No abstract

Myelodysplastic syndrome (MDS) is characterized by an ineffective hematopoiesis with production of aberrant clones and a high cell apoptosis rate in bone marrow (BM). Macrophages are in charge of phagocytosis. Innate Immune cells and specific T cells are in charge of immunosurveillance. Little is known on BM cell recruitment and activity as BM aspirate is frequently contaminated with peripheral blood. But evidences suggest an active role of immune cells in protection against MDS and secondary leukemia. BM CD8+ CD28− CD57+ T cells are directly cytotoxic and have a distinct cytokine signature in MDS, producing TNF-α, IL-6, CCL3, CCL4, IL-1RA, TNFα, FAS-L, TRAIL, and so on. These tools promote apoptosis of aberrant cells. On the other hand, they also increase MDS-related cytopenia and myelofibrosis together with TGFβ. IL-32 produced by stromal cells amplifies NK cytotoxicity but also the vicious circle of TNFα production. Myeloid-derived suppressing cells (MDSC) are increased in MDS and have ambiguous role in protection/progression of the diseases. CD33 is expressed on hematopoietic stem cells on MDS and might be a potential target for biotherapy. MDS also has impact on immunity and can favor chronic inflammation and emergence of autoimmune disorders. BM is the site of hematopoiesis and thus contains a complex population of cells at different stages of differentiation from stem cells and early engaged precursors up to almost mature cells of each lineage including erythrocytes, megakaryocytes, myelo-monocytic cells (monocyte/macrophage and granulocytes), NK cells, and B cells. Monocytes and B cell finalize their maturation in peripheral tissues or lymph nodes after migration through the blood. On the other hand, T cells develop in thymus and are present in BM only as mature cells, just like other well vascularized tissues. BM precursors have a strong proliferative capacity, which is usually associated with a high risk for genetic errors, cell dysfunction, and consequent cell death. Abnormal cells are prone to destruction through spontaneous apoptosis or because of the immunosurveillance that needs to stay highly vigilant. High rates of proliferation or differentiation failures lead to a high rate of cell death and massive release of debris to be captured and destroyed (1). Numerous macrophages reside in BM in charge of home-keeping. They have a high capacity of phagocytosis required for clearing all these debris.

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Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis and dysplasia in at least one myeloid cell lineage. This persistent monocytosis should be distinguished from the reactive monocytosis which is sometimes observed in a context of infections or solid tumors. In 2015, Selimoglu-Buet et al. observed an increased percentage of classical monocytes (CD14+/CD16− >94%) in the peripheral blood (PB) of CMML patients. In this study, using multiparametric flow cytometry (MFC), we assessed the monocytic distribution in PB samples and in bone marrow aspirates from 63 patients with monocytosis or CMML suspicion, and in seven follow-up blood samples from CMML patients treated with hypomethylating agents (HMA). A control group of 12 healthy age-matched donors was evaluated in parallel in order to validate the analysis template. The CMML diagnosis was established in 15 cases in correlation with other clinical manifestations and biological tests. The MFC test for the evaluation of the repartition of monocyte subsets, as previously described by Selimoglu-Buet et al. showed a specificity of 97% in blood and 100% in marrow samples. Additional information regarding the expression of intermediate MO2 monocytes percentage improved the specificity to 100% in blood samples allowing the screening of abnormal monocytosis. The indicative thresholds of CMML monocytosis were different in PB compared to BM samples (classical monocytes >95% for PB and >93% for BM). A decrease of monocyte levels in PB and BM, along with a normalization of monocytes distribution, was observed after treatment in 4/7 CMML patients with favorable evolution. No significant changes were observed in 3/7 patients who did not respond to HMA therapy and also presented unfavorable molecular prognostic factors at diagnosis (ASXL1, TET2, and IDH2 mutations). Considering its simplicity and robustness, the monocyte subsets evaluation by MFC provides relevant information for CMML diagnosis.

Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34 + CD38 − and CD34 + CD38 + ) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34 + CD38 − than in CD34 + CD38 + subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.

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Flow cytometry is broadly used for the identification, characterization, and monitoring of hematological malignancies. However, the use of clinical flow cytometry is restricted by its lack of reproducibility across multiple centers. Since 2006, the EuroFlow consortium has been developing a standardized procedure detailing the whole process from instrument settings to data analysis. The FranceFlow group was created in 2010 with the intention to educate participating centers in France about the standardized instrument setting protocol (SOP) developed by the EuroFlow consortium and to organise several rounds of quality controls (QCs) in order to evaluate the feasibility of its application and its results. Here, we report the 5 year experience of the FranceFlow group and the results of the seven QCs of 23 instruments, involving up to 19 centers, in France and in Belgium. The FranceFlow group demonstrates that both the distribution and applicability of the SOP have been successful. Intercenter reproducibility was evaluated using both normal and pathological blood samples. Coefficients of variation (CVs) across the centers were <7% for the percentages of cell subsets and <30% for the median fluorescence intensities (MFIs) of the markers tested. Intracenter reproducibility provided similar results with CVs of <3% for the percentages of the majority of cell subsets, and CVs of <20% for the MFI values for the majority of markers. Altogether, the FranceFlow group show that the 19 participating labs might be considered as one unique laboratory with 23 identical flow cytometers able to reproduce identical results. Therefore, SOP significantly improves reproducibility of clinical flow in hematology and opens new avenues by providing a robust companion diagnostic tool for clinical trials in hematology.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that exhibit heterogeneous clinical presentation and morphological findings, which complicates diagnosis, especially in early stages. Recently, refined definitions and standards in the diagnosis and treatment of MDS were proposed, but numerous questions remain. Multiparameter flow cytometry (MFC) is a helpful tool for the diagnostic workup of patients with suspected MDS, and various scores using MFC data have been developed. However, none of these methods have achieved the sensitivity that is required for a reassuring diagnosis in the absence of morphological abnormalities. One reason may be that each score evaluates one or two lineages without offering a broad view of the dysplastic process. The combination of two scores (e.g., Ogata and Red Score) improved the sensitivity from 50–60 to 88%, but the positive (PPV) and negative predictive values (NPV) must be improved. There are prominent differences between study groups when these scores are tested. Further research is needed to maximize the sensitivity of flow cytometric analysis in MDS. This review focuses on the application of flow cytometry for MDS diagnosis and discusses the advantages and limitations of different approaches.