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Purpose Delayed intensification (DI) is an integral part of treatment of childhood acute lymphoblastic leukemia (ALL), but it is associated with relevant toxicity. Therefore, standard-risk patients of trial AIEOP-BFM ALL 2000 (Combination Chemotherapy Based on Risk of Relapse in Treating Young Patients With ALL) were investigated with the specific aim to reduce treatment intensity. Patients and Methods Between July 2000 and July 2006, 1,164 patients (1 to 17 years of age) with standard-risk ALL (defined as the absence of high-risk cytogenetics and undetectable minimal residual disease on days 33 and 78) were randomly assigned to either experimental reduced-intensity DI (protocol III; P-III) or standard DI (protocol II; P-II). Cumulative drug doses of P-III were reduced by 30% for dexamethasone and 50% for vincristine, doxorubicin, and cyclophosphamide, which shortened the treatment duration from 49 to 29 days. The study aimed at noninferiority of reduced-intensity P-III; analyses were performed according to treatment given. Results For P-III and P-II, respectively, the 8-year rate of disease-free survival (6 SE) was 89.2 6 1.3% and 92.3 6 1.2% (P = .04); cumulative incidence of relapse, 8.7 6 1.2% and 6.4 6 1.1% (P = .09); and overall survival, 96.1 6 0.8% and 98.0 6 0.6% (P = .06). Patients with ETV6-RUNX1–positive ALL and patients 1 to 6 years of age performed equally well in both arms. The incidence of death during remission was comparable, which indicates equivalent toxicity. The 8-year cumulative incidence rate of secondary malignancies was 1.3 6 0.5% and 0.6 6 0.4% for P-III and P-II, respectively (P = .37). Conclusion Although the criteria used for the standard-risk definition in this trial identified patients with exceptionally good prognosis, reduction of chemotherapy was not successful mainly because of an increased rate of relapse. The data suggest that treatment reduction is feasible in specific subgroups, which underlines the biologic heterogeneity o

Background Flow cytometric analysis of leukemia-associated immunophenotypes and polymerase chain reaction-based amplification of antigen-receptor genes rearrangements are reliable methods for monitoring minimal residual disease. The aim of this study was to compare the performances of these two methodologies in the detection of minimal residual disease in childhood acute lymphoblastic leukemia. Design and Methods Polymerase chain reaction and flow cytometry were simultaneously applied for prospective minimal residual disease measurements at days 15, 33 and 78 of induction therapy on 3565 samples from 1547 children with acute lymphoblastic leukemia enrolled into the AIEOP-BFM ALL 2000 trial. Results The overall concordance was 80%, but different results were observed according to the time point. Most discordances were found at day 33 (concordance rate 70%) in samples that had significantly lower minimal residual disease. However, the discordance was not due to different starting materials (total versus mononucleated cells), but rather to cell input number. At day 33, cases with minimal residual disease below or above the 0.01% cut-off by both methods showed a very good outcome (5-year event-free survival, 91.6%) or a poor one (5-year eventfree survival, 50.9%), respectively, whereas discordant cases showed similar event-free survival rates (around 80%). Conclusions Within the current BFM-based protocols, flow cytometry and polymerase chain reaction cannot simply substitute each other at single time points, and the concordance rates between their results depend largely on the time at which they are used. Our findings suggest a potential complementary role of the two technologies in optimizing risk stratification in future clinical trials.

The Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000) study has for the first time introduced standardized quantitative assessment of minimal residual disease (MRD) based on immunoglobulin and T-cell receptor gene rearrangements as polymerase chain reaction targets (PCR-MRD), at 2 time points (TPs), to stratify patients in a large prospective study. Patients with precursor B (pB) ALL (n = 3184) were considered MRD standard risk (MRD-SR) if MRD was already negative at day 33 (analyzed by 2 markers, with a sensitivity of at least 10-4); MRD high risk (MRD-HR) if 10-3 or more at day 78 and MRD intermediate risk (MRD-IR): others. MRD-SR patients were 42% (1348): 5-year event-free survival (EFS, standard error) is 92.3% (0.9). Fifty-two percent (1647) were MRD-IR: EFS 77.6% (1.3). Six percent of patients (189) were MRD-HR: EFS 50.1% (4.1; P < .001). PCR-MRD discriminated prognosis even on top of white blood cell count, age, early response to prednisone, and genotype. MRD response detected by sensitive quantitative PCR at 2 predefined TPs is highly predictive for relapse in childhood pB-ALL. The study is registered at http://clinicaltrials.gov: NCT00430118 for BFM and NCT00613457 for AIEOP. (Blood. 2010;115(16):3206-3214) © 2010 by The American Society of Hematology.

Background Minimally differentiated acute myeloid leukemia is heterogeneous in karyotype and is defined by immature morphological and molecular characteristics. This originally French-American-British classification is still used in the new World Health Organization classification when other criteria are not met. Apart from RUNX1 mutation, no characteristic molecular aberrations are recognized. Design and Methods We performed whole genome single nucleotide polymorphism analysis and extensive molecular analysis in a cohort of 52 patients with minimally differentiated acute myeloid leukemia. Results Many recurring and potentially relevant regions of loss of heterozygosity were revealed. These point towards a variety of candidate genes that could contribute to the pathogenesis of minimally differentiated acute myeloid leukemia, including the tumor suppressor genes TP53 and NF1, and reinforced the importance of RUNX1 in this leukemia . Furthermore, for the first time in this minimally differentiated form of leukemia we detected mutations in the transactivation domain of RUNX1. Mutations in other acute myeloid leukemia associated transcriptions factors were infrequent. In contrast, FLT3, RAS, PTPN11 and JAK2 were often mutated. Irrespective of the RUNX1 mutation status, our results show that RAS signaling is the most important pathway for proliferation in minimally differentiated acute myeloid leukemia. Importantly, we found that high terminal deoxynucleotidyl transferase expression is closely associated with RUNX1 mutation, which could allow an easier diagnosis of RUNX1 mutation in this hematologic malignancy. Conclusions Our results suggest that in patients without RUNX1 mutation, several other molecular aberrations, separately or in combination, contribute to a common minimally differentiated phenotype.

Background: Single-laboratory experience showed that flow cytometric (FCM) assessment of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) is feasible in most patients and gives independent prognostic information. It is, however, not known whether FCM analysis can reliably be standardized for multicentric application. Methods: An extensive standardization program was installed in four collaborating laboratories, which study FCM-MRD in children treated with the AIEOP-BFM-ALL 2000 protocol. This included methodological alignment, continuous quality monitoring, as well as personnel education by exchange and performance feed-back. Results: Blinded inter-laboratory tests of list-mode data interpretation concordance (n = 202 blood and bone marrow samples from follow-up during induction of 31 randomly selected patients of a total series of n = 395) showed a very high degree of inter-rater agreement among the four centers despite differences in cytometers and software usage (intraclass correlation coef.cient [ICC] 0.979 based on n = 800 single values). Lower concordance was reached with amounts of MRD below 0.1%. Comparing data from sample exchange experiments (n = 42 samples; ICC 0.98) and from independent patient cohorts from the four centers (regarding positive samples per time-point of follow-up as well as risk estimates) concordance was also good. Conclusion: MRD-evaluation by FCM in ALL can be standardized for reliable multicentric assessment in large trials. © 2008 Clinical Cytometry Society.

Expression of CD99 is higher on immature than on mature T cells. We postulated that this marker could be used to assess minimal residual disease (MRD) in T-lineage acute lymphoblastic leukemia (T-ALL). In diagnostic bone marrow (BM) samples from 27 children with T-ALL, expression of CD99 on leukemic lymphoblasts by flow cytometry was in median 7.7 times higher than on normal T lymphocytes from within the same sample. In 85% of cases, leukemic MFI values were higher than the mean MFI + 2 s.d. of normal populations. We applied CD99 to study MRD in 39 follow-up samples from 15 consecutive T-ALL patients, and compared the results with those obtained with the well-established MRD-marker terminal deoxynucleotidyl transferase (TdT). Either antibody was combined in four-color flow cytometry with CD7, surfaceCD3, and cytoplasmicCD3. We found that CD99 was a valid complement to TdT in quantifying T-ALL MRD. Given a considerable interpatient variability, CD99 could be favorably used in nine patients, and TdT in other five patients. Both approaches showed a similar very low non-specific background throughout 12 weeks from diagnosis (in median 0.002% of nucleated BM cells in patients with non-T ALL). We conclude that CD99 is a highly informative tool for MRD detection In T-ALL, bearing the advantage of surface expression in contrast to TdT. © 2004 Nature Publishing Group All rights reserved.

The molecular approach for the analysis of leukemia associated chromosomal translocations has led to the identification of prognostic relevant subgroups. In pediatric acute lymphoblastic leukemia (ALL), the most common translocations, t(9;22) and t(4;11), have been associated with a poorer clinical outcome. Recently the TEL gene at chromosome 12p13 and the AML1 gene at chromosome 21q22 were found to be involved in the translocation t(12;21)(p13;q22). By conventional cytogenetics, however, this chromosomal abnormality is barely detectable and occurs in less than 0.05% of childhood ALL. To investigate the frequency of the molecular equivalent of the t(12;21), the TEL/AML1 gene fusion, we have undertaken a prospective screening in the running German Berlin-Frankfurt-Münster (BFM) and Italian Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) multicenter ALL therapy trials. We have analyzed 334 unselected cases of pediatric ALL patients consecutively referred over a period of 5 and 9 months, respectively. The overall incidence of the t(12;21) in pediatric ALL is 18.9%. The 63 cases positive for the TEL/AML1 chimeric products ranged in age between 1 and 12 years, and all but one showed CD10 and pre-B immunophenotype. Interestingly, one case displayed a pre-pre-B immunophenotype. Among the B-lineage subgroup, the t(12;21) occurs in 22.0% of the cases. Fifteen of 61 (24.6%) cases coexpressed at least two myeloid antigens (CD13, CD33, or CDw65) in more than 20% of the gated blast cells. DNA index was available for 59 of the 63 TEL/AML1 positive cases; a hyperdiploid DNA content (> or = 1.16) was detected in only four patients, being nonhyperdiploid in the remaining 55. Based on this prospective analysis, we retrospectively evaluated the impact of TEL/AML1 in prognosis by identifying the subset of B-lineage ALL children enrolled in the closed German ALL-BFM-90 and Italian ALL-AIEOP-91 protocols who had sufficient material for analysis. A total of 342 children wer