Leukemia Research 28 (2004) 121–126

Lack of KIT or FMS internal tandem duplications butco-expression with ligands in AML

Rui Zhenga, Kendra Klanga, Norbert C. Gorinb, Donald Smalla,∗a Department of Pediatric Oncology, Johns Hopkins University School of Medicine, Room 253,

Bunting-Blaustein Cancer Research Building, Baltimore, MD 21231-1000, USAb Department of Hematology, Hospital Saint-Antoine, Paris 75012, France

Received 21 February 2003; accepted 26 May 2003

Abstract

KIT and FMS, members of the class III receptor tyrosine kinase family, are expressed on normal hematopoietic cells and have impor-tant roles in normal hematopoiesis. FLT3 is also a member of the class III receptor tyrosine kinase family and plays important role inhematopoietic stem/progenitor cells, NK, and dendritic cells. Recently, internal tandem duplication (ITDs) mutations have been foundin the juxtamembrane (JM) region of FLT3 receptor expressed by patients with acute myeloid leukemia (AML) and myelodysplasticsyndrome (MDS). The mutations result in the constitutive dimerization and activation of the receptor, contributing to leukemic trans-formation. KIT and FMS are also frequently expressed in AML and are closely related to FLT3. Thus, similar ITD mutations couldalso occur in the KIT and/orFMS gene of patients with AML. To explore this possibility, 13 human leukemia-lymphoma cell lines and44 AML patient samples were examined by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of ITD muta-tions in the JM region of the KIT or FMS receptor. None of the 13 human leukemia-lymphoma cell lines or 44 AML primary bonemarrow samples express ITDs in either KIT or FMS in the JM region that is involved in FLT3 mutations. The 13 cell lines and 44AML samples were also examined for the possible co-expression of KIT and/or FMS receptors with their respective ligands, as wehave seen for FLT3 and its ligand, FL. This co-expression could contribute to leukemic transformation through autocrine, paracrine, orintracrine activation mechanisms. And 6/13 cell lines and 27/44 primary AML samples exhibit co-expression of the KIT receptor andligand (SCF) while 10/13 cell lines and 35/44 primary AML samples exhibit co-expression of the FMS receptor and ligand (CSF-1).Therefore, while ITD mutations were not found, the findings of co-expression of KIT and/or FMS with their respective ligands impliesthese receptors might contribute to leukemogenesis in some patients with AML through autocrine, paracrine, or intracrine interactivestimulation.© 2003 Elsevier Ltd. All rights reserved.

Keywords: KIT; FMS; Internal tandem duplication; Mutation; Acute myeloid leukemia; SCF; CSF-1

1. Introduction

KIT, FMS and FLT3 encode members of the classIII receptor tyrosine kinase family that are expressed byand play important roles in normal hematopoietic cells[1–3]. Common features of the receptors include: fiveimmunoglobulin-like domains in the extracellular re-gion, a single transmembrane domain, a juxtamembrane(JM) region, an interrupted tyrosine kinase domain and aC-terminal domain[4]. Binding of their growth factors (FL,

Abbreviations: JM, juxtamembrane; AML, acute myeloid leukemia;RT-PCR, reverse transcription-polymerase chain reaction; ITD, internaltandem duplication

∗ Corresponding author. Tel.:+1-410-614-0994; fax:+1-410-955-8897.E-mail address: donsmall@jhmi.edu (D. Small).

SCF/KL and CSF-1, respectively) results in dimerization,activation of the tyrosine kinase activity, and autophos-phorylation of tyrosine residues of the receptors, as wellas transphosphorylation of other proteins involved in thesignal transduction cascade[5]. Signal transduction alsoinvolves docking of downstream signaling proteins contain-ing SH2 domains that recognize phosphorylated tyrosineson the receptors[6,7]. Ultimately, signal transductionthrough these receptors results in the proliferation and dif-ferentiation of hematopoietic cells expressing the receptors[8,9].

Within the human hematopoietic system, the KIT re-ceptor is expressed by approximately 70% of CD34+cells in bone marrow, as well as mast cells[10,11]. In60–100% of acute myeloid leukemia (AML) patients, KITis expressed by myeloblasts as assessed by cell surface

0145-2126/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0145-2126(03)00184-X

122 R. Zheng et al. / Leukemia Research 28 (2004) 121–126

immunostaining, ribonuclease protection assay or RT-PCR[12–17]. The addition of SCF stimulates the proliferationof some leukemia derived cell lines, as well as the leukemicblasts from most cases of KIT+ AML [13,15,18]. Simi-larly, the activation of wild-type FLT3 receptor by FL alsostimulates the proliferation of primary AML[19]. FMS isexpressed by cells of the monocyte and macrophage lin-eage, as well as by 46–100% of AML samples measuredby Northern blot [20–22]. Single nucleotide mutations,which constitutively activate the receptors, have been foundin both KIT and FMS in AML [23,24]. Cellular transfor-mation, inhibition of differentiation, survival advantages,and expansion are all potential consequences of mutationsor autocrine ligand-stimulation of these receptors and canlead to the development of leukemia[25–27]. Other typesof KIT mutations, deletion plus insertion mutations, occurin approximately one third of cases of AML with inv(16)karyotype[28]. In addition, both activating and silent pointmutations have been identified in patients with mastocy-tosis [29–31]. Spontaneously arising mastocytomas havealso been reported in dogs. In these dog tumors, mutations,deletions, and one case of an insertion in the JM region ofKIT have been identified[32]. The insertion consists of a48 bp internal tandem duplication (ITD) which results inthe addition of repeated and novel amino acids to the JMdomain of KIT. All of the KIT alterations found in dogmastocytosis induce spontaneous phosphorylation of KITin the absence of exogenous SCF.

A new mechanism of mutation by which the FLT3 recep-tor becomes constitutively activated has been reported. Small(3–400 bp) ITD mutations of the JM region of FLT3 havebeen found in 17–34% of patients with AML[33,34]. TheJM domain inhibits the FLT3 kinase activation, and the elon-gation mutation of the JM domain due to the ITD mutationappears to abrogate the inhibitory function of this region inthe absence of FL resulting in constitutive tyrosine kinase ac-tivation[35]. In vitro studies have shown that FLT3 receptorscontaining ITD mutations dimerize in a ligand-independentmanner, leading to autophopsphorylation of the recep-tor through constitutive activation of the tyrosine kinasedomain [36]. Constitutively activated FLT3 appears tocontribute to leukemic transformation and portends an es-pecially poor prognosis for patients with this mutation[37–39].

Due to the fact that KIT and FMS belong to the samereceptor tyrosine kinase family as FLT3 and have the samegeneral structure, it is very possible that ITD mutations ofKIT or FMS could occur in AML. The purpose of this studyis to determine whether or not KIT and/or FMS exhibit ITDmutations analogous to those found in the FLT3 receptor ineither leukemia cell lines or AML patient samples. In ad-dition, the possibility of co-expression of KIT and/or FMSwith their respective ligands, SCF and CSF-1, was investi-gated to determine whether autocrine stimulation of thesereceptors might occur, which could contribute to leukemo-genesis.

2. Materials and methods

2.1. Patients and samples

Bone marrow samples from 44 AML patients were ob-tained prior to therapy with patient consent under IRBapproved protocols at the Johns Hopkins Hospital and Hos-pital Saint-Antoine (Paris, France). FAB classification wasavailable for 39 samples (1 M0, 9 M1, 14 M2, 8 M4, 1 M5,and six developed from myelodysplastic syndrome (MDS)).Leukemic blast cells from fresh or frozen samples were sep-arated by Ficoll-Pague (Amersham, Piscataway, NJ) densitygradient centrifugation and low-density cells were used[40].

2.2. Cell lines

Established human leukemia and lymphoma-derived celllines including EM3, HEL, K562, HL-60, U937, LAZ 221,REH, ML-1, MOLM-1, EOL-1, BV173, MV4-11, andWSU-NHL, were cultured in RPMI 1640, supplementedwith 10% heat-inactivated fetal calf serum (FCS) and antibi-otics (penicillin and streptomycin) at 37◦C with 5% CO2.

2.3. RT-PCR

Total cellular RNA was extracted from cultured cell linesand patients’ blast cells using RNeasy columns (Qiagen, Va-lencia, CA) following the manufacturer’s instructions. And50–100 ng RNA were reverse transcribed and amplified us-ing the one-step RT-PCR KIT (Invitrogen, Carlsbad, CA).PCR was performed with the following primer pairs.

KIT forward primer: 5′-CTGTTCACTCCTTTGCTG-AT-3′.

KIT reverse primer: 5′-CACCCAGGGTTTTCCCAAA-ACT-3′.

FMS forward primer: 5′-GCATGTCCATCATGGCCT-TG-3′.

FMS reverse primer: 5′-CTCCGAGGGTCTTACCAAA-CTG-3′.

The primers for KIT and FMS were designed to cover thehomologous region of the FLT3 JM region. The expectedamplification product of KIT and FMS using these primersis 229 and 204 bp, respectively.

Primers designed for SCF and CSF-1 were used to detecttranscripts of KIT and FMS ligand, respectively.

SCF forward primer: 5′-CAGAACAGCTAAACGGAG-TC-3′.

SCF reverse primer: 5′-CTCCACAAGGTCATCGAC-TA-3′.

CSF-1 forward primer: 5′-TCGGAGTACTGTAGCCAC-AT-3′.

CSF-1 reverse primer: 5′-CAACTGGAGAGGTGTCTC-AT-3′.

R. Zheng et al. / Leukemia Research 28 (2004) 121–126 123

The expected amplification product of SCF and CSF-1using these primers is 405 and 330 bp, respectively.

To enable discrimination between possible genomic DNAcontamination and cDNA amplification, all primer pairsspanned sequences encoded in different exons. Reversetranscription was performed at 50◦C for 30 min. Thirty fivecycles of amplification were then performed at 94◦C for30 s for denaturation, at 55◦C for 30 s for annealing andfinally at 72◦C for 1 min for extension on a Bio-Rad DNAiCycler (Hercules, CA). PCR products were resolved in a5% polyacrylamide gel which has enabled us to easily re-solve FLT3 ITDs differing by as little as 18 base pairs fromthe wild type FLT3 sequence in this region. If a KIT/ITDor FMS/ITD mutation is present, a higher molecular weightband than wild-type receptor would be visualized.

2.4. Immunoprecipitation and Western blot analysis

Cells were washed twice with ice-cold phosphate bufferedsaline (PBS) and lysed for 30 min in ice-cold NP-40 ly-sis buffer (20 mmol/l Tris–HCl, pH 7.4, 150 mmol/l NaCl,100 mmol/l NaF, 10% glycerol, 1% NP-40 and 10 mmol/lEDTA) containing protease and phosphatase inhibitors(2 mmol/l sodium orthovanadate, 50�g/ml antipain, 5�g/mlaprotinin, 1�g/ml leupeptin and 10�g/ml phenylmethyl-sulfony fluoride; Sigma, St. Louis, MO). Clarified lysates(500�g) were incubated with rabbit polyclonal antibody tohuman KIT or FMS (SantaCruz Biotechnology, Santa Cruz,CA) followed by protein A-agarose (Upstate Biotechnology,Inc, Lake Placid, NY) at 4◦C. The immunoprecipitates werewashed 3 times with ice-cold TBS-T (10 mmol/l Tris–HCl,pH 7.4, 100 mmol/l NaCl, 0.1% Tween-20), resuspended inSDS sample buffer, heated, and separated by SDS-PAGEin 8% gels. Gels were transferred onto polyvinylidene flu-oride (PVDF) membrane (Millipore, Bedford, MA) andincubated with monoclonal mouse anti-phosphotyrosineantibody (4G10) (Upstate Biotechnology, Inc, Lake Placid,NY). Antibody binding was detected by incubation witha horseradish peroxidase-conjugated secondary antibody,followed by chemiluminescence detection (Amersham, Ar-lington Heights, IL). Blots were then stripped with strippingbuffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mMTris–HCl, pH 6.7) and incubated with KIT or FMS antibody.

3. Results and discussion

3.1. Absence of ITD mutations in the JM region of KITor FMS expressed by leukemia and lymphoma derivedcell lines and primary AML cells

KIT and FMS expression in 13 leukemia and lymphomaderived cell lines and 44 blast samples from AML patientswas tested by amplification with primers that cover the JMdomain of these receptors. Samples which have visibly de-tectable PCR product of the expected size were defined as

Fig. 1. . Expression of KIT, FMS, SCF, and CSF-1 transcripts in humanleukemia-and lymphoma-derived cell lines and primary blast samples fromAML patients. Total cellular RNA was extracted from leukemia-lymphomacell lines (A) and patients’ blast cells (B). 50–100 ng RNA was reversetranscribed and amplified using primer pairs for KIT, FMS, SCF, CSF-1and actin. Reverse transcription negative control was performed by omit-ting the reverse transcriptase in the reaction to verify the absence ofgenomic DNA in RNA preparation. PCR products were resolved on 5%polyacrylamide gel in the presence of ethidium bromide and photographswere taken under UV transillumination. If a KIT/ITD or FMS/ITD mu-tation was present, a higher molecular weight band than wild-type KITor FMS would be visualized.

positive expression of receptors. The expected KIT PCRfragment of 229 bp was detected in 12/13 of the cell linesand 42/44 AML samples. Among these KIT+ samples, 8/12leukemia and lymphoma derived cell lines and 41/42 pri-mary AML samples have readily detectable KIT expression.The expected FMS PCR fragment of 204 bp was observedin 10/13 of the cell lines and 44/44 AML samples (Fig. 1,Tables 1 and 2). Among these FMS+ samples, 8/10 leukemiaand lymphoma cell lines and 44/44 AML samples have read-ily detectable FMS expression. The percentage of KIT andFMS positive AML cases is consistent with the report fromprevious studies[16,22].

124 R. Zheng et al. / Leukemia Research 28 (2004) 121–126

Table 1Expression and phosphorylation of KIT and FMS by human leukemia and lymphoma derived cell lines

Cell line KIT SCF transcript FMS CSF-1transcript

KIT transcript KIT protein FMS transcript FMS protein

WTa ITD KIT P-KIT b WT ITD FMS P-FMSc

WSU-NHL + − − − − − − − − +EM3 + − − − + + − − − +HEL + − + + − + − − − +K562 + − − − − + − − − +HL-60 + − − − + − − − − +U937 + − − − + + − − − +LAZ221 + − − − − + − − − +REH + − + − − − − − − +ML-1 + − − − + + − − − +MOLM-1 + − + − + + − + + +EOL-1 + − − − − + − − − +BV173 − − − − − + − − − +MV4-11 + − − − + + − + − +

a WT: wild-type.b P-KIT: phosphorylated KIT.c P-FMS: phosphorylated FMS.

Higher molecular weight bands, indicating the possibilityof ITD mutations, were not identified in any of the 13 celllines or 44 primary AML samples. This is somewhat sur-prising given their expression in AML and close homologyto FLT3, which frequently does undergo ITD mutation. Thismight be a reflection of the mechanism by which FLT3/ITDmutations form. One hypothesis relates ITD mutations tothe formation of a palindromic intermediate in the JM re-gion of FLT3. If a lagging strand makes a hairpin duringDNA replication and the following mismatch repair systemis impaired, the tandemly duplicated fragment will be fixedin DNA. If FLT3/ITD occurs in an out-of-frame manner, theleukemia cell carrying it does not acquire a growth advan-tage and will not be selected[36]. Perhaps KIT and FMSare not able to form those structures because of differencesin the DNA sequence coding for their JM domains. An addi-tional possibility is that such mutations do form in KIT andFMS but they do not give much of a growth or anti-apoptoticadvantage to hematopoietic cells. This could reflect a lackof constitutive tyrosine kinase activation upon ITD mutationin KIT and FMS. This possibility appears less likely be-

Table 2Expression of KIT, SCF, FMS, and CSF-1 by primary AML blasts from44 patients

Positive cases

KIT transcript 42KIT/ITD 0SCF transcript 27Co-expression of KIT and SCF transcripts 27FMS transcript 44FMS/ITD 0CSF-1 transcript 35Co-expression of FMS and CSF-1 transcripts 35

cause deletion mutations do occur in the JM region of KITin a murine mast cell line (FMA3) and human gastrointesti-nal stromal tumours resulting in KIT activation[41,42]. Itcould also be a result of signaling differences between thereceptors. While they do share many elements of the signaltransduction pathways, there are known and probably manyunknown differences as well.

3.2. Expression and phosphorylation of KIT and FMSreceptor in leukemia and lymphoma derived cell lines

We further analyzed the expression and phosphorylation(activation) state of KIT and FMS at the protein level byimmunoprecipitation and immunoblotting in the same 13leukemia and lymphoma cell lines. KIT is expressed in threecell lines and phosphorylated in one cell line, while FMSis expressed in two cell lines and phosphorylated in 1 cellline (Fig. 2 and Table 1). In contrast, KIT and FMS tran-scripts were positive in 12 and 10 of the 13 cell lines, re-spectively. The difference in expression between the assayspossibly reflects the great sensitivity of RT-PCR comparedto Western blotting. The finding of phosphorylation of KITin the absence of added ligand is consistent with a previousreport that KIT receptor was tyrosine phophorylated in theabsence of SCF in 7/12 of KIT+ AML cases, implicatingKIT activating mutations or autocrine stimulation[13].

3.3. Co-expression of SCF or CSF-1 with their respectivereceptors in leukemia-lymphoma derived cell lines andAML blast cells

Because ligand binding can lead to activation and phos-phorylation of KIT and FMS and that such autocrine ligand-stimulation is likely to provide cells with a proliferative

R. Zheng et al. / Leukemia Research 28 (2004) 121–126 125

Fig. 2. . Expression and phosphorylation of KIT and FMS in humanleukemia-lymphoma cell lines. Total cellular protein extracts derived from1×107 cells were immunoprecipitated with anti-KIT or anti-FMS antibody.The immunoprecipitates were resolved by 8% SDS-PAGE and subjectedto immunoblot analysis with anti-phosphotyrosine antibody (4G10). Thesame blot was stripped and reprobed with anti-KIT or anti-FMS antibody.KIT expressing BAF3 cells stimulated by SCF and FMS expressing BAF3cells stimulated by CSF-1 were used as positive controls.

and anti-apoptotic advantage[5,13,15,18], we investigatedthe possibility of SCF or CSF-1 co-expression with theirrespective receptors. RT-PCR revealed that 6/13 leukemiaand lymphoma cell lines and 27/44 AML blasts samplesco-expressed SCF and KIT (Fig. 1, Tables 1 and 2). And10/13 cell lines and 35/44 AML samples co-expressedCSF-1 and FMS. Previously, co-expression of CSF-1 andFMS assessed by Northern blot was reported in five of 15AML cases[21]. Co-expression of SCF and KIT was shownin 17 of 30 human myeloid leukemia cell lines detected byRT-PCR and immunostaining, as well as in 15 of 80 primaryadult AML specimens detected by ribonuclease protectionassay[43,44]. Our findings, together with those previousresults, demonstrate co-expression of KIT and FMS withtheir respective ligands occurs at a fairly high rate in AML.This suggests the possibility that autocrine and/or intracrinesignaling loops through these receptors could play a role inleukemogenesis. Of note, 42/44 AML samples expressedboth KIT and FMS receptor implying a tendency for AMLblasts to express multiple cytokine receptors.

Despite the frequent finding of receptor/ligand co-expression, constitutively activated KIT or FMS was onlyseen in two out of the 13 leukemia and lymphoma derivedcell lines tested in this study. Thus, simple co-expressionof receptor and ligand is not sufficient to predict whetheror not receptor will be activated. Levels of receptor andligand expression, culture conditions, and cell surface ver-sus secreted ligand expression could all contribute to theobserved results.

ITD mutations of FLT3 are somatic genetic alterationsthat occur frequently in AML[45]. However, ITD mu-tations of KIT and FMS were not present in any of theAML cases that were screened in this study. Thus, KIT/ITDand FMS/ITD mutations, if they occur at all, must be rareevents in AML. The role of KIT/SCF and FMS/CSF-1 co-expression in leukemia requires further investigation to val-idate the wild-type receptors as possible molecular targetsfor therapy of AML.

Acknowledgements

The authors thank Matthew T Malehorn for his technicalsupport for the RNA preparation. This work was supportedby grants from the NCI (CA91177 and CA90668, to D.S.),Leukemia & Lymphoma Society (to D.S.), and Children’sCancer Foundation (to D.S.). D.S. is the Douglas Kroll Re-search Foundation Translational Researcher of the Leukemiaand Lymphoma Society.

Contributions. R. Zheng contributed to the concept anddesign, interpreted and analyzed the data, provided draftingof the article, provided critical revisions and important intel-lectual content, gave final approval, collected and assembledthe data. K. Klang contributed to the concept and design, in-terpreted and analyzed the data, collected and assembled thedata. N. Gorin contributed to the concept and design, inter-preted and analyzed the data, gave final approval, and pro-vided study materials/patients. D. Small contributed to theconcept and design, interpreted and analyzed the data, pro-vided drafting of the article, provided critical revisions andimportant intellectual content, gave final approval, providedstudy materials/patients, and obtained a funding source.

References

[1] Rosen O, Stephenson D, Mattei M-G, Marchetto S, Shibuya M,Chapman V, et al. Close physical linkage of theFLT1 and FLT3genes on chromosome 13 in man and chromosome 5 in mouse.Oncogene 1993;82:173–9.

[2] Yarden Y, Kuang WJ, Yang-Feng T, Coussens L, Munemitsu S, DullTJ, et al. Human proto-oncogenec-kit: a new cell surface receptortyrosine kinase for an unidentified ligand. EMBO J 1987;6:3341–51.

[3] Coussens L, Van Beveren C, Smith D, Chen E, Mitchell RL, IsackeCM, et al. Structural alteration of viral homologue of receptorproto-oncogenefms at carboxyl terminus. Nature 1986;320:277–80.

[4] Ullrich A, Schlessinger J. Signal transduction by receptors withtyrosine kinase activity. Cell 1990;61:203–12.

[5] Weiss A, Schlessinger J. Switching signals on or off by receptordimerization. Cell 1998;94:277–80.

[6] Rosnet O, Buhring HJ, deLapeyriere O, Beslu N, Lavagna C,Marchetto S, et al. Expression and signal transduction of the FLT3tyrosine kinse receptor. Acta Haematol 1996;95:218–23.

[7] Porter AC, Vaillancourt RR. Tyrosine kinase receptor-activatedsignal transduction pathways which lead to oncogenesis. Oncogene1998;17:1343–52.

[8] Drexler HG. Expression of FLT3 receptor and response to FLT3ligand by leukemic cells. Leukemia 1996;10:588–99.

126 R. Zheng et al. / Leukemia Research 28 (2004) 121–126

[9] Lyman SD, Jacobsen SE. c-kit ligand and Flt3 ligand: stem/progenitorcell factors with overlapping yet distinct activities. Blood1998;91:1101–34.

[10] Ashman LK, Cambareri AC, To L, Levinsky RL. Expression ofthe YB5.B8 antigen (c-kit protooncogene product) in normal humanbone marrow. Blood 1991;78:30–7.

[11] Papayannopoulou T, Brice M, Broudy VC, Zsebo KM. Isolation ofc-kit receptor-expressing cells from bone marrow, peripheral blood,and fetal liver: functional properties and composite antigenic profile.Blood 1991;78:1403–12.

[12] Sperling C, Schwartz S, Buchner T, Thiel E, Ludwig WD. Expressionof the stem cell factor receptor C-KIT (CD117) in acute leukemias.Haematologica 1997;82:617–21.

[13] Ikeda H, Kanakura Y, Tamaki T, Kuriu A, Kitayama H, Ishikawa J,et al. Expression and functional role of the proto-oncogenec-kit inacute myeloblastic leukemia cells. Blood 1991;78:2962–8.

[14] Wells SJ, Bray RA, Stempora LL, Farhi DC. CD117/CD34 expressionin leukemic blasts. Am J Clin Pathol 1996;106:192–5.

[15] Broudy VC, Smith FO, Lin N, Zsebo KM, Egrie J, BernsteinID. Blasts from patients with acute myelogenous leukemia expressfunctional receptors for stem cell factor. Blood 1992;80:60–7.

[16] Piao X, Curtis JE, Minkin S, Minden MD, Bernstein A. Expressionof the Kit and KitA receptor isoforms in human acute myelogenousleukemia. Blood 1994;83:476–81.

[17] Crosier PS, Ricciardi ST. Expression of isoforms of the humanreceptor tyrosine kinase c-kit in leukemic cell lines and acute myeloidleukemia. Blood 1993;82:1151–8.

[18] Wang CC, Curtis JE, Giessler EN, McCulloch EA, Minden MD.The expression of the proto-oncogenec-kit in the blast cells of acutemyeloblastic leukemia. Leukemia 1989;3:699–702.

[19] Lisovsky M, Estrov Z, Zhang X, Consoli U, Sanchez-WilliamsG, Snell V, et al. Flt3 ligand stimulates proliferation and inhibitsapoptosis of acute myeloid leukemia cells: regulation of Bcl-2 andBax. Blood 1996;88:3987–97.

[20] Parwaresch MR, Kriepe H, Felgner J, Heidorn K, Jaquet K,Bodewadt-Radzun S, et al.M-CSF and M-CSF receptor geneexpression in acute myelomonocytic leukemias. Leuk Res 1990;4:27–37.

[21] Rambaldi A, Wakamiya N, Vellenga E, Horiguchi J, Warren MK,Kufe D, et al. Expression of the macrophage colony-stimulatingfactor andc-fms genes in human acute myeloblastic leukemia cells.J Clin Invest 1988;1:1030–5.

[22] Dubreuil P, Torres H, Courcoul M-A, Birg F, Mannoni P. c-fmsexpression is a molecular marker of human acuter myeloid leukemias.Blood 1988;72:1081–5.

[23] Ferrao P, Gonda T, Ashman L. Expression of constitively activatedhuman c-kit in Myb transformed early myeloid vells leads to factorindependence, histolytic differentiation, and tumorigenicity. Blood1997;90:4539–52.

[24] Ridge SA, Worwood M, Oscier D, Jacobs A, Padua RA. FMSmutations in myelodysplastic, leukemic, and normal subjects. ProcNatl Acad Sci USA 1990;87:1377–80.

[25] Kitayama H, Tsujimura T, Matsumura I, Oritani K, Ikeda H, IshikawaJ, et al. Neoplastic transformation of normal hematopoietic cells byconstitutively activating mutations of c-kit receptor tyrosine kinase.Blood 1996;88:995–1004.

[26] Roussel MF, Downing JR, Rettenmier CW, Sherr CJ. A pointmutation in the extracellular domain of the human CSF-1 receptor(c-fms proto-oncogene product) activates its transforming potential.Cell 1988;55:979–88.

[27] Roussel MF, Downing JR, Sherr CJ. Transforming activities of humanCSF-1 receptors with different point mutations at codon 301 in theirextracellular domains. Oncogene 1990;5:25–30.

[28] Gari M, Goodeve A, Wilson G, Winship P, Langabeer S, Linch D,et al. c-kit proto-oncogene exon 8 in-frame deletion plus insertionin acute myeloid leukaemia. Br J Haematol 1999;105:894–900.

[29] Longley BJ, Tyrrell L, Lu SZ, Ma YS, Langley K, Ding TG, etal. Somatic c-KIT activating mutation in urticaria pigmentosa andaggressive mastocytosis: establishment of clonality in a human mastcell neoplasm. Nat Genet 1996;12:312–4.

[30] Pignon JM, Giraudier S, Duquesnoy P, Jouault H, Imbert M,Vainchenker W, et al. A new c-kit mutation in a case of aggressivemast cell disease. Br J Haematol 1997;96:374–6.

[31] Nagata H, Worobec AS, Oh CK, Chowdhury BA, Tannenbaum S,Suzuki Y, et al. Identification of a point mutation in the catalyticdomain of the protooncogene c-kit in peripheral blood mononuclearcells of patients who have mastocytosis with an associatedhematologic disorder. Proc Natl Acad Sci USA 1995;92:10560–4.

[32] Ma Y, Longley BJ, Wang X, Blount JL, Langley K, CaugheyGH. Clustering of activating mutations in c-KIT’s juxtamembranecoding region in canine mast cell neoplasms. J Invest Dermatol1999;112:165–70.

[33] Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, etal. Internal tandem duplication of theFLT3 gene found in acutemyeloid leukemia. Leukemia 1996;10:1911–8.

[34] Schnittger S, Schoch C, Dugas M, Kern W, Staib P, WuchterC. Analysis of FLT3 length mutations in 1003 patients withacute myeloid leukemia: correlation to cytogenetics, FAB subtype,and prognosis in the AMLCG study and usefulness as a markerfor the detection of minimal residual disease. Blood 2002;100:59–66.

[35] Kiyoi H, Ohno R, Ueda R, Saito H, Naoe T. Mechanism ofconstitutive activation of FLT3 with internal tandem duplication inthe juxtamembrane domain. Oncogene 2002;21:2555–63.

[36] Kiyoi H, Towatari M, Yokota S, Hamaguchi M, Ohno R, Saito H, etal. Internal tandem duplication of theFLT3 gene is a novel modalityof elongation mutation which causes constitutive activation of theproduct. Leukemia 1998;12:1333–7.

[37] Fenski R, Flesch K, Serve S, Mizuki M, Oelmann E, Kratz-AlbersK, et al. Constitutive activation of FLT3 in acute myeloid leukaemiaand its consequences for growth of 32D cells. Br J Haematol2000;108:322–30.

[38] Iwai T, Yokota S, Nakao M, Okamoto T, Taniwaki M, Onodera N,et al. Internal tandem duplication of theFLT3 gene and clinicalevaluation in childhood acute myeloid leukemia. The Children’sCancer and Leukemia Study Group, Japan. Leukemia 1999;13:38–43.

[39] Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S, etal. Prognostic implication ofFLT3 and N-RAS gene mutations inacute myeloid leukemia. Blood 1999;93:3074–80.

[40] Levis M, Tse KF, Smith BD, Garrett E, Small D. A FLT3 tyrosinekinase inhibitor is selectively cytotoxic to acute myeloid leukemiablasts harboring FLT3 internal tandem duplication mutations. Blood2001;98:885–7.

[41] Tsujimura T, Morimoto M, Hashimoto K, Moriyama Y, KitayamaH, Matsuzawa Y, et al. Constitutive activation of c-kit in FMA3murine mastocytoma cells caused by deletion of seven amino acidsat the juxtamembrane domain. Blood 1996;87:273–83.

[42] Hirota S, Isozaki K, Moriyama Y, Hashimoto K, Nishida T, IshiguroS, et al. Gain-of-function mutations of c-kit in human gastrointestinalstromal tumors. Science 1998;279:577–80.

[43] Cole SR, Aylett GW, Harvey NL, Cambareri AC, Ashman LK.Increased expression of c-Kit or its ligand steel factor is nota common feature of adult acute myeloid leukaemia. Leukemia1996;10:288–96.

[44] Pietsch T, Kyas U, Yakisan E, Hadam M, Ludwig W, ZseboK, et al. Effects of human stem cell factor (c-kit ligand) onproliferation of myeloid leukemia cells: heterogeneity in responseand synergy with other hematopoietic growth factors. Blood 1992;80:1199–206.

[45] Stirewalt DL, Kopecky KJ, Meshinchi S, Appelbaum FR, Slovak ML,Willman CL. FLT3, RAS, and TP53 mutations in elderly patientswith acute myeloid leukemia. Blood 2002;97:3589–95.