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.

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.

Treatment and outcome of Philadelphia chromosome-positive acute lymphoblastic leukemia in adults after relapse.

INTRODUCTION: Despite the significant progress that has been made over the last years in the front-line treatment of Philadelphia (Ph) chromosome-positive acute lymphoblastic leukemia (ALL), relapses are frequent and their treatment remains a challenge, especially among patients with resistant BCR-ABL1 mutations. AREAS COVERED: This manuscript reviews available data for the treatment of adult patients with relapsed/refractory Ph-positive ALL, with a focus on the role of tyrosine kinase inhibitors (TKIs), monoclonal antibodies, and immunotherapy. EXPERT OPINION: Although a majority of patients with first relapsed Ph-positive ALL respond to subsequent salvage chemotherapy plus TKI combination, their outcomes remain poor. The main predictor of survival is the achievement of major molecular response anytime during the morphological response. More treatment strategies to improve survival are under investigation. Monoclonal antibodies and bispecific antibody constructs hold considerable promise in improving the outcomes of patients with relapsed ALL including Ph-positive ALL.

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.

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.

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.

An miRNA-DNMT1 Axis Is Involved in Azacitidine Resistance and Predicts Survival in Higher-Risk Myelodysplastic Syndrome and Low Blast Count Acute Myeloid Leukemia.

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.

Impact of ATG Dose on the Outcome of Patients Undergoing Reduced Intensity Conditioning Followed by Allogeneic Hematopoietic Stem Cell Transplantation for Hematological Malignancies.

Reduced intensity conditioning for allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often proposed for patients with comorbidities. To enhance engraftment and limit graft-versus-host disease (GVHD), antithymoglobulin (ATG) is usually used. However, the dose needed remains unclear unlike myeloablative conditioning. In order to clarify this point, we conducted a retrospective study on patients who received a reduced intensity conditioning allo-HSCT based on a 2-day fludarabine and busulfan treatment with either 1 or 2 days of ATG treatment. One hundred and eight patients received 2.5 mg/kg (ATG2.5) and another 60 patients 5 mg/kg (ATG5). The median follow-up was 36 months. The median overall survival was 39 months and the median disease-free survival 45 months. In multivariate analysis, overall nonrelapse mortality (NRM) was independently influenced by the acute GVHD grade III-IV (p < 0.001) and ATG dose (30 vs. 21% for ATG5; p = 0.008). Despite heterogeneity of populations, using proportional-hazard assumptions, we have been able to observe in multivariate analysis a lower NRM in the ATG5 group. This leads to a statistically higher overall survival for the ATG5 group. In conclusion, 2 days of ATG decrease NRM independently without increasing the risk of relapse or infectious disease.

Impact of fluconazole versus posaconazole prophylaxis on the incidence of fungal infections in patients receiving induction chemotherapy for acute myeloid leukemia.

BACKGROUND: Invasive fungal infections (IFIs) remain one of the worrying complications in patients with acute myeloid leukemia (AML) due to their incidence and high level of attributable mortality. In light of these risks, antifungal prophylaxis has always been debated. We conducted a single-center retrospective study of two prophylactic antifungal agents (fluconazole/posaconazole) in 91 consecutive patients receiving induction chemotherapy for AML between 2005 and 2009, in order to evaluate the impact on the incidence of IFI and on the mycological flora of the patients. METHODS: In total, 39 patients received prophylactic fluconazole versus 52 who received posaconazole. The baseline characteristics of the two groups were comparable. RESULTS: Overall, 17 patients developed an IFI, with no difference in frequency between the two groups. Utilization of empirical or pre-emptive therapy was similar irrespective of the type of prophylaxis used. Mycological examination of stools revealed an increase in non-albicans Candida colonization in the fluconazole group during hospitalization and the appearance of Saccharomyces cerevisiae colonization in patients receiving posaconazole. CONCLUSION: The present study does not distinguish between fluconazole and posaconazole as a primary effective prevention against fungal infections. More prospective studies and meta-analyses are warranted.

Prognostic value of CXCR4 and FAK expression in acute myelogenous leukemia.

We analysed, by flow cytometry, the "adhesive" phenotype of acute myelogenous leukemia (AML) cells from 36 patients treated in our institution. In univariate analysis, the main prognostic factor for CR achievement was lower CXCR4 expression (p=0.03). Overall survival (OS) was negatively influenced by higher CXC chemokine receptor 4 (CXCR4) (p=0.01), very late antigen-4 (VLA-4) (p=0.01), and focal adhesion kinase (FAK) expression (p=0.04). Combination of these markers allowed to distinguish two prognostic groups: patients overexpressing 2 or 3 factors had a significantly shorter OS (p=0.015). CXCR4, VLA-4 and FAK are new phenotypic markers which could be helpful to establish risk-stratified therapeutic strategies.