Endogenous FLT-3 ligand serum levels are associated with ... - Nature

Leukemia (1999) 13, 553–557  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu

Endogenous FLT-3 ligand serum levels are associated with disease stage in patients with myelodysplastic syndromes H Zwierzina1, JE Anderson2, I Rollinger-Holzinger1, B Torok-Storb2, V Nuessler3 and SD Lyman4 1

Medizinische Universita¨tsklinik Innsbruck, Innsbruck, Austria; 2Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA, USA; 3Medizinische Klinik III, Klinikum Grosshadern, Munich, Germany; and 4Immunex Corporation, Seattle, WA, USA

Myelodysplastic syndromes (MDS) caused by a clonal hematopoietic stem cell disorder progress to either overt leukemia or cytopenia, which leads to lethal infection or bleeding. Although several clinical trials have attempted to reverse cytopenia by using hematopoietic growth factors (HGF), success has been limited due in part to a limited understanding of the role of HGF in MDS progression. The FLT3 ligand, which binds to and activates the FLT3 receptor, does not have a stimulatory effect on hematopoietic cells, but can synergize with other HGF to support the expansion of both immature and committed progenitors. Using ELISA technology we measured endogenous serum levels in 93 patients with MDS: 29 RA, 1 RARS, 31 RAEB, 23 RAEBt, 9 CMML. 48.3% of RA patients’ sera had significantly elevated FLT3 ligand levels ranging from 404 to 5735 pg/ml, whereas none of the RAEB, RAEBt, or CMML patients sera had levels different from controls. No significant correlation was found between FLT3 ligand levels and peripheral blood counts, bone marrow cellularity, age, cytogenetic abnormalities, or survival. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS, which may represent an appropriate response to a decreased number of normal progenitors, or alternatively a dysregulated HGF system. Keywords: myelodysplastic syndromes (MDS); FLT3 ligand; serum level

Introduction Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders and lead to abnormal maturation of hematopoietic cells and to progressive cytopenia. With expansion of the malignant clone the tendency for transition to overt leukemia increases. The course of MDS is highly variable, with some patients remaining stable over years or progressing rather slowly and others with a rapid transition to acute myeloid leukemia. In addition to an initial malignant transformation at the hemopoietic stem cell level, a further event seems necessary before growth control mechanisms are altered sufficiently to give the new clone a proliferative advantage over normal hemopoietic cells. Such an advantage and the consequent clonal expansion may be related to increased sensitivity of the malignant clone to growth factors.1 Prognosis in MDS is closely correlated with bone marrow blast cell count,2 cytogenetic abnormalities3 and with cytopenia,4 causing an increased risk of lethal bleeding or infection. In the majority of cases, the need for therapy is dictated not by development of overt leukemia, but by dyshematopoiesis leading to anemia, neutropenia and/or thrombocytopenia. To date, no generally accepted treatment is available for MDS except allogeneic bone marrow transplantation. Although several clinical trials have attempted to overcome cytopenias by using hematopoietic growth factors (HGF),5–8 success has been limited in part due to an insufficient underCorrespondence: H Zwierzina, Universita¨tsklinik fu¨r Innere Medizin, A-6020 Innsbruck, Austria; Fax: 43 51 25 04 42 09 Received 20 August 1998; accepted 16 December 1998

standing of the role of HGF in the pathogenesis and the course of MDS. In the group of patients suffering from refractory anemia (RA) who were treated with G-CSF,5 GM-CSF6 or IL-3,7,8 no increased rate of irreversible transition to overt leukemia occurred as compared to the natural course of the disease. Although it can not be ruled out that long-term exposure to these HGF may cause an irreversible growth advantage of the malignant clone, excessive supply does not seem to be the exclusive stimulatory mechanism. Other factors acting on the early stem cell level, however, may be involved in clonal expansion either singly or in synergy with later-acting factors. The hemopoietic growth promoting effects of HGF are signaled through their receptors. Flt3/flk-2 (FLT3) is a tyrosine kinase receptor, preferentially expressed on primitive hemopoietic progenitor cells, suggesting a role in early hematopoiesis.9 In normal bone marrow, FLT3 is expressed primarily on stem and progenitor cells (including CD34+ cells), but in acute myeloid leukemia it is also expressed on leukemic blast cells irrespective of CD34 expression.10 This suggests that overexpression of FLT3 may contribute to the malignant phenotype and may be involved in the proliferation of the malignant clone in AML or MDS. The ligand of the human FLT3 receptor was cloned11 and shown to stimulate the proliferation of hemopoietic progenitor cells isolated from mouse fetal liver or adult mouse bone marrow. It is functionally active on human CD34+ bone marrow cells enriched for hemopoietic stem cells and synergizes with later-acting HGF in order to support expansion of both immature and committed progenitors.12 FLT3 ligand is a transmembrane protein that is produced by a wide variety of cell types including stromal cells. It can be proteolytically cleaved to generate a soluble, biologically active form.11 Like many other hemopoietic growth factors, FLT3 ligand stimulates the proliferation of some leukemic blasts without any relation to the French–American–British (FAB) subtype13 and thus seems to be another important factor for the growth of myeloid leukemia cells, either as a direct stimulator or as a synergistic factor with other cytokines. Whether coexpression of FLT3 and FLT3 ligand results in an autocrine or paracrine loop and plays a role in leukemia and potentially myelodysplastic syndromes remains to be investigated. Data presented in this report begin to address this issue by evaluating the serum levels of FLT3 ligand in patients presenting with different stages of MDS. Patients and methods

Patient characteristics Serum samples were collected from 93 patients suffering from myelodysplastic syndromes after obtaining informed consent. Classification of MDS was performed according to FAB criteria.2 Twenty-nine patients presented with refractory anemia (RA), one with RA with ringed sideroblasts (RARS), 31 with

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refractory anemia with excess of blasts (RAEB), 23 with refractory anemia with excess of blasts in transition (RAEBt), and nine with chronic myelomonocytic leukemia (CMML). All collected sera were stored at −20°C until use. Blood count including differential count was determined at the same date. None of the patients suffered from infection or had received cytotoxic agents and/or cytokines during the previous 3 months. Cytogenetic evaluation had been performed in 59 of the 93 patients (63.4%). Characteristics of patients suffering from RA are shown in Table 1.

Bone marrow cellularity In MDS patients, bone marrow cellularity can be equally well assessed in biopsy and aspirate provided a sufficient number of bone marrow particles can be detected.14 As bone marrow biopsy is not always routinely done, we therefore evaluated megakaryopoiesis in the bone marrow smears. In order to obtain objective data and exclude smears with an insufficient number of bone marrow particles, cellularity was evaluated by two independent blinded reviewers in 10 fields of view using 10-fold magnification. According to published criteria,14 results were scored according to three categories: hypercellular, normal, hypocellular. In our series bone marrow smears with sufficient quality for evaluation of cellularity were available from 83 patients (90.2%) in whom endogenous serum FLT3 ligand concentrations had been measured. Of these patients 29 suffered from RA, 27 from RAEB, 21 from RAEBt, and 7 from CMML.

Detection of endogenous FLT3 ligand concentrations Serum concentrations of FLT3 ligand in MDS patients were determined using ELISA technology as described elsewhere.15 In brief, wells of polystyrene microtiter plates were coated with 100 ␮l of a 0.1 ␮g/ml solution of ascites-produced human FLT3 ligand-specific monoclonal antibody in 0.01 mol/l PBS, pH 7.2, and incubated at 2°C to 8°C overnight. A CHO-derived human FLT3 ligand standard and human sera were diluted in a sample buffer comprised of PBS with 0.05% Tween-20 (PBST) with an additional 0.5 mol/l NaCl and 5% normal rat serum. All reactants were dispensed in 100 ␮l per well volumes and the wells were washed with PBST before the addition and incubation of each successive reactant. Standards and sera were incubated for 1 h at RT, followed by a 1 h RT incubation of a solution of rabbit antihuman FLT3 ligand polyclonal antibody, followed by a 1 h RT incubation of a solution of peroxidase-conjugated donkey anti-rabbit IgG. Color was developed with 3,3⬘,5,5⬘-tetramethylbenzidine (TMB) peroxidase substrate/chromogen solution for 10 min at RT. The reaction was stopped with 1 mol/l H3PO4, and optical densities were determined at a wavelength of 450 nm. The lower detection limit of the assay was 100 pg/ml.

Statistical analysis Results of single groups are summarized as median and range values. Non-parametric tests were used for statistical evalu-

Table 1 Patient characteristics: refractory anemia

UPN

71 99 110 123 142 143 145 157 160 167 237 257 265 286 288 302 319 357 415 418 442 462 6089 6540 6650 9679 9787 9966 10780

Age/Sex

flt3 pg/ml

Cellularity

Platelets 109/l

hb g/dl

leukocytes 109/l

Cytogenetics

Survival

75/m 74/m 53/m 41/f 66/m 75/m 54/m 33/f 65/m 53/f 54/f 26/m 22/f 67/m 60/m 27/f 49/f 66/m 69/m 67/m 75/f 93/f 23/m 21/f 23/m 60/f 32/f 16/m 48/f

4844 lts lts 522 lts lts lts 496 lts 438 749 2036 lts lts lts 2991 lts 2350 2084 lts lts lts 5735 522 408 129 986 404 lts

1 3 3 1 1 2 3 2 3 3 3 1 2 2 3 2 3 2 3 1 3 2 2 3 3 2 1 3 1

133 196 526 20 147 181 463 150 183 46 93 15 21 80 121 61 22 24 16 2 375 136 82 25 120 425 71 13 54

13.5 8.6 9.3 9.3 6.2 9.4 7.1 7.2 10.2 14.2 12.6 7.6 13.3 7.3 9.3 9.9 8.5 10.7 8.1 6.5 10.1 9.6 12.3 7.7 5.1 11.9 11.2 7.6 8.4

0.8 3.3 4.4 7.9 3.3 3.7 5.3 3.6 2.4 2.3 5.6 2 3.6 1 2.2 2.8 4.1 0.9 0.6 9 19.7 1.7 2.9 1.9 2.2 4.5 2.6 3.4 2.1

ND ND 46 XY, 5q− complex 46 XY, 5q− ND 46 XY, 5q− ND ND 46 XX 46 XX 46 XY 46 XX 46 XY, 5q− ND 46 XX 46 XX, 4p− 46 XY 46 XY ND 46XX, 5q− ND 46 XY 46XX, 20q− ND 46 XX, 20q− 46 XX 46XY ND

4 11 65 29 16 7 62 84+ 24 81+ 73+ 1 92+ 1 31 68+ 13 3 2 2 16 2 1+ 59+ 7+ 15 5+ 7 10

UPN, unique patient number; cellularity (1 = decreased; 2 = normal, 3 = increased); lts, less than standard; ND, not done.


ation and calculated with the StatView software package. Comparisons between the groups were performed with the Mann–Whitney U test or Chi-square test, correlation analysis with Spearman’s rank correlation test. A P value of 0.05 or less was deemed a significant correlation. Results

Thirteen smears demonstrated a hypercellular, nine a normocellular, and seven a hypocellular marrow. No correlation was detected between cellularity and FLT3 ligand serum level. Cytogenetics had been performed in 19 of the 29 RA patients (65.5%). No correlation was found between the presence of cytogenetic abnormalities and FLT3 ligand serum level. It is interesting to note, however, that none of the five patients with deletion of the long arm of chromosome 5 (5q−) had a detectable FLT3 ligand serum level.

FLT3 ligand serum concentrations In our series of 93 MDS patients (Figure 1), elevated endogenous FLT3 ligand serum concentrations between 404 and 5735 pg/ml (median 1838 pg/ml, s.e.m. 359.4 pg/ml) were detected in 15 patients. Four patients demonstrated detectable levels within the normal range (1 RA, 129 pg/ml; 2 RAEB, 139 and 140 pg/ml; 1 RAEBt, 114 pg/ml). No significant correlation was found between FLT3 ligand levels and peripheral blood counts, age, or the presence of cytogenetic abnormalities. Also no correlation was detected between FLT3 ligand concentration and neutrophil, monocyte or lymphocyte count (data not shown). Refractory anemia (RA) and RA with ringed sideroblasts (RARS): In 29 patients with refractory anaemia (RA) (Table 1), platelet counts ranged from 2 to 526 × 109/l (median: 93 × 109/l; s.e.m. 29 × 109/l), leukocytes from 0.6 to 19.7 × 109/l (median: 3.3 × 109/l; s.e.m. 0.8 × 109/l), and hemoglobin levels from 5.1 to 14.2 g/dl (median: 9.4 g/dl; s.e.m. 0.5 g/dl). Of the 29 serum samples of RA patients, 15 (48.3%) showed FLT3 ligand concentrations greater than 100 pg/ml; 14 of those would be considered elevated with values between 404 and 5735 pg/ml (median: 2036 pg/ml; s.e.m. 394 pg/ml). No correlation was found with survival. One patient with RARS was included in our study and demonstrated an FLT3 ligand level of 2190 pg/ml. Bone marrow (BM) cellularity represents an objective parameter for its overall activity and can be equally well assessed in MDS patients by using biopsy or bone marrow aspirate.14

Figure 1 Levels of FLT3 ligand in serum of 93 patients with various forms of MDS. The numbers shown below the 100 pg/ml line are the numbers of individuals whose serum levels measured below the limit of detection in the ELISA. Thus, the total number of samples analyzed for each category of MDS were: RA, 29; RARS, 1; RAEB, 31; RAEBt, 23; CMML, 9.

Refractory anemia with excess of blasts (RAEB), RAEB in transition (RAEBt), chronic myelomonocytic leukemia (CMML): Low but measurable FLT3 ligand serum levels were detected in two of the 31 RAEB and one of the 23 RAEBt patients. In all the other RAEB/RAEBt patients and in nine CMML patients serum levels were not measurable. Discussion Despite extensive research during the past decade, the pathogenesis of MDS is still not completely understood. As a fascinating general concept for the origin and nature of cancer, the autocrine/paracrine model envisages an affected cell or its neighborhood producing an appropriate level of a growth factor to which it can respond, thus stimulating proliferation.16 On the basis of this model, it has been argued that defects in hemopoietic growth factor (HGF) regulation might be involved in malignant myeloid disorders. Studies have shown that AML blasts may produce and secrete GM-CSF17 or interleukin-318 in vitro and also express the receptors of these cytokines on their surface.19 GM-CSF serum levels were found to be elevated in about a quarter of MDS patients independent of their peripheral neutrophil or monocyte count,20 and endogenous serum IL-6 levels in MDS patients correlated with blast cell count and prognosis.21 These data suggest that HGF may be involved in autocrine/paracrine growth stimulation not only in AML but also in myelodysplastic syndromes. No correlation between FAB stage and serum level, however, was found for any of these HGF. As the majority of MDS patients do not die from acute leukemia but from the consequences of cytopenia, clinical trials have attempted to overcome cytopenia with the use of HGF,5–8 despite the potential risk of stimulating the malignant clone. Although factors like G-CSF,5 GM-CSF6 or IL-37,8 were shown to improve cytopenia for the duration of treatment in a significant number of patients, success in general has been limited. The clinical trials demonstrated, however, that in the group of patients suffering from refractory anemia (RA) no increased rate of irreversible transition to overt leukemia occurred as compared to the natural course of the disease. Therefore even excessive exogenous supply of HGF does not seem to be the exclusive stimulatory mechanism of clonal expansion. On the other hand, it cannot be ruled out that factors acting on an earlier stem cell level like stem cell factor (SCF) or FLT3 ligand22 may be involved in malignant growth alone or in combination with later-acting factors. SCF is an essential regulator of hematopoiesis and acts mainly on primitive hematopoietic progenitor cells.23 On its own, SCF exerts only limited colony-stimulating activity but synergistically stimulates in vitro colony formation of progenitor cells in combination with other hemopoietic growth factors (HGFs) like G-CSF, GM-CSF or IL-3.24,25 SCF signals through the c-kit tyrosine kinase receptor,26 which shows

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homology to the flt3/flk-2 receptor.9 The FLT3 ligand was cloned11 and although its range of activities has not yet been established in detail, it is apparent that, like SCF, it can synergize with multiple HGF to enhance growth and expansion of very primitive hematopoietic progenitor cells.11,27 In spite of the relatively similar effects which both factors exert, a comparison of the levels of FLT3 ligand and SCF in normal individuals and in those with hematopoietic disorders reveals some striking differences. Levels of SCF in normal human serum average several nanograms per milliliter,28,29 while FLT3 ligand serum levels are usually below 100 pg/ml. With regard to SCF, which like FLT3 ligand is also produced by bone marrow stromal cells,23 it cannot be ruled out that its overexpression might be involved in the pathogenesis of MDS by acting as a paracrine growth factor for the malignant clone. On the other hand, SCF deficiency may prevent lateracting hemopoietic growth factors from triggering cells along the maturation and differentiation pathways. In MDS patients, however, SCF serum levels were shown to be within the normal range produced by physiological stimuli and to not correlate with peripheral blood counts or FAB stage.29 SCF overexpression or deficiency thus does not seem to be involved in the pathogenesis of MDS although minor changes in the hemopoietic microenvironment cannot be completely ruled out. Aiming to characterize the potential of the bone marrow to release FLT3 ligand in myelodysplasia and detect a possible role in the pathogenesis of MDS, we measured the endogenous FLT3 ligand serum level in MDS patients. In our series FLT3 ligand serum levels were elevated in almost half of refractory anemia patients, while in the FAB stages with increased bone marrow blast cell count, none of the patients showed an elevated level. Although FLT3 ligand serum level was associated with disease stage, no correlation was detected in refractory anemia patients with survival or presence of cytogenetic abnormalities. The observation that none of the five patients with loss of the long arm of chromosome 5 demonstrated measurable FLT3 ligand serum levels may need further investigation. Our data suggest that FLT3 ligand levels can be upregulated early in the course of MDS. This may represent an appropriate response of the bone marrow stroma to a decreased number of normal progenitors. Similar data have been described in severe aplastic anemia and Fanconi anemia, where FLT3 serum levels were significantly greater than in healthy volunteers.15 In the more advanced stages of MDS when the malignant clone has further progressed, FLT3 ligand production may be downregulated due to further increasing dyshematopoiesis and accelerated turn-over of bone marrow cells caused by the malignant clone. According to this hypothesis one can argue that FLT3 overexpression is simply a secondary phenomenon and not necessarily involved in the growth advantage of the malignant clone. It is well-documented that FLT3 ligand is able to expand dendritic cells and cytolytic NK cells.30 As dendritic cells are very potent antigen-presenting cells, it may also be argued that upregulation of FLT3 ligand at an early stage of MDS may be a physiological reaction of the immune system with the aim to suppress the malignant process. On the other hand, it cannot be ruled out that upregulation of FLT3 ligand might provide the malignant clone with a growth advantage and be causally involved in its progression. In the presence of further hematopoietic growth factors, the malignant clone may profit from increased FLT3 production caused by physiological stimuli and progress to more

advanced MDS stages and over leukemia. Possible explanations for the normal FLT3 ligand serum levels in MDS stages with excess of blasts are dysregulation in cytokine production or binding of the ligand to its receptor. Based on published data,30 levels of FLT3 receptor are known to be low, generally less than several thousand sites per cell. In MDS, however, FLT3 receptor expression is expected to be higher due to its increased expression on malignant hematopoietic cells.31 Therefore enough receptors may be present to bind free FLT3 ligand in patients with elevated bone marrow blast cell count. In conclusion, our data demonstrate that FLT3 ligand is the only known growth factor with a distinct expression pattern in MDS stages. Further investigations are necessary to define its potential role in the pathogenesis of MDS. References 1 Koeffler Ph. Myelodysplastic syndromes (preleukemia). Semin Hematol 1986; 4: 284–291. 2 Bennett J, Catovsky D, Flandrin G, Daniel MT, Galton DAG, Gralnick H, Sultan C. Proposals for the classification of the myelodysplastic syndromes. Br J Haematol 1982; 1: 189–199. 3 Yunis J, Rydell RE, Oken MM, Arnesen M, Mayer M, Lobell M. Refined chromosome analysis as an independent prognostic indicator in de novo myelodysplastic syndromes. Blood 1986; 67: 1721–1730. 4 Mufti GJ, Stevens J, Oscier D, Hamblin TG. Myelodysplastic syndromes: a scoring system with prognostic significance. Br J Haematol 1986; 59: 425–432. 5 Negrin RS, Stein R, Vardiman J, Doherty K, Cornwell J, Krantz S, Greenberg PL. Treatment of the anemia of myelodysplastic syndromes using recombinant human granulocyte colony-stimulating factor in combination with erythropoietin. Blood 1993; 82: 737–743. 6 Willemze R, van der Lely N, Zwierzina H, Suciu S, Solbu G, Gerhartz H, Labar B, Visani G, Peetermans ME, Jacobs A, Stryckmans P, Fenaux P, Haak HL, Ribeiro MM, Baumelou E, Baccarani M, Mandelli F, Jaksic B, Louwagie A, Thyss A, Hayat M, de Cataldo F, Stern AC, Zittoun R on behalf of the EORTC Leukemia Cooperative Group. A randomized phase I/II multicenter study of recombinant human GM-CSF therapy for patients with myelodysplastic syndromes and a relatively low risk of acute leukemia. Ann Hematol 1992; 64: 173–180. 7 Ganser A, Vo¨lkers B, Greher J, Ottmann O, Waltherm F, Becherm R, Bergmann L, Schulz G, Hoelzer D. Recombinant human granulocyte–macrophage colony-stimulating factor in patients with myelodysplastic syndromes – a phase I/II trial. Blood 1989; 73: 31–36. 8 Willemze R, Fenaux P, Gerhartz H, Zwierzina H, de Witte T, Stryckmans P, Labar B, Sklenar I, Suciu S, Solbu G, Dardenne M, Hoffbrand V, Jacobs A, Josten C, Zittoun R. A randomized phase I/II multicenter study of rhIL-3 in patients with myelodysplastic syndromes at relatively low risk of developing leukemia. Blood 1992; 80 (Suppl 1): 86a. 9 Matthews W, Jordan CT, Wiegand GW, Pardoll D, Lemischka IR. A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations. Cell 1991; 65: 1143–1148. 10 Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, Rockwell P, Witte L, Borowitz MJ, Civin Cl, Small D. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. Blood 1996; 87: 1089–1096. 11 Lyman SD, James L, Van den Bos T, de Vries P, Brasel K, Gliniak B, Hollingsworth LT, Picha KS, McKenna HJ, Splett RR, Fletcher FF, Maraskovsky E, Farrah F, Foxworthe D, Williams DE, Beckmann MP. Molecular cloning of the ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 1993; 75: 1157–1167. 12 Rusten LS, Lyman SD, Veiby OP, Jacobsen SEW. The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro. Blood 1996; 87: 1317–1323. 13 Piacibello W, Fubine L, Sanavio F, Brizzi MF, Severino A, Garetto


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