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C. Rizzari et al.
analyses were focused on these two time points only but with the following adjustments: (i) CSF samples were considered analyz- able when collected at a distance of 7±3 days and of 19±3 days after the second PEG-asparaginase dose (day +26) of protocol IA; (ii) the serum samples had to be collected the same day (±1) as the CSF samples. In the text, tables and figures of this report the CSF asparagine values obtained at day +33 ±3 and day +45 ±3 are sim- ply referred to as day +33 and day +45.
Sample collection
Serum and CSF sample collection started on June 1st, 2010; CSF collection ended on December 31st, 2012. Samples were collected from patients treated according to the AIEOP-BFM ALL 2009 pro- tocol in Italy (AIEOP), Germany (BFM-G), Austria (BFM-A), and the Czech Republic (CPH). CSF samples were immediately frozen at -80°C, shipped on dried ice and stocked at -80°C until amino acid analysis. Blood samples from a peripheral vein or central venous catheter were collected according to the treatment sched- ule. Serum was separated in 2 mL tubes and immediately frozen at -20°C until shipment. CSF asparagine levels and asparaginase serum activity were determined in the Laboratory of Cancer Pharmacology at the Department of Oncology of the “Mario Negri” Pharmacology Research Institute IRCCS (Milan, Italy) for the AIEOP samples, in the Clinical Pharmacology Laboratory of the Department of Pediatric Hematology and Oncology at the University Hospital of Muenster (Germany) for the BFM-G and CPH samples and in the Department of Pediatrics and Adolescent Medicine of the Medical University of Vienna (Austria) for the BFM-A CSF samples.
Determination of serum pegylated-asparaginase activity
PEG-asparaginase activity was evaluated with the commercially available enzymatic medac asparaginase activity test (MAAT) (medac GmbH, Hamburg, Germany) or with the L-aspartic β- hydroxamate (AHA) test.17
The medac asparaginase activity test
Briefly, the MAAT is an IVD-CE-certified test which is commer- cially available. It is a homogeneous microplate assay that analy- ses the catalytically active asparaginase in serum by detecting the amount of hydrolyzed substrate analogue of asparagine, quanti- fied by photometric reading at 700 nm. The assay uses calibrators containing a native enzyme preparation from E. coli (ASP medac) and has a lower limit of quantification (LLOQ) of 30 U/L. All the values below the LLOQ were considered 0 U/L for the statistical analysis. The MAAT was used for the determination of asparagi- nase activity in the serum samples of AIEOP patients.
The L-aspartic β-hydroxamate test
AHA is the substrate for the quantification of native E. coli,
pegylated E. coli, and Erwinia chrysanthemii asparaginase in human serum. Asparaginase hydrolyzes AHA to L-aspartic acid and hydroxylamine, which is determined at 710 nm after condensa- tion with 8-hydroxyquinoline and oxidation to indooxine. The LLOQ is 5 U/L.17 All the values below the LLOQ were considered 0 U/L for the statistical analysis. The AHA test was used for the determination of asparaginase activity in serum of CPH and BFM- G samples. Since the AHA test calibrates against known amounts of PEG-asparaginase in contrast to the MAAT, which uses native E. coli asparaginase as the calibrator, it considers the different sub- strate turnover rates of PEG-asparaginase compared to native E. coli asparaginase under the assay conditions. Thus, the PEG- asparaginase activity determined by the MAAT is a mean of 1.42 higher than that determined by the AHA test, as recently demon- strated.18
Determination of cerebrospinal fluid asparagine levels
CSF asparagine levels were measured using a high performance liquid chromatographic technique after derivatization with o- phthaldialdehyde as described by Turnell and Cooper19 and already used in previous pharmacological studies performed by the AIEOP-BFM group.15,20 The LLOQ was 0.2 μmol/L and all the analyzed data with results below this limit were considered 0 μmol/L for the statistical analysis. Since bloody CSF punctures might have altered the quantification of asparagine, either though the release of asparagine present in the erythrocytes or through possible contamination by asparaginase, CSF samples contaminat- ed with blood were excluded from the analysis.
Informed consent
All patients and their parents or legal guardians signed appropri- ate informed consent for the biological study procedure encom- passed in the AIEOP-BFM ALL 2009 study for the asparaginase therapeutic drug monitoring. Assent was given by patients accord- ing to ethical standards and national guidelines. Protocol studies were approved by each national and local review board, in accor- dance with the Declaration of Helsinki and national laws.
Statistical analysis
Descriptive analyses include the distribution of patients’ charac- teristics and dot plots on CSF asparagine concentration and PEG- asparaginase serum activity. Box plots and scatter plots were used to describe continuous values, with the Wilcoxon test to compare medians. Data are presented separately on the original scale according to the type of enzymatic test used, which was MAAT for AIEOP samples and AHA for all other samples, collectively identified as BFM samples.
Results
Between June 2010 and December 2012 1,764 patients were unselectively enrolled in Italian, German, Czech, and Austrian centers adopting the AIEOP-BFM ALL 2009 study protocol. Overall, 736 patients were included in the present study, 245 of whom belonged to the AIEOP cohort and 491 to the BFM cohort. Their main biological and clinical characteristics are presented in Table 1. The distribution of these characteristics is superimposable to that of the entire cohort of 1,764 patients enrolled in the study in the same period (data not shown).
The total number of CSF samples collected in the two groups was 903. Overall, 903 CSF samples were collected on days 33 and/or 45, of which 314 in the AIEOP cohort and 589 in the BFM cohort.
Asparagine levels in cerebrospinal fluid
Of the 903 CSF samples analyzed for asparagine levels, 686 (AIEOP n=230 and BFM n=456) were collected on protocol day +33 and 217 (AIEOP n=84 and BFM n=133) on protocol day +45. The distribution of different CSF asparagine levels detected at the CSF punctures (on days +33 and +45) is presented in Table 2 and Figure 1 (A and B for the AIEOP and BFM cohorts, respectively). Given that the physiological concentration of asparagine in the CSF of children and adolescents ranges between 4 and 10 μmol/L, the CSF asparagine levels found in this study were overall quite consistently reduced at both time points, as depicted in Figure 1. Independently of the levels of asparaginase activity, CSF asparagine levels were signifi- cantly reduced during the investigated study phase but
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haematologica | 2019; 104(9)