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 coefficient [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.

Immunophenotyping by flow cytometry (FCM) is a worldwide mainstay in leukemia diagnostics. For concordant multicentric application, however, a gap exists between available classification systems, technologic standardization, and clinical needs. The AIEOP‐BFM consortium induced an extensive standardization and validation effort between its nine national reference laboratories collaborating in immunophenotyping of pediatric acute lymphoblastic leukemia (ALL). We elaborated common guidelines which take advantage of the possibilities of multi‐color FCM: marker panel requirements, immunological blast gating, in‐sample controls, tri‐partite antigen expression rating (negative vs. weak or strong positive) with capturing of blast cell heterogeneities and subclone formation, refined ALL subclassification, and a dominant lineage assignment algorithm able to distinguish “simple” from bilineal/“complex” mixed phenotype acute leukemia (MPAL) cases, which is essential for choice of treatment. These guidelines are a first step toward necessary inter‐laboratory standardization of pediatric leukemia immunophenotyping for a concordant multicentric application. © 2017 International Clinical Cytometry Society

CD20 is expressed in approximately one- half of pediatric acute lymphoblastic leukemia (ALL) cases with B-cell precursor (BCP) origin. We observed that it is occasionally up-regulated during treatment. To understand the impact of this on the potential effectiveness of anti-CD20 immunotherapy, we studied 237 CD10+ pediatric BCP-ALL patients with Berlin-Frankfurt-Munster (BFM)–type therapy. We analyzed CD20 expression changes from diagnosis to end-induction, focusing on sample pairs with more than or equal to 0.1% residual leukemic blasts, and assessed complement-induced cytotoxicity by CD20-targeting with rituximab in vitro. CD20-positivity significantly increased from 45% in initial samples to 81% at end-induction (day 15, 71%). The levels of expression also increased; 52% of cases at end-induction had at least 90% CD20pos leukemic cells, as opposed to 5% at diagnosis (day 15, 20%). CD20 up-regulation was frequent in high-risk patients, patients with high minimal residual disease at end-induction, and patients who suffered later from relapse, but not in TEL/AML1 cases. Notably, up-regulation occurred in viable cells sustaining chemotherapy. In vitro, CD20 up-regulation significantly enhanced rituximab cytotoxicity and could be elicited on prednisolone incubation. In conclusion, CD20 up-regulation is frequently induced in BCP-ALL during induction, and this translates into an acquired state of higher sensitivity to rituximab. This study was registered at http://www.clinicaltrials.gov as #NCT00430118.

Despite attempts to improve the definitions of ambiguous leukemia (ALAL) during the last two decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study iBFM-AMBI2012 on 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (5yEFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80±4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatments (36±7.2% and 50±12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5yEFS of CD19^pos leukemia was 83±5.3% on ALL-type primary treatment compared to 0±0% and 28±14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using WHO and/or EGIL definitions), and in the bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19^pos ALAL. AML-type of treatment is preferred in a minority of ALAL cases with CD19^neg and no other lymphoid features ALALs. No overall benefit of transplantation was documented and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change to the treatment type, this is to be considered only in cases with 5% blast or greater after remission induction. The results provide a basis for a prospective trial. Key points The largest cohort of ambiguous leukemias to date reveals a better prognosis of children who started on lymphoid-directed treatment. Myeloid type of primary treatment correlated with dismal outcomes in CD19pos leukemias.

PURPOSE: The International Berlin-Frankfurt-Münster (BFM) study group conducted a study on pediatric acute lymphoblastic leukemia (ALL). Minimal residual disease (MRD) was assessed using flow cytometry (FCM), and the impact of early intensification and methotrexate (MTX) dose on survival was evaluated. PATIENTS AND METHODS: We included 6,187 patients younger than 19 years. MRD by FCM refined the risk group definition previously used in the ALL intercontinental-BFM 2002 study on the basis of age, WBC count, unfavorable genetic aberrations, and treatment response measured morphologically. Patients at intermediate risk (IR) and high risk (HR) were randomly assigned to protocol augmented protocol I phase B (IB) versus IB regimen. MTX doses of 2 versus 5 g/m² every 2 weeks, four times, were evaluated in precursor B-cell-ALL (pcB-ALL) IR. RESULTS: The 5-year event-free survival (EFS ± SE) and overall survival (OS ± SE) rates were 75.2% ± 0.6% and 82.6% ± 0.5%, respectively. Their values in risk groups were standard risk (n = 624), 90.7% ± 1.4% and 94.7% ± 1.1%; IR (n = 4,111), 77.9% ± 0.7% and 85.7% ± 0.6%; and HR (n = 1,452), 60.8% ± 1.5% and 68.4% ± 1.4%, respectively. MRD by FCM was available in 82.6% of cases. The 5-year EFS rates in patients randomly assigned to protocol IB (n = 1,669) and augmented IB (n = 1,620) were 73.6% ± 1.2% and 72.8% ± 1.2%, respectively ( P = .55), while those in patients receiving MTX doses of 2 g/m² (n = 1,056) and MTX 5 g/m² (n = 1,027) were 78.8% ± 1.4% and 78.9% ± 1.4%, respectively ( P = .84). CONCLUSION: The MRDs were successfully assessed using FCM. An MTX dose of 2 g/m² was effective in preventing relapse in non-HR pcB-ALL. Augmented IB showed no advantages over the standard IB. [Media: see text]

Post-relapse therapy remains critical for survival in children with acute myeloid leukemia (AML). We evaluated survival, response and prognostic variables following relapse in independent cooperative group studies conducted by COG and the population-based AML-BFM study group. BFM included 197 patients who relapsed after closure of the last I-BFM relapse trial until 2017, while COG included 852 patients who relapsed on the last Phase 3 trials (AAML0531, AAML1031). Overall survival at 5 years (OS) was 42 ± 4% (BFM) and 35 ± 2% (COG). Initial high-risk features (BFM 32 ± 6%, COG 26 ± 4%) and short time to relapse (BFM 29 ± 4%, COG 25 ± 2%) predicted diminished survival. In the BFM dataset, there was no difference in OS for patients who had a complete remission with full hematopoietic recovery (CR) following post-relapse re-induction compared to those with partial neutrophil and platelet recovery (CRp and CRi) only (52 ± 7% vs. 63 ± 10%, p = 0.39). Among 90 patients alive at last follow-up, 87 had received a post-relapse hematopoietic stem cell transplant (HSCT). OS for patients with post-relapse HSCT was 54 ± 4%. In conclusion, initial high-risk features and early relapse remain prognostic. Response assessment with full hematopoietic recovery following initial relapse therapy does not predict survival. These data indicate the need for post-relapse risk stratification in future studies of relapse therapies.