Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Validation of a 30-year-old process for the manufacture of L-asparaginase from Erwinia chrysanthemi.

A 30-year-old manufacturing process for the biologic product L-asparaginase from the plant pathogen Erwinia chrysanthemi was rigorously qualified and validated, with a high level of agreement between validation data and the 6-year process database. L-Asparaginase exists in its native state as a tetrameric protein and is used as a chemotherapeutic agent in the treatment regimen for Acute Lymphoblastic Leukaemia (ALL). The manufacturing process involves fermentation of the production organism, extraction and purification of the L-asparaginase to make drug substance (DS), and finally formulation and lyophilisation to generate drug product (DP). The extensive manufacturing experience with the product was used to establish ranges for all process parameters and product quality attributes. The product and in-process intermediates were rigorously characterised, and new assays, such as size-exclusion and reversed-phase UPLC, were developed, validated, and used to analyse several pre-validation batches. Finally, three prospective process validation batches were manufactured and product quality data generated using both the existing and the new analytical methods. These data demonstrated the process to be robust, highly reproducible and consistent, and the validation was successful, contributing to the granting of an FDA product license in November, 2011.

Pharmacokinetic/pharmacodynamic relationships of asparaginase formulations: the past, the present and recommendations for the future.

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 0.4-0.7 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.

Discovery of why acute lymphoblastic leukaemia cells are killed by asparaginase: Adventures of a young post-doctoral student, Bertha K Madras.

A surprising finding was made by JG Kidd (1909-1991) that guinea pig serum could make tumours disappear in mice. A later finding made by JD Broome (1939-) showed that asparaginase could suppress or kill tumour cells. However, the major mystery was why were only tumour cells but not normal cells affected by the asparaginase? The biology underlying this mechanism was unravelled by a young post-doctoral student, Bertha K Madras (1942-) who hypothesized that cells with low asparagine synthetase are those that die following treatment with asparaginase. To test her theory, Madras developed an assay for asparagine synthetase. The hypothesis was supported by the results that cells with normal asparagine synthetase were protected, while cells with low levels of this enzyme were killed by asparaginase. The findings provide a clinical guide for the use of asparaginase in acute lymphoblastic leukaemia in children and adults.