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Introduction The current understanding of prognostic factors of functional recovery after a proximal femoral fracture is limited, and enhancements could improve the prognostic accuracy and target subgroups for additional care strategies. This systematic review aims to identify all studied factors with an independent prognostic value for the long-term functional recovery of patients with a proximal femoral fracture. Materials and methods Observational studies with multivariate analyses on prognostic factors of long-term functional outcome after proximal femoral fractures were obtained through an electronic search performed on November 9, 2018. Results In the 31 included articles, thirteen prognostic factors were studied by at least two independent studies and an additional ten by only one study. Age, comorbidity, functionality and cognition were factors for which the majority of studies indicated a significant effect. The majority of studies which included sex as a factor found no significant effect. The level of evidence for the remaining factors was deemed too low to be conclusive on their relevance for long-term functional outcome. Conclusion The identified factors showed overlap with prognostic factors of short-term functional outcomes and mortality. The validity and applicability of prognostic models based on these factors may be of interest for future research.

We prospectively studied the incidence and clinical course of hypertriglyceridemia and hypercholesterolemia during very prolonged use of asparaginase in relation to levels of asparaginase activity in children with acute lymphoblastic leukemia. We also evaluated the incidence of pancreatitis, thrombosis, hyperammonemia and central neurotoxicity and their association with asparaginase activity levels. Eighty-nine patients were treated according to the Dutch Childhood Oncology Group Acute Lymphoblastic Leukemia 10 medium-risk intensification proto-col, which includes 15 doses of PEGasparaginase (2,500 IU/m(2)) over 30 weeks. Erwinia asparaginase (20,000 IU/m(2)) was administered when allergy to or silent inactivation of PEGasparaginase occurred. Triglyceride, cholesterol and ammonia levels increased rapidly in children treated with PEGasparaginase and remained temporarily elevated, but normalized after administration of the last asparaginase dose. Among the patients treated with PEGasparaginase, hypertriglyceridemia and hypercholesterolemia (grade 3/4) were found in 47% and 25%, respectively. The correlation between PEGasparaginase activity levels and triglyceride levels was strongest at week 5 (Spearman correlation coefficient=0.36, P=0.005). The triglyceride levels were higher in children >= 10 years old than in younger patients (

Malignant cells infiltrating the bone marrow (BM) interfere with normal cellular behaviour of supporting cells, thereby creating a malignant niche. We found that CXCR4-receptor expression was increased in paediatric precursor B-cell acute lymphoblastic leukaemia (BCP-ALL) cells compared with normal mononuclear haematopoietic cells (P < 0·0001). Furthermore, high CXCR4-expression correlated with an unfavourable outcome in BCP-ALL (5-year cumulative incidence of relapse ± standard error: 38·4% ± 6·9% in CXCR4-high versus 12% ± 4·6% in CXCR4-low expressing cases, P < 0·0001). Interestingly, BM levels of the CXCR4-ligand (CXCL12) were 2·7-fold lower (P = 0·005) in diagnostic BCP-ALL samples compared with non-leukaemic controls. Induction chemotherapy restored CXCL12 levels to normal. Blocking the CXCR4-receptor with Plerixafor showed that the lower CXCL12 serum levels at diagnosis could not be explained by consumption by the leukaemic cells, nor did we observe an altered CXCL12-production capacity of BM-mesenchymal stromal cells (BM-MSC) at this time-point. We rather observed that a very high density of leukaemic cells negatively affected CXCL12-production by the BM-MSC while stimulating the secretion levels of granulocyte colony-stimulating factor (G-CSF). These results suggest that highly proliferative leukaemic cells are able to down-regulate secretion of cytokines involved in homing (CXCL12), while simultaneously up-regulating those involved in haematopoietic mobilization (G-CSF). Therefore, interference with the CXCR4/CXCL12 axis may be an effective way to mobilize BCP-ALL cells.

Background Mesenchymal cells (MSCs) in bone marrow (BM) may produce asparagine and form protective niches for leukemic cells. In vitro, this led to high levels of asparagine and conferred asparaginase resistance to acute lymphoblastic leukemia (ALL) cells. The aim of this study was to investigate whether MSCs or other cells in BM indeed produce such significant amounts of asparagine in vivo as to result in clinical asparaginase resistance. Procedure Twenty-six patients with newly diagnosed ALL were enrolled. All children received induction chemotherapy according to the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase was administered from days 12–33. Asparaginase, asparagine, aspartic acid, glutamine, and glutamic acid levels were measured in BM and blood at diagnosis, days 15, 33, and 79. Results Median asparaginase trough levels were not significantly different at days 15 and 33. Only at diagnosis, asparagine level was significantly higher in BM than in blood (P = 0.001). Asparagine levels were all below the lower limit of quantification in BM and blood at days 15 and 33. However, aspartic acid level in BM was significantly higher than in blood (P

Asparaginase antibodies (AAAs) neutralize asparaginase and are often accompanied by a clinically overt allergic reaction, but in a proportion of the patients these antibodies occur without any signs of hypersensitivity, termed as silent inactivation.[1][1],[2][2] Vrooman et al. and Panosyan et al.

This study prospectively analyzed the efficacy of very prolonged courses of pegylated Escherichia coli asparaginase (PEGasparaginase) and Erwinia asparaginase in pediatric acute lymphoblastic leukemia (ALL) patients. Patients received 15 PEGasparaginase infusions (2500 IU/m(2) every 2 weeks) in intensification after receiving native E coli asparaginase in induction. In case of allergy to or silent inactivation of PEGasparaginase, Erwinia asparaginase (20 000 IU/m(2) 2-3 times weekly) was given. Eighty-nine patients were enrolled in the PEGasparaginase study. Twenty (22%) of the PEGasparaginase-treated patients developed an allergy; 7 (8%) showed silent inactivation. The PEGasparaginase level was 0 in all allergic patients (grade 1-4). Patients without hypersensitivity to PEGasparaginase had serum mean trough levels of 899 U/L. Fifty-nine patients were included in the Erwinia asparaginase study; 2 (3%) developed an allergy and none silent inactivation. Ninety-six percent had at least 1 trough level >= 100 U/L. The serum asparagine level was not always completely depleted with Erwinia asparaginase in contrast to PEGasparaginase. The presence of asparaginase antibodies was related to allergies and silent inactivation, but with low specificity (64%). Use of native E coli asparaginase in induction leads to high hypersensitivity rates to PEGasparaginase in intensification. Therefore, PEGasparaginase should be used upfront in induction, and we suggest that the dose could be lowered. Switching to Erwinia asparaginase leads to effective asparaginase levels in most patients. Therapeutic drug monitoring has been added to our ALL-11 protocol to individualize asparaginase therapy.

_To the Editor:_ Asparaginase is an enzyme which hydrolyses asparagine into aspartic acid and ammonia. Levels of asparaginase activity are relatively easy to measure, and a trough level >100 U/L is associated with complete asparagine depletion. So, the measurement of levels of asparaginase activity is a very useful tool to monitor asparaginase activities in clinical practice. However, these assays are not available in all countries in contrast to ammonia measurements. Recently, a Polish study group published ammonia levels of 87 children during acute lymphoblastic leukemia (ALL) induction therapy. They concluded that the ammonia concentration reflects the levels of asparaginase activity and could be useful when asparaginase activity assays are not available. We evaluated if ammonia levels could be used as a surrogate marker of levels of asparaginase activity. [...]

The diferrous oxidation level of carboxylate-bridged non-heme diiron proteins is pivotal because only it reacts directly with dioxygen. In this state each ferrous ion has ST = 2. Such integer-spin, so-called “Non-Kramers” (NK) systems typically are difficult to access by paramagnetic resonance techniques, and this property has precluded the application of ENDOR and ESEEM spectroscopies to this state. Integer-spin systems frequently exhibit a ground state “Non-Kramers doublet”, however, which is split in zero applied field by an energy within the microwave range, thus rendering the center EPR active. Recently, we showed that ENDOR and ESEEM spectroscopies also can be applied to NK doublets, in preliminary measurements on diferrous methane monoxygenase hydroxylases (MMOHred) Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b and azido-hemerythrin (N3Hrred) (Hoffman, B. M.; Sturgeon, B. E.; Doan, P. E.; DeRose, V. J.; Liu, K. E.; Lippard, S. J. J. Am. Chem. Soc. 1994, 116, 6023−6024). Buildi...

Asparaginase is an expensive drug, but important in childhood acute lymphoblastic leukemia. In order to compare costs of PEGasparaginase, Erwinia asparaginase and native E. coli asparaginase, we performed a cost-analysis in the Dutch Childhood Oncology Group ALL-10 medium-risk group intensification protocol. Treatment costs were calculated based on patient level data of 84 subjects, and were related to the occurrence of allergy to PEGasparaginase. Simultaneously, decision tree and sensitivity analyses were conducted. The total costs of the intensification course of 30 weeks were $57,893 in patients without PEGasparaginase allergy (n=64). The costs were significantly higher ($113,558) in case of allergy (n=20) necessitating a switch to Erwinia asparaginase. Simulated scenarios (decision tree analysis) using native E. coli asparaginase in intensification showed that the costs of PEGasparaginase were equal to those of native E. coli asparaginase. Also after sensitivity analyses, the costs for PEGasparaginase were equal to those of native E. coli asparaginase. Intensification treatment with native E. coli asparaginase, followed by a switch to PEGasparaginase, and subsequently to Erwinia asparaginase in case of allergy had similar overall costs compared to the treatment with PEGasparaginase as the first-line drug (followed by Erwinia asparaginase in the case of allergy). PEGasparaginase is preferred over native E. coli asparaginase, because it is administered less frequently, with less day care visits. PEGasparaginase is less immunogenic than native E. coli asparaginase and is not more expensive. Asparaginase costs are mainly determined by the percentage of patients who are allergic and require a switch to Erwinia asparaginase.

Mesenchymal cells (MSCs) in bone marrow (BM) may produce asparagine and form protective niches for leukemic cells. In vitro, this led to high levels of asparagine and conferred asparaginase resistance to acute lymphoblastic leukemia (ALL) cells. The aim of this study was to investigate whether MSCs or other cells in BM indeed produce such significant amounts of asparagine in vivo as to result in clinical asparaginase resistance.Twenty-six patients with newly diagnosed ALL were enrolled. All children received induction chemotherapy according to the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase was administered from days 12-33. Asparaginase, asparagine, aspartic acid, glutamine, and glutamic acid levels were measured in BM and blood at diagnosis, days 15, 33, and 79.Median asparaginase trough levels were not significantly different at days 15 and 33. Only at diagnosis, asparagine level was significantly higher in BM than in blood (P = 0.001). Asparagine levels were all below the lower limit of quantification in BM and blood at days 15 and 33. However, aspartic acid level in BM was significantly higher than in blood (P 0.001) at diagnosis, and also at days 15, 33, and 79.We demonstrate higher aspartic acid levels in BM compared to blood; however, no increased asparagine levels were seen during induction therapy containing asparaginase in BM when compared to blood. Therefore, increased asparagine synthesis by MSCs is of relevance for resistance to asparaginase of leukemic cells in vitro, but it is questionable whether this leads to asparaginase resistance in childhood ALL patients.

Purpose We prospectively studied the incidence and clinical course of hypertriglyceridemia and hypercholesterolemia during very prolonged use of PEGasparaginase or Erwinia asparaginase in relation to asparaginase activity levels in children with acute lymphoblastic leukemia (ALL). Also, the incidence of pancreatitis, thrombosis, hyperammonemia and central neurotoxicity and their association with asparaginase activity levels were evaluated. Patients and Methods Patients were treated according to Dutch Childhood Oncology Group (DCOG) ALL-10 medium risk intensification protocol, which includes 15 doses of PEGasparaginase (2,500 IU/m2) for 30 weeks. Erwinia asparaginase (20,000 IU/m2) was administered when an allergy to or silent inactivation of PEGasparaginase occurred. Definitions of silent inactivation of PEGasparaginase and Erwinia asparaginase were previously described (Tong et al., Blood, 2014 Mar;123(13):2026-33). Hypertriglyceridemia, hypercholesterolemia, hyperammonemia, pancreatitis, thrombosis and central neurotoxicity were graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE). Changes over time of triglyceride, cholesterol, and ammonia levels were evaluated using mixed models analysis of variance (ANOVA). Changes related to age and gender were also investigated using mixed models ANOVA. The incidence of toxicities (pancreatitis, thrombosis, central neurotoxicity) related to treatment (PEGasparaginase or Erwinia asparaginase) was investigated with the Fisher's exact tests. Finally, Spearman correlation coefficients were used to evaluate the relations between triglyceride, cholesterol, and asparaginase activity levels. Results In total, 89 patients were enrolled from two pediatric oncology centers. Triglyceride, cholesterol and ammonia levels increased rapidly in children with PEGasparaginase and remained temporary elevated, but normalized after the finishing the last asparaginase dose. Hypertriglyceridemia and hypercholesterolemia (grade 3/4) were found in 47% and in 25%, respectively, of the patients treated with PEGasparaginase. Studying the correlations between PEGasparaginase activity levels and triglyceride levels showed the strongest correlation at week 5 (Rs = 0.36, p=0.005). Children >= 10 years had higher triglyceride levels as compared to younger patients (< 10 years) adjusted for asparaginase preparations: median levels of 4.9 mmol/L versus 1.6 mmol/L (p In patients receiving Erwinia asparaginase, triglyceride levels increased in the first weeks as well, but no hypertriglyceridemia and hypercholesterolemia (grade 3/4) were found. Hyperammonemia (grade 3/4) was only found in Erwinia asparaginase treated patients (9%). No associations were found between pancreatitis and hypertriglyceridemia nor between ammonia and central neurotoxicity. Thrombosis occurred in 4.5%, pancreatitis in 7% and central neurotoxicity in 9% of the patients using each of both asparaginase agents; these toxicities were not related to asparaginase activity levels nor to triglyceride levels. Conclusions Severe dyslipidemia occurred frequently, but was temporary and was not associated with relevant clinical events and therefore should not be considered a reason for asparaginase treatment modifications. We show that high asparaginase activity levels are associated with high triglyceride and high cholesterol levels. However, pancreatitis, thrombosis and central neurotoxicity appear unrelated to asparaginase activity levels. Also, no associations were found between pancreatitis and hypertriglyceridemia and between ammonia level and central neurotoxicity. Table 1 Toxicity table, p-values are given for comparisons of grade 3/4 toxicities between both asparaginase agents, ns; not significant. PEGasparaginase (n=67) Erwinia asparaginase (n=22) p-value Grade 1/2 Grade 3/4 Grade 1/2 Grade 3/4 n % n % n % n % Pancreatitis 0 0 4 6 1 5 2 9 ns Hypertriglyceridemia 15 22 31 47 7 32 0 0 p Disclosures Tong: EUSA Pharma: Research Funding.

Purpose Previous studies have shown that children with silent inactivation of asparaginase had poorer outcomes as they were not switched to another asparaginase preparation that retained its activity. Recently, a case report was published that described the successful use of desensitization courses in a patient with severe hypersensitivity reaction to asparaginase. We analyzed whether continuation of asparaginase in case of silent inactivation may result in desensitization, disappearance of asparaginase antibodies (AAA) and recovery of asparaginase activity levels in children with newly diagnosed acute lymphoblastic leukemia (ALL). Patients and Methods Children who received intensified PEGasparaginase or Erwinia asparaginase for 30 weeks according to the intensification phase of the Dutch Childhood Oncology Group-ALL-10 medium-risk protocol were studied. All children had received native E.coli asparaginase in induction and all asparaginase preparations were administered intravenously in one hour. AAA against native E.coli asparaginase (Coli-AAA), PEGasparaginase (PEG-AAA), and Erwinia asparaginase (Erwinia-AAA) and PEGasparaginase and Erwiniaasparaginase activity levels were analyzed in serum. Results 7/89 patients had silent inactivation of PEGasparaginase. Two were detected by real-time asparaginase activity measurements and were switched to Erwinia asparaginase. Five patients continued PEGasparaginase because no real-time asparaginase measurements were available at the starting phase of our drug monitoring program. Those patients with silent inactivation were, therefore, not recognized in time. PEGasparaginase activity levels recovered in all 5 patients after 2-7 PEGasparaginase infusions. In all 5 patients, Coli-AAA were present at start of the intensification phase which declined over time coinciding with the rise of PEGasparaginase activity levels. PEG-AAA were absent at the start of intensification but also increased after 1-2 doses of PEGasparaginase, and declined thereafter also coinciding with recovery of the PEGasparaginase activity levels. 29% of the PEGasparaginase patients without an allergy and without silent inactivation were positive for Coli-AAA. Also in this group, the Coli-AAA gradually decreased to undetectable levels after 5 PEGasparaginase courses. In a different cohort of 59 patients treated with Erwinia asparaginase, there were no cases of silent inactivation and two developed allergic reactions. In 50% of the non-allergic patients, the Erwinia-AAA were absent at start of therapy, gradually increased and decreased to absent baseline values during 30 weeks of Erwiniaasparaginase therapy. Conclusion This unintended desensitization program applied in five patients with silent inactivation of PEGasparaginase leads to recovery of PEGasparaginase activity levels. However, this takes an unpredictable and sometimes long time period. Therefore, we do not advise such desensitization approaches, but recommend switching to Erwinia asparaginase. A significant proportion of patients treated for prolonged period with PEGasparaginase or Erwinia asparaginase develops antibodies without influencing asparaginase activity levels that disappear with continued use of the same asparaginase product. Disclosures: No relevant conflicts of interest to declare.

Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p The data presented here suggest that interference with the CXCR4/CXCL12 axis (for instance by using Plerixafor) may be an effective way to mobilize BCP-ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. In proof-of concept studies, this hypothesis needs to be validated to pave the way for implementation in future treatment protocols for children with ALL. Disclosures: No relevant conflicts of interest to declare.

Purpose A prospective drug monitoring study was performed to analyse the efficacy of very prolonged use of PEGasparaginase and Erwiniaasparaginase by assessing asparaginase activity, asparagine, glutamine levels and asparaginase antibodies in children with newly diagnosed acute lymphoblastic leukemia (ALL). Patients and Methods Children received 15 PEGasparaginase infusions (2,500 IU/m2 every other week) according to the Dutch Childhood Oncology Group (DCOG)-ALL-10 medium risk intensification protocol after having received native E.coli asparaginase (5,000 IU/m2 every 3 days, 8 doses in total) in the induction course. In case of an allergy to or silent inactivation of PEGasparaginase, Erwinia asparaginase (20,000 IU/m22x-3x per week) was given. All asparaginase preparations were administered intravenously in one hour. Serum asparaginase activity, asparagine, glutamine levels and asparaginase antibodies were measured. Results 89 patients were enrolled in two centers to monitor the PEGasparaginase courses. 62/89 (70%) patients without clinical allergy to and without silent inactivation of PEGasparaginase had serum mean trough activity levels of 899 U/L which were much higher than requested. 20/89 (22%) of the patients showed an allergy and 7/89 (8%) silent inactivation in intensification. All 20 allergic patients (grade 1-4 Common Terminology Criteria Adverse Events) showed PEGasparaginase activity levels of zero. This was not due to the fact that the PEGasparaginase infusion was stopped, as 18 patients showed their allergic reactions at the second dose whereas the serum asparaginase activity level after the first full dose already appeared to be zero in all 18 cases. Moreover, in 4 patients with grade 1 allergy, the second full PEGasparaginase dose was given with pre-treatment of clemastine and hydrocortisone, also resulting in unmeasurable serum activity levels of PEGasparaginase. 59 children from 7 centers with allergy to or silent inactivation of PEGasparaginase who were switched to Erwinia asparaginase were enrolled to monitor the Erwiniaasparaginase courses. Only 2/59 (3%) of the patients developed an allergy to Erwinia asparaginase. No patients with silent inactivation of Erwinia asparaginase were seen. Of the non-allergic Erwinia asparaginase patients, 55/57 (96%) had at least one serum Erwinia asparaginase trough activity level ≥ 100 U/L and 57/57 (100%) ≥ 50 U/L. In 65% and 85% of all samples had serum trough activity levels ≥ 100 U/L and ≥ 50 U/L, respectively. In 33% of patients, the administration frequency could be reduced from 3 times to 2 times per week based upon serum Erwinia asparaginase activity levels ≥ 100 U/L at 72 hours. Serum asparagine level was strongly depleted, but not always completely depleted in Erwinia asparaginase treated patients in contrast to PEGasparaginase. Serum glutamine level was slightly lowered by Erwiniaasparaginase, but no glutamine depletion was observed with both compounds. The presence of serum asparaginase antibodies is related to allergy to and silent inactivation of asparaginase, but predicting asparaginase allergy or silent inactivation is clinically not applicable because of the low specificity, 64% (95%-CI: 43%-82%). Conclusion The use of native E.coli asparaginase in induction leads to 22% allergy and 8% silent inactivation rates of PEGasparaginase in intensification. Therefore, PEGasparaginase should be used upfront already in the induction course instead of native E.coli asparaginase. The dose of PEGasparaginase of 2,500 IU/m2 can be lowered. Switching to Erwinia asparaginase in case of allergy to or silent inactivation of PEGasparaginase leads to effective asparaginase activity levels in the majority of patients. Measuring serum asparaginase activity levels to monitor efficacy of asparaginase is preferred over serum asparagine levels and serum asparaginase antibodies. Therapeutic drug monitoring has now been implemented to individualize PEGasparaginase and Erwinia asparaginase dose and to detect silent inactivation in the current DCOG-ALL-11 protocol. Disclosures: No relevant conflicts of interest to declare.

Abstract 4227 Asparaginase is an expensive drug, but important in childhood acute lymphoblastic leukemia (ALL). Due to increasing costs of treatment of childhood ALL more insight in costs of asparaginase preparations is desired. In order to compare pharmacoeconomic aspects of PEGasparaginase, Erwinia asparaginase and native E.coli asparaginase, we performed a cost-effectiveness analysis in Dutch Childhood Oncology Group (DCOG) ALL-10 medium risk group (MRG) intensification protocol. Between April 2005 and October 2009, MRG patients were included in this multi-center study. Treatment costs were calculated based on patient level data of 84 subjects (33 female), and were related to allergy to asparaginase. We have used 3 treatment scenarios for asparaginase of which 2 scenarios were hypothetical: PEGasparaginase as first line preparation and Erwinia asparaginase as second line used as scenario 1 (similar to the actual ALL-10 treatment schedule); native E.coli asparaginase as first line preparation and Erwinia asparaginase as second line used as hypothetical scenario 2; or native E.coli asparaginase as first line preparation, PEGasparaginase as second line and Erwinia asparaginase as third line preparation used as hypothetical scenario 3. Medical technology assessments techniques were used for this cost-effectiveness analysis. Decision tree analysis was used to compare costs of PEGasparaginase or Erwinia asparaginase to native E.coli asparaginase, while taking into account the incidence of allergy to asparaginase and the different associated costs. Sensitivity analyses (one-way and two-way) were conducted to account for uncertainty in the used prices and calculated costs. The total costs of the ALL-10 MRG intensification course of 30 weeks were $71,147 per patient. Subgroup analysis revealed that the costs were $57,893 in patients without PEGasparaginase allergy (N=64). The costs were significantly higher ($113,558) in case of PEGasparaginase allergy (N=20) necessitating a switch to Erwinia asparaginase. The total costs were also calculated based on two weeks of asparaginase exposure; in case of PEGasparaginase the costs were $ 1,930 and for Erwinia asparaginase $ 3,785 per two weeks of treatment. Decision tree analysis showed that the treatment costs were $ 70,402 when using native E.coli asparaginase as first line preparation in intensification (scenario 3) and $ 71,809 when using PEGasparaginase as first line preparation (scenario 1). Treatment costs using native E.coli asparaginase as first line followed by Erwinia asparaginase as second line (scenario 2) the costs would be significantly higher ($ 103,474). One-way sensitivity analysis showed that the subgroup with allergy to native E.coli asparaginase (scenario 2) had the largest range in treatment costs. When treatment costs were calculated with the new European price of Erwinia asparaginase (the price has been doubled in March 2011), both treatments with PEGasparaginase as first line preparation (scenario 1) or native E.coli asparaginase as first line preparation (scenario 3) would be less expensive ($ 100,199 or $ 103,089, respectively) compared to the native E.coli asparaginase scenario 2 ($ 190,284). Two-way sensitivity analysis revealed that treatment with PEGasparaginase (scenario 1) is less expensive than treatment with native E.coli asparaginase (scenarios 2) for allergy probabilities of PEGasparaginase ranging from zero to 0.8 with a fixed allergy rate of 0.65 (Veerman et al. (Lancet Oncol. 2009)). This also holds true if a fixed allergy rate for native E.coli asparaginase of 0.4 is used, which is frequently found in studies using less native E.coli asparaginase in intensification after native E.coli asparaginase in induction (Nachman et al. (JCO 1997) and Willer et al. (Blood 2011)). In conclusion, treatment with native E.coli asparaginase, followed by a switch to PEGasparaginase, and subsequently to Erwinia asparaginase in case of allergy carried similar overall costs compared to the treatment with PEGasparaginase as first line drug (followed by Erwinia asparaginase in case of allergy). However, PEGasparaginase is preferred, because it is administered less frequently, with less outpatient care visits and is less immunogenic compared to native E.coli asparaginase. The costs with PEGasparaginase are much lower compared to Erwinia asparaginase. Disclosures: No relevant conflicts of interest to declare.

Abstract 1505 Asparaginase is an essential component of combination chemotherapy of acute lymphoblastic leukemia (ALL). Asparaginase breaks down asparagine into aspartic acid and ammonia. Because asparagine is necessary for protein synthesis, its depletion leads to cell death. Recently, it has been suggested that mesenchymal cells in the bone marrow may produce asparagine and form ‘protective niches’ for leukemic cells. In vitro, this led to high levels of asparagine and asparaginase resistance of the ALL cells (Iwamoto et al. (J Clin Invest. 2007)). However, it is unknown if this holds true for the clinical in vivo situation. The aim of our study is to analyse whether mesenchymal cells or other cells in the bone marrow indeed produce significant amounts of asparagine in vivo that may lead to clinical asparaginase resistance. Ten de novo ALL patients were enrolled in this study. All children received induction chemotherapy according to protocol 1-A and 1-B of the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase levels and amino acid levels (asparagine, aspartic acid, glutamine and glutamic acid) were measured in bone marrow (BM) and peripheral blood at diagnosis (day 1), days 15, 33 and 79. On days that asparaginase was administered (days 15 and 33) it was ensured that study material was obtained before the E-coli L-asparaginase infusions. Changes over time of asparaginase trough levels in BM and peripheral blood were evaluated using Mixed models ANOVA. The amino acids levels in 0.5 ml BM, 3 ml BM and peripheral blood at days 15 and 33 were also compared using Mixed models ANOVA. All these analyses were done after log transformation of measured values to get approximate normal distributions. A two-sided p-value < 0.05 was considered statistically significant. The asparaginase levels were all below detection limit (< 5 IU/L) in BM and peripheral blood at days 1 and 79. In both compartments, the median asparaginase trough levels were not significantly different at days 15 and 33. At diagnosis, no significant difference in asparagine level between 3 ml BM and peripheral blood was found (median: 44.5 μM (range 20.6–59.6 μM) and 43.9 μM (range 18.4 –58.5 μM), respectively). However, the median level of aspartic acid at diagnosis in 3 ml BM (19.2 μM; range 6.2–52.6 μM) was significantly higher as compared to median level of peripheral blood (5.7 μM; range 2.4–10.1 μM) (p=0.002). The aspartic acid levels were also higher in BM compared to peripheral blood at days 15 and 33 (both p=0.001) and at day 79 (p=0.002). Aspartic acid levels were significantly higher in 0.5 ml versus 3 ml BM (p=0.001) and this difference was also found when comparing 0.5 ml BM versus peripheral blood (p In conclusion, we demonstrate higher aspartic acid levels in bone marrow compared to peripheral blood. The higher aspartic acid levels are detected at diagnosis, during asparaginase therapy at days 15 and 33, and also at day 79 at complete remission, showing that these do not originate from leukemic cells nor from asparagine breakdown by asparaginase but from cells in the microenvironment of the bone marrow. However, there is no increased asparagine synthesis in vivo in the bone marrow of ALL patients. Therefore, increased asparagine synthesis by mesenchymal cells may be of relevance for resistance to asparaginase of leukemic cells in vitro but not in vivo. Disclosures: No relevant conflicts of interest to declare.