CD371-positive pediatric B-cell acute lymphoblastic leukemia: propensity to lineage switch and slow early response to treatment.

ABSTRACT: In the effort to improve immunophenotyping and minimal residual disease (MRD) assessment in acute lymphoblastic leukemia (ALL), the international Berlin-Frankfurt-Münster (iBFM) Flow Network introduced the myelomonocytic marker CD371 for a large prospective characterization with a long follow-up. In the present study, we aimed to investigate the clinical and biological features of CD371-positive (CD371pos) pediatric B-cell precursor ALL (BCP-ALL). From June 2014 to February 2017, 1812 pediatric patients with newly diagnosed BCP-ALLs enrolled in trial AIEOP-BFM ALL 2009 were evaluated as part of either a screening (n = 843, Italian centers) or validation cohort (n = 969, other iBFM centers). Laboratory assessment at diagnosis consisted of morphological, immunophenotypic, and genetic analysis. Response assessment relied on morphology, multiparametric flow cytometry (MFC), and polymerase chain reaction (PCR)-MRD. At diagnosis, 160 of 1812 (8.8%) BCP-ALLs were CD371pos. This correlated with older age, lower ETV6::RUNX1 frequency, immunophenotypic immaturity (all P < .001), and strong expression of CD34 and of CD45 (P < .05). During induction therapy, CD371pos BCP-ALLs showed a transient myelomonocytic switch (mm-SW: up to 65.4% of samples at day 15) and an inferior response to chemotherapy (slow early response, P < .001). However, the 5-year event-free survival was 88.3%. Among 420 patients from the validation cohort, 27 of 28 (96.4%) cases positive for DUX4-fusions were CD371pos. In conclusion, in the largest pediatric cohort, CD371 is the most sensitive marker of transient mm-SW, whose recognition is essential for proper MFC MRD assessment. CD371pos is associated to poor early treatment response, although a good outcome can be reached after MRD-based ALL-related therapies.

A New View on Minimal Residual Disease Quantification in Acute Lymphoblastic Leukemia using Droplet Digital PCR.

Real-time quantitative PCR (qPCR) using immunoglobulin/T-cell receptor gene rearrangements has been used as the gold standard for minimal residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) for >20 years. Recently, new PCR-based technologies have emerged, such as droplet digital PCR (ddPCR), which could offer several methodologic advances for MRD monitoring. In the current work, qPCR and ddPCR were compared in an unbiased blinded prospective study (n = 88 measurements) and in a retrospective study with selected critical low positive samples (n = 65 measurements). The former included flow cytometry (Flow; n = 31 measurements) as a third MRD detection method. Published guidelines (qPCR) and the latest, revised evaluation criteria (ie, ddPCR, Flow) have been applied for data analysis. The prospective study shows that ddPCR outperforms qPCR with a significantly better quantitative limit of detection and sensitivity. The number of critical MRD estimates below quantitative limit was reduced by sixfold and by threefold in the retrospective and prospective cohorts, respectively. Furthermore, the concordance of quantitative values between ddPCR and Flow was higher than between ddPCR and qPCR, probably because ddPCR and Flow are absolute quantification methods independent of the diagnostic sample, unlike qPCR. In summary, our data highlight the advantages of ddPCR as a more precise and sensitive technology that may be used to refine response monitoring in ALL.

Automated identification of cell populations in flow cytometry data with transformers.

Acute Lymphoblastic Leukemia (ALL) is the most frequent hematologic malignancy in children and adolescents. A strong prognostic factor in ALL is given by the Minimal Residual Disease (MRD), which is a measure for the number of leukemic cells persistent in a patient. Manual MRD assessment from Multiparameter Flow Cytometry (FCM) data after treatment is time-consuming and subjective. In this work, we present an automated method to compute the MRD value directly from FCM data. We present a novel neural network approach based on the transformer architecture that learns to directly identify blast cells in a sample. We train our method in a supervised manner and evaluate it on publicly available ALL FCM data from three different clinical centers. Our method reaches a median F1 score of ≈0.94 when evaluated on 519 B-ALL samples and shows better results than existing methods on 4 different datasets.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Automated Flow Cytometric MRD Assessment in Childhood Acute B- Lymphoblastic Leukemia Using Supervised Machine Learning.

Minimal residual disease (MRD) as measured by multiparameter flow cytometry (FCM) is an independent and strong prognostic factor in B-cell acute lymphoblastic leukemia (B-ALL). However, reliable flow cytometric detection of MRD strongly depends on operator skills and expert knowledge. Hence, an objective, automated tool for reliable FCM-MRD quantification, able to overcome the technical diversity and analytical subjectivity, would be most helpful. We developed a supervised machine learning approach using a combination of multiple Gaussian Mixture Models (GMM) as a parametric density model. The approach was used for finding the weights of a linear combination of multiple GMMs to represent new, "unseen" samples by an interpolation of stored samples. The experimental data set contained FCM-MRD data of 337 bone marrow samples collected at day 15 of induction therapy in three different laboratories from pediatric patients with B-ALL for which accurate, expert-set gates existed. We compared MRD quantification by our proposed GMM approach to operator assessments, its performance on data from different laboratories, as well as to other state-of-the-art automated read-out methods. Our proposed GMM-combination approach proved superior over support vector machines, deep neural networks, and a single GMM approach in terms of precision and average F 1 -scores. A high correlation of expert operator-based and automated MRD assessment was achieved with reliable automated MRD quantification (F 1 -scores >0.5 in more than 95% of samples) in the clinically relevant range. Although best performance was found, if test and training samples were from the same system (i.e., flow cytometer and staining panel; lowest median F 1 -score 0.92), cross-system performance remained high with a median F 1 -score above 0.85 in all settings. In conclusion, our proposed automated approach could potentially be used to assess FCM-MRD in B-ALL in an objective and standardized manner across different laboratories. © 2019 International Society for Advancement of Cytometry.

Aneuploidy in children with relapsed B-cell precursor acute lymphoblastic leukaemia: clinical importance of detecting a hypodiploid origin of relapse.

Aneuploidy is common in paediatric B-cell precursor acute lymphoblastic leukaemia (ALL). Specific subgroups, such as high hyperdiploidy (>50 chromosomes or DNA Index ≥1·16) and hypodiploidy (<45 chromosomes), predict outcome of patients after primary treatment. Whether aneuploidy has a prognostic value for relapsed disease is yet to be determined. Using DNA index and centromere screening by multiplex ligation-dependent probe amplification, we investigated aneuploidy in 413 children treated for first relapse of B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. Ten-year event-free survival of patients with high hyperdiploid relapses approached 70%, whereas it was only 40% in low hyperdiploid relapses. Three patients with apparent hyperdiploid relapse had TP53 mutations. In these cases, array-based allelotyping revealed a hypodiploid origin with absence of the hypodiploid founder clone (masked hypodiploidy). Collectively, patients with evident or masked hypodiploid relapses showed an extremely low event-free survival rate of 9%. Importantly, the current relapse risk stratification did not identify cases with masked hypodiploidy as high-risk patients, due to their favourable clinical presentation. In multivariate analysis, hypodiploidy proved to be an independent prognostic factor. This finding supports stratification of relapses with hypodiploid origin into high-risk arms in future trials or allocation of patients to alternative treatment approaches.

Flow-cytometric minimal residual disease monitoring in blood predicts relapse risk in pediatric B-cell precursor acute lymphoblastic leukemia in trial AIEOP-BFM-ALL 2000.

BACKGROUND: Flow-cytometric monitoring of minimal residual disease (MRD) in bone marrow (BM) during induction of pediatric patients with acute lymphoblastic leukemia (ALL) is widely used for outcome prognostication and treatment stratification. Utilizing peripheral blood (PB) instead of BM might be favorable, but data on its usefulness are scarce. PROCEDURE: We investigated 1303 PB samples (days 0, 8, 15, 33, and 52) and 285 BMs (day 15) from 288 pediatric ALL patients treated in trial AIEOP-BFM ALL 2000. MRD was assessed by four-color flow cytometry and evaluated as relative, absolute, and kinetic result. RESULTS: In B-ALL only, PB measures from early time points correlated with relapse incidence (CIR). Best separation occurred at threshold <1 blast/µL at day 8 (5-year CIR 0.02 ± 0.02 vs 0.12 ± 0.03; P = 0.044). Patients with highest relapse risk were not distinguishable, but PB-MRD at days 33 and 52 correlated with prednisone response and postinduction BM-MRD by PCR (P < 0.001). Kinetic assessment did not convey any advantage. In multivariate analysis including day 15 BM-MRD, PB-MRD measures lost statistical power. CONCLUSIONS: In summary, PB-MRD in pediatric B-ALL correlates with outcome and risk parameters, but its prognostic significance is not strong enough to substitute for BM assessment in AIEOP-BFM trials. It might, however, be valuable in treatment environments not using multifaceted risk stratification with other MRD measures.

Expression of CD56 defines a distinct subgroup in childhood T-ALL with inferior outcome. Results of the ALL-BFM 2000 trial.

This study reports the prognostic impact of the expression of the natural killer cell marker CD56 in a large series of risk-adapted paediatric patients with T cell acute lymphoblastic leukaemia (T-ALL; n = 493) treated within the ALL-Berlin-Frankfurt-Münster (BFM) 2000 protocol. The immunophenotype was analysed centrally at diagnosis using flow cytometry and correlated with clinical parameters and outcome. CD56 expression was detected in 7·1% and early T-cell precursor (ETP) phenotype in 6·7% of all T-ALL patients. The percentage of ETP in the CD56+ T-ALL cohort was 4-fold higher than in the whole cohort. CD56+ T-ALL frequently expressed the progenitor marker CD34 and myeloid antigens CD13 and CD33. The 5-year event-free survival (EFS) rates for the European Group for the Immunological classification of Leukaemias/World Health Organization subgroups and the ETP phenotype were not statistically different. By contrast, patients with CD56 expression had a significantly reduced EFS (60 ± 8%) and overall survival (60 ± 8%) at 5 years, with a hazard ratio of 2·46 (P = 0·002) and 2·99 (P < 0·001), respectively. Moreover, CD56 expression in combination with the minimal residual disease (MRD)-based high risk assignment defined a population with a 'very-high' risk probability of relapse in the ALL-BFM 2000 trial. The CD56 marker has the potential to augment MRD-based risk stratification and may serve as a molecular target for antibody-based treatment strategies in childhood T-ALL.

AIEOP-BFM consensus guidelines 2016 for flow cytometric immunophenotyping of Pediatric acute lymphoblastic leukemia.

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.

Minimal residual disease analysis by eight-color flow cytometry in relapsed childhood acute lymphoblastic leukemia.

Multiparametric flow cytometry is an alternative approach to the polymerase chain reaction method for evaluating minimal residual disease in treatment protocols for primary acute lymphoblastic leukemia. Given considerable differences between primary and relapsed acute lymphoblastic leukemia treatment regimens, flow cytometric assessment of minimal residual disease in relapsed leukemia requires an independent comprehensive investigation. In the present study we addressed evaluation of minimal residual disease by flow cytometry in the clinical trial for childhood relapsed acute lymphoblastic leukemia using eight-color flow cytometry. The major challenge of the study was to reliably identify low amounts of residual leukemic cells against the complex background of regeneration, characteristic of follow-up samples during relapse treatment. In a prospective study of 263 follow-up bone marrow samples from 122 patients with B-cell precursor acute lymphoblastic leukemia, we tested various B-cell markers, adapted the antibody panel to the treatment protocol, and evaluated its performance by a blinded parallel comparison with the polymerase chain reaction data. The resulting eight-color single-tube panel showed a consistently high overall concordance (P<0.001) and, under optimal conditions, sensitivity similar to that of the reference polymerase chain reaction method. Overall, evaluation of minimal residual disease by flow cytometry can be successfully integrated into the clinical management of relapsed childhood acute lymphoblastic leukemia either as complementary to the polymerase chain reaction or as an independent risk stratification tool. ALL-REZ BFM 2002 clinical trial information: NCT00114348.

Flow diagnostics essential code: a simple and brief format for the summary of leukemia phenotyping.

BACKGROUND: Flow cytometry is a valuable part in the routine diagnostics of acute leukemia (AL). Although internationally recognized definitions of main AL subsets are available, there is currently no consensus format for the short summary of clinical flow cytometry reports. Since clinical reports are too long for most database purposes, there is a need for a standardized format of their short summaries. METHODS: The Associazione Italiana Ematologia Oncologia Pediatrica--Berlin Frankfurt Muenster (AIEOP-BFM) Flow Network that encompasses reference diagnostics laboratories in Australia, Austria, Czechia, Germany, Israel, Italy, and Switzerland have designed a pro-forma for the summary of flow cytometry results in the diagnosis of leukemia. The process involved several meetings and other communications, during which the group established a consensus on the essentials that lead to the diagnostic conclusions in childhood AL. RESULTS: The "Flow Diagnostics Essential (FDE) Code" is a result from an agreement within the AIEOP-BFM Flow Network. In a standardized format, it reports the extent of the infiltration by a malignant clone, followed by description antigen expression as strong, weak or negative, and a diagnostic conclusion. CONCLUSIONS: A consensus brief format (the "FDE Code") has been designed as a brief summary of the diagnostic immunophenotype of childhood AL. It is also applicable for the diagnostic investigation of other malignancies by flow cytometry. The FDE code may be included in the final clinical report and/or used in the setting of a multicenter clinical trial database.

High CD45 surface expression determines relapse risk in children with precursor B-cell and T-cell acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol.

Further improvement of outcome in childhood acute lymphoblastic leukemia could be achieved by identifying additional high-risk patients who may benefit from intensified treatment. We earlier identified PTPRC (CD45) gene expression as a potential new stratification marker and now analyzed the prognostic relevance of CD45 protein expression. CD45 was measured by flow cytometry in 1065 patients treated according to the ALL-BFM-2000 protocol. The 75(th) percentile was used as cut-off to distinguish a CD45-high from a CD45-low group. As mean CD45 expression was significantly higher in T-cell acute lymphoblastic leukemia than in B-cell-precursor acute lymphoblastic leukemia (P<0.0001), the analysis was performed separately in both groups. In B-cell-precursor acute lymphoblastic leukemia we observed a significant association of a high CD45 expression with older age, high initial white blood cell count, ETV6/RUNX1 negativity, absence of high hyperdiploidy (P<0.0001), MLL/AF4 positivity (P=0.002), BCR/ABL1 positivity (P=0.007), prednisone poor response (P=0.002) and minimal residual disease (P<0.0001). In T-cell acute lymphoblastic leukemia we observed a significant association with initial white blood cell count (P=0.0003), prednisone poor response (P=0.01), and minimal residual disease (P=0.02). Compared to CD45-low patients, CD45-high patients had a lower event-free survival rate (B-cell-precursor acute lymphoblastic leukemia: 72 ± 3% versus 86 ± 1%, P<0.0001; T-cell acute lymphoblastic leukemia: 60 ± 8% versus 78 ± 4%, P=0.02), which was mainly attributable to a higher cumulative relapse incidence (B-cell-precursor acute lymphoblastic leukemia: 22 ± 3% versus 11 ± 1%, P<0.0001; T-cell acute lymphoblastic leukemia: 31 ± 8% versus 11 ± 3%, P=0.003) and kept its significance in multivariate analysis considering sex, age, initial white blood cell count, and minimal residual disease in B-cell-precursor- and T-cell acute lymphoblastic leukemia, and additionally presence of ETV6/RUNX1, MLL/AF4 and BCR/ABL1 rearrangements in B-cell-precursor acute lymphoblastic leukemia (P=0.002 and P=0.025, respectively). Consideration of CD45 expression may serve as an additional stratification tool in BFM-based protocols. (ClinicalTrials.gov identifier: NCT00430118).

Time point-dependent concordance of flow cytometry and real-time quantitative polymerase chain reaction for minimal residual disease detection in childhood acute lymphoblastic leukemia.

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 event-free 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.

Mutations and deletions of the TP53 gene predict nonresponse to treatment and poor outcome in first relapse of childhood acute lymphoblastic leukemia.

PURPOSE: In the clinical management of children with relapsed acute lymphoblastic leukemia (ALL), treatment resistance remains a major challenge. Alterations of the TP53 gene are frequently associated with resistance to chemotherapy, but their significance in relapsed childhood ALL has remained controversial because of small studies. PATIENTS AND METHODS: Therefore, we systematically studied 265 first-relapse patients enrolled in the German Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Mü nster 2002 (ALL-REZ BFM 2002) trial for sequence and copy number alterations of the TP53 gene by using direct sequencing and multiplex ligation-dependent probe amplification. RESULTS: We observed copy number and sequence alterations of TP53 in 12.4% (27 of 218) of patients with B-cell precursor ALL and 6.4% (three of 47) of patients with T-cell ALL relapse. Backtracking to initial ALL in 23 matched samples revealed that 54% of all TP53 alterations were gained at relapse. Within B-cell precursor ALL, TP53 alterations were consistently associated with nonresponse to chemotherapy (P < .001) and poor event-free survival (P < .001) and overall survival rates (P = .002). TP53 alterations also had a significant impact on survival within intermediate-risk (S2) and high-risk (S3/S4) relapse patients (P = .007 and P = .019, respectively). This prognostic significance of TP53 alterations was confirmed in multivariate analysis. Besides their clinical impact, TP53 alterations were associated with a higher fraction of leukemic cells in S/G(2)-M phase of the cell cycle at relapse diagnosis. CONCLUSION: Alterations of the TP53 gene are of particular importance in the relapse stage of childhood ALL, in which they independently predict high risk of treatment failure in a significant number of patients. Therefore, they will aid in future risk assessment of children with ALL relapse.

CD11b is a therapy resistance- and minimal residual disease-specific marker in precursor B-cell acute lymphoblastic leukemia.

A consistently increased mRNA expression of the adhesion receptor CD11b is a hallmark of the reported genomewide gene expression changes in precursor B-cell acute lymphoblastic leukemia (PBC-ALL) after 1 week of induction therapy. To investigate its clinical relevance, CD11b protein expression in leukemic blasts has been prospectively measured at diagnosis (159 patients) and during therapy (53 patients). The initially heterogeneous expression of CD11b inversely correlated with cytoreduction rates measured at clinically significant time points of induction therapy in the ALL-Berlin-Frankfurt-Münster 2000 protocol. CD11b positivity conferred a 5-fold increased risk of minimal residual disease (MRD) after induction therapy (day 33) and of high-risk group assignment after consolidation therapy (day 78). In the multivariate analysis CD11b expression was an independent prognostic factor compared with other clinically relevant parameters at diagnosis. During therapy, CD11b expression increased early in most ALL cases and remained consistently increased during induction/consolidation therapy. In more than 30% of MRD-positive cases, the CD11b expression on blast cells exceeded that of mature memory B cells and improved the discrimination of residual leukemic cells from regenerating bone marrow. Taken together, CD11b expression has considerable implications for prognosis, treatment response monitoring, and MRD detection in childhood PBC-ALL.

Modulation of antigen expression in B-cell precursor acute lymphoblastic leukemia during induction therapy is partly transient: evidence for a drug-induced regulatory phenomenon. Results of the AIEOP-BFM-ALL-FLOW-MRD-Study Group.

BACKGROUND: Changes of antigen expression on residual blast cells of acute lymphoblastic leukemia (ALL) occur during induction treatment. Many markers used for phenotyping and minimal residual disease (MRD) monitoring are affected. Glucocorticoid (GC)-induced expression modulation has been causally suspected, however, subclone selection may also cause the phenomenon. METHODS: We investigated this by following the phenotypic evolution of leukemic cells with flow cytometry from diagnosis to four time points during and after GC containing chemotherapy in the 20 (of 360 consecutive) B-cell precursor patients with ALL who had persistent MRD throughout. RESULTS: The early expression changes of CD10 and CD34 were reversible after stop of GC containing chemotherapy. Modulation of CD20 and CD45 occurred mostly during the GC phase, whereas CD11a also changed later on. Blast cells at diagnosis falling into gates designed according to "shifted" phenotypes from follow-up did not form clusters and were frequently less numerous than later on. CONCLUSIONS: Our data support the idea that drug-induced modulation rather than selection causes the phenomenon. The good message for MRD assessment is that modulation is transient in at least two (CD10 and CD34) of the five prominent antigens investigated and reverts to initial aberrant patterns after stop of GC therapy, whereas CD20 expression gains new aberrations exploitable for MRD detection.

Standardization of flow cytometric minimal residual disease evaluation in acute lymphoblastic leukemia: Multicentric assessment is feasible.

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.