- Email: [email protected]
P092 Granulocyte subpopulations in phenotypic pattern of MDS
5. Diagnostic and prognostic tools I – Morphology and new diagnostic tools Results: We found that B7.1 expression levels on blasts in patients with MDS and AL-MDS was significantly higher than those in patients with de novo AML (p = 0.039 and 0.013, respectively). However, there was no difference in the B7 expression levels between MDS and AL-MDS patients. A majority of F36P cells did and normal CD34+ cells did not express B7.1 constitutively, and B7.1 expression on these cells was induced in cultures supplemented with interleukin-1beta or interferon-g. The percentages of cells in S and G2/M phase were higher in B7.1-positive F36P cells compared with B7.1-negative F36P cells. Consistent with this finding, the percentages of apoptotic cells induced by cytarabine in vitro were significantly higher in B7.1-positive F36P cells compared with B7.1-negative F36P cells. Discussion: Our results suggest that B7.1 expression may be induced by cytokines, which can be derived from the BM environment in MDS, and that the cell cycle of B7.1positive blasts is stimulated. Furthermore, B7.1 expression on blasts might be useful in the differential diagnosis of de novo AML and MDS. P091 B7-H1 molecules on blasts in myelodysplastic syndromes A. Kondo ° , T. Yamashita, H. Tamura, C. Sato, R. Jo, T. Tsuji, K. Dan, K. Ogata. Nippon Medical School; Tokyo University of Science, Japan *E-mail: [email protected] Introduction: B7-H1 (CD274) is a costimulatory/ coinhibitory molecule that plays a crucial role in T cell regulation in various immune responses. B7-H1 expression is detected not only on antigen-presenting cells but also on some tumor cells including leukemia. It was reported that B7-H1 molecules on tumor cells inhibit tumor-specific cytotoxic T lymphocytes and is associated with poor prognosis in patients with some tumors such as renal cancer and breast cancer. We reported that B7-H1 expression on blasts is rare in de novo acute myeloid leukemia (AML). In this study, we investigated whether B7-H1 is expressed on blasts in myelodysplastic syndromes (MDS). Methods: Using flow cytometry (FCM), we analyzed B7H1 expression in MDS cell lines (OIH-1, SKM-1, and F36P) and on blasts in samples of bone marrow or peripheral blood obtained from 40 MDS patients and 19 patients with AML transformed from MDS (AL-MDS). We also investigated whether some cytokines, which might be involved in the pathogenesis of MDS, induced B7-H1 expression on MDS blasts. The cell cycle phase of B7-H1-positive and B7-H1negative F36P cells was determined using FCM. Results: Among the cell lines, F36P cells showed a high level of B7-H1 expression. B7-H1 expression on F36P cells was increased further in cell cultures supplemented with interferon-g or tumor necrosis factor-a. The percentages of cells in G2/M phase were significantly higher in B7H1-positive compared with B7-H1-negative F36P cells. In
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patient samples, B7-H1 expression levels on blasts tended to be the highest in patients with AL-MDS, followed by refractory anemia with excess of blasts (RAEB) in transformation and RA/RAEB (P < 0.1). Discussion: Our results suggest that in MDS B7-H1 expression may be induced by cytokines, which can be derived from the bone marrow environment, that the cell cycle of B7-H1-positive blasts is stimulated, and that high B7-H1 expression may be associated with disease progression. B7-H1 molecules on blasts might contribute to the proliferation of MDS blasts by a mechanism different from the known inhibitory function of B7-H1 on T cell immune responses.
P092 Granulocyte subpopulations in phenotypic pattern of MDS M. Vikentiou1 ° , K. Psarra2 , V. Kapsimali2 , K. Liapis2 , K. Tsionos3 , E. Lianidou1 , C. Papasteriades2 . 1 National University of Athens, 2 Evangelismos Hospital, 3 251 General Hospital, Athens, Greece *E-mail: [email protected] Introduction: Flow cytometric immunophenotypic analysis of neutrophil granulocytes offers important diagnostic information in patients with myelodysplastic syndrome (MDS). The purpose of this study was the investigation, in patients with MDS, of total neutrophil granulocytes population and of the two distinct subpopulations as defined by distinct side scatter and CD45 fluorescence intensity antigen expression patterns. Methods: BM from 70 patients with MDS and 15 controls were analyzed by multiparametric 3- and 4-color flow cytometry using an extensive combination panel in the gate of total granulocytes population using the histogram of SSC = f (CD45 expression). The two subpopulations were analysed in gates of dimmer CD45 (subpopulation A) and stronger CD45 (subpopulation B) in the dotplot of SSC = f (CD45 expression), in 39 MDS and 7 Controls BM samples. Results: Comparing the total granulocytes immunophenotype of MDS and Control group: (a) a decrease of the percentage of CD16 expression and of MPO/LF co-expression and (b) an increase of CD34 expression and CD34/HLADR co-expression were noted. Comparing the two granulocytes subpopulations of MDS patients, subpopulation A was characterized by the following statistical significant findings compared to subpopulation B: (a) a decrease of the percentage and the fluorescence intensity of CD11b, CD116, (b) a decrease of the percentage of CD16, CD10, KORSA, CD36 and MPO/LF(+/+), (c) a decrease of CD13 fluorescence intensity, (d) an increase of the percentage and fluorescence intensity of CD15, CD33, CD38, (e) an increase of the percentage of HLADR and (f) an increase of MPO, LF, MPO/LF(+/+), KORSA, CD36, and CD10 fluorescence intensity. The statistically
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significant differences between the two subpopulations in Controls were limited. More significantly, Controls granulocytes subpopulation A compared to subpopulation B was characterized by (a) a decrease of CD15, CD16, CD11b fluorescence intensity, (b) a decrease of MPO/LF(+/+) percentage and (c) an increase of the CD11b percentage. It should be noted that only a small percentage (5%) of apoptotic cells was detected only on population B. Discussion: The two granulocyte subpopulations show characteristics of dysplasia, subpopulation B being more mature than A. Given the heterogeneity of MDS patients, the immunophenotype study of the two populations offers important information indicative of dysplasia, as it permits the individual investigation of each particular patient.
P093 Changes in the flowcytometric dysplasia score during treatment with erythropoietin/G-CSF reflect responses in low/int-I risk myelodysplastic syndromes T.M. Westers ° , M.E.D. Chamuleau, A.H. Westra, A.M. Dr¨ager, G.J. Ossenkoppele, A.A. van de Loosdrecht. Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, Amsterdam, The Netherlands *E-mail: [email protected] Introduction: Flowcytometry in MDS is recognized as a useful tool in identifying aberrancies in expression of differentiation antigens on immature and mature bone marrow cells of the erythroid and myelomonocytic lineages. Recently, we showed that certain aberrancies in pure RA±RS patients (acc. to WHO), i.e. CD7 on myeloblasts, predict transfusion dependency and disease progression independent of classical prognostic parameters such as WHO, cytogenetics and IPSS. Methods: We evaluated the MDS flow-score in 13 patients with low or int-I risk MDS (6 RA±RS; 6 RCMD±RS; 1 RAEB1) at diagnosis and after 1 year of treatment with a standardized regimen of Epo (NeoRecormon®) and G-CSF. Results: The median flow-score before treatment was 3 in RA±RS (range 2−4), 5.5 in RCMD±RS (range 4−9) and 7 in RAEB1. In 13 normal bone marrow samples the median flow-score was 0 (range 0−2). No aberrancies were detected on the myeloid blasts in the RA±RS patients, normal and G-CSF-treated controls; 4 out of 6 RCMD±RS patients had lineage infidelity marker expression on their myeloid blasts (CD7, n = 3; CD56, n = 1) at diagnosis. Four patients (all RA±RS) showed hematological improvement (HI) upon Epo/G-CSF treatment. In these patients the number of aberrancies in the myelomonocytic lineages as assessed by flowcytometry decreased resulting in a decline in the MDS flow-score (median 3 and 1, at diagnosis and follow-up, respectively). In 5 patients with stable disease, no significant changes in the flow-score could be
detected (median 5 at diagnosis and follow-up). Whereas in 3 patients with progressive disease during Epo/G-CSF and 1 highly transfusion-dependent patient (3 RCMD±RS, 1 RAEB1) percentages of myeloid blasts and hence flowscores increased (median 6 and 8, at diagnosis and followup, respectively). Discussion: From these data we hypothesize that flowcytometry can be used as a sensitive disease monitoring parameter in low/int-I risk MDS. Furthermore, in a subset of these patients treatment with Epo/G-CSF may have altered disease progression, which was reflected by normalization or stabilization of aberrancies on myelomonocytic cells. This underscores observations that Epo/G-CSF improves overall survival and/or leukemia free survival. Prospective studies are being conducted to validate flowcytometric analysis in the diagnosis, prognostication and disease monitoring of low-int-I risk MDS during Epo/G-CSF and lenalidomide.
P094 Erythroleukemia might be a possible erythroblastic transformation from myelodysplastic syndrome: molecular cytogenetic study on the clonality of erythroid lineage by immunomagnetic bead and immunophenotyping M.Y. Kim1 , S.H. Kang1 , H.J. Min1,2 , T.Y. Kim1,2 , B.R. Oh1,2 , I.H. Kim1 , H.I. Cho1 , D.S. Lee1,2 ° . 1 Seoul National University College of Medicine, Seoul; 2 Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea *E-mail: [email protected] Background: Myelodysplastic syndrome is a clonal disorder of stem cells, eventually progressing to acute myelogenous leukemia (AML). Erythroleukemia is a neoplastic proliferation of erythroid and myeloid precursors, even though a pure erythroid proliferation may occur rarely. To characterize the clonal property of erythoid lineage in myelodysplastic syndrome and to find whether there is any similarity of clonal change between erythroleukemia and MDS, we investigated the molecular cytogenetic changes by immunomagnetic enrichment of erythroid & lymphoid cells and fluorescent in situ hybridization combined by immunophenotyping (FICTION) method. Methods: FISH [AML/ETO, PML/RARA, MLL, inv(16) FISH] study was done in newly diagnosed 314 patients with AML (M0: 10, M1: 37, M2: 103, M3: 38, M4: 63, M5: 16, M6: 21, M7: 7) and FISH study for 5q deletion, 7q deletion, trisomy 8, gain of 1q was dodne in 76 patients with MDS. Bone marrow aspiates of patients with MDS are subjected to immunomagnetic enrichment with the MACS system, using an CD3 and CD20 antibody directly conjugated to magnetic beads to obtain an lymphoid lineage population, and the subsequent FISH [5q deletion, 7q deletion, gain of 1q, trisomy 8] was performed. For
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patient samples, B7-H1 expression levels on blasts tended to be the highest in patients with AL-MDS, followed by refractory anemia with excess of blasts (RAEB) in transformation and RA/RAEB (P < 0.1). Discussion: Our results suggest that in MDS B7-H1 expression may be induced by cytokines, which can be derived from the bone marrow environment, that the cell cycle of B7-H1-positive blasts is stimulated, and that high B7-H1 expression may be associated with disease progression. B7-H1 molecules on blasts might contribute to the proliferation of MDS blasts by a mechanism different from the known inhibitory function of B7-H1 on T cell immune responses.
P092 Granulocyte subpopulations in phenotypic pattern of MDS M. Vikentiou1 ° , K. Psarra2 , V. Kapsimali2 , K. Liapis2 , K. Tsionos3 , E. Lianidou1 , C. Papasteriades2 . 1 National University of Athens, 2 Evangelismos Hospital, 3 251 General Hospital, Athens, Greece *E-mail: [email protected] Introduction: Flow cytometric immunophenotypic analysis of neutrophil granulocytes offers important diagnostic information in patients with myelodysplastic syndrome (MDS). The purpose of this study was the investigation, in patients with MDS, of total neutrophil granulocytes population and of the two distinct subpopulations as defined by distinct side scatter and CD45 fluorescence intensity antigen expression patterns. Methods: BM from 70 patients with MDS and 15 controls were analyzed by multiparametric 3- and 4-color flow cytometry using an extensive combination panel in the gate of total granulocytes population using the histogram of SSC = f (CD45 expression). The two subpopulations were analysed in gates of dimmer CD45 (subpopulation A) and stronger CD45 (subpopulation B) in the dotplot of SSC = f (CD45 expression), in 39 MDS and 7 Controls BM samples. Results: Comparing the total granulocytes immunophenotype of MDS and Control group: (a) a decrease of the percentage of CD16 expression and of MPO/LF co-expression and (b) an increase of CD34 expression and CD34/HLADR co-expression were noted. Comparing the two granulocytes subpopulations of MDS patients, subpopulation A was characterized by the following statistical significant findings compared to subpopulation B: (a) a decrease of the percentage and the fluorescence intensity of CD11b, CD116, (b) a decrease of the percentage of CD16, CD10, KORSA, CD36 and MPO/LF(+/+), (c) a decrease of CD13 fluorescence intensity, (d) an increase of the percentage and fluorescence intensity of CD15, CD33, CD38, (e) an increase of the percentage of HLADR and (f) an increase of MPO, LF, MPO/LF(+/+), KORSA, CD36, and CD10 fluorescence intensity. The statistically
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significant differences between the two subpopulations in Controls were limited. More significantly, Controls granulocytes subpopulation A compared to subpopulation B was characterized by (a) a decrease of CD15, CD16, CD11b fluorescence intensity, (b) a decrease of MPO/LF(+/+) percentage and (c) an increase of the CD11b percentage. It should be noted that only a small percentage (5%) of apoptotic cells was detected only on population B. Discussion: The two granulocyte subpopulations show characteristics of dysplasia, subpopulation B being more mature than A. Given the heterogeneity of MDS patients, the immunophenotype study of the two populations offers important information indicative of dysplasia, as it permits the individual investigation of each particular patient.
P093 Changes in the flowcytometric dysplasia score during treatment with erythropoietin/G-CSF reflect responses in low/int-I risk myelodysplastic syndromes T.M. Westers ° , M.E.D. Chamuleau, A.H. Westra, A.M. Dr¨ager, G.J. Ossenkoppele, A.A. van de Loosdrecht. Department of Hematology, VU Institute for Cancer and Immunology, VU University Medical Center, Amsterdam, The Netherlands *E-mail: [email protected] Introduction: Flowcytometry in MDS is recognized as a useful tool in identifying aberrancies in expression of differentiation antigens on immature and mature bone marrow cells of the erythroid and myelomonocytic lineages. Recently, we showed that certain aberrancies in pure RA±RS patients (acc. to WHO), i.e. CD7 on myeloblasts, predict transfusion dependency and disease progression independent of classical prognostic parameters such as WHO, cytogenetics and IPSS. Methods: We evaluated the MDS flow-score in 13 patients with low or int-I risk MDS (6 RA±RS; 6 RCMD±RS; 1 RAEB1) at diagnosis and after 1 year of treatment with a standardized regimen of Epo (NeoRecormon®) and G-CSF. Results: The median flow-score before treatment was 3 in RA±RS (range 2−4), 5.5 in RCMD±RS (range 4−9) and 7 in RAEB1. In 13 normal bone marrow samples the median flow-score was 0 (range 0−2). No aberrancies were detected on the myeloid blasts in the RA±RS patients, normal and G-CSF-treated controls; 4 out of 6 RCMD±RS patients had lineage infidelity marker expression on their myeloid blasts (CD7, n = 3; CD56, n = 1) at diagnosis. Four patients (all RA±RS) showed hematological improvement (HI) upon Epo/G-CSF treatment. In these patients the number of aberrancies in the myelomonocytic lineages as assessed by flowcytometry decreased resulting in a decline in the MDS flow-score (median 3 and 1, at diagnosis and follow-up, respectively). In 5 patients with stable disease, no significant changes in the flow-score could be
detected (median 5 at diagnosis and follow-up). Whereas in 3 patients with progressive disease during Epo/G-CSF and 1 highly transfusion-dependent patient (3 RCMD±RS, 1 RAEB1) percentages of myeloid blasts and hence flowscores increased (median 6 and 8, at diagnosis and followup, respectively). Discussion: From these data we hypothesize that flowcytometry can be used as a sensitive disease monitoring parameter in low/int-I risk MDS. Furthermore, in a subset of these patients treatment with Epo/G-CSF may have altered disease progression, which was reflected by normalization or stabilization of aberrancies on myelomonocytic cells. This underscores observations that Epo/G-CSF improves overall survival and/or leukemia free survival. Prospective studies are being conducted to validate flowcytometric analysis in the diagnosis, prognostication and disease monitoring of low-int-I risk MDS during Epo/G-CSF and lenalidomide.
P094 Erythroleukemia might be a possible erythroblastic transformation from myelodysplastic syndrome: molecular cytogenetic study on the clonality of erythroid lineage by immunomagnetic bead and immunophenotyping M.Y. Kim1 , S.H. Kang1 , H.J. Min1,2 , T.Y. Kim1,2 , B.R. Oh1,2 , I.H. Kim1 , H.I. Cho1 , D.S. Lee1,2 ° . 1 Seoul National University College of Medicine, Seoul; 2 Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea *E-mail: [email protected] Background: Myelodysplastic syndrome is a clonal disorder of stem cells, eventually progressing to acute myelogenous leukemia (AML). Erythroleukemia is a neoplastic proliferation of erythroid and myeloid precursors, even though a pure erythroid proliferation may occur rarely. To characterize the clonal property of erythoid lineage in myelodysplastic syndrome and to find whether there is any similarity of clonal change between erythroleukemia and MDS, we investigated the molecular cytogenetic changes by immunomagnetic enrichment of erythroid & lymphoid cells and fluorescent in situ hybridization combined by immunophenotyping (FICTION) method. Methods: FISH [AML/ETO, PML/RARA, MLL, inv(16) FISH] study was done in newly diagnosed 314 patients with AML (M0: 10, M1: 37, M2: 103, M3: 38, M4: 63, M5: 16, M6: 21, M7: 7) and FISH study for 5q deletion, 7q deletion, trisomy 8, gain of 1q was dodne in 76 patients with MDS. Bone marrow aspiates of patients with MDS are subjected to immunomagnetic enrichment with the MACS system, using an CD3 and CD20 antibody directly conjugated to magnetic beads to obtain an lymphoid lineage population, and the subsequent FISH [5q deletion, 7q deletion, gain of 1q, trisomy 8] was performed. For