Dutch Childhood Oncology Group Research Articles

Development of pediatric oncology supportive care indicators: Evaluation of febrile neutropenia care in the north of the Netherlands.

Febrile neutropenia (FN) is a common complication of the intensive treatment strategies used in pediatric oncology. By close adherence to high-quality guidelines, which can be evaluated by indicators, the burden of FN can potentially be reduced. The aims of this study were tripartite-(1) to develop structure, process, and outcome indicators, (2) to evaluate the implementation of the Dutch Childhood Oncology Group (DCOG) guideline on FN, and (3) to produce baseline measures on local quality of FN care (in the north of the Netherlands). Seven indicators derived from the DCOG guideline were developed. Regarding structure indicators, we gathered information from all local centers providing care for children with cancer (n=9). Regarding process and outcome indicators, we collected individual patient data from one academic and two shared-care hospitals. Children (<18 years) were included if they had been diagnosed with cancer in 2014 or 2015 and had suffered from FN. Six out of nine hospitals used the DCOG guideline on FN and three hospitals used an outdated supportive care handbook. Regarding individual patient data, we included 119 FN episodes in 59 patients. All FN episodes without focus were initially treated with guideline-based antibiotics. Of all FN episodes, 18.5% resulted in intensive care unit (ICU) admittance. Cumulative incidence of death during FN was 1.74%. Adherence to the DCOG guideline at the individual patient level was excellent. However, indicators concerning mortality and ICU admittances showed that FN still has devastating consequences. Subsequently, we will implement these indicators nationwide in order to improve FN care.

Evolution of Asymptomatic Left Ventricular Dysfunction in 380 Long-term Survivors of Childhood Cancer

Introduction Long-term childhood cancer survivors (CCS) treated with cardiotoxic cancer therapies (CTCT) are screened for LV dysfunction (LVD). The intervals of 2 to 5 years in the Dutch Childhood Oncology Group guideline (Annals of Oncology 2012) depend on anthracycline (300 mg/m2) and radiotherapy dose (30 Gy). There is a gap between detection of LVD, defined as an ejection fraction (EF) Methods Patient and treatment characteristics were collected from a CCS cohort diagnosed with cancer between 1966 and 1997 who received anthracyclines and/or chest radiotherapy. Patients with congenital heart disease and heart failure before baseline echocardiogram were excluded. The first available echocardiogram at >5 years since cancer diagnosis was the baseline echocardiogram. All subsequent echocardiograms were collected. FS was measured by M-mode echocardiography. EF was calculated from two apical chamber views by the Simpson formula. If no EF was available, EF was calculated using the Teicholz formula. Patients with an available baseline echocardiogram were stratified into three subgroups: Reduced (EF Results Of 671 CCS eligible, 448 had a baseline echocardiogram at a median of 17years since cancer diagnosis and 381 had at least one follow-up echocardiogram. At baseline, 6, 41, and 334 CCS were in the reduced, grey zone and preserved EF group respectively. CCS in the grey zone received higher anthracycline dose (median 350 mg/m2) compared to the preserved group (median 200 mg/m2), and more CCS with anthracycline dose >300 mg/m2 were present in the grey zone (51.2% vs 26.6%). In the grey zone group 4.9% and 7.3% developed an EF Conclusion Long-term CCS treated with CTCT with an EF in the grey zone are at an increased risk of developing an EF

Efficacy and safety of recombinant E. coli asparaginase in children with previously untreated acute lymphoblastic leukemia: A randomized multicenter study of the Dutch Childhood Oncology Group.

The efficacy and safety of recombinant Escherichia coli-asparaginase (rASNase) was compared to native E.coli asparaginase (Asparaginase medac). One hundred and ninety-nine children with newly diagnosed acute lymphoblastic leukemia were randomized to receive one of both agents at a dose of 5,000 U/m² during induction (eight doses) and 10,000 U/m² during the postinduction phase (only high-risk patients; standard- and medium-risk patients received pegaspargase). Median trough serum asparaginase activity levels were comparable between both groups; they ranged from 143 to 182U/l during induction and were above the target value of 100U/l. Complete asparagine depletion in serum was achieved in 97.9% of patients, with no significant differences between both groups. On day 33 (end of induction), only two (2%) evaluable patients in each group had measurable asparagine serum levels, and complete asparagine depletion in the cerebrospinal fluid was achieved in 98.8% and 93.6% of the patients with rASNase and Asparaginase medac, respectively. During induction, 2.1% and 5% of patients developed an allergic reaction to rASNase or Asparaginase medac, respectively. Approximately 41% of the patients in both groups had a clinical allergy or enzyme inactivation to the first dose of any asparaginase preparation in postinduction. A comparable proportion of patients in both groups developed anti-asparaginase antibodies (57%) during repeated administration of asparaginase. Minimal residual disease levels at the end of induction, 5-year event-free survival, and 5-year cumulative incidence of relapse did not differ between both groups. The efficacy, safety, and immunogenicity of both asparaginase preparations are comparable. This trial was registered at www.clinicaltrials.gov as #NCT00784017; EudraCT number 2006-003180-31.

Site of childhood cancer care in the Netherlands

BackgroundDue to the complexity of diagnosis and treatment, care for children and young adolescents with cancer preferably occurs in specialised paediatric oncology centres with potentially better cure rates and minimal late effects. This study assessed where children with cancer in the Netherlands were treated since 2004. MethodsAll patients aged under 18 diagnosed with cancer between 2004 and 2013 were selected from the Netherlands Cancer Registry (NCR) and linked with the Dutch Childhood Oncology Group (DCOG) database. Associations between patient and tumour characteristics and site of care were tested statistically with logistic regression analyses. ResultsThis population-based study of 6021 children diagnosed with cancer showed that 82% of them were treated in a paediatric oncology centre. Ninety-four percent of the patients under 10 years of age, 85% of the patients aged 10–14 and 48% of the patients aged 15–17 were treated in a paediatric oncology centre. All International Classification of Childhood Cancers (ICCC), 3rd edition, ICCC-3 categories, except embryonal tumours, were associated with a higher risk of treatment outside a paediatric oncology centre compared to leukaemia. Multivariable analyses by ICCC-3 category revealed that specific tumour types such as chronic myelogenous leukaemia (CML), embryonal carcinomas, bone tumours other type than osteosarcoma, non-rhabdomyosarcomas, thyroid carcinomas, melanomas and skin carcinomas as well as lower-staged tumours were associated with treatment outside a paediatric oncology centre. ConclusionThe site of childhood cancer care in the Netherlands depends on the age of the cancer patient, type of tumour and stage at diagnosis. Collaboration between paediatric oncology centre(s), other academic units is needed to ensure most up-to-date paediatric cancer care for childhood cancer patients at the short and long term.

Central nervous system disease in pediatric acute myeloid leukemia.

Central nervous system disease in pediatric acute myeloid leukemia.

A cost analysis of individualized asparaginase treatment in pediatric acute lymphoblastic leukemia.

Therapeutic drug monitoring (TDM) of asparaginase is necessary to respond to variability in asparaginase activity levels, detect silent inactivation, and distinguish between real allergies and allergic-like reactions with and without asparaginase neutralization, respectively. In this study, the costs of an individualized and fixed asparaginase dosing schedule were compared. Patients, treated according to the Dutch Childhood Oncology Group ALL-11 protocol (individualized PEGasparaginase treatment, starting dose: 1,500 IU/m2 ) or ALL-10 protocol (native Escherichia coli asparaginase followed by 2,500 IU/m2 PEGasparaginase), were included. To focus on TDM of PEGasparaginase, the costs were also calculated excluding patients treated with Erwinia asparaginase and compared to a hypothetical protocol with a fixed dose of 1,500 IU/m2 PEGasparaginase. Direct asparaginase-related medical costs, including costs for asparaginase use (calculated with the absolute dose), TDM, laboratory tests, daycare treatment, and outpatient clinic visits, were calculated. Eighty-three ALL-10 patients and 51 ALL-11 patients were included. The asparaginase-related costs were 30.8% lower in ALL-11 than in ALL-10 ($29,048 vs. $41,960). The ALL-11 costs of nonallergic patients were 20.4% lower, when using TDM, than the hypothetical protocol with a fixed dose of 1,500 IU/m2 ($13,178 vs. $16,551). TDM accounted for 12.4% of the costs. Including asparaginase waste, TDM in ALL-11 will be cost saving if three doses can be prepared out of one vial compared to a fixed dose of 1,500 IU/m2 . TDM of asparaginase is cost saving if calculated with the absolute asparaginase dose and will be if the waste is minimalized by preparing multiple doses out of one vial.

Therapy reduction in patients with Down syndrome and myeloid leukemia: the international ML-DS 2006 trial

AbstractChildren with myeloid leukemia associated with Down syndrome (ML-DS) have superior outcome compared with non-DS patients, but suffer from higher constitutional cytotoxic drug susceptibility. We analyzed the outcome of 170 pediatric patients with ML-DS enrolled in the prospective, multicenter, open-label, nonrandomized ML-DS 2006 trial by Nordic Society for Pediatric Hematology and Oncology (NOPHO), Dutch Childhood Oncology Group (DCOG), and Acute Myeloid Leukemia–Berlin-Frankfurt-Münster (AML-BFM) study group. Compared with the historical control arm (reduced-intensity protocol for ML-DS patients from the AML-BFM 98 trial), treatment intensity was reduced by lowering the cumulative dose of etoposide (950 to 450 mg/m2) and intrathecal central nervous system prophylaxis while omitting maintenance therapy. Still, 5-year overall survival (89% ± 3% vs 90% ± 4%; Plog-rank = .64), event-free survival (EFS; 87% ± 3% vs 89% ± 4%; Plog-rank = .71), and cumulative incidence of relapse/nonresponse (CIR/NR; 6% ± 3% vs 6% ± 2%; PGray = .03) did not significantly differ between the ML-DS 2006 trial and the historical control arm. Poor early treatment response (5-year EFS, 58% ± 16% vs 88% ± 3%; Plog rank = .0008) and gain of chromosome 8 (CIR/NR, 16% ± 7% vs 3% ± 2%, PGray = .02; 5-year EFS, 73% ± 8% vs 91% ± 4%, Plog rank = .018) were identified as independent prognostic factors predicting a worse EFS. Five of 7 relapsed patients (71%) with cytogenetic data had trisomy 8. Our study reveals prognostic markers for children with ML-DS and illustrates that reducing therapy did not impair excellent outcome. The trial was registered at EudraCT as #2007-006219-2.

TropicALL study: Thromboprophylaxis in Children treated for Acute Lymphoblastic Leukemia with Low-molecular-weight heparin: a multicenter randomized controlled trial

BackgroundVenous thromboembolism (VTE) is a common and severe complication during treatment of acute lymphoblastic leukemia (ALL). An important cause is the intensive use of asparaginase. Prospective cohort studies in which prophylactic low-molecular-weight heparin (LMWH) was used to prevent VTE showed lower VTE risk than in historic control cohorts, with a negligible bleeding risk. However, the efficacy of thromboprophylaxis with LMWH during ALL treatment has never been investigated in a randomized design. Here, we present the protocol of a randomized controlled trial in which the efficacy and safety of thromboprophylaxis with high prophylactic dose LMWH versus no thromboprophylaxis will be assessed in children treated for primary ALL with asparaginase.Methods/DesignThromboprophylaxis in Children treated for Acute Lymphoblastic Leukemia with Low-molecular-weight heparin (TropicALL) is a multicenter, randomized controlled open-label trial conducted in the Netherlands. Patients between 1 and 19 years of age with primary ALL, who are treated within the Dutch Childhood Oncology Group (DCOG) ALL-11 or 12 study will be randomized to thromboprophylaxis with LMWH once daily, (dose of 85 IU/kg (intervention arm A)), or to no thromboprophylaxis (arm B, standard of care) during asparaginase courses of ALL treatment. Primary efficacy endpoint is symptomatic objectified VTE during ALL treatment; secondary efficacy endpoints are overall survival and the composite of symptomatic and asymptomatic objectified VTE. Primary safety endpoints are major bleeding, clinically relevant non-major bleeding and minor bleeding. A total of 324 patients will be included to obtain a relative risk reduction of 75% with a power of 80%, using a two-sided test with significance level α = 0.05.DiscussionThis trial will be the first to assess efficacy and safety of thromboprophylaxis with LMWH during asparaginase treatment for ALL in children in a randomized design.Trail registrationNederlands Trial Register NTR4707. Registered 30 July 2014.

Population pharmacokinetics of intravenous Erwinia asparaginase in pediatric acute lymphoblastic leukemia patients.

Erwinia asparaginase is an important component in the treatment of pediatric acute lymphoblastic leukemia. A large variability in serum concentrations has been observed after intravenous Erwinia asparaginase. Currently, Dutch Childhood Oncology Group protocols dose alterations are based on trough concentrations to ensure adequate asparaginase activity (≥100 IU/L). The aim of this study was to describe the population pharmacokinetics of intravenous Erwinia asparaginase to quantify and gather insight into inter-individual and inter-occasion variability. The starting dose was evaluated on the basis of the derived population pharmacokinetic parameters. In a multicenter prospective observational study, a total of 714 blood samples were collected from 51 children (age 1–17 years) with acute lymphoblastic leukemia. The starting dose was 20,000 IU/m2 three times a week and adjusted according to trough levels from week three onwards. A population pharmacokinetic model was developed using NONMEM®. A 2-compartment linear model with allometric scaling best described the data. Inter-individual and inter-occasion variability of clearance were 33% and 13%, respectively. Clearance in the first month of treatment was 14% higher (P<0.01). Monte Carlo simulations with our pharmacokinetic model demonstrated that patients with a low weight might require higher doses to achieve similar concentrations compared to patients with high weight. The current starting dose of 20,000 IU/m2 might result in inadequate concentrations, especially for smaller, lower weight patients, hence dose adjustments based on individual clearance are recommended. The protocols were approved by the institutional review boards. (Registered at NTR 3379 Dutch Trial Register; www.trialregister.nl).

Allergic-like reactions to asparaginase: Atypical allergies without asparaginase inactivation.

Asparaginase is an important component of pediatric acute lymphoblastic leukemia (ALL) therapy. Unfortunately, this treatment is hampered by hypersensitivity reactions. In general, allergies - regardless of severity - cause complete inactivation of the drug. However, we report atypical allergic reactions without inactivation of asparaginase, here called allergic-like reactions. Patients with an allergic-like reaction, who were treated according to the Dutch Childhood Oncology Group ALL-11 or the CoALL 08-09 protocol, were described. The reactions were identified by continual measurement of asparaginase activity levels. Characteristics, including timing of occurrence, symptoms, grade, and the presence of antiasparaginase antibodies, were compared to those of real allergies. Fourteen allergic-like reactions occurred in nine patients. Five reactions were to PEGasparaginase and nine to Erwinia asparaginase. Allergic-like reactions occurred relatively late after the start of infusion compared to real allergies. Antibodies were absent in all but one patient with an allergic-like reaction, while they were detected in all patients with a real allergy. Symptoms and grade did not differ between the groups. Asparaginase was continued with the same formulation in six patients of whom four finished treatment with adequate activity levels. In conclusion, allergic-like reactions occur relatively late after the start of infusion and without antibodies. Despite these clinical differences, allergic-like reactions can only be distinguished from real allergies by continually measuring asparaginase activity levels. If clinically tolerated, formulations should not be switched in case of allergic-like reactions. Moreover, failure to recognize these reactions may lead to a less favorable prognosis if asparaginase therapy is terminated unnecessarily.

Hydrocortisone as an Intervention for Dexamethasone-Induced Adverse Effects in Pediatric Patients With Acute Lymphoblastic Leukemia: Results of a Double-Blind, Randomized Controlled Trial.

Dexamethasone is a key component in the treatment of pediatric acute lymphoblastic leukemia (ALL), but can induce serious adverse effects. Recent studies have led to the hypothesis that neuropsychological adverse effects may be a result of cortisol depletion of the cerebral mineralocorticoid receptors. We examined whether including a physiologic dose of hydrocortisone in dexamethasone treatment can reduce neuropsychologic and metabolic adverse effects in children with ALL. We performed a multicenter, double-blind, randomized controlled trial with a crossover design. Of 116 potentially eligible patients (age 3 to 16 years), 50 were enrolled and were treated with two consecutive courses of dexamethasone in accordance with Dutch Childhood Oncology Group ALL protocols. Patients were randomly assigned to receive either hydrocortisone or placebo in a circadian rhythm (10 mg/m(2)/d) during both dexamethasone courses. Primary outcome measure was parent-reported Strength and Difficulties Questionnaire in Dutch, which assesses psychosocial problems. Other end points included questionnaires, neuropsychological tests, and metabolic parameters. Of 48 patients who completed both courses, hydrocortisone had no significant effect on outcome; however, a more detailed analysis revealed that in 16 patients who developed clinically relevant psychosocial adverse effects, addition of hydrocortisone substantially reduced their Strength and Difficulties Questionnaire in Dutch scores in the following domains: total difficulties, emotional symptoms, conduct problems, and impact of difficulties. Moreover, in nine patients who developed clinically relevant, sleep-related difficulties, addition of hydrocortisone reduced total sleeping problems and disorders of initiating and maintaining sleep. In contrast, hydrocortisone had no effect on metabolic parameters. Our results suggest that adding a physiologic dose of hydrocortisone to dexamethasone treatment can reduce the occurrence of serious neuropsychological adverse effects and sleep-related difficulties in pediatric patients with ALL.

Recurrent abnormalities can be used for risk group stratification in pediatric AMKL: a retrospective intergroup study

AbstractGenetic abnormalities and early treatment response are the main prognostic factors in acute myeloid leukemia (AML). Acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML. Deep sequencing has identified CBFA2T3/GLIS2 and NUP98/KDM5A as recurrent aberrations, occurring in similar frequencies as RBM15/MKL1 and KMT2A-rearrangements. We studied whether these cytogenetic aberrations can be used for risk group stratification. To assess frequencies and outcome parameters of recurrent cytogenetic aberrations in AMKL, samples and clinical data of patients treated by the Associazione Italiana Ematologia Oncologia Pediatrica, Berlin-Frankfurt-Munster Study Group, Children's Oncology Group, Dutch Childhood Oncology Group, and the Saint Louis Hôpital were collected, enabling us to screen 153 newly diagnosed pediatric AMKL cases for the aforementioned aberrations and to study their clinical characteristics and outcome. CBFA2T3/GLIS2 was identified in 16% of the cases; RBM15/MKL1, in 12%; NUP98/KDM5A and KMT2A rearrangements, in 9% each; and monosomy 7, in 6%. These aberrations were mutually exclusive. RBM15/MKL1-rearranged patients were significantly younger. No significant differences in sex and white blood cell count were found. NUP98/KDM5A, CBFA2T3/GLIS2, KMT2A–rearranged lesions and monosomy 7 (NCK-7) independently predicted a poor outcome, compared with RBM15/MKL1-rearranged patients and those with AMKL not carrying these molecular lesions. NCK-7-patients (n = 61) showed a 4-year probability of overall survival of 35 ± 6% vs 70 ± 5% in the RBM15/MKL1-other groups (n = 92, P < .0001) and 4-year probability of event-free survival of 33 ± 6% vs 62 ± 5% (P = .0013), the 4-year cumulative incidence of relapse being 42 ± 7% and 19 ± 4% (P = .003), respectively. We conclude that these genetic aberrations may be used for risk group stratification of pediatric AMKL and for treatment tailoring.

Acute Activation of Metabolic Syndrome Components in Pediatric Acute Lymphoblastic Leukemia Patients Treated with Dexamethasone.

Although dexamethasone is highly effective in the treatment of pediatric acute lymphoblastic leukemia (ALL), it can cause serious metabolic side effects. Because studies regarding the effects of dexamethasone are limited by their small scale, we prospectively studied the direct effects of treating pediatric ALL with dexamethasone administration with respect to activation of components of metabolic syndrome (MetS); in addition, we investigated whether these side effects were correlated with the level of dexamethasone. Fifty pediatric patients (3–16 years of age) with ALL were studied during a 5-day dexamethasone course during the maintenance phase of the Dutch Childhood Oncology Group ALL-10 and ALL-11 protocols. Fasting insulin, glucose, total cholesterol, HDL, LDL, and triglycerides levels were measured at baseline (before the start of dexamethasone; T1) and on the fifth day of treatment (T2). Dexamethasone trough levels were measured at T2. We found that dexamethasone treatment significantly increased the following fasting serum levels (P<0.05): HDL, LDL, total cholesterol, triglycerides, glucose, and insulin. In addition, dexamethasone increased insulin resistance (HOMA-IR>3.4) from 8% to 85% (P<0.01). Dexamethasone treatment also significantly increased the diastolic and systolic blood pressure. Lastly, dexamethasone trough levels (N = 24) were directly correlated with high glucose levels at T2, but not with other parameters. These results indicate that dexamethasone treatment acutely induces three components of the MetS. Together with the weight gain typically associated with dexamethasone treatment, these factors may contribute to the higher prevalence of MetS and cardiovascular risk among survivors of childhood leukemia who received dexamethasone treatment.

Successful Therapy Reduction and Intensification for Childhood Acute Lymphoblastic Leukemia Based on Minimal Residual Disease Monitoring: Study ALL10 From the Dutch Childhood Oncology Group.

Outcome of childhood acute lymphoblastic leukemia (ALL) improved greatly by intensifying chemotherapy for all patients. Minimal residual disease (MRD) levels during the first months predict outcome and may select patients for therapy reduction or intensification. Patients 1 to 18 years old with ALL were stratified on the basis of MRD levels after the first and second course of chemotherapy. Thereafter, therapy was substantially reduced in patients with undetectable MRD (standard risk) and intensified in patients with intermediate (medium risk) and high (high risk) levels of MRD. Seven hundred seventy-eight consecutive patients were enrolled. The method of analysis was intention-to-treat. Outcome was compared with historical controls. In MRD-based standard-risk patients, the 5-year event-free survival (EFS) rate was 93% (SE 2%), the 5-year survival rate was 99% (SE 1%), and the 5-year cumulative incidence of relapse rate was 6% (SE 2%). The safety upper limit of number of observation years was reached and therapy reduction was declared safe.MRD-based medium-risk patients had a significantly higher 5-year EFS rate (88%, SE 2%) with therapy intensification (including 30 weeks of asparaginase exposure and dexamethasone/vincristine pulses) compared with historical controls (76%, SE 6%). Intensive chemotherapy and stem cell transplantation in MRD-based high-risk patients resulted in a significantly better 5-year EFS rate (78%, SE 8% v 16%, SE 8% in controls). Overall outcome improved significantly (5-year EFS rate 87%, 5-year survival rate 92%, and 5-year cumulative incidence of relapse rate 8%) compared with preceding Dutch Childhood Oncology Group protocols. Chemotherapy was substantially reduced safely in one-quarter of children with ALL who were selected on the basis of undetectable MRD levels, without jeopardizing the survival rate. Outcomes of patients with intermediate and high levels of MRD improved with therapy intensification.

Genetic Architecture of Relapsed Pediatric B Cell Precursor Acute Lymphoblastic Leukemia Are Related to the Intensity of Upfront Therapy

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common malignancies in children. In the period 1991-2013, the Dutch Childhood Oncology Group (DCOG) has completed three treatment trials for childhood ALL: ALL8, 9 and 10, each protocol with stratifications into risk-groups (details: www.skion.nl). Although the cure rates increased in these subsequent trials, relapses still occurred in a significant number of children. Since consecutive upfront treatment protocols usually change at multiple levels, genomic alterations that are associated with relapse may also be variable, which could provide insight into the biology underlying therapy failure and relapse. In this study, we characterized the genetic architecture of relapsed BCP-ALL patients within the context of these three Dutch upfront protocols.We identified 3 patient groups based on upfront treatment as follows: Group-1: patients treated upfront with high-amounts of corticosteroids (CS) and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). The number of high-risk patients that relapsed after ALL8 HR and ALL10 HR chemotherapy courses was too low to be included for analysis.We determined, at relapse, the presence of copy number alterations and sequence mutations in 21 recurrently affected genes involved in B-cell development, cell cycle regulation and RAS signaling, in 123 patients that relapsed after treatment in group-1 (n=56), group-2 (n=20) and group-3 (n=47). The number of CREBBP mutations in patients that relapsed after treatment according to group-1 (ALL9) was significantly lower compared to the other two groups, whereas B-cell development alterations were most common in patients that relapsed after treatment according to group-1, mainly due to a higher number of IKZF1 alterations (Figure 1).The high number of relapsed patients with leukemic clones carrying IKZF1 alterations in patients treated with high-amounts of CS and relatively mild additional chemotherapy is in line with our recent finding that IKZF1 is a key determinant of GC-induced apoptosis in normal and leukemic B-cells, and that loss of IKZF1 function confers resistance to dexamethasone, the major treatment component in group-1 (Marke et al., submitted). Additionally, in the group-2 patients treated with high-amounts of CS and highly intensive additional chemotherapy, a lower percentage IKZF1-deleted clones was detected at relapse, indicating that more GC-resistant, IKZF1-deleted clones are killed by the intense chemotherapy given in addition to CS in group-2 patients. Similarly, in the group-3 patients relapsing after treatment with lower amounts of CS and moderately-intensive additional chemotherapy, the percentage of surviving IKZF1-deleted clones was lower than in patients treated with high-amounts of CS.Taken together, our data indicate that the genetic architecture of relapsed BCP-ALL patients depends on the upfront treatment and, in addition, that the poor-prognostic feature of IKZF1-deletions may be more prominent in upfront treatment with high-amounts of CS and relatively mild additional chemotherapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CNOT1 Microrna Single Nucleotide Polymorphism (SNP) Determines the Occurrence of Methotrexate Related Mucositis in Pediatric Acute Lymphoblastic Leukemia

BACKGROUNDCure rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity due to chemotherapeutic regimens occurs frequently but with great heterogeneity. This suggests that genetic variation is involved. In order to identify determinants of adverse effects, recent studies have investigated pharmacogenetic features in relation to toxicity. Most of these studies examined coding regions of the genome. Recently, it has been described that epi genetic regulators, such as micro-RNA's (miRNA), might also have an important regulatory function in genes involved in drug related toxicity. In a recent study 25 miRNA SNPs were found to be related to toxicity in pediatric ALL treatment (Lopez-Lopez, PLoS ONE, 2014). In pediatric ALL mucositis is one of the most frequent side effects during high dose methotrexate (MTX) treatment.AIMThe aim of this study was to detect novel, epigenetic biomarkers that predict MTX related oral mucositis in pediatric acute lymphoblastic leukemia (B- and T cell) by studying single nucleotide polymorphisms (SNP) involved in miRNA levels and function.METHODSDNA was isolated from whole blood of 118 pediatric ALL patients that were treated with high dose MTX (5 gr/m2) according to the Dutch Childhood Oncology Group ALL-10 protocol. The recently published 25 SNPs, involved in miRNA function and located in the DROSHA, CNOT1, CNOT4, EIF2C1, GEMIN3, GEMIN4, MIR604, MIR453, MIR2110, MIR2053, MIR1294, MIR1206, DICER, XPO5 and TNRC6B genes, were selected for genotyping. Toxicity data during the consolidation phase were prospectively collected and documented according to the National Cancer Institute (NCI) v.3.0 score system. Mucositis NCI grade ≥ 3 (grade 3: confluent ulcerations, bleeding with minor trauma), was considered as clinical significant toxicity and was used as endpoint.RESULTSMucositis was the only recurring toxicity in this prospectively well-documented cohort and therefore used as endpoint of this study. A selection of 20 of the previously identified 25 candidate SNPs was studied based on technical feasibility. In addition, 1 SNP in the XPO 5 gene was not considered for analysis because it was not in Hardy Weinberg equilibrium. Mucositis occurred in 19% of the patients in at least one of the MTX courses. Only the TT genotype of rs11866002 in the CNOT1 (CCR4-NOT complex, subunit 1) gene was associated with a higher risk of developing mucositis (NCI ≥ 3) compared to carries of CC/CT. The other 18 candidate SNPs analyzed did not show statistically significant associations.CONCLUSIONThe inter-patient variability of mucosal toxicity was not associated with most of our investigated SNPs which are involved in miRNA transcription and function. CNOT1 rs11866002 C>T was the only single nucleotide polymorphism associated with the occurrence of oral mucositis during pediatric acute lymphoblastic leukemia treatment.We acknowledge the Foundation Children Cancerfree (KiKa), Amstelveen, The Netherlands, for funding this research. DisclosuresNo relevant conflicts of interest to declare.

Is a New Protocol for Acute Lymphoblastic Leukemia Research or Standard Therapy?

In the United States, doctors generally develop new cancer chemotherapy for children by testing innovative chemotherapy protocols against existing protocols in prospective randomized trials. In the Netherlands, children with leukemia are treated by protocols that are agreed upon by the Dutch Childhood Oncology Group. Periodically, the Dutch Childhood Oncology Group revises its protocols. Sometimes, these revisions are categorized as research, sometimes as treatment. In this Ethics Rounds, we analyze whether enrollment in a new protocol ought to be considered research and, if so, we discuss the implications of that designation. Our discussion highlights the different ways different countries approach complex issues of research ethics.

ARID5B Regulates Leukemia Sensitivity to Antimetabolites in Children with Acute Lymphoblastic Leukemia Via Effects on Cell Cycle Progression

▪Acute lymphoblastic leukemia (ALL) in children is a prototype of cancer that can be cured by chemotherapy alone. However, the molecular mechanisms for anti-leukemic drug sensitivity and genetic basis of inter-patient variability in treatment response are not fully understood. Taking a genome-wide approach, we recently identified genetic variants in the ARID5B gene that strongly predispose children to developing ALL and also a high risk of relapse following therapy (J Clin Oncol 2012 30:751, Nat Genet 2009 41:1001). To understand the mechanisms by which ARID5B is linked to treatment outcome in childhood ALL, we sought to 1) characterize ARID5B expression in different genetic subtypes of ALL, 2) determine the effects of ARID5B expression on cytotoxicity of chemotherapeutic agents commonly used in ALL therapy, and 3) describe molecular pathways linking ARID5B to anti-leukemic drug sensitivity. In 567 children with newly diagnosed ALL treated at St. Jude Children’s Research Hospital (GSE33315), ARID5B expression was highest in cases with hyperdiploid karyotype (>50 chromosomes) and lowest in T-cell ALL and cases with MLL rearrangements. This pattern was validated in an independent cohort of 106 children from the Dutch Childhood Oncology Group (GSE13351). In 59 patients treated on the Children’s Oncology Group (COG) CCG1961 trial, lower ARID5B expression was associated with higher rates of relapse (P=0.01, GSE7440). Importantly, when we compared matched newly-diagnosed vs. relapsed ALL blasts from a cohort of 60 patients enrolled in COG trials (GSE28460), ARID5B expression was further downregulated at disease recurrence (P=0.0009). shRNA-mediated ARID5B knockdown in 3 ALL cell lines (Nalm6, SEM, and UOCB-1) substantially increased resistance to antimetabolites (an average of 5.16 and 35.3-fold increase in IC50 for methotrexate [MTX] and 6-mercaptopurine [6MP], respectively), with minimal effects on glucocorticoids, vincristine, asparaginase, and daunorubicin. Because cytotoxic effects of MTX and 6MP are highly dependent on the rate of cell proliferation, we postulate that ARID5B directly influences cell cycle entry. In all 3 cell lines, ARID5B knockdown led to significant blockade at the G1/S checkpoint, increasing the percent of cells in G0/G1 phase. At the molecular level, downregulation of ARID5B resulted in higher levels of p21 and reduction in phosphorylated Rb, consistent with the retention at G0/G1 phase. ARID5B expression was restricted to nucleus but affected both nuclear and cytoplasmic p21 expression in a time-dependent fashion. Interestingly, there was a highly significant negative correlation between p21 expression and MTX- and 6MP-induced apoptosis in all 3 ALL cell lines. Taken together, we hypothesize that lower expression of ARID5B impairs ALL cell cycling by upregulating p21, contributing to resistance to MTX and 6MP and eventually leukemia relapse. Finally, we compared global gene expression in ARID5B knockdown vs. control ALL cells, and via the Connectivity Map analysis we identified histone deacetylase (HDAC) inhibitors as promising agents for overcoming ARID5B-related drug resistance. Indeed, ARID5B knockdown cells were significantly more sensitive to panobinostat than controls, suggesting HDAC inhibitors as potential therapeutic options for patients with ARID5B-deficient and drug resistant ALL. DisclosuresNo relevant conflicts of interest to declare.

The Negative Impact of Underweight and Weight Loss on Survival of Children with Acute Lymphoblastic Leukemia

Background: Body mass index (BMI: kg/m2) and change in BMI during treatment might influence treatment outcome of pediatric patients with acute lymphoblastic leukemia (ALL). However, previous studies in pediatric acute lymphoblastic leukemia reported contradictory results. Therefore, we studiedthe influence of (change in) BMI on treatment outcome in pediatric ALL patients who were treated according to a dexamethasone-based protocol (Dutch Childhood Oncology Group [DCOG] ALL-9).Patients and Methods: Data on body composition were prospectively collected from a cohort of newly diagnosed Dutch pediatric ALL patients (N=762, age 2-17 years), treated from 1997-2004. BMI at diagnosis was expressed as standard deviation scores (SDS) and categorized into underweight (≤–1.8SDS), or normal weight and overweight (>–1.8SDS). BMI loss was defined after 32 weeks of treatment. Dual X-ray absorptiometry scans were performed in a nested single center cohort (n=106) to assess the contribution of %fat and lean body mass to BMI. Multivariate analyses were corrected for age at diagnosis and risk treatment grpup.Results: Multivariate analyses showed that patients with underweight (8%) had an increased risk of relapse (Hazard Ratio (HR) 1.79, 95% CI: [1.04-3.10], and a similar overall survival (HR 1.10 [0.57-2.10]). BMI loss during the first 32 weeks of treatment was associated with a decreased overall survival (HR 2.10 [1.14-3.87]), and a similar risk of relapse (HR 1.27 [0.70-2.30]) compared to patients without BMI loss. Dual X-ray absorptiometry revealed that BMI loss mainly consisted of a loss of lean body mass and gain in %fat.Conclusion: Underweight at diagnosis is associated with an increased risk of relapse and a BMI loss early during treatment is associated with an increased mortality. This suggests that these patients might benefit from exercise interventions and a high-quality nutrient diet during therapy. DisclosuresNo relevant conflicts of interest to declare.

Toxicity of Very Prolonged Pegasparaginase and Erwinia Asparaginase Courses in Relation to Asparaginase Activity Levels with a Special Focus on Dyslipidemia

▪PurposeWe 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 MethodsPatients 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.ResultsIn 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<0.001).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.ConclusionsSevere 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 1Toxicity 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-valueGrade 1/2Grade 3/4Grade 1/2Grade 3/4n%n%n%n%Pancreatitis00461529nsHypertriglyceridemia1522314773200p<0.001Hypercholesterolemia69172583700p=0.01Hyperammonemia34510094129nsThrombosis00230029nsCentral neurotoxicity007100015ns DisclosuresTong:EUSA Pharma: Research Funding.