Detailed immunophenotyping of B-cell precursors in regenerating bone marrow of acute lymphoblastic leukaemia patients: implications for minimal residual disease detection

Flow cytometric detection of minimal residual disease (MRD) in children with B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) requires immunophenotypic discrimination between residual leukaemic cells and B-cell precursors (BCPs) which regenerate during therapy intervals. In this study, EuroFlow-based 8-colour flow cytometry and innovative analysis tools were used to first characterize the immunophenotypic maturation of normal BCPs in bone marrow (BM) from healthy children, resulting in a continuous multiparametric pathway including transition stages. This pathway was subsequently used as a reference to characterize the immunophenotypic maturation of regenerating BCPs in BM from children treated for BCP-ALL. We identified pre-B-I cells that expressed low or dim CD34 levels, in contrast to the classical CD34 pre-B-I cell immunophenotype. These CD34 pre-B-I cells were relatively abundant in regenerating BM (11–85% within pre-B-I subset), while hardly present in healthy control BM (9–13% within pre-B-I subset; P = 0·0037). Furthermore, we showed that some of the BCP-ALL diagnosis immunophenotypes (23%) overlapped with CD34 pre-B-I cells. Our results indicate that newly identified CD34 pre-B-I cells can be mistaken for residual BCP-ALL cells, potentially resulting in false-positive MRD outcomes. Therefore, regenerating BM, in which CD34 pre-B-I cells are relatively abundant, should be used as reference frame in flow cytometric MRD measurements.The research for this manuscript was (in part) performed within the framework of the Erasmus Postgraduate School Molecular Medicine and was financially supported by PrioMedChild, project 40-41800-98-027. This study was performed within the EuroFlow Consortium, the EU-supported LSHB-CT2006-018708 FP6 Specific Targeted Research Project, which obtained sustainability based on income from the development of intellectual property and related patents, which have been licensed to industry. The work was supported through AZV (15-28525A) to EM and to MN.Peer Reviewe