A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of ≤10-5, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients. Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (≤10-5), if sufficient cells (>4 × 106, preferably more) are evaluated.

• MeSH
• Child MeSH
• Adult MeSH
• Gene Rearrangement MeSH
• Infant MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Infant, Newborn MeSH
• Precursor B-Cell Lymphoblastic Leukemia-Lymphoma diagnosis   MeSH
• Child, Preschool MeSH
• Flow Cytometry methods   standards   MeSH
• Receptors, Antigen, B-Cell genetics   MeSH
• Neoplasm, Residual diagnosis   MeSH
• Aged MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Middle Aged MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, Antigen, B-Cell MeSH

GATA-2 deficiency was recently described as common cause of overlapping syndromes of immunodeficiency, lymphedema, familiar myelodysplastic syndrome or acute myeloid leukemia. The aim of our study was to analyze bone marrow and peripheral blood samples of children with myelodysplastic syndrome or aplastic anemia to define prevalence of the GATA2 mutation and to assess whether mutations in GATA-2 transcription factor exhibit specific immunophenotypic features. The prevalence of a GATA2 mutation in a consecutively diagnosed cohort of children was 14% in advanced forms of myelodysplastic syndrome (refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and myelodysplasia-related acute myeloid leukemia), 17% in refractory cytopenia of childhood, and 0% in aplastic anemia. In GATA-2-deficient cases, we found the most profound B-cell lymphopenia, including its progenitors in blood and bone marrow, which correlated with significantly diminished intronRSS-Kde recombination excision circles in comparison to other myelodysplastic syndrome/aplastic anemia cases. The other typical features of GATA-2 deficiency (monocytopenia and natural killer cell lymphopenia) were less discriminative. In conclusion, we suggest screening for GATA2 mutations in pediatric myelodysplastic syndrome, preferentially in patients with impaired B-cell homeostasis in bone marrow and peripheral blood (low number of progenitors, intronRSS-Kde recombination excision circles and naïve cells).

• MeSH
• Anemia, Aplastic diagnosis   etiology   MeSH
• B-Lymphocytes metabolism   MeSH
• Biomarkers MeSH
• Bone Marrow Cells metabolism   pathology   MeSH
• Diagnosis, Differential MeSH
• Child MeSH
• Phenotype MeSH
• Immunophenotyping MeSH
• Infant MeSH
• Bone Marrow metabolism   pathology   MeSH
• Humans MeSH
• Lymphopenia diagnosis   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Mutation MeSH
• Myelodysplastic Syndromes diagnosis   genetics   MeSH
• Myeloid Cells metabolism   MeSH
• Lymphocyte Count MeSH
• Child, Preschool MeSH
• Precursor Cells, B-Lymphoid metabolism   MeSH
• ROC Curve MeSH
• T-Lymphocyte Subsets immunology   metabolism   MeSH
• GATA2 Transcription Factor deficiency   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Child, Preschool MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• GATA2 Transcription Factor MeSH