocytes by in vivo chemotherapy. Blood 2001; 98: 3066–3073.

    CAS  Article  PubMed  Google Scholar 

  37. Durrieu F, Belloc F, Lacoste L, Dumain P, Chabrol J, Dachary-Prigent J et al. Caspase activation is an early event in anthracycline-induced apoptosis and allows detection of apoptotic cells before they are ingested by phagocytes. Exp Cell Res 1998; 240: 165–175.

    CAS  Article  PubMed  Google Scholar 

  38. Boos J, Werber G, Ahlke E, Schulze-Westhoff P, Nowak-Gottl U, Wurthwein G et al. Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations. Eur J Cancer 1996; 32A: 1544–1550.

    CAS  Article  PubMed  Google Scholar 

  39. Franek F, Fismolova I, Eckschlager T . Antiapoptotic and proapoptotic action of various amino acids and analogs in starving MOLT-4 cells. Arch Biochem Biophys 2002; 398: 141–146.

    CAS  Article  PubMed  Google Scholar 

  40. Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M . In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnol Bioeng 1998; 59: 90–98.

    CAS  Article  PubMed  Google Scholar 

  41. Iwamoto S, Mihara K, Downing JR, Pui CH, Campana D . Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase. J Clin Invest 2007; 117: 1049–1057.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Rotoli BM, Uggeri J, Dall’Asta V, Visigalli R, Barilli A, Gatti R et al. Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells. Cell Physiol Biochem 2005; 15: 281–292.

    CAS  Article  PubMed  Google Scholar 

  43. Tardito S, Uggeri J, Bozzetti C, Bianchi MG, Rotoli BM, Franchi-Gazzola R et al. The inhibition of glutamine synthetase sensitizes human sarcoma cells to L-asparaginase. Cancer Chemother Pharmacol 2007; 60: 751–758.

    CAS  Article  PubMed  Google Scholar 

  44. Wagner A, Boos J . Unphysiological effects contributing to asparaginase toxicity in vitro. Am J Physiol 1998; 274 (4 Part 1): C1185–C1186.

    CAS  Article  PubMed  Google Scholar 

  45. Fine BM, Kaspers GJ, Ho M, Loonen AH, Boxer LM . A genome-wide view of the in vitro response to L-asparaginase in acute lymphoblastic leukemia. Cancer Res 2005; 65: 291–299.

    CAS  PubMed  Google Scholar 

  46. Newsholme EA, Crabtree B, Ardawi MS . Glutamine metabolism in lymphocytes: its biochemical, physiological and clinical importance. Q J Exp Physiol 1985; 70: 473–489.

    CAS  Article  PubMed  Google Scholar 

  47. Iiboshi Y, Papst PJ, Hunger SP, Terada N . -asparaginase inhibits the rapamycin-targeted signaling pathway. Biochem Biophys Res Commun 1999; 260: 534–539.

    CAS  Article  PubMed  Google Scholar 

  48. Wakayama K, Besa EC, Baskin SI . Changes in intracellular taurine content of human leukemic cells. Nagoya J Med Sci 1983; 45: 89–96.

    CAS  PubMed  Google Scholar 

  49. Chakrabarti R, Schuster SM . -asparaginase: perspectives on the mechanism of action and resistance. Int J Ped Hem/Oncol 1997; 4: 597–611.

    Google Scholar 

  50. Ryan WL, Sornson HC . Glycine inhibition of asparaginase. Science 1970; 167: 1512–1513.

    CAS  Article  PubMed  Google Scholar 

  51. Keefer JF, Moraga DA, Schuster SM . Comparison of glycine metabolism in mouse lymphoma cells either sensitive or resistant to L-asparaginase. Biochem Pharmacol 1985; 34: 559–565.

    CAS  Article  PubMed  Google Scholar 

  52. Sobin LH, Kidd JG . A metabolic difference between two lines of lymphoma 6c3hed cells in relation to asparagine. Proc Soc Exp Biol Med 1965; 119: 325–327.

    CAS  Article  PubMed  Google Scholar 

  53. Bushman JE, Palmieri D, Whinna HC, Church FC . Insight into the mechanism of asparaginase-induced depletion of antithrombin III in treatment of childhood acute lymphoblastic leukemia. Leuk Res 2000; 24: 559–565.

    CAS  Article  PubMed  Google Scholar 

  54. Reinert RB, Oberle LM, Wek SA, Bunpo P, Wang XP, Mileva I et al. Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase. J Biol Chem 2006; 281: 31222–31233.

    CAS  Article  PubMed  Google Scholar 

Download references

Author information

Affiliations

  1. Department of Pediatric Oncology/Hematology, Erasmus MC/Sophia Children's Hospital, Erasmus University Medical Center, Rotterdam, The Netherlands

    I M Appel, K M Kazemier, J Huijmans, M L den Boer & R Pieters

  2. Department of Pediatric Hematology/Oncology, University Children's Hospital Muenster, Muenster, Germany

    J Boos & C Lanvers

  3. Department of Pediatrics, University Hospital, Vrije Universiteit, Amsterdam, The Netherlands

    A J P Veerman

  4. Dutch Childhood Oncology Group, The Hague, The Netherlands

    A J P Veerman & E van Wering

Authors
  1. I M Appel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K M Kazemier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Boos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C Lanvers
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Huijmans
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A J P Veerman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. E van Wering
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M L den Boer
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R Pieters
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to I M Appel.

Additional information

Authorship

IMA designed and analyzed experiments and wrote the paper; KMK performed the research on the laboratory and analyzed the data; JB contributed vital tools for experiments on serum L-asparaginase activity and serum amino-acid analyses; CL performed experiments and contributed vital tools for experiments on serum L-asparaginase activity and serum amino-acid analyses; JH contributed vital tools for experiments on intracellular amino-acid analyses; AJPV is chairman of the DCOG-ALL-9 protocol; EvW contributed vital tools on behalf of the DCOG; MLdB designed and analyzed experiments and contributed to writing the paper; RP designed and analyzed experiments and contributed to writing the paper.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Appel, I., Kazemier, K., Boos, J. et al. Pharmacokinetic, pharmacodynamic and intracellular effects of PEG-asparaginase in newly diagnosed childhood acute lymphoblastic leukemia: results from a single agent window study. Leukemia 22, 1665–1679 (2008). https://doi.org/10.1038/leu.2008.165

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2008.165

Keywords

  • PEG-asparaginase
  • intracellular effects
  • window response
  • drug resistance
  • ALL

Further reading

Search

Advanced search

Quick links