Leukemia Research Vol, 14, No. 3, pp. 247-253, 1990. Printed in Great Britain.

CLONAL FROM

GROWTH

0145-2126/90 $3,00 + .00 Pergamon Press plc

OF HAEMOPOIETIC

MYELODYSPLASTIC RECOMBINANT

MARROW

PROGENITOR

CELLS

IN RESPONSE

TO

HAEMOPOIETINS

PAUL BAINES, DAVID BOWEN and ALLANJACOBS Department of Haematology, University Hospital of Wales, Cardiff, U.K.

(Received 21 August 1989. Accepted 30 September 1989) Abstract--The growth factor requirements of granulocyte-macrophage (GM) and erythroid marrow progenitor cells from 12 myelodysplastic (MDS) patients have been analysed. GM progenitors from two of six patients who grew normal numbers of colonies in response to conditioned medium + erythropoietin (5637CM + Epo) showed defective responses to either GMCSF and/or IL-3. Of all the recombinant factors tested (IL-3, IL-1, GCSF, GMCSF, MCSF), GMCSF was the strongest stimulator of myeloid clonal growth, inducing normal numbers of GM colonies from marrow of six patients (two of whom were neutropenic). Erythroid colonies were low in 5637CM + Epo-supplemented cultures of marrow from all but one patient and remained poor in the presence of any of the haemopoietins tested. Supraoptimal doses (for normal marrow) of these haemopoietins improved colony growth in only one patient (GM colonies in response to IL-3). Combinations of factors were also largely ineffective at raising myeloid or erythroid colony numbers. These data indicate that the defective response of MDS progenitor cells to growth factors is not amenable to experimental manipulation of recombinant factor levels or combinations. Clonal assays might suggest a role for GMCSF therapy in a subpopulation of neutropenic MDS patients but their potential now needs to be evaluated in association with clinical trials.

Key words: Myelodysplastic syndrome, clonogenic cells, recombinant growth factors.

INTRODUCTION

granulocyte-macrophage colony stimulating factor (GMCSF) or granulocyte (G) CSF might be beneficial, additional treatment where neutropenia is an extra complication. Interleukin 1 (IL-1) can augment receptors for other CSFs on early progenitor cells [5] and might move MDS progenitors to a stage where they could respond to other factors. Evidently there is a need for a test to determine appropriate therapy in individual patients. Semisolid, clonal assays provide one way of assessing the responses of haemopoietic cells to haemopoietins. The effect of growth factors in vitro might predict the efficacy of sustained systemic growth factor levels. Consequently we have investigated the in-vitro growth response of MDS marrow progenitor cells to the recombinant factors GMCSF, GCSF, IL-3, IL-1 and macrophage (M) CSF either alone or in combinations. The study has also helped to answer three specific questions: (i) Which factors induce the (albeit limited) colony formation previously observed in cultures supplemented with conditioned media? (ii) Can high concentrations, or combinations, of these factors improve clonal growth? (iii) Do those patients with apparently normal growth in the presence of conditioned medium (5637CM) and erythropoietin

THERAPEUTIC regimes for myelodysplasia [1] have frequently involved the use of agents likely to overcome the differentiation block manifested by the abnormal clone of stem cells which comes to dominate haemopoiesis in this disorder. Recent attention has focused on the use of recombinant haemopoietins [2]. While these may not exhaust the preleukemic clone, they do show early promise in alleviating the peripheral cytopenias [3, 4] which should diminish the incidence of infection, reduce bleeding and correct the anaemia. Which particular haemopoietin, or combination of haemopoietins, should be administered to a particular patient would seem to depend on exactly which lineages are affected. For example, a patient presenting with anaemia might require only interleukin 3 (IL-3) or erythropoietin (Epo), whereas

Abbreviations: MDS, myelodysplastic; G, granulocyte; M, macrophage; CSF, colony-stimulating factor; Epo, erythropoietin; 1L, interleukin; 5637CM, medium conditioned by 5637 cells. Correspondence to: Dr Paul Baines, Department of Haematology, University Hospital of Wales, Cardiff CF4 4XW, U.K. 247

248

P. BAINES et al. TABLE 1. CLINICAL AND HAEMATOLOGICAL DATA ON PATIENTS AT TIME OF EXPERIMENTATION

Sex

Age

Hb g/dl

WBC x 109/1

Mon x 109/1

Neu x 109/1

Plat x 109/1

MCV fl

Marrow blasts

1

F

2 3 4 5 6 7 8 9 10 11 12

M M M F M M F F M M M

39 73 72 58 34 82 79 22 83 66 68 79

12.2 7.6 12.3 7.1 12.3 8.2 10.5 8.8 10.5 6.0 10.5 9.3

2.0 3.1 2.0 2.6 3.7 21.8 3.7 2.9 8.1 1.8 1.6 4.4

0.12 0.03 0.01 0.18 0.1 1.09 0.7 0.18 0.3 0.05 0.08 0.35

0.5 2.23 0.82 1.61 1.7 17.66 0.78 1.49 4.37 1.22 0.91 2.11

315 71 204 108 84 444 140 52 420 38 210 211

90 90 78 92 107 93 96 110 86 77 102 92

1 0 16 0 3 0 3 1 0 2 21 0

Patient

Diagnosis* RA SA RAEB SA RA SA RA RA SA RA RAEB-t SA

WBC, white blood cells; Mon, monocytes; Neu, neutrophils; Plat, platelets; MCV, mean cell volume. * Diagnosis at time of first presentation.

( E p o ) , s h o w d e f e c t i v e g r o w t h in r e s p o n s e to individual factors? METHODS

Normal marrow donors Marrows from haematologically normal donors were obtained, with informed consent, from the sternum of patients underoing cardiac surgery or from donors for allogeneic marrow transplant.

MDS patients All patients studied fulfilled the diagnostic criteria defined by Bennett et al. [6]. Marrow aspirates were obtained during routine diagnostic sampling from the iliac crest or sternum. Their clinical presentations are given in Table 1.

Recombinant factors GMCSF was kindly provided by Dr Gillis, Immunex, Seattle at a specific activity of >4 × 107 u/mg protein and was diluted to 500 ng/ml in single strength Iscove's modified Dulbecco's medium (IMDM, Gibco) containing 1% deionised bovine serum albumin (BSA fraction V, Sigma). GCSF and MCSF were generously provided by Dr S. C. Clark, Genetics Institute, Cambridge, M A as CHO conditioned media for suggested dilution of 1:30,000 and 1:3,000-30,000 for half-maximal colony formation, respectively. Both preparations were diluted 1:100 in IMDM + 1% BSA for use in culture. IL-3 (Genetics Inst.) was obtained as a 0.37 mg/ml solution at a specific activity of 2-4 x 106 u/ml and further diluted in I M D M + 1% BSA to 500 ng/ml for use in culture. IL-1 was given by Roche Products Limited (Welwyn, Herts, U.K.) as a 107 units/ mg solution and was diluted in I M D M + 1% BSA to 104 units/ml for use in culture.

to 1 ml of IMDM (Gibco) containing 0.3% Noble Agar (Difco), 1% deionised B SA (Sigma), 20% foetal calf serum (Imperial Labs), 5 x 10 -5 M fl-mercaptoethanol, 2.5-5% RPMI-1640 medium (Flow Labs) conditioned by 5637 bladder carcinoma cells (5637CM) and 2 u/ml ofTFL-1 erythropoietin (Epo, Terry Fox Labs). Cultures were performed in duplicate and incubated at 37 ° for 14 days in a fully humidified air atmosphere containing 5% CO2.

Scoring colonies Myeloid and erythroid colonies (>50 cells) and clusters (20-50 cells) were scored in the same dish using a dissecting microscope. Red (haemogtobinised) clones were scored as erythroid.

Statistics Probabilities were determined by the non parametric Mann-Whitney or 'paired' Wilcoxon tests.

Calculation of confidence limits for variation between duplicates In order to determine whether colony numbers in the presence of supraoptimal dose haemopoietin or combinations of haemopoietins, differed significantly from colony numbers in optimal dose haemopoietin/single haemopoietins, we employed the following statistical procedure which enabled us to examine individual patient's responses. Confidence limits for the reproducibility of the assay system (Tennant & Jacobs, unpublished data) were constructed from a series of 95% confidence intervals calculated from the difference between duplicate counts having a common mean value. These subpopulations were drawn from results of several hundred duplicates having mean values between 2 and 90 counts per dish. The composite confidence limits (-+ 1.96 S.E.) are shown in Fig. 2. Where combined data is shown (Fig. 3) the confidence intervals were derived from standard errors based on twice the variance. RESULTS

Colony Assays Marrows were collected into heparinized (15u/ml) Hepes-buffered, modified Eagle's medium (MEM, Wellcome, U.K.). Bony particles were teased apart with a scalpel and flushed with medium. The cells were washed once and resuspended in MEM before addition of 105 cells

Growth of granulocyte-macrophage (GM) progenitors from normal and M D S marrows in the presence of recombinant haemopoietins Each

of t h e r e c o m b i n a n t

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FIG. 1. A. Myeloid (GM) colonies; B. Erythroid colonies; in cultures supplemented with different haemopoietins. Normal marrow (©) and MDS marrow (O). Marked ranges for normals calculated from mean +- 2 S.D. Epo, erythropoietin; CM, 5637 conditioned medium; GM, GMCSF; IL-3, interleukin 3; G, GCSF; M, MCSF; IL-1, interleukin 1. routinely added to cultures of the same normal marrows (supplemented with Epo), at concentrations already shown to be optimal for normal progenitor growth. Few GM colonies (Fig. 1A---open circles) developed in the absence of any supplements or in the presence of Epo alone. The addition of 5637CM (5%), GMCSF (200 units) or IL-3 (13 units) significantly augmented GM colony numbers in cultures containing Epo (p = 0.0034, 0.0015, 0.0022 for 5637CM, GMCSF, IL-3 respectively vs Epo alone). GMCSF supported significantly more GM colonies than IL-3 (medians for GMCSF, IL-3 respectively = 48, 18; p = 0.0032). GMCSF also supported large numbers of clusters. The remaining haemopoietins (GCSF, MCSF and IL-1) did not induce more colonies or clusters than Epo alone. As in normal marrow, 5637CM or GMCSF or IL-3 all increased GM colony numbers (p = 0.0073,0.008, 0.025 respectively) in Epo-supplemented cultures of MDS marrow (Fig. 1A---closed circles). Although colony growth in 5637CM + Epo was not subnormal, in the MDS group as a whole (medians for normal, MDS = 31, 21, respectively; p = 0.075), GM colony numbers fell below the normal range in 5/12 of our MDS patients. Of the remaining seven patients, six formed normal numbers of colonies in response to plateau dose GMCSF. Two of these six patients were neutropenic (patients 1 and 10). Over the MDS group as a whole, GMCSF induced subnormal numbers of GM colonies (medians for normals, MDS = 48, 18 respectively; p = 0.041). As in normals,

GMCSF induced more GM colonies than IL-3 (medians = 18, 8.5 respectively; p = 0.014) over the MDS group as a whole. Of the patients who grew normal numbers of GM colonies in 5637CM-supplemented cultures, colony numbers fell below the normal range in GMCSF or IL-3-supplemented cultures of marrow from patient 7 and in IL-3-supplemented cultures of marrow from patient 10 (individual data not shown). Cluster numbers in the MDS group did not exceed the numbers seen in normal marrow cultures--under any conditions, and the cluster-colony ratio was not significantly raised (median normal vs MDS ratios = 1.1 vs 1.4; p = 0.56) in these patients.

Growth of erythroid progenitors from normal and MDS marrows in the presence of various recombinant haemopoietins No erythroid colonies were recorded in the absence of Epo (Fig. 1B). A mean of 16 erythroid colonies formed when Epo was added to normal marrow cultures and this was further increased in the presence of 5637CM, GMCSF or IL-3 (p = 0.027, 0.036, 0.027, respectively). The median values for 5637CM, GMCSF and IL-3 (51, 32, 62, respectively) did not differ significantly. The remaining factors did not augment erythroid colony formation. Epo-stimulated erythroid growth in cultures of most MDS marrows was poor in the presence of 5637CM or any of the recombinant haemopoietins (medians for MDS vs normal: 2, 51; p = 0.0012:

P. BARNESet al.

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FIG. 2. Myeloid (O) and erythroid (0) colonies in cultures of MDS marrow supplemented with high dose compared to low dose GMCSF (A), IL-3 (B), GCSF (C), IL-1 (D). Solid lines represent 95% confidence limits calculated as described in Methods. Values above or below these confidence limits represent significant departures from the line of equality.

5, 32; p = 0.0016: 4, 62; p = 0.0032; for 5637CM, GMCSF and IL-3 respectively). None of the recombinant haemopoietins raised erythroid colony numbers significantly above those observed in cultures containing erythropoietin alone (median --- 2).

MDS progenitor growth in high-dose haemopoietins Augmentation of colony numbers at high concentrations of haemopoietin was rarely observed for either myeloid or erythroid lineages (Fig. 2 A - D ) ; compared with lower (optimal, plateau) dose of IL-3, one patient (No. 10) showed an augmentation of GM colony numbers which fell outside the 95% confidence limit curves constructed from the variation observed over a large number of duplicates. No significant changes were seen over the MDS group as a whole, for any of the haemopoietins at high dose.

MDS progenitor growth in combinations of haemopoietins When patients' progenitor responses to a combination of haemopoietins were analysed individually, there was no case where the observed number of GM or erythroid colonies exceeded the number expected if the two factors had acted additively (Fig. 3A, B). By comparing colony numbers in combinations of factors with numbers in the presence of the 'dominant' haemopoietin, for the MDS group as a whole, only in one combination (IL-1 + IL-3) was a significant rise in (GM) colony numbers recorded. Conversely, there were no instances where colony numbers, in factor-combinations, were significantly lower than in the 'dominant' haemopoietin, indicating a lack of inhibitory effects. DISCUSSION Before investigating the effects of recombinant

MDS, CFC and recombinant growth factors

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FIG. 3. Myeloid (closed symbols) and erythroid (open symbols) colony numbers in MDS marrow cultures: A, containing GMCSF and either IL-3 (O O), GCSF (& A) or IL-1 (11 D); B, containing IL-3 and either GCSF (O O), IL-1 (A A) or MCSF (B D), compared with the sum of colony numbers in cultures containing each factor alone; solid lines representing 95% confidence limits calculated as described under Methods. Values above or below these limits represent significant departures from the line of equality. haemopoietins on MDS progenitors we have determined the responses of normal progenitors to these factors in our culture system. That recombinant GMCSF is a stronger myeloid stimulus than IL-3 is consistent with published data [7, 8] although we would have expected IL-3 to induce more erythroid colonies than GMCSF: this may reflect the small sample size. G-CSF, MCSF or IL-1 failed to induce significant numbers of myeloid or erythroid clones. In the case of GCSF its effects may be obscured by factors added to culture in the form of the foetal calf serum supplement and which support some myeloid growth. Both MCSF and IL-1 are known to be poor inducers of clonal growth on their own, requiring the presence of other CSFs for full expression of colonystimulating activity [9, 10]. Myeloid (GM) colony growth in 5637CM-supplemented cultures of MDS marrows was extremely variable, being particularly poor in 5 patients. Erythroid growth was more severely impaired and was reduced in all but one of our patients. These growth defects were not corrected by either of the two 'dominant' haemopoietins (GMCSF and IL-3), even when added at supra-optimal dose, with the exception of patient 10 whose marrow grew more GM colonies in high-dose IL-3. These findings are inconsistent with data collected by us on the effects of high-dose GMCSF on MDS progenitors in an earlier study [11] and which had prompted this latest investigation.

However, the previous cohort contained many more MDS patients with a shorter clinical history. Progenitors from more recently diagnosed patients may be more responsive to exogenous growth factors [12]. The current batch of patients contained far fewer RAEB patients and did not exhibit an inverted cluster: colony ratio, indicating that the two populations differed considerably. In cultures of MDS marrow, combinations of haemopoietins rarely augmented colony numbers above the sum of the colonies formed in the presence of the two factors alone. Furthermore, since colony numbers in combinations of factors seldom exceeded the scores in the presence of the dominant haemopoietin, these haemopoietins appear to have often recruited the same progenitors into clonal growth. We are unable to say whether these responses to factor-combinations are abnormal since we have not investigated the behaviour of normal progenitors under similar conditions. Of six MDS marrows that grew normal numbers of GM colonies in the presence of 5637CM, which contains GMCSF, GCSF and IL-1 [10, 13, 14], two (patients 7 and 10) failed to generate normal numbers of colonies in response to an individual haemopoietin. GM progenitors from patient 7 responded poorly to GMCSF or IL-3, and those from patient 10 required a supraoptimal dose of IL-3, or the coaddition of IL-1 and IL-3 to grow fully in culture.

252

P. BAINESet al.

IL-3 is not present in 5637CM and progenitors from these patients may express a clonally-specified growth response defect to this factor. The poor response of progenitors from patient 7 to GMCSF is harder to interpret since mixtures of GMCSF with either GCSF or IL-1 did not significantly augment colony numbers. Unfortunately, we did not mix all three factors together, for this patient. We also examined clonal morphology (data not shown) in stained, dried-agar cultures of normal and MDS marrows, using the technique described by Baines [15]. Compared with cultures from five normal marrows, there was a tendancy towards reduced monocytic and erythroid colony size in seven MDS marrow cultures. There were no marked morphological changes in cultures of MDS marrow supplemented with high dose haemopoietin or with combinations of haemopoietins. There was no evidence that clones of blasts were increased by any of the haemopoietins, as has been reported for overtly leukaemic cells in culture [16, 17] and following GMCSF therapy of R A E B patients [18], although none of the patients examined in this way presented with more than 5% of blasts in the marrow. Whether these in-vitro results can, in fact, be extrapolated to the likely in-vivo effects of these factors must await the result of clinical trials. GM progenitors from half the MDS marrows responded to GMCSF; two of these patients were neutropenic and so might have been candiates for GMCSF therapy. Myeloid progenitors from the three other neutropenic patients responded poorly to GMCSF (as well as to the other factors) even at supraoptimal doses. Published data on G M C S F therapy indicates a better response rate than this [4, 19]. This disparity may reflect the fact that clonal assays do not parallel the situation in vivo very accurately. For example, continuous therapy with G M C S F allows for the accumulation of GM progenitors with associated increased neutrophil production. In clonal assays however, G M C S F can only act on progenitors already present. Further, although we have specifically used unfractionated marrow in order to preserve the natural balance of cells within the marrow, these cells are no longer in the close association which may be critical for some of the indirect, accessory effects known to be induced by haemopoietins. A disappointing feature was the unresponsiveness of myelodysplastic erythroid progenitors to growth factors in vitro and it is difficult to see how clonal assays could be useful in suggesting corrective therapy for the anaemia. The defective response by MDS progenitors to growth factors has been intensively investigated in cultures containing crude conditioned media [20] and

more recently (over a restricted range of patients) in response to recombinant factors [21]. Our current findings indicate that manipulation in vitro of growth factors alone is unlikely to improve our knowledge of the biological defect and may be of only limited value in suggesting appropriate therapy in longstanding MDS. Acknowledgements--I wish to thank Dr G. Tennant and Mrs Tessa Lewis for their advice on statistical analysis of these data, Dr M. A. Bains for advice on the morphology of cells stained with Jenner-Giemsa, and Dr Gillis (Immunex, Seattle), Dr S. C. Clark (Genetics Institute, Cambridge, MA) and Dr Westmacott (Roche, U.K.) for their generous gifts of recombinant factors.

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MDS, CFC and recombinant growth factors

11.

12.

13.

14.

15.

ascribed to hemopoietin 1. Proc. natn. Acad. Sci., U.S.A. 84, 5267. Mayani H., Baines P., Bowen D. & Jacobs A. (1989) In-vitro growth of myeloid and erythroid progenitor cells from myelodysplastic patients in response to recombinant human granulocyte-macrophage colonystimulating factor. Leukemia 3, 29. Tennant G. & Jacobs A. (1989) Effect of 5637-conditioned medium on peripheral blood granulocytemacrophage progenitors in normal subjects and patients with the myelodysplastic syndrome. Leukemia Res. 13, 385. Gabrilove J. L., Welte K., Harris P., Platzer E., Lu L., Levi E., Mertelsman R. & Moore M. A. S. (1986) Pluripoietin: a second human hematopoietic colonystimulating factor produced by the human bladder carcinoma cell line 5637. Proc. natn. Acad. Sci. U.S.A. 83, 2478. Welte K. E., Platzer E., Lu L., Gabrilove J. L., Levi E., Mertelsman R. & Moore M. A. S. (1985) Purification and biochemical characterization of human pluripotent haemopoietic colony stimulating factor. Proc. natn. Acad. o f Sci., U.S.A. 82, 1526. Baines P. (1989). Drying and staining soft-agar cultures of haemopoietic colonies. Int. J. Cell Cloning, 7, 136.

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16. Vellenga E., Ostapovicz D., O'Rourke B. & Griffin J. D. (1987) Effects of recombinant IL-3, GMCSF and GCSF on proliferation of leukaemic clonogenic cells in short-term and long-term cultures. Leukemia 1, 584. 17. Murohashi I., Nagata K., Suzuki T., Maruyma Y. & Nara N. (1988) Effects of recombinant G-CSF and GMCSF on the growth in methylcellulose and suspension of the blast cells in acute myeloblastic leukemia. Leukemia Res. 12, 433. 18. Hoelzer D., Ganser A., Volkers B., Greher J. & Walther F. (1988) In-vitro and in-vivo action of recombinant human GMCSF (rhGM-CSF) in patients with myelodysplastic syndromes. Blood Cells 14, 551. 19. Antin J. H., Smith B. R., Holmes W. & Rosenthal D. S. (1988) Phase I/II study of recombinant human granulocyte-macrophage colony stimulating factor in aplastic anaemia and myelodysplastic syndrome. Blood 72, 705. 20. Koeffler H. P. (1986) Myelodysplastic syndromes (Preleukemia). Semin. Hemat. 23, 284. 21. Shouten H. C., Delwel R., Bot F. J., Hagemeijer A., Touw I. P. & Lowenberg B. (1989) Characterization of clonogenic cells in refractory anemia with excess of blasts (RAEB-CFU): response to recombinant hematopoietic growth factors and maturation phenotypes. Leukemia Res. 13, 245.

Clonal growth of haemopoietic progenitor cells from myelodysplastic marrow in response to recombinant haemopoietins.

The growth factor requirements of granulocyte-macrophage (GM) and erythroid marrow progenitor cells from 12 myelodysplastic (MDS) patients have been a...
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