Structural Characterisation of Non-Deamidated Acidic Variants of Erwinia chrysanthemi L-asparaginase Using Small-Angle X-ray Scattering and Ion-Mobility Mass Spectrometry.

PURPOSE: Erwinia chrysanthemi L-asparaginase (ErA) is an enzyme commonly used in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). Biopharmaceutical products such as ErA must be monitored for modifications such as deamidation, typically using ion-exchange chromatography (IEX). Analysis of clinical-grade ErA using native IEX resolves a number of enzymatically-active, acidic variants that were poorly characterised. METHODS: ErA IEX variants were isolated and fully characterised using capillary electrophoresis (cIEF), LC-MS and LC-MS/MS of proteolytic digests, and structural techniques including circular dichroism, small-angle X-ray scattering (SAXS) and ion-mobility mass spectrometry (IM-MS). RESULTS: LC-MS, MS/MS and cIEF demonstrated that all ErA isolates consist mainly of enzyme lacking primary-sequence modifications (such as deamidation). Both SAXS and IM-MS revealed a different conformational state in the most prominent acidic IEX peak. However, SAXS data also suggested conformational differences between the main peak and major acidic variant were minor, based on comparisons with crystal structures. CONCLUSIONS: IEX data for biopharmaceuticals such as ErA should be thoroughly characterised, as the most common modifications, such as deamidation, may be absent.

Comparison of Escherichia coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer-Children's Leukemia Group phase 3 trial.

Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and lymphoblastic lymphoma in children. It has minimal bone marrow toxicity. Its major side effects are anaphylaxis, pancreatitis, diabetes, coagulation abnormalities, and thrombosis, especially intracranial. It is derived from 2 different sources: Escherichia coli and Erwinia chrysanthemi. Nonrandomized clinical studies have suggested a similar efficacy of these 2 types of asparaginases and a lower toxicity for Erwinia-asparaginase. The European Organisation for Research and Treatment of Cancer-Children's Leukemia Group (EORTC-CLG) 58881 trial randomized 700 children with acute lymphoblastic leukemia or lymphoblastic lymphoma to either E coli- or Erwinia-asparaginase at the same dosage of 10 000 IU/m(2) twice weekly to compare toxicity and efficacy. Coagulation abnormalities were more frequent in the E coli-asparaginase than in the Erwinia-asparaginase arm of the study (30.2% versus 11.9%, P <.0001). The incidence of other toxicity was not significantly different. In the Erwinia-asparaginase arm, more patients failed to achieve complete remission (4.9% versus 2.0%; P =.038) and the relapse rate was higher, leading to shorter event-free survival (hazard ratio,1.59; 95% CI, 1.23-2.06; P =.0004). The estimate of event-free survival rate (SE) at 6 years was 59.8% (2.6%) versus 73.4% (2.4%). Overall survival rate at 6 years was also lower in the Erwinia-asparaginase arm at 75.1% (2.3%) versus 83.9% (2.0%), P =.002. With the dose scheduling used in this protocol, E coli-asparaginase induced more coagulation abnormalities but was superior to Erwinia-asparaginase for the treatment of childhood lymphoid malignancies.

Cryoprecipitate-induced mesenteric venous thrombosis during L-asparaginase therapy for acute lymphoblastic leukaemia.

We present a case of fatal mesenteric vein thrombosis (MVT) associated with L-asparaginase (L-asp) therapy and temporally related to cryoprecipitate infusion, in an adult with acute lymphoblastic leukaemia (ALL). Cryoprecipitate was given on two consecutive days to raise a low fibrinogen level of 0.7 g/L, in the presence of severe thrombocytopenia and mucocutaneous bleeding. The thrombotic event presented as sudden abdominal pain a day after the second cryoprecipitate infusion, which raised the fibrinogen to 1.5 g/L. Concurrent levels of antithrombin III (AT III), protein C (PC) and protein S (PS) were very low. The patient died after laparotomy and wide resection of gangrenous bowel. We believe this is the first reported case in the English literature of a patient who developed mesenteric venous thrombosis during L-asp therapy, and once more we advise caution in using conventional blood products, especially cryoprecipitate, and recommend restricting the use of cryoprecipitate and fresh frozen plasma (FFP) to the treatment of serious hemorrhagic manifestations, until new effective and safe therapies are available.

Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91-01.

The Dana-Farber Cancer Institute (DFCI) acute lymphoblastic leukemia (ALL) Consortium Protocol 91-01 was designed to improve the outcome of children with newly diagnosed ALL while minimizing toxicity. Compared with prior protocols, post-remission therapy was intensified by substituting dexamethasone for prednisone and prolonging the asparaginase intensification from 20 to 30 weeks. Between 1991 and 1995, 377 patients (age, 0-18 years) were enrolled; 137 patients were considered standard risk (SR), and 240 patients were high risk (HR). Following a 5.0-year median follow-up, the estimated 5-year event-free survival (EFS) +/- SE for all patients was 83% +/- 2%, which is superior to prior DFCI ALL Consortium protocols conducted between 1981 and 1991 (P =.03). There was no significant difference in 5-year EFS based upon risk group (87% +/- 3% for SR and 81% +/- 3% for HR, P =.24). Age at diagnosis was a statistically significant prognostic factor (P =.03), with inferior outcomes observed in infants and children 9 years or older. Patients who tolerated 25 or fewer weeks of asparaginase had a significantly worse outcome than those who received at least 26 weeks of asparaginase (P <.01, both univariate and multivariate). Older children (at least 9 years of age) were significantly more likely to have tolerated 25 or fewer weeks of asparaginase (P <.01). Treatment on Protocol 91-01 significantly improved the outcome of children with ALL, perhaps due to the prolonged asparaginase intensification and/or the use of dexamethasone. The inferior outcome of older children may be due, in part, to increased intolerance of intensive therapy.