Erwinia Asparaginase Research Articles

Results of the Dana-Farber Cancer Institute ALL Consortium Protocol 95-01 for children with acute lymphoblastic leukemia

AbstractThe Dana-Farber Cancer Institute (DFCI) Childhood ALL Consortium Protocol 95-01 was designed to minimize therapy-related morbidity for children with newly diagnosed ALL without compromising efficacy. Patients participated in randomized comparisons of (1) doxorubicin given with or without dexrazoxane, a cardioprotectant (high-risk patients), (2) intensive intrathecal chemotherapy and cranial radiation (standard-risk patients), and (3) Erwinia and Escherichia coli asparaginase (all patients). Between 1996 and 2000, 491 patients (aged 0-18 years) were enrolled (272 standard risk and 219 high risk). With a median of 5.7 years of follow-up, the estimated 5-year event-free survival (EFS) for all patients was 82% ± 2%. Dexrazoxane did not have a significant impact on the 5-year EFS of high-risk patients (P = .99), and there was no significant difference in outcome of standard-risk patients based on type of central nervous system (CNS) treatment (P = .26). Compared with E coli asparaginase, Erwinia asparaginase was associated with a lower incidence of toxicity (10% versus 24%), but also an inferior 5-year EFS (78% ± 4% versus 89% ± 3%, P = .01). We conclude that (1) dexrazoxane does not interfere with the antileukemic effect of doxorubicin, (2) intensive intrathecal chemotherapy is as effective as cranial radiation in preventing CNS relapse in standard-risk patients, and (3) once-weekly Erwinia is less toxic than E coli asparaginase, but also less efficacious.

Pharmacodynamics and Tolerability of Twice-Weekly Erwinia Asparaginase after E. coli Asparaginase Allergy in Children with ALL.

BACKGROUND: E.coli L-asparaginase (ASP) is an important component of multiagent chemotherapy for childhood ALL; however, allergic symptoms develop in 25–30% of patients (pts). Erwinia ASP is an alternative preparation which has been used after E.coli ASP allergy; however, it has not been previously shown how well E.coli ASP-allergic pts tolerate Erwinia ASP or whether they achieve therapeutic ASP levels.METHODS: On DFCI ALL Consortium Protocol 00–01 (2000–2005), newly diagnosed children with ALL (aged 1–18 years) received 30 weeks (wks) of IM ASP during consolidation beginning 7 weeks after diagnosis. All pts initially received weekly E.coli ASP. Nadir serum ASP concentrations were measured every 3 wks by a validated biochemical assay and antibodies to E.coli and Erwinia ASP were measured by ELISA every 6 wks. If E.coli ASP allergy developed, children received Erwinia ASP (25000 IU/m2/dose twice weekly) until 2003, when Erwinia ASP became unavailable.RESULTS: We analyzed the data of all 44 patients treated between 2000–2002 who received Erwinia ASP after E.coli ASP-allergy. Median age at diagnosis was 5.5 years. Median duration of E.coli ASP prior to development of allergy was 5 wks (range 1–23). 26 (59%) pts were positive for E.coli ASP antibodies. Erwinia ASP toxicities included: allergy in 15 (34%) pts (which occurred a median 8 wks after starting Erwinia, range 2–15.5), pancreatitis in 1 (2%) and insulin-requiring hyperglycemia in 1 (2%) pt. 18 (41%) pts became positive for Erwinia ASP antibodies, 4 of whom developed Erwinia ASP allergy. Highest nadir ASP concentrations observed after Erwinia ASP in the 44 E.coli-allergic pts are displayed in the Table (below). Prior data has suggested that a nadir serum ASP level of >=0.1 IU/mL is associated with asparagine depletion (therapeutic level).Nadir Serum ASP Levels after Erwinia ASP in E.coli ASP-allergic ptsNMedian (range) ASP Level (IU/mL)% with ASP Level >= 0.1 IU/mLAll E.coli ASP allergic pts440.231(0.00–3.33)84%Subsequent allergy to ErwiniaYes150.418 (0.00–3.33)80%No290.215 (0.00–0.93)86%E.coli ASP antibodypositive260.171 (0.00–3.33)73%negative180.318 (0.10–0.93)100%Erwinia ASP antibodyPositive180.171 (0.00–3.33)72%negative260.322 (0.00–0.93)92%Highest ASP concentration with E.coli ASP prior to allergySubtherapeutic(<0.1 IU/mL)200.221 (0.00–1.04)85%Therapeutic(>=0.1 IU/mL)240.323 (0.00–3.33)83%Excluding pts who switched to PEG when Erwinia became unavailable in 2003 (N=11), pts remained on Erwinia ASP for a median of 16 wks (range 2–28). 31 pts (70%) ultimately completed all planned 30 wks of ASP consolidation.CONCLUSIONS: We conclude that twice-weekly Erwinia ASP is well-tolerated and achieves detectable and potentially therapeutic serum ASP levels in the majority of E.coli ASP-allergic pts, including those with ASP antibodies (E.coli and/or Erwinia), pts who developed subsequent allergy to Erwinia ASP and pts who never achieved therapeutic nadir levels with prior E.coli ASP. Erwinia ASP should be considered as alternative therapy for pts with ALL who develop E.coli ASP allergy.

THROMBOTIC EFFECTS OF ASPARAGINASE IN TWO ACUTE LYMPHOBLASTIC LEUKEMIA PROTOCOLS (NOPHO ALL-1992 VERSUS NOPHO ALL-2000): A Single-Institution Study

Asparaginase is essential in the treatment of lymphoproliferative malignancies, but it is associated with several side effects. The objective of this study was to compare asparaginase-induced alterations of the coagulation inhibitors and the impact on central line-associated thrombosis in children treated according to 2 different asparaginase regimens. The study enrolled 30 children treated for acute lymphoblastic leukemia, and they were divided into 2 groups with respect to asparaginase preparation and protocol (NOPHO ALL-1992 versus NOPHO ALL-2000). The coagulation inhibitors antithrombin, protein C, and proteins S were measured prior to and during asparaginase therapy, and incidence of central line-associated thromboses was compared to evaluate the protocols' thrombogenicity. Thirteen children received Erwinia asparaginase and 17 children received E. coli asparaginase. Independent of protocol, the coagulation inhibitors were significantly reduced during asparaginase therapy (p < .001), and central line-associated thromboses were frequent. Four children developed thrombosis in the course of asparaginase therapy, and there was a correlation between asparaginase-induced fall of antithrombin and occurrence of new thromboses (p = .01).

Variables Affecting Kinetics of Minimal Residual Disease Clearance in Children with Lymphoblastic Leukaemia; Results of the United Kingdom Medical Research Council (UK MRC) Protocols ALL97, ALL97/99 and ALL2003.

We studied the influence of patient, leukaemia and treatment characteristics on the kinetics of Minimal Residual Disease (MRD) clearance in children with lymphoblastic leukaemia treated using an intensive risk stratified approach. UK MRC protocol ALL97 (1997–1999), and its amended version ALL 97/99 (1999–2002), compared the efficacy and toxicity of dexamethasone (DEX) with prednisolone (PRED), and 6-thioguanine (TG) with 6-Mercaptopurine (MP) in a randomised fashion. The trial produced a 5 year event-free survival (EFS) of 80%, with better systemic and Central Nervous System outcomes in DEX compared with PRED recipients but no difference between TG and MP recipients. Several changes to the risk stratification and treatment regimens during the period of the trial provided an opportunity to determine their impact on MRD clearance. We compared this with clearance in those treated on the successor trial ALL 2003 (more intensive induction containing DEX and Pegylated Asparaginase). The variables investigated for their potential influence on MRD status at the end of induction (EOI) were: NCI Risk; Asparaginase intensity (Erwinia Asparaginase [ERW] in ALL 97 and early part of ALL97/99 vs native or Pegylated E. Coli Asparaginase [E. Coli] in later part of ALL 97/99 and ALL2003); DEX vs PRED; and marrow response at day 8/15 of induction (Slow Early Response [SER] >25% blasts vs Rapid Early Response [RER] ⩽ 25% blasts). MRD was assessed using either a semi-quantitative sequence-specific PCR (ALL97) or Real-Time Quantitative PCR (ALL99 and ALL 2003) of antigen receptor gene re-arrangements at EOI. MRD status was defined as NEG if no MRD was detected by two markers sensitive to 10−4; POS if > 10−4, and Positive Outside Quantitative Range (POQR) if positive < 10−4. Results were available from retrospective testing in 66 ALL97 and 76 ALL97/99 patients, and 204 ALL2003 patients monitored prospectively. There was no significant difference in the proportions of patients MRD NEG, POS or POQR in steroid or NCI sub-groups. Significantly more ERW Asparaginase recipients were MRD POS compared with E.Coli (p< 0.0005). Although SER was predictive of slow MRD clearance, 80/235 (34%) RERs were MRD POS and 9/27(33%) SERs were MRD NEG. Relapse risk at 4 years is 8% in NEG v 28% in POS group in ALL97, 0% in NEG/POQR, versus 15% in POS group in ALL97/99 (p=.001) 2 of the ALL2003 patients have relapsed; one was POS and the other POQR. In the context of intensive risk stratified treatment of childhood ALL, type of Asparaginase influenced rapidity of MRD clearance, whilst NCI risk and type of steroid had no significant influence. Patients with detectable MRD < 10−4 at EOI have the same low risk of relapse as those who have undetectable MRD.

Pharmacokinetic/Pharmacodynamic Relationships of Asparaginase Formulations

The discovery of the tumour-inhibitory properties of asparaginase began 50 years ago with the observation that guinea-pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. Soon afterwards, the asparaginase of bacterial origin was isolated. The asparaginases of bacterial origin induce anti-asparaginase neutralising antibodies in a large proportion of patients (44-60%), thus negating the specific enzymatic activity and resulting in failure of the target amino acid deamination in serum. There is immunological cross-reaction between the antibodies against various formulations of native Escherichia coli-asparaginase and polyethylene glycol (PEG)-asparaginases, but not to Erwinia asparaginase, as suggested by laboratory preclinical findings. This evidence was strongly inferred from the interim analyses in the Children's Cancer Group (CCG)-1961 study. Thus, anti-E. coli or PEG-asparaginase antibodies seropositive patients may benefit from the Erwinia asparaginase. The inter-relationships between asparaginase activity, asparagine (ASN) and glutamine deamination remain largely unexplored in patients. Studies have shown that ASN depletion is insufficient to induce apoptosis in T lymphoblasts in vitro and that the inhibitory concentration of CEM T-cell line is correlated with the asparaginase concentration responsible for 50% glutamine deamination. The optimal catalysis of ASN and glutamine deamination in serum by asparaginase induces apoptosis of leukaemic lymphoblasts. The percentage of ASN and glutamine deamination was predicted by asparaginase activity. Asparaginase activity of 0.1 IU/mL provided insufficient depletion of both amino acids in high-risk acute lymphoblastic leukaemia (ALL) patients. With increasing glutamine deamination, mean asparaginase activities and percentages of post-treatment samples with effective ASN depletion (<3 micromol/L) increase. Both glutamine and ASN deamination are predicted by asparaginase activity. Further population analyses resulted in identification of sigmoid relationships between asparaginase levels and post-treatment glutamine and ASN deamination.Furthermore, pharmacodynamic analyses strongly suggested that >/=90% deamination of glutamine must occur before optimal ASN deamination takes place, due to the de novo ASN biosynthesis by the liver. These pharmacodynamic results from the best-fit population pharmacokinetic/pharmacodynamic model obtained from nonlinear mixed effects model pharmacodynamic analyses for standard-risk ALL patients are similar. These analyses produced the following results: (i) asparaginase activity </=0.4 IU/mL provided insufficient deamination of ASN, whereas >0.4-0.7 IU/mL was required for optimal (90%) ASN and glutamine deamination; and (ii) deamination of glutamine is dependent on asparaginase activity and it correlates with enhanced serum ASN deamination. Thus, glutamine deamination enhances asparaginase efficacy in ALL patients. Deamination of ASN >/=90% of control or ASN concentration <3 micromol/L may be associated with improved survival in this subset of patients. Our findings support the pharmacodynamic mechanism of PEG-asparaginase for disease control in ALL patients. These results taken together strongly support new experimental approaches for application of population pharmacokinetic/pharmacodynamic analyses to further enhance survival of leukaemia patients.

Outcome of Dana-Farber Cancer Institute (DFCI) Consortium Protocol 95–01 for Children with Newly Diagnosed Acute Lymphoblastic Leukemia (ALL).

DFCI Consortium Protocol 95–01 was designed to minimize therapy-related morbidity for children with newly diagnosed ALL without compromising efficacy. Patients were considered high risk (HR) if they met any of the following criteria: 1) white blood cell count (WBC) ≥ 50,000/microliter, 2) age between < 1.00 years or ≥ 10.00 years, 3) presence of leukemia blasts at diagnosis in a cytocentrifuged cerebrospinal fluid specimen, 4) presence of a mediastinal mass, 5) T-cell immunophenotype, or 6) presence of the Philadelphia chromosome. All other patients were considered standard risk (SR). The protocol included three randomizations designed to evaluate whether acute and late toxicities could be reduced, including comparisons of 1) Erwinia and E.coli asparaginase given weekly for 20 weeks during post-remission consolidation (SR and HR patients), 2) intrathecal chemotherapy given with or without 18 Gy cranial radiation as central nervous system (CNS)-directed treatment (SR patients only), and 3) doxorubicin given with or without dexrazoxane, a potential cardioprotectant agent, during remission induction and post-remission consolidation (HR patients only). Between 1996 and 2000, 491 eligible patients (ages 0-18 years) were enrolled, 272 of whom were classified as SR and 219 HR. With 4.6 years median follow-up, the estimated 5-year event-free survival (EFS) was 81 ± 2% for all patients. The 5-year EFS according to risk group was 86 ± 2% for SR and 76 ± 3% for HR patients (p<0.01). Erwinia asparaginase was associated with a decreased incidence of asparaginase-related toxicities compared with E.coli asparaginase (10 % versus 24%, p<0.01), but also significantly more relapses (19% versus 8%, p<0.01), including relapses involving the CNS (6% versus 1%, p<0.01). The 5-year EFS for the 139 patients randomized to Erwinia asparaginase was 78 ± 4% compared with 89 ± 3% for the 148 patients randomized to E.coli asparaginase (p<0.01). The 5-year EFS for SR patients randomized to CNS treatment without radiation (IT-only) was 82 ± 5% compared with 87 ± 4% for those randomized to receive cranial radiation (p=0.35). There were four relapses involving the CNS in the 83 SR patients randomized to IT-only (4.8%) and none in the 80 irradiated patients (0%) (p=0.12). However, three of the four CNS relapses in IT-only SR patients occurred in those who had also been randomized to Erwinia asparaginase. CNS relapses (isolated or combined with other sites) occurred in 1.9% of randomized IT-only SR patients who received E.coli asparaginase. Dexrazoxane did not significantly impact the EFS of HR patients, with a 5-year EFS of 75 ± 5% for the 105 patients randomized to doxorubicin with dexrazoxane compared with 77 ± 4% for the 101 patients randomized to receive doxorubicin alone (p=0.85). We conclude that 1) Erwinia asparaginase is less toxic but also less efficacious than E.coli asparaginase when dosed once weekly during post-remission consolidation, 2) Intensive intrathecal chemotherapy without cranial radiation provides adequate CNS prophylaxis in SR patients in the setting of effective systemic chemotherapy, and 3) Dexrazoxane does not appear to interfere with the anti-leukemic effect of Doxorubicin.

Anti-Erwinia asparaginase antibodies during treatment of childhood acute lymphoblastic leukemia and their relationship to outcome: a case-control study.

A case-control study was performed to determine whether patients who had been treated with Erwinia asparaginase as part of their treatment for childhood acute lymphoblastic leukemia (ALL) and who showed relapsed of their disease more often developed anti-asparaginase antibodies than patients who remained in remission. A group of 13 patients who showed relapsed of their disease (median follow-up 35 months) were randomly matched with control patients of the same risk group (two control patients to each case), who had received therapy of the same intensity during the same period (median follow-up 70 months). Anti- Erwinia asparaginase antibodies were measured (ELISA method) during maintenance therapy after asparaginase treatment (30,000 IU/m(2) daily for 10 days in all patients plus twice weekly for 2 weeks in intermediate-risk and high-risk ALL patients). The overall incidence of anti- Erwinia asparaginase antibodies was 8% (3 of 39 patients). There was no statistically significant difference in the incidence of antibody formation between patients who had suffered relapse (1 of 13) and those who had not (2 of 26). In two of the three patients who developed antibodies, the antibodies disappeared after some time, whereas one patient had measurable antibody levels for more than a year after asparaginase therapy. In this study, the development of anti-Erwinia asparaginase antibodies was rare and was unrelated to the risk of relapse.

Antibody formation during intravenous and intramuscular therapy with Erwinia asparaginase.

Determination of the frequency of antibody formation during first and second exposure to Erwinia asparaginase after i.v. and i.m. administration. Thirty-nine children with newly diagnosed acute lymphoblastic leukemia (ALL) were included in this prospective study. Antibodies were determined (ELISA method) in plasma from these patients on specific days during and after therapy with 30,000 IU/m(2) i.v. or i.m. every day for ten days during the induction phase (first exposure). For 19 children, antibodies were measured in plasma during and after the re-induction phase (second exposure) following treatment with 30,000 IU/m(2) i.v. or i.m. twice a week for two weeks (Mondays and Thursdays). On the same days of therapy, enzyme activity (spectrophotometric method) and the concentration of asparagine (HPLC) was determined. During the first exposure, none of the patients developed anti-Erwinia asparaginase antibodies. During the second exposure, one patient (1 of 8 patients) treated intravenously developed antibodies, which were associated with disappearance of enzyme activity and reappearance of asparagine. Three of eleven patients developed antibodies of pharmacokinetic importance after i.m. therapy. None of the children had any clinical symptoms of hypersensitivity. The formation of antibodies and subsequently altered pharmacokinetics of Erwinia asparaginase seemed to be of importance only during a second period of asparaginase therapy.

Changes in coagulation and fibrinolysis in childhood acute lymphoblastic leukaemia re-induction therapy using three different asparaginase preparations.

Recently we reported the influence of two different Escherichia coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood acute lymphoblastic leukaemia (ALL) demonstrating a significant association between ASP activity and haemostatic alterations. The present study was designed for prospective evaluation of coagulation and fibrinolytic parameters in leukaemic children receiving different ASP preparations during the course of re-induction. Forty leukaemic children receiving ASP (Medac: n = 13; Bayer: n = 10; Erwinia: n = 17) at 3-day intervals during re-induction were enrolled in this study. Blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring. Compared with Medac ASP 10,000 IU/m2, patients receiving Bayer ASP or Erwinia ASP showed significantly higher fibrinogen values. Antithrombin and plasminogen showed normal values in children after Erwinia ASP. Alpha2-antiplasmin and D-Dimer were no different in the groups studied. Neither side-effects, nor sustained asparagine depletion was observed in the majority of children treated with Erwinia ASP. Data of this study show a down-regulation of coagulation proteins in children treated with Medac ASP, less pronounced in patients after Bayer or Erwinia ASP. Since children treated with Erwinia ASP showed no adequate asparagine depletion during the course of ASP therapy, a dose adjustment should be discussed to guarantee asparagine depletion, the specific metabolic therapy for ALL.

Effects of Erwinia-asparaginase on the coagulation system.

L-Asparaginase treatment during induction therapy in acute lymphoblastic leukaemia (ALL) is known to be frequently complicated by thromboembolic events. It was recently suggested that L-asparaginase derived from Erwinia chrysanthemi alters the coagulation system less severely than does Escherichia coli asparaginase. In a series of 11 adult patients with ALL, we investigated some parameters of the coagulation system during treatment with Erwinia asparaginase. The doses employed were rather high; all patients below the age of 60 years received 15,000 U/m2 daily over 14 days. In accordance with what is known from treatment with E. coli asparaginase, we observed significant lowering of antithrombin as well as of fibrinogen. However, as to fibrinogen indeed a significant decrease had occurred prior to the institution of Erwinia asparaginase treatment. The most striking observation in the present study was that the levels of prothrombin complex, reflecting the function of K-vitamin dependent coagulation factors II, VII and X, remained within normal ranges during treatment. This indicates that these coagulation factors were not affected by Erwinia asparaginase, an observation at variance with several reports where E. coli asparaginase was investigated. This latter observation was the only finding which could lend support to the view that Erwinia asparaginase affects the coagulation system less than E. coli asparaginase. Finally, one of our patients developed a sinus thrombosis, a severe thrombotic complication.

L-Asparaginase from Erwinia carotovora: PHYSICOCHEMICAL PROPERTIES OF THE NATIVE AND SUCCINYLATED ENZYME

Abstract The physicochemical properties of the antileukemic l-asparaginase from Erwinia carotovora have been studied and compared with those of the corresponding clinically effective enzyme from Escherichia coli B. Although both enzymes are tetramers with similar molecular weight, the amino acid compositions are distinctly different and the Erwinia enzyme is more basic as judged by polyacrylamide electrophoresis. Of the 48 to 50 tyrosyl residues in native Erwinia asparaginase, only about 10% ionize with a normal pK, while 30% of these residues in the E. coli enzyme titrate normally. The native tetramer (s20,w = 7.2) is considerably more stable than the corresponding tetramer from E. coli. Asparaginase from Erwinia is only partially dissociated in 8 m urea, whereas the E. coli enzyme is completely converted to the 1.7 S monomer under the same conditions. Guanidinium chloride at concentrations of 3.5 m completely dissociates Erwinia asparaginase. Dissociation is accompanied by the appearance of an ultraviolet difference spectrum with a maximum at 288 nm. The rate of dissociation is markedly increased by the addition of alcohols to the denaturant. Dissociation in the presence of alcohols occurs in two distinct steps whose rates increase as the length of the alkyl side chain of the alcohol increases. Succinylation of the lysyl residues results in marked increases in mobility of the modified enzyme on polyacrylamide gels, but does not affect the state of aggregation of the tetramer or its catalytic activity. In addition succinylation does not affect either the rate of dissociation in guanidinium chloride or the extent of reconstitution to the active enzyme. The presence of five bands on polyacrylamide gel electrophoresis of hybrids prepared from the native and succinylated enzyme indicates that Erwinia carotovora asparaginase is composed of four identical subunits.

Erwinia carotovora asparaginase in patients with prior anaphylaxis to asparaginase from E. coli.

Asparaginase prepared from Erwinia carotovora was well tolerated when given to four patients with acute lymphocytic leukemia who previously had had anaphylactic reactions from E. coli asparaginase. The Erwinia enzyme was cleared from plasma with a half life of 7 to 13 hours, a time similar to some preparations of E. coli asparaginase in patients who had not sustained anaphylaxis. Serum asparagine levels decreased to unmeasurable levels after Erwinia asparaginase in a manner reminiscent of the E. coli enzyme. No positive precipitin reaction or skin test to either E. coli or Erwinia enzyme was detected. These results indicate that there is no great degree of immunologic cross reaction to prevent the use of the Erwinia enzyme in patients who sustained anaphylaxis to the E. coli preparation. The availability of a second active asparaginase from a different microbiological source extends the usefulness of asparaginase in clinical therapy. Stabilization and protective effect of Erwinia enzyme activity by human plasma was demonstrated.