Background: The development of multiparameter flow cytometry (FCM) and increasingly sophisticated analysis software has considerably improved the exploration of hematological disorders. These tools have been widely applied in leukaemias, lymphomas, and myelodysplasias, yet with very heterogeneous approaches. Consequently, there is no extensive reference document reporting on the characteristics of normal human bone marrow (BM) in multiparameter FCM. Here, we report a reference analysis procedure using relevant antibody combinations in normal human BM.Methods: A first panel of 23 antibodies, constructed after literature review, was tested in four-color combinations (including CD45 in each) on 30 samples of BM. After evaluation of the data, a second set of 22 antibodies was further applied to another 35 BM samples. All list-modes from the 65 bone marrow samples were reviewed collectively. A systematised protocol for data analysis was established including biparametric representations and color codes for the three major lineages and undifferentiated cells.Results: This strategy has allowed to obtain a reference atlas of relevant patterns of differentiation antigens expression in normal human BM that is available within the European LeukemiaNet. This manuscript describes how this atlas was constructed.Conclusions: Both the strategy and atlas could prove very useful as a reference of normality, for the determination of leukemia-associated immunophenotypic patterns, analysis of myelodysplasia and, ultimately, investigation of minimal residual disease in the BM. V C 2009 Clinical Cytometry Society

Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. IntroductionT-cell acute lymphoblastic leukemias (T-ALL) are malignant proliferations of T-cell precursors that represent 10% of pediatric and 25% of adult ALL. 1 Although treatment outcome has significantly improved in the last decade, ϳ 30% of patients relapse and remain of dismal prognosis, stressing the critical importance of gaining further insights on the molecular pathways controlling malignant transformation and drug resistance. However, a major obstacle in deciphering such pathways and implementing targeted therapy strategies resides in the fact that T-ALLs constitute a particularly heterogeneous and complex group of disease, resulting from numerous combinations of multigenic aberrations and oncogenic cooperation. 2 To date, the deregulation of Ͼ 30 distinct oncogenes and tumor suppressors (TS) has been reported, occurring through a large diversity of genomic aberrations and epigenetic deregulations. All such mechanisms are not functionally equivalent, 3 and distinct modes of oncogenic activation may drive different oncogenic processes, and generate distinct subtypes of prognostic significance. Some oncogenes (eg, TLX1, TAL1) appear to be mutually exclusive (type A) and delineate distinct subgroups of prognostic significance, correlating with specific stages of thymocyte developmental arrest (immature/DN, intermediate/ pre-␣␤, and mature/T-cell receptor␣␤ ϩ , respectively). [4][5] By contrast, other deregulations, such as loss of CDKN2A/p14ARF, or constitutive NOTCH1 activation, are found in a large proportion of cases and irrespective of subgroups (type B), 2 indicating a more universal role for these alterations in T-ALL pathogenesis, and pointing to attractive therapeutic targets. One such target is NOTCH1 and downstream pathways. Indeed, the key finding that Ͼ 50% of T-ALL cases display gain-of-function NOTCH1 mutations (NOTCH1 m ) initially held great promise for targeted therapy through the use of ␥-secretase inhibitors (GSI). 6 However, the frequent occurrence of GSI-resistance (GSI R ) has revealed an unsuspected complexity of the oncogenic network signaling downstream of NOTCH1. 6-8 Among the numerous target genes and pa...

Minimal residual disease (MRD) quantification is widely used for therapeutic stratification in pediatric acute lymphoblastic leukemia (ALL). A robust, reproducible, sensitivity of at least 0.01% has been achieved for IG/TCR clonal rearrangements using allele-specific quantitative PCR (IG/TCR-QPCR) within the EuroMRD consortium. Whether multiparameter flow cytometry (MFC) can reach such inter-center performance in ALL MRD monitoring remains unclear. In a multicenter study, MRD was measured prospectively on 598 follow-up bone marrow samples from 102 high-risk children and 136 adult ALL patients, using IG/TCR-QPCR and 4/5 color MFC. At diagnosis, all 238 patients (100%) had at least one suitable MRD marker with 0.01% sensitivity, including 205/238 samples (86%) by using IG/TCR-QPCR and 223/238 samples (94%) by using MFC. QPCR and MFC were evaluable in 495/598 (83%) samples. Qualitative results (<0.01% or ≥0.01%) concurred in 96% of samples and overall positivity (including <0.01% and nonquantifiable positivity) was concurrent in 84%. MRD values ≥0.01% correlated highly (r(2)=0.87) and 69% clustered within half-a-log(10). QPCR and MFC can therefore be comparable if properly standardized, and are highly complementary. MFC strategies will benefit from a concerted approach, as does molecular MRD monitoring, and will contribute significantly to the achievement of 100% MRD informativity in adult and pediatric ALL.

These normal values are proposed for use in everyday practice. They make it possible to distinguish, without ambiguity, a normal situation from a pathological situation. Moreover, they might be used over all mainland France.

Background: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10 . Here we report the MFC methodological aspects from this multi-center experience. Methods: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. Results: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10 cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. Conclusions: Measurement of MRD by MFC at the 10 cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL. © 2014 Clinical Cytometry Society.