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In previously untreated, medically fit patients with chronic lymphocytic leukemia, research is focused on developing fixed-duration strategies to improve long-term outcomes whilst sparing patients from serious toxicities. The ICLL-07 trial evaluated a fixed-duration (15-month) immunochemotherapy approach where, following obinutuzumab-ibrutinib induction for 9 months, patients (n=10) in complete remission (CR) with bone marrow (BM) measurable residual disease (MRD)

DNMT1 protein expression in SKM1 AZA-sensitive (-S) versus AZA-resistant clones (-R). The Figure corresponds to the Figure 1B with signal quantification performed with ImageJ software. The data are expressed as mean {plus minus} SD of three independent experiments, with each performed in triplicate (Mann-Whitney test, p

Pathway enrichment analysis. For ontology analysis, gene lists were analyzed using DAVID software (KEGG pathways). The complete set of genes featured in the microarrays was used as the reference background. The three numbers on the right represent the number of deregulated mRNAs, the overall number of genes within the pathway and the p value, respectively. Data are presented for genes targeted by at least one of the 7 miRNAs and that were found repressed in SKM1 AZA resistant cells.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro. We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3′ UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance.Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025–34. ©2016 AACR.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)–like, acute lymphoid leukemia (ALL)–like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])–like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.

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.

We report a 38-year-old woman who presented with a subdural hematoma after minor facial trauma in a stressful situation. The laboratory data showed a subnormal platelet count (166×10 9 /L), VWF:RCo activity was 45% and VWF:Ag was 53% with a VWF:RCo/VWF Ag ratio of 0.79. Hemostasis results and gene analysis revealed von Willebrand disease (VWD) type 2B with normal multimers and a novel mutation c.4136 G>T (R1379L), which appears to be a novel mutation of VWD type 2B that is mainly diagnosed with hypersensitivity to ristocetin and an hyperfixation of platelet Willebrand to a recombinant Gp1b. SIMILAR CASES PUBLISHED: None.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR-ABL1 transcript as a result of reciprocal translocation between chromosome 9 and 22. The most common transcripts subtypes are e13a2 (b2a2) and e14a2 (b3a2). The prognostic impact of the type of BCR-ABL1 transcript has been the subject of controversies over time. In the imatinib era, several studies have suggested a deeper and faster response in patients expressing e14a2. However, the impact on response after first line therapy with a second-generation tyrosine kinase inhibitor, nilotinib, is unknown. We retrospectively evaluated 118 patients newly diagnosed with chronic phase CML and treated frontline with nilotinib inside or outside clinical trial in five French centers. Only patients expressing e14a2 or e13a2 transcripts alone were analyzed. At baseline, 55.3% expressed e14a2, 44.7% expressed e13a2. The median age was 51 years and median follow-up was 49 months. Relative risks of CML at diagnosis were similar according to the ELTS score (p = .87). Complete hematological response and complete cytogenetic response rates were similar among groups. Patients expressing e14a2 transcripts compared to e13a2 transcripts had deeper and faster molecular responses, when considering MMR (100% vs 84.1%, p = .007) with a median time of 6.7 and 17.1 months or MR4.5 (100% vs 59.9%, p = .005) with a median time of 39.7 and 70.9 months, respectively. A sustained treatment free remission was observed in 10/10 patients with e14a2 versus 1/3 with e13a2 transcript (p = .04). In conclusion, even treated with nilotinib first line, patients with chronic phase CML expressing BCR-ABL1 e13a2 transcript have a lower rate of deep molecular responses.

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&rsquo, 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&rsquo, 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&rsquo, homeostasis.

Background Studies of normal bone marrow (BM) cell composition by flow cytometry are scarce. Presently, we aimed to quantify 14 cell subsets from infants to elderly patients. Methods Cell subsets in BM samples from 180 individuals without morphologically abnormal leukocytes were analyzed using a single combination of eight antibodies: CD3/CD10/CD38/CD19/CD36/CD16/CD34/CD45. Results By comparison with the Holdrinet score, we first validated the immature granulocyte/neutrophil (IGRA/N) ratio as a readily obtainable criterion of BM sample purity in 145 cases. Then, the 115 highly pure samples were selected (IGRA/N ≥ 1.2) and analyzed according to age group. CD34+ myeloblasts became progressively more infrequent with age: median 1.4% in infancy to 0.5% in the elderly. Neutrophils increased: 10.7% to 22.8%; all other myeloid subsets, IGRA, eosinophils, basophils and monocytes remained stable: respectively 40.3% to 46.7%, 2.0% to 2.8%, 0.2% to 0.3%, and 4.4% to 5.0% throughout life. Erythroblasts were lower in children (8.4% to 10.3%) than in adults (12.5% to 15.1%). For lymphoid cells, hematogones and transitional B-cells decreased: 15.5% to 0.6% and 3.6% to 0.1%, respectively; mature lymphocytes remained stable: B-cells: 1.4% to 2.8%, T-cells: 5.8% to 8.7%, and NK-cells: 0.7% to 1.4%. Plasma cells varied slightly: 0.1% to 0.5%. Differences of about 40% were seen in moderately pure (IGRA/N: 0.5 to 1.2) BM samples. Conclusion We thus provide the first values for 14 myeloid and lymphoid subsets characterizing BM cell composition in 5 age ranges. They should provide important information when screening patients for hematological disorders or abnormal bone marrow development. © 2018 International Clinical Cytometry Society.

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.

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.

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p

International audience; Suspicion of myelodysplastic syndromes is one of the commonest reasons for bone marrow aspirate in elderly patients presenting with persistent peripheral blood cytopenia of unclear etiology. A peripheral blood assay that accurately rules out myelodysplastic syndromes would have major benefits. The diagnostic accuracy of the intraindividual robust coefficient of variation for neutrophil myeloperoxidase expression measured by flow cytometric analysis in peripheral blood was evaluated in a retrospective derivation study (44 myelodysplastic syndrome cases and 44 controls) and a prospective validation study (68 consecutive patients with suspected myelodysplastic syndromes). Compared with controls, myelodysplastic syndrome cases had higher median robust coefficient of variation values for neutrophil myeloperoxidase expression (40.2% versus 30.9%, P

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR-ABL1 transcript as a result of reciprocal translocation between chromosome 9 and 22. The most common transcripts subtypes are e13a2 (b2a2) and e14a2 (b3a2). The prognostic impact of the type of BCR-ABL1 transcript has been the subject of controversies over time. In the imatinib era, several studies have suggested a deeper and faster response in patients expressing e14a2. However, the impact on response after first line therapy with a second-generation tyrosine kinase inhibitor, nilotinib, is unknown. We retrospectively evaluated 118 patients newly diagnosed with chronic phase CML and treated frontline with nilotinib inside or outside clinical trial in five French centers. Only patients expressing e14a2 or e13a2 transcripts alone were analyzed. At baseline, 55.3% expressed e14a2, 44.7% expressed e13a2. The median age was 51 years and median follow-up was 49 months. Relative risks of CML at diagnosis were similar according to the ELTS score (p = .87). Complete hematological response and complete cytogenetic response rates were similar among groups. Patients expressing e14a2 transcripts compared to e13a2 transcripts had deeper and faster molecular responses, when considering MMR (100% vs 84.1%, p = .007) with a median time of 6.7 and 17.1 months or MR4.5 (100% vs 59.9%, p = .005) with a median time of 39.7 and 70.9 months, respectively. A sustained treatment free remission was observed in 10/10 patients with e14a2 versus 1/3 with e13a2 transcript (p = .04). In conclusion, even treated with nilotinib first line, patients with chronic phase CML expressing BCR-ABL1 e13a2 transcript have a lower rate of deep molecular responses.

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

Background Bone marrow analysis by flow cytometry is part of the routine diagnosis of hematological disorders in medical laboratories. Differential leukocyte count and identification of abnormal cell subsets is currently performed through morphological examination on bone marrow smears by skilled cytologists. In this work, we propose a single 8-color tube for providing equivalent information, using flow cytometry. Methods 99 bone marrow samples were classified into 2 groups, (i) 51 samples, obtained from either healthy donors (n = 4) or patients with various diseases at diagnosis or during remission that did not present a hematological malignancy (n = 47), and (ii) 48 pathological samples with quantitative and/or qualitative abnormalities. A panel of eight antibodies-CD3-FITC/CD10-PE/CD38-PerCP-Cy5.5/CD19-PECy7/CD36-APC/CD16-APC-H7/CD34-BV421/CD45-V500-was tested to identify the main cell subsets at different stages of maturation using a FACSCanto-II analyzer. Results We first proposed a strategy of sequential gating leading to the identification of 14 leukocyte subsets, that is, erythroblasts, monocytes, B-lymphoid cells from hematogones to plasma-cells (5 subsets), T- and NK-cells, polymorphonuclear cells (neutrophils, eosinophils, and basophils), myeloblasts and other immature granular cells. This approach was validated by comparing flow cytometry and microscopic morphological examination, both in cases of normal and abnormal samples. Interestingly, cell identification, and numeration by flow cytometry was easy to perform and highly reproducible. Conclusion A very simple, rapid, and reproducible flow cytometric approach, using a combination of eight antibodies allows determination of the cellular composition of bone marrow with high precision. © 2016 International Clinical Cytometry Society.

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.

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.

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.

IF 3.118; International audience; The assessment of minimal residual disease (MRD) in acute myeloblastic leukemia is of growing interest as a prognostic marker of patients' outcome. Multiparameter flow cytometry (MFC), tracking leukemia-associated immunophenotypic patterns, has been shown in several studies to be a useful tool to investigate MRD. Here, we report a multicenter prospective study which allowed to define a harmonized analysis strategy, as well as the efficacy of MFC MRD to predict outcome. This study included 276 patients, in 10 different MFC centers, of whom 268 had at least 1 MRD check point. The combination of a CD45, CD34, and CD33 backbone, with the addition of CD117, CD13, CD7, and CD15 in 2 five-color tubes allowed to define each patient's multiparameter immunophenotypic characteristics at diagnosis, according to a Boolean combination of gates. The same individual diagnosis gating strategy was then applied at each MRD time point for each patient. MRD levels were stratified according to log by log thresholds, from 5 × 10-2 (the classical morphological threshold to define remission) down to

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.

// Aminetou Mint Mohamed 1 , Marie Balsat 1 , Morgan Thenoz 1 , Catherine Koering 1 , Lea Payen-Gay 2 , Meyling Cheok 3 , Hussein Mortada 4 , Didier Auboeuf 4 , Christiane Pinatel 5 , Mohamed El-Hamri 6 , Charles Dumontet 7 , Emeline Cros 7 , Pascale Flandrin-Gresta 1, 8 , Olivier Nibourel 4 , Claude Preudhomme 4 , Mauricette Michallet 1, 6 , Xavier Thomas 6 , Franck Nicolini 6 , Francoise Solly 1, 8 , Denis Guyotat 1, 9 , Lydia Campos 1, 8 , Eric Wattel 1, 6, * , Franck Mortreux 1, * 1 Universite Lyon 1, CNRS UMR5239, Oncovirologie et Biotherapies, Faculte de Medecine Lyon Sud, ENS – HCL, Pierre Benite, France 2 INSERM, UMR-S1052, Centre de Recherche en Cancerologie de Lyon, Lyon, France 3 Jean-Pierre Aubert Center, INSERM U837, Facteurs de persistance des cellules leucemiques, Institute for Cancer Research in Lille, Lille cedex, France 4 Centre de Recherche sur le Cancer de Lyon, Inserm, Epissage alternatif et progression tumorale, Lyon, France 5 Centre de Recherche sur le Cancer de Lyon, Inserm, Echappement aux systemes de sauvegarde et plasticite cellulaire, Lyon, France 6 Universite Lyon I, Service d’Hematologie, Pavillon Marcel Berard, Centre Hospitalier Lyon-Sud, Pierre Benite, France 7 Centre de Recherche sur le Cancer de Lyon, Inserm, Anticorps anticancer, Lyon, France 8 Universite de Saint Etienne, Laboratoire d’Hematologie, CHU de Saint-Etienne, Saint-Etienne, France 9 Institut de Cancerologie de la Loire, CHU de Saint-Etienne, Saint Priest en Jarez, France * These authors have contributed equally to this work Correspondence to: Eric Wattel, e-mail: eric.wattel@chu-lyon.fr Franck Mortreux, e-mail: franck.mortreux@ens-lyon.fr Keywords: acute myeloid leukemia, alternative splicing, WT1, DEK, multidrug resistance Received: March 16, 2015 Accepted: April 28, 2015 Published: May 12, 2015 ABSTRACT In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro , 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

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.

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.

CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. Recent flow cytometry analyses reported that CXCR4 expression levels have a major prognostic impact in acute myeloid leukaemia (AML). CXCR4 expression is associated with poor prognosis and can be useful to stratify patients, according to their phenotype, in order to establish risk-adapted strategies. Newly diagnosed AML are now routinely stratified according to molecular markers which guide prognosis and treatment. Many leukaemia are composed of multiples subclones with differential susceptibility to treatment and specific targeted therapies are missing. Despite therapeutic improvements for the treatment of AML, long term survival remains poor. Targeting CXCR4 is a novel promising approach of therapy. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies. This review summarises our current knowledge regarding the key role of CXCR4 in AML and discusses how targeting this pathway could provide an interesting approach to eradicate the LSC.

Flow cytometry (FC) instruments settings classically rely on local establishment of photomultipliers (PMT) voltages adapted to the measurements expected to be performed. In the era of multiparameter FC (MFC), it appears more and more desirable that comparable patterns of fluorescence are obtained in different settings. This relies on a harmonization of settings between instruments. Although this has been shown to be feasible within a given brand of flow cytometers, little information is available about broader comparisons in a given center or in a multicenter fashion. Here, we report a two-phase series of experiments first performed between a Canto II (BD Biosciences) and a Navios (Beckman Coulter) instruments in the same center. PMT values adjusted on the reference instrument (RI) Canto II were used to establish target values for PMT settings on the paired Navios practice instrument (PI). This allowed to show the good correlation of all but peaks 1 and 2 of Rainbow(®) beads between RI and PI. Using 4- or 8-color stained leukocytes, the similitude of the settings was further confirmed. A complex set of matrices was then established between five centers all equipped with both instruments. Using Bland & Altman difference comparisons for median fluorescence values, it was shown that using either Rainbow beads or CD16 stained polymorphonuclears to set-up target values on the RI CantoII, highly superimposable results could be obtained on all 9 PI. The latter were obtained using Rainbow beads or Compbeads(®) for comparisons. In summary, this two-phase study demonstrates the feasibility of different methods allowing for a robust harmonization of settings for MFC.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML). Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro. We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML. Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3′ UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance. Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025–34. ©2016 AACR.

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.

The Innovance VWF:Ac test (Siemens) has the particularity to assess the binding capacity of von Willebrand factor (VWF) to recombinant platelet GPIb mutated in the absence of ristocetin. Our study aimed to evaluate and validate according to standard NF EN ISO 15189 the original protocol adaptation on STA-R Evolution series analyser (Diagnostica Stago). We evaluated the performance in terms of imprecision and we validate additional parameters necessary in range B as recommended by the SH GTA 04 (Cofrac). We compared the new assay with the reference assay: ristocetin cofactor activity (VWF:RCo) performed on the BCS-XP analyser by testing retrospectively samples from 82 healthy normal subjects and 61 patients with von Willebrand disease (VWD). This new assay is consistent with objectives set in terms of imprecision with CV around 4%. Excepted limit of quantification higher, additional parameters evaluated in range B have been validated. The Innovance VWF: Ac assay allowed the detection of all deficits of VWF already detected by the VWF:RCo test on the BCS-XP. This adjustment on STA-R analyser therefore has satisfactory analytical performance criteria. Apart from the limit of quantification, this reagent can be used according to the recommendations specified in the original protocol adaptation. Its performance and compatibility with the spot measurement allow the diagnosis and therapeutic monitoring of VWD according to current requirements and guidelines.

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2S