Dutch Childhood Oncology Group Research Articles

The toxicity of very prolonged courses of PEGasparaginase or Erwinia asparaginase in relation to asparaginase activity, with a special focus on dyslipidemia.

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 protocol, 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 (<10 years old) after adjustment for type of asparaginase preparation: median 4.9 mmol/L versus 1.6 mmol/L (P<0.001). In patients receiving Erwinia asparaginase, triglyceride levels increased in the first weeks as well, but no grade 3/4 dyslipidemia was found. Hyperammonemia (grade 3/4) was only found in patients treated with Erwinia asparaginase (9%). Thrombosis occurred in 4.5%, pancreatitis in 7%, and central neurotoxicity in 9% of patients using either of the two agents; these toxicities were not related to levels of asparaginase activity or to triglyceride levels. In conclusion, severe dyslipidemia occurred frequently, but was temporary and was not associated with relevant clinical events and should not, therefore, be considered a reason for modifying asparaginase treatment. Dyslipidemia was the only toxicity related to levels of asparaginase activity.

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events

AbstractPhosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression.

Fertility studies in female childhood cancer survivors: selecting appropriate comparison groups

Little information is available on the use of appropriate comparison groups for studies investigating late effects of childhood cancer. Two comparison groups in a nationwide study on reproductive function and ovarian reserve in female childhood cancer survivors were recruited (The Dutch Childhood Oncology Group Long-Term Effects After Childhood Cancer Cohort Study). Experiences of this process are reported. Two types of comparison groups were used: sisters of participating survivors and controls from the general population. A total of 352 out of 580 (61%) of the participating survivors who had a sister gave permission to invite them for the study. The participation rate of sisters was much higher than control participants from the general population (74% versus 21%, respectively), whereas considerably more effort was involved in recruiting controls from the general population. Participants in this group were significantly older and more highly educated than sister controls (P < 0.001 for both groups). No significant differences were observed between both types of comparison groups in several fertility-related characteristics, suggesting minimal bias owing to selective participation. Researchers setting up a study to investigate late effects among survivors of childhood cancer should carefully consider the advantages and disadvantages of using various types of comparison groups.

BCOR and BCORL1 Mutations In Pediatric Acute Myeloid Leukemia

IntroductionIn pediatric acute myeloid leukemia (AML) current survival rates are approximately 70%, but further improvements are required to improve disease outcome. Prognosis is correlated to early response to treatment and genetic aberrations (Creutzig et al, 2012). In approximately 20% no cytogenetic aberrations can be identified. In some of these cases repetitive aberrations, such as NPM1 mutations or cryptic translocations including NUP98-translocations (Hollink, 2011 and De Rooij, 2013) have been found. Recently, mutations in BCOR and BCORL1, both located on the X-chromosome, were found in adult AML using next generation sequencing. They both are transcriptional co-repressors, although with distinct binding targets (Tiacci, Heamatologica, 2012), and are thought to represent a novel mechanism of leukemogenesis. Somatic inactivating BCOR mutations were identified in 4% of adult cytogenetically normal (CN-) AML patients, predominantly located in exon 4, but also in other exons (Grossmann et al, 2011). Of interest, germline BCOR mutations cause the X-linked oculo-facio-cardio-dental genetic syndrome, which may occur due to its function as a co-repressor of the BCL6 gene. Somatic inactivating BCORL1mutations were found in 6% of adult AML patients (Li et al, 2011); all mutations were located in exon 4. Their exact role in AML and the targets of their co-repressive transcriptional activity has not been elucidated as yet (Tiacci, Haematologica, 2012). MethodsWe screened newly diagnosed pediatric AML patients for the presence of BCOR and BCORL1 mutations using direct sequencing of the complete coding sequence of both genes starting with a cohort of 86 patients including all cytogenetic subgroups patients, and later expanding this with an additional 146 patients for BCORL1screening of exon 4. This cohort was enriched for samples from CN-AML patients (56% and 21% respectively). Samples were obtained from the Dutch Childhood Oncology Group (DCOG; The Hague, The Netherlands), the AML-BFM-SG; Hannover, Germany and Prague, Czech Republic, and the Hôpital Robert Debré (Paris, France). ResultsA single BCOR mutation was found in 1 patient only with CN-AML. The mutation, p.A854T, was located in exon 4. The patient was a 4 year old boy with a FAB M1, WBC 354 x 109/L, who is alive 45 months after diagnosis. In addition, only 1 patient carried a BCORL1 mutation. The mutation, located in exon 4, p.G158X, caused a premature stop-codon. The male patient was diagnosed with secondary AML, aged 17 years, with normal cytogenetics and a WBC of 9.4 x 109/L, FAB M1, and died 3 months after diagnosis. Multiple recurrent SNPs were observed for both BCOR (rs5917933: 7/86 pts (91.9%); rs6520618: 15/86 (17.4%); rs144606152: 6/86 (7.0%)) and BCORL1 (rs4830173: 232/232 (100%); rs5932715: 36/232 (15.5%)), all in exon 4. No relation could be found between the presence of SNPs and disease outcome. ConclusionsBCOR and BCORL1 mutations occur in less than 1% of pediatric AML patients. These data provide further evidence for the differences in genetic background between pediatric and adult AML. Separate next-generation studies should be performed to elucidate the genetic background of pediatric CN-AML.This project was funded by KIKA, project number 64, entitled: Aberrant signal transduction profiling in pediatric AML. Disclosures:No relevant conflicts of interest to declare.

A Prospective Study On Drug Monitoring Of Pegasparaginase and Erwinia Asparaginase and Asparaginase Antibodies In Pediatric Acute Lymphoblastic Leukemia

PurposeA 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 MethodsChildren 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. Results89 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%). ConclusionThe 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.

BCR-ABL1-Like Cases In Pediatric Acute Lymphoblastic Leukemia: A Comparison Between COG/St. Jude and Dutch DCOG Signatures

BackgroundPediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) with the BCR-ABL1 translocation forms a small group with a poor prognosis. Approximately 15% of BCP-ALL cases are characterized by a gene expression signature similar to BCR-ABL1-positive disease. This so-called BCR-ABL1-like group shares the poor prognosis of the BCR-ABL1-positive subtype but without presence of a BCR-ABL1 translocation. Using different cohorts and methods, two different BCR-ABL1-like signatures have been identified (Den Boer et al., Lancet Oncol 2009; Mullighan et al., New Engl J Med 2009). Both signatures identify groups of patients with a poor prognosis and with deletions in B-cell development genes (Den Boer et al., Lancet Oncol 2009; Roberts et al., Cancer Cell 2012). The aim of this study was to compare these two signatures and the association to molecular aberrations and clinical outcome in diagnostic samples of the Dutch Childhood Oncology Group (DCOG) and the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL). Patients and methodsThe BCP-ALL study cohort consisted of 452 patients, including 146 patients with B-precursor ALL without known recurrent aberrations (BCR-ABL1 translocation, ETV6-RUNX1 translocation, MLL rearrangements, TCF3 rearrangements, hyperdiploidy). BCR-ABL1-like cases were identified in these 146 B-precursor cases using Prediction Analysis of Microarrays (PAM) with 257 probe sets (Roberts et al., Cancer Cell 2012) and separately by Hierarchical Clustering (HC) with 110 probe sets (Den Boer et al., Lancet Oncol 2009), using Affymetrix U133Plus2 expression data. Frequencies of molecular aberrations detected by multiplex ligation-dependent probe amplification, array-comparative genomic hybridization and Sanger sequencing, including IKZF1 deletions, JAK2 mutations and CRLF2 aberrations, were compared between groups of patient samples classified by the two prediction algorithms using Fisher's Exact test. We analyzed the cumulative incidence of relapse (CIR) with death as a competing event. ResultsIn the DCOG/COALL cohort, which included patients from all ALL risk groups, we identified 33 BCR-ABL1-like cases with the PAM signature, of which 24 (73%) were high-risk according to the NCI-Rome criteria (WBC ≥ 50 cells/nl and/or age ≥ 10 years), and 79 cases with the HC signature, of which 57 (72%) were high-risk. Compared with 59 B-other cases not identified by either of the two BCR-ABL1-like signatures, the most enriched molecular aberrations in the PAM-identified group were IKZF1 deletions (52% vs 19%, p=0.002), high CRLF2 expression (39% vs 8%, p<0.001), intrachromosomal amplification of chromosome 21 (iAMP21; 32% vs 0%, p<0.0001), and RB1 deletions (23% vs 6%, p=0.04). Enriched aberrations in the HC-identified group included IKZF1 deletions (38% vs 19%, p=0.02), dic(9;20) (28% vs 4%, p<0.001), and iAMP21 (15% vs 0%, p=0.002), while BTG1 deletions occurred less frequently (6% vs 17%, p=0.04). Directly comparing PAM and HC-identified BCR-ABL-like cases indicated significantly higher proportions of CRLF2-high (p=0.004) and JAK2 mutations (p=0.02) in the PAM group, and higher dic(9;20) in the HC group (p=0.03). Next, we compared the overlap between cases identified by the two signatures. PAM identified 32% (25/79) of the BCR-ABL1-like cases identified by HC, and vice versa HC identified 76% (25/33) of the cases identified by PAM. The cumulative incidence of relapse at 5-years in the BCR-ABL1-like groups identified by PAM (20%, p=0.04), HC (27%, p<0.0001), or both methods (18%, p=0.15), was worse than in the non-BCR-ABL1-like/BCR-ABL1-negative/MLL wild-type BCP-ALL reference group (8.1%). Cases identified only by HC (31%, p<0.0001) or only by PAM (25%, p=0.05) also had higher 5-year cumulative relapse incidences than the reference group. ConclusionsBoth BCR-ABL1-like expression signatures defined by HC or PAM identified a group of patients with an unfavorable prognosis in the DCOG/COALL study. The two groups largely overlapped, but each signature also identified unique patients not identified by the other signature. Importantly, each of the signatures was associated with poor outcome, while patients identified by both signatures (PAM/HC-double positive) did not have a worse outcome than those identified only by PAM or HC. Since the two signatures are partially complementary, we suggest that both should be used. Disclosures:Evans:St. Jude: In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property., In accordance with institutional policy, St. Jude allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating this intellectual property. Patents & Royalties, Under this policy, I and/or my spouse have in the past received a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms as clinical diagnostics. Other.

NADH dehydrogenase subunit 4 variant sequences in childhood acute myeloid leukaemia

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Cost-analysis of treatment of childhood acute lymphoblastic leukemia with asparaginase preparations: the impact of expensive chemotherapy

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.

Widespread and diffuse pattern of metastatses at 123I-MIBG scans correlates with MYCN single copy tumors in neuroblastoma

Gitta Bleeker (1,2), Huib N. Caron HN (1,3), Berthe L. van Eck (4), Rogier Versteeg (2), Susan G. Kreissman SG (5,6), Gregory Yanik (6,7), Godelieve A. Tytgat (1,3), 11 collaborators (1) Department of Pediatric Oncology, Academic Medical Center/Emma Children’s Hospital, Amsterdam, Netherlands (2) Department of Oncogenomics, Academic Medical Center, Amsterdam, Netherlands. (3) Dutch Childhood Oncology Group (DCOG) (4) Department of Nuclear Medicine, Academic Medical Center, Amsterdam, Netherlands. (5) Duke University Medical Center, Durham, North Carolina (6) Children’s Oncology Group (COG). 7. Department of Pediatrics, Division of Hematology and Oncology, University of Michigan, Michigan, USA

Existing radiotherapy dose quantification methods in published late effects studies: a review of the literature

s ingediend voor het Amsterdam Kindersymposium 2013 67 Existing radiotherapy dose quantification methods in published late effects studies: a review of the literature Judith L. Kok MSc (1,12), Wil V. Dolsma MD PhD (2,12), Flora E. van Leeuwen PhD (3,12), Marry M. van den Heuvel-Eibrink MD PhD (4,12), Wim J.E. Tissing MD PhD (5,12), Eline van Dulmen-den Broeder PhD (6,12), Jacqueline Loonen MD PhD (7,12), Dorine Bresters MD PhD (8,12), Birgitta A. Versluys MD (9,12), Irma W.E.M. van Dijk MSc (10), Berthe M.P. Aleman MD PhD (11), Huib N. Caron MD PhD (1,12), Arjen van der Schaaf PhD (2), Leontien C.M. Kremer MD PhD (1,12) and Cecile M. Ronckers PhD (1,12), for the Dutch Childhood Oncology Group (DCOG) LATER Dosimetry Research Group (1) Dept. of Pediatric Oncology, Emma Children’s Hospital/Academic Medical Center, Amsterdam, the Netherlands (2) Dept. of Radiation Oncology, University Medical Center Groningen, Groningen, the Netherlands (3) Dept. of Psychosocial Research and Epidemiology, the Netherlands Cancer Institute, Amsterdam, the Netherlands (4) Dept. of Pediatric Oncology, Sophia Children’s Hospital/Erasmus Medical Center, Rotterdam, the Netherlands (5) Dept. of Pediatric Oncology and Hematology, University Medical Center Groningen, Groningen, the Netherlands (6) Dept. of Pediatric Oncology, Free university Medical Center, Amsterdam, the Netherlands (7) Dept. of Pediatric Oncology and Hematology, Radboud University Medical Center, Nijmegen, the Netherlands (8) Willem Alexander Children’s Hospital, Leiden University Medical Center, Leiden, the Netherlands (9) Dept. of Pediatric Oncology and Hematology, University Medical Center Utrecht, Utrecht, the Netherlands (10) Dept. of Radiation Oncology, Academic Medical Center, Amsterdam, the Netherlands (11) Dept. of Radiotherapy, the Netherlands Cancer Institute, Amsterdam, the Netherlands (12) Dutch Childhood Oncology Group Late Eff ects after Childhood Cancer (DCOG-LATER) Consortium INTRODUCTION Radiotherapy (RT) is widely used in childhood cancer (CC) therapy. Combined therapies, including RT, greatly increased survival, but also cause adverse health eff ects because healthy organs (organs at risk: OAR) absorb varying levels of the prescribed tumour dose. Risk estimation for late eff ects in retrospective studies requires appropriate dose estimation. We aim to provide a review of various RT dose quantifi cation methods used in late eff ects studies, including advantages and drawbacks. METHODS A Pubmed search was performed to identify eligible studies, i.e. late eff ects studies published between 2002 and 2012, describing methods that quantify RT dose and including at least 100 long-term CC survivors. Titles and abstracts were screened by two independent reviewers (JK, CR). RESULTS The search resulted in 1782 studies; of which 70 fulfi lled the inclusion criteria. We categorized RT dose quantifi cation methods into three main groups (Table 1). Retrospective dose reconstruction (1) provides information about absorbed dose at OAR. Eff ects of various Table 1. Categorization of RT dose quantifi cation methods Category Explanation Advantages Drawbacks 1. Retrospective dose reconstruction Dose calculation by mathematical phantom Absorbed dose at OAR Volume and fraction size typically not reported 2. Radiobiological dose metrics Recalculation of total dose and dose per fraction according to biological eff ectiveness Uniform comparison of various fractionation schedules Prescribed tumour dose as surrogate of absorbed dose 3. Prospective RT registration Documentation of absorbed dose to OAR as calculated by modern RT planning systems Dose and volume at OAR available Requires high-quality long-term prospective, clinical follow-up

Cost-Effectiveness of Treatment of Childhood Acute Lymphoblastic Leukemia with Pegasparaginase and Erwinia Asparaginase: The Impact of Expensive Chemotherapy

Abstract 4227Asparaginase 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.

NADH Dehydrogenase Subunit 4 (ND4) Mutations in Pediatric Acute Myeloid Leukemia

AbstractAbstract 1380NADH dehydrogenase subunit 4 (ND4) is encoded by mitochondrial DNA and is an integral component of Complex I, one of the core enzymatic complexes critical for mitochondrial oxidative phosphorylation and regulation of the balance between NADH and NAD+. ND4 mutations have recently been described in adult acute myeloid leukemia (AML). In the current study, we investigated the frequency and prognostic impact of ND4 mutations in 289 pediatric leukemia patients (<= 18 years). Total cellular DNA was isolated from bone marrow or peripheral blood samples at diagnosis (n=289) and at complete remission (n=6) for children treated uniformly within multicenter treatment trials AML-Berlin-Frankfurt-Münster (BFM, n=180) and Dutch Childhood Oncology Group (DCOG, n=109).ND4 mutations were detected by direct sequencing in 13 of 289 (4.5 %) pediatric AML patients. Mutations occurred throughout the ND4 sequence, and included missense mutations (n=10), deletions (n=2) and a nonsense mutation. The most commonly detected mutations were S86N (n=2), delA 11,032–11,038 (n=2), and F50L (n=2). All other mutations were detected in single cases. Four (30.8 %) ND4 mutations were heteroplasmic (i.e. both wild-type and mutated ND4 were detected) and 9 (69.2 %) were homoplasmic (i.e. only mutated ND4 was detected), which is similar to the distribution we previously observed for adult AML patients (37.9% and 62.1%, respectively). Of the 4 heteroplasmic mutations detected in the pediatric AML cohort, 3 are predicted to result in a truncated ND4 protein. The remaining heteroplasmic mutation, which results in an L72P substitution, is predicted to be damaging (PolyPhen2 score = 0.999). Thus all 4 heteroplasmic mutations are expected to interfere with ND4 protein function. In contrast, 3 of the 9 (33.3 %) homoplasmic mutations are within transmembrane regions and only 1 (11.1 %) is predicted to be damaging (S459Y, PolyPhen2 score = 0.906). The 11 predicted transmembrane domains (TMD) of ND4 may be important for mitochondrial proton transport. However, like in adult AML, the presence of ND4 mutations affecting or not affecting a TMD had no impact on pediatric AML patient outcome.Non-tumoral DNA available through samples collected in routine follow-up examinations during complete remission allowed determination of mutation origin (e.g. somatic or germ-line) in 6 cases. Interestingly, the homoplasmic substitutions resulting in F50L, S86N and A131T were each defined to be germline mutations in both adult and pediatric AML samples. The heteroplasmic one base-pair deletion in a stretch of seven adenine residues (11,032–11,038) detected in two pediatric leukemia samples was determined to be somatic in the one case for whom a sample obtained during complete remission was available for analyses. Patient characteristics including age, FAB-subtype, WBC count, cytogenetic subgroup or presence of FLT3-ITD were similar regardless of ND4 mutation status.In accordance with our earlier observations in adult AML, comparison of ND4mutated with ND4wildtype patients demonstrated no significant difference on overall survival (OS, P=.67). In the adult study, a survival advantage was observed for patients with somatic heteroplasmic ND4 mutations. No survival advantage was observed for children with heteroplasmic ND4 mutations, possibly due to limited numbers of ND4mutated patients treated in the BFM and DCOG study groups.Gene expression profiles (GEP) for ND4mutated (n=11) and ND4wild-type (n=188) pediatric AML patients revealed no significant differences. However, 8 probe sets were found to be differentially regulated when GEP for heteroplasmic ND4mutated (n=4) and ND4wildtype (n=187) were compared. Two of these probe sets annotated the SETDB2 (CLLD8, KMT1F) gene, which encodes a histone H3 methyltransferase. Quantitative RT-PCR validated the lower SETDB2 expression as a characteristic of ND4mutated cases (P=.02). SETDB2 contributes to several important cellular functions, including heterochromatin formation, chromatin condensation and transcriptional repression.In summary, ND4 mutations were not predictive for outcome in pediatric AML, but were significantly associated with decreased SETDB2 expression, providing a link between mitochondrial gene mutation and epigenetic control of gene expression. Disclosures:No relevant conflicts of interest to declare.

The Effect of Fresh Frozen Plasma On the Incidence of Venous Thromboembolism and Haemorrhages in Children with Acute Lymphoblastic Leukaemia

Abstract 1471▪▪This icon denotes a clinically relevant abstract Background:Venous thromboembolism (VTE) and haemorrhages frequently occur during asparaginase (ASP) treatment in paediatric patients treated for acute lymphoblastic leukaemia (ALL). These complications cause significant morbidity and mortality. The effect of fresh frozen plasma (FFP) to prevent these complications in ALL patients is not entirely clear. The aims of this study were to assess the effect of FFP on the incidence of VTE and haemorrhages and the plasma levels of antithrombin (AT) and fibrinogen (FG) during the induction phase in paediatric ALL patients treated according to the Dutch Childhood Oncology Group ALL IX or ALL X protocol. In addition, we compared the incidences of VTE and haemorrhages and the plasma levels of AT and FG between both ALL protocols. Methods:A retrospective observational cohort study was performed among paediatric patients, aging from 0 to 18 years, with newly diagnosed ALL receiving induction therapy according to the ALL IX or ALL X protocol between February 1997 and January 2012. Treatment during the induction phase of the ALL IX protocol, day 0–49, included dexamethasone and 4 doses of 6 000 IU/m2 ASP intravenously over a period of 2 weeks (day 29–40). FFP supplementation was recommended if FG levels were < 1.0 g/L. Treatment during the induction phase of the ALL X protocol, day 0–64, included prednisone and 8 doses of 5 000 IU/m2ASP intravenously over a period of 3 weeks (day 12–33). In the ALL X protocol, FFP supplementation was recommended if FG levels were < 0.6 g/L. Medical records were reviewed and all VTE and haemorrhages during induction treatment were recorded. Plasma levels of AT (normal value: 80–140%) and FG (normal value: 0.7–4 g/L) and the frequency of FFP supplementation during the induction phase were also recorded. Results:In this study, 199 patients were included. During the induction phase, VTE and haemorrhages occurred in 7 (3.5%; 95% CI: 1.0–6.2%) and 2 (1%; 95% CI: 0.4–2.4%) of the 199 paediatric ALL patients treated according to both ALL protocols, respectively. Four VTE occurred in the central nervous system (57%) and 3 VTE were central venous line (CVL) related (43%) and occurred in the subclavian vein. Both haemorrhages were intracranial. 42% of all paediatric ALL patients received FFP supplementation. No statistical significant effect of FFP supplementation was seen on the incidence of VTE and haemorrhages. The mean AT and FG levels significantly decreased after ASP administration from 130% to a minimum of 78% and from 1.6 g/l to a minimum of 0.9 g/l, respectively. In general, the AT and FG levels did not increase by FFP supplementation.The paediatric patients treated according to the ALL IX protocol (n=94) consisted of significantly less male, younger patients, and less T-ALL patients than patients of the ALL X protocol (n=105). The incidence of VTE during the induction phase was significantly lower in children treated according to the ALL IX protocol (n=1), including dexamethasone and 24 000 IU/m2 ASP than in those included in the ALL X protocol (n=6) treated with prednisone and 40 000 IU/m2ASP (p=0.038). 25% of the ALL IX patients and 62% of the ALL X patients received FFP. After the third and fourth administration of ASP, the FG plasma levels were significant higher in the ALL IX protocol than in the ALL X protocol (p= 0.034 and 0.01, respectively). Conclusion:FFP has no significant effect on the incidence of VTE and haemorrhages and the plasma level of AT and FG during the induction phase in paediatric ALL patients treated according to the Dutch Childhood Oncology Group ALL IX or ALL X protocol. VTE occurred significantly less often in ALL IX patients treated with dexamethasone and 24 000 IU/m2 ASP than in ALL X patients treated with prednisone and 40 000 IU/m2 ASP. Disclosures:No relevant conflicts of interest to declare.

Leukemia Cells with a BCR-ABL1-Like signature and/or IKZF1 deletions, but Not High CRLF2 Expression, Are Predictive of an Unfavorable Prognosis in Childhood B Cell Precursor Acute Lymphoblastic Leukemia

AbstractAbstract 880 BackgroundThe prognosis of childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) has improved enormously due to risk-adapted therapeutic stratification of patients. Still, most relapses occur in the group that is supposed to have an intermediate relapse risk. To overcome this problem, new prognostic markers are needed. Recently, new high-risk molecular features were identified in BCP-ALL: the BCR-ABL1-like signature, IKZF1 deletions and high CRLF2 expression (CRLF2-high). These features can occur together; however, whether they confer independent prognostic value is currently unknown. In this study we determined the frequency, co-occurrence and prognostic relevance of these new molecular features in four independent cohorts of children with ALL. MethodsBCR-ABL1-like gene signature (110-gene probe classifier), IKZF1 deletions (Multiplex Ligation-dependent Probe Amplification) and CRLF2 expression levels (Affymetrix U133 plus 2.0) were determined in leukemic cells (>90% blasts) from 1128 children with newly diagnosed ALL enrolled in three consecutive Dutch Childhood Oncology Group trials (DCOG ALL-8/-9/-10) and in the German COALL-97/03 trial. The cumulative incidence of relapse (CIR) was calculated using death as a competing event. ResultsBCR-ABL1-like, IKZF1-deleted and CRLF2-high cases were found in 16%, 17% and 10% of the BCP-ALL cases, respectively. Collectively, these three features constitute 34% of BCP-ALL cases for which 4% of those had all three, 29% had any two and 67% had only one these features. The BCR-ABL1-like signature and IKZF1-deletions co-occurred the most frequent of these three features (22%). The BCR-ABL1-like signature was only found in B-other cases negative for the genetic abnormalities ETV6-RUNX1, BCR-ABL1, hyperdiploid, TCF3- and MLL-rearrangement, whereas IKZF1-deleted and CRLF2-high cases were also found in other subtypes of BCP-ALL. The frequency of IKZF1- deletions was highest in BCR-ABL1- positive (70%) and BCR-ABL1-like (40%) cases. The BCR-ABL1-like signature, IKZF1 deletions and CLRF2-high were associated with prognosis in four, three and one out of 4 studied treatment protocols, respectively. Protocol-stratified analysis revealed that five-year CIR was higher in BCR-ABL1-like (32%, p<0.001) and IKZF1- deleted (34%, p<0.001) compared to other BCP-ALL cases (11% and 13%, respectively), whereas no unfavorable CIR was associated with the CRLF2-high feature. IKZF1- deleted cases also were at higher risk of relapse in the hyperdipoid (5-year CIR 34%, p=0.04) and non-BCR-ABL1- like B-other group (5-year CIR 40%, p=0.02) compared to 9% for non-deleted BCP-ALL cases whereas IKZF1-deletions had no (additive) prognostic value in ETV6-RUNX1- positive or BCR-ABL1-like cases. Cases with only the BCR-ABL1-like signature (HR 5.3) or only an IKZF1 deletion (HR 4.4) as well as those cases positive for both features (HR 3.7) were at higher risk to develop an adverse event than those cases negative for both features (p<0.001). Together these two features accounted for 62.9% of all relapses in BCP-ALL whereas no relapse occurred in the cases with high CRLF2 expression as single feature. The adverse prognosis associated with BCR-ABL1-like signature and/or IKZF1-deletions was independent of other risk factors including age and white blood cell count. Moreover, the BCR-ABL1-like signature (HR=3.7 p=0.026) and IKZF1- deletions (HR 2.7, p=0.043) were predictive of an unfavorable prognosis in the group of BCP-ALL cases with intermediate minimal residual disease levels at day 79 of treatment. ConclusionsHigh CRLF2 expression is the least predictive, whereas the BCR-ABL1-like signature and IKZF1- deletions –both individually and combined- are strong independent prognostic factors and have additional prognostic value on top of intermediate minimal residual disease levels in children with BCP-ALL. These findings demonstrate that these two molecular features are important clinically for identifying high-risk patients who may require more intensive therapy. Disclosures:No relevant conflicts of interest to declare.

No evidence of increased asparagine levels in the bone marrow of patients with acute lymphoblastic leukemia during asparaginase therapy

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.

The Dutch Childhood Oncology Group guideline for follow-up of asymptomatic cardiac dysfunction in childhood cancer survivors

The Late Effects of Childhood Cancer task force of the Dutch Childhood Oncology Group (DCOG LATER) developed a guideline for follow-up of asymptomatic cardiac dysfunction in childhood cancer survivors (CCS). In this paper, we present the methods, available evidence and final recommendations of our guideline. A multidisciplinary working group specified clinical questions that should be answered to get to recommendations for the guideline. We carried out short or extensive evidence summaries and determined methodological quality of studies and levels of evidence in order to answer all clinical questions. When evidence was lacking for CCS, we carefully extrapolated evidence from other populations. Final recommendations were based on evidence and consensus. There was high-level evidence for the increased risk of cardiac dysfunction in CCS and its main risk factors. Evidence was lacking regarding the prognosis, diagnosis and treatment of cardiac dysfunction in CCS. We recommended echocardiographic screening for asymptomatic cardiac dysfunction in CCS treated with cardiotoxic treatments and counseling about potential advantages and disadvantages of our screening recommendations. The DCOG LATER guideline recommends risk-based screening for asymptomatic cardiac dysfunction in CCS, but it should be noted that recommendations are not completely supported by evidence in CCS.

High frequency of copy number alterations in myeloid leukaemia of Down syndrome

High frequency of copy number alterations in myeloid leukaemia of <scp>D</scp>own syndrome

Motor performance and functional exercise capacity in survivors of pediatric acute lymphoblastic leukemia

Impaired motor performance and reduced maximum exercise capacity during and after treatment of acute lymphoblastic leukemia (ALL) has been shown. However, no longitudinal study monitoring motor performance after cessation of treatment has been published. Whether sub-maximal exercise capacity is reduced is unknown. Motor performance of pediatric ALL survivors, treated with Dutch Childhood Oncology Group ALL-9 protocol was measured with the movement-ABC at stop treatment and ≥5 years later. At follow-up functional exercise capacity was also investigated using the 6-minute walk test (6MWT). Heart rate and oxygen saturation were measured with a portable pulse oximeter before and after the 6MWT. Nineteen boys and 15 girls, median age 12.3 years (range: 9.0-18.7), median time since completion of chemotherapy 5.2 years (5.0-7.1), participated. Mean height/age and weight/age were within the norm, whereas mean BMI/age was significantly increased (mean SDS 0.38, SEM 0.17, P = 0.04). Motor performance had improved significantly (P = 0.001). In contrast, functional exercise capacity at follow-up was significantly impaired (mean SDS -2.05, SEM 0.13, P < 0.001). At ≥5 years after completion of ALL treatment motor performance had improved significantly, but functional exercise capacity was significantly impaired. The exact underlying cause of this late effect needs further study.

Outcome in children with Down's syndrome and acute lymphoblastic leukemia: role of IKZF1 deletions and CRLF2 aberrations

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had ≥1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year event-free survival (EFS) 45 ± 16% vs 95 ± 4%; P=0.002), which was confirmed in the validation cohort (6-year EFS 21 ± 12% vs 58 ± 11%; P=0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P=0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL.

Late effects clinics and follow-up within DCOG LATER in the Netherlands

On behalf of the Taskforce Late Effects of the Dutch Childhood Oncology Group (DCOG LATER); board: Kremer LC1 (chair), Van Dulmen-den Broeder E3, Van den Heuvel-Eibrink MM4, and Tissing WJ5