Loss of hematopoietic progenitors heterogeneity is an adverse prognostic factor in lower-risk myelodysplastic neoplasms.

Myelodysplastic neoplasms (MDS) are characterized by clonal evolution starting from the compartment of hematopoietic stem and progenitors cells (HSPCs), leading in some cases to leukemic transformation. We hypothesized that deciphering the diversity of the HSPCs compartment may allow for the early detection of an emergent sub-clone that drives disease progression. Deep analysis of HSPCs repartition by multiparametric flow cytometry revealed a strong disorder of the hematopoietic branching system in most patients at diagnosis with different phenotypic signatures closely related to specific MDS features. In two independent cohorts of 131 and 584 MDS, the HSPCs heterogeneity quantified through entropy calculation was decreased in 47% and 46% of cases, reflecting a more advanced state of the disease with deeper cytopenias, higher IPSS-R risk and accumulation of somatic mutations. We demonstrated that patients with lower-risk MDS and low CD34 + CD38+HSPCs entropy had an adverse outcome and that this parameter is as an independent predictive biomarker for progression free survival, leukemia free survival and overall survival. Analysis of HSPCs repartition at diagnosis represents therefore a very powerful tool to identify lower-risk MDS patients with a worse outcome and valuable for clinical decision-making, which could be fully integrated in the MDS diagnostic workflow.

PCR-Fluo-ASXL1-FA: A fast, sensitive and inexpensive complementary method to detect ASXL1 mutations in haematological malignancies.

INTRODUCTION: The additional sex combs like 1 (ASXL1) gene is frequently mutated in a number of haematological neoplasms. The c.1934dupG, known to be the most common alteration in ASXL1, is associated with poor clinical outcome. A systematic determination of ASXL1 mutational status in myeloid malignancies is therefore necessary for prognostic stratification. METHODS: Because direct sequencing is not sensitive and next-generation sequencing (NGS) is time-consuming, expensive and sometimes does not allow the detection of the c.1934dupG, we have developed a fragment analysis assay, complementary to NGS, that allows the detection of c.1934dupG mutation in addition to other nearby insertions/deletions of ASXL1 located close to it. We called this assay the "PCR-Fluo-ASXL1-FA." RESULTS: First, we evaluated the efficiency of our approach compared to NGS and Sanger. We showed that "PCR-Fluo-ASXL1-FA" could detect all insertional mutations of ASXL1 located on its area, with a high sensitivity (1.5%). Then, we have illustrated the interest of this technique by three concrete cases. DISCUSSION: In summary, we have established a fragment analysis approach, which can detect most ASXL1 mutations, in particular the c.1934dupG, in a sensitive, fast and inexpensive manner. We therefore recommend the synchronous use of this method with NGS, to ensure complete detection of all clinically relevant ASXL1 mutations in patients suffering with myeloid neoplasms.

Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs are associated with myeloid malignancies: most frequently Chronic MyeloMonocytic Leukemia (CMML) but also Acute Myeloid Leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs in pDC-AML harbor a clearly different phenotype from BPDCN: CD4+ CD56- in 100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a pre-pDC stage. In all cases, pDCs, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.