Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study.

Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB, n = 41) and bone marrow (BM; n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r2 > 0.95 for all cell types in PB and r2 > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a "sample-in and result-out" approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures.

Flow cytometric analysis of neutrophil myeloperoxidase expression in peripheral blood for ruling out myelodysplastic syndromes: a diagnostic accuracy study.

Suspicion of myelodysplastic syndromes (MDS) is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood (PB) cytopenia of unclear etiology. A PB assay that accurately rules out MDS would have major benefits. The diagnostic accuracy of the intra-individual robust coefficient of variation (RCV) for neutrophil myeloperoxidase (MPO) expression measured by flow cytometric analysis in PB was evaluated in a retrospective derivation study (44 MDS cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected MDS). Compared with controls, MDS cases had higher median RCV values for neutrophil MPO expression (40.2% vs 30.9%; P<0.001). The area under the receiver operating characteristic curve estimates were 0.94 [95% confidence interval (CI): 0.86-0.97] and 0.87 (95%CI: 0.76-0.94) in the derivation and validation studies, respectively. A RCV lower than 30% ruled out MDS with 100% sensitivity (95%CI: 78-100%) and 100% negative predictive value (95%CI: 83-100%) in the prospective validation study. Neutrophil MPO expression measured by flow cytometric analysis in PB might obviate the need for invasive bone marrow aspirate and biopsy for up to 29% of patients with suspected MDS.

Evaluation of bone marrow microenvironment could change how myelodysplastic syndromes are diagnosed and treated.

Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic disorders. However, the therapies used against the hematopoietic stem cells clones have limited efficacy; they slow the evolution toward acute myeloid leukemia rather than stop clonal evolution and eradicate the disease. The progress made in recent years regarding the role of the bone marrow microenvironment in disease evolution may contribute to progress in this area. This review presents the recent updates on the role of the bone marrow microenvironment in myelodysplastic syndromes pathogenesis and tries to find answers regarding how this information could improve myelodysplastic syndromes diagnosis and therapy.

Epigallocatechin-3-O-gallate alleviates the malignant phenotype in A-431 epidermoid and SK-BR-3 breast cancer cell lines.

In this study, we evaluated the effects of epigallocatechin-3-O-gallate (EGCG) in two cancer cell lines, A-431 overexpressing ErbB1 and SK-BR-3, overexpressing ErbB2. EGCG treatment showed dose-dependent collapse of mitochondrial membrane potential (Δψm), increase in reactive oxygen species (ROS) production, changes in nuclear morphology and reduced viability. Flow cytometry data indicated that EGCG partially decreases the phosphorylation of several proteins involved in cell proliferation and survival: pErbB1(Y1173, Y1068), pAkt(S473) and pERK(Y204). EGCG affected the clonogenic growth in both cell lines with an EC50 of 2.5 and 5.4 µM for A-431 and SK-BR-3, respectively. Wound scratch assay demonstrated that EGCG inhibited the healing in dose-dependent manner and the effect was correlated with partial reduction in phosphorylation of pFAK(S910). Our data suggest that EGCG administration might reduce the unfavourable traits, particularly associated with ErbB1/EGFR overexpression.

Expression of embryonic stem cell markers in acute myeloid leukemia.

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.

High-Dimensional Mass Cytometry Analysis of Embryonic Antigens and Their Signaling Pathways in Myeloid Cells from Bone Marrow Aspirates in AML Patients at Diagnosis.

BACKGROUND: Embryonic antigens (EA) regulate pluripotency, self-renewal, and differentiation in embryonic stem (ES) cells during their development. In adult somatic cells, EA expression is normally inhibited; however, EAs can be re-expressed by cancer cells and are involved in the deregulation of different signaling pathways (SPs). In the context of AML, data concerning the expression of EAs are scarce and contradictory. METHODS: We used mass cytometry to explore the expression of EAs and three SPs in myeloid cells from AML patients and normal bone marrow (NBM). Imaging flow cytometry was used for morphological assessment of cells in association with their OCT3/4 expression status (positive vs. negative). RESULTS: An overall reduction in or absence of EA expression was observed in immature myeloid cells from AML patients compared to their normal counterparts. Stage-specific embryonic antigen-3 (SSEA-3) was consistently expressed at low levels in immature myeloid cells, whereas SSEA-1 was overexpressed in hematopoietic stem cells (HSCs) and myeloblasts from AML with monocytic differentiation (AML M4/M5). Therefore, these markers are valuable for distinguishing between normal and abnormal myeloid cells. These preliminary results show that the exploration of myeloid cell intracellular SPs in the setting of AML is very informative. Deregulation of three important leukemogenic SPs was also observed in myeloid cells from AML. CONCLUSIONS: Exploring EAs and SPs in myeloid cells from AML patients by mass cytometry may help identify characteristic phenotypes and facilitate AML follow-up.

Combined flow cytometry natural killer immunophenotyping and KIR/HLA-C genotyping reveal remarkable differences in acute myeloid leukemia patients, but suggest an overall impairment of the natural killer response.

Introduction: Natural killer (NK) cells are key anti-tumor effectors of the innate immunity. Phenotypic differences allow us to discriminate in between three functional stages of maturation, named immature, mature and hypermature that are distinctive in terms of receptor expression, cytokine secretion, cytotoxic properties and organ trafficking. NKs display an impressive repertoire of highly polymorphic germline encoded receptors that can be either activating, triggering the effector's function, or inhibitory, limiting the immune response. In our study, we have investigated peripheral blood NK cells of acute myeloid leukemia (AML) patients. Methods: The Killer Immunoglobulin-like receptors (KIRs) and the HLA-C genotypes were assessed, as HLA-C molecules are cognate antigens for inhibitory KIRs. Results: The AA mainly inhibitory KIR haplotype was found in a higher proportion in AML, while a striking low frequency of the 2DS3 characterized the mainly activating Bx haplotype. Flow cytometry immunophenotyping evidenced a lower overall count of NK cells in AML versus healthy controls, with lower percentages of the immature and mature subpopulations, but with a markedly increase of the hypermature NKs. The analysis of the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A inhibitory receptors surface expression revealed a remarkable heterogeneity. However, an overall trend for a higher expression in AML patients could be noticed in all maturation subpopulations. Some of the AML patients with complex karyotypes or displaying a FLT3 gene mutation proved to be extreme outliers in terms of NK cells percentages or inhibitory receptors expression. Discussion: We conclude that while the genetic background investigation in AML offers important pieces of information regarding susceptibility to disease or prognosis, it is flow cytometry that is able to offer details of finesse in terms of NK numbers and phenotypes, necessary for an adequate individual evaluation of these patients.

Flow Cytometry of CD5-Positive Hairy Cell Leukemia.

BACKGROUND AND OBJECTIVE: Hairy cell leukemia (HCL) is a chronic lymphoproliferative disorder for which diagnosis is typically straightforward, based on bone marrow morphology and flow cytometry (FC) or immunohistochemistry. Nevertheless, variants present atypical expressions of cell surface markers, as is the case of CD5, for which the differential diagnosis can be more difficult. The aim of the current paper was to describe diagnosis of HCL with atypical CD5 expression, with an emphasis on FC. METHODS: The detailed diagnostic methodology for HCL with atypical CD5 expression is presented, including differential diagnosis from other lymphoproliferative diseases with similar pathologic features, by FC analysis of the bone marrow aspirate. RESULTS: Diagnosis of HCL by means of FC started by gating all events based on side scatter (SSC) versus CD45 and B lymphocytes were selected from the lymphocytes gate as CD45/CD19 positive. The gated cells were positive for CD25, CD11c, CD20, and CD103, while CD10 proved to be dim to negative. Moreover, cells positive for CD3, CD4, and CD8, the three pan-T markers, as well as CD19, showed a bright expression of CD5. The atypical CD5 expression is usually correlated with a negative prognosis and thus chemotherapy with cladribine should be initiated. CONCLUSION: HCL is an indolent chronic lymphoproliferative disorder and diagnosis is usually straightforward. However, atypical expression of CD5 renders its differential diagnosis more difficult, but FC is a useful tool that allows an optimal classification of the disease and allows initiation of timely satisfactory therapy.

Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells.

Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y(397)], and HSP90α/ß and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y(397)], and HSP90α/ß formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y(397)], and HSP90α/ß and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90α/ß client protein, these results suggest the utility of HSP90α/ß inhibition as a target for adjuvant therapy for myelodysplasia.

Quality Assessment of a Large Multi-Center Flow Cytometric Dataset of Acute Myeloid Leukemia Patients-A EuroFlow Study.

Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a dataset and developed a QA methodology of the EuroFlow Acute Myeloid Leukemia (AML) database, based on the eight-color EuroFlow AML panel consisting of six different antibody combinations, including four backbone markers. In total, 1142 AML cases and 42 normal bone marrow samples were included in this analysis. QA was performed on 803 AML cases using multidimensional analysis of backbone markers, as well as tube-specific markers, and data were compared using classical analysis employing median and peak expression values. Validation of the QA procedure was performed by re-analysis of >300 cases and by running an independent cohort of 339 AML cases. Initial evaluation of the final cohort confirmed specific immunophenotypic patterns in AML subgroups; the dataset therefore can reliably be used for more detailed exploration of the immunophenotypic variability of AML. Our data show the potential pitfalls and provide possible solutions for constructing large flowcytometric databases. In addition, the provided approach may facilitate the building of other databases and thereby support the development of novel tools for (semi)automated QA and subsequent data analysis.

Advanced Flow Cytometry Analysis Algorithms for Optimizing the Detection of "Different From Normal" Immunophenotypes in Acute Myeloid Blasts.

Acute myeloid leukemias (AMLs) are a group of hematologic malignancies that are heterogeneous in their molecular and immunophenotypic profiles. Identification of the immunophenotypic differences between AML blasts and normal myeloid hematopoietic precursors (myHPCs) is a prerequisite to achieving better performance in AML measurable residual disease follow-ups. In the present study, we applied high-dimensional analysis algorithms provided by the Infinicyt 2.0 and Cytobank software to evaluate the efficacy of antibody combinations of the EuroFlow AML/myelodysplastic syndrome panel to distinguish AML blasts with recurrent genetic abnormalities (n = 39 AML samples) from normal CD45low CD117+ myHPCs (n = 23 normal bone marrow samples). Two types of scores were established to evaluate the abilities of the various methods to identify the most useful parameters/markers for distinguishing between AML blasts and normal myHPCs, as well as to distinguish between different AML groups. The Infinicyt Compass database-guided analysis was found to be a more user-friendly tool than other analysis methods implemented in the Cytobank software. According to the developed scoring systems, the principal component analysis based algorithms resulted in better discrimination between AML blasts and myHPCs, as well as between blasts from different AML groups. The most informative markers for the discrimination between myHPCs and AML blasts were CD34, CD36, human leukocyte antigen-DR (HLA-DR), CD13, CD105, CD71, and SSC, which were highly rated by all evaluated analysis algorithms. The HLA-DR, CD34, CD13, CD64, CD33, CD117, CD71, CD36, CD11b, SSC, and FSC were found to be useful for the distinction between blasts from different AML groups associated with recurrent genetic abnormalities. This study identified both benefits and the drawbacks of integrating multiple high-dimensional algorithms to gain complementary insights into the flow-cytometry data.

Database-Guided Analysis for Immunophenotypic Diagnosis and Follow-Up of Acute Myeloid Leukemia With Recurrent Genetic Abnormalities.

Acute myeloid leukemias (AMLs) are hematologic malignancies with varied molecular and immunophenotypic profiles, making them difficult to diagnose and classify. High-dimensional analysis algorithms might increase the utility of multicolor flow cytometry for AML diagnosis and follow-up. The objective of the present study was to assess whether a Compass database-guided analysis can be used to achieve rapid and accurate diagnoses. We conducted this study to determine whether this method could be employed to pilote the genetic and molecular tests and to objectively identify different-from-normal (DfN) patterns to improve measurable residual disease follow-up in AML. Three Compass databases were built using Infinicyt 2.0 software, including normal myeloid-committed hematopoietic precursors (n = 20) and AML blasts harboring the most frequent recurrent genetic abnormalities (n = 50). The diagnostic accuracy of the Compass database-guided analysis was evaluated in a prospective validation study (125 suspected AML patients). This method excluded AML associated with the following genetic abnormalities: t(8;21), t(15;17), inv(16), and KMT2A translocation, with 92% sensitivity [95% confidence interval (CI): 78.6%-98.3%] and a 98.5% negative predictive value (95% CI: 90.6%-99.8%). Our data showed that the Compass database-guided analysis could identify phenotypic differences between AML groups, representing a useful tool for the identification of DfN patterns.

Natural Killer Cell Subpopulations and Inhibitory Receptor Dynamics in Myelodysplastic Syndromes and Acute Myeloid Leukemia.

Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.

FAK Deficiency in Bone Marrow Stromal Cells Alters Their Homeostasis and Drives Abnormal Proliferation and Differentiation of Haematopoietic Stem Cells.

Emerging evidence indicates that in myelodysplastic syndromes (MDS), the bone marrow (BM) microenvironment may also contribute to the ineffective, malignant haematopoiesis in addition to the intrinsic abnormalities of haematopoietic stem precursor cells (HSPCs). The BM microenvironment influences malignant haematopoiesis through indirect mechanisms, but the processes by which the BM microenvironment directly contributes to MDS initiation and progression have not yet been elucidated. Our previous data showed that BM-derived stromal cells (BMSCs) from MDS patients have an abnormal expression of focal adhesion kinase (FAK). In this study, we characterise the morpho-phenotypic features and the functional alterations of BMSCs from MDS patients and in FAK knock-downed HS-5 cells. The decreased expression of FAK or its phosphorylated form in BMSCs from low-risk (LR) MDS directly correlates with BMSCs' functional deficiency and is associated with a reduced level of haemoglobin. The downregulation of FAK in HS-5 cells alters their morphology, proliferation, and differentiation capabilities and impairs the expression of several adhesion molecules. In addition, we examine the CD34+ healthy donor (HD)-derived HSPCs' properties when co-cultured with FAK-deficient BMSCs. Both abnormal proliferation and the impaired erythroid differentiation capacity of HD-HSPCs were observed. Together, these results demonstrate that stromal adhesion mechanisms mediated by FAK are crucial for regulating HSPCs' homeostasis.

Parathyroid Hormone Remodels Bone Transitional Vessels and the Leptin Receptor-Positive Pericyte Network in Mice.

Intermittent parathyroid hormone (iPTH) is anti-osteoporotic and affects bone vessels. Transitional capillaries close to the bone surface, which express both endomucin (Edm) and CD31, bear leptin receptor-expressing (LepR) perivascular cells that may differentiate into osteoblasts. Increased numbers of type H endothelial cells (THEC; ie, Edmhi /CD31hi cells assessed by flow cytometry, FACS) are associated with higher bone formation in young mice. We hypothesized that iPTH administration impacts transitional vessels by expanding THECs. Four-month-old C57/Bl6J female mice were injected with PTH 1-84 (100 µg/kg/d) or saline (CT) for 7 or 14 days. We quantified LepR+ , CD31+ , Edm+ cells and THECs by FACS in hindlimb bone marrow, and Edm/LepR double immunolabelings on tibia cryosections. Additionally, we analyzed bone mRNA expression of 87 angiogenesis-related genes in mice treated with either intermittent or continuous PTH (iPTH/cPTH) or saline (CT) for 7, 14, and 28 days. iPTH dramatically decreased the percentage of THECs by 78% and 90% at days 7 and 14, respectively, and of LepR+ cells at day 14 (-46%) versus CT. Immunolabeling quantification showed that the intracortical Edm+ -vessel density increased at day 14 under iPTH. In the bone marrow, perivascular LepR+ cells, connected to each other via a dendrite network, were sparser under iPTH at day 14 (-58%) versus CT. iPTH decreased LepR+ cell coverage of transitional vessels only (-51%), whereas the number of LepR+ cells not attached to vessels increased in the endocortical area only (+ 49%). Transcriptomic analyses showed that iPTH consistently upregulated PEDF, Collagen-18α1, and TIMP-1 mRNA expression compared with CT and cPTH. Finally, iPTH increased immunolabeling of endostatin, a Collagen-18 domain that can be cleaved and become antiangiogenic, in both endocortical (79%) and peritrabecular transitional microvessels at day 14. Our results show that iPTH specifically remodels transitional vessels and suggest that it promotes LepR+ cell mobilization from these vessels close to the bone surface. © 2019 American Society for Bone and Mineral Research.

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

Evaluation by Flow Cytometry of Mature Monocyte Subpopulations for the Diagnosis and Follow-Up of Chronic Myelomonocytic Leukemia.

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.

Impaired Expression of Focal Adhesion Kinase in Mesenchymal Stromal Cells from Low-Risk Myelodysplastic Syndrome Patients.

The pathogenic role of mesenchymal stromal cells (MSCs) in myelodysplastic syndromes (MDS) development and progression has been investigated by numerous studies, yet, it remains controversial in some aspects (1, 2). In the present study, we found distinct features of MSCs from low-risk (LR)-MDS stromal microenvironment as compared to those from healthy subjects. At the molecular level, focal adhesion kinase, a key tyrosine kinase in control of cell proliferation, survival, and adhesion process, was found profoundly suppressed in expression and activation in LR-MDS MSC. At a functional level, LR-MDS MSCs showed impaired growth and clonogenic capacity, which were independent of cellular senescence and apoptosis. The pro-adipogenic differentiation and attenuated osteogenic capacity along with reduced SDF-1 expression could be involved in creating an unfavorable microenvironment for hematopoiesis. In conclusion, our experiments support the theory that the stromal microenvironment is fundamentally altered in LR-MDS, and these preliminary data offer a new perspective on LR-MDS pathophysiology.

Potential Role of OCT4 in Leukemogenesis.

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.

Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes.

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.