Leukemia Research Vol. 14, No. 1, pp. 85--89, 1990. Printed in Great Britain.
0145--2126/90 $3.00 + .00 Pergamon Press plc
B O N E M A R R O W C U L T U R E S A N D P R O G N O S I S IN P R I M A R Y MYELODYSPLASTIC SYNDROMES MARCEL KORTHOUT, ROBRECHT DE BOCK, DIRK VAN BOCKSTAELE and MARC PEETERMANS Laboratory of Experimental Haematology, University of Antwerp, Wilrijk, Belgium
(Received 24 May 1989. Revision accepted 3 September 1989) Abstract--Bone marrow cultures and survival time were studied in 39 patients with primary myelodysplastic syndromes. We divided the patients into two groups according to the CFU-GM numbers on day 10: type I with low colony (CFU-GM < 30) and type II with normal to high colony formation (CFU-GM ~> 30). The median survival time was shorter for patients with an in vitro growth type II (5 months) than it was for patients with an in vitro growth type I (>36 months). No relation was found between growth types and FAB-type, Bournemouth score or initial karyotype. The initial bone marrow blast percentage correlated well with the in vitro growth number.
Key words: Myelodysplastic syndrome, CFU-GM, survival, prognosis.
were refractory anaemia (RA) in eight, refractory anaemia with ringed sideroblasts (RARS) in eight, refractory anaemia with excess of blasts (RAEB) in 12, RAEB in transformation (RAEB-t) in five and chronic myelomonocytic leukemia (CMML) in six patients. Laboratory data at diagnosis of these patients are shown in Table 1. Only a minority of patients was given chemotherapy and treatment had no significant effect on survival time [11]. There were 26 males and 13 females. The median age was 68 years (range 38-84).
INTRODUCTION THE MYELODYSPLASTIC syndromes (MDS) are a group of haematological disorders characterized by dysplasia in the h a e m a t o p o i e t i c cell lines [1]. The clinical course is extremely variable and a lot of efforts have been m a d e to define p a r a m e t e r s that predict prognosis [2, 3]. Beside bone m a r r o w morphology and k a r y o t y p e [4, 5, 6], bone m a r r o w cultures have attracted great interest [7-10], since the disturbed haematopoiesis in M D S patients is reflected in their in vitro bone m a r r o w growth patterns. Granulocyte-macrophage progenitors ( C F U - G M ) have been studied very extensively. We studied 39 patients with p r i m a r y M D S and classified them according to FAB-criteria. We distinguished two groups based on C F U - G M numbers. This system permits further separation of patients into good and p o o r prognostic groups within the existing classification and scoring systems.
Preparation of cells Bone marrow cells were obtained by aspiration into sterile tubes containing 2 ml Iscove's modified Dulbecco's medium (IMDM) (Gibco) with 200 IU preservative-free heparin. The cell suspensions were separated over Lymphocyte Separation Medium (LSM), density 1.077g/ml (Boehringer). The low density cell fraction was harvested and washed once with RPMI-1640 (Flow). CFU-GM assay CFU-GM were cultured in a medium containing 20% fetal calf serum (FCS), 10% conditioned medium from the human bladder carcinoma cell line 5637 [12] or 10% human placental conditioned medium (HPCM) as a source of CSF [13], 0.3% agar and IMDM in a fully humidified atmosphere at 37°C and 5% CO 2. Two replicates of 2 ml, each containing 1 x 105 bone marrow mononuclear cells (BM MNC) were plated into 35 mm Petri dishes. The culture dishes were examined under an inverted microscope for colony (/>40 cells) and cluster (3-39 cells) growth after 7 days of incubation, with an additional examination for colonies after 10 days. The CFU-GM classification in type I and type II was based on the number of colonies on day 10: above or below 30. Survival was calculated from the day of first bone marrow culture (i.e. presentation at the hospital) until death. The
PATIENTS AND METHODS
Patient population All patients met the diagnostic criteria for MDS according to the FAB Co-operative Group. Witheld diagnoses Abbreviations: MDS, myelodysplastic syndrome; CFUGM, granulocyte-macrophage colony forming unit; BM MNC, bone marrow mononuclear cells; FCS, fetal calf serum; HPCM, human placental conditioned medium; IMDM, Iscove's modified Dulbecco's medium. Correspondence to: M. Korthout, Haematology and Bloodtransfusion Unit, University of Antwerp (UZA), Wilrijkstraat 10, B-2520 EDEGEM, Belgium. 85
86
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0145--2126/90 $3.00 + .00 Pergamon Press plc
B O N E M A R R O W C U L T U R E S A N D P R O G N O S I S IN P R I M A R Y MYELODYSPLASTIC SYNDROMES MARCEL KORTHOUT, ROBRECHT DE BOCK, DIRK VAN BOCKSTAELE and MARC PEETERMANS Laboratory of Experimental Haematology, University of Antwerp, Wilrijk, Belgium
(Received 24 May 1989. Revision accepted 3 September 1989) Abstract--Bone marrow cultures and survival time were studied in 39 patients with primary myelodysplastic syndromes. We divided the patients into two groups according to the CFU-GM numbers on day 10: type I with low colony (CFU-GM < 30) and type II with normal to high colony formation (CFU-GM ~> 30). The median survival time was shorter for patients with an in vitro growth type II (5 months) than it was for patients with an in vitro growth type I (>36 months). No relation was found between growth types and FAB-type, Bournemouth score or initial karyotype. The initial bone marrow blast percentage correlated well with the in vitro growth number.
Key words: Myelodysplastic syndrome, CFU-GM, survival, prognosis.
were refractory anaemia (RA) in eight, refractory anaemia with ringed sideroblasts (RARS) in eight, refractory anaemia with excess of blasts (RAEB) in 12, RAEB in transformation (RAEB-t) in five and chronic myelomonocytic leukemia (CMML) in six patients. Laboratory data at diagnosis of these patients are shown in Table 1. Only a minority of patients was given chemotherapy and treatment had no significant effect on survival time [11]. There were 26 males and 13 females. The median age was 68 years (range 38-84).
INTRODUCTION THE MYELODYSPLASTIC syndromes (MDS) are a group of haematological disorders characterized by dysplasia in the h a e m a t o p o i e t i c cell lines [1]. The clinical course is extremely variable and a lot of efforts have been m a d e to define p a r a m e t e r s that predict prognosis [2, 3]. Beside bone m a r r o w morphology and k a r y o t y p e [4, 5, 6], bone m a r r o w cultures have attracted great interest [7-10], since the disturbed haematopoiesis in M D S patients is reflected in their in vitro bone m a r r o w growth patterns. Granulocyte-macrophage progenitors ( C F U - G M ) have been studied very extensively. We studied 39 patients with p r i m a r y M D S and classified them according to FAB-criteria. We distinguished two groups based on C F U - G M numbers. This system permits further separation of patients into good and p o o r prognostic groups within the existing classification and scoring systems.
Preparation of cells Bone marrow cells were obtained by aspiration into sterile tubes containing 2 ml Iscove's modified Dulbecco's medium (IMDM) (Gibco) with 200 IU preservative-free heparin. The cell suspensions were separated over Lymphocyte Separation Medium (LSM), density 1.077g/ml (Boehringer). The low density cell fraction was harvested and washed once with RPMI-1640 (Flow). CFU-GM assay CFU-GM were cultured in a medium containing 20% fetal calf serum (FCS), 10% conditioned medium from the human bladder carcinoma cell line 5637 [12] or 10% human placental conditioned medium (HPCM) as a source of CSF [13], 0.3% agar and IMDM in a fully humidified atmosphere at 37°C and 5% CO 2. Two replicates of 2 ml, each containing 1 x 105 bone marrow mononuclear cells (BM MNC) were plated into 35 mm Petri dishes. The culture dishes were examined under an inverted microscope for colony (/>40 cells) and cluster (3-39 cells) growth after 7 days of incubation, with an additional examination for colonies after 10 days. The CFU-GM classification in type I and type II was based on the number of colonies on day 10: above or below 30. Survival was calculated from the day of first bone marrow culture (i.e. presentation at the hospital) until death. The
PATIENTS AND METHODS
Patient population All patients met the diagnostic criteria for MDS according to the FAB Co-operative Group. Witheld diagnoses Abbreviations: MDS, myelodysplastic syndrome; CFUGM, granulocyte-macrophage colony forming unit; BM MNC, bone marrow mononuclear cells; FCS, fetal calf serum; HPCM, human placental conditioned medium; IMDM, Iscove's modified Dulbecco's medium. Correspondence to: M. Korthout, Haematology and Bloodtransfusion Unit, University of Antwerp (UZA), Wilrijkstraat 10, B-2520 EDEGEM, Belgium. 85
86
MARCEL KORTHOUT et al.
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