Characterization of clonogenic cells in refractory anemia with excess of blasts (RAEB-CFU): Response to recombinant hematopoietic growth factors and maturation phenotypes

Leukemia Research Vol. 13, No. 3, pp. 245-251, 1989. Printed in Great Britain.

0145-212~89 $3.00 + .00 Pergamon Press plc

CHARACTERIZATION REFRACTORY

ANEMIA

CFU)" RESPONSE GROWTH

OF CLONOGENIC WITH

EXCESS

TO RECOMBINANT

FACTORS

AND

CELLS

OF BLASTS

IN (RAEB-

HEMATOPOIETIC

MATURATION

PHENOTYPES

HARRY C. SCHOUTEN,* RUUD DELWEL,* FREEK J. BOT,* ANNE HAGEMEIJER,t I v o P. TOUW* and BOB LOWENBERG* *The Dr Daniel den Hoed Cancer Center, Rotterdam and tDept of Cell Biology and Genetics, Erasmus University, Rotterdam, The Netherlands (Received 21 September 1988. Accepted 24 October 1988) Abstract--The proliferative and maturation abilities of bone marrow progenitors in patients with

refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T) have previously been investigated in vitro using impure sources of colony stimulating activity. Here we report studies that were concerned with defining growth factor responses of RAEB progenitors (RAEB-CFU) in colony culture using pure hematopoietic growth factors. Marrow cells of 10 RAEB patients were cultured with recombinant IL3, GM-CSF, G-CSF, M-CSF and EPO. Factor dependent colony growth of four patients was examined in detail cytologically. The analysis revealed notable deficiencies in the colony forming spectrum as compared with normal marrow: although granulocytic colonies were formed in all of these four RAEB cases, macrophage colonies could not be induced in 1/4 cases and eosinophilic and erythroid colony formation could not be propagated in 2/4 cases with the proper stimuli. These findings are indicative of the intrinsic incapabilities of RAEB-CFU to mature along certain differentiation pathways in response to the growth factors. We then determined the surface phenotypes of RAEB-CFU using MoAbs Vim-2 (myelomonocytic) and BI3C5 (CD34) following dual labeling and fluorescence activated cell sorting and subsequent culture of the separately sorted BI3C5+/Vim-2 +, BIC5+/Vim-2 -, BI3C5-/Vim-2 ÷ and BIC5-/Vim-2- cells. In normal marrow most clonogenic cells were recovered from the BI3C5+/Vim-2 - fraction. In contrast, in RAEB marrow increased proportions of the colony forming cells were BI3C5+/Vim-2 +, BI3C5-/Vim-2 ÷, or BI3C5-. The altered distribution of surface immunophenotypes of RAEB-CFU provides further evidence for the imbalance of maturation in the progenitor cell compartment. The results are discussed in view of the concept that the inabilities of the RAEB hematopoietic precursors to mature in response to the hematopoietic growth factors are partial and variable, but may culminate in a progressive loss of the differentiation competence of the progenitors when leukemia evolves. Key words: RAEB-CFU, CSFs, immunophenotyping.

INTRODUCTION

kemia (AML), and are therefore considered preleukemic conditions. The in-vitro growth characteristics of the MDS-colony forming cells (MDS-CFU) have previously been examined in colony systems containing conditioned media as impure sources of hematopoietic growth factors (HGFs) [2]. Recently several human H G F s have been produced by recombinant D N A techniques. The in-vitro activities of these molecules, i.e. interleukin-3 (IL3), erythropoietin (EPO), granulocyte-macrophagecolony stimulating factor (GM-CSF), granulocyteCSF (G-CSF) and macrophage-CSF (M-CSF) on normal bone marrow progenitor cells have been reported [3-8]. H e r e we present experiments that were concerned with investigating the response of R A E B - C F U to the

THE MYELODYSPLASTICsyndromes comprise a group of diseases characterized by the restricted ability of the hematopoietic precursor cells to mature, resulting in cytopenia, cellular dysfunction and morphological abnormalities of bone marrow cells [1]. Refractory anemia with excess of blasts and R A E B in transformation ( R A E B - T ) represent two subtypes of MDS that frequently evolve to acute myeloid leuAbbreviations: MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess of blasts; CFU, colony forming unit; IL, interleukin; CSF, colony stimulating factor; EPO, erythropoietin. Correspondence to: Dr B. L6wenberg, The Dr Daniel den Hoed Cancer Center, P.O. Box 5201, 3008 AE Rotterdam, The Netherlands. 245

246

HARRY C. SCriOUTENet al.

r e c o m b i n a n t H G F s in o r d e r to assess t h e i r p r o liferative a n d m a t u r a t i o n c a p a c i t i e s as c o m p a r e d to n o r m a l m a r r o w C F U . In a d d i t i o n we e m p l o y e d two m o n o c l o n a l a n t i b o d i e s t h a t h a v e p r e v i o u s l y b e e n useful in s u r f a c e m a r k e r analysis of h u m a n A M L p r e c u r s o r s in o r d e r to c o m p a r e the p h e n o t y p e s of RAEB-CFU with t h o s e o f the n o r m a l m a r r o w precursors.

MATERIALS

AND METHODS

Patients Bone marrow cells of seven patients with R A E B and three with R A E B - T were studied (Table 1). In control experiments bone marrow specimens from hematologically normal adults were used after informed consent.

background (negative) fluorescence. Analysis and cell sorting were performed under sterile conditions using a FACS 440 (Becton-Dickinson, Mountain View, CA) at a maximum rate of 2000 cells/s as described [13]. The cells were separated into Vim-2+/BI3C5 +, Vim-2+/BI3C5 - , V i m - 2 - / BI3C5 ÷ and Vim-2-/BI3C5- fractions and then cultured (for example see Fig. 1). Recombinant H G F preparations Recombinant human IL-3, GM-CSF, G-CSF and MCSF preparations were generously provided by Genetics Institute (Cambridge, MA) and I1-1cr by Immunex (Seattle, WA). EPO was purchased from Kirin-Amgen (Thousand Oaks, CA). The structure and biological characteristics of these H G F s have been reported [3-8, 14]. These factors were added to the cultures in optimal concentrations, i.e. IL-3 3% v/v, GM-CSF 100 U/ml; G-CSF and M-CSF 0.1% v/v; EPO 1 U/ml; and IL-lol 1000 U/ml.

Preparation o f cells Bone marrow aspirates were collected in Hank's balanced salt solution (HBSS) with heparin and separated over a Ficoll gradient (Nycomed, Oslo, Norway). In two patients marrow mononuelear cells were obtained after bovine serum albumin (BSA) density gradient centrifugation. In addition, contaminating T-lymphocytes were removed from the cells of two patients following Ficoll gradient by E-rosette sedimentation as described [9, 10]. The mononuclear cells were cryopreserved and stored in liquid nitrogen. Fluorescence activated cell sorting The cells were double labeled with the monoclonal antibody (MoAb) BI3C5 (CD34, IgG1 Sera-lab, Crawley Down, U.K.) [11] at a final dilution of 1:100 and Moab Vim-2 (IgM, reactive with myelomonocytic cells; kindly provided by Dr W. Knapp, Vienna, Austria) at a 1:50 dilution [12]. Biotinilation of Vim-2 was performed in 0.2 M borate buffer, p H 8.5. A 1-ml disposable syringe was filled with Sephadex G25 (Pharmacia, Uppsala, Sweden) in borate buffer. Glass wool was used to plug the bottom hole of the syringe. The syringe was placed in a 50-ml falcon tube (Becton-Dickinson) and then centrifugated for l m i n at 700g, to settle the gel. A 100Hxl sample, containing 1 mg/ ml protein, was applied on top of the gel and the syringe, placed in a second 50-ml tube, was centrifugated again. The sample was collected and the M o A b was biotinilated following the addition of 10 Ixl (1 mg/ml) N-hydroxy-succinimidobiotin (NHS-biotin, Sigma, St Louis, MO) in DMSO and gently stirring for 3 h at room temperature. Excess NHS-biotin was removed by centrifugation as described above but with sephadex-G25 in phosphate buffered saline (PBS). The cells were incubated with the two MoAbs in phosphate buffered saline (PBS) with 5% fetal calf serum (FCS) for 30 min on ice. After washing in PBS and 5% FCS, the cells were further incubated with IgG1 subclass specific goat-anti-mouse-FITC (GAM-IgG1-FITC, Nordic; Tilburg, The Netherlands) at a dilution of 1 : 40 and avidin phycoerythrin (Becton-Dickinson, Mountain View, CA) for another 30 min on ice. The cells were then washed twice and resuspended in PBS at a concentration of 106 nucleated cells per ml. A control sample was incubated with G A M - I g G 1 - F I T C and avidin phycoerythrin to assess

unlabeled

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LOG FLUORESCENCEINTENSITY FIG. 1. Double labelling with MoAbs BI3C5(CD34) and Vim-2 and cell sorting. Fluorescence histograms of marrow cells of a patient with R A E B - T (P5) after CD34 and GAMIgG 1-FITC staining (upper diagram) and biotinilated Vim2 and avidin phycoerythrin labeling (lower diagram). Background fluorescence (negative) is indicated by the dotted curves and was determined following incubation with GAM-IgG1-FITC (upper diagram) or incubation with avidin phycoerythrin (lower section) alone. The inset shows an example of the dot plot analysis of the labelled cells and the fractions into which they have been separated. The cells were sorted in the four fractions according to the broken lines. The four phenotypically distinct fractions thus obtained were cultured with the complete cocktail of growth factors, i.e. IL-3, GM-CSF, G-CSF, M-CSF and EPO.

RAEB and recombinant CSFs

Clonogenic assays Cells (0.5 x 105) were cultured in a 1-ml mixture of Iscove's modified Dulbecco's medium (IMDM) (Gibco, Paisley, UK), 1.1% methylcellulose, 30% heparinized plasma of normal AB donors, bovine serum albumin (BSA), transferrin, lecithin, sodium selenite and 2mercaptoethanol. Duplicate dishes were incubated at 37°C and 100% humidity in an atmosphere of 5% CO 2 in air. Colonies (consisting of more than 50 cells) were scored between days 12 and 15. Individual colonies from six patients and the normal subjects were plucked from the plates with a finely-drawn Pasteur pipette, transferred to a cytocentrifuge slide, stained with May-Gri~nwald-Giemsa, and examined cytologically by differential counting.

Cytogenetic analysis The cells from two patients with clonal chromosomal abnormalities were examined cytogenetically after 5 days of suspension culture to document proliferation of the MDS cell clone. Spread metaphases of the cultured cells were examined with Q-, R- and G-banding techniques for cytogenetic abnormalities [10]. RESULTS

Colony forming capacity of RAEB-CFU in response to the combined CSFs The colony forming capacity of R A E B marrow was determined in 10 patients following stimulation with a cocktail of IL-3, GM-CSF, G-CSF, M-CSF and E P O in comparison with that of normal bone marrow (Table 1). In three patients (P3, P4, P7) no or minimal numbers of colonies appeared in vitro. Considerable numbers of colonies arose in culture following stimulation of the marrow cells of the seven other cases of R A E B .

247

Colony formation of RAEB-CFU in response to individual CSFs The bone marrow cells from four patients (P1, P2, P8, P10) were then cultured with each of the individual CSFs, and colony numbers and colony types (cytological analysis) were assessed in comparison to the results of a series of normal marrow cultures (Fig. 2). IL-3 failed to induce considerable numbers of colonies in P8. Mainly granulocytic colonies were produced in P1 whereas no eosinophilic colonies were formed in P2 and P10. In contrast Ficoll-Isopaque isolated normal marrow cells gave rise to significant numbers of granulocytic, monocytic and eosinophilic colonies upon IL-3 stimulation. In response to GM-CSF in P1 and P8 no monocytic colonies were formed, whereas in P2 and P10 no eosinophilic colonies and in P1 and P8 no GM-colonies grew. The response of R A E B marrow to GCSF revealed the formation of granulocytic colonies, exactly as was seen following G-CSF stimulation of normal marrow. After stimulation with M-CSF, in only two cases (P2, P10) considerable numbers of monocytic colonies appeared in culture while in the other two cases G M and G (P1) or no colonies (P8) were generated. In contrast to the normal marrow response granulomonocytic or granulocytic colonies were induced by M-CSF in P10. Finally, the marrow cells incubated in vitro with E P O produced erythroid colonies in two cases only (P2, P10). When no growth factors were added the cells from two patients (P1 and P2) showed colony formation.

TABLE 1. IN VITROCOLONYFORMATIONOF MARROWCELLSOF 10 MDS PATIENTS AND FOURHEALTHYDONORSIN RESPONSETO A COCKTAILOF IL-3, GM-CSF, GCSF, M-CSF AND EPO Patient/Normal P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 N1 N2 N3 N4

Diagnosis*

Colony numberst

RAEB-T RAEB RAEB RAEB RAEB-T RAEB RAEB RAEB RAEB after M. Hodgkin RAEB-T Normal marrow Normal marrow Normal marrow Normal marrow

1170 86 20 0 1460 368 5 2215 3175 >2000:~ 360 603 891 514

* According to FAB classification (see [1]). t Expressed per 105 plated Ficoll or BSA separated bone marrow cells; colonies were scored on days 12-15 of culture and contained at least 50 cells. ~: Also IL-lol (1000 U/ml) included in the mixed growth factor supplement.

248

HARRYC. SCHOUTENet al. P1

P2

P8

Normal

P10

1L 3 (7)

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(192)

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NONE •

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FIG. 2. Colony formation in vitro of RAEB marrow following stimulation with the single growth factors IL-3, GM-CSF, G-CSF, M-CSF, or EPO. The length of the bars indicate the relative proportions of GM-, G-, M-, Eo- or E-colonies grown from the marrow specimens of the four patients with RAEB or RAEB-T (P1, P2, P8, P10). The total numbers of colonies formed per l0 s plated cells are given in parentheses. For comparison the results of the normal bone marrow experiments, i.e. relative proportions of colony types, are also shown. The mean colony numbers +--S.D. and the number of experiments are indicated.

In P1 GM, G and M colony types and in P2 limited numbers of M-colonies appeared in culture. The normal control marrow specimens did not exhibit spontaneous growth (n = 10).

Surface phenotypes of RAEB-CFU: expression of C D 3 4 and Vim-2 antigens

The surface i m m u n o p h e n o t y p e s of R A E B - C F U were determined following dual labelling with the

M o A b s BI3C5 (CD34) and Vim-2, employing the marrow cells of six patients and two normal controls. The cells were separated into four phenotypically distinct fractions (BI3C5+/Vim-2+; BI3C5+/Vim-2-; B I 3 C 5 - / V i m - 2 +, and B I 3 C 5 - / V i m - 2 - ) (see Fig. 1) and then plated in culture supplemented with a complete mixture of IL-3, GM-CSF, G-CSF, M-CSF and EPO. In the normal marrow the major portion, i.e. 75%

RAEB and recombinant CSFs

colony number

P1

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FIG. 3. Expression of Vim-2 and BI3C5 (CD34) antigens on RAEB-CFU. The cells were sorted in the four fractions as indicated in Fig. 1. Total colony number indicates the sum of colonies recovered from the four separate fractions and is set at 100%. The length of the bars indicates the percentage of RAEB-CFU, recovered from each of the fractions, i.e. BI3C5÷/Vim-2 -; BI3C5-/Vim-2-; BI3C5-/ Vim-2÷; BI3C5+/Vim-2 +. The distribution of different colony types within each fraction is presented graphically in the bars. Data of six patients (P1-P7) and two normal subjects (N1, N2) are given. or more, of the colony forming cells were recovered from the BI3CS+/Vim-2 - fraction and minor proportions (less than 25%) of those distributed among the other three, i.e. BI3CS+/Vim-2 +, BI3CS-/Vim2 +, and BI3C5-/Vim-2- populations (Fig. 3). It appeared that the hematopoietic progenitor cells with the latter three surface phenotypes were increased in the R A E B patients. Increased proportions of the R A E B colony forming cells were shown to be BI3CS+/Vim-2 + (P1, P2, P5, P6), BI3CS-/Vim-2 + (P6) or BI3C5 /Vim-2- (P1, P3, PS, P6, PT). Thus, in these patients the phenotypic distributions of the hematopoietic precursors had significantly altered as compared to normal. The colonies were collected from the plates for cytological analysis in order to differentiate these colonies as G E M M (mixed myelo-mono-erythroid), GM, G, M, Eo or E types. This allowed to assess the phenotypic distributions for each of the cell lineage progenitors (Fig. 3). The relative distribution of these

precursors in the patients exhibited a great variability. It was evident that large proportions of R A E B - C F U - G M , RAEB-CFU-G, RAEB-CFU-M, R A E B - C F U - E o and RAEB-BFU-E expressed surface phenotypes that were discrepant from those of their normal counterparts. C F U - G E M M were not demonstrated in marrow cultures of any of the R A E B patients.

Cytogenetic analysis In one patient (P9), the cells incubated with each of the single CSFs were karyotyped after suspension culture. The cells grown following stimulation with any of those CSFs revealed cytogenetic abnormalities, confirming the outgrowth of cells belonging to the R A E B cell clone (Table 2). In a second patient (P3) the cells from the unseparated and the four sorted fractions cultured with the cocktail of the four CSFs plus EPO, were examined cytogenetically. Cells from each of BI3C5+/Vim-2 +,

250

HARRY C. SCHOUTEN et al. TABLE 2. CYTOGENETIC ANALYSIS OF I N - V I T R O CULTURED BONE MARROW CELLS FROM PATIENTS WITH R A E B

Cultured cells and culture conditions

Abnormal mitosis after culture*

unsorted fractiont Vim-2+/BI3C5 + Vim-2+/BI3C5 Vim-2-/BI3C5 + Vim-Z-/BI3C5 IL-35 GM-CSF G-CSF M-CSF

9/11" 7/7 4/4 15/15 8/13 16/16 15/16 16/16 16/16

Cytogenetic abnormality P3 45, XY, 5p-, -7, -19, -21, +M1, +M2

P9 45, XX, - 7 , t (10;18)

* Quotient of the number of metaphases with cytogenetic abnormality of the total number of metaphases analysed. t Unseparated and four sorted fractions were inoculated in cultures supplemented with a cocktail of IL-3, GM-CSF, G-CSF, M-CSF and EPO for karyotypic analysis. $ Mononuclear bone marrow cells were incubated with either one of four different CSFs to perform cytogenetic analysis.

BI3C5+/Vim-2 -, BI3C5-/Vim-2 + and BI3C5-/Vim2- fractions revealed the cytogenetic abnormality in all or the majority of mitoses (Table 2). DISCUSSION The in-vitro proliferation abilities of MDS precursors have previously been investigated in the presence of impure stimulating factors or conditioned media [2]. H e r e we report on the proliferative and maturation abilities of hematopoietic precursors in patients with MDS (seven with R A E B and three with R A E B - T ) in response to the recombinant colony stimulating factors and erythropoietin. We demonstrate that in four patients with R A E B the hematopoietic cells had lost their abilities to form colonies of certain differentiation lineages following stimulation with the appropriate factors (Fig. 2). Of note was the absence of CFU-M in one case (P1), B F U - E in two patients (P1, P8), C F U - E o in two other patients (P2, P10). These gaps in the spectrum of colony forming cells may reflect specific cellular alterations and suggest deficient abilities of immature precursors stages to differentiate along certain lineages. In contrast to acute myeloid leukemia (AML), R A E B - C F U had retained the ability to form colonies of mature granulocytes or eosinophils indicating that maturation competence of the cells along particular pathways had remained intact. These data would fit a model in which maturation defects affect restricted hematopoietic precursors in R A E B that progress to involve a broader scale of progenitors when A M L evolves.

The immunophenotypical characteristics of the clonogenic cells varied among the patients with R A E B . These phenotypes often differed from those of normal marrow progenitors. In the normal controls the majority of hematopoietic progenitors are known to be BI3C5+/Vim-2 - and minor subpopulations of clonogenic cells with other phenotypes coexisL In R A E B marrow a similar subset of clonogenic cells with the BI3C5+/Vim-2 phenotype is demonstrable but in general the population of progenitor cells exhibit phenotypes significantly shifted towards BI3C5+/Vim-2 +, BI3C5 /Vim-2- and BI3C5-/Vim-2 + compartments. These discrepant surface phenotype distributions of the hematopoietic progenitors of R A E B marrow are comparable to those seen in A M L [15-17]. Apparently, abnormal maturation of both human A M L and R A E B progenitors results in an expansion in the number of progenitor cells with uncommon phenotypes. This p h e n o m e n o n is suggestive of a maturation arrest at the progenitor cell level and may reflect a relative accumulation of progenitor cell types in the bone marrow of patients with R A E B that are normally infrequent in the marrow. The findings reported here are indicative of MDS being a condition which is intermediate between normal and AML. While the concurrent expression of early and late maturation surface antigens upon the R A E B - C F U and a loss of maturation responsiveness of certain progenitor cell stages to the proper CSFs is typical of A M L , other lineage precursors in R A E B appeared normally stimulable to proliferate and mature to terminally differentiated cells. One may

RAEB and recombinant CSFs assume that progressive changes develop that ultimately result in an extended maturation arrest and that these alterations beyond a critical level mark the difference between R A E B and A M L .

7.

Acknowledgements--This work was supported by The Netherlands Cancer Foundation "Koningin Wilhelmina Fonds". The technical assistance of Ruud van Gurp, Loes van Eijk, Pauline Schipper, Lianne Broeders, Hans Hoogerbrugge and Carin van Buitenen is appreciated. We thank Aloysia Sugiarsi for preparing the manuscript.

8.

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10.

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