Bruce A. Chabner
Alison M. Friedmann
The growth of malignant as well as normal cells depends on the availability of specific nutrients and cofactors used in protein synthesis. Some of these nutrients can be synthesized within the cell, but others such as essential amino acids are required from external sources. Nutritional therapy for cancer has been directed at identifying differences between the host and malignant cells that might be exploited in treatment; these attempts have been largely unsuccessful because of difficulties in producing a deficiency state by dietary means and a lack of clear differences in the nutritional requirements of rapidly proliferating host cells and the tumor. The only exception has been the use of L-asparaginase in the treatment of childhood acute leukemia.
L-Asparagine is a nonessential amino acid synthesized by transamination of L-aspartic acid (Fig. 20.1). The amine group is donated by glutamine, and the reaction is catalyzed by the enzyme L-asparagine synthetase. This enzyme is constitutive in many tissues, which accounts for the modest toxicity of asparagine depletion from the plasma, but the capacity to synthesize asparagine is lacking in certain human malignancies, particularly those of lymphocytic derivation. In tumor cells lacking L-asparagine synthetase, such as L5178Y murine leukemia cells,1 the amino acid can be obtained only from a culture medium or, in vivo, from plasma.
The enzyme L-ASP (L-asparagine amidohydrolase, EC 22.214.171.124), which catalyzes the hydrolysis of asparagine to aspartic acid and ammonia as end products, is found in many plants and microorganisms and in the plasma of certain animals. General interest in L-ASP as a therapeutic agent was the result of an unexplained observation by Kidd,2 who in 1953 reported that the growth of transplantable lymphomas in rodents was inhibited by guinea pig serum but not by rabbit, horse, or human serum. Ten years later, Broome3 demonstrated that the responsible factor was the enzyme L-ASP. Subsequently, highly purified preparations of enzyme from Escherichia coli4 and Erwinia carotovora (also known as Erwinia chrysanthemi)5 showed significant activity against childhood acute lymphocytic leukemia (ALL) and have become standard components of remission induction, consolidation, and reinduction therapy in this disease and in adult ALL, contributing to the 80% or greater 5-year disease-free survival in childhood ALL and 35 to the 50% 5-year disease-free survival in adult ALL.6 A chemically modified enzyme, pegaspargase, having a longer half-life and reduced immunogenicity, is approved for use in patients hypersensitive to the native E. coli enzyme. Pegaspargase is being used increasingly in pediatric ALL regimens because it has a significantly lower incidence of allergic reactions and reduces the frequency of painful intramuscular injections for the child. The clinical and biochemical features of L-ASP chemotherapy have been summarized in several comprehensive reviews.7 The key features of L-ASP pharmacology are listed in Table 20.1.
PROPERTIES AND MECHANISM OF ACTION
L-ASP (L-Asp) purified from E. coli8 has been used most widely in both basic and clinical research, although L-ASP obtained from other sources, including E. chrysanthemi, Serratia marcescens, guinea pig serum, and the serum of other members of the species Caviodea, also possesses antitumor activity. The purified bacterial enzyme has a molecular weight of 133,000 to 141,000 Da and is composed of four subunits, each with one active site.9 The gene coding for the E. chrysanthemi10 enzyme has been cloned and sequenced and expressed in E. coli.11 Preparations of enzyme from different bacterial strains and by different purification methods show slight differences in enzyme characteristics. For the bacterial enzymes, the specific activity of purified enzyme is usually 300 to 400 µmol of substrate cleaved per minute per milligram of protein; the isoelectric point lies between pH 4.6 and 5.5 for the E. coli enzyme and is 8.6 for the Erwinia protein; and the Km (Michaelis-Menten constant) for asparagine is usually 1 × 10-5 mol per liter.9, 12 The E. coli enzyme contains 321 amino acids in each subunit (molecular weight 34,080 Da),13 and the Erwinia subunit has a molecular weight of 32,000 Da.14 (See reference 9 for the amino acid sequence of the E. coli enzyme.) The crystal structure of the E. coli enzyme has been solved.13 It has only a 46% homology with the E. chrysanthemi enzyme; the two enzymes lack antigenic cross-reactivity and differ in biochemical properties. For example, ammonia activates E. coli asparaginase, whereas oxygen represses its synthesis; neither affects the Erwinia enzyme.14
Figure 20.1 Sources of L-asparagine for peripheral tissues. The amino acid may be obtained directly from the circulating blood pool of L-asparagine or may be synthesized by transamination of L-aspartic acid, with L-glutamine acting as the NH2 donor in a reaction catalyzed by L-asparagine synthetase. The liver is a major source of L-asparagine found in plasma.
The E. coli and Erwinia enzymes are highly specific for L-asparagine as substrates and have less than 10% activity for the D-isomer, for N-acylated derivatives, or for L-asparagine in peptide linkage. In contrast, the enzyme from Saccharomyces cerevisiae has equal or greater activity with D-asparagine and with N-substituted substrates.15
The hydrolysis of L-asparagine proceeds according to a reaction mechanism that involves an initial displacement of the amino acid NH2 group during the formation of an enzyme-aspartyl intermediate, followed by hydrolytic cleavage of the latter bond to generate free L-aspartate and active enzyme. The reaction may be summarized E + Asn ↔ NH3E • Asp → E + Asp + NH3′, where E • Asp represents the enzyme-aspartyl intermediate.15 The reaction is irreversibly inhibited by the L-asparagine analog 5-diazo-4-oxo-l-norvaline, which binds covalently to the enzyme's active site.16
CELLULAR PHARMACOLOGY AND RESISTANCE
The enzyme L-ASP owes its antitumor effects to the rapid and complete depletion of circulating pools of L-asparagine. In clinical practice, hyperdiploid subtypes of ALL display marked sensitivity to treatment, for unexplained reasons.17 Similarly, intensive therapy with L-ASP appears to be important in the effective treatment of the less common T cell variety of ALL.18 Plasma L-asparagine levels (usually in the range of 4 × 10-5 mol/L) are more than sufficient for L-asparagine–requiring tumor cells, which can grow at a normal rate in tissue culture medium containing 1 × 10-6 mol/L asparagine.19 Because the Km of the E. coli enzyme for L-asparagine is 1 × 10-5 mol/L, the hydrolysis of L-asparagine proceeds at less than maximal velocity once plasma levels fall below this concentration, and considerable excess L-ASP is required in plasma to degrade L-asparagine to sufficiently low concentrations to halt tumor growth. The critical enzyme concentration for maintaining depletion of L-asparagine appears to be at least 0.03 u/mL, but it is possibly 10-fold higher.20
The cellular effects of L-ASP result from inhibition of protein synthesis. Cytotoxicity correlates well with inhibition of protein synthesis. Inhibition of nucleic acid synthesis is also observed in sensitive cells but is believed to be secondary to the block in protein synthesis. Cells insensitive to asparagine depletion from growth medium in vitro are also insensitive to L-ASP and show little inhibition of protein synthesis in the presence of the enzyme. These resistant cells have high endogenous activity of asparagine synthetase.21 The dependence on asparagine exhibited by sensitive cells may be related not only to the requirement for the amino acid itself as a constituent of protein but also to its role as a donor of the NH2 group in the synthesis of glycine.22 The mechanism of cell death may be the activation of programmed cell death, or apoptosis, as suggested by both in vitro and in vivo experiments.23
Resistance to L-Asparaginase
Resistance emerges rapidly when L-ASP is employed as a single agent, both in animal tumor systems and in humans. Early studies in cell culture21 and cells taken from resistant leukemia patients24 demonstrated elevated levels of asparagine synthetase (AspS), indicating the selection of cells that up-regulate the synthesis of asparagine in the presence of the enzyme. Subsequent studies showed that up-regulation was associated with hypomethylation of the AspS gene.25 However, there is, as yet, no clear correlation of AspS expression and sensitivity to L-ASP in tissue culture studies or prospective clinical trials of human ALL.26 For example, AspS levels are high in the cells from patients with the (12:21) TEL/AML1 translocation, a type of ALL that exhibits high sensitivity to L-ASP both in vitro and in patients.26 Further, there is no evidence for greater AspS induction after treatment in resistant versus sensitive ALL cells. Definitive studies of the role of AspS are not yet available.27 Other possible mechanisms of resistance in ALL have been reported, including the development of neutralizing antibodies (referred to as “silent hypersensitivity”)29 and defective induction of apoptosis, a change that confers resistance to glucocorticoids as well.30 A 35-gene expression profile highly predictive of L-ASP resistance in vitro and in clinical outcomes has been reported but does not include AspS as a contributor.28
TABLE 20.1 KEY FEATURES OF ESCHERICHIA COLI L-ASP PHARMACOLOGY
In an attempt to reduce the immunogenicity of L-ASP, to eliminate L-glutaminase activity from the molecule, and to prolong the enzyme's plasma half-life, the E. coli asparaginase has been subjected to various modifications. Most bacterial L-ASP preparations contain significant L-glutaminase activity (3 to 5% of the L-ASP activity), activity linked to immunosuppression and cerebral dysfunction. Attempts to eliminate the L-glutaminase activity31 have met with limited success; the nitrated enzyme has little L-glutaminase but also has reduced L-ASP action.
A second objective has been to reduce immunogenicity. The E. coli enzyme, modified by conjugation with 5,000 Da of monomethoxypolyethylene glycol (PEG), displays a similar decrease in immunogenicity and a 5- to 10-fold increase in plasma half-life and retains 50% of its initial activity.32 The PEG-asparaginase (pegaspargase) is active and nonimmunogenic in about 70% of patients hypersensitive to the native enzyme. A copolymer of asparaginase with albumin has markedly reduced immunoreactivity and “satisfactory” activity in mice.33 PEG-asparaginase is an effective alternative for patients hypersensitive to the E. coli enzyme and is increasingly employed in primary treatment regimens.34
The in vivo clearance rate of the enzyme and its Km are two important factors that may play roles in determining the efficacy of asparaginase as an antitumor agent. L-ASPs isolated from Bacillus coagulans, Fusarium tricinctum, and Candida albicans are devoid of antitumor activity and are almost completely cleared from the circulation in 30 minutes to 1 hour after intravenous administration into mice. On the other hand, enzymes derived from guinea pig serum and from E. coli and Erwinia exhibit antitumor activity and have a much longer half-life.35, 36
The affinity of the enzyme for L-asparagine is another important factor that affects the antitumor activity of L-ASP.37 Serum concentrations of L-asparagine are 30 to 50 µmol/L, which exceeds the Km of the bacterial enzymes used in clinical chemotherapy. Thus substrate hydrolysis occurs rapidly under physiologic conditions. As serum levels of the amino acid fall, they approach and then fall below the Km, which slows the rate of hydrolysis. E. coli L-asparaginase and Erwinia L-asparaginase, which possess strong antitumor activity, have Km values of 1 to 1.25 × 10-5 mol/L, whereas lower-affinity L-ASPs from agouti or guinea pig serum37 or other bacterial sources38 have only moderate or no antitumor activity.39
Drug Assay and Pharmacokinetics
L-ASP is easily measured in biologic fluids by assays that detect ammonia release40 or by a coupled enzymatic assay.41 The drug is given subcutaneously, intramuscularly, or intravenously; the intramuscular and subcutaneous routes produce peak blood levels 50% lower than the intravenous route but may be less immunogenic. For the E. coli enzyme, the usual dosages are a single dose of up to 25,000 IU/m2 weekly, 5,000 to 10,000 IU/m2 every other day or every third day for 2 to 4 weeks, or daily doses of 1,000 to 10,000 IU/m2 for 10 to 20 days. A comparison of the clinical effectiveness of various doses of L-ASP given three times per week demonstrated a higher complete remission rate for doses of 6,000 IU/m2 or higher than for doses 3,000 IU/m2 or less42 (Fig. 20.2). L-ASP activity is detectable in the bloodstream for 2 to 3 weeks after large single doses of the E. coli enzyme (25,000 IU/m2), but depletion of asparagine lasts for only 1 week or less.42 Thus asparagine levels return toward normal even in the presence of low levels of the enzyme. The threshold at which recovery takes place appears to be 0.03 IU/mL of plasma. However, accurate measurement of serum asparagine levels requires that blood be collected and stored in the presence of an L-ASP inhibitor, such as 5-diazo-4-oxo-l-nor valine.43 The enzyme distributes primarily within the intravascular space. The cerebrospinal fluid (CSF) concentration of asparagine falls rapidly, however, and an antileukemic effect is exerted in this sanctuary, despite the poor penetration of enzyme into the CSF.21 The drug can be given directly into the CSF but exits rapidly from this site, and use of this route seems to have no clear therapeutic advantage.
The concentration of L-ASP in plasma is proportional to dose for doses up to 200,000 IU/m2 and has a primary half-life of 30 hours.44 The Erwinia enzyme, although preserving activity in patients hypersensitive to the E. coli preparation, has the disadvantage of a shorter half-life in plasma (16 hours)44 and does not give equivalent therapeutic results when used with the same dose and schedule as the E. coli enzyme (5,000 U/m2 three times per week). A doubling of the dosage of Erwinia enzyme (10,000 U/m2 per day or 20,000 IU/m2 3 days per week) is recommended to maintain continuous asparagine depletion and equivalent antitumor effects.45 In patients who develop neutralizing antibodies to the enzyme, plasma clearance is greatly accelerated, and enzyme activity may be undetectable in plasma as soon as 4 hours after administration.46
Figure 20.2 Relationship between dose of L-ASP and response in the treatment of acute lymphoblastic leukemia. Patients received the indicated doses every other day, three doses per week, for a maximum of 6 weeks. Successful induction is judged by achievement of an M1 bone marrow status. (From Ertel IJ, Nesbit ME, Hammond D, et al. Effective dose of L-ASP for induction of remission in previously treated children with acute lymphocytic leukemia: a report from Children's Cancer Study Group. Cancer Res 1979;39:3893.)
Covalent linkage of L-ASP with PEG has succeeded in markedly reducing the clearance of the enzyme, whereas the volume of distribution remains equivalent to the average plasma volume in humans. In plasma, pegaspargase has a half-life of 6 days, considerably longer than that of the native enzyme; the total clearance is 5.3 ± 3.1 mL/hour per m2 or 0.13 ± 0.08 mL/hour per kg, and the apparent volume of distribution is 2.1 ± 0.6 L/m2 or 52.3 ± 16.1 mL/kg.47 The recommended dosage of pegaspargase is 2,500 U per m2 every week or every two weeks.21 This dose results in plasma L-ASP activity of more than 0.1 µmL for at least 7 days. In patients showing hypersensitivity to E. coli asparaginase, both native and PEG-linked enzyme may have a shorter half-life, although the PEG enzyme remains active in hypersensitive patients.
The primary toxicities of L-ASP, listed in Table 20.2, fall into two main groups: those related to immunologic sensitization to the foreign protein and those resulting from depletion of asparagine pools and inhibition of protein synthesis. Hypersensitivity reactions to L-ASP are of great concern because they are a common and potentially fatal complication of therapy, particularly when the drug is used as a single agent.46 Up to 40% of patients receiving single-agent treatment develop some evidence of sensitization.48 Possibly because of the immunosuppressive effect of corticosteroids, 6-mercaptopurine, and other antileukemic agents, the incidence of hypersensitivity reactions falls to less than 20% in patients receiving combination chemotherapy. Other factors that increase the incidence of reactions include the use of dosages above 6,000 IU/m2 per day, intravenous as opposed to intramuscular administration, and repeated courses of treatment.49 Reactions to an initial dose rarely occur; more commonly, hypersensitivity phenomena appear during the second week of treatment or later.50
TABLE 20.2 TOXICITY OF L-ASP
The clinical manifestations of hypersensitivity vary from urticaria (approximately two thirds of reported reactions) to true anaphylactic reactions (hypotension, laryngospasm, cardiac arrest). Rarely, serum sickness–type responses—with arthralgias, proteinuria, and fever—may develop several weeks after an extended course of treatment.50 Fatal reactions occur in less than 1% of patients treated, but evidence of hypersensitivity should prompt a change in treatment to L-ASP derived from Erwinia5 or to pegaspargase. (The Erwinia drug is not sold commercially, but for treatment of ALL it may be available through Ipsen Pharmaceuticals, Ltd, and its US distributor, McKesson BioService Corp; phone 301-315-8460.) Allergic reactions to Erwinia L-ASP may occur as an independent phenomenon in patients who have not previously received E. coli enzyme51 and may ultimately develop in 5 to 20% of patients receiving multiple courses of this enzyme. PEG-asparaginase can also be used in hypersensitive patients, among whom a 30% incidence of allergy to the new drug can be expected.
Because of the frequency and severity of allergic reactions to L-ASP, routine skin testing was recommended for prediction of allergy before the first dose of drug. Allergic reactions may occur in patients with negative skin tests,52 however, and positive skin tests are not invariably predictive of reactions. Hypersensitive patients usually have both immunoglobulin E and immunoglobulin G antibodies to L-ASP in serum,53 but more than half the patients with such antibodies do not display an allergic reaction to the drug clinically. Thus the antibody tests have limited value for predicting which patients will have an allergic reaction. Routine skin testing is not recommended for pegaspargase. However, only personnel trained in and prepared for the management of anaphylaxis should administer L-ASP, and patients are generally observed closely for a minimum of 1 hour following its administration.
Other toxic effects result from inhibition of protein synthesis; these include hypoalbuminemia, decrease in clotting factors, decreased serum insulin with hyperglycemia, and decreased serum lipoproteins. Abnormalities in clotting function are regularly observed in association with L-ASP therapy and can lead to thromboembolism in 2 to 11% of ALL patients, most frequently during induction therapy and when glucocorticoids are being administered concurrently.54, 55,56, 57 Hemorrhagic events occur less frequently and are probably secondary to decreased synthesis of vitamin K–dependent factors, with prolongation of the prothrombin time, partial thromboplastin time, and thrombin time50, 58 and decrease in factors IX and X.59 Platelets from L-ASP–treated subjects display deficient aggregation in response to collagen but not to adenosine diphosphate, arachidonic acid, or epinephrine.60 Two instances have been reported of a spontaneous intracranial hemorrhage in a child with marked hypofibrinogenemia.61
Inhibition of the synthesis of anticoagulant proteins is likely responsible for thrombotic events. L-ASP decreases the synthesis of antithrombin III, a physiologic anticoagulant and protease inhibitor. Circulating levels of this factor fall to 50% or less compared with levels in controls after single large doses of L-ASP.50 Also inhibited are the syntheses of vitamin K–dependent inhibitors of clotting, protein C, and its cofactor protein S.
Thrombosis in the central nervous system is a particularly problematic complication of therapy with L-ASP.62, 63, 64 This occurred in 1% of patients receiving 30 weeks of continuous L-ASP therapy in a Dana Farber Cancer Institute pediatric ALL trial.65 It typically involves the transverse or sagittal sinus circulation of the brain, where it causes seizures, headache, confusion, and stroke symptoms. Subclinical sinus occlusions can be detected by magnetic resonance imaging in patients with modest complaints of headache and undoubtedly occur more frequently than recognized clinically. Catheter-related venous thrombosis may give rise to superior vena cava or internal jugular vein thrombosis.55
Interestingly, L-ASP–associated thromboses are thought to occur with increased frequency in patients with underlying inherited disorders of clotting.54 A survey of 289 children with ALL treated in a German cooperative group (Bonn, Frankfurt, Munster group) trial disclosed events in 32 patients, of whom 27 (85%) had one or more defects predisposing to thrombosis. These defects included the TT677 mutation in methylene tetrahydrofolate reductase (which causes homocysteine elevation in plasma), factor V Leiden, deficiency in protein C or protein S, elevated lipoprotein(a), and the G20210A variant of prothrombin. In this study, 27 of 58 patients (47%) with one or more of these defects experienced a thrombotic event, compared with only 5 of 231 patients (2%) with no prothrombotic defect. The overall incidence of prothrombotic abnormalities in a White population is approximately 20%. Prophylactic anticoagulation for patients at high risk may be effective but requires more extensive study in view of the possibility of hemorrhagic side effects of anticoagulation.55 A careful family history should be obtained prior to the initiation of L-ASP therapy, with consideration given to obtaining laboratory tests to screen for prothrombotic conditions.
In an attempt to prevent thrombosis, pilot trials have used prophylactic replacement of antithrombin III in children undergoing L-ASP treatment66 and have observed no thrombotic episodes in the small numbers of children thus treated. A second study of 17 adult patients receiving L-ASP with recombinant antithrombin III (AT) found no posttreatment episodes of thrombosis, as compared to 10 episodes in 54 patients not receiving AT.67
Other toxicities are not as easily explained by the drug's mode of action. In 25% of patients, cerebral dysfunction with confusion, stupor, or frank coma may develop. The latter syndrome resembles ammonia toxicity and has, in some cases, been associated with elevated serum ammonia levels.63 Some of these mental status changes probably represent incompletely evaluated episodes of cortical sinus thrombosis, which is visualized by MRI.64
Acute pancreatitis is an infrequent complication that occurs in fewer than 15% of patients, but it may progress to severe hemorrhagic pancreatitis. In most of the affected individuals, a transient increase in serum amylase concentration may coincide with mild nausea, vomiting, and abdominal pain, and these signs of pancreatitis quickly resolve with discontinuation of the drug. L-ASP is frequently the cause of abnormal liver function test results, including increased serum levels of bilirubin, serum glutamic-oxaloacetic transaminase, and alkaline phosphatase. Liver biopsy reveals fatty metamorphosis that is probably due to decreased mobilization of lipids.
Approximately two thirds of patients receiving L-ASP experience nausea, vomiting, and chills as an immediate reaction, but these side effects can be mitigated by administration of antiemetics, antihistamines, or, in extreme cases, corticosteroids. Close attention should be given to any symptoms that may be mediated by allergy, as a local allergic reaction frequently heralds a subsequent life-threatening systemic hypersensitivity reaction. L-ASP has no known toxicity to gastrointestinal mucosa or bone marrow and is thus a favorable agent for use in combination chemotherapy.
The only well-established drug interaction of L-ASP is its ability to terminate the action of methotrexate.68 The antagonism of L-asparaginase when given before methotrexate is possibly the result of inhibition of protein synthesis, with consequent prevention of cell entry into the vulnerable S phase of the cell cycle. An alternative explanation for antagonism is derived from the inhibition of methotrexate polyglutamylation by L-asparaginase pretreatment,69 with decreased retention of methotrexate by tumor cells. After a single intravenous dose of L-ASP, inhibition of DNA synthesis lasts for approximately 10 days, a period during which cells are refractory to methotrexate. This interval is followed by a period of increased DNA synthetic activity as cells recover from the block in protein synthesis; during this recovery period, cells are thought to be particularly vulnerable to methotrexate.70 These considerations form the rationale for clinical trials that use an initial dose of L-ASP, followed in 10 to 14 days by methotrexate, and then a second dose of L-ASP to abbreviate methotrexate toxicity.71 The results of this approach are inconclusive.
The immunosuppressive properties of L-ASP have been demonstrated in animals and may contribute to high rates of infection with bacteria and fungal organisms, as reported in certain ALL trials in which patients were randomized to receive or not receive high doses of E. coli L-ASP.72 Hyperglycemia, hypoalbuminemia, and catheter-related thrombosis in patients treated with the drug may also contribute to the risk of infection.
1. Haley EE, Fischer GA, Welch AD. The requirement for L-asparagine of mouse leukemic cells L5178Y in culture. Cancer Res 1961;21:532.
2. Kidd JG. Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum, I: course of transplanted cancers of various kinds in mice and rats given guinea pig serum, horse serum, or rabbit serum. J Exp Med 1953;98:565.
3. Broome JD. Evidence that the L-ASP of guinea pig serum is responsible for its antilymphoma effects, I: properties of the L-ASP of guinea pig serum in relation to those of the antilymphoma substance. J Exp Med 1963;118:99.
4. Hill JM, Loeb E, MacLellan A, et al. Response to highly purified L-ASP during therapy of acute leukemia. Cancer Res 1969;29:1574.
5. Ohnuma T, Holland JF, Meyer P. Erwinia carotovora asparaginase in patients with prior anaphylaxis to asparaginase from E. coli. Cancer 1972;30:376.
6. Todeschini G, Tecchio C, Meneghini V, et al. Estimated 6-year event-free survival of 55% in 60 consecutive adult acute lymphoblastic leukemia patients treated with an intensive phase II protocol based on a high induction dose of daunorubicin. Leukemia 1998;12:144.
7. Daenen S, van Imhoff GW, van den Berg E, et al. Improved outcome of adult acute lymphoblastic leukaemia by moderately intensified chemotherapy which includes a pre-induction course for rapid tumour reduction: preliminary results on 66 patients. Br J Haematol 1998;100:273.
8. Braun S, Schlimok G, Heumos I, et al. ErbB2 overexpression on occult metastatic cells in bone marrow predicts poor clinical outcome of stage I-III breast cancer patients. Cancer Res 2001;61:1890.
9. Ho PK, Milikin EB, Bobbitt JL, et al. Crystalline L-ASP from E. coli B: purification and chemical characterization. J Biol Chem 1970; 245:3703.
10. Maita T, Matsuda G. The primary structure of L-ASP from Escherichia coli. Hoppe Seylers Z Physiol Chem 1980;361:105.
11. Gilbert HJ, Blazek R, Bullman HMS, et al. Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora. J Gen Microbiol 1986;132:151.
12. Minton NP, Bullman HMS, Scawen MD, et al. Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene. Gene 1986;46:25.
13. Howard JB, Carpenter FH. L-ASP from Erwinia carotovora: substrate specificity and enzymatic properties. J Biol Chem 1972;247:1020.
14. Swain AL, Jaskolski M, Housset D, et al. Crystal structure of Escherichia coli L-ASP, an enzyme used in cancer therapy. Proc Natl Acad Sci USA 1993;90:1474.
15. Wade HE, Robinson HK, Phillips BW. L-ASP and glutaminase activities of bacteria. J Gen Microbiol 1971;69:249.
16. Dunlop PC, Meyer GM, Roon RJ. Reactions of asparaginase II of Saccharomyces cerevisiae: a mechanistic analysis of hydrolysis and hydroxylaminolysis. J Biol Chem 1980;255:1542.
17. Lachman LB, Handschumacher RE. The active site of L-asparaginase: dimethylsulfoxide effect of 5-diazo-4-oxo-l-norvaline interactions. Biochem Biophys Res Commun 1976;73:1094.
18. Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med. 2004;350:1535.
19. Amylon MD, Shuster J, Pullen J, et al. Intensive high-dose asparaginase consolidation improves survival for pediatric patients with T cell acute lymphoblastic leukemia and advanced stage lymphoblastic lymphoma: a Pediatric Oncology Group study. Leukemia 1999;13:335.
20. Haley EE, Fischer GA, Welch AD. The requirement for L-asparagine of mouse leukemia cells L5178Y in culture. Cancer Res 1961;21:532.
21. Hawkins DS, Park JR, Thomson BG, et al. Asparaginase pharmacokinetics after intensive polyethylene glycol-conjugated L-asparaginase therapy for children with relapsed acute lymphoblastic leukemia. Clin Cancer Res 2004;10:5335.
22. Horowitz B, Madras BK, Meister A, et al. Asparagine synthetase activity of mouse leukemia. Science 1968;160:533.
23. Keefer JF, Moraga DA, Schuster SM. Comparison of glycine metabolism in mouse lymphoma cells either sensitive or resistant to L-ASP. Biochem Pharmacol 1985;34:559.
24. Story MD, Voehringer DW, Stephens LC, et al. L-ASP kills lymphoma cells by apoptosis. Cancer Chemother Pharmacol 1993;32:129.
25. Haskell CM, Canellos GP. L-ASP resistance in human leukemia-asparagine synthetase. Biochem Pharmacol 1969;18:2578.
26. Worton KS, Kerbel RS, Andrulis IL. Hypomethylation and reactivation of the asparagine synthetase gene induced by L-asparaginase and ethyl methanesulfonate. Cancer Res 1991;51:985.
27. Stams WAG, Den Boer ML, Beverloo HB, et al. Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21) pediatric ALL. Blood 2003;101:2743.
28. Irino T, Kitoh T, Koami K, et al. Establishment of real-time polymerase chain reaction method for quantitative analysis of asparagine synthetase expression. J Mol Diagnostics 2004;6:217.
29. Asselin BL. The three asparaginases: comparative pharmacology and optimal use in childhood leukemia. Adv Exp Med Biol 1999;456:621.
30. Holleman A, Den Boer ML, Kazemier KM, et al. Resistance to different classes of drugs is associated with impaired apoptosis in childhood acute lymphoblastic leukemia. Blood 2003; 102:4541.
31. Liu YP, Handschumacher RE. Nitroasparaginase: subunit cross-linkage and altered substrate specificity. J Biol Chem 1972;247:66.
32. Keating MJ, Holmes R, Lerner S, et al. L-ASP and PEG asparaginase: past present and future. Leuk Lymphoma 1993;10(Suppl):153.
33. Yasura T, Kamisaki Y, Wada H, et al. Immunological studies on modified enzymes, I: soluble L-ASP/mouse albumin copolymer with enzyme activity and substantial loss of immunosensitivity. Int Arch Allergy Appl Immunol 1981;64:11.
34. Molino A, Pelosi G, Micciolo R, et al. Bone marrow micrometastases in 109 breast cancer patients: correlations with clinical and pathological features and prognosis. Breast Cancer Res Treat 1997;42:23.
35. Campbell HA, Mashburn LT, Boyse SE, et al. Two L-ASPs from E. coli B: their separation, purification, and antitumor activity. Biochemistry 1967;6:721.
36. Mashburn LT, Landin LM. Some physiochemical aspects of L-ASP therapy. Recent Results Cancer Res 1970;33:48.
37. Broome JD. Factors which may influence the effectiveness of L-ASPs as tumor inhibitors. Br J Cancer 1968;22:595.
38. Yellin TO, Wriston JC Jr. Purification and properties of guinea pig serum asparaginase. Biochemistry 1966;5:1605.
39. Law AS, Wriston JC Jr. Purification and properties of Bacillus coagulans L-ASP. Arch Biochem Biophys 1971;147:744.
40. Roberts J, Holcenberg JS, Dolowy WC. Isolation, crystallization, and properties of Achromobacteraceae glutaminase-asparaginase with antitumor activity. J Biol Chem 1972;247:84.
41. Cooney DA, Capizzi RI, Handschumacher RE. Evaluation of L-asparagine metabolism in animals and man. Cancer Res 1970; 30:929.
42. 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 Children's Cancer Study Group. Cancer Res 1979;39:3893.
43. Asselin BL, Lorenson MY, Whitin JC, et al. Measurement of serum L-asparagine in the presence of L-ASP requires the presence of an L-ASP inhibitor. Cancer Res 1991;51:6568.
44. Asselin BL, Whitin JC, Coppola DJ, et al. Comparative pharmacokinetic studies of three asparaginase preparations. J Clin Oncol 1993;11:1780.
45. Otten J, Suciu S, Lutz P, et al. The importance of L-ASP (A'ASE) in the treatment of acute lymphoblastic leukemia in children: results of the EORTC 58881 randomized phase trial showing greater efficiency of Escherichia coli as compared to Erwinia A'ASE [abstract]. Blood 1996;88(Suppl 1):669. Abstract 2663.
46. Peterson RC, Handschumacher RF, Mitchell MS. Immunological responses to L-ASP. J Clin Invest 1971;50:1080.
47. Ho DH, Brown NS, Yen A, et al. Clinical pharmacology of polyethylene glycol-L-ASP. Drug Metab Disp 1986;14:349.
48. Clavell LA, Gelber RD, Cohen HJ, et al. Four-agent induction and intensive asparaginase therapy for treatment of childhood acute lymphoblastic leukemia. N Engl J Med 1986;315:657.
49. Jones B, Holland JF, Glidewell O, et al. Optimal use of L-asparaginase (NSC-109229) in acute lymphocytic leukemia. Med Pediatr Oncol 1977;3:387.
50. Rutter DA. Toxicity of asparaginases. Lancet 1975;1:1293.
51. Land VJ, Sutow WW, Fernbach DJ, et al. Toxicity of L-asparaginase in children with advanced leukemia. Cancer 1972;40:339.
52. Khan A, Hill JM. Atopic hypersensitivity to L-ASP. Int Arch Allergy 1971;40:463.
53. Killander D, Dohlwitz A, Engstedt L, et al. Hypersensitive reactions and antibody formation during L-ASP treatment of children and adults with acute leukemia. Cancer 1976;37:220.
54. Nowak-Gottl U, Wermes C, Junker R, et al. Prospective evaluation of the thrombotic risk in children with acute lymphoblastic leukemia carrying the MTHFR TT 677 genotype, the prothrombin G20210A variant, and further prothrombotic risk factors. Blood 1999;93:1595.
55. Sills RH, Nelson DA, Stockman JA III. L-ASP-induced coagulopathy during therapy of acute lymphocytic leukemia. Med Pediatr Oncol 1978;4:311.
56. Sutor AH, Mall V, Thomas KB. Bleeding and thrombosis in children with acute lymphoblastic leukaemia, treated according to the ALL-BFM-90 protocol. Klin Padiatr 1999;211:201.
57. Nowak-Gottl U, Ahlke E, Fleischhack G, et al. Thromboembolic events in children with acute lymphoblastic leukemia (BFM protocols): prednisone versus dexamethasone administration. Blood 2003;101:2529.
58. Gralnick HR, Henderson E. Hypofibrinogenemia and coagulation factor deficiencies with L-ASP treatment. Cancer 1970;27:1313.
59. Ramsay NKC, Coccia PF, Krivit W, et al. The effect of L-asparaginase on plasma coagulation factors in acute lymphoblastic leukemia. Cancer 1977;40:1398.
60. Shapiro RS, Gerrard JM, Ramsay NK, et al. Selective deficiency in collagen-induced platelet aggregation during L-asparaginase therapy. Am J Pediatr Hematol Oncol 1980;2:207.
61. Cairo MS, Lazarus K, Gilmore RL, et al. Intracranial hemorrhage and focal seizures secondary to use of L-ASP during induction therapy of acute lymphocytic leukemia. J Pediatr 1980;97:829.
62. Mitchell L, Hoogendoorn H, Giles AR, et al. Increased endogenous thrombin generation in children with acute lymphoblastic leukemia: risk of thrombotic complications in L-ASP-induced antithrombin III deficiency. Blood 1994;83:386.
63. Leonard JV, Kay JDS. Acute encephalopathy and hyperammonaemia complicating treatment of acute lymphoblastic leukaemia with asparaginase. Lancet 1986;1:162.
64. Bushara KO, Rust RS. Reversible MRI lesions due to pegaspargase treatment of non-Hodgkin's lymphoma. Pediatr Neurol 1997;17: 185.
65. Silverman LB, Gelber RD, Dalton VK, et al. Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91-01. Blood 2001;97:1211.
66. Alberts SR, Bretscher M, Wiltsie JC, et al. Thrombosis related to the use of L-ASP in adults with acute lymphoblastic leukemia: a need to consider coagulation monitoring and clotting factor replacement. Leuk Lymphoma 1999;32:489.
67. Elliott MA, Wolf RC, Hook CC, et al. Thromboembolism in adults with acute lymphoblastic leukemia during induction with L-asparaginase-containing multi-agent regimens: incidence, risk factors, and possible role of antithrombin. Leuk Lymphoma 2004;45:1545.
68. Capizzi RL. Schedule-dependent synergism and antagonism between methotrexate and L-ASP. Biochem Pharmacol 1974;23:151.
69. Jolivet J, Cole DE, Holcenberg JS, et al. Prevention of methotrexate polyglutamate formation. Cancer Res 1985;45:217.
70. Lobel JS, O'Brien RT, McIntosh S, et al. Methotrexate and asparaginase combination chemotherapy in refractory acute lymphoblastic leukemia of childhood. Cancer 1979;43:1089.
71. Harris RE, McCallister JA, Provisor DS, et al. Methotrexate/L-ASP combination chemotherapy for patients with acute leukemia in relapse: a study of 36 children. Cancer 1980;46:2004–2008.
72. Liang DC, Hung IJ, Yang CP, et al. Unexpected mortality from the use of E. coli L-ASP during remission induction therapy for childhood acute lymphoblastic leukemia: a report from the Taiwan Pediatric Oncology Group. Leukemia 1999;13:155.