Colony growth characteristics in chronic myelomonocytic leukemia

Vol. 12, No. 5, pp. 373-377, 1988. Printed in Great Britain.

0145-2126/88 $3.00 + .00 Pergamon Press plc

Leukemia Research

COLONY G R O W T H CHARACTERISTICS IN CHRONIC MYELOMONOCYTIC LEUKEMIA K L A U S G E I S S L E R , W O L F G A N G H I N T E R B E R G E R , P E T E R BE'VFELHEIM, O S K A R H A A S * KLAUS LECHNER

and

1st Dept. of Medicine, University of Vienna and *St Anna Children Hospital, Vienna, Austria (Received 11 November 1987. Revision accepted 13 January 1988) Abstract--Using cell culture studies specific in-vitro characteristics have been reported for Philadelphia chromosome positive myelogenous leukemia (Ph+ CML) and for juvenile chronic myelogenous leukemia (JCML) previously. We performed cell culture studies in four patients with chronic myelomonocytic leukemia (CMML) and demonstrated the following in-vitro features: excessively increased circulating CFU-C, while BFU-E and CFU-mix were either moderately increased or not detectable; CFU-C colony formation from CMML mononuclear cells (MNC) without addition of exogenous colony stimulating activity (CSA), even after depletion from adherent cells; failing inhibition of CMML MNC on normal BFU-E colony formation. These in-vitro characteristics point to CMML as a distinct entity. In two CMML-patients investigated CFU-C proliferation appeared to some extent inhibited by the addition of IFN-tr, IFN-y and TNF-o: to cell cultures. Key words: Chronic myelomonocytic leukemia, CFU-C, BFU-E, CFU-mix.

INTRODUCTION

for further characterization of hemopoietic malignancies. Such investigations have shown basically different in-vitro characteristics in P h + CML and J C M L [13], but C M M L has not been analysed by these means yet. We used a panel of in-vitro techniques in four CMML-patients and we demonstrated in-vitro characteristics different from that having been reported in P h + CML and JCML.

CHRONIC myelomonocytic leukemia (CMML) is a hemopoietic malignancy of the elderly which is characterized by leucocytosis with monocytes and granulocytic cells in all stages of development, marked dysmyelopoiesis, a chronic course and unresponsiveness to aggressive chemotherapy [1-4]. Because of these clinical symptoms C M M L may be included among atypical myeloproliferative syndromes (MPS) [5] or among myelodysplastic syndromes (MDS) [3]. While C M M L can be distinguished from the remaining MDS-groups by the demonstration of increased CFU-C growth in vitro [2, 6-8], it shares this feature with the MPS, particularly with Philadelphia chromosome positive chronic myelogenous leukemia ( P h + CML) [9-11] and with juvenile chronic myelogenous leukemia (JCML) [12, 13]. Detailed culture studies such as spontaneous colony formation and co-culture studies may be helpful

PATIENTS AND METHODS Patients All four patients met the diagnostic criteria for CMML according to the FAB-group [3], i.e. dyspoietic features of myelodysplastic syndromes associated with a blood monocytosis over 109/1, an increase in BM monocyte precursors, and a blast cell percentage less than 5% in the peripheral blood (PB) and less than 30% in BM. Clinical and laboratory data of these patients are shown in Table 1. None of the patients had received chemotherapy within 6 weeks before the study or during it. Fifteen normal individuals aged 20-74yr served as control. Cytogenetic studies were performed according to a method previously described [14].

Abbreviations: Ph + CML, Philadelphia positive chronic myelogenous leukemia; JCML, juvenile chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; CFU-C, granulocyte-macrophage committed progenitor cell; BFU-E, erythroid committed progenitor cell; CFU-mix, pluripotent hemopoietic progenitor cell; MNC, mononuclear cell; CSA, colony stimulating activity; IFN, interferon; TNF, tumor necrosis factor. Correspondence to: Dr K. Geissler, 1st Dept. of Medicine, Division of Hematology, University of Vienna, Lazarettg. 14, A-1090 Vienna, Austria.

Preparation of cells PB (16ml) was collected into sterile tubes containing 4 ml EDTA. BM samples were obtained by aspiration into sterile tubes containing heparin with no preservative (Seromed). PB MNC and BM MNC were harvested after a Ficoll-Hypaque gradient centrifugation (400g; 40 min; 1.077 g/ml). 373

KLAUS GEISSLER el al.

374 TABLE

1.

C L I N I C A L A N D L A B O R A T O R Y D A T A IN F O U R PATIENTS WITH C H R O N I C MYELOMONOCYTIC LEUKEMIA

Patient Sex Age (yr) Duration of disease (months) Hemoglobin (g/dl) WBC (× 109/1) Platelets (x 109/1) PB monocytes (%) PB blasts (%) Serum lysozyme (p.g/ml) Karyotype

1

2

3

4

M 63 8 10.1 161 153 31 4 65 47XY, + 21

M 54 5 9.4 48 86 50 2 607 46XY

F 59 40 9.6 20.1 6 12 4 165 46XX

M 84 4 7.8 62.5 90 30 1 123 46XY

PB, peripheral blood.

CFU-C assay The CFU-C assay was performed as previously described [15]. In brief: BM MNC or PB MNC were cultured in 0.8% methylcellulose, 30% fetal calf serum (FCS), 20% colony stimulating activity (CSA)(Seromed) and re-medium. Cultures were plated in duplicate at 2.5 x 103-1.0 x 105 MNC/ ml. After a culture period of 10 days (37°, 5% CO2, full humidity) cultures were examined under an inverted microscope. Aggregates with at least 40 cells were counted as CFU-C colonies. CFU-mix assay The CFU-mix assay was performed as previously described [16]. Each plate contained 0.9% methylcellulose, 30% FCS, 10% bovine serum albumin (Behring), 1 U/ml erythropoietin (Toyobo), 5% phytohemagglutinineleucocyte-conditioned medium and Iscove's modified Dulbecco's medium (Gibco). Cells were plated and incubated as described above but cultures were examined after a period of 14 days. Aggregates with at least 50 translucent, dispersed cells were counted as CFU-C colonies. Bursts containing more than 100 red coloured cells were scored as BFU-E. CFU-mix were identified by their heterogeneous composition of translucent and hemoglobinized cells. The colony numbers per 1 ml of PB were calculated on the basis of the number of colonies per number of PB MNC plated, the white blood cell (WBC) count, the percentage of MNC in the blood and the percentage of MNC in the population of cells obtained by Ficoll-Hypaque gradient centrifugation.

Human interferons (IFN), human tumor necrosis factor (TNF) Natural crude IFN-o~ (specific activity 1.59 x 107 U/mg protein) and natural crude IFN-~, (8.2 x 107 U/mg) were obtained from Interferon Sciences, Inc., New Brunswick, NJ. Pure recombinant TNF-o: was a generous gift from Dr G. Adolf, Ernst Boehringer Inst., Vienna, Austria. A stock solution was prepared at 3000 U/ml in mmedium with 10% FCS and stored in small aliquots at -20°C. IFNo:, IFN-7 or TNF-o~ were added to MNC 30 min before plating. RESULTS As shown in Table 2, PB C F U - C (examined by the CFU-mix assay) were extremely increased per ml in all four CMML-patients as compared to controls. If expressed per number of plated cells, C F U - C were highly increased as well (in 15 controls 1.3---0.6 (mean --- S.D.) CFU-C/2.5 × 104 MNC vs 1525 +2515 in CMML-patients). PB B F U - E and CFU-mix were moderately increased in two cases and in one case, respectively, but were not detectable in the remaining cases. In average 47% (range 32--66) of C F U - C colonies obtained from PB of CMML-patients contained monocyte-macrophages whereas the percentage of

TABLE 2. P R O G E N I T O R C E L L N U M B E R S IN T H E P E R I P H E R A L B L O O D F R O M PATIENTS WITH

CMML Patient

CFU-C/ml

BFU-E/ml

CFU-mix/ml

1 2 3 4 Normal ranges f f o m l 5 controls

12,244,050 130,378 124,740 84,610 38-256

0 2233 4050 0 120-720

0 0 405 0 12-101

Colony growth in CMML

375

TABLE 3. IN V I T R O CULTURES FROM PATIENTS WITH C M M L USING THE C F U - C ASSAY

With CSA

Without CSA

Source

CFU-C/2.5 x 104MNC

Pt 1 BM MNC 6 Controls BM MNC

910 19.8 -+ 8.5

Pt 2 PB MNC 6 Controls PB MNC

23.0 0.36 -+ 0.15

Pt 1 BM MNC 6 Controls BM MNC

815 0.0 - 0.0

Pt2 PB MNC 6 Controls PB MNC

27.0 0.0 -+ 0.0

CMML, chronic myelomonocytic leukemia; CSA, colony-stimulating activity; BM, bone marrow; PB, peripheral blood; MNC, mononuclear cells

monocyte-macrophage containing colonies in controls was low (mean = 21%, range 16-30) (p < 0.05 by the U-test). These findings were confirmed by morphological, cytochemical and immunological analysis of individual colonies plucked from the culture and by examination of the cellular composition of entire harvested culture plates. Using the CFU-C assay growth requirements of C M M L C F U - C colonies were studied (Table 3). As shown in two CMML-patients excessive plating efficiency persisted if CSA was omitted from the cultures. Furthermore, PB MNC from patient 2 were depleted of adherent cells and cultured without CSA. Even in this case excessive plating efficiency persisted (272 CFU-C per 2.5 x 104 non-adherent MNC). PB MNC from patient 1 were cocultured in varying ratios with control PB MNC in order to detect a possible inhibition of C M M L cells on erythropoiesis (Table 4). With increasing number of C M M L cells in co-culture, no significant change in the B F U - E number from control PB MNC could be observed.

TABLE4. EFFECTOF CMML PB MNC ONBFU-E COLONY FORMATIONFROM CONTROLPB MNC Number of PB MNC from patient 1 added to control PB MNC

BFU-E number/150,000 control PB MNC

0 1000 10,000 50,000 75,000

72.5 73.0 70.0 71.0 71.5

(BFU-E number/75,000 CMML PB MNC -- 1.5) CMML, chronic myelomonocytic leukemia; PB, peripheral blood; MNC, mononuclear cells.

TABLE5. IN-VITRO INFLUENCEOF IFN-c~, IFN- 7 ANDTNFa' ON CFU-C COLONYFORMATIONIN CMML PATIENTS Percent inhibition of colony formation Patient

IFN-ac (400 U/ml)

IFN-y (400 U/ml)

TNF-o~ (12.5 U/ml)

47 -+ 7* 32-+ 8*

53 - 9*

2

41 -+ 12"

63 -+ 11" 30-+ 5*

3 Controls

54 - 12"

72 - 7*

68 -+ 10"

1

Results are expressed as percentage inhibition (mean S.D.) compared with control medium; *p < 0.05 by the ttest.

To CFU-C cultures from two CMML-patients IFN0:, IFN- 7 and TNF-a~ were added in dosages which have been found to be moderately suppressive on normal CFU-C colony formation in previous experiments. As shown in Table 5, all three substances were able to inhibit C M M L CFU-C colony formation. However, in no case a preferential inhibition of abnormal CFU-C was observed.

DISCUSSION In previous studies BM CFU-C concentrations have been shown to be increased in the majority of CMML-patients whereas erythroid progenitor cells (BFU-E, CFU-E) were either normal or decreased [2, 6-8]. In all four C M M L patients in our study circulating CFU-C were increased and the increment was much more pronounced than the increment in BM in the before mentioned studies. The reason for this finding may be differences in the patient

376

KLAUS GEISSLER et al.

characteristics but may also reflect a preferential expansion of the CFU-C compartment in PB as has been demonstrated in P h + CML previously [9-11]. B F U - E and CFU-mix were found to be either moderately increased or not detectable in our CMMLpatients. In contrast B F U - E and CFU-mix are usually increased in P h + CML-patients although these progenitor cells may also be undetectable in some cases [9-11]. In three JCML-patients studied no BFU-E or CFU-mix were seen in culture [13]. The percentage of pure monocyte-macrophage colonies or monocyte-macrophage containing colonies is low (21%) in PB from normal individuals [17]. Not unexpectedly, the percentage of monocyte-macrophage containing colonies in CMML-patients was considerably higher (47%). Most CFU-C from PB of patients with P h + CML are of granulocytic lineage [13, 18] whereas the cellular composition of CFU-C in JCML is exclusively monocyte-macrophage [12, 13]. Thus our findings in C M M L differ from both entities. Atypical growth requirements for C M M L CFU-C were found in our study. For proliferation of normal BM or PB CFU-C colonies in vitro, a humoral growth factor is essential and can be provided by the addition of exogenous CSA [19]. This factor is also necessary for CFU-C growth in P h + CML but not in JCML [13]. As has been shown previously, CSA is released by normal monocytes and by activated T cells [20]. In our study CFU-C growth from normal individuals was entirely CSA dependent. In sharp contrast, addition of CSA was not essential for CMML CFUC to proliferate into colonies. One may speculate, that spontaneous CFU-C growth in CMML may be caused by increased endogenous CSA production due to the higher percentage of monocytes among plated cells. This however could not be substantiated since appropriate CFU-C growth was also observed, if monocytes were removed by adherence. There still remains the possibility that non adherent leukemic cells could be the source of CSA production as has been shown in a recent study where non adherent, T-depleted mononuclear cells from 11 of 22 cases of acute myeloid leukemia expressed granulocytemacrophage colony stimulating factor (GM-CSF) transcripts [21]. Anemia is usually observed in CMML [1-4] and has been also found in our CMML-patients. The pathogenesis of it is still obscure. In JCML it has been found that J C M L cells are able to excrete a monokine that can suppress normal hemopoiesis in vitro [13] and which may be identical to the LIA described by Broxmeyer et al. [22]. We wondered if a similar mechanism could cause inhibition of erythropoiesis in CMML. However, we could not

detect any inhibition of normal B F U - E growth by adding various numbers of CMML cells to cultures. In-vitro a n d / o r in-vivo data suggest that IFN-tr, IFN-), and TNF-o~ may have inhibitory effects on clonal proliferation of cells from patients with acute myeloid leukemia and CML [23-27]. Preliminary observations in two CMML-patients suggest that the excessive in-vitro proliferation of CFU-C may be significantly but, as compared to control CFU-C, not predominantly inhibited by the addition of IFN-oc, IFN-y and TNF-tr to cell cultures. The clinical significance of this finding remains to be established.

Acknowledgements--These investigations were supported by Grant No. 4100 of the "Fond zur F6rderung der wissenschaftlichen Forschung in Osterreich" and by the "Kommission ftir Leuk~imieforschung und Knochenmarkstransplantation" of the Austrian Academy of Sciences.

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