Suivi précose de la blastose sanguine dans les leucémies aiguës myéloïdes par une méthode standardisée de cytométrie en flux

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

RECONSTITUTION IMMUNITAIRE ET ACQUISITION DU REPERTOIRE T APRES GREFFE DE CELLULES SOUCHES PERIPHERIQUES

AIX-MARSEILLE2-BU Pharmacie (130552105) / SudocSudocFranceF

All-trans retinoic acid skews monocyte differentiation into interleukin-12-secreting dendritic-like cells

All-trans retinoic acid (ATRA) and retinoid derivatives are essential agents for multiple biological processes. Numerous immune system dysfunctions can occur in the case of retinoid deficiency. Because of the central role of dendritic cells (DCs) in controlling immunity and the wide effects of retinoids on the immune system homeostasis, we investigated the ability of ATRA to influence the differentiation of DCs from circulating peripheral blood monocytes. Human peripheral blood monocytes were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and various concentrations of ATRA. Differentiated cells were assayed for their morphology, phenotype, antigen uptake, allostimulatory capacity and cytokine secretion profile. ATRA (10(-12) mol/l) and GM-CSF drove the differentiation of monocytes into dendritic-like cells (ATRA-DC). ATRA-DCs exhibited DC morphology, had a phenotype of immature DCs, with the expression of CD1a, and upregulation of adhesion and co-stimulatory molecules. ATRA-DCs could induce a proliferative response in naive CD4+ T cells. Although ATRA-DCs retained their antigen-capture capacity, they secreted interleukin (IL)-12p70 without the need for any maturation agent. In addition, ATRA-DCs could drive T cells towards an IL-12-dependent T-helper cell type 1 response with secretion of interferon-gamma. DCs appear to be potential targets for ATRA, giving new insights into the immunomodulatory function of retinoids, with implications potentially related to immunotherapy

Four- and five-color flow cytometry analysis of leukocyte differentiation pathways in normal bone marrow: a reference document based on a systematic approach by the GTLLF and GEIL.

International audienceBACKGROUND: 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

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts.

Background. Chronic myelomonocytic leukemia is close to, but separate from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. Design and Methods. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Results. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to the former, the latter overexpressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated by using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Conclusions. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not identical mutation spectrum

Multicenter study of ZAP-70 expression in patients with B-cell chronic lymphocytic leukemia using an optimized flow cytometry method.

International audienceBACKGROUND: Flow cytometry allows specific assessment of the expression of ZAP-70, a promising new prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL), but suffers from a lack of multicenter standardization. DESIGN AND METHODS: An optimized method for direct detection of ZAP-70 in flow cytometry was tested in a multicenter fashion. Adapted for frozen cells, this method includes a normalization step by addition of B cells from a pool of peripheral blood mononuclear cells collected from normal donors. ZAP-70 expression levels were assessed for 153 patients with typical B-cell chronic lymphocytic leukemia chronic lymphocytic leukemia. Results were expressed as the ratio of ZAP-70 mean fluorescence intensity between B-CLL cells and normal B cells. RESULTS: The statistically optimized cut-off of ZAP-70 positivity was a ratio of 1.4. Concordance between ZAP-70 and CD38 expression was 67%. Concordance between the mutational status of IgVH genes and ZAP-70 or CD38 expression was 87% and 65%, respectively. ZAP-70 was significantly expressed in 28%, 54% and 61% of patients with Binet stages A, B and C B-cell chronic lymphocytic leukemia, respectively (p=0.008). The absence of ZAP-70 expression was associated with isolated del(13q14), a cytogenetic abnormality with a good prognosis, while most patients with the del(17p13) poor prognosis cytogenetic marker expressed ZAP-70 (p<10(-5)). ZAP-70 expression was not related to the other poor prognosis cytogenetic abnormality del(11q22.3) nor to trisomy 12. CONCLUSIONS: This new technique provides highly reliable results well correlated with the mutational status of IgVH genes, CD38 expression, Binet stage and cytogenetic abnormalities. This robust discriminative technique appears of particular interest for routine diagnosis and assessment of ZAP-70 expression in large, prospective, multicenter therapeutic trials