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C019 Functional abnormalities of neutrophils in patients with myelodysplastic syndromes
2. Molecular Mechanisms and Pathophysiology support the idea that MDS may be fundamentally a disease of disordered HSC self-renewal, and point toward the potential utility of NR2F6 ligands as therapeutic agents in MDS.
C019 Functional abnormalities of neutrophils in patients with myelodysplastic syndromes H. Papadaki ° , M. Velegraki, A. Kozana, A. Zamboulaki, N. Kanellias, G. Gvazava, P. Kanellou, M. Kaparou, M. Psyllaki, C. Kalpadakis. Department of Haematology, University of Crete School of Medicine, Heraklion, Greece *E-mail: [email protected] Purpose: Functional and/or quantitative abnormalities of neutrophils have been associated with increased susceptibility to infections in a variety of disease states. In patients with myelodysplastic syndromes (MDS), infection vulnerability does not always reflect the severity of neutropenia. This observation suggests the possible existence of defect(s) in neutrophil function, a hypothesis which has not been extensively studied. The purpose of this study was to examine the phagocytic/chemotactic activity of peripheral blood (PB) neutrophils in patients with MDS as well as the expression of the respective neutrophil receptors. Patients and Methods: 18 patients with low/intermediate-I MDS and 12 healthy individuals were studied after informed consent. The phagocytic activity of PB neutrophils was estimated by measuring the percentage of granulocytes that ingest fluorescein (FITC)-labeled opsonized bacteria (E.coli-FITC) using a commercially available “phagotest kit” and a whole blood lysis technique. Results were evaluated by flow-cytometry. A commercially available “migratest kit” was used for the quantitative flow-cytometric determination of the chemotactic function of granulocytes by assessing: (a) the number of neutrophils migrating through cell culture inserts towards a concentration gradient of the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP), (b) the decrease in expression of the cell adhesion molecule LECAM-1and (c) the changes of the forward scatter Mean Florescence Intensity (MFI) which reflect the changes in the shape of the activated for chemotaxis neutrophils. Flow-cytometry was also used for the evaluation of the proportion and MFI of cells expressing the CD35, CD11b and CD11c complement receptors, the CD16 Fc-gamma receptor and the Toll-like receptors (TLR) TLR1-TLR4 and TLR9 recognizing microbial structures, in the gate of granulocytes. The number of infections per patient during the last year was also recorded. Results: The proportion of granulocytes that had ingested opsonised bacteria was significantly lower in MDS patients (66.26%±22.5%) compared to healthy controls (87.1%±8.4%) (P = 0.0041). Patient neutrophils had also an altered chemotactic function compared to controls as was demonstrated by the number of
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cells that migrated towards the fMLP (1.98±0.28 and 10.91±0.28 respectively) (P = 0.03) and by the proportion of the LECAM negative granulocytes (68.2%±14.39% and 95.42%±4.02% respectively) (P = 0.018). No statistically significant differences were documented between patients and controls in the expression of CD35, CD11b, TLR1TLR4 and TLR9. However, MDS patients had statistically significant lower proportion of CD16+ and CD11c+ neutrophils (85.67%±11.69% and 56.16%±27.95% respectively) compared to healthy individuals (94.75%±4.09% and 77.6%±16.02%, respectively) (P = 0.002 and P = 0.010, respectively). Patient neutrophils displayed decreased MFI of CD16 and CD11c (145.24±107.34 and 4.64±2.81 respectively) compared to controls (370.61±108.34 and 6.43±1.72 respectively, P < 0.0001 and P = 0.0063 respectively). A positive correlation was found between the neutrophil phagocytic capacity and MFI of both CD16 (r = 0.826, P = 0.006) and CD11c (r = 0.7553, P = 0.0186). The phagocytic activity of patient granulocytes inversely correlated with the number of infections (r = −0.7003, P = 0.005). Conclusions: Polymorphonuclear neutrophils of MDS patients display impaired phagocytic and chemotactic capacity. These functional defects are associated with decreased expression of the CD16 Fc-gamma receptor and the CD11c complement receptor and may constitute an independent risk factor for infections in patients with MDS. C020 Prevalence of TET2 mutations in MDS O. Kosmider ° , V. Gelsi-Boyer, M. Cheok, S. Grabar, P. Guardiola, O. Beyne-Rauzy, B. Quesnel, E. Solary, N. Vey, M. Hunault-Berger, P. Fenaux, V. Mansat-de Mas, M. Guesnu, F. Viguie, C. Lacombe, W. Vainchenker, C. Preudhomme, D. Birnbaum, F. Dreyfus, O. Bernard, M. Fontenay, on behalf of the Groupe Francophone des My´elodysplasies (GFM) *E-mail: [email protected] Background: Genetic pathways involved in the development and progression of MDS remain poorly known. In addition, no gene mutation has demonstrated independent prognostic values in MDS. We recently showed TET2 gene, a member of Ten-eleven translocation family located at the 4q24 locus, to be frequently mutated in myeloid malignancies (F. Delhommeau, N. Engl. J. Med., in press) Aim: To establish the prevalence and prognostic impact of TET2 mutations in MDS. Methods: We retrospectively analyzed TET 2 mutations and their prognostic value in 206 MDS and AML post MDS enrolled in GFM multicenter trials (41 RA/RCMD/MDSU/5q−, 18 RCMD, 28 RARS/RCMD-RS/RARS-T, 43 RAEB 1, 32 RAEB 2, 44 AML post MDS). Comparison between categorical variables were performed by Chisquared statistics. Univariate analyses were conducted using logistic regression models.
C019 Functional abnormalities of neutrophils in patients with myelodysplastic syndromes H. Papadaki ° , M. Velegraki, A. Kozana, A. Zamboulaki, N. Kanellias, G. Gvazava, P. Kanellou, M. Kaparou, M. Psyllaki, C. Kalpadakis. Department of Haematology, University of Crete School of Medicine, Heraklion, Greece *E-mail: [email protected] Purpose: Functional and/or quantitative abnormalities of neutrophils have been associated with increased susceptibility to infections in a variety of disease states. In patients with myelodysplastic syndromes (MDS), infection vulnerability does not always reflect the severity of neutropenia. This observation suggests the possible existence of defect(s) in neutrophil function, a hypothesis which has not been extensively studied. The purpose of this study was to examine the phagocytic/chemotactic activity of peripheral blood (PB) neutrophils in patients with MDS as well as the expression of the respective neutrophil receptors. Patients and Methods: 18 patients with low/intermediate-I MDS and 12 healthy individuals were studied after informed consent. The phagocytic activity of PB neutrophils was estimated by measuring the percentage of granulocytes that ingest fluorescein (FITC)-labeled opsonized bacteria (E.coli-FITC) using a commercially available “phagotest kit” and a whole blood lysis technique. Results were evaluated by flow-cytometry. A commercially available “migratest kit” was used for the quantitative flow-cytometric determination of the chemotactic function of granulocytes by assessing: (a) the number of neutrophils migrating through cell culture inserts towards a concentration gradient of the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP), (b) the decrease in expression of the cell adhesion molecule LECAM-1and (c) the changes of the forward scatter Mean Florescence Intensity (MFI) which reflect the changes in the shape of the activated for chemotaxis neutrophils. Flow-cytometry was also used for the evaluation of the proportion and MFI of cells expressing the CD35, CD11b and CD11c complement receptors, the CD16 Fc-gamma receptor and the Toll-like receptors (TLR) TLR1-TLR4 and TLR9 recognizing microbial structures, in the gate of granulocytes. The number of infections per patient during the last year was also recorded. Results: The proportion of granulocytes that had ingested opsonised bacteria was significantly lower in MDS patients (66.26%±22.5%) compared to healthy controls (87.1%±8.4%) (P = 0.0041). Patient neutrophils had also an altered chemotactic function compared to controls as was demonstrated by the number of
S43
cells that migrated towards the fMLP (1.98±0.28 and 10.91±0.28 respectively) (P = 0.03) and by the proportion of the LECAM negative granulocytes (68.2%±14.39% and 95.42%±4.02% respectively) (P = 0.018). No statistically significant differences were documented between patients and controls in the expression of CD35, CD11b, TLR1TLR4 and TLR9. However, MDS patients had statistically significant lower proportion of CD16+ and CD11c+ neutrophils (85.67%±11.69% and 56.16%±27.95% respectively) compared to healthy individuals (94.75%±4.09% and 77.6%±16.02%, respectively) (P = 0.002 and P = 0.010, respectively). Patient neutrophils displayed decreased MFI of CD16 and CD11c (145.24±107.34 and 4.64±2.81 respectively) compared to controls (370.61±108.34 and 6.43±1.72 respectively, P < 0.0001 and P = 0.0063 respectively). A positive correlation was found between the neutrophil phagocytic capacity and MFI of both CD16 (r = 0.826, P = 0.006) and CD11c (r = 0.7553, P = 0.0186). The phagocytic activity of patient granulocytes inversely correlated with the number of infections (r = −0.7003, P = 0.005). Conclusions: Polymorphonuclear neutrophils of MDS patients display impaired phagocytic and chemotactic capacity. These functional defects are associated with decreased expression of the CD16 Fc-gamma receptor and the CD11c complement receptor and may constitute an independent risk factor for infections in patients with MDS. C020 Prevalence of TET2 mutations in MDS O. Kosmider ° , V. Gelsi-Boyer, M. Cheok, S. Grabar, P. Guardiola, O. Beyne-Rauzy, B. Quesnel, E. Solary, N. Vey, M. Hunault-Berger, P. Fenaux, V. Mansat-de Mas, M. Guesnu, F. Viguie, C. Lacombe, W. Vainchenker, C. Preudhomme, D. Birnbaum, F. Dreyfus, O. Bernard, M. Fontenay, on behalf of the Groupe Francophone des My´elodysplasies (GFM) *E-mail: [email protected] Background: Genetic pathways involved in the development and progression of MDS remain poorly known. In addition, no gene mutation has demonstrated independent prognostic values in MDS. We recently showed TET2 gene, a member of Ten-eleven translocation family located at the 4q24 locus, to be frequently mutated in myeloid malignancies (F. Delhommeau, N. Engl. J. Med., in press) Aim: To establish the prevalence and prognostic impact of TET2 mutations in MDS. Methods: We retrospectively analyzed TET 2 mutations and their prognostic value in 206 MDS and AML post MDS enrolled in GFM multicenter trials (41 RA/RCMD/MDSU/5q−, 18 RCMD, 28 RARS/RCMD-RS/RARS-T, 43 RAEB 1, 32 RAEB 2, 44 AML post MDS). Comparison between categorical variables were performed by Chisquared statistics. Univariate analyses were conducted using logistic regression models.