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1.
Cell Commun Signal ; 17(1): 89, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31382969

RESUMO

Escape from immune control must be important in the natural course of B-cell lymphomas, especially for those with activation of NF-κB. The pre-clinical LMP1/CD40-expressing transgenic mouse model is characterized by B-cell specific CD40 signaling responsible for NF-κB continuous activation with a spleen monoclonal B-cell tumor after 1 year in 60% of cases. LMP1/CD40 tumors B-cells expressed high levels of PD-L1. This expression was dependent on activation of either NF-κB, JAK1/JAK2 or BTK pathways since these pathways were activated in tumor B-cells and ex vivo treatment with the inhibitory molecules PHA-408, ruxolitinib and ibrutinib led to decrease of its expression. Treatment of LMP1/CD40-expressing lymphomatous mice with an anti-PD-L1 monoclonal antibody induced tumor regression with decreased spleen content, activation and proliferation rate of B-cells as well as a marked increase in T-cell activation, as assessed by CD62L and CD44 expression. These results highlight the interest of therapies targeting the PD-1/PD-L1 axis in activated lymphomas with PD-L1 expression, with possible synergies with tyrosine kinase inhibitors.

2.
J Immunol ; 203(6): 1665-1674, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31434708

RESUMO

EBV infects and immortalizes B cells in vitro and in vivo. It is the causative agent of most immune deficiency-related lymphoproliferative disorders and is associated with various lymphomas. EBV latency III-transformed B cells are known to express two immunosuppressive molecules, IL-10 and PD-L1, two characteristics of regulatory B cells (Bregs). In this study, we show that, in addition to secretion of the Breg immunosuppressive cytokines IL-10, IL-35, and TGF-ß1, EBV latency III-transformed B cells were able to repress proliferation of their autologous T cells preactivated by CD2, CD3, and CD28. This inhibitory effect was likely caused by CD4+ T cells because EBV latency III-transformed B cells induced a strong proliferation of isolated autologous CD8 T cells. Indeed, EBV was able to promote expansion of autologous FOXP3+ CD39high CTLA4+, Helios+, GITR+, LAG3+ CD4 T cells (i.e., regulatory T cells [Tregs]). Two types of Tregs were induced: unconventional CD25neg and conventional CD25pos Tregs. These Tregs expressed both the latency-associated peptide (LAP) and the PD-1 receptor, two markers of functional Tregs. Expansion of both Treg subtypes depended on PD-L1, whose expression was under the control of LMP1, the main EBV oncogene. These results demonstrate that, like Bregs, EBV latency III-transformed B cells exhibit strong immunoregulatory properties. These data provide clues to the understanding of how after EBV primo-infection, EBV-proliferating B cells can survive in an aggressive immunological environment and later emerge to give rise to EBV-associated B cell lymphomas such as in elderly patients.

6.
Artigo em Inglês | MEDLINE | ID: mdl-30450798

RESUMO

BACKGROUND: The different B-cell subsets in human bone marrow result from a dynamic equilibrium between endogenous production, B-cell bone marrow reentry and terminal plasma cell differentiation. Our aim was to define and quantify the different medullary B-cell subsets. METHODS: A series of 32 normal adult bone marrows plus 15 normal adult blood samples was studied by nine color flow cytometry (CD10, CD19, CD24, CD27, CD34, CD38, CD45, IgM, and IgD). With the Kaluza software radar plots, two 2D triple parametric histograms (CD10/CD34/CD45 and CD27/IgM/IgD) were set-up to identify six progenitor and five mature B-cell subsets. RESULTS: Very early B-cell progenitors were CD19neg/CD10pos/CD34pos. B-cell progenitors were split into five subsets on the CD10/CD34/CD45 triple parametric histogram, sequentially ordered according to the loss of CD34 and CD10 and acquisition of surface IgM and IgD. CD19pos/CD38low mature B-cells were divided into four subsets on the CD27/IgM/IgD triple parametric histogram, with two stages of naïve B-cells and two CD27hi marginal zone and switched memory B-cell compartments. CD19pos/CD34neg/CD10low immature B-cells were the main bone marrow B-cell subset, accounting for one third of bone marrow B-cells. Transitional B-cells were the only immature bone marrow stage found in the blood. Compared to blood, the bone marrow was enriched in both marginal zone and switched B-cells. CONCLUSION: We provide the first analysis of 3D B-cell differentiation by multicolor flow cytometry leading to propose reference values for each bone marrow and blood B-cell compartment. This warrants further exploration of normal and pathological human B-cell maturation. © 2018 International Clinical Cytometry Society.

7.
Artigo em Inglês | MEDLINE | ID: mdl-30328273

RESUMO

BACKGROUND: Similarly, to sepsis, cardiac surgery with cardiopulmonary bypass (CPB) induces major changes in leukocyte subsets. Immature granulocytes (IGs) increase both in sepsis and after open-heart surgery. Secondary infections are a major complication of cardiac surgery with CPB. We hypothesized that the assessment of leukocyte subsets with multicolor flow cytometry (FCM) could help the front-line clinician to better identify patients at high risk of infectious complications in this clinical setting. METHODS: In this single-center observational pilot study, we identified 26 leukocyte subsets using three combinations of antibodies (from 5 to 10 colors per combinations): one devoted to granulocytes, one to lymphocyte subpopulations and one for rare cells (plasma cells and dendritic cells). Blood samples were obtained preoperatively and immediately after open-heart surgery under CPB in 59 patients without immuno-depression, chronic or neoplastic inflammatory disease, and immunosuppressive treatment. Secondary infections during hospital stay were recorded. RESULTS: Patients exhibited postoperative NK and T-cell lymphopenia, increased levels of IGs and monocytes with low levels of surface HLA DR. Twelve patients developed secondary infectious complications. Only immediate postoperative IG levels were significantly higher in these patient (6.6 [6; 7.39] G/L vs. 3.8 [2.67; 5.72] G/L, P = 0.01). Patients with immediate postoperative increase of IGs developed more frequently infectious complication (10/22 [46%] vs. 2/37 [5%]: P < 0.001). CONCLUSIONS: This study suggests that postoperative increase of IGs is related to postoperative organ failure and promises to help in early identification of patients at risk of infectious complications after open-heart surgery under CPB.

8.
J Med Chem ; 61(15): 6574-6591, 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30004697

RESUMO

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

10.
Cancer Med ; 7(6): 2621-2628, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29745034

RESUMO

More than 35 years after the Binet classification, there is still a need for simple prognostic markers in chronic lymphocytic leukemia (CLL). Here, we studied the treatment-free survival (TFS) impact of normal serum protein electrophoresis (SPE) at diagnosis. One hundred twelve patients with CLL were analyzed. The main prognostic factors (Binet stage; lymphocytosis; IGHV mutation status; TP53, SF3B1, NOTCH1, and BIRC3 mutations; and cytogenetic abnormalities) were studied. The frequencies of IGHV mutation status, cytogenetic abnormalities, and TP53, SF3B1, NOTCH1, and BIRC3 mutations were not significantly different between normal and abnormal SPE. Normal SPE was associated with Binet stage A, nonprogressive disease for 6 months, lymphocytosis below 30 G/L, and the absence of the IGHV3-21 gene rearrangement which is associated with poor prognosis. The TFS of patients with normal SPE was significantly longer than that of patients with abnormal SPE (log-rank test: P = 0.0015, with 51% untreated patients at 5.6 years and a perfect plateau afterward vs. a median TFS at 2.64 years for abnormal SPE with no plateau). Multivariate analysis using two different Cox models and bootstrapping showed that normal SPE was an independent good prognostic marker for either Binet stage, lymphocytosis, or IGHV mutation status. TFS was further increased when both normal SPE and mutated IGHV were present (log-rank test: P = 0.008, median not reached, plateau at 5.6 years and 66% untreated patients). A comparison with other prognostic markers suggested that normal SPE could reflect slowly advancing CLL disease. Altogether, our results show that a combination of normal SPE and mutated IGHV genes defines a subgroup of patients with CLL who evolve very slowly and who might never need treatment.

11.
Chest ; 154(3): 617-627, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29705219

RESUMO

BACKGROUND: In this study, we primarily sought to assess the ability of flow cytometry to predict early clinical deterioration and overall survival in patients with sepsis admitted in the ED and ICU. METHODS: Patients admitted for community-acquired acute sepsis from 11 hospital centers were eligible. Early (day 7) and late (day 28) deaths were notified. Levels of CD64pos granulocytes, CD16pos monocytes, CD16dim immature granulocytes (IGs), and T and B lymphocytes were assessed by flow cytometry using an identical, cross-validated, robust, and simple consensus standardized protocol in each center. RESULTS: Among 1,062 patients screened, 781 patients with confirmed sepsis were studied (age, 67 ± 48 years; Simplified Acute Physiology Score II, 36 ± 17; Sequential Organ Failure Assessment, 5 ± 4). Patients were divided into three groups (sepsis, severe sepsis, and septic shock) on day 0 and on day 2. On day 0, patients with sepsis exhibited increased levels of CD64pos granulocytes, CD16pos monocytes, and IGs with T-cell lymphopenia. Clinical severity was associated with higher percentages of IGs and deeper T-cell lymphopenia. IG percentages tended to be higher in patients whose clinical status worsened on day 2 (35.1 ± 35.6 vs 43.5 ± 35.2, P = .07). Increased IG percentages were also related to occurrence of new organ failures on day 2. Increased IG percentages, especially when associated with T-cell lymphopenia, were independently associated with early (P < .01) and late (P < .01) death. CONCLUSIONS: Increased circulating IGs at the acute phase of sepsis are linked to clinical worsening, especially when associated with T-cell lymphopenia. Early flow cytometry could help clinicians to target patients at high risk of clinical deterioration. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01995448; URL: www.clinicaltrials.gov.

12.
Hematol Oncol ; 36(2): 422-428, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29218734

RESUMO

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 <5 × 10-5 . MRD was found to be constantly negative (<5 × 10-5 ) for 148 patients. Survival analyses significantly associated MRD negativity with a good prognosis and any positive value with poorer outcome. All P values were <0.0001 both for disease-free and overall survival at the earliest time point (post-induction, MRD1) as well as when considering all time points together. Finally, MRD levels were independent of cytogenetics and allowed in fact to further stratify all cytogenetics risk groups. In summary, this multicenter study demonstrates that a simple combination of immunophenotypic markers successfully allows for the detection of MRD in acute myeloblastic leukemia patients, with a strong correlation to outcome.


Assuntos
Citometria de Fluxo/métodos , Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Imunofenotipagem , Lactente , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Adulto Jovem
13.
J Dermatol Sci ; 88(2): 238-246, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28838616

RESUMO

BACKGROUND: Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-). OBJECTIVES: As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin. METHODS: Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model. RESULTS: A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch µ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression. CONCLUSION: These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell.


Assuntos
Genes de Imunoglobulinas/genética , Centro Germinativo/patologia , Região Variável de Imunoglobulina/genética , Linfoma de Células B/genética , Neoplasias Cutâneas/genética , Superantígenos/imunologia , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Linfócitos B/patologia , Biópsia , Evolução Clonal/genética , Evolução Clonal/imunologia , Análise Mutacional de DNA/métodos , Feminino , Genes de Imunoglobulinas/imunologia , Centro Germinativo/imunologia , Humanos , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/imunologia , Perna (Membro) , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Plasmócitos/imunologia , Plasmócitos/patologia , Estudos Retrospectivos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia
14.
Haematologica ; 102(5): 883-894, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28232371

RESUMO

While c-Myc dysregulation is constantly associated with highly proliferating B-cell tumors, nuclear factor (NF)-κB addiction is found in indolent lymphomas as well as diffuse large B-cell lymphomas, either with an activated B-cell like phenotype or associated with the Epstein-Barr virus. We raised the question of the effect of c-Myc in B cells with NF-κB activated by three different inducers: Epstein-Barr virus-latency III program, TLR9 and CD40. Induction of c-Myc overexpression increased proliferation of Epstein-Barr virus-latency III immortalized B cells, an effect that was dependent on NF-κB. Results from transcriptomic signatures and functional studies showed that c-Myc overexpression increased Epstein-Barr virus-latency III-driven proliferation depending on NF-κB. In vitro, induction of c-Myc increased proliferation of B cells with TLR9-dependant activation of MyD88, with decreased apoptosis. In the transgenic λc-Myc mouse model with c-Myc overexpression in B cells, in vivo activation of MyD88 by TLR9 induced splenomegaly related to an increased synthesis phase (S-phase) entry of B cells. Transgenic mice with both continuous CD40 signaling in B cells and the λc-Myc transgene developed very aggressive lymphomas with characteristics of activated diffuse large B-cell lymphomas. The main characteristic gene expression profile signatures of these tumors were those of proliferation and energetic metabolism. These results suggest that c-Myc is an NF-κB co-transforming event in aggressive lymphomas with an activated phenotype, activated B-cell like diffuse large B-cell lymphomas. This would explain why NF-κB is associated with both indolent and aggressive lymphomas, and opens new perspectives on the possibility of combinatory therapies targeting both the c-Myc proliferating program and NF-κB activation pathways in diffuse large B-cell lymphomas.


Assuntos
Linfócitos B/metabolismo , Transformação Celular Viral , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Apoptose/genética , Linfócitos B/virologia , Antígenos CD40/genética , Antígenos CD40/metabolismo , Linhagem Celular Transformada , Proliferação de Células/genética , Perfilação da Expressão Gênica/métodos , Herpesvirus Humano 4/fisiologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética
15.
Am J Hematol ; 92(1): 68-76, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27737507

RESUMO

Little is known on the phylogenetic relationship between diagnostic and relapse clones of diffuse large B-cell lymphoma (DLBCL). We applied high throughput sequencing (HTS) of the VDJ locus of Immunoglobulin heavy chain (IGHV) on 14 DLBCL patients with serial samples, including tumor biopsies and/or peripheral blood mononuclear cells (PBMC). Phylogenetic data were consolidated with targeted sequencing and cytogenetics. Phylogeny clearly showed that DLBCL relapse could occur according either an early or a late divergent mode. These two modes of divergence were independent from the elapsed time between diagnosis and relapse. We found no significant features for antigen selection pressure in complementary determining region both at diagnosis and relapse for 9/12 pairs and a conserved negative selection pressure for the three remaining cases. Targeted HTS and conventional cytogenetics revealed a branched vs. linear evolution for 5/5 IGHV early divergent cases, but unexpected such "oncogenetic" branched evolution could be found in at least 2/7 IGHV late divergent cases. Thus, if BCR signaling is mandatory for DLBCL emergence, oncogenetic events under chemotherapy selection pressure may be the main driving forces at relapse. Finally, circulating subclones with divergent IGHV somatic hypermutations patterns from initial biopsy could be detected in PBMC at diagnosis for 4/6 patients and, for two of them, at least one was similar to the ones found at relapse. This study highlights that oncogenetic intraclonal diversity of DLBCL should be evaluated beyond the scope a single biopsy and represents a rationale for future investigations using peripheral blood for lymphoid malignancies genotyping. Am. J. Hematol. 92:68-76, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Evolução Clonal , Cadeias Pesadas de Imunoglobulinas/genética , Linfoma Difuso de Grandes Células B/genética , Recidiva Local de Neoplasia/genética , Recombinação V(D)J , Genes de Cadeia Pesada de Imunoglobulina , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma Difuso de Grandes Células B/sangue , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/patologia , Recidiva Local de Neoplasia/imunologia , Filogenia , Estudos Retrospectivos , Análise de Sequência de DNA
16.
Haematologica ; 102(2): 308-319, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27758818

RESUMO

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.


Assuntos
Células Eritroides/metabolismo , Células Eritroides/patologia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Adulto Jovem
17.
Int J Cancer ; 139(4): 793-802, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27061907

RESUMO

Extranodal natural killer/T-cell lymphomas (NK/TL), rare in Europe, are Epstein-Barr virus (EBV) associated lymphomas with poor outcomes. Here, we determined the virus type and analyzed the EBV latent membrane protein-1 (LMP1) gene sequence in NK/TL from French patients. Six clones of viral LMP1 were sequenced by Sanger technology in blood from 13 patients before treatment with an l-asparaginase based regimen and, for 8 of them, throughout the treatment. Blood LMP1 sequences from 21 patients without any known malignancy were tested as controls. EBV Type A was identified for 11/13 patients and for all controls. Before treatment, a clonal LMP1 gene containing a 30 bp deletion (del30) was found in 46.1% of NK/TL and only in 4.8% of controls. Treatment was less effective in these patients who died more rapidly than the others. Patients with a deleted strain evolving toward a wild-type strain during treatment reached complete remission. The LMP1 gene was sequenced by highly sensitive next-generation sequencing technology in five NK/TL nasopharyngeal biopsies, two of them originating from the previous patients. Del30 was present in 100% of the biopsies; two viruses at least coexisted in three biopsies. These results suggest that del30 may be associated with poor prognosis NK/TL and that strain evolution could be used as a potential marker to monitor treatment.


Assuntos
Evolução Clonal , Infecções por Vírus Epstein-Barr/complicações , Deleção de Genes , Herpesvirus Humano 4/genética , Linfoma Extranodal de Células T-NK/etiologia , Proteínas da Matriz Viral/genética , Adulto , Idoso , Linhagem Celular Transformada , Infecções por Vírus Epstein-Barr/virologia , Europa (Continente) , Feminino , Humanos , Linfoma Extranodal de Células T-NK/terapia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
19.
Haematologica ; 100(4): 472-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25637056

RESUMO

Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative.


Assuntos
Antígenos CD7/metabolismo , Antígenos CD5/metabolismo , Antígeno CD56/metabolismo , Imunofenotipagem , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/metabolismo , Doenças Mieloproliferativas-Mielodisplásicas/diagnóstico , Doenças Mieloproliferativas-Mielodisplásicas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD7/genética , Antígenos CD5/genética , Antígeno CD56/genética , Diagnóstico Diferencial , Citometria de Fluxo , Expressão Gênica , Humanos , Imunofenotipagem/métodos , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/genética , Doenças Mieloproliferativas-Mielodisplásicas/genética , Sensibilidade e Especificidade
20.
Crit Care Med ; 42(9): 2007-18, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24942511

RESUMO

OBJECTIVES: Primary objective was to identify leukocyte subsets that could predict the early evolution of sepsis at 48 hours (i.e., deterioration or stability/improvement). Secondary objectives were to evaluate the prognostic value of leukocyte subsets on mortality and immunosuppressive properties of immature granulocytes. DESIGN: Twenty-three peripheral blood leukocyte subsets were analyzed using a new-generation 10-color flow cytometry. T-cell killing activity of immature granulocytes was explored using a sorting method specifically developed. SETTING: ICUs and emergency departments. PATIENTS: All patients admitted to emergency department and ICU for sepsis ongoing for less than 24 hours were eligible. Exclusion criteria were pregnancy, age less than 18 years, solid tumors, HIV infection, hematological or inflammatory conditions, and immunosuppressive drugs. Finally, 177 patients were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The two most salient features of sepsis were decreased CD10 (CD10) and CD16 (CD16) expressions on granulocytes. With a threshold of 90% of CD10 and 15% of CD16 granulocytes, these immunophenotypic features, which are those of immature granulocytes, predicted sepsis deterioration at 48 hours with a sensitivity of 57% and 70% and a specificity of 78% and 82%, respectively. Survival rate at day 30 was 99% for patients without CD10 and CD16, 85% for patients with increased CD16 only, and 63% for patients with increased CD16 and CD10 granulocytes (p < 0.001). Among CD16 immature granulocytes, we identified a CD14/CD24 myeloid-derived suppressor cell subset with the capability of killing activated T cells. Consistently, an excess of CD16 immature granulocytes was associated with both CD3 and CD4 T-cell lymphopenia in deteriorating patients. CONCLUSIONS: Circulating immature granulocytes predicted early sepsis deterioration and were enriched in myeloid-derived suppressor cells which could be responsible for immunosuppression through the induction of T-cell lymphopenia.


Assuntos
Granulócitos/imunologia , Sepse/imunologia , Sepse/mortalidade , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Prognóstico , Sepse/sangue , Análise de Sobrevida
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