- Email: [email protected]
P-057 Myelodysplastic syndromes (MDS) with a deletion 5q display a specific immunophenotypic profile
Poster Presentations – 12th International Symposium on Myelodysplastic Syndromes / Leukemia Research 37 S1 (2013) S1–S117
mined using ELISA. (3) CD25 expression was induced by retinoic acid (RA) in the MDS cell line F-36P. Then, the cells were treated with LMB-2 (Pseudomonas exotoxin A conjugated anti-CD25) 0.001 to 1000 ng/ml and subjected to a cell viability assay. Results: (1) Plasma sIL-2Ra levels were significantly elevated in patients with IPSS-high and AL-MDS compared with patients with IPSS-low and INT-1. (2) In univariate analysis, high levels of sIL2Ra (>1500 pg/ml) were associated with shorter overall survival in MDS patients (P=0.0001). Patients with high levels of sIL-2Ra (>1500 pg/ml) had higher percentages of bone marrow blasts and higher leukocyte counts compared with other patients. (3) In multivariate analysis, high levels of sIL-2Ra (>1500 pg/ml) was an independent prognostic factor for overall survival (P=0.0185). (4) CD25 expression was mainly observed on blasts, not on lymphocytes. Furthermore, percentages of CD25+ blasts correlated positively with plasma concentrations of sIL-2Ra (r=0.743, P=0.032). Moreover, when CD25positive blasts were cultured, sIL-2Ra concentrations in culture supernatants increased with the length of culture. (5) Untreated F-36P cells lacked CD25 expression, but 30% of RA-treated F-36P cells expressed CD25. Cell viability of RA-treated F-36P cells was inhibited by LMB-2 in a dose-dependent manner. Conclusions: Our data indicate that sIL-2Ra in the peripheral blood of MDS patients is derived from CD25 molecules expressed on blasts. Soluble IL-2Ra would be a useful prognostic biomarker in MDS. Moreover, CD25 molecules on MDS blasts are potential targets of future pharmacotherapy.
P-056 The significance of abnormal co-expressions of CD34+ for the diagnosis and prognosis in MDS Reis-Alves 1 ,
Rocha 2 ,
Pereira-Cunha 2 ,
Saad 1 ,
F.F. F.G. S.T.O. S.C. F. Traina 1 , I. Lorand-Metze 1 . 1 Internal Medicine, University of Campinas, Campinas, Brazil; 2 Hematology/Hemotherapy, University of Campinas, Campinas, Brazil Background: Immunophenotyping has been recognized as important for diagnosis of MDS. Introduction: Recently a flow score (FCSS) was described, that is useful for the differential diagnosis as well as prognosis. Purpose: We examined if the inclusion of quantitative values of abnormal expressions in CD34+ cells and monocytes in this index could improve its diagnostic and prognostic significance. Materials and Methods: Immunophenotypic analyzes were performed on bone marrow (BM) cells of patients with MDS and cases of non-clonal cytopenias. The two scores were applied to all cases. Controls: 41 normal BMs from BMT donors. The modified score covers 9 granulocytic, 8 monocytic and 6 CD34+ cell abnormalities. Antibody combinations: HLA-DR/CD14/CD45/CD33, CD16/CD11b/CD45/ CD13, CD13/CD117/CD45/CD34, CD10/CD19/CD45/CD34 and CD7/ CD56/CD45/CD34. Results: We analyzed 32 reactive (deficiency anemias and drug myelotoxicity) and 48 cases of MDS (mean age: 60 and 69 years respectively). WHO types: 6 RA, 30 RCMD, 5 RAEB-I and 7 RAEB-II. IPSSR: 5 very low risk, 12 low, 15 intermediate, 9 high and 3 very high risk. Table 1 Granulocytes series
Monocyte series
IMF Pattern HLA- CD56+ CD34+ HLASSC CD13/CD16 DR/CD33 DR/CD33 MDS 429 Reactive 522 p 0.001
25/48 4/32 <0.0001
31/44 8/30 0.001
10/48 10/48 1/32 1/32 0.05 0.05
21/46 4/30 0.007
CD34+
CD56+ CD7+
22/48 14/48 14/47 1/32 2/32 1/32 <0.0001 0.02 0.009
MDS patients had a significantly higher number of CD34+ (2.41 vs 0.66 respectively), CD34+ CD13+ CD117+ (1.76 vs 0.31) and CD34+/
S47
CD56+ cells (0.70 vs 0.02) and decrease of hematogones (0.04 vs 0.10) as non-clonal PB cytopenias. The median FCSS was significantly higher in MDS (4 vs 1) as well as our score (7 vs 2), but only our score presented a correlation with IPSS-R (r=0.30). In the univariate Cox analysis, were related to a shorter overall survival: IPSS-R (p=0.005), %CD34+ (p=0.001), %CD34+/CD13+/CD117+ cells (p=0.001), anomalous expression of %CD34+/CD7+ (p=0.004) and %CD34+/CD56+ cells (p=0.007), FCSS (p=0.015) and our score (p=0.008). In the multivariate analysis, considering isolated phenotypic features, only CD34+/CD7+ and CD34+/CD56+ cells were independent risk factors. In a model containing both scores, only the modified score was an independent risk factor. Considering both scores and IPSS-R, only IPSS-R was the independent risk factor. Conclusions: Abnormal co-expressions in CD34 cells are more specific of SMD. They had also a prognostic impact. Adding the number of these abnormal precursor cells to the FCSS increases its potential as prognostic factor. Supported: FAPESP, CNPq, MDS Foundation.
P-057 Myelodysplastic syndromes (MDS) with a deletion 5q display a specific immunophenotypic profile U. Oelschlaegel 1 , T.M. Westers 2 , B. Mohr 1 , S. Parmentier 1 , K. Sockel 1 , M. Bornhäuser 1 , A.A. Van de Loosdrecht 2 , U. Platzbecker 1 . 1 Medical Clinic and Policlinic I, University Hospital Dresden, Dresden, Germany; 2 Department of Hematlogy, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands Background: Patients (pts) with MDS harbouring a deletion of the long arm of chromosome 5 [del(5q)] often display characteristic morphological features in the bone marrow (BM). Introduction: Besides, 4-color flow cytometry (FCM) has become a valuable tool in the diagnostics of MDS, although it is still unknown, whether aberrations detected by FCM differ between certain cytogenetic subtypes. Purpose: The aim of this prospective study was to compare the immunophenotype of blasts, granulocytes, monocytes, and erythroid cells in MDS patients with and without del(5q). Materials and Methods: An 8-color-lyse-stain-wash FCM procedure was performed in a “test” (n=157) as well as a “validation” cohort (Dresden/Amsterdam n=40) of de novo-MDS [either with or without del(5q)], non-clonal cytopenias (n=90) and normal bone marrow (n=50). First, immunophenotype of single del(5q) and non-del(5q) MDS (30 vs. 88 pts) was compared measuring an antigen panel capable of scoring according to Ogata et al. 2009 and Wells et al. 2003, modified by Della Porta et al. 2012 and van de Loosdrecht et al. 2008. Thereafter, a parameter combination most representing MDS with del(5q) was assessed using ROC analyses. Additionally, thirty-one del(5q) pts were analyzed repeatedly during disease course. Results: Discovering a large amount of significant immunophenotypic differences in the test cohort, we built up a pattern of immunophenotypic characteristics able to distinguish between del(5q) and all other controls (non-del(5q) MDS, non-MDS pts) with a high sensitivity (82%) and specificity (91%). The del(5q)-specific FCM features included e.g. increased myeloid progenitors (>2%), aberrant CD7 expression (>15% of progenitors), normal CD10 (>25%) and CD71 (≤20%) on granulocytes, the absence of aberrant CD56 expression (≤20%) on monocytes, and decreased CD71 expression on erythroid cells. Second, we were able to set up the del(5q) diseasespecific FCM-score with a comparable robustness in the validation cohort (sensitivity 95%, specificity 94%). Most patients with del(5q) but a non-del(5q) immunophenotypic profile (12/16 pts) at diagnosis or during disease course showed a mutation in the TP53 gene or a del(17p). Of note, the disappearance of the del(5q) phenotypic char-
S48
Poster Presentations – 12th International Symposium on Myelodysplastic Syndromes / Leukemia Research 37 S1 (2013) S1–S117
acteristics could be shown in patients achieving a complete cytogenetic remission in response to lenalidomide. Conclusions: In summary, we demonstrate for the first time a strong association of a cytogenetic abnormality – del(5q) – with a specific immunophenotypic profile in the bone marrow. These data implicate the use of this method as rapid accessible tool for diagnosis. Its value in disease monitoring will be revealed in future studies.
P-058 Implementation of an extended Ogata Flow Cytometry Myelodysplasia Score at King’s College Hospital V. Tindell, T. Milne, A. Douri, G.J. Mufti, R.M. Ireland. Haematology, King’s College London, London, United Kingdom Background: The diagnosis of MDS is currently dependent on morphological assessment of the blood and bone marrow, especially when the blast count is low and cytogenetics are normal. Introduction: Multiparameter flow cytometry (FCM) is frequently routinely requested at initial investigation to add certainty to diagnosis of MDS and non clonal cytopenias. Purpose: The aid of FCM discriminatory value in the diagnosis of non clonal pathological controls (ncp) and RA/RCMD and unilineage dysplasia. Materials and Methods: From 06/2011 samples received in the diagnostic laboratory for FCM labelled query MDS were analysed using an extended Ogata score: • % CD34+ myeloblasts (mb) in all nucleated cells (nc) • % CD34+ B cell progenitors in all CD34+ cells • % Ly/MB CD45 ratio (mean of CD45 on lymphocytes divided mean of CD45 on CD34+ mb) • %Granulocyte/Lymphocyte side scatter median • % CD34+/11b+ mb nc • % CD34+/56+mb nc • % CD34+/15+ mb nc • % CD34+/7+ mb nc • % CD34+/19+ mb nc Clinical and pathological information was retrospectively gathered. Results: 300 samples collected so far have clinical and pathological
Graph 2. RAEBI+II vs non clonal pathological controls.
correlation, of them 66 patients had MDS and 40 patients had non clonal pathological controls. In the MDS group the mean age was 68 (range 27-90 years old) with the WHO categories RA (n=2) RARS (n=1) 5q- (n=1) RCMD (n=34) RCMD+RS (n=3) RAEB I (n=8) RAEB II (n=17). The non clonal pathological control group the mean age eas 62 (range 21-81 years old) with samples from normal transplanted marrow (n=3), aplastic anaemia (n=1) haemolytic anaemia (n=3) alcohol (n=3) immune (n=10)normal morphology (n=4) haematinic deficiency (n=2)reactive (n=4) metastatic carcinoma (n=2) drug induced (n=5) anaemia chronic disorder (n=4). Conclusions: The FCM parameters in the panel are cost neutral, easilyy reproducable, automated and simple to interpret in a busy diagnostic laboratory. The ROC curve hs specificity and sensitivity to discriminate RAEBI+II from ncp but was poor at discriminating RA/RARS/RCMD/5q- from ncp. The number of samples analysed was small and we are currently unable to discrimate low grade MDS and non clonal cytopenias with certainty. We intend to examine a weighted system scoring difference from normal for some parameters.
P-059 Distribution of clonal cells in flow cytometry-defined subpopulations of patients with myelodysplastic syndromes (MDS) M. Röhnert, U. Oelschlaegel, K. Sockel, S. Parmentier, M. Wermke, M. Von Bonin, C. Klotsche, C. Thiede, G. Ehninger, M. Bornhäuser, B. Mohr, U. Platzbecker. Department of Medicine I, University Hospital Carl-Gustav-Carus, Dresden, Germany
Graph 1. RA/RARS/5q-/RCMD vs non clonal.
Background: Patients (pts) with MDS display characteristic morphological and cytogenetic features in the bone marrow (BM). Introduction: Flow cytometry (FCM) has become a valuable tool in the diagnostics allowing for a distinct separation and identification of abnormal antigen expression (e.g. CD56 on myeloid progenitors). Purpose: It is still unknown whether FCM-defined aberrant cells are a predominant part of the clonal hematopoiesis in MDS. Materials and Methods: BM of 27 pts with del(5q) (IPSS low/int-
mined using ELISA. (3) CD25 expression was induced by retinoic acid (RA) in the MDS cell line F-36P. Then, the cells were treated with LMB-2 (Pseudomonas exotoxin A conjugated anti-CD25) 0.001 to 1000 ng/ml and subjected to a cell viability assay. Results: (1) Plasma sIL-2Ra levels were significantly elevated in patients with IPSS-high and AL-MDS compared with patients with IPSS-low and INT-1. (2) In univariate analysis, high levels of sIL2Ra (>1500 pg/ml) were associated with shorter overall survival in MDS patients (P=0.0001). Patients with high levels of sIL-2Ra (>1500 pg/ml) had higher percentages of bone marrow blasts and higher leukocyte counts compared with other patients. (3) In multivariate analysis, high levels of sIL-2Ra (>1500 pg/ml) was an independent prognostic factor for overall survival (P=0.0185). (4) CD25 expression was mainly observed on blasts, not on lymphocytes. Furthermore, percentages of CD25+ blasts correlated positively with plasma concentrations of sIL-2Ra (r=0.743, P=0.032). Moreover, when CD25positive blasts were cultured, sIL-2Ra concentrations in culture supernatants increased with the length of culture. (5) Untreated F-36P cells lacked CD25 expression, but 30% of RA-treated F-36P cells expressed CD25. Cell viability of RA-treated F-36P cells was inhibited by LMB-2 in a dose-dependent manner. Conclusions: Our data indicate that sIL-2Ra in the peripheral blood of MDS patients is derived from CD25 molecules expressed on blasts. Soluble IL-2Ra would be a useful prognostic biomarker in MDS. Moreover, CD25 molecules on MDS blasts are potential targets of future pharmacotherapy.
P-056 The significance of abnormal co-expressions of CD34+ for the diagnosis and prognosis in MDS Reis-Alves 1 ,
Rocha 2 ,
Pereira-Cunha 2 ,
Saad 1 ,
F.F. F.G. S.T.O. S.C. F. Traina 1 , I. Lorand-Metze 1 . 1 Internal Medicine, University of Campinas, Campinas, Brazil; 2 Hematology/Hemotherapy, University of Campinas, Campinas, Brazil Background: Immunophenotyping has been recognized as important for diagnosis of MDS. Introduction: Recently a flow score (FCSS) was described, that is useful for the differential diagnosis as well as prognosis. Purpose: We examined if the inclusion of quantitative values of abnormal expressions in CD34+ cells and monocytes in this index could improve its diagnostic and prognostic significance. Materials and Methods: Immunophenotypic analyzes were performed on bone marrow (BM) cells of patients with MDS and cases of non-clonal cytopenias. The two scores were applied to all cases. Controls: 41 normal BMs from BMT donors. The modified score covers 9 granulocytic, 8 monocytic and 6 CD34+ cell abnormalities. Antibody combinations: HLA-DR/CD14/CD45/CD33, CD16/CD11b/CD45/ CD13, CD13/CD117/CD45/CD34, CD10/CD19/CD45/CD34 and CD7/ CD56/CD45/CD34. Results: We analyzed 32 reactive (deficiency anemias and drug myelotoxicity) and 48 cases of MDS (mean age: 60 and 69 years respectively). WHO types: 6 RA, 30 RCMD, 5 RAEB-I and 7 RAEB-II. IPSSR: 5 very low risk, 12 low, 15 intermediate, 9 high and 3 very high risk. Table 1 Granulocytes series
Monocyte series
IMF Pattern HLA- CD56+ CD34+ HLASSC CD13/CD16 DR/CD33 DR/CD33 MDS 429 Reactive 522 p 0.001
25/48 4/32 <0.0001
31/44 8/30 0.001
10/48 10/48 1/32 1/32 0.05 0.05
21/46 4/30 0.007
CD34+
CD56+ CD7+
22/48 14/48 14/47 1/32 2/32 1/32 <0.0001 0.02 0.009
MDS patients had a significantly higher number of CD34+ (2.41 vs 0.66 respectively), CD34+ CD13+ CD117+ (1.76 vs 0.31) and CD34+/
S47
CD56+ cells (0.70 vs 0.02) and decrease of hematogones (0.04 vs 0.10) as non-clonal PB cytopenias. The median FCSS was significantly higher in MDS (4 vs 1) as well as our score (7 vs 2), but only our score presented a correlation with IPSS-R (r=0.30). In the univariate Cox analysis, were related to a shorter overall survival: IPSS-R (p=0.005), %CD34+ (p=0.001), %CD34+/CD13+/CD117+ cells (p=0.001), anomalous expression of %CD34+/CD7+ (p=0.004) and %CD34+/CD56+ cells (p=0.007), FCSS (p=0.015) and our score (p=0.008). In the multivariate analysis, considering isolated phenotypic features, only CD34+/CD7+ and CD34+/CD56+ cells were independent risk factors. In a model containing both scores, only the modified score was an independent risk factor. Considering both scores and IPSS-R, only IPSS-R was the independent risk factor. Conclusions: Abnormal co-expressions in CD34 cells are more specific of SMD. They had also a prognostic impact. Adding the number of these abnormal precursor cells to the FCSS increases its potential as prognostic factor. Supported: FAPESP, CNPq, MDS Foundation.
P-057 Myelodysplastic syndromes (MDS) with a deletion 5q display a specific immunophenotypic profile U. Oelschlaegel 1 , T.M. Westers 2 , B. Mohr 1 , S. Parmentier 1 , K. Sockel 1 , M. Bornhäuser 1 , A.A. Van de Loosdrecht 2 , U. Platzbecker 1 . 1 Medical Clinic and Policlinic I, University Hospital Dresden, Dresden, Germany; 2 Department of Hematlogy, Cancer Center Amsterdam, VU University Medical Center, Amsterdam, Netherlands Background: Patients (pts) with MDS harbouring a deletion of the long arm of chromosome 5 [del(5q)] often display characteristic morphological features in the bone marrow (BM). Introduction: Besides, 4-color flow cytometry (FCM) has become a valuable tool in the diagnostics of MDS, although it is still unknown, whether aberrations detected by FCM differ between certain cytogenetic subtypes. Purpose: The aim of this prospective study was to compare the immunophenotype of blasts, granulocytes, monocytes, and erythroid cells in MDS patients with and without del(5q). Materials and Methods: An 8-color-lyse-stain-wash FCM procedure was performed in a “test” (n=157) as well as a “validation” cohort (Dresden/Amsterdam n=40) of de novo-MDS [either with or without del(5q)], non-clonal cytopenias (n=90) and normal bone marrow (n=50). First, immunophenotype of single del(5q) and non-del(5q) MDS (30 vs. 88 pts) was compared measuring an antigen panel capable of scoring according to Ogata et al. 2009 and Wells et al. 2003, modified by Della Porta et al. 2012 and van de Loosdrecht et al. 2008. Thereafter, a parameter combination most representing MDS with del(5q) was assessed using ROC analyses. Additionally, thirty-one del(5q) pts were analyzed repeatedly during disease course. Results: Discovering a large amount of significant immunophenotypic differences in the test cohort, we built up a pattern of immunophenotypic characteristics able to distinguish between del(5q) and all other controls (non-del(5q) MDS, non-MDS pts) with a high sensitivity (82%) and specificity (91%). The del(5q)-specific FCM features included e.g. increased myeloid progenitors (>2%), aberrant CD7 expression (>15% of progenitors), normal CD10 (>25%) and CD71 (≤20%) on granulocytes, the absence of aberrant CD56 expression (≤20%) on monocytes, and decreased CD71 expression on erythroid cells. Second, we were able to set up the del(5q) diseasespecific FCM-score with a comparable robustness in the validation cohort (sensitivity 95%, specificity 94%). Most patients with del(5q) but a non-del(5q) immunophenotypic profile (12/16 pts) at diagnosis or during disease course showed a mutation in the TP53 gene or a del(17p). Of note, the disappearance of the del(5q) phenotypic char-
S48
Poster Presentations – 12th International Symposium on Myelodysplastic Syndromes / Leukemia Research 37 S1 (2013) S1–S117
acteristics could be shown in patients achieving a complete cytogenetic remission in response to lenalidomide. Conclusions: In summary, we demonstrate for the first time a strong association of a cytogenetic abnormality – del(5q) – with a specific immunophenotypic profile in the bone marrow. These data implicate the use of this method as rapid accessible tool for diagnosis. Its value in disease monitoring will be revealed in future studies.
P-058 Implementation of an extended Ogata Flow Cytometry Myelodysplasia Score at King’s College Hospital V. Tindell, T. Milne, A. Douri, G.J. Mufti, R.M. Ireland. Haematology, King’s College London, London, United Kingdom Background: The diagnosis of MDS is currently dependent on morphological assessment of the blood and bone marrow, especially when the blast count is low and cytogenetics are normal. Introduction: Multiparameter flow cytometry (FCM) is frequently routinely requested at initial investigation to add certainty to diagnosis of MDS and non clonal cytopenias. Purpose: The aid of FCM discriminatory value in the diagnosis of non clonal pathological controls (ncp) and RA/RCMD and unilineage dysplasia. Materials and Methods: From 06/2011 samples received in the diagnostic laboratory for FCM labelled query MDS were analysed using an extended Ogata score: • % CD34+ myeloblasts (mb) in all nucleated cells (nc) • % CD34+ B cell progenitors in all CD34+ cells • % Ly/MB CD45 ratio (mean of CD45 on lymphocytes divided mean of CD45 on CD34+ mb) • %Granulocyte/Lymphocyte side scatter median • % CD34+/11b+ mb nc • % CD34+/56+mb nc • % CD34+/15+ mb nc • % CD34+/7+ mb nc • % CD34+/19+ mb nc Clinical and pathological information was retrospectively gathered. Results: 300 samples collected so far have clinical and pathological
Graph 2. RAEBI+II vs non clonal pathological controls.
correlation, of them 66 patients had MDS and 40 patients had non clonal pathological controls. In the MDS group the mean age was 68 (range 27-90 years old) with the WHO categories RA (n=2) RARS (n=1) 5q- (n=1) RCMD (n=34) RCMD+RS (n=3) RAEB I (n=8) RAEB II (n=17). The non clonal pathological control group the mean age eas 62 (range 21-81 years old) with samples from normal transplanted marrow (n=3), aplastic anaemia (n=1) haemolytic anaemia (n=3) alcohol (n=3) immune (n=10)normal morphology (n=4) haematinic deficiency (n=2)reactive (n=4) metastatic carcinoma (n=2) drug induced (n=5) anaemia chronic disorder (n=4). Conclusions: The FCM parameters in the panel are cost neutral, easilyy reproducable, automated and simple to interpret in a busy diagnostic laboratory. The ROC curve hs specificity and sensitivity to discriminate RAEBI+II from ncp but was poor at discriminating RA/RARS/RCMD/5q- from ncp. The number of samples analysed was small and we are currently unable to discrimate low grade MDS and non clonal cytopenias with certainty. We intend to examine a weighted system scoring difference from normal for some parameters.
P-059 Distribution of clonal cells in flow cytometry-defined subpopulations of patients with myelodysplastic syndromes (MDS) M. Röhnert, U. Oelschlaegel, K. Sockel, S. Parmentier, M. Wermke, M. Von Bonin, C. Klotsche, C. Thiede, G. Ehninger, M. Bornhäuser, B. Mohr, U. Platzbecker. Department of Medicine I, University Hospital Carl-Gustav-Carus, Dresden, Germany
Graph 1. RA/RARS/5q-/RCMD vs non clonal.
Background: Patients (pts) with MDS display characteristic morphological and cytogenetic features in the bone marrow (BM). Introduction: Flow cytometry (FCM) has become a valuable tool in the diagnostics allowing for a distinct separation and identification of abnormal antigen expression (e.g. CD56 on myeloid progenitors). Purpose: It is still unknown whether FCM-defined aberrant cells are a predominant part of the clonal hematopoiesis in MDS. Materials and Methods: BM of 27 pts with del(5q) (IPSS low/int-