Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia.

Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A assay, and immunophenotypic, cytogenetic, molecular, and targeted next-generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novo-type AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-free survival (P=0.0370). JC-1 +/- cyclosporine A assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.

CD28/4-1BB CD123 CAR T cells in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is associated with a remarkably poor prognosis and with no treatment consensus. The identification of relevant therapeutic targets is challenging. Here, we investigated the immune functions, antileukemia efficacy and safety of CD28/4-1BB CAR T cells targeting CD123 the interleukin (IL)-3 receptor alpha chain which is overexpressed on BPDCN. We demonstrated that both retroviral and lentiviral engineering CD28/4-1BB CD123 CAR T cells exhibit effector functions against BPDCN cells through CD123 antigen recognition and that they efficiently kill BPDCN cell lines and BPDCN-derived PDX cells. In vivo, CD28/4-1BB CD123 CAR T-cell therapy displayed strong efficacy by promoting a decrease of BPDCN blast burden. Furthermore we showed that T cells from BPDCN patient transduced with CD28/4-1BB CD123 CAR successfully eliminate autologous BPDCN blasts in vitro. Finally, we demonstrated in humanized mouse models that these effector CAR T cells exert low or no cytotoxicity against various subsets of normal cells with low CD123 expression, indicating a potentially low on-target/off-tumor toxicity effect. Collectively, our data support the further evaluation for clinical assessment of CD28/4-1BB CD123 CAR T cells in BPDCN neoplasm.

Proportion of Cytotoxic Peripheral Blood Natural Killer Cells and T-Cell Large Granular Lymphocytes in Recurrent Miscarriage and Repeated Implantation Failure: Case-Control Study and Meta-analysis.

We aimed to compare the proportion of peripheral blood natural killer (NK) cells (CD3-CD56+) and T-cell large granular lymphocytes (CD8+CD57+) during preconception in a homogenous group of women with unexplained well-defined recurrent miscarriage (RM) and repeated implantation failure (RIF) vs healthy controls in relation to pregnancy outcomes. This case-control study followed by a literature review and meta-analysis was conducted in three university hospitals. Patients and controls were consecutively recruited from December 2015 to October 2017. In total, 115 women were included in the study: 54 with RM, 41 with RIF and 20 healthy controls with ≥ 2 term births. Percentages of CD3-CD56+ and CD8+CD57+ cells and sub-populations of CD3-CD56+ cells did not differ between cases and controls. The results for women with subsequent miscarriage did not differ from those with live births. The meta-analysis of the literature showed higher NK-cell proportions in RM [mean difference 3.47 (95% CI 2.94-4.00); p < 0.001] and RIF [mean difference 1.64 (95% CI 0.82-2.45); p < 0.001] than controls. However, the heterogeneity between the different studies was high. The proportion of peripheral blood CD3-CD56+ and CD8+CD57+ cells in the preconception period does not reflect the risk of implantation failure or miscarriage and should not be recommended indicators for the management of RM and RIF. Further prospective large studies are needed to develop a reliable peripheral blood marker of immune deregulation.

T-cell prolymphocytic leukemia and tuberculosis: a puzzling association.

T-cell prolymphocytic leukemia can result in severe immune T-cell deficiency. Clinicians should be aware of this complication in this rare lymphoid malignancy, and opportunistic infections should be ruled out before the use of usual immunosuppressive procedures such as alemtuzumab and hematopoietic stem cell transplantation.

Efficacy of a rituximab regimen based on B cell depletion in thrombotic thrombocytopenic purpura with suboptimal response to standard treatment: Results of a phase II, multicenter noncomparative study.

The standard four-rituximab infusions treatment in acquired thrombotic thrombocytopenic purpura (TTP) remains empirical. Peripheral B cell depletion is correlated with the decrease in serum concentrations of anti-ADAMTS13 and associated with clinical response. To assess the efficacy of a rituximab regimen based on B cell depletion, 24 TTP patients were enrolled in this prospective multicentre single arm phase II study and then compared to patients from a previous study. Patients with a suboptimal response to a plasma exchange-based regimen received two infusions of rituximab 375 mg m-2 within 4 days, and a third dose at day +15 of the first infusion if peripheral B cells were still detectable. Primary endpoint was the assessment of the time required to platelet count recovery from the first plasma exchange. Three patients died after the first rituximab administration. In the remaining patients, the B cell-driven treatment hastened remission and ADAMTS13 activity recovery as a result of rapid anti-ADAMTS13 depletion in a similar manner to the standard four-rituximab infusions schedule. The 1-year relapse-free survival was also comparable between both groups. A rituximab regimen based on B cell depletion is feasible and provides comparable results than with the four-rituximab infusions schedule. This regimen could represent a new standard in TTP. This trial was registered at www.clinicaltrials.gov (NCT00907751). Am. J. Hematol. 91:1246-1251, 2016. © 2016 Wiley Periodicals, Inc.

Genetic hierarchy and temporal variegation in the clonal history of acute myeloid leukaemia.

In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner.

The spectrum of chronic CD8+ T-cell expansions: clinical features in 14 patients.

Chronic CD8(+) T-cell expansions can result in parotid gland swelling and other organ infiltration in HIV-infected patients, or in persistent cytopenias. We report 14 patients with a CD8+ T-cell expansion to better characterize the clinical spectrum of this ill-defined entity. Patients (9 women/5 men) were 65 year-old (range, 25-74). Six patients had ≥ 1 symptomatic organ infiltration, and 9 had ≥ 1 cytopenia with a CD8(+) (>50% of total lymphocyte count) and/or a CD8(+)/CD57(+) (>30% of total lymphocyte count) T-cell expansion for at least 3 months. One patient had both manifestations. A STAT3 mutation, consistent with the diagnosis of large granular lymphocyte leukemia, was found in 2 patients with cytopenia. Organ infiltration involved lymph nodes, the liver, the colon, the kidneys, the skin and the central nervous system. Three patients had a HIV infection for 8 years (range, 0.5-20 years). Two non-HIV patients with hypogammaglobulinemia had been treated with a B-cell depleting monoclonal antibody (rituximab) for a lymphoma. One patient had a myelodysplastic syndrome with colon infiltration and agranulocytosis. The outcome was favorable with efficient antiretroviral therapy and steroids in HIV-infected patients and intravenous immunoglobulins in 2/3 non-HIV patients. Six patients had an agranulocytosis of favorable outcome with granulocyte-colony stimulating factor only (3 cases), cyclophosphamide, methotrexate and cyclosporine A, or no treatment (1 case each). Three patients had a pure red cell aplasia, of favorable outcome in 2 cases with methotrexate and cyclosporine A; one patient was unresponsive. Chronic CD8(+) T-cell expansions with organ infiltration in immunocompromised patients may involve other organs than parotid glands; they are non clonal and of favorable outcome after correction of the immune deficiency and/or steroids. In patients with bone marrow infiltration and unexplained cytopenia, CD8(+) T-cell expansions can be clonal or not; their identification suggests that cytopenias are immune-mediated. Our results extend the clinical spectrum of chronic CD8(+) T-cell expansions.

[Polyclonal CD8+/CD57+ T cell expansions: clinical significance]. / Signification clinique des expansions polyclonales lymphocytaires T CD8+/CD57+.

Polyclonal CD8(+)/CD57(+)T cell lymphocytosis can be observed in various conditions such as chronic viral infections, autoimmune cytopenias, connective tissue diseases, chronic graft-versus-host disease and primary or secondary immune deficiencies. This population results from the chronic stimulation of CD8(+)/CD28(+)/CD57(-)lymphocytes by exogenous (mostly infection-related), autologous or allogeneic antigens. Paralleling chronic antigen stimulation, these CD8(+) T cells acquire a poor capacity to proliferate in standard conditions in relation with the loss of CD28, whereas CD57 antigen becomes expressed at their surface. CD8(+)/CD57(+)T cells represent activated cytotoxic T lymphocytes at a terminal stage of their differentiation with evidence of immunological senescence, which have usually lost their cytotoxic properties to become "regulatory" T cells. Patients with a CD8(+)/CD57(+)T cell lymphocytes expansion can display features of organ infiltration, as well as chronic idiopathic neutropenia. The search of this population has therefore a diagnostic value in clinical practice. A CD8(+)/CD57(+)T cell lymphocytes expansion must be suggested in patients with an organomegaly or organ(s) infiltration, particularly in patients infected by the human immunodeficiency virus or in the setting of allogeneic hematopoietic stem cell transplantation, as well as in patients with a neutropenia of unexplained origin. The identification of a CD8(+)/CD57(+)T cell lymphocytes expansion also has therapeutical consequences since patients with an organ infiltration or a neutropenia may respond remarkably to immunomodulatory therapies. The search of a CD8(+)/CD57(+) T cell expansion thus represents a useful and still poorly known diagnostic tool which clinical interest deserves further evaluation.

Mast cell leukemia: identification of a new c-Kit mutation, dup(501-502), and response to masitinib, a c-Kit tyrosine kinase inhibitor.

OBJECTIVE: Most patients with systemic mastocytosis bear mutations in the tyrosine kinase receptor gene c-Kit. Limited treatment options exist for mast cell leukemia, a rare form of systemic mastocytosis associated with a dire prognosis. Our aim was to investigate c-Kit mutations associated with mast cell leukemia and find new treatment for this severe form of mastocytosis. PATIENT AND METHODS: We describe here a patient with mast cell leukemia characterized by 42% of circulating mast cells associated with a previously unidentified c-Kit mutation in adult mastocytosis: dup(501-502). MAIN FINDINGS: This patient was treated with masitinib, a novel c-Kit tyrosine kinase inhibitor, with a dramatic response observed following 3 months of treatment, including clinical improvement, disappearance of circulating mast cells, and decrease in both serum histamine and tryptase levels. In vitro and ex vivo research was performed on the patient's cells and revealed constitutive c-Kit phosphorylation in mast cell leukemia. CONCLUSIONS: This case highlights the importance of sequencing all c-Kit exons when the classical D816V c-Kit mutation is not found, even in adults with SM. It also indicates that masitinib may be safe and effective for the treatment for some mast cell leukemia.

Blood CD34-c-Kit+ cell rate correlates with aggressive forms of systemic mastocytosis and behaves like a mast cell precursor.

Mastocytosis is a heterogeneous disease characterized by the accumulation of mast cells in one or more organs. Our objective was to identify a peripheral mast cell precursor and assess its variation rate in mastocytosis. A peripheral blood phenotypic analysis was performed among 50 patients with mastocytosis who were enrolled in a prospective multicentric French study, and the phenotypic analysis results of the patients were compared with those of healthy donors. The rate of peripheral blood CD34(-)c-Kit(+) cells correlated with the severity of mastocytosis. This cellular population was isolated from healthy donors as well as from patients with systemic mastocytosis. After 30 days of culture, the CD34(-)c-Kit(+) cells gave birth to mature mast cells, indicating that this cellular population constitutes a mast cell circulating precursor. Monitoring peripheral CD34(-)c-Kit(+) cells by flow cytometry could be a useful and low-invasive tool to determine the disease severity and the relapses and to assess treatment efficiency.

Pediatric mastocytosis is a clonal disease associated with D816V and other activating c-KIT mutations.

Adult mastocytosis is an incurable clonal disease associated with c-KIT mutations, mostly in exon 17 (D816V). In contrast, pediatric mastocytosis often spontaneously regresses and is considered a reactive disease. Previous studies on childhood mastocytosis assessed only a few patients and focused primarily on codon 816 mutations, with various results. In this study, we analyzed the entire c-KIT sequence from cutaneous biopsies of 50 children with mastocytosis (ages 0-16 years). A mutation of codon 816 (exon 17) was found in 42% of cases, and mutations outside exon 17 were observed in 44%. Unexpectedly, half of the mutations were located in the fifth Ig loop of c-KIT's extracellular domain, which is encoded by exons 8 and 9. All mutations identified in this study were somatic and caused a constitutive activation of c-KIT. There was no clear phenotype-genotype correlation, no clear relationship between the mutations and familial versus spontaneous disease, and no significant change in the relative expression of the c-KIT GNNK+ and GNNK isoforms. These findings strongly support the idea that, although pediatric mastocytosis can spontaneously regress, it is a clonal disease most commonly associated with activating mutations in c-KIT.

Acute left ventricular dilatation and shock-induced myocardial dysfunction.

OBJECTIVE: Whether cardiac ventricles can acutely dilate during septic myocardial dysfunction. DESIGN: A prospective echocardiographic study was performed to assess changes of left ventricular dimensions over time in patients with septic shock. SETTINGS: A 20-bed surgical intensive care unit of Pitié-Salpêtrière university hospital in Paris. PATIENTS: Forty-five patients were studied over the first 10 days of septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Left ventricular end-diastolic area (LVEDA), fractional area change (FAC), velocity time integral of the aortic flow, echocardiographic indices of left ventricular relaxation, and cardiac troponin I (cTnI) were measured at day 1, 2, 3, 4, 7, and 10. Three groups were defined: 29 patients without increased cTnI and cardiac impairment (group 1), eight patients with increased cTnI and left systolic ventricular dysfunction (group 2), and eight patients with increased cTnI and isolated impairment of left ventricular relaxation (group 3). At day 1, LVEDA was significantly higher in group 2 (13 +/- 3 cm/m, p < 0.05) compared with groups 1 (10 +/- 2 cm/m) and 3 (11 +/- 2 cm/m). LVEDA did not change in groups 1 and 3. In group 2, LVEDA and FAC returned within 10 days to values observed in groups 1 and 2. A significant correlation was found between aortic velocity time integral and LVDEA (r =.78, p = 0.022) and FAC (r =.89, p = 0.003) only in group 2. CONCLUSIONS: Acute and reversible left ventricular dilation accompanies septic shock-induced systolic left ventricular dysfunction. When septic myocardial abnormalities are limited to reversible impairment of left ventricular relaxation, left ventricular dimensions remain unchanged.

Phenotypic and genotypic characteristics of mastocytosis according to the age of onset.

Adult's mastocytosis is usually associated with persistent systemic involvement and c-kit 816 mutation, while pediatrics disease is mostly limited to the skin and often resolves spontaneously. We prospectively included 142 adult patients with histologically proven mastocytosis. We compared phenotypic and genotypic features of adults patients whose disease started during childhood (Group 1, n = 28) with those of patients whose disease started at adult's age (Group 2, n = 114). Genotypic analysis was performed on skin biopsy by sequencing of c-kit exons 17 and 8 to 13. According to WHO classification, the percentage of systemic disease was similar (75 vs. 73%) in 2 groups. C-kit 816 mutation was found in 42% and 77% of patients in groups 1 and 2, respectively (p<0.001). 816 c-kit mutation was associated with systemic mastocytosis in group 2 (87% of patients with systemic mastocytosis vs. 45% with cutaneous mastocytosis, p = 0.0001). Other c-kit activating mutations were found in 23% of patients with mastocytosis' onset before the age of 5, 0% between 6 and 15 years and 2% at adults' age (p<0.001). In conclusion, pathogenesis of mastocytosis significantly differs according to the age of disease's onset. Our data may have major therapeutic relevance when considering c-kit-targeted therapy.

Isolated and reversible impairment of ventricular relaxation in patients with septic shock.

OBJECTIVE: Many patients with septic shock and increased cardiac troponin I (cTnI) do not exhibit significant left ventricular systolic dysfunction. We hypothesized that an isolated and reversible impairment of ventricular relaxation may be associated with the increase in cTnI. DESIGN: Prospective, observational study. SETTING: Surgical intensive care unit in a university hospital. PATIENTS: Total of 54 patients with septic shock. INTERVENTIONS: Fractional area change, early diastolic velocity of mitral annulus, flow propagation velocity of early diastolic mitral inflow, cTnI, tumor necrosis factor-alpha, interleukin (IL)-6, -1beta, -8, and -10 were measured at days 1, 2, 3, 4, 7, and 10 after onset of septic shock. Patients were classified into three groups: normal cTnI (group 1), increased cTnI and fractional area change <50% (group 2), and increased cTnI and fractional area change >50% (group 3). MEASUREMENTS AND MAIN RESULTS: A total of 22 patients had an increase in cTnI, 11 with both systolic and diastolic dysfunctions and 11 with isolated impairment of left ventricular relaxation. At day 1, early diastolic velocity of mitral annulus and flow propagation velocity of early diastolic mitral inflow were significantly lower and tumor necrosis factor-alpha, IL-8, and IL-10 significantly higher in groups 2 and 3 compared with group 1. With resolution of septic shock, early diastolic velocity of mitral annulus and flow propagation velocity of early diastolic mitral inflow measured in patients of groups 2 and 3 returned progressively to values observed in group 1, with a parallel normalization of tumor necrosis factor-alpha, IL-8, and IL-10. CONCLUSIONS: Isolated and reversible impairment of left ventricular relaxation, associated with transient increases in cTnI, tumor necrosis factor-alpha, IL-8, and IL-10, was observed in 20% of patients with septic shock.

Solid-phase synthesis and evaluation of libraries of substituted 4,5-dihydropyridazinones as vasodilator agents.

The solid-phase parallel preparation of a library of 4,5-dihydropyridazin-3(2H)-one derivatives substituted at position 6 with piperazinylmethyl or tetrahydroquinolinylmethyl groups and analogues (3) is reported. Polymer-supported gamma-keto-delta-aminoesters prepared from Wang resin reacted with hydrazine or methylhydrazine to afford pyridazinones in good yields after a cyclization cleavage approach. We have evaluated these novel analogues and several compounds of other series (1, 2) for their vasorelaxant effect. Among the products tested, 3l and 3d proved to be efficacious and potent relaxant agents of the isolated rat aorta. Inhibitors of phosphodiesterase (PDE3), responsible for the breakdown of cyclic AMP in the vascular smooth muscle, are currently developed for cardiac heart failure because of their inotropic effect and coronary vasodilatation. We had expected that the vasodilatation induced by 3l, as efficient as reference PDE3 inhibitors, milrinone or CI-930, to be due to PDE3 inhibition. However 3l and 3d exhibited a low inhibitory effect against PDE3 isoenzyme activity. These compounds induced a significant vasorelaxation, which could be of therapeutic interest even if their mechanism of action remains to be determined.

Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells.

In the present report we have analyzed whether human normal cord blood-derived mast cells (CBMC) could interact with bacterial products, especially lipopolysaccharide (LPS) from Escherichia coli and peptidoglycan (PGN) from Staphylococcus aureus, known as Toll-like receptor (TLR) 4 and TLR2 agonists, respectively. We found that both LPS and PGN induced significant release of not only tumor necrosis factor-alpha (TNF-alpha), but also IL-5, IL-10 and IL-13 by human mast cells (MC). We also established that the stimulation of CBMC with LPS or with PGN is mediated through interactions with TLR4 or with TLR2, respectively. Thus, our data indicate that activation of either TLR2 or TLR4 pathway may lead to a pro-Th2 immune response. However, the release of TNF-alpha induced by LPS, conversely to PGN, required the priming of CBMC by IL-4 and the presence of serum components, in particular soluble CD14. Of interest, stimulation by PGN, but not by LPS, induced release of histamine by human MC. Altogether, these findings provide the first evidence that human MC differentially respond towards bacterial components, and that their responses depend on TLR pathways and reveal human specificities in the pattern of cytokine production.

Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.

Systemic mastocytosis (SM) is a rare disease caused by an abnormal mast cell accumulation in various tissues. Two classes of constitutive activating c-kit mutations are found in SM. The most frequent class occurs in the catalytic pocket coding region with substitutions at codon 816 and the other in the intracellular juxtamembrane coding region. Therefore, kinase inhibitors that block mutated c-kit activity might be used as therapeutic agents in SM. Here, we show that STI571 inhibits both wild-type and juxtamembrane mutant c-kit kinase activity, but has no effect on the activity of the D816 V mutant. Accordingly, STI571 selectively decreases the survival of normal mast cell and of mast cell lines either with juxtamembrane c-kit mutations, but not that of tumoral mast cell from patient with SM or of mast cell lines with the D816 V mutation. Therefore, STI571 is not a good candidate to treat SM and specific kinase inhibitors should be designed to inhibit constitutive activating mutations at codon 816.

Kit and c-kit mutations in mastocytosis: a short overview with special reference to novel molecular and diagnostic concepts.

Mastocytosis is a heterogeneous group of hematopoietic disorders characterized by abnormal growth and accumulation of mast cells (MC) in one or more organs. Clinical symptoms occur as a result of the release of chemical mediators and/or of pathologic infiltration of MC in various tissues. Although the initial events leading to mastocytosis have not yet been unraveled, acquired alterations in the c-kit gene coding for the receptor of stem cell factor (SCF), a major cytokine involved in MC growth, have been described in a significant number of patients. Of particular interest are point mutations resulting in a constitutively activated SCF receptor. Such mutations are probably involved in the abnormal (SCF-independent) proliferation of MC in these patients. New therapeutic strategies may be envisaged to inhibit the deregulated kinase activity of these mutant forms of c-kit.

The role of mast cells in host defense and their subversion by bacterial pathogens.

Mast cells (MCs) play a prominent role in the early immune response to invading pathogenic bacteria. This newly discovered role for MCs involves the release of chemoattractants that recruit neutrophils and the direct phagocytosis and killing of opsonized bacteria. Whereas these activities are clearly beneficial to the host, certain pathogens have evolved mechanisms to evoke anomalous MC responses to the detriment of the host. These include evoking phagocytosis without killing of unopsonized bacteria and the production of toxins that corrupt the release of mediators by MCs. Elucidating how pathogens subvert the activities of MCs could provide clues to limiting the pathological activities of these cells during infectious diseases.