.Effect of Stem Cell Factor on Colony Growth From Acquired and Constitutional (Fanconi) Aplastic Anemia

By G.P. Bagnara, P. Strippoli, L. Bonsi, M.F. Brizzi, G.C. Avanzi, F. Timeus, U. Rarnenghi, G. Piaggio, J. Tong, M. Podesta, G. Paolucci, L. Pegoraro, V. Gabutti, and A. Bacigalupo

The aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hemato- poietic progenitors from patients with acquired severe aplas- tic anemia (AA) or Fanconi’s anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony- forming unit granulocyte-macrophage (CFU-GM) (in the pres- ence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU- GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 t o 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/105 cells plated. In patients with acquired AA, SCF induced a significant enhance- ment of BFU-E growth (8 to 29; P = .01) and allowed the formation of GEMM colonies that were not scored in baseline

OLONY FORMATION from marrow cells of patients C with both acquired or congenital (Fanconi’s) aplastic anemia (AA) is consistently low.’“ In standard culture conditions, colony growth is unaffected by the addition of colony stimulating factors (CSFs) such as granulocyte- macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF), interleukin-3 (1L-3), and, only rarely, by IL-1.5 However, in some cases of acquired aplasia, T-lymphocyte depletion partially restores the in vitro response of progenitor cells to these cytokines.6 Intrinsic defects of the hematopoietic progenitor cells and/or derangement of the bone marrow microenvironment are believed to be involved in the patho- genesis of these disorders, although their respective roles are still poorly understood.

Stem cell factor (SCF) has been shown to stimulate the more primitive hematopoietic progenitor cells in combina- tion with other cyt~kines?-~ Mutations in the Steel locus (Sl), which encodes the SCF, and in the white spotting locus

From the Divisione di Ematologia, Ospedale San Martino, Genova; the Istituto di Istologia ed Embriologia, Centro Interdipartimentale di Ricerca sul Cancro Giorgio Prodi, Universitd Bologna, Bologna, and the Istituto di Clinica Pediatrica e Dipartimento di Scienze Bwmediche e Oncologia Umana, Universita Torino, Torino, Italy.

Submitted November 15,1991; accepted March 25,1992. Supported by the Italian National Research Council (C.N.R)

Ematologia CT No. 88.00739.44 and 89.02407.04, and by Associazi- one Ricerca Trapianto Midollo Osseo (A.RI.T.M.0.) f0rA.B. and by Italian Association for Cancer Research (AIRC) grants to G.P.B. and L.P. M.E.B. is a fellow of the AIRC.

Address reprint requests to A. Bacigalupo, MD, Diviswne Ematolo- gia 2, Ospedale San Martino, 16132 Genova, Italy.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

0 1992 by The American Society of Hematology. 0006-4971 l92i8002-0014$3.00/0

culture conditions (0 to 8; P = .01). CFU-GM growth was enhanced (4 to 20). but not significantly (P = .3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion-dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hemato- poietic progenitor cells from FA patients t o GM-CSF + SCF or IL-3 + SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for a- and p-chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progeni- tors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder. 0 1992 by The American Society of Hematology.

(W), encoding the SCF receptor (c-kit) of the mouse, are known to cause a vast array of phenotypic alterations mainly in the coat color, gonadal development, and hemato- poiesis.10J Anemia in Steel locus (Sl/Sld) mutant mice, which are defective in the production of SCF, can be reversed by the in vivo administration of this cytokine.I2 In Blackfan Diamond anemia (DBA) patients, which share several clinical similarities with S1 mutant mice, weI3 and other~’~J5 have shown that the addition of SCF can partly restore in vitro erythropoiesis. However, more recently, a higher than normal serum level of immunoreactive SCF as well as normal nucleotide sequences for both the SCF and c-kit coding regionsI6 have been shown in DBA patients.

Fanconi’s anemia (FA) is an autosomal recessive disease exhibiting a variable degree of severity and an average age of onset of 8 to 9 years. The disease, which is associated with several deformities and increased spontaneous chromo- some damage, shares some clinical features with the ane- mia in W-defective mice. In these mice, an array of mutations in the transmembrane and intracellular domains of the c-kit receptor gene have been described correspond- ing to different degrees of anemia.lOJ1

The present study was undertaken to investigate the effect of SCF on colony growth, and the expression of GM-CSF/IL-3 receptor and c-kit messenger RNAs (m- RNAs) in bone marrow cells from patients with acquired or constitutional AA.

MATERIALS AND METHODS

We studied 12 patients with acquired severe AA17: six at diagnosis and six after immunosuppressive therapy consisting of antilymphocyte globulin (ALG) or cyclosporin A (CyA). We also studied 14 patients with FA who had a hypoplastic marrow and no evidence of leukemia and nine healthy donors. The main clinical data of the patients are summarized in Table 1.

All patients were studied for chromosomal abnor- malities. All patients under the age of 20 were also tested for

Patients.

Cytogenetics.

382 Blood, Vol80, No 2 (July 15). 1992: pp 382-387

STEM CELL FACTOR AND APLASTIC ANEMIA 383

Table 1. Clinical Data of Patients at the Time of Study

Blood Counts at This Study

Age/ Etiology Years From Last Hct PMN Plts Transfusion Case Sex of SAA Diagnosis Treatment Response (X) ( X 10SlL) (X10~1L) at Study

1 2 3 4 5 6 7 8 9

10 1 1 12’ 13 14 15 16 17 18 19 20 21 22 23 24 25’ 26’

56/F 1?/M 15/M 35/M

2/F 49/F 42/M 24/M 48/M 23/M 11/M 16/M 14/M 16/M 9/M 8/F 7/M

13/F 8/F

15/M 12/M 4/M 6/M 4/M ?/M

21/M

ID 2 ID 9 ID 7 ID At Dx ID At Dx ID At Dx 5q- At Dx ID 1 ID At Dx ID 0.5 ID 0.5 ID At Dx FA 2 FA 11 FA 6 FA 3 FA 3 FA 8 FA 1 FA 4 FA 5 FA 4 FA 2 FA 1 FA 4 FA 1

HALG HALG + An RALG-CyA - - -. -

CYA

CYA HALG

- An An An An An

An

An

PR

No No

PR 42 PR 32 Re1 26

21 22 20 22 32 25 25 25 22 35 24 24 20 28 30 32 25 30 20 32 23 27 26

2.0 1.2 2.2 0.8 0.1 0.1 0.3 1.2 1.5 1.3 0.15 1.5 0.6 0.72 0.38 0.8 0.36 0.87 0.8 0.58 1.26 0.21 3.04 1.16 1.8 0.5

90 48 12 8 5 1 5

100 10 31

5 17

1 1 1 19 35 10 15 84 54 40 93 19

100 54 52

123

Abbreviations: SAA, severe AA; 5q-, patients referred with SAA but formed to have a 5q- myelodisplasia; ID, idiopathic SAA; At Dx, at diagnosis; Rel, relapse of the aplasia after response; PR, partial remission; HALG + An, horse ALG plus androgen; RALG, rabbit ALG; Ind, independent; Dep, dependent.

‘Only molecular study performed.

chromosomal breakage after exposure to diepoxybutane (DEB).18 Patients defined as acquired AA had normal karyotypes. One patient (no. 7) had a clonal abnormality in all examined metaphases (del 5q-). All FA patients showed abnormal chromosomal break- age with the DEB test.

Bone marrow was aspirated from the posterior iliac crests in heparinized syringes. The marrow was then run on Ficoll-Hypaque gradient (Pharmacia, Uppsala, Sweden) and the light-density cells were recovered and freed of adherent cells by two sequential plastic adherence (each for 1 hour at 37°C). Light-density, nonadherent mononuclear cells (MNAC) were fur- ther freed of T, B, and natural killer (NK) cells by passage in immunomagnetic beads coated with antimouse IgG (M-450 Dyna- beads; Dynal, Oslo, Norway) after incubation with anti-CD2, -CD4, -CD8, -CD19, -CD56, -CD57, and -CD14 (Becton Dickinson Italia, Milan, Italy).

Colony growth. Non-T, non-B, non-NK MNAC cells were cultured in vitro for burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte, erythroid, monocyte, megakaryo- cyte (CFU-GEMM) according to Iscove et a1.19 Briefly, 105 cells were plated in 1 mL mixture of Iscove’s modified Dulbecco’s medium (IMDM) containing 30% fetal calf serum (FCS), 2 x W4 mol/L Hemin, 5 X mol/L P-mercaptoethanol, 1% bovine serum albumin (BSA; fraction V, Sigma, St Louis, MO), 0.9% methylcellulose in the presence of 2 U recombinant human erythropoietin (rhEpo; Cilag Chemi, Cologno Monzese, Milan, Italy) and 100 U of rhIL-3 (Genzyme Corporation, Cambridge, MA) with or without 20 ng/mL SCF (Genzyme). The 14-day

Cell separation.

CFU-GM assay was performed as previously described.zo Briefly, 105 non-T, non-B, non-NK MNAC cells were plated in 35-mm Petric dishes in 1 mL of IMDM medium containing 20% heat- inactivated FCS, 0.3% noble agar, 200 U of rhGM-CSF (Gen- zyme), and 100 U of rhIL-3 with or without 20 ng of SCF. Petri dishes were incubated at 37°C in 5% COz and 14-day colonies were scored with a Leitz (Germany). Triplicate wells were counted and the results are expressed as colonies/105 cells plated.

Whole bone marrow samples from three human healthy donors, from three FA patients (case nos. 16, 25, and 26), and from two AA patients (case nos. 9 and 12) were processed for RNA extraction. M07e cell linez1 and the human bladder carcinoma cell line 5637 (kindly provided by Dr G. Rovera, Wistar Institute, Philadelphia, PA) were used as positive and negative controls, respectively, for cytokine receptor mRNA expres- sion. Total RNA was extracted with guanidinum thiocyanate and isolated by acid phenol-chloroform extraction.z2 Twenty-micro- gram RNA samples were fractionated on 6% formaldehyde-1.2% agarose gel and blotted onto nylon membrane (Hybond-N+; Amersham, Buckinghamshire, UK). DNA probes were 3zP-labeled by random-priming according to Sambrook et al.23 The filter was hybridized and washed by the method of Church and Gilbertz4 and exposed to X-ray film for 2 to 4 days. The following probes were used: c-kit was a 1.2-kb 5’ human cDNA fragment:5 myeloperoxi- dase was a nearly full-length human c D N & ~ ~ human GM-CSF receptor a-chain was a 1.2-kb nearly full-length human CDNA?’ human GM-CSF/IL-3 receptor P-chain was a human full-length

Northem blot analysis.

384 BAGNARA ET AL

c D N A , ~ and human IL-3 receptor a-chain was a full-length human cDNA."

RESULTS

The in vitro growth of bone marrow progenitor cells was studied in 11 AA, 12 FA patients, and ninc normal controls. In the latter, the addition of SCF to baseline culture conditions enhanced CFU-GM, BFU-E, and CFU-GEMM growth (Table 2).

Marrow cclls from patients with acquired AA in standard culture conditions showcd greatly reduced growth of CFU-GM and BFU-E with respect to normal controls (Table 2) and CFU-GEMM growth was totally absent (Fig 1).

The addition of SCFenhanced CFU-GM growth (P = .3) (Table 2 and Fig 1). This cnhancing effect was more pronounced on BFU-E growth (P = .01) (Table 2 and Fig 1). Finally, in fivc patients, SCF allowed the formation of GEMM colonics, which were totally absent in all baseline cultures. This was seen in patients at diagnosis and after immunosuppressive trcatmcnt.

In FA paticnts, the growth of CFU-GM, BFU-E, and CFU-GEMM in basclinc conditions was nearly absent. In one case (case no. 17), SCF was found to stimulate

Colony growth.

Table 2 Colony Growth In vitro From Non-T, Non-B, Non-NK Light-Density Nonadherent Mononuclear Bone Marrow Cells in

Patients With SAA, FA, and Normal Controls

CFU.GMIl0 EFU-EI105 CFU.GEMMI10'

-SCF +SCF -SCF +SCF -SCF +SCF

Acquired SAA 1 ND ND 10 103 0 16 2 15 20 8 64 0 14 3 ND ND 18 78 ND ND 4 50 46 16 66 ND ND 5 2 14 0 18 ND ND 6 8 36 8 29 0 8 7. 0 0 0 0 0 0 8 4 32 16 28 0 8 9 0 0 16 36 0 8

lot 27 32 0 0 0 0 l l t 0 0 0 0 0 0

13 0 0 0 0 0 0 14 0 0 0 0 0 0 15 0 0 0 0 0 0 16 0 0 0 0 0 0 17 4 27 13 93 0 0 18t 0 0 0 0 0 0 19t ND 0 0 ND 20 0 0 0 0 0 0 21t 0 0 0 0 0 0 22 0 0 8 0 0 4 23 0 0 4 8 0 0 24t 0 0 4 60 ND

FA

Normal controls

(mean f SD) 106 f 24 230 2 43 206 2 47 407 2 72 4 f 2 43 f 10 9 cases

Abbreviation: ND, not done. .Clonal cytogenetic abnormality 5q-. tMNAC, further purification was not possible.

N.colonles p- 0.01 36

I

i 1 p' 0.01 j

- 8 - , ni -, ;I j - i 0 0 ~ 0 , o 0 0 0 !-A- - "

Fmconl Acsutred SAA Fanconl A c w r e a SAA Fmconl ACaulraa SAA

CFU-GM BFU-E CFU-GEMM

Fig 1. Colony growth of manow cells from patients with acquired AA and FA in the (W) absence or in the presence (Z) of SCF. Patients with FA show no improvement of colony formation. On the contrary, patients with acquired AA show a clear improvement of CFU-GM, BFU-E, and CFU-GEMM growth. Numbers represent median numbers of colonies xlW/cells plated; Pvalues above bars.

CFU-GM and BFU-E growth. The patient was transfusion- dependent and on androgens therapy. In another case (case no. 24, transfusion-indcpcndent), only BFU-E growth was stimulated by thc addition of SCF.

Morphologic examination of colonies developed both in AA and in FA paticnts showed normal terminal cell diffcrcntiation.

Of six patients studicd at diagnosis of the diseasc, four were treated with immunosupprcssion and are evaluable. AI1 four showed enhanced colony growth with thc addition of SCF. One dicd early of infection (case no. 6) (day +20), two re- sponded (case nos. 4 and s), and one is 4 months posttreat- mcnt transfusion-dependent (case no. 9).

Molccular analysis was performed to evaluate whethcr the abscnt or dcfcctive in vitro response of AA and FA bone marrow progenitor cells to GM-CSF, IL-3, and SCF was due to an abnormal expression of thcir receptor transcripts. For this purpose, c-kit, GM-CSF, and IL-3 receptor p-chain, GM-CSF rcceptor a-chain, and IL-3 receptor a-chain transcripts were analyzed by Northern blot analysis in whole bonc marrow samples from three FA patients (patient nos. 16. 27, and 28). two AA patients (patient nos. 9 and 12), and four normal controls. To document the presencc of myeloid cells in the samples, the expression of the myeloperoxidase gene was also studied. As shown in Fig 2A, c-kit and myeloperoxydase transcripts were dctccted in normal as wcll as in all FA and AA samples. A considerable degree of variability was observed not only among patients but also among normal bonc marrow samples. As shown in Fig 2B, thc transcript for GM-CSF and IL-3 receptor p-chain was clearly exprcssed, although in variable amounts, in all FA and AA patients and in the controls. Thc transcript of GM-CSF and IL-3 rcccptor a-chains appeared to be weakly expressed. How- ever, we were ablc to dctcct transcripts for GM-CSF receptor a-chain in all FA and AA patients (Fig 2C). Thc expression of IL-3 receptor a-chain gcnc (Fig 2D) was well dctectablc in one FA paticnt and in onc normal control

Clinical response to immunosuppression.

Molecular studies.

STEM CELL FACTOR AND APLASTIC ANEMIA

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

Fig 2 Nocthem blot analysis of RNA from whole bone marrow sampler of four normal controls (lanes 1 through 4). three FA pa- tients (lane 9, case no. 25; lane 10, case no. 26; lane 12, case no. 16). and two AA patients (lane 12, case no. 9; lane 13, case no. 12). The RNA from cell lines 5837 (lane 6) and M97e (lane 7) are included as negative and posi- tive controls. The same filter was hybridized sequentially wtth the c-kif and the myeloperoxidare probes (A), with the GM-CSF and IL-3 receptor p-chain probe (B), with the GM-CSF receptor a-chain probe (C), with the IL-3 receptor a-chain probe (D), and with the p-actin probe (E). The latter was Included as a control for the amounts of RNA loaded in each lane.

c - K I T

MPOX

6M -CSF / 11-3 R fl -chi in

GM-CSF R a-chiin

11-3 R a -chain

- ACTIN

.* . a*

(lanes 3 and 9 of Fig 2D), barely detectable in another FA patient and in two normal controls (lanes 1.2, and 1 l), and undetectable in the other samples.

DISCUSSION

The aim of the present study was to test the effect of SCF on colony growth of bone marrow cells from patients with acquired or constitutional severe AA. Our results indicate that SCF significantly improves the in vitro colony growth of patients with acquired AA over baseline colony formation with GM-CSF, IL-3, and Epo. This is true especially for BFU-E and for CFU-GEMM, both for patients at diagnosis of the disease and after ALG treatment. It is of interest that SCF improved colony formation even in untreated patients with very severe AA (neutrophil counts below 0.2 x 10p/L and platelet count below 5 x 109/L), suggesting that, de- spite severe pancytopenia and an empty marrow, residual stem cells are still surviving. This concept is in line with the observation of autologous hematopoietic reconstitution after rejection of an allogenic bone marrow graftM and with the notion that 50% of patients treated with immunosup- pressive therapy may recover to self-sustaining hematopoie-

385

A

B

C

D

E

sis.31.32 Despite these clinical findings, it has been extremely difficult so far to grow and quantitate hematopoietic progen- itors from these patients, not only at diagnosis of the disease, but also after hematologic reco~ery.~.~~” The finding that SCF enhances the in vitro growth both with and without T cells (data not shown) suggests that the effects of SCF are not dependent on the presence/absence of acces- sory cells. This finding is in keeping with a direct effect of SCF on hematopoietic progenitor^.^^ In contrast with the data obtained in AA, in almost all

FA patients no colony growth was observed with or without the addition of SCF. There were only two exceptions: in case no. 17, a partial responder to androgen treatment, and case no. 21, a transfusion-independent subject.

The clinical appearance of FA is heterogenous and there has been a recent attempt to classify FA patients into several categories of severity according to their in vitro colony growth.M The failure to promote the formation of hematopoietic colonies in vitro from FA patients in the presence of GM-CSF, IL-3, and SCF, alone or in combina- tion, could be related to the depletion of progenitor cells or to their intrinsic inability to respond to the cytokines. The

386 BAGNARA ET AL

former hypothesis is disproved by experiments in which FA patients’ CD34+ cells recovered and cultured failed to form colonies in the presence of SCF, IL-3, and GM-CSF (data not shown).

To test the possibility that the lack of response of hematopoietic progenitor cells could be due to an intrinsic defect of GM-CSF/IL-3 and SCF receptors, we studied the expression of their mRNAs. GM-CSF/IL-3 receptors are formed by a and p subunits that must be coexpressed to form high-affinity binding sitesa The a subunits specifically recognize each ligand, whereas the p subunit is common to both receptors. Our results show that both AA and FA bone marrow cells express the transcripts for the a and p subunits of these receptors. Furthermore, in FA patients, a c-kit mRNA of the same molecular weight observed in normal subjects and AA patients was detected. Preliminary results from our laboratory, obtained with an antis-kit antiserum, indicate that SCF protein is expressed on CD34+ cells from normal as well as from AA and FA bone marrow samples.

However, these findings do not rule out the possibility that an abnormal c-kit protein, either in the ligand binding region or in the intracellular signal transduction domain, could be responsible for the hematologic defect. Mutations in the W locus affect various aspects of hematopoiesis and the proliferation and migration of primodrial germ cells and melanoblasts during development.lOJ1 In addition, a large number of independent mutations at this locus give rise to phenotypes that vary in severity in the heterozygous and homozygous states. Some of these mutations give rise to a c-kit protein product normally expressed but showing a defective thyrosine kinase activity3’ or a defective interac- tion of the kit receptor with the cytoplasmic signaling proteins.38 Because of the comparable clinical features between the W defect in mouse and FA, it can also be

speculated that the human disease could be the conse- quence of point mutations in the receptor gene. Sequencing of the c-kit gene in FA will allow us to validate this hypothesis.

It is premature to raise the question of the potential effect of SCF in vivo, although preclinical studies have shown promising re~ults.3~ Growth factors are being used increasingly in patients with pancytopenia, although there is at present no convincing evidence of a long-lasting effect on the marrow function of patients with AA and a transi- tory increase in peripheral blood counts has been the rule, with few Actually, the administration of growth factors in vivo could be hazardous, because it may force residual stem cells to differentiate, and we have recently proposed that refractoriness to high doses of GM-CSF in patients with AA may be one “protective” mechanism allowing for the gradual expansion of the stem cell pool? The possibility of growing mixed colonies from marrow cells of AA patients in the presence of SCF is certainly encouraging, but does not guarantee hematopoi- etic reconstitution. We may be looking at forced differenti- ation of a small number of cells with limited proliferative capacity.

Our results point out the intrinsic nature of the stem cell defect in FA patients, possibly associated with the multiple genetic defects described in this disease,” and this, in turn, may explain the major difference between acquired and constitutional aplastic patients.

ACKNOWLEDGMENT

We are grateful to Dr T Kitamura (DNAX, Palo Alto, CA) for kindly providing the GM-CSF and IL-3 receptor cDNA probes. We thank the Italian Research Association on Fanconi’s Anemia (A.I.R.F.A.) and the Stem Cell Study Group of Italian Association of Pediatric Ematology and Oncology for their collaboration.

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