Acute Lymphoblastic Leukemia IC-BFM Research Articles

Diagnosis of Acute Lymphoblastic Leukemia Originating From T-Lineage Precursors and Approaches to Minimal Residual Disease Monitoring

Background. Minimal residual disease (MRD) is an independent prognostic factor in acute lymphoblastic leukemia (ALL) in children. The immunological assessment of MRD cell count is based on aberrant immunophenotype of tumor lymphoblasts. However, in the case of ALL originating from T-lineage precursor cells (T-ALL) no clear aberrancy criteria have been defined, yet. Flow-cytometric MRD assessment in T-ALL can be based on characteristics of normal T-cell ontogenesis, i.e. the absence of normal T-lineage precursor cells (T-LP) in bone marrow. Aim. To assess the feasibility of immunological method of flow cytometry for MRD detection based on T-LP immunophenotype on Days 15 and 33 of treatment of T-ALL children. Materials & Methods. The analysis included the data on primary immunophenotype and MRD assessment on Days 15 and 33 of treatment of 31 T-ALL patients in the age of 2-17 years. In the majority of cases (61.3 %) the cortical/ thymic immuno-subvariant of ALL was detected, in the rest of cases (38.7 %) it was the pre-T-cell one. Diagnosis was based on cumulative results of morphocytochemical and immunological bone marrow analyses. Assessing the MRD state the morphological and immunological analyses of bone marrow aspirate were carried out in parallel with one and the same tube. All patients enrolled in the trial were treated at Scientific Research Institute of Pediatric Oncology and Hematology of NN Blokhin National Medical Cancer Research Center according to the ALL IC-BFM 2009 protocol. Results. Our study demonstrated that at all therapy stages MRD can be assessed by the unified immunological method based on detecting cyCD3<sup>+</sup>CD7<sup>+/++</sup>smCD3<sup>-</sup> (T-LP) immunophenotype cells. It is important to ensure that the correct clones of monoclonal antibodies are used for detecting CD3 cytoplasmic and membrane molecules (UCHT1 and SK7, respectively). Standard risk group included no patients. The majority of patients (76.2 %) treated according to ALL IC-BFM 2009 protocol were assigned to medium risk group on Day 15 of treatment. By Day 33 a quarter of them (25 %) was included into high risk group. Conclusion. The capabilities of multicolor flow cytometry allow for the most complete characterization of primary immu-nophenotype of tumor T-cell lymphoblasts for further search of leukemia-associated immunophenotypes. Specific ontogenesis features of normal T-cells enable unification of immunological approaches to MRD assessment at all stages of T-ALL therapy.

Antithrombin III as the Indicator of L-Asparaginase Activity in Children Treated for Acute Lymphoblastic Leukemia.

L-asparaginase (ASP) is widely used in the treatment of acute lymphoblastic leukemia (ALL) in children. Monitoring its activity is necessary because of the risk of drug inactivation as the result of an immune reaction. Besides allergic reactions, another frequent side effect of ASP treatment is coagulopathy, especially deficiency of antithrombin III (ATIII). The aim of this study was to analyze the relationship between ASP and ATIII activities and the possibility of ATIII activity use in an indirect ASP activity assessment. ASP and ATIII activity was measured in 76 children with ALL treated according to the ALL IC BFM 2002 protocol. A correlation between ASP and ATIII activities was found (R=-0.43, P=0.0001). ROC curve analysis revealed some utility regarding the determination of ATIII in identifying patients with low or undetectable ASP activity (area under the curve=0.87 [95% confidence interval, 0.77-0.96], P<0.0001 and 0.93 [95% confidence interval, 0.85-1.0], P<0.0001, respectively). Higher ATIII activity is associated with a higher probability of a decline in ASP activity. Examination of ATIII activity cannot replace a direct determination of ASP activity, but in the case of unavailability of the direct test, it can be a helpful surrogate parameter of drug activity.

Assessment of Hematological Toxicity in Children with Acute Lymphoblastic Leukemia, Receiving Treatment with ALL IC-BFM 2009 Protocol

The most common childhood cancer is acute lymphoblastic leukemia (ALL). Chemotherapy-associated hematological toxicity is well-known; however, there are few studies on hematologic toxicity incidence in children with ALL. We investigated the severity and incidence of hematologic toxicity during intense chemotherapy processes in children treated with ALL IC-BFM 2009 protocol. The study included 41 leukemic children in standard (SR) and intermediate risk (IR) groups treated between 2011 and 2015. During the induction period, the incidence of grade 4 toxicity in neutrophil count was 60%; the incidence of grade ≥ 3 toxicity in hemoglobin level was 34%; and the incidence of grade ≥ 3 toxicity in the platelet count was 51%. Deep neutropenia duration was 36.6 ± 12.7 (18-68) days during the induction. 53% of the febrile neutropenic (FEN) episodes developed during the induction period. There were no statistical differences between SR and IR risk groups with respect to hemogram values deep neutropenia duration and the number of FEN episodes (p > 0.05, all). There was a positive correlation between the number of FEN episodes and duration of neutropenia. During the induction, the mean neutrophil count remained between 0.5-1 × 109/L. FEN episodes most commonly developed during the induction phase.

Chromosomal aberrations in childhood acute lymphoblastic leukemia: 15-year single center experience

Genetic analysis of leukemic cells significantly impacts prognosis and treatment stratification in childhood acute lymphoblastic leukemia (ALL). Our retrospective single center study of 86 children with ALL enrolled into three consecutive treatment protocols (ALL-BFM 90, ALL-BFM 95 and ALL IC-BFM 2002) between 1991 and 2007 demonstrates the importance of conventional cytogenetics and fluorescence in situ hybridization (FISH). Cytogenetic and FISH examinations were performed successfully in 82/86 (95.3%) patients and chromosomal changes were detected in 78 of the 82 (95.1%) patients: in 69/73 patients with B-cell precursor (BCP)-ALL and in 9/9 patients with T-lineage ALL (T-ALL). The most frequent chromosomal changes in subgroups divided according to WHO classification independent of treatment protocol and leukemia subtype were hyperdiploidy in 36 patients (with ≥50 chromosomes in 23 patients, with 47-49 chromosomes 13 patients) followed by translocation t(12;21) with ETV6/RUNX1 fusion detected by FISH in 18 (22%) patients. Additional changes were detected in 16/18 (88.8%) ETV6/RUNX1-positive ALL patients with predominant deletion or rearrangement of untranslocated ETV6 allele. Unique aberrations were detected in 4 patients and dicentric chromosomes in 8 patients, one with T-ALL. These results demonstrate that cytogenetics and FISH successfully provided important prognostic information and revealed not only recurrent but also new and rare rearrangements requiring further investigation in terms of prognostic significance.

Treatment and prognosis of childhood acute lymphoblastic leukemia on protocols ALL-BFM 90, 95 and ALL IC-BFM 2002: a retrospective single-center study from Olomouc, Czech Republic

Great progress has been made in the diagnostics and treatment of childhood acute lymphoblastic leukemia (ALL) over the past decades. The vast majority of children are cured, however, there is need for further improvement, especially in specific patient subgroups. Our aim was to retrospectively evaluate disease characteristics and treatment outcomes of children with ALL enrolled in asingle center into consecutive treatment protocols (ALL-BFM 90, ALL-BFM 95 and ALL IC-BFM 2002) between years 1990 and 2007 and comprehensively summarize diagnostic and therapeutic advances between protocols. In total, 97 patients aged 0 to 18 years were treated for ALL at University Hospital Olomouc in the Czech Republic and steadily high relapse-free survival (RFS), event-free survival (EFS) and overall survival (OS) were observed during the evaluated time period without significant difference between the protocols (RFS 80-86%, EFS 75-83% and OS 84-92%). In conclusion, our center has demonstrated survival rates comparable to leading international study groups for childhood ALL over asubstantial period of time. This has been achieved namely due to advances in diagnostics, excellent collaboration on regional, national and international level, quality assurance and high overall standard of care. The acquired experience has been crucial for current participation in the best performing Berlin-Frankfurt-Münster (BFM)-based international trials for childhood ALL.

TREATMENT RESULTS OF ALL IC-BFM 2002 PROTOCOL IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Beneficial chemotherapy programs along with improved supportive care methods promoted acute lymphoblastic leukemia (ALL) from fatal to potentially curable disease. Leukemogenesis study and prognostic factor analysis became the basis for identification of prognostic groups and design of riskadapted chemotherapy for ALL. One of the modern treatment protocols for ALL is ALL IC-BFM 2002, its treatment results are presented in the current article. This protocol occurs to be highly-effective and results in high long-term survival rates: 10-year overall survival was 90,4±2,6%, event-free — 82,5±3,4%, and relapse-free — 87,3±3,1%.

Outcome in pediatric acute lymphoblastic leukemia in children and adolescents using contemporary protocols of chemotherapy – experience of a single Pediatric Hematology-Oncology Center

The authors studied the outcome of a cohort of pediatric acute lymphoblastic leukemia (ALL) patients (33 children and adolescence) diagnosed and treated conforming to modern Chemotherapy Protocols (ALL ICBFM 2002, Interfant 06) in a single Center – Pediatric Clinic, Fundeni Clinical Institute, Bucharest, Romania. They analyzed the factors which determine the prognosis and the outcome of these patients in the course of multi-agent systemic chemotherapy to stand at the base of these Protocols: initial age, initial leukocyte count, blasts immunophenotype, cytogenetic and molecular abnormalities, initial response to cortisone, risk groups, time to obtain the complet remission, etc. Among the factors they discussed, a great value was proven to have the minimal residual disease (MRD) determination in certain check points of Protocols and revaluation of patients risk conforming to MRD values. Using the modern Protocols and continuously watching the evolution on therapy enable the authors to obtain results close to those of European and North American Pediatric Hematology Oncology Centers: OS 90.9% by 40 Mo and EFS 72.7% at the end of the same period of time.

Do We Need the Chelation Therapy during Intensive Treatment of Acute Lymphoblastic Leukemia (ALL) in Children?

IntroductionChildren with ALL during the intensive chemotherapy receive multiple transfusions of packed red blood cell (pRBC) that may lead to iron overload. With each transfusion of pRBC the body is supplied with an about 200-250 mg of iron, which excessive accumulation in tissues, due to the lack of mechanism for its active excretion, may cause toxic organ damage.MethodsThe aim of the study was to evaluate the serum ferritin concentration in children with ALL, depending on the amount of transfused pRBCs and to determine the group of patients with a risk of iron overload. The study patients included 54 children with newly diagnosed ALL treated at the Department of Pediatrics, Hematology and Oncology between 2008 and 2011, according to the ALL IC BFM 2002 therapy protocol. Prior to initiation the treatment and during the intensive chemotherapy, serum ferritin concentration and the number of pRBC transfusions (ml/kg) were assessed separately for each of the three ALL risk groups-standard risk (SR), intermediate risk (IR) and high risk (HR).ResultsAfter the intensive chemotherapy the mean ± standard deviation (SD) of serum ferritin concentration in group HR (2770 ± 1175 ng/ml) was significantly higher compared to the median in group SR 844.4 ng/ml (452.5; 1316) (p = 0.0007) and the mean ± SD in group IR-1270 ± 673.1 ng/ml (p = 0.0040).Throughout the intensive chemotherapy children in HR group received the largest volume of pRBC transfusions (ml/kg) (156.2 ± 68.31 ml/kg). In IR and SR groups the amounts of transfused pRBCs were comparable, respectively 113.5 ± 39.86 and 113.8 ± 29.56 ml/kg.Significant positive correlation was found between the serum ferritin concentration and the total amount of transfused pRBCs (ml/kg) after intensive chemotherapy (p <0.0001).After intensive treatment the concentration of serum ferritin exceeding 1000 ng/ml, that has traditionally been used as a trigger for chelation therapy, was found in 30 of 54 patients, for a prevalence in the entire cohort of 55,6% including 6 out of 6 patients in HR group (100%), 14 out of 22 patients in IR group (63,6%) and 10 out of 24 patients in SR group (41,7%).The group of patients with post-treatment serum ferritin concentration exceeding 1000 ng/mL, received significantly more pRBC transfusions (ml/kg) (139.8 ± 44.92) than the groups with serum ferritin levels between 500-1000 ng/mL (103.6 ± 18.96) (p <0.001) and <500 ng/mL-83.52 ± 17.66 (p <0.05).ConclusionsThese observations indicate a need of monitoring the cumulative volumes of pRBC transfusions, especially in children with ALL HR group. There is a need of routine screening for iron overload using serum ferritin in patients during intensive chemotherapy, in order to identify patients with indications for early iron chelation therapy. This is particularly important because some of them will be candidates to a hematopoietic stem cell transplantation, whereas iron overload adversely affects outcome of transplantation. DisclosuresNo relevant conflicts of interest to declare.

Functional variants of gene encoding folate metabolizing enzyme and methotrexate-related toxicity in children with acute lymphoblastic leukemia

Methotrexate (MTX) is commonly used agent in therapy of malignancies, including acute lymphoblastic leukemia (ALL). Based on the literature data it is known that MTX elimination and toxicity can be affected by polymorphisms in genes encoding enzymes involved in MTX metabolism.The aim of our study was to investigate the influence of C677T and A1298C polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene on MTX-induced toxicity during treatment of children with ALL. We also tried to answer the question whether simultaneous occurrence of these two polymorphisms has a clinical significance.MTHFR polymorphisms were assessed in 47 pediatric ALL patients, treated according to intensive chemotherapy for childhood ALL, ALL IC BFM 2009.Prolonged MTX elimination and higher incidence of toxicity were observed for patients with 677T-1298A haplotype. On the other hand, occurrence of 677C-1298A haplotype had protective effect on MTX clearance and toxicity, that was not observed in carriers of 677C-1298C haplotype. In patients with coexistence of studied variants 677CT/1298AC heterozygotes as well as in 677TT/1298AA homozygotes more frequently toxicity incidents were noted. The obtained results suggest that occurrence of 677T allele and coexistence of 677T and 1298C alleles may be associated with lower MTX clearance and elevated risk of adverse effects during MTX-treatment of pediatric ALL patients.

Improved outcome for children and adolescent with acute lymphoblastic leukemia in the first decade of the 21st century: Areport from the Slovak Republic.

Our aim was to analyze event-free (EFS) and overall survival (OS) among children and adolescents with acute lymphoblastic leukemia (ALL) treated with International BFM Intercontinental trial (ALL IC 2002) therapy in the Slovak Republic. In total, 280 children and adolescent age 1 to 18 years were treated with ALL IC BFM 2002 based therapy from 2002 to 2012, which was divided into two periods. During 2002-2007, when patients were actively enrolled in the ALL IC-BFM 2002 trial, and during 2008-2012 when the trial was closed and patients were treated with the same therapy without randomization. Five-year EFS and OS rates were 79% (+/- 2.6%) and 86% (+/- 2.1%), respectively, similar to results obtained in the ALL-BFM 95 trial, which was the basis for ALL IC BFM 2002 therapy. The EFS (p<0.012) and OS (p<0.003) were significantly better than the prior Slovak experience in 1997-2001. Survival is improved in standard and intermediate risk groups, including those age 1 to 6 years, and older; with B-cell or T-cell immunophenotype, and is also excellent for those with good early response. The rate of death in induction, cumulative incidence of death in complete remission and of relapse decreased. However, outcome was suboptimal for patients in the high risk group. Current EFS and OS rates for children and adolescents with ALL in the Slovak Republic resembled those obtained in Western Europe as aresult of clinical trial participation, and clinical experience acquired with intensive BFM type treatment.

Associations of novel genetic variations in the folate‐related and ARID5B genes with the pharmacokinetics and toxicity of high‐dose methotrexate in paediatric acute lymphoblastic leukaemia

High-dose methotrexate (HD-MTX) plays an important role in the consolidation therapy of acute lymphoblastic leukaemia (ALL) in many treatment regimens worldwide. However, there is a large interpatient variability in the pharmacokinetics and toxicity of the drug. We investigated the influence of single nucleotide polymorphisms (SNPs) in genes of the folate metabolic pathway, transporter molecules and transcription proteins on the pharmacokinetics and toxicity of MTX and 7-hydroxy-methotrexate (7-OH-MTX). 63 SNPs of 14 genes were genotyped and a total of 463 HD-MTX courses (administered according to the ALL-BFM 95 and ALL IC-BFM 2002 protocols) were analysed. Haematological, hepatic and renal toxicities, estimated by routine laboratory parameters were evaluated. Random forest and regression trees were used for variable selection and model building. Linear mixed models were established to prove the significance of the selected variables. SNPs (rs4948502, rs4948496, rs4948487) of the ARID5B gene were associated with the serum levels of MTX (P < 0·02), serum levels and area under the curve of 7-OH-MTX (P < 0·02) and with hypoproteinaemia (P = 0·004). SLCO1B1 rs4149056 also showed a significant association with serum MTX levels (P < 0·001). Our findings confirm the association of novel genetic variations in folate-related and ARID5B genes with the serum MTX levels and acute toxicity.

Expanding Clinical Trial Networks in Pediatric Acute Lymphoblastic Leukemia

Expanding Clinical Trial Networks in Pediatric Acute Lymphoblastic Leukemia

Intensive Chemotherapy for Childhood Acute Lymphoblastic Leukemia: Results of the Randomized Intercontinental Trial ALL IC-BFM 2002

From 2002 to 2007, the International Berlin-Frankfurt-Münster Study Group conducted a prospective randomized clinical trial (ALL IC-BFM 2002) for the management of childhood acute lymphoblastic leukemia (ALL) in 15 countries on three continents. The aim of this trial was to explore the impact of differential delayed intensification (DI) on outcome in all risk groups. For this trial, 5,060 eligible patients were divided into three risk groups according to age, WBC, early treatment response, and unfavorable genetic aberrations. DI was randomized as follows: standard risk (SR), two 4-week intensive elements (protocol III) versus one 7-week protocol II; intermediate risk (IR), protocol III × 3 versus protocol II × 1; high risk (HR), protocol III × 3 versus either protocol II × 2 (Associazione Italiana Ematologia Oncologia Pediatrica [AIEOP] option), or 3 HR blocks plus single protocol II (Berlin-Frankfurt-Münster [BFM] option). At 5 years, the probabilities of event-free survival and survival were 74% (± 1%) and 82% (± 1%) for all 5,060 eligible patients, 81% and 90% for the SR (n = 1,564), 75% and 83% for the IR (n = 2,650), and 55% and 62% for the HR (n = 846) groups, respectively. No improvement was accomplished by more intense and/or prolonged DI. The ALL IC-BFM 2002 trial is a good example of international collaboration in pediatric oncology. A wide platform of countries able to run randomized studies in ALL has been established. Although the alternative DI did not improve outcome compared with standard treatment and the overall results are worse than those achieved by longer established leukemia groups, the national results have generally improved.

Comparison of pharmacokinetics and toxicity after high-dose methotrexate treatments in children with acute lymphoblastic leukemia

We carried out a detailed comparative study of the pharmacokinetics and toxicity of methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) after high-dose intravenous methotrexate (HD-MTX) in children with acute lymphoblastic leukemia (ALL). Overall, 65 children were treated with 5 g/m2/24 h MTX and 88 children were treated with 2 g/m2/24 h MTX according to ALL-BFM 95 and ALL IC-BFM 2002 protocols (mean age: 6.4 years, range 1.0-17.9 years). A total of 583 HD-MTX courses were analyzed. Serum MTX and 7-OH-MTX levels were measured at 24, 36, and 48 h, and cerebrospinal fluid (CSF) MTX levels were determined 24 h after the initiation of the infusion. The area under the concentration-time curve was calculated. Hepatotoxicity, nephrotoxicity, and bone marrow toxicity were estimated by routine laboratory tests. We investigated pharmacokinetics and toxicity in distinct age groups (< 6 and > 14 years). 5 g/m2/24 h treatments resulted in higher serum and CSF MTX and 7-OH-MTX levels (P < 0.05). The CSF penetration rate of MTX was independent of the MTX dose [2.3% (95% confidence interval: 1.7-2.5%) vs. 2.8% (95% confidence interval: 2.4-3%)]. The CSF MTX concentration was correlated with the 24 h MTX serum level (r = 0.38, P < 0.0001). Repeated treatments did not alter MTX or 7-OH-MTX levels. 7-OH-MTX levels were correlated with nephrotoxicity (r = 0.36, P < 0.0001). Higher MTX levels and toxicity occurred more frequently in children aged older than 14 years (P < 0.05). Therapeutic serum and CSF MTX concentrations can be achieved more reliably with 5 g/m2/24 h treatments. To predict the development of toxicity, monitoring of the level of the 7-OH-MTX is useful. Monitoring of pharmacokinetics is essential to prevent the development of severe adverse events in adolescents.

Ikaros (IKZF1) alterations and minimal residual disease at day 15 assessed by flow cytometry predict prognosis of childhood BCR/ABL‐negative acute lymphoblastic leukemia

Recently, several studies have demonstrated a negative prognostic impact of Ikaros (IKZF1) gene alterations in acute lymphoblastic leukemia (ALL). However, controversies still exist regarding the impact of IKZF1 in current treatment protocols. We simultaneously detected IKZF1 gene deletions by multiplex ligation-dependent probe amplification and gene expression of IKZF1 isoforms in 206 children with BCR/ABL-negative ALL treated with ALL IC-BFM 2002 protocol, in which risk stratification was not based on minimal residual disease (MRD), and validated the results on a cohort of 189 patients treated with MRD-directed ALL-BFM 2000 protocol. Deletion of IKZF1 was present in 14 of 206 (7%) ALL IC patients. Interestingly, gene expression did not completely correlate with the deletion status in either cohort. Deletions were not always reflected in the gene expression of dominant-negative isoforms, and conversely, 7 of 395 (2%) non-deleted cases overexpressed dominant-negative isoform Ik6. IKZF1 deletions significantly affected event-free survival (EFS) of the ALL IC cohort (41 ± 14% vs. 86 ± 3%, P < 0.0001). Regarding IKZF1 isoforms, only Ik6 overexpression had negative prognostic impact (EFS 50 ± 16% vs. 85 ± 3%, P = 0.003). In multivariate analysis, which included ALL IC risk criteria, flow-cytometric MRD and IKZF1 alterations, day 15 MRD and IKZF1 deletion status displayed an independent prognostic impact. We show that MRD-directed treatment diminishes prognostic impact of IKZF1 alterations. However, IKZF1 status alone or combined with day 15 flow cytometry can significantly improve risk stratification within BFM protocols at centers that do not perform antigen-receptor-based MRD monitoring.

1104 - Improved Therapeutic Outcome in Children with Acute Lymphoblastic Leukemia in Croatia

ABSTRACT Therapeutic outcome in children with acute lymphoblastic leukemia (ALL) has greatly improved with more intensive chemotherapy, risk adapted strategies and better supportive care. Aim Analysis of therapeutic results recorded by the National Group for Pediatric Hematology in children with ALL in Croatia treated with pediatric trial ALL IC-BFM 2002. Patients and methods From November 1,2002 until November 2,2010, 208 ALL children i.e. 119 (58%) male and 89 (42%) female, mean age 75.4 months, were treated according to ALL IC-BFM 2002 protocol: SRG – 74 (35.5%); IRG -104 (50.6%); and HRG -30 (13.9%), at four Croatian pediatric hematology-oncology centers: Zagreb University Hospital Center 145 (69.7%); Split University Hospital – 30 (14.4%); Rijeka University Hospital Center – 17 (8.2%); Zagreb University Children's Hospital S.Milosrdnice – 16 (7.7%). Results The first complete remission (CR) was achieved in 208(100.0%) patients, whereas relapse occured in 23(11.2%), hematologic relapse (BM) in 17(8.2%), meningeal relapse (CNS) in 3(1.4%), hematologic and meningeal relapse (BM and CNS) in 2(0.97%) and hematologic and testicular relapse (BM and TR) in 1(0.48%). 2 (0.9%) patients dropped out from our records. 16(7.6%) of 206 patients died: 3(1.4%)in the first complete remission, 2(0.9%) after bone marrow transplantation and 11 (5.3%) in relapse. 190(92%) patients have survived to the present; 72(97.7%) from SRG, 97(91.8%) in IRG and 21(64.7%) from HRG. Secondary tumor disease was not recorded. The 4-year event free survival (EFS) was 88%; overall free survival (OFS) was 89%. Conclusion These results were nearly identical to those reported from the leading European centers for the treatment children with ALL. Disclosure All authors have declared no conflicts of interest.

Repeated Bone Marrow Aspiration At the End of Induction Therapy: Implications for Treatment Stratification in Paediatric Acute Lymphoblastic Leukaemia

Abstract 756▪FN2▪This icon denotes a clinically relevant abstractBone marrow (BM) aspiration at the end of induction therapy plays a crucial role for the evaluation of remission and the minimal residual disease (MRD), both critical for treatment stratification in modern treatment protocols for paediatric acute lymphoblastic leukaemia (ALL). However, the aspiration is repeated in 15–20% of patients, either due to non-representative morphology or to insufficient material needed for MRD analysis.We prospectively analysed 320 paediatric ALL patients treated according to ALL-BFM 2000 (n=301) or ALL IC-BFM 2002 (n=19) protocols with repeated BM aspiration at the end of induction therapy, on treatment day 33. Fourteen patients had more than one re-puncture. The median follow-up was 69 months, 45 (14%) patients had an event (relapse/death). The cause for the repeated BM aspiration was non-representative morphology (32%), insufficient material for MRD analysis (33%) or both (35% cases). In order to evaluate prognostic significance of the re-punctures and to determine which of the repeated samples should be used for the final treatment stratification we analysed MRD levels and MRD stratification, morphology, leukocyte count (WBC) and the length of treatment delay caused by waiting for the repeated aspiration. MRD data were collected and interpreted according to the EuroMRD guidelines in one central reference laboratory per each participating country. Morphology was evaluated centrally using an own scoring system (with a max value of 26 points).Treatment delay between the original and the last aspiration was one-third longer in patients with subsequent event compared to patients remaining in complete remission (CR) (median 8 (range 2 – 21) vs. 6 (1 - 28) days, respectively; p=0.020). Patients with a subsequent event had significantly higher WBC at the time of the last repeated BM aspiration, compared to patients without event (p=0.019), while there was no difference relative to the original aspiration (p=0.9).Analysis of the BM morphology at the original aspiration showed no significant difference between patients with an event vs. those in CR. However, the repeated aspiration of patients with a subsequent event had significantly better morphology (median 18.5/26 vs. 15/26 points, p=0.0012) mainly due to higher cellularity (p=0.003) and number of megakaryocytes (p=0.048).MRD levels were identical or decreased in 88% and increased in 12% of cases comparing the original aspiration to the repeated aspiration. In 63 patients (20%) the different MRD levels would lead to different treatment stratification. Higher MRD was associated with treatment failure; the best predictive values for subsequent event were obtained using the MRD results of the original aspiration (p=3.1e-07) or the highest of the detected MRD levels (p=6.0e-07). The last aspiration before proceeding with treatment had the lowest, though still a highly significant predictive value (p=8.6e-06). Corresponding results are obtained when MRD levels are substituted by final MRD risk stratification into standard, medium or high risk (p<0.0001, p=0.0005 and p=0.0008 for the prediction of treatment failure using the MRD level in the original, the highest and the last aspiration, respectively).In conclusion, our data show that the original BM aspiration – independently of the quality of morphology – is not inferior for MRD treatment stratification, and that it actually has the best predictive value. In cases where sample quality precludes MRD analysis, the repeated sample with the highest MRD level should be used for stratification in order to not underestimate the putative risk of treatment failure.Longer treatment delay caused by waiting for a more representative sample seems to worsen the outcome. Notion that patients with a subsequent event need more time for BM regeneration is not justified, as their cellularity, overall morphology and also WBC before proceeding with treatment are better than in patients remaining in long-term CR.Any decision to perform a re-puncture at the end of induction therapy due to a non-representative morphology should be critically weighed. If possible, any unnecessary prolongation of treatment delay should be avoided unless being inevitable for other reasons, and therapy should be continued as soon as possible. Support:Deutsche Krebshilfe, Germany (Projects 50–2698 Schr1; 50–2722 BA6/7); St. Anna Kinderkrebsforschung, Austria; MSM0021620813; IGA NS/1000-4. Disclosures:No relevant conflicts of interest to declare.

Role of base-excision repair in the treatment of childhood acute lymphoblastic leukaemia with 6-mercaptopurine and high doses of methotrexate

Methotrexate (MTX) and 6-mercaptopurine (6MP) are the most commonly used drugs in the therapy of childhood acute lymphoblastic leukaemia (ALL). The main genotoxic effect of MTX resulting from inhibition of thymidylate synthase is mis-incorporation of uracil into DNA, which is considered essential for the effectiveness of the Protocol M in ALL IC BFM 2002/EURO LB 2002 regimens. In this study, we investigated the level of basal and induced DNA damage as well as the effectiveness of DNA repair in lymphocytes of children with ALL at four time-points during therapy with MTX and 6MP. To assess DNA damage and the efficacy of DNA repair we used the modified alkaline comet assay with uracil DNA glycosylase (Udg) and endonuclease III (EndoIII). In addition, we examined the induction of apoptosis in the lymphocytes of the patients during treatment. Finally, we compared the activity of base-excision repair (BER), involved in removal of both uracil and oxidized bases from DNA in lymphocytes of children with ALL and lymphocytes of healthy children. BER efficiency was estimated in an in vitro assay with cellular extracts and plasmid substrates of heteroduplex DNA with an AP-site. Our results indicate that there is a significant decrease in the efficacy of DNA repair associated with an increased level of uracil in DNA and induction of apoptosis during therapy. Moreover, it was found that the BER capacity was decreased in the lymphocytes of ALL patients in contrast to that in lymphocytes of healthy children. Thus, we suggest that an impairment of the BER pathway may play a role in the pathogenesis and therapy of childhood ALL.

Results of acute lymphoblastic leukemia treatment in children in the Slovak Republic

Treatment of childhood acute lymphoblastic leukemia (ALL) according to the 1st Slovak protocol started in the Slovak Republic in 1971 in eight pediatric departments. Gradually, two other protocols were written, and 496 children were treated with these three protocols from 1971 to 1992. Since 1992, all children have been treated in three pediatric oncological departments. From 1992 to 1996, protocols for standard and high-risk ALL were developed that were used for 111 children in Bratislava, while the centres in Banska Bystrica and Kosice had started to use BFM protocols. Since 1997, all patients with ALL have been treated according to ALL BFM 95, and since 2002, they have been included in the ALL IC BFM 2002 study. We evaluated treatment results in the standard arm of ALL BFM 95 and compared it with previous Slovak protocols. Rates of complete remission increased from 90.6% in the first Slovak protocol to 97.1% in BFM 95; EFS and OS increased from 0.46 and 0.50 to 0.67 and 0.72, respectively. Relapse rates decreased from 42% to 20.5%. EFS was significantly worse in children with leukocyte counts >100×109/l and in the HR group in comparison to standard- and medium-risk groups. OS was also significantly worse in T-cell ALL than in B-cell ALL. Death rates in the 1st complete remission decreased gradually in the Slovak protocols from 10.4% to 1.8%, but were high in BFM 95 (10.8%) due to more intensive therapy and initial unsatisfactory experiences with BFM protocols. All parameters improved in the ALL IC BFM 2002 study.

History of treatment and long-term outcome in children with acute lymphoblastic leukemia in the Czech Republic

PURPOSE: Treatment of childhood acute lymphoblastic leukemia (ALL) was unified in the year 1986 in the Czech Republic using BFM protocols. PATIENTS AND METHODS: Children were treated in 10 and later on in 8 centers localized at the pediatric departments of the biggest hospitals. More than 1.000 children and adolescents up to the age of 18 years were treated between 1986 and 2002 on three consecutive studies ALL-BFM 83, ALL-BFM 90 and ALL-BFM 95. RESULTS: Event-free survival and overall survival improved gradually from 58/62% on ALL-BFM 83 through 70.5/76.6% on ALL-BFM 90 until 72.1/80.2% on ALL-BFM 95. At the same time the incidence of toxic deaths was decreasing and the success in the achievement of complete remission was increasing. Diagnostics in the Czech Republic improved remarkably in the 1990 with gradual introduction of centralized flow cytometry and molecular genetic analysis in one reference laboratory (CLIP = Childhood Leukemia Investigation Prague), which since then has become an internationally respected research center. CONCLUSIONS: Building up a network of closely collaborating leukemia centers covering the whole country, together with the establishment of reference and research laboratories has paved the way for the implementation of the Czech Pediatric Hematology Working Group (CPH) into the international studies Interfant 99, EsPhALL and for the active role in ALL IC-BFM 2002, the I-BFM-SG international randomized trial for treatment of children and adolescents with non-B ALL starting in 2002.