Assessment of Aspergillus-specific PCR as a screening method for invasive aspergillosis in paediatric cancer patients and allogeneic haematopoietic stem cell recipients with suspected infections.

Invasive aspergillosis (IA) remains difficult to diagnose in immunocompromised patients, because diagnostic EORTC/MSG criteria are often not met. As biomarkers might elucidate the pathogen, we analysed the performance of an Aspergillus PCR assay in blood for diagnosis of IA in immunocompromised paediatric patients with suspected infections. Ninety-five haemato-oncological paediatric patients were included over a period of 3 years, the underlying diseases consisting of acute leukaemia, solid tumours, non-malignant immunocompromising disorders and haematopoietic stem cell transplantation recipients. We retrospectively analysed 253 consecutive episodes of suspected infections. Thirty-eight patients had possible IA, none of the patients fulfilled EORTC/MSG criteria of probable/proven IA. PCR positivity was observed in 97/967 analyses. Sensitivity, specificity, positive and negative predictive value of the PCR per episode were 34%, 78%, 31% and 81% using possible IA as endpoint. Taken together, an undirected blood screening by Aspergillus-specific PCR is of little diagnostic value in a heterogenous paediatric patient cohort. Harnessing PCR for diagnosis of IA should thus be focused on blood analyses of more homogenous high-risk patients and/or analyses of bronchoalveolar lavage, tissue or cerebrospinal fluid specimens.

Pharmacokinetic dose adjustment of Erwinia asparaginase in protocol II of the paediatric ALL/NHL-BFM treatment protocols.

Native forms of asparaginase stem from different biological sources. Previously reported data from children treated with Erwinase showed significantly lower trough levels and pharmacokinetic dose intensity than after E. coli-derived preparations. Hence, schedule optimization was initiated to achieve relevant serum activities. 21 children on reinduction therapy received Erwinase on Mondays, Wednesdays and Fridays for 3 weeks (9 x 20000 IU/m2 i.v.) instead of 4x 10 000IU/m2 of E. coli asparaginase (twice weekly for 2 weeks). Asparaginase trough activities were measured as the primary parameter, targeting 100-200 IU/I after 2 d and >50 IU/l after 3 d. Concurrently, asparagine trough concentrations were monitored. The mean trough activity was 156+/-99 IU/l, with 2/108 samples showing no detectable activity. Regarding trough levels per individual (three or more measurements/patient), means ranged from 52+/-29 to 276+/-114 IU/l (20 patients, 106 samples), with nine, six, and five children inside, below, and above the target range, respectively. The mean 3 d trough activity was 50+/-39 IU/l (20 patients, 51 samples). In 11 of these samples no activity was measurable. Mean trough activities calculated per individual ranged from < 20-84+/-30 IU/l (14 patients, 42 samples) with seven children below the target limit of 50 IU/l and asparagine concentrations <0.2 - 1.5microM. We concluded that an increased dose of 9x20000 IU/m2 of Erwinia asparaginase within 3 weeks resulted in a pharmacokinetic dose intensity comparable to former observations made with 4 x 10 000IU/m2 of the E. coli product Crasnitin which is no longer marketed. High interindividual variability and the phenomenon of 'silent' inactivation necessitate monitoring wherever possible.

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

UNLABELLED: 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. CONCLUSION: 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.

Dose reduction of asparaginase under pharmacokinetic and pharmacodynamic control during induction therapy in children with acute lymphoblastic leukaemia.

The enzyme asparaginase is an important element in the therapy of acute lymphoblastic leukaemia (ALL). The usual asparaginase dose as prescribed in the ALL-BFM-86/90 treatment protocol for the therapy of ALL is 10,000 IU/m2 at 3 d intervals and had been developed on the basis of the E. coli asparaginase preparation Crasnitin from the Bayer company. Using the described schedule the E. coli asparaginase preparation from the Medac company shows significantly higher biological activity than the Bayer preparation. These findings prompted an attempt to reduce the dose of the Asparaginase medac under careful pharmacokinetic and pharmacodynamic monitoring. At the first step of dose reduction in ALL treatment protocol I, 11 children received 5000 IU/m2 of Asparaginase medac. Another 15 children were given 2500 IU/m2 of the enzyme at the second step of dose reduction. Prior to each asparaginase dose, blood samples were taken to determine amino acids and trough enzyme activity. Concurrent with the asparaginase monitoring, the coagulation parameters were measured. 96% of samples from the first step of dose reduction (5000 IU/m2 every third day) showed complete L-asparagine depletion (< 0.1 microM), the median trough enzyme activity was 265 IU/l. At the second step of dose reduction (2500 IU/m2) complete L-asparagine depletion was seen in 97% of samples, and the median trough enzyme activity was 102 IU/l. Cerebrospinal fluid (CSF) depletion was complete in all samples tested (11/11). We concluded that an Asparaginase medac dose reduced from the usual 10000 IU/m2 down to 5000 IU/ m2 or 2500 IU/m2, applied at 3 d intervals, was sufficient to achieve complete L-asparagine depletion in serum. Changes of the fibrinogen levels was significantly less pronounced in the group on 2500 IU.

Changes in coagulation and fibrinolysis in childhood ALL: a two-step dose reduction of one E. coli asparaginase preparation.

The influence of two different E. coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood ALL was recently reported, demonstrating a clearly significant association between ASP activity and haemostatic changes. Since the Bayer preparation is no longer available for treatment of large series of patients with ALL, the present study was designed to prospectively evaluate coagulation and fibrinolytic changes in leukaemic children receiving different doses of Medac ASP, which is now available for treatment of childhood ALL. Leukaemic children in whom ASP Medac was administered at 3 d intervals in a two-step dose reduction (5000 IU/m2, n = 10; 2500 IU/m2, n = 15) were compared with children who had received Bayer ASP 10,000 IU/m2 in the same time schedule in a former randomized trial; at the same venipuncture, blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring. Compared with Bayer ASP 10,000 IU/m2, patients receiving Medac ASP 5000 IU/m2 showed significantly decreased values of fibrinogen, plasminogen, and alpha 2-antiplasmin, along with significantly enhanced thrombin generation. Improvement occurred in children treated with 2500 IU/m2 Medac ASP; alpha 2-antiplasmin and D-dimer no longer differed from the Bayer group. Since both patient groups showed complete asparagine depletion during the course of ASP administration, the lower dosage of 2500 IU/m2 administered at 3 d intervals should guarantee the specific metabolic therapy for ALL, leading to depletion of the circulating pool of asparagine.

Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations.

The antileukaemic enzyme L-asparaginase is used to achieve the greatest possible reduction in blood levels of the amino acid asparagine, an essential factor for the growth of leukaemic blasts. There are two main sources of the enzyme, E. coli and Erwinia. Faced with increasing reports of treatment complications, we established a programme to monitor enzyme activity and asparagine levels in serum, in children receiving treatment for acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL). Trough asparagine and asparaginase levels were measured in 49 children on induction treatment with different E. coli preparations (Asparaginase medac, Crasnitin) and in 52 children on re-induction (Asparaginase medac, Crasnitin, and, in the event of allergic reactions, Erwinase) just prior to each sequential application of 10000 U/m2 of asparaginase. Measurements were made by an enzyme assay and an HPLC method. During induction, both Escherichia coli preparations induced the desired reduction in asparagine, but the asparaginase activity with Asparaginase medac was significantly higher than with Crasnitin (median of trough levels 475 versus 74 U/l). Under re-induction treatment (median, Asparaginase medac 528 U/l, Crasnitin 49 U/l, and Erwinase < 20 U/l) complete asparagine depletion was recorded on day 3 in more than 90% of Asparaginase medac samples, more than 60% of Crasnitin samples and in 26% of Erwinase samples. The latter two groups included some children with unchanged asparagine levels and no measurable enzyme activity. Different asparaginase preparations are not readily interchangeable. When Asparaginase medac is used instead of Crasnitin, and identical dose will be associated with significantly higher enzyme activity, well above the level required for complete asparagine depletion. Clinical studies will need to specify both the preparation and the dose to be used. When substitution of an alternative drug is mandatory owing to allergic reactions, monitoring is advisable.

Influence of two different Escherichia coli asparaginase preparations on fibrinolytic proteins in childhood ALL.

BACKGROUND: Alterations in hemostasis have frequently been observed in patients with leukemia, and thrombotic events are well documented in patients receiving L-asparaginase (ASP) as a single agent or in combination with vincristine, prednisone (sometimes complemented by an anthracycline). The present study was designed to evaluate prospectively fibrinolytic parameters in leukemic children receiving different E. coli ASP preparations (Kyowa ASP, n = 20; Bayer ASP, n = 20), and to relate changes in the fibrinolytic system to serum ASP activity. MATERIALS AND METHODS: Blood samples for coagulation studies were obtained together with serum samples for pharmacokinetic monitoring in the same venipuncture (before the first and 6th-7th doses of ASP). RESULTS: Patients receiving Kyowa ASP showed significantly (0.0001) enhanced ASP-activity compared to children treated with the Bayer preparation. Significantly decreased values of fibrinogen (p < 0.001), plasminogen (p < 0.0002) and alpha 2-antiplasmin (p < 0.0003) were found in the Kyowa group, along with significantly enhanced thrombin generation (F1 + 2; p < 0.001), t-P (p < 0.01) and D-dimer levels (p < 0.05). In contrast, PAI 1 activity demonstrated no significant difference in the two E. coli ASP administered. CONCLUSIONS: Changes in fibrinogen, plasminogen, alpha 2-antiplasmin and D-dimer are clearly associated with ASP activity during the course of ASP administration in children with ALL.

Asparaginase decreases clotting factors in vitro: a possible pitfall?

L-Asparaginase treatment of leukemia patients causes hemostatic problems. To investigate whether L-asparaginase influences coagulation studies, 63 blood samples of 21 healthy male donors were incubated with L-asparaginase for 30 min at room temperature. After treatment with 100 IU/ml L-asparaginase plasma fibrinogen (P = 0.002), plasma antithrombin (P = 0.0002), plasma protein C (P = 0.0004), and plasma plasminogen (P = 0.0039) were decreased compared with controls. In contrast, a significant increase in plasma von Willebrand factor antigen (P = 0.08) and plasma thromboglobulin (P = 0.005) was observed. The decrease in plasma anti-thrombin (P = 0.001), plasma protein C (P = 0.0003), and plasma plasminogen (P = 0.0043) was also measurable after 0.05 IU/ml asparaginase treatment. The incubation with L-asparaginase was similar to the normal time from blood sampling to testing and hence the results suggest that L-asparaginase may directly attack proteins of the coagulation system during the interval between sampling and assay.