ALL IC-BFM Research Articles

ANALYSIS OF PSYCHO-EMOTIONAL STATE AND THE SEVERITY OF ASTHENIC SYNDROME IN CHILDREN WITH ACUTE LEUKEMIA AFTER THE FIRST STAGE OF CHEMOTHERAPY

Children and adolescents with hematological diseases are constantly in a state of prolonged psychological stress caused by hospitalization, debilitating treatment, lifestyle changes, which ultimately leads to a significant reduction in the quality of life. In this connection it is necessary to study the dynamics of mental and emotional condition of the child at different stages of chemotherapy and the timing of formation of asthenic syndrome. Determine the presence of psycho-emotional disorders and the severity of asthenic syndrome in children with acute leukemia after the first stage of chemotherapy in order to further predict the development of this pathology and develop a package of solutions that minimize its manifestations. A survey of 36 children was conducted, of them 21 people aged 6–10 years old, 15 people aged 11–16 years old. There are 27 boys (75%) and 9 girls (25%). The survey was conducted on the 78th day of the ALL IC-BFM 2009 protocol, which corresponds to the end of the first phase of chemotherapy. In the survey did not participate children with the syndrome of multiple organ failure, as well as with the presence of background mental disorders. To assess the psycho-emotional state, a questionnaire was used: “state of health, activity, mood”, adapted for children. For the study of asthenia was used questionnaire I. K. Schatz. The findings suggest that absolutely all children receiving chemotherapy at the end of the first phase have been violations of the psycho-emotional background. The index of state of health suffers the most, and to a lesser extent - activity. Indicators of state of health had lower values in the older group - from 11 to 16 years. Thus according to the data in the group of children aged 6 to 10 years being the average was 4.02 ± 0.09 (p <0.05), in children age 11 - 16 years - 3.83 ± 0.11 (p <0.05). The average activity substantially the same in groups and indicates a favorable condition. Mood indicators are also within the framework of a favorable state in both groups. Also during the investigation revealed that the general for all children is a symptom asthenic. According to the data, fatigue reaction (less than 7 points) was present only in 9 children (25%). The most numerous manifestations were moderate asthenia - 21 children (58%). Severe asthenia (13 - 18 points) was observed only in 6 children (17%). This study will allow to develop a package of solutions for the prevention and minimization of asthenic syndrome in children with acute leukemia receiving chemotherapy.

Clinical characteristics and analysis of treatment result in children with Ph-positive acute lymphoblastic leukaemia in Poland between 2005 and 2017.

The aim of this study was to analyse the clinical characteristics and outcome of children diagnosed with Ph+ ALL. A total of 2591 newly diagnosed ALL children were treated in Poland between the years 2005 and 2017. Of those, 44 were diagnosed with Ph(+) ALL. The patients were treated according to protocols: ALL IC-BFM 2002 and 2009 (26 patients), EsPhALL (12 patients), initially ALL IC-BFM and then EsPhALL (6 patients). The median of follow-up in the observed group was 3years. Overall survival (OS) and event-free survival (EFS) of Ph+ ALL group were 0.73 and 0.64. OS and EFS of patients after HSCT were 0.78 and 0.66, while without HSCT were 0.6 and 0.6, P=0.27 and 0.63. OS was 0.8 for patients treated with chemotherapy plus imatinib and 0.61 for chemotherapy alone, P=0.22, while EFS was 0.66 (imatinib therapy) and 0. 61 (without imatinib), P=0.41. Our study suggests that adding imatinib to intensive chemotherapy seems to improve outcome. However, this study was limited by a small number of patients and a variety of chemotherapy regimens with or without imatinib.

Variants in TPMT, ITPA, ABCC4 and ABCB1 Genes As Predictors of 6-mercaptopurine Induced Toxicity in Children with Acute Lymphoblastic Leukemia.

SummaryBackgroundAcute lymphoblastic leukemia is the most common childhood malignancy. Optimal use of anti leukemic drugs has led to less toxicity and adverse reactions, and a higher survival rate. Thiopurine drugs, including 6-mercaptopurine, are mostly used as antileukemic medications in the maintenance phase of treatment for children with acute lymphoblastic leukemia. For those patients, TPMT genotype- tailored 6-mercaptopurine therapy is already implemented in the treatment protocols. We investigated the role of TPMT, ITPA, ABCC4 and ABCB1 genetic variants as predictors of outcome and 6-mercaptopurine induced toxicity during the maintenance phase of treatment in pediatric acute lymphoblastic leukemia.MethodsSixty-eight children with acute lymphoblastic leukemia were enrolled in this study. Patients have been treated according to ALL IC-BFM 2002 or ALL IC-BFM 2009 protocols. Toxicity and adverse events have been monitored via surrogate markers (off-therapy weeks, episodes of leu - ko penia and average 6-mercaptopurine dose) and a prob- abilistic model was employed to predict overall 6-mercaptopurine related toxicity.ResultsWe confirmed that patients with acute lymphoblastic leukemia that carry inactive TPMT allele(s) require 6- mercaptopurine dose reduction. ITPA and ABCC4 genetic variants failed to show an association with 6-mercapto - purine induced toxicity during the maintenance phase. Carriers of ABCB1 variant allele experienced greater hepatotoxicity. The probabilistic model Neural net which considered all the analysed genetic variants was assessed to be the best prediction model. It was able to discriminate ALL patients with good and poor 6-mercaptopurin tolerance in 71% of cases (AUC=0.71).ConclusionsThis study contributes to the design of a panel of pharmacogenetic markers for predicting thiopurineinduced toxicity in pediatric ALL.

Long-term brain structural magnetic resonance imaging and cognitive functioning in children treated for acute lymphoblastic leukemia with high-dose methotrexate chemotherapy alone or combined with CNS radiotherapy at reduced total dose to 12\xa0Gy

IntroductionThe aim of this study was to assess the long-term side effects of central nervous system prophylaxis (high-dose chemotherapy alone vs chemotherapy and CNS radiotherapy) according to the ALL IC-BFM 2002.MethodsThirty-tree children aged 6.7–19.9 years have been studied. The control group consisted of 12 children newly diagnosed with acute lymphoblastic leukemia. We assessed subcortical gray matter volume using automatic MRI segmentation and cognitive performance to identify differences between two therapeutic schemes and patients prior to treatment.ResultsPatients treated with chemotherapy and CNS radiotherapy had smaller hippocampi than two other subgroups and lower IQ score than patients treated with chemotherapy alone. Both treated groups, whether with chemotherapy only or in combination with CNS radiotherapy, had significantly lower volumes of caudate nucleus and performed significantly worse on measures of verbal fluency in comparison with patients prior to treatment. There were no differences in the mean volumes of total white matter, total gray matter, thalamus, putamen, and amygdala between the studied groups.ConclusionIn all children treated according to the ALL IC-BFM 2002 with high-dose chemotherapy, both decreased volume of selected subcortical structures and cognitive impairment was observed, especially in children who received chemotherapy in combination with reduced dose CNS radiotherapy. In all children treated according to the ALL IC-BFM 2002 with high-dose chemotherapy, both decreased volume of selected subcortical structures and cognitive impairment were observed, especially in children who received chemotherapy in combination with CNS radiotherapy.

Non-Hodgkin’s Lymphomas in Children: 25-Year Clinical Experience

Background &amp; Aims. Current polychemotherapeutic protocols based on differentiated and risk-adopted approaches permitted to consider non-Hodgkin's lymphomas (NHL) potentially curable diseases although they had been considered fatal previously. The aim of this study is to summarize and analyze outcomes of NHL therapy over a 25-year period. Methods. 246 patients were enrolled in the study. They were treated in the department of chemotherapy of hemoblastoses in the Scientific Research Institute of Pediatric Oncology and Hematology under the NN Blokhin Russian Cancer Research Center over the period of 25 years: from April 1, 1991, till June 1, 2016. B-NHL-BFM 90/95 protocols and a modified B-NHL-BFM 95 protocol (with rituximab) were used for B-cell NHLs (n = 130). Patients with lymphocytic leukemia (n = 75) were treated using ALL-mBFM 90/95 and ALL IC-BFM 2002 protocols. 21 patients with anaplastic large cell lymphomas (ALCL) received treatment according to the B-NHL-BFM 90/95 protocol, and 20 patients received the НИИ ДОГ-АККЛ-2007 protocol. Results. Taking into account clinical and immunological characteristics of ALCL, the authors invented an original НИИ Д0Г-АККЛ-2007 protocol. Special attention was paid to potential modification of standard treatment regimens for B-cell NHL by adding rituximab. The article demonstrates the evolution in prescription of rituximab for B-cell NHL and possibilities for reduction of the total number of polychemotherapy cycles for late-stage tumors without deterioration of treatment outcomes. Conclusion. The obtained results permit to conclude that introduction of achievements of oncoimmunology, molecular biology, and cytogenetics will become the basis for further modification of existing treatment options for NHL.

Impact of mTOR expression on clinical outcome in paediatric patients with B-cell acute lymphoblastic leukaemia - preliminary report.

Aim of the studyTo characterise expression of mTOR (mammalian target of rapamycin) in childhood B-cell acute lymphoblastic leukaemia (ALL), and to evaluate a possible link between mTOR and clinical characteristics.Material and methodsThe examined group consisted of 21 consecutive patients, aged 1–18 years, diagnosed with B-cell ALL in 2010, and 10 relapsed B-cell ALL patients diagnosed for the first time between 2009 and 2011, who developed relapse before 2014. All subjects were treated in the Department of Paediatric Haematology and Oncology of the Medical University of Warsaw according to the ALL-IC BFM 2002 Protocol. We evaluated mTOR and phospho-mTOR expression by immunohistochemistry using rabbit monoclonal antibodies.ResultsmTOR expression was found to be significantly associated with the risk of relapse and was more frequent in ALL recurrence. No significant relationship was detected between mTOR expression and other features of high-risk disease in paediatric ALL.ConclusionsmTOR activity could be considered a high-risk feature in paediatric B-cell ALL. Expression of mTOR kinase is observed remarkably more frequently in disease recurrence than at first diagnosis, indicating higher proliferative and survival potential of leukaemic cells in relapse. Routine analysis of mTOR activity could be performed to select patients that may potentially benefit from mTOR inhibitors (MTI) treatment.

Impact of single nucleotide polymorphisms of cytarabine metabolic genes on drug toxicity in childhood acute lymphoblastic leukemia.

Cytarabine (cytosine arabinoside, ara-C) is a chemotherapeutical agent used in the treatment of pediatric acute lymphoblastic leukemia (ALL). Adverse drug reactions, such as interpatient variability in sensitivity to ara-C, are considerable and may cause difficulties during chemotherapy. Single nucleotide polymorphisms (SNPs) can play a significant role in modifying nucleoside-drug pharmacokinetics and pharmacodynamics and thus the development of adverse effects. Our aim was to determine whether polymorphisms in genes encoding transporters and enzymes responsible for the metabolism of ara-C are associated with toxicity and clinical outcome in a patient population with childhood ALL. We studied 8 SNPs in the CDA, DCK, DCTD, SLC28A3, and SLC29A1 genes in 144 patients with childhood acute lymphoblastic leukemia treated according to ALLIC BFM 1990, 1995 and 2002 protocols. DCK rs12648166 and DCK rs4694362 SNPs were associated with hematologic toxicity (OR = 2.63, CI 95% = 1.37-5.04, P = 0.0036 and OR = 2.53, CI 95% = 1.34-4.80, P = 0.0044, respectively). Our results indicate that DCK polymorphisms might be important genetic risk factors for hematologic toxicity during ALL treatment with ara-C. Individualized chemotherapy based on genetic profiling may help to optimize ara-C dosing, leading to improvements in clinical outcome and reduced toxicity.

The Prevalence of Congenital Trombophilia in Children Treated for Acute Lymphoblastic Leukemia with Thrombosis As a Complication

The aim of the study was to evaluate factors predisposing to thrombosis, especially congenital trombophilia, in children treated for acute lymphoblastic leukemia according to ALL IC BFM 2002 and 2009.An analysis of 36 cases of thrombosis in 242 children (15%) treated due to ALL in the Department in years 2007-2013 was carried out. Age at onset, location, cause and treatment methods were taken into consideration. All analyzed patients underwent screening for congenital thrombophilia.The age of patients ranged from 1.5 to 17 years with the median of 8.5 years. Arterial thrombosis occurred in 2 persons (1%), venous thrombosis in 34 (99%) patients. Thromboembolism was usually related to the central venous catheter (n = 31, 91%). Massive thrombosis occurred in 7 patients (20%). Relapses occurred in 3 children.The most common causes of thrombosis were: the presence of the central catheter (n = 31, 91%) and L-asparaginase treatment (n = 19, 55%). The factor V Leiden mutation was diagnosed in 1 patient, protein C deficiency in 1 patient as well, and elevated levels of factor VIII were detected in 4 cases.The mean concentrations of factor VIII, von Willebrand factor and antithrombin were not different in the groups with and without thrombosis (table 1 ). The concentration of fibrinogen measured after finishing the chemotherapy was similar in both groups.Conclusions 1. A risk factor for thrombosis in children with ALL is the treatment of L-asparaginase and the presence of central venous catheter.2. Screening for congenital thrombophilia in children treated for ALL should not be done routinely because the coexistence of this two conditions is rare. Table 1FVIII (%)Fibrinogen ( g/l)AT (%)Patients with thrombosis1012,776Patients without thrombosis892,4382p0,45 ( ns)0,2 (ns)0,34 (ns) DisclosuresNo relevant conflicts of interest to declare.

Identification of novel NOTCH1 mutations: Increasing our knowledge of the NOTCH signaling pathway

Alterations in the NOTCH1 signaling pathway are found in about 60% of pediatric T-ALL, but its impact on prognosis remains unclear. We extended the previously published CoALL cohort (n = 74) to a larger cohort (n = 127) and additionally included 38 Argentine patients from ALL IC-BFM to potentially identify novel mutations and decipher a stronger discriminatory effect on the genotype/phenotype relationship with regard to early treatment response and long-term outcome. Overall, 101 out of 165 (61.2%) T-ALL samples revealed at least one NOTCH1 mutation, 28 of whom had combined NOTCH1 and FBXW7 mutations. Eight T-ALL samples (4.8%) exclusively revealed FBXW7 mutations. Fifty-six T-ALL (33.9%) exhibited a wild-type configuration of either gene. Four novel NOTCH1 mutations were identified localized in the C-terminal PEST domain, in the rarely affected LNR repeat domain and in the ankyrin domain. Novel LNR mutations may contribute to a better understanding of the structure of the NOTCH1 negative regulatory region (NRR) and the R1946 mutation in the ankyrin domain may represent an unusual loss-of-function mutation. Overall, NOTCH1 pathway mutations did not affect the relapse rate and outcome of the extended T-ALL cohort uniformly treated according to CoALL protocols, although NOTCH1 mutations were associated with good response to induction therapy (P = 0.009). Individually, HD and PEST domain mutations might exert distinct functional effects on cellular homeostasis under treatment NOTCH1 pathway activity with prognostic implications.

Long-Term Brain Status and Cognitive Functioning In Children With Acute Lymphoblastic Leukemia Treated According To ALL IC-BFM 2002

BackgroundAcute lymphoblastic leukemia (ALL) comprises about 85% of all leukemias in children and is the most frequent malignancy of childhood. Long-term side effects of applied therapy according to ALL IC-BFM 2002 (with prophylactic CNS radiotherapy dose reduced to 12 Gy given in combination with high-dose methotrexate intra venous and methotrexate alone or in combination with cytarabine and prednisolone intra thecally) are still unknown. AimThe aim of this cross-sectional study is to assess the long-term side effects of central nervous system (CNS) prophylaxis (high-dose chemotherapy i.v. alone vs. high dose chemotherapy i.v. combined with prophylactic CNS radiotherapy) according to ALL IC-BFM 2002 by the whole brain magnetic resonance and assessment the neuropsychological functioning. Patients and MethodsOut of 150 children treated in 2002-2010 because of ALL, 58 children (23 girls and 35 boys) aged 6.4 - 19.9 years (median: 14.4) have been previously studied, including 26 children treated with chemotherapy alone, 21 - treated with chemo- and radiotherapy, and 11 - before treatment (control group). The structural assessment included evaluation of the segmentation of brain and calculation of the volume of white and gray matter as well as segmentation of subcortical structures. The neuropsychological assessment included the Wechsler Intelligence Test for Children (WISC-R; IQ assessment and long-term memory), Rey Auditory Verbal Learning Test (RAVLT; evaluation of short term memory), Benton Visual Retention Test (evaluation of visual memory), Verbal Fluency Test (evaluation of verbal memory), the Stroop test (evaluation of reaction time and executive) and Wisconsin Card Sorting Test (WSCT; evaluation of executive functions). ResultsPatients treated with high-dose chemotherapy and CNS radiotherapy had a lower volume of the hippocampus (6.7 cm³) and basal ganglia (29 cm³) compared to the group treated with chemotherapy alone (8 cm³ and 31 cm³, respectively) and the control group (8 cm³ and 32 cm³). All patients treated due to ALL had smaller total volume of brain ventricles (12 cm³ in the group treated with chemo-and radiotherapy, and 13 cm³ in the group treated with chemotherapy alone), compared to the control group (18 cm³).In patients, who that received chemo-and radiotherapy the average intelligence quotient was lower (IQ - 101), visual-spatial memory was worse (Benton test – 6 correct reproductions), auditory-verbal memory was worse (6, 7, 9, 9, 11) and lower verbal fluency (10 for phonemic category and 13 for semantic one) as compared to patients treated with chemotherapy alone (respectively: IQ - 116, Benton test - 7; RAVLT -7, 7, 10, 12, 13; verbal fluency – 12 and 16) and to patients in the control group (IQ - 116, Benton test - 9; RAVLT - 9, 11 , 13, 13, 14; verbal fluency – 15 and 19). The group treated with chemo-and radiotherapy (WSCT - 3.2 classified category, the rate of correct answers - 0.48, Stroop test - 3.2) and the group treated with chemotherapy alone (WSCT - 4.1 category, the rate of correct answers - 0.59; Stroop test - 2.9) performed significantly worse on measures of executive functioning in comparison to the control group (WSCT - 5.2 category, the rate of correct answers - 0.71; Stroop test - 2.5). ConclusionsIn children treated for ALL according to the ALL IC-BFM 2002 both CNS structural changes and cognitive impairment may be observed. These abnormalities are even more pronounced in children who received chemotherapy in combination with CNS radiotherapy.This work was supported by grant from the National Science Centre (DEC-2012/05/N/NZ5/00879). Disclosures:No relevant conflicts of interest to declare.

Thrombosis Risk Factors Assessment In Children Treated For Acute Lymphoblastic Leukemia

The aim of the study was to evaluate factors predisposing to thrombosis in children treated for acute lymphoblastic leukemia according to ALL IC BFM 2002. An analysis of 30 cases of thrombosis in 210 children (14%) treated due to ALL in the Department in years 2007-2011 was carried out. Age at onset, location, cause and treatment methods were taken into consideration. All analyzed patients underwent screening for congenital thrombophilia. The age of patients ranged from 1.5 to 16 years with the median of 7.5 years. Arterial thrombosis occurred in 1 person (3%), venous thrombosis in 29 (97%) patients. Thromboembolism was usually related to the central venous catheter (n = 27, 90%). Massive thrombosis occurred in 6 patients (20%). Relapses occurred in 2 children. The most common causes of thrombosis were: the presence of the central catheter (n = 27, 90%) and L-asparaginase treatment (n = 16, 53%). The factor V Leiden mutation was diagnosed in 1 patient, protein C deficiency in 1 patient as well, and elevated levels of factor VIII were detected in 3 cases. Five children (16%) underwent systemic thrombolysis with recombinant tissue plasminogen activator and 17 patients (52%) had local thrombolysis. Anticoagulation with warfarin following low-molecular-weight heparin discontinuation was used in 6 patients (20%). The complete resolution of the thrombus was observed in the whole group of patients. Conclusions 1. A risk factor for thrombosis in children with ALL is the treatment of L-asparaginase and the presence of central venous catheter. 2. Screening for congenital thrombophilia in children treated for ALL should not be done routinely since the coexistence of this two conditions is rare. Thrombosis in children with ALL responds well to treatment. Disclosures: Klukowska: Octapharma AG: Investigator Other.

BCL11B, FLT3, NOTCH1 and FBXW7 mutation status in T-cell acute lymphoblastic leukemia patients

T-cell acute lymphoblastic leukemia is a heterogeneous malignancy originating from developing lymphocyte precursors likely due to mutations in genes regulating thymocyte differentiation. Here, we characterized mutation status of BCL11B and FLT3 genes, presumably involved in T-ALL, together with FBXW7 and NOTCH1 as known players in T-ALL in 65 pediatric T-cell acute lymphoblastic leukemia patients. We also aimed at the assessment of prognostic value of NOTCH1 and FBXW7 mutations in ALL-IC BFM 2002 protocol. FLT3 and BCL11B mutations were detected in 3% and 2% of patients, respectively. FBXW7 mutations were observed in 8% of patients, while NOTCH1 was mutated in 40%. No correlation was found between NOTCH1 and FBXW7 mutations and traditionally used clinical factors or molecular features. In total we have detected nine mutations, which have not been previously described by others. Eight of them were found in NOTCH1 and one in BCL11B gene. Observed frequencies of NOTCH1 and FBXW7 are in line with previous reports, thus confirming postulated participation of these two genes in T-ALL pathomechanism. Moreover, we report on mutation frequency of FLT3 and BCL11B, not extensively studied in T-ALL so far. Finally, we suggest a putative role of BLC11B as an oncogene in T-ALL pathogenesis.

Transient hyperglycaemia – an underestimated problem of paediatric oncohaematology

IntroductionThe majority of hyperglycaemic incidents in oncohaematological patients treated with glucocorticosteroids remain undiagnosed. The aim of our study was to work out a detailed protocol for the control of carbohydrate metabolism and to evaluate whether such a protocol can help in diagnosis of carbohydrate metabolism disturbances in oncohaematological paediatric patients.Material and methodsA one hundred and twenty-eight children treated for proliferative diseases of the haematopoietic system and severe aplastic anaemia with therapeutic protocols including glucocorticosteroids were divided into two groups. Group I consisted of 70 children, whose blood glucose was evaluated on random occasions (retrospective analysis). Group II consisted of 58 children included in the programme of intensive carbohydrate metabolism control (prospective analysis). We compared the incidence of hyperglycaemia in both groups as well as the number of hyperglycaemic incidents per individual therapeutic protocol.ResultsA significantly higher incidence of transient hyperglycaemia was noted in oncohaematological patients in the programme of early carbohydrate metabolism disturbances diagnosis than in the other group (22.4% vs. 5.7% respectively; p = 0.008), especially in patients treated with the ALL IC-BFM 2002 protocol for the high risk group (arm A and B), the ALL-REZ BFM 2002 protocol, and in a heterogenous group of children (protocols ALCL 99, Euro-LB02, Interfant-06, WPSAA) (p = 0.042, 0.021 and 0.002, respectively).ConclusionsThe improvement of transient hyperglycaemia detection may constitute the first step towards the reduction of unfavourable consequences of hyperglycaemia. Prospective studies are required to demonstrate the influence of normal carbohydrate metabolism on the frequency of infectious complications in this group.

Results of the Randomized I-BFM-SG Trial „Acute Lymphoblastic Leukemia Intercontinental-BFM 2002“ in 5060 Children Diagnosed in 15 Countries on 3 Continents

Abstract 872 Introduction: ALL IC-BFM 2002 is one of the most successful projects developed by the I-BFM-SG, which is one of the world largest societies involving national leukemia groups. The trial evaluated in a randomized manner the impact on outcome of intensified late reinduction in the context of a newly developed risk stratification. The main goal of the trial was improvement of outcome of children with ALL in each of the 3 risk groups (RG). Patients & methods: From Nov 2002-Nov 2007, 5060 eligible pts aged 0–18 yrs with newly diagnosed ALL from Argentina (1270), Chile (558), Croatia (122), Cuba (151), Czech Republic (291), Hong Kong (155), Hungary (259), Israel (292), Poland (908), Serbia (266), Slovakia (137), Slovenia (36), Ukraine (421), Uruguay (96), and Moscow (98) were enrolled in the trial (http:clinicaltrials.gov “NCT00764907”). Stratification into 3 RGs was based on early treatment response (evaluated in PB on day 8 and in BM on days 15 and 33), age, initial WBC, and presence/absence of BCR/ABL or MLL/AF4. Standard Risk (SR) criteria were: Results: With median follow-up 4.9 yr, the 5-yr EFS was 74 ± 1% and 5-yr OS 82 ± 1% for the whole group of 5060 pts. The CR rate was 97%, 255 (5%) children died in remission. The 5-yr EFS/OS was 81 ± 1%/90 ± 1% in 1564 SR pts (30.9% of all pts), 75 ± 1%/83 ± 1% in 2650 IR pts (52.4%) and 55 ± 2%/62 ± 2% in 846 HR pts (16.7%). Randomization rate was 79% of those patients who survived for at least 20 weeks (planned timepoint of randomization). None of the experimental arms achieved significantly better EFS compared to standard treatment. CI of relapses at 5 yr was 18 ± 1% overall, CI for isolated BM relapse was 12 ± 1%, isolated and combined CNS relapse 4 ± 0.3%, isolated and combined testicular relapse 2 ± 0.2%. Secondary malignancy was diagnosed so far in 26 patients (5-yr CI 0.6 ± 0.1%). Significantly better EFS was achieved in BCP-ALL(75 ± 1%) in comparison with T-ALL (69 ± 1%), girls vs. boys (76 ± 1% vs 72 ± 1%), children aged Conclusions: Although the experimental arm was no better than the traditional one across individual RGs, the majority of participating countries, many of them were new-comers to this intensive therapy, improved their treatment results against previous national studies. The trial confirmed the validity and feasibility of a simple risk stratification of ALL applied in a complex and heterogeneous multinational environment. Despite the great differences between individual countries, the trial set a firm stage for willing national leukemia groups to run collaborative clinical trials in ALL under the umbrella of I-BFM-SG. Supported by MSM0021620813 and MZ0FNM2005. Disclosures: No relevant conflicts of interest to declare.

Minimal residual disease in peripheral blood at day 15 identifies a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with superior prognosis

Most minimal residual disease-directed treatment interventions in current treatment protocols for acute lymphoblastic leukemia are based on bone marrow testing, which is a consequence of previous studies showing the superiority of bone marrow over peripheral blood as an investigational material. Those studies typically did not explore the prognostic impact of peripheral blood involvement and lacked samples from very early time points of induction. In this study, we employed real-time quantitative polymerase chain reaction analysis to examine minimal residual disease in 398 pairs of blood and bone marrow follow-up samples taken from 95 children with B-cell precursor acute lymphoblastic leukemia treated with the ALL IC-BFM 2002 protocol. We confirmed the previously published poor correlation between minimal residual disease in blood and marrow at early treatment time points, with levels in bone marrow being higher than in blood in most samples (median 7.9-fold, range 0.04-8,293-fold). A greater involvement of peripheral blood at diagnosis was associated with a higher white blood cell count at diagnosis (P=0.003) and with enlargement of the spleen (P=0.0004) and liver (P=0.05). At day 15, a level of minimal residual disease in blood lower than 10(-4) was associated with an excellent 5-year relapse-free survival in 78 investigated patients (100% versus 69 ± 7%; P=0.0003). Subgroups defined by the level of minimal residual disease in blood at day 15 (high-risk: ≥ 10(-2), intermediate-risk: <10(-2) and ≥ 10(-4), standard-risk: <10(-4)) partially correlated with bone marrow-based stratification described previously, but the risk groups did not match completely. No other time point analyses were predictive of outcome in peripheral blood, except for a weak association at day 8. Minimal residual disease in peripheral blood at day 15 identified a large group of patients with an excellent prognosis and added prognostic information to the risk stratification based on minimal residual disease at day 33 and week 12.

Minimal residual disease detection using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: Slovak experience.

Acute lymphoblastic leukemia is the most common form of cancer in children. The 10-year event-free survival ranged from 77 to 85% after having achieved complete remission rates of 93% or higher. The main cause of treatment failure is relapse arising from outgrowth of residual leukemic cells that are refractory to therapy. An intense effort has been made to develop methods to determine the degree of minimal residual leukemia cells present in patients considered to be in morphological remission. Because of the strong correlation between minimal residual disease (MRD) levels and risk of relapse, monitoring of MRD provides unique information regarding treatment response. The MRD monitoring based on real-time quantitative PCR detection of patient-specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements is currently considered to be the most reliable tool for MRD-based diagnosis in ALL. Because the significance of MRD monitoring has been strongly supported by several studies and because it has been implemented in the latest protocols, there has been a significant effort to develop MRD monitoring in the Slovak Republic since 2005. Between October 2006 and December 2009, 50 children with ALL who were treated at three Slovak centers were included in the RQ PCR MRD pilot project. A total of 40 patients with BCP-ALL ( B cell precursor ALL) and 4 patients with T ALL were analyzed for Ig/TCR rearrangement. We identified 106 different rearrangements in the 44 ALL patients analyzed. Based on MRD stratification, we identified 26 patients who were stratified into the HRG ( high risk group) (n = 3; 11.5%), IRG ( intermediate risk group) (n = 14; 54%) and SRG ) standard risk group) (n = 9; 34.5%). Morphology-based risk stratification allows the identification of most HRG patients identified also by MRD-based stratification, but fails to discriminate the IRG assigned to therapy reduction. Patients in the SRG and the IRG could profit from MRD-based risk assignment

Relative Expression of Ikaros Isoforms Has a Prognostic Impact in Phildelphia-Negative Childhood Acute Lymphoblastic Leukemia.

Abstract 1282Poster Board I-304Ikaros, encoded by the IKZF1 gene, is a zinc-finger transcription factor crucial for normal differentiation of the lymphoid lineage. Multiple isoforms of Ikaros are generated by alternative splicing in lymphoid progenitors. Recent studies based primarily on high-risk (HR) patients with ALL, including Philadelphia-positive cases, have shown an inferior prognosis of patients with Ikaros alterations, leading in most cases to the expression of short, non-DNA binding isoforms of IKZF1. There is only a limited information about the overall frequency and prognostic impact of IKZF1 alterations in non-selected, BCR/ABL-negative ALL cohorts. Using a simplified yet efficient approach based on expression assay described by Iaccobucci et al. together with Agilent-on-chip semi-quantitative electrophoresis, we examined the expression of IKZF1 transcript variants in diagnostic bone marow (BM) samples from 94 children with Ph- ALL. The patients were diagnosed between November 2002 and December 2004 and treated by ALL IC-BFM 2002 protocol. Based on the analysis of peripheral blood from healthy donors and remission BM samples, we determined physiological range for relative expression of IKZF1 isoforms. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK8) and functional IK1 and IK2 isoforms was significantly elevated in 26 of 94 patients (28%). There were no associations between elevated short/long isoforms ratio and age, WBC, ALL IC risk group, TEL/AML1 or hyperdiploidy. Considering the key role of Ikaros in lymphoid lineage specification, we tested whether its altered expression was related to the expression of myeloid markers on leukemic blasts. Neither short/long isoforms ratio, nor single transcript variant expression had any relation to the level of myeloperoxidase (MPO), CD13, CD33, CD65, CD117, CD14 or CD15 expression estimated by flow cytometry. Patients having the short/long isoforms ratio more than 80% had a 5-year RFS 66.7±13.6% compared to 87.5±4.1% in other patients (p=0.04). The main difference between leukemic and normal samples was observed in the relative expression of IK6 dominant-negative isoform. Using a cut-off of 10%, 14 of 94 (15%) ALL samples had increased IK6 expression in relation to other isoforms. Only 2 of 94 patients (2%) expressed IK6 alone. Elevated relative IK6 expression in the range 10-20% (6 patients) had no prognostic impact. Using a cut-off of 20%, 5-year RFS survival was 90.2± 3.3% in the group with low IK6 expression compared to 37.5±17.1% in patients with the high expression (p<0.0001). With the cut-off of 50%, RFS was 20±17.9% in the IK6 high expression group compared to 89.4±3.3% in the group with low expression (p<0.0001). Of the 5 patients with IK6 expression higher than 50%, two were treated in ALL IC HR group (based on poor prednisone response), one in the intermediate risk and two in the standard risk group, based on WBC, age and BM status at day 15. Only 1 of 4 patients with available MRD would be classified as MRD-HR based on MRD higher than 10−3 at week 12. Surprisingly, elevated relative expression of IK4, IK4del, IK4A and IK8 (all non-DNA binding isoforms) had no prognostic impact. The absolute level of IKZF1 expression, which might have indicated IKZF1 haploinsufficiency, had no prognostic impact either. In conclusion, we showed that a substantial proportion of childhood Ph-negative ALL cases had an increased expression of short, non-DNA binding Ikaros transcripts. However, only elevated expression of the IK6 isoform had prognostic impact. Patients with IK6 expression higher than 50% (5% of all patients) had very poor prognosis. The method used in this study could serve as a rapid screening for a new subgroup of HR patients with ALL.Supported by GA UK 7393/2007, MSMT NPV 2B06064, VZ MSM 0021620813 and MZ 000064203. DisclosuresNo relevant conflicts of interest to declare.

Thrombin Generation in Pediatric Patients Treated with L-asparaginase.

Abstract 3983Poster Board III-919 IntroductionTreatment with L-asparaginase is associated with an disturbance of coagulation cascade. Increased thrombin generation is the most frequently cited coagulation anomaly associated with L-asparaginase treatment. Until now, almost all studies aimed on estimation of thrombin generation, have used circulating procoagulant markers such are thrombin/antithrombin complexes(TAT),D dimers(DD),prothrombin fragments (F1,F2).Calibrated automated thrombogram (CAT) allows the precise estimation of the whole amount of thrombin generated in vivo at present time. We show the first data on thrombin generation measured by CAT among pediatric patients with acute lymphoblastic leukemia treated with L-asparaginase. Patients and methodsThrombin generation was measured in platelet poor plasma of 18 patients (age 7-17years) by means of CAT. Samples were obtained at pre-defined time points during the induction and reinduction phase of protocol ALL-IC BFM 2000. We simultaneously checked the APTT,PT,AT III,fibrinogen and DD from each blood sample. Detailed patientsx data were collected prospectively. ResultsWe collected 22 series of CAT measurements from 18 patients. The average endogenous thrombin potential (ETP) value was 1237,64±334,49nM/min during protocol I(PI) and 892,31±405,03 during protocol II resp.III(PII/III) (p .000013). Maximum ETP was reached on day 24 in PI (1310,15±256,3nM/min) and on day 15 in PII/III (978,20±262,30 nM/ min). The average ETP value in 20 healthy controls was 1218,12±129,00 nM/min. Three patients developed deep venous thrombosis (DVT) during the study period, all of them on PI. Their average ETP value on the last measurement before thrombosis was 1510,54±78,6 nM/min. Four patient had an infection gr. III/IV, according WHO, during the study period. Their average ETP value after the infection was 1711,43±106,0 nM/min. Two of those four patient developed DVT within 5 resp.8 days after the first sign of infection. Fibrinogen level <1g/l was detected in 19 samples out of 104. From those 19 samples with fibrinogen < 1,0,the ETP value from the same sample was normal in 14 cases and low in 5 cases. There were no bleeding episodes in that time. ConclusionsThrombin generation is preserved in majority of patients treated with L-asparaginase in accordance with ALL-IC BFM 2000 protocol, despite the associated hypofibrinogenemia. Patients on induction treatment generate significantly more thrombin than patients on reinduction treatment. Our results show the sharp increase in ETP right after the infection with risk of DVT. Our data should be proved on larger group of patients. Disclosures:No relevant conflicts of interest to declare.

Prognosis of ProB ALL in Children

ProB ALL is considered an unfavorable subset of ALL both in children and adults. It is currently defined as B precursor ALL with no expression of CD10. MLL/AF4pos cases, who are almost never express CD10, contribute to the poorer prognosis of proB ALL. Until now, it has not been solved whether the prognosis of proB children remains poorer even after removal of MLL/AF4pos cases. We have analyzed the prognosis of proB ALL in comparison to the other types of B precursor ALL in a population-wide cohort of Czech children tested in our lab between 9/1996 and 8/2006; we asked whether the prognosis was affected by MLL/AF4pos cases. Concurrently, we asked whether a more sensitive definition of proB ALL uncovers additional patients with a similar prognosis. We retrospectively analyzed all 505 patients tested in our reference lab, age below 18 years at diagnosis of B precursor ALL. All patients have been treated in one of the Czech Pediatric Hematology working group centers according to the standard treatment protocols BFM95 (n=290), Interfant99 (n=12), Interfant2006 (n=3), ALL-IC BFM 2002 (n=192) or POG9407 (n=5); 3 children were treated/followed off-protocol. ProB ALL (n=42) was associated with a poorer prognosis in the entire cohort (p= 0.0084, 5y EFS 62%±7.9%). After removing MLL/AF4pos ALL (n=10), 5y EFS is 66% ± 8.8% and the difference from CD10pos patients is no longer significant (p=0.09). Next, we tested a hypothesis that patients with a “partial proB” ALL (i.e., those containing CD10pos cells in addition to a substantial proportion of CD10neg lymphoblasts) have a poorer prognosis. The percentage of CD10neg cells was defined in all as a difference between CD19pos and CD10pos gated lymphoblasts; cases with greater than 20% of CD10neg cells who did not fulfill the current proB ALL definition were categorized as “partial proB”. The prognosis of “partial proB” ALL (n=45; 8.9%) is similar (5y EFS 63% ± 7.7%) to the classical proB ALL. With the exception of MLL/AF4, none of which expresses CD10, the distribution of main genotype categories is not significantly different between proB ALL and “partial proB” ALL. In contrast, the genotype distribution differs between “partial proB” ALL and other non-proB cases (p=0.0072). Response of the “partial proB” patients to prednisone is not different from the “classical” proB but poorer than that of non-proB ALL (pFisher=0.0039). Combining the “partial proB” and proB ALL cases into one category (n=87) defines a subset with a poor prognosis (n=77, 5y EFS 64%±5.9%), which is significantly poorer than that of other B precursor ALL cases even after excluding MLL/AF4pos children (5y EFS 78%±2.2%; p=0.011). Therefore, the definition of proB ALL should be based on the percentage of CD10neg blasts, rather than on a complete absence of CD10 in all blasts–such criteria define patients with a new ALL subtype whose prognosis is poorer even after excluding MLL/AF4pos ALL.Supported by MSM0021620813, MZdNR/9531–3. Czech Pediatric Hematology Working Group. [Display omitted]

Minimal residual disease (MRD) analysis in the non-MRD-based ALL IC-BFM 2002 protocol for childhood ALL: is it possible to avoid MRD testing?

The ALL IC-BFM 2002 protocol was created as an alternative to the MRD-based AIEOP-BFM ALL 2000 study, to integrate early response criteria into risk-group stratification in countries not performing routine PCR-based MRD testing. ALL IC stratification comprises the response to prednisone, bone marrow (BM) morphology at days 15 and 33, age, WBC and BCR/ABL or MLL/AF4 presence. Here, we compared this stratification to the MRD-based criteria using MRD evaluation in 163 patients from four ALL IC member countries at days 8, 15 and 33 and week 12. MRD negativity at day 33 was associated with an age of 1-5 years, WBC<20,000 microl(-1), non-T immunophenotype, good prednisone response and non-M3 morphology at day 15. There were no significant associations with gender or hyperdiploidy in the study group, or with TEL/AML1 fusion within BCP-ALL. Patients with M1/2 BM at day 8 tended to be MRD negative at week 12. Patients stratified into the standard-risk group had a better response than intermediate-risk group patients. However, 34% of them were MRD positive at day 33 and/or week 12. Our findings revealed that morphology-based ALL IC risk-group stratification allows the identification of most MRD high-risk patients, but fails to discriminate the MRD low-risk group assigned to therapy reduction.