American Journal of Hematology 39:96-101 (1992)
Circulating CD34+ Cells: An Adverse Prognostic Factor in the Myelodysplastic Syndromes Sally A. Sullivan, Katherine A. Marsden, Raymond M. Lowenthal, David M. Jupe, and Michael E. Jones Department of Medicine, University of Tasmania (S.A.S., K.A.M.); Departments of Medical Oncology (R.M.L.) and Haematology (D.M.J.), Royal Hobart Hospital; and the Menzies Centre for Population Health Research (M.E.J.), Hobart, Tasmania, Australia
As part of an epidemiological survey of myelodysplastic syndromes (MDS) in southern Tasmania, 62 MDS patients identified over a 2 year period were tested for the presence of CD34, the human progenitor cell antigen (HPCA), in their peripheral blood. The results were correlatedwith transformation to acute myeloid leukemia(AML) and patient survival, and CD34+ status was compared as a prognostic indicator with Bournemouth score, cytogenetics, and CFU-GM colony growth which were also assessed. Circulating CD34+ cells were found in 23 of the 62 MDS patients; 9 of the 23 patients with circulating CD34+ cells transformed to AML, as compared with none of the 39 CD34 negative patients (P < 0.0001); and 11 of the 23 patients with circulating CD34+ cells were dead at the end of the 2 year period, as opposed to 6 of the 39 with no CD34+ cells (P < 0.03). The Bournemouth score was also significantly associated with transformation to AML (P < 0.0001) and poor survival (P < 0.04). These were the only significant associations of the possible prognostic factors studied with either transformation or survival. In summary, the presence of circulating CD34+ cells was significantly associated with both progression to AML and poor survival and was found to be a better prognostic indicator than cytogenetics or CFU-GM colony growth.
Key words: myelodysplastic syndromes, CD34, acute myeloid leukemia
INTRODUCTION
The myelodysplastic syndromes (MDS) comprise a group of hematological disorders classified by the French- American-British (FAB) group into five categories: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMML). These categories have in common bone marrow dysplasia with varying degrees of anemia, leucopenia and thrombocytopenia [I]. MDS was previously referred to as “preleukemia” and has been considered as essentially a premalignant condition which in many cases transforms to acute myeloid leukemia (AML). The incidence is greatest in the elderly and the clinical course variable with the median survival being 2 years [2]. A variety of laboratory features has been studied by a number of investigators in order to identify prognostic indicators. The prognostic features Often ‘Onsidered are FAB classification, blood count pardmeters, percentage of blasts in blood and marrow, presence of abnormal 0 1992 Wiley-Liss, Inc.
localizations of abnormal precursors (ALIPs), abnormal cytogenetics, in vitro marrow culture, and DNA ploidy [2]. Various scoring systems, such as the Bournemouth score [ 3 ] have been devised with the aim of providing a more objective assessment of the disease. While it is sometimes possible to assess the likely course of the disease in a given patient, with reference in particular to percentage of blasts in the marrow, FAB classification, and Bournemouth score, there are limitations with all of the prognostic indicators presently used and the course of the disease is often unpredictable. Some patients die shortly after diagnosis either from progression to leukemia or as result of pancytopenia, while in others the disease remains stable and fairly benign for
Received for publication April 3, 1991; accepted August 15, 1091. Address reprint requests to Mrs. Sally Sullivan, Department of Medicine, University of Tasmania, 43 Collins Street. Hobart, Tasmania, Australia 7000.
Circulating CD34+ Cells in MDS
years. Some patients only progress to AML 10 or more years after the diagnosis of MDS [4]. Guyotat et al. [5]have recently reported a significant association of the presence of CD34+ cells in bone marrow with both progression to leukemia and poor survival in MDS. Other studies of AML have linked CD34 expression in bone marrow with significantly lower remission rates [6-81. CD34 is the human progenitor cell antigen (HPCA) which is present on both unipotent and multipotent progenitor cells but not on normal mature peripheral blood cells. In this study we have tested 62 MDS patients for the presence of CD34+ cells in their peripheral blood and correlated the results with transformation to AML and survival. We have also compared CD34 expression as a prognostic indicator with Bournemouth score and with cytogenetics and bone marrow CFU-GM colony growth. MATERIAL AND METHODS Patients
The 62 MDS cases tested were identified as part of a Tasmanian epidemiological survey and informed consent was obtained in all cases. They represent 78% of all prevalent and incident cases of MDS identified in southem Tasmania from May 1988 through November 1990, by means of screening all blood counts performed in any hospital or private laboratory in the region. The population of the catchment area is about 210,000. The patients were diagnosed and classified according to the criteria used by the FAB Group [ 11 as amended by May et al. [9] to include a sixth category, idiopathic macrocytosis. Both primary and secondary MDS were included. The two secondary MDS cases had both been previously treated with chlorambucil.
97
lmmunofluorescence
Standard indirect immunofluorescence techniques were used using Anti-HPCA-1 (Becton Dickinson) and fluorescein conjugated sheep-anti-mouse F(ab'), fragments (FITC-SAM, Silenus); 75 p1 mononuclear cells at 1 X 107/ml were incubated with 10 p l anti-HPCA-1 for 5 min at room temperature (RT), washed twice in cold PBS-azide (4"C), and incubated with 10 p1 FITC-SAM for 5 min at RT. The cells were then washed three times with cold PBS-azide, air-dried on slides, fixed in ethanol, and mounted with buffered glycerol. Fluorescence was read on a Leitz Dialux EB 20 UV microscope and the presence of 1% or more CD34 cells was counted as positive. Only one of the 20 controls had an occasional positive cell (40 cells) used as controls. (normal range: 35-200 colonies, c1uster:colony ratio Cell Separation
Circulating CD34+ Cells: An Adverse Prognostic Factor in the Myelodysplastic Syndromes Sally A. Sullivan, Katherine A. Marsden, Raymond M. Lowenthal, David M. Jupe, and Michael E. Jones Department of Medicine, University of Tasmania (S.A.S., K.A.M.); Departments of Medical Oncology (R.M.L.) and Haematology (D.M.J.), Royal Hobart Hospital; and the Menzies Centre for Population Health Research (M.E.J.), Hobart, Tasmania, Australia
As part of an epidemiological survey of myelodysplastic syndromes (MDS) in southern Tasmania, 62 MDS patients identified over a 2 year period were tested for the presence of CD34, the human progenitor cell antigen (HPCA), in their peripheral blood. The results were correlatedwith transformation to acute myeloid leukemia(AML) and patient survival, and CD34+ status was compared as a prognostic indicator with Bournemouth score, cytogenetics, and CFU-GM colony growth which were also assessed. Circulating CD34+ cells were found in 23 of the 62 MDS patients; 9 of the 23 patients with circulating CD34+ cells transformed to AML, as compared with none of the 39 CD34 negative patients (P < 0.0001); and 11 of the 23 patients with circulating CD34+ cells were dead at the end of the 2 year period, as opposed to 6 of the 39 with no CD34+ cells (P < 0.03). The Bournemouth score was also significantly associated with transformation to AML (P < 0.0001) and poor survival (P < 0.04). These were the only significant associations of the possible prognostic factors studied with either transformation or survival. In summary, the presence of circulating CD34+ cells was significantly associated with both progression to AML and poor survival and was found to be a better prognostic indicator than cytogenetics or CFU-GM colony growth.
Key words: myelodysplastic syndromes, CD34, acute myeloid leukemia
INTRODUCTION
The myelodysplastic syndromes (MDS) comprise a group of hematological disorders classified by the French- American-British (FAB) group into five categories: refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMML). These categories have in common bone marrow dysplasia with varying degrees of anemia, leucopenia and thrombocytopenia [I]. MDS was previously referred to as “preleukemia” and has been considered as essentially a premalignant condition which in many cases transforms to acute myeloid leukemia (AML). The incidence is greatest in the elderly and the clinical course variable with the median survival being 2 years [2]. A variety of laboratory features has been studied by a number of investigators in order to identify prognostic indicators. The prognostic features Often ‘Onsidered are FAB classification, blood count pardmeters, percentage of blasts in blood and marrow, presence of abnormal 0 1992 Wiley-Liss, Inc.
localizations of abnormal precursors (ALIPs), abnormal cytogenetics, in vitro marrow culture, and DNA ploidy [2]. Various scoring systems, such as the Bournemouth score [ 3 ] have been devised with the aim of providing a more objective assessment of the disease. While it is sometimes possible to assess the likely course of the disease in a given patient, with reference in particular to percentage of blasts in the marrow, FAB classification, and Bournemouth score, there are limitations with all of the prognostic indicators presently used and the course of the disease is often unpredictable. Some patients die shortly after diagnosis either from progression to leukemia or as result of pancytopenia, while in others the disease remains stable and fairly benign for
Received for publication April 3, 1991; accepted August 15, 1091. Address reprint requests to Mrs. Sally Sullivan, Department of Medicine, University of Tasmania, 43 Collins Street. Hobart, Tasmania, Australia 7000.
Circulating CD34+ Cells in MDS
years. Some patients only progress to AML 10 or more years after the diagnosis of MDS [4]. Guyotat et al. [5]have recently reported a significant association of the presence of CD34+ cells in bone marrow with both progression to leukemia and poor survival in MDS. Other studies of AML have linked CD34 expression in bone marrow with significantly lower remission rates [6-81. CD34 is the human progenitor cell antigen (HPCA) which is present on both unipotent and multipotent progenitor cells but not on normal mature peripheral blood cells. In this study we have tested 62 MDS patients for the presence of CD34+ cells in their peripheral blood and correlated the results with transformation to AML and survival. We have also compared CD34 expression as a prognostic indicator with Bournemouth score and with cytogenetics and bone marrow CFU-GM colony growth. MATERIAL AND METHODS Patients
The 62 MDS cases tested were identified as part of a Tasmanian epidemiological survey and informed consent was obtained in all cases. They represent 78% of all prevalent and incident cases of MDS identified in southem Tasmania from May 1988 through November 1990, by means of screening all blood counts performed in any hospital or private laboratory in the region. The population of the catchment area is about 210,000. The patients were diagnosed and classified according to the criteria used by the FAB Group [ 11 as amended by May et al. [9] to include a sixth category, idiopathic macrocytosis. Both primary and secondary MDS were included. The two secondary MDS cases had both been previously treated with chlorambucil.
97
lmmunofluorescence
Standard indirect immunofluorescence techniques were used using Anti-HPCA-1 (Becton Dickinson) and fluorescein conjugated sheep-anti-mouse F(ab'), fragments (FITC-SAM, Silenus); 75 p1 mononuclear cells at 1 X 107/ml were incubated with 10 p l anti-HPCA-1 for 5 min at room temperature (RT), washed twice in cold PBS-azide (4"C), and incubated with 10 p1 FITC-SAM for 5 min at RT. The cells were then washed three times with cold PBS-azide, air-dried on slides, fixed in ethanol, and mounted with buffered glycerol. Fluorescence was read on a Leitz Dialux EB 20 UV microscope and the presence of 1% or more CD34 cells was counted as positive. Only one of the 20 controls had an occasional positive cell (40 cells) used as controls. (normal range: 35-200 colonies, c1uster:colony ratio Cell Separation
