Interleukin (IL)-3/granulocyte macrophage-colony stimulatingfactor/IL-5 receptor alpha and beta chains are preferentiallyexpressed in acute myeloid leukaemias with mutatedFMS-related tyrosine kinase 3 receptor

Acute myeloid leukaemia (AML) is a clonal disorder of

haematopoiesis characterized by the accumulation of large

numbers of myeloid blast cells that fail to differentiate into

functional blood cells (Fialkow et al, 1987). Although most

patients with newly diagnosed AML achieve a complete

remission with conventional chemotherapy, the majority

relapse and die with chemotherapy-resistant disease (Rowe

et al, 1994; Bennet et al, 1997). New therapeutic approaches

are therefore needed in this disease.

Several lines of evidence suggest that multiple molecular

mechanisms are involved in the pathogenesis of AMLs (Hahn &

Weinberg, 2002), such as chromosome translocations, muta-

tions and activated cell signalling pathways that may be

responsible for the proliferation and/or apoptosis escape of

leukaemic blasts. In recent years, it has been reported that

constitutive extracellular signal-regulated kinase (ERK)1/2

activation is frequently found in solid tumours as well as in

haematological diseases, particularly AML (Milella et al, 2001;

Ricciardi et al, 2005). In this context, a key role is played by

mutations or alterations in the level of expression of membrane

receptors involved in the control of the proliferation of

haemopoietic cells, such as FMS-related tyrosine kinase 3

(FLT3), cellular-receptor tyrosine kinase (c-kit) and interleu-

kin-3 receptor (IL-3R). IL-3R is a heterodimeric membrane

receptor (Chang et al, 2003). The a subunit is essential for ligand

binding and confers ligand specificity to the receptor. The

Roberta Riccioni,1 Daniela Diverio,2

Viviana Riti, 2 Sonia Buffolino,2

Gualtiero Mariani,1 Alessandra Boe,1

Michele Cedrone,3 Tiziana Ottone,4

Robin Foa2 and Ugo Testa1

1Department of Haematology, Oncology and

Molecular Medicine, Istituto Superiore di Sanita,2Department of Cellular Biotechnology and

Haematology, University ‘‘La Sapienza’’,3Haematology Section Ospedale San Giovanni,

and 4Department of Biopathology, Universita

‘‘Tor Vergata’’, Rome, Italy

Received 15 July 2008; accepted for publication

16 September 2008

Correspondence: Dr Ugo Testa, Department of

Haematology, Oncology and Molecular

Medicine, Istituto Superiore di Sanita, Viale

Regina Elena 299, Rome 00161, Italy.

E-mail: ugo.testa@iss.it

Summary

The common beta chain subunit (bc), also known as CDw131, shared by the

interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor

(GM-CSF) and IL-5 receptors, is required for high-affinity ligand binding

and signal transduction. The present study explored the expression of CDw131

in 105 de novo cases of acute myeloid leukaemia (AML). The levels of CDw131

expression were used to identify two AML subgroups characterized by low (75/

105) and high (30/105) expression of this receptor chain. It was observed that

(i) the level of CDw131 expression strictly correlated with the level of CD116

(GM-CSFa receptor chain) and CD123 (IL-3Ra chain); (ii) AMLs with high

CDw131 expression were characterized by low CD34 expression and usually

high CD11b, CD14 expression; (iii) AMLs with high CDw131 expression

frequently co-expressed receptors for angiogenic growth factors (vascular

endothelial growth factor R2, Tie-2); (iv) AMLs with high CDw131 expression

were more cycling than those with low CDw131 expression; (v) AMLs with

high CDw131 frequently displayed Feline Murine Sarcoma (FMS-related)

tyrosine kinase 3 (FLT3) internal tandem duplication and constitutively

activated Signal Transducer and Activator of Transcription-5 (STAT5). In

conclusion, the analysis of the level of CDw131 expression enabled the

identification of a subset of AMLs characterized by a high cycling status, the

expression of myelo-monocytic markers, mutated FLT3 and the co-expression

of receptors for angiogenic growth factors. These findings are of value for the

development of new therapeutic strategies for the treatment of these AMLs.

Keywords: leukaemia, growth factor receptors, interleukin-3, granulo-

monocytic colony stimulating factor, mutations.

research paper

First published online 25 November 2008 ª 2008 The Authorsdoi:10.1111/j.1365-2141.2008.07491.x Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387

common beta chain (bc) subunit, also known as CDw131, which

is shared by the granulocyte macrophage-colony stimulating

factor (GM-CSF), IL-3 and IL-5 receptors, is required for high-

affinity ligand binding and signal transduction. IL-3R is

expressed on leukaemic blasts from the large majority of

patients with AML (Jordan et al, 2000; Testa et al, 2002). In

particular, the a subunit (also known as CD123) is often

expressed at much higher levels in AML than in normal

haematopoietic progenitor and precursor cells and it is also

detected on subpopulations of AML blasts enriched for malig-

nant progenitors, leukaemic stem cells that engraft non-obese

diabetic/severe combined immunodeficient mice (Shultz et al,

1995; Bonnett & Dick, 1997; Jordan et al, 2000). Interestingly,

recent microarray studies confirmed that IL3Ra is amongst the

seven genes that are more overexpressed in AMLs (Stirewalt

et al, 2008). Thus, IL-3R may be an appropriate target for

cytotoxic drugs (i.e., recombinant IL-3 fused with diphtheria

toxin) designed to selectively kill AML cells, whilst sparing their

normal haematopoietic cell counterparts (Testa et al, 2005;

Yalcintepe et al, 2006). IL-3R and GM-CSFR exert their

biological activity through interaction with cell surface receptors

that consist of two subunits, the alpha subunit specific to each

and the common beta chain (b chain or bc) (Miyajima et al,

1993; Bagley et al, 1997). The interaction of the a chain with the

b chain leads to the formation of a high-affinity receptor

complex, able to bind the respective ligand in the range of its

physiological concentrations and to transduce proliferative,

antiapoptotic and differentiative signals (Gearing et al, 1989;

Hayashida et al, 1990; Kitamura et al, 1991). The b chain

expressed alone in the absence of a specific a chain confers little

binding affinity for either IL-3 or GM-CSF (Tavernier et al,

1991; Hara & Miyajima, 1992). The bc chain is not only required

for high-affinity binding of ligands, but is also crucial for the

signalling of downstream pathways through its intracytoplas-

matic tail. In common with other receptors, the membrane

proximal region of bc appears to be both essential and sufficient

for proliferation (Sakamaki et al, 1992). This region is known to

bind the family of Janus Protein Kinase-2 (JAK2), and the

activation of JAK2 by tyrosine phosphorylation correlates

closely with the induction of mitogenesis (Quelle et al, 1994).

It is also essential for the induction of MYC, PIM1 and CIS.

Membrane-distal domains that are responsible for the major

tyrosine phosphorylation of proteins, induction of FOS and JUN

transcription, activation of Ras pathway, and prevention of

apoptosis (Sakamaki et al, 1992; Sato et al, 1993; Kinoshita et al,

1995) have been identified. In addition, the membrane-distal

region has been associated with the induction of differentiation

and negative regulation of receptor signals (Itoh et al, 1996).

Accumulated evidence suggest a role for bc in modulating signal

transduction of other haematopoietic cytokines, including GM-

CSF, erythropoietin (Epo) and stem cell factor (SCF) (Walker

et al, 1985). In addition to phosphorylation and physical

interaction, IL-3R and GM-CSFR are known to co-operate with

EpoR in erythropoiesis (Goodman et al, 1985; Sieff et al, 1989),

and common signal transduction pathways involving STAT5 are

used by these receptors (Pallard et al, 1995). STAT5 is activated

by a variety of ligands in different tissues. In primary leukaemic

cells a constitutive activation of the JAK-STAT pathway has been

demonstrated and the essential role of this pathway for the

malignant transformation of haematopoietic cells has been

suggested (Lin et al, 2000). In addition, bc has been implicated

in signalling by SCF via its receptor, c-kit, and SCF is able to

induce serine/threonine phosphorylation of bc (Liu et al, 1994),

even though there are no evidences about the physiological

significance of this receptor cross-talk in primary haemopoietic

cells (Scott et al, 2000).

The link between endothelial and haematopoietic systems is

well known because they share a common stem cell, the

haemangioblast. However, the role of vascular endothelial

growth factor (VEGF) receptors in myeloid leukaemias is

unclear. Two-thirds of AML patients show VEGF and VEGF

receptor 2 (VEGFR-2) expression, with concomitant increased

marrow microvascular density (Padro et al, 2002). Prolifera-

tion and survival of leukaemic blasts may be sustained by a

paracrine loop. Myeloid blasts release VEGF, which stimulates

adjacent marrow endothelial cells to secrete myeloid growth

factors (Bellamy et al, 1999; Dias et al, 2002). Furthermore, we

have recently shown that Tie-2, the receptor for angiopoietin-1

and -2, is expressed on monocytic and myelomonocytic AMLs

(Riccioni et al, 2007). On the other hand, AML blasts release

angiopoietins that may sustain leukaemic survival through an

autocrine/paracrine mechanism (Wakabayashi et al, 2004).

The current study examined the expression of bc in AML. In

this context, we were particularly interested in exploring

whether bc expression on AMLs might be associated with

specific cellular phenotypes. The level of bc expression was

measured by fluorescent-activated cell sorting (FACS) analysis

in 105 AML samples, which were divided into a group with

low and in a group with high bc expression. AMLs with high bc

expression were found to share myelomonocytic markers.

Furthermore, high bc expression was frequently associated

with the expression of VEGF-R2 and Tie-2. Finally, AMLs with

high bc expression very frequently displayed FLT3-internal

tandem duplication (ITD) mutations.

Materials and methods

Cells

Fresh leukaemic blasts from 105 patients with AML, obtained

after informed consent, were isolated from either bone marrow

or peripheral blood by Ficoll-Hypaque density gradient

centrifugation and immediately processed. All patients were

diagnosed at the Divisions of Haematology of the University

‘La Sapienza’, of the University ‘Tor Vergata’ and of the

Hospital ‘San Giovanni’, Rome, Italy. Leukaemias were

classified by morphological and cytochemical criteria

according to the French–American–British (FAB) classification

(2% corresponded to M0, 19% to M1, 24% to M2, 9% to M3,

26% to M4 and 20% to M5).

Expression of IL-3/GM-CSF/IL-5 in AML

ª 2008 The AuthorsJournal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387 377

The following criteria were fulfiled in all cases diagnosed as

FAB M5 (acute monocytic leukaemia): (i) 70–80% of the

leukaemic cells were morphologically of the monocytic lineage,

including monoblasts, promonocytes and monocytes; (ii) the

leukaemic population contained a population, a percentage of

CD14+ cells >40%; (iii) a minor granulocytic component

(<20%); (iv) in FAB M5a cases the percentage of monoblasts is

predominant (>60–70%), whereas (v) in FAB M5b cases the

percentage of promonocytes and monocytes was predominant

(>70%); 6); finally, the large majority of the leukaemic

population showed intense non-specific esterase activity,

inhibited by sodium fluoride.

All analysed samples contained infiltration by more than

70% leukaemic blasts. Approval for these studies was obtained

from the Institutional Review Board of the Istituto Superiore

di Sanita, Rome, Italy. Informed consent was obtained in

accordance with the Declaration of Helsinki.

Immunophenotypic analysis of leukaemic cells

Analysis of cell surface antigens was performed by flow

cytometry using a FACScan Flow Cytometer (Becton Dickinson,

Bedford, MA, USA). The following antibodies directed to

membrane antigens were used for standard immunophenotypic

analysis of AML blasts: anti-CD3, -CD7, -CD11a, CD11b, -

CD11c, -CD13, -CD14, -CD15, -CD18, -CD19, -CD33, -CD34, -

CD36, -CD38, -CD41, -CD45, -CD61, -CD64, -CD71, -CD90, -

CD116, -CD117, -CD235 and -HLA-DR (all from Pharmingen/

Becton Dickinson). In addition, the following monoclonal

antibodies (mAbs) were used to characterize AML blasts: anti-

CDw123 (Pharmingen/Becton Dickinson) -CD116, CDw131

(Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VEGF-

R1, -VEGF-R2, VEGF-R3, -Tie-2, -M-CSFR (Macrophage

Colony Stimulating Factor), -c-met, -IGF-1R (Insulin-like

Growth Factor-1 Receptor) all purchased from R & D Systems,

Minneapolis, MN, USA) -CD133 (Milteny, Bergisch Gladbach,

Germany), anti-Flt3 mAb purchased from Serotec (Oxford,

UK). Cells were labelled and analysed as previously reported

(Testa et al, 2002).

For quantitative evaluation of CDw123, CD116, CDw131

and CD135 expression, leukaemic cells were incubated with

specific antibodies and analysed by flow cytometry. Cell

fluorescence emission was evaluated, maintaining a fixed

photomultiplier tube (PMT) voltage to allow a quantitative

comparison between various samples. Fluorescence data were

evaluated in terms of mean fluorescence intensity (MFI)

calculated as the ratio between MFI observed in cells incubated

with anti-CDw123, CD116, CDw131, CD135 and the MFI

observed for the cells incubated with control IgG.

RNA extraction and RT-PCR analysis

Total RNA was extracted from leukaemic blasts using the

method of Chomczynsky and Sacchi (1987). One microgram

of total RNA was retro-transcribed using random hexamers

primers as published by Biomed Concerted Action Protocol

(van Dongen et al, 1999). To evaluate the expression of FLT3,

2 ll of reverse transcriptase was amplified within the linear

range by 35 PCR cycles: denaturation at 94�C for 30 s,

annealing at 56�C for 45 s, extension at 72�C for 30 s, followed

by 10 min at 72�C for final extension, in a mixture of 50 ll

volume containing 1· PCR buffer, 30 pmol of each specific

primer, 0Æ2 nmol/l dNTPs mix, 1Æ5 mmol/l MgCl2 and 1Æ25 U

Taq gold DNA polymerase. With the aim of simultaneously

analysing both ITD and D835, a multiplex PCR strategy was

adopted using the following four oligonucleotide primers: R5:

5¢-TGTCGAGCAGTACTCTAAACA-3¢ and R6: 5¢-ATCCTA-

GTACCTTCCCAAACTC-3¢, which amplify the entire trans-

membrane domain and the Juxta Membrane (JM) domain of

the gene; and 17F: 5¢-CCGCCAGGAACGTGCTTG-3¢ and

17R: 5¢-GCAGCCTCACATTGCCCC-3¢, which explore the

region containing exon 17 where the D835 mutation is

located. At the end of the PCR, 1Æ5 ll of these amplification

products were digested using the EcoRV restriction endonu-

clease and electrophoresed onto a 3% agarose gel containing

ethidium bromide. The quality of cDNA was assessed by

amplification of Hydroxymethylbilane Synthase (HMBS) using

the same conditions and cycling parameters and furthermore

RNA contamination was excluded using appropriate negative

controls.

Cell cycle analysis

Cell cycle analysis was carried out on nuclei stained with

propidium iodide (PI) using the Cycle TESTTM PLUS DNA

reagent of Becton-Dickinson (San Jose, CA, USA). Briefly,

100000 cells were first washed in buffer solution containing

sodium citrate, sucrose, dimethyl sulphoxide, and then resus-

pended in: 250 ll solution A to digest cell membrane and

cytoskeletons, 200 ll solution B containing trypsin inhibitor

and ribonuclease A to digest the RNA, 200 ll solution C

containing PI and spermine to stabilize nuclear chromatin.

The PI stoichiometrically binds to the DNA at a final

concentration of at least 125 lg/ml. Cells were then analysed

by flow cytometry using a FACScan equipped with software for

cell cycle analysis (Vindelov et al, 1983a,b).

Western blot analysis

To prepare total extracts, the cells were washed twice with cold

phosphate-buffered saline and lysed on ice for 30 min with 1%

Nonidet P40 lysis buffer (20 mmol/l Tris–HCl pH 8Æ0,

137 mmol/l NaCl, 10% glycerol, 2 mmol/l EDTA) in the

presence of 1 mmol/l phenylmethylsulfonylfluoride, 1 mmol/l

dithiothreitol, 1 mmol/l sodium orthovanadate, 2 lg/ml leu-

peptin and 2 lg/ml aprotinin. Cell debris was removed by

centrifugation at 3250 g for 10 min at 4�C, and protein

concentration of supernatants was determined using the Bio-

Rad protein assay (Richmond, CA, USA, http://www.biorad.

com). Aliquots of cells extracts containing 40 lg of total

R. Riccioni et al

ª 2008 The Authors378 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387

protein were resolved by 10% sodium dodecyl sulphate–

polyacrylamide gel electrophoresis under reducing and dena-

turing conditions and transferred onto Hybond-C extra

nitrocellulose membrane (Amersham Pharmacia Biotech,

Buckinghamshire, UK). Filters were blocked for 1 h at room

temperature in 5% non-fat dry milk dissolved in Tris-buffered

saline-Tween (TBS-T; 10 mmol/l Tris–HCl pH 8Æ0, 150 mmol/

l NaCl, 0,2% Tween 20) followed by incubation with primary

antibodies. After washing in TBS-T buffer, the filters were

incubated for 1 h at room temperature in 5% non-fat dry milk

dissolved in TBS-T containing a 1:4000 dilution of corre-

sponding peroxidase-conjugated secondary antibodies. Pro-

teins were visualized with the enhanced chemiluminescence

technique according to the manufacturer’s instructions (Super

Signal West Pico; Pierce, Rockford, IL, USA, http://

www.piercenet.com). Anti-ERK1/2 mAb was purchased from

Cell Signaling (Danvers, MA, USA), anti-pSTAT5 mAb was

purchased from R&D System Inc (Minneapolis, MN, USA),

anti-pERK mAb was purchased from Immunological Science

(Rome, Italy), anti-STAT5 mAb was purchased from Upstate

Biotechnology (Lake Placid, NY, USA, http://www.upstate.

com), anti-actin mAb was purchased from Oncogene Research

Products (Cambridge, MA, USA, http://www.oncogene.com)

and used as loading control.

Cell sorting

Leukaemic blasts stained with phycoerythrin-labelled anti IL-

3Rbc antibody were sorted according to fluorescence intensity

into IL-3Rbcdim and IL-3Rbc

bright fractions using a FACS

Vantage (Becton Dickinson).

Statistical analysis

The statistical significance of various parameters was evaluated

by the Student’s t-test. P values <0Æ05 were considered

significant.

Results

CDw131 expression in AML

The expression of CDw131, CD123 and CD116 on leukae-

mic blasts derived from 105 AML patients was investigated

by flow cytometry using specific mAbs directly conju-

gated with PE. In the majority of patients all the three

membrane receptor chains were detectable (representative

examples are shown in Fig 1). MFI analysis showed a

considerable variation in the label intensity for each of

Fig 1. Flow cytometric analysis of CDw131 (b-chain), CD123 (IL3R-a), CD116 (GM-CSFRa), Tie-2 and KDR in acute myeloid leukaemias (AMLs).

Flow cytometry histograms of three representative AMLs. Top middle and bottom panels correspond to AML cases displaying high, intermediate and

low CDw131 expression respectively. In each histogram the grey area corresponds to the fluorescence observed for cells incubated with control

irrelevant immunoglobulins, whilst the white area indicates the fluorescence observed for cells incubated with the indicated monoclonal antibodies.

IL, interleukin; GM-CSFR, granulocyte macrophage colony-stimulating factor; KDR, kinase domain receptor.

Expression of IL-3/GM-CSF/IL-5 in AML

ª 2008 The AuthorsJournal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387 379

these receptor chains amongst the various AML patients

(Fig 2A).

It is known that both CD123 and CD116 exert their biological

activity through interaction with CDw131 (b-chain) to form the

high-affinity receptor complex IL-3R and GM-CSFR, respec-

tively. Given this association between CDw131 (b-chain) and

CD123 or CD116 we evaluated whether their expression on AML

blasts is correlated. Interestingly, an extremely significant and

direct correlation was observed between the level of CDw131 and

the level of CD123 (P < 0Æ01) or the level of CD116

(P < 0Æ0001), thus suggesting that leukaemic cells have a

co-ordinated control of the amount of these three receptor

chains expressed on the cell membrane (Fig 2B and C).

Molecular abnormalities of AML blasts subdivided intotwo groups according to CDw131 expression levels

According to the reactivity of AML blasts with the anti-

CDw131 (b-chain) mAb, the 105 AML patients analysed were

divided into two groups: CDw131 low (75/105 AMLs express-

ing CDw131 with a MFI <10, including also some CDw131-

negative cases) and CDw131 high (30/105 AMLs expressing

CDw131 with a MFI > 10). The distribution of white blood

cell counts at diagnosis was similar (P = 0Æ6) in the two groups

(Fig 3). The CDw131 high cases were mostly specimens from

M3, M4 and M5 AMLs (Fig 3). The analysis of CDw131

expression of amongst 105 AML patients clearly showed an

higher MFI in the M3, M4 and M5 FAB subtypes, suggesting

an important role of this receptor in the granulocytic-

monocytic differentiation.

Fig 2. Flow cytometry analysis of CDw131 (b-chain), CD123 (IL3R-a)

and CD116 (GM-CSFRa) in acute myeloid leukaemias (AMLs). (Top

Panel) Expression of the IL-3Ra, b-chain and GM-CSFRa expressed as

mean fluorescence intensity (MFI) in 105 AMLs. All the three mem-

brane receptors are detectable in the majority of patients. (Middle and

Bottom Panels). Correlation of b-chain with GM-CSFRa or IL-3Ra in

105 AML patients: the correlation is significant with P < 0Æ0001

between b-chain and GM-CSFRa and P < 0Æ05 between b-chain and

IL-3Ra. IL3R-a, interleukin 3 receptor a; GM-CSFR, granulocyte

macrophage colony-stimulating factor receptor.

Fig 3. White blood cells (WBC) and FAB subtypes in acute myeloid

leukaemia (AML) patients subdivided into two groups according to

the level of CDw131 expression (A). WBC counts at diagnosis observed

in 79 AMLs; 55 patients with low expression of CDw131 and 24

patients with high expression of CDw131. (B) CDw131 expression level

in 105 specimens of AML patients subdivided into six groups (M0, M1,

M2, M3, M4, M5 and M6) according to the FAB classification.

MFIRAU, mean fluorescence intensity relative arbitrary units; FAB,

French–American–British.

R. Riccioni et al

ª 2008 The Authors380 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387

The World Health Organization (WHO) classification for

tumours of haematopoietic tissues has linked recurrent

chromosomal abnormalities and other molecular abnormali-

ties to morphology, immunophenotype and other biological

features of AMLs. We have therefore analysed our patients for

the presence of some recurrent chromosomal rearrange-

ments associated with AMLs (Table I). The analysis was

separately extended to the two different groups with low

and high expression of CDw131 according to most fre-

quent AML chromosome translocation t(15;17) (q22:q21),

t (8;21) (q22:q22), inv16(p13:q22), t (6;9) (p23:q34), t(9;22)

(q34:q11) and t(11:q23; var). The frequency of these alterations

in the present AML series is shown in Table I, no significant

difference was observed between the two groups; however,

AMLs with high CDw131 expression were more positive for

the presence of PML/RARA and CBFB transcripts.

Incidence of FLT3-ITD and D835 mutations

Another important molecular abnormality observed in AMLs

is represented by the mutations of FLT3. To investigate

whether FLT3 mutations were associated with CDw131

expression, the distribution/ frequency of FLT3-ITD and

mutations at aspartic acid residue 835 (Flt3-D835) were

compared amongst 75 patients with de novo AML divided into

CDw131 low and CDw131 high: 57 AMLs low versus 18 AML

high. 20/57 (35%) of CDw131 low AMLs displayed FLT3-ITD/

D835 abnormalities, compared with 11/18 (61%) of CDw131

high AMLs. We further subdivided AMLs into three sub-

groups, FLT3 wild type (WT; 45 cases) FLT3-ITD (25 cases)

and FLT3 D835 (5 cases), and analysed CD123, CD116 and

CDw131 levels in these three groups. The results of this

analysis (Fig 4) showed that: (i) CD123 and CDw131 expres-

sion levels were clearly higher in FLT3-ITD than in FLT3-WT

(wild-type) AML (P £ 0Æ01 for CDw131 and <0Æ001 for

CD123); (ii) CD116 was expressed at higher levels in

FLT3-ITD than in FLT3-WT AML (P £ 0Æ05).

Analysis of immunophenotype

The distribution of the CDw131-positive AMLs along the

various FAB subtypes prompted a careful immunophenotypic

analysis of these AMLs. Expression of surface markers on

leukaemic cells from patients divided in the two CDw131 low

and CDw131 high groups is shown in Fig 5.

CD34, CD133 and CD117 (c-kit) present on haematopoietic

stem/progenitor cells were expressed as the percentage of

positive cells and were detected in most AMLs analysed, with

different patterns. An inverse relationship was observed

between CDw131 high expression and the CD34 marker. The

mean percentage of CD34+ cells in the CDw131 low group was

41 ± 3Æ98 (P < 0Æ01) compared with 24, 15 ± 6, 10 in the

CDw131 high group. CD133 and CD117 markers showed no

differences in the two groups: the mean percentage of CD133

and CD117 was 22Æ87 ± 3Æ45, 51Æ03 ± 3Æ63 for the CDw131 low

group and 27Æ22 ± 5Æ82, 56Æ54 ± 5Æ96 for the CDw131 high

group. CD135 level, expressed as MFI, was higher in the

CDw131 high group than in the CDw131 group (Fig 5).

Differences were observed for the expression of membrane

granulo-monocytic markers CD11b and CD14. These markers

were expressed in the two groups, but differences were

observed in terms of percentage of positive cells for each

marker: the mean percentage of CD11b and CD14 was

28Æ8 ± 2Æ83 and 15Æ13 ± 2Æ24 for CDw131 low group compared

with 48Æ3 ± 5Æ42 and 27Æ77 ± 5Æ14 for CDw131 high group.

The ensemble of these analyses provided several interesting

findings: (i) the majority of the cases in the CDw131 high

group showed a low proportion of CD34+ cells, whilst the

majority of CDw131 low group showed a high proportion of

CD34+ cells (Fig 4); (ii) a significant proportion of AMLs

pertaining to the CDw131 high group exhibited a high

percentage of CD11b and CD14 positive cells, whilst the

frequency of these cells was usually low amongst the CDw131

low group (Fig 5); (iii) FLT3 expression was constantly

elevated amongst the two groups with a higher MFI in the

CDw131 high group than in the CDw131 low group (Fig 5).

The higher CDw131 expression, associated with myelomono-

cytic markers, indicated that bc is involved in the myeloid

differentiation machinery.

Expression of VEGFR-2 (KDR)/Tie-2 angiogenic receptors

We explored also the expression of the receptors for the

angiogenic growth factors, VEGFR-2 Kinase Domain Receptor

Table I. Distribution of chromosomal and

molecular abnormalities amongst 59 AMLs with

CDw131 high and low expression. Chromosome

translocations Fusion gene

Frequency of

alteration CDw131

AML low (42 samples)

Frequency of alteration

CDw131 AML high

(17 samples)

t (15:17)(q22;q21) PML/RARA 6Æ6% 13Æ3%

t (8:21)(q22;q22) RUNX1/RUNX1T1 2Æ4% 0%

inv16 (p13;q22) CBFB/MYH11 19% 11Æ8%

t (6:9)(p23;q34) DEK/NUP214 2Æ4% 0%

t (9:22)(q34;q11) BCR/ABL1 0% 0%

t (11 q23:var.) MLL/var 2Æ4% 0%

AML, acute myeloid leukaemia.

Expression of IL-3/GM-CSF/IL-5 in AML

ª 2008 The AuthorsJournal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387 381

(KDR) and Tie-2. VEGFR-2 was expressed as the percentage of

positive cells and Tie-2 as MFI (Fig 6). The mean percentage of

VEGFR-2 (KDR) was 9Æ73 ± 1Æ47 for the CDw131 low group

and 21 ± 3Æ68 for the CDw131 high group (P < 0Æ01), whilst

the MFI of Tie-2 was 4Æ67 ± 0Æ53 for the CDw131 low group

and 6Æ73 ± 1Æ22 for the CDw131 high group (P < 0Æ05). In the

CDw131 high group 13/24 (54%), AMLs showed >20% KDR+

cells, whilst in the CDw131 group only 14/81 (17%) AMLs

exhibited >20% KDR+ cells. These observations indicated that

receptors for angiogenic growth factors are frequently

expressed amongst AMLs with high CDw131 level. Further-

more, a comparable pattern was also observed for two other

receptors, VEGF-R1 and VEGF-R3 (data not shown).

Leukaemic blasts with elevated CDw131 expression arecycling

To evaluate whether the elevated expression of CDw131

observed in some AMLs may offer a growth advantage to

leukaemic blasts, we evaluated the proportion of cycling cells

in 18 AMLs, of which 10 belonged to the group exhibiting high

CDw131 expression (i.e. MFI > 10) and 8 to the group

exhibiting low CDw131 expression (i.e. MFI < 10). The results

of this analysis (Fig 7A) clearly showed that the proportion of

cycling leukaemic blasts was significantly higher amongst the

CDw131 high group (S+G2/M = 7Æ05 ± 0Æ94%) than in the

CDw131 low group (S+G2/M = 2Æ8 ± 0Æ81%) (P = 0Æ0015). In

a second set of experiments, cells within the leukaemic

population displaying strong and low reactivity with anti-

CDw131 mAb were sorted. The proportion of cycling cells was

determined within the CDw131 bright and the CDw131 dim

populations. This analysis performed in seven AML patients

(Fig 7B) showed that CDw131 bright blasts displayed a

significantly higher proportion of cycling cells (S+G2/

M = 7Æ5 ± 1Æ8%) than CDw131 dim blasts (S+G2/

M = 3Æ25 ± 0Æ63%) (P < 0Æ05).

STAT5, ERK1/2 expression and activation in AML

It is well known that downstream signals engaged by growth

factors such as IL-3 and GM-CSF involve the activation of

STAT5; moreover, a number of studies have shown that

activation of mitogen-activated protein kinase (MAPK) is

involved in a wide set of responses affecting cell proliferation

and differentiation.

Western blot analysis was performed to investigate whether

the regulation of myeloid cell proliferation of the two groups

of AMLs with low and high expression of b-chain could be

associated with constitutive activation of MAPK pathway

(ERK1/2) and STAT5 (Fig 8). Total cell extracts were prepared

from 26 patients (10 pertaining to the high b-chain group and

16 to the low b-chain group patients) with de novo AML and

analysed by phosphotyrosin-specific antibodies. Constitutive

ERK1/2 phosphorylation was observed in most of patients

analysed (about 90%). Constitutive tyrosine phosphorylation

of STAT5 was found in 6/16 (38%) AMLs pertaining to the low

CDw131 group, and in 7/10 (70%) AMLs pertaining to the

high CDw131 group. Interestingly, in the high b-chain group

amongst the seven pSTAT5+ AMLs, five displayed FLT3

Fig 4. CD123, CDw131 and CD116 expression in FMS-related tyro-

sine kinase 3 (FLT3) mutant acute myeloid leukaemias (AMLs). AMLs

were subdivided into three groups according to the presence of

FLT3-WT (wild-type), FLT3-ITD or FLT3-D835 and the levels of

CD123 (A), CDw131 (B) and CD116 (C) in these AMLs are shown.

ITD, internal tandem duplication; D835, mutations at aspartic acid

residue 835.

R. Riccioni et al

ª 2008 The Authors382 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387

Fig 5. Analysis of immunophenotypic features of acute myeloid leukaemias (AMLs) subdivided into two groups (CD131 low and CDw131 high)

according to the level of CDw131 expression. Membrane antigen expression was explored by flow cytometry, and results are expressed as the

percentage of positive cells for CD34, CD133, CD117, CD11b and CD14 and as mean fluorescence intensity (MFI) for FMS-related tyrosine kinase 3

(FLT-3). Differences between the two groups, CDw131 low and CDw131 high, were observed for the expression of CD34 (P < 0Æ01) myelo-monocytic

markers CD11b (P < 0Æ01) and CD14 (P < 0Æ01).

Fig 6. Expression of Tie-2 and vascular endothelial growth factor

receptor (VEGFR-2) (KDR) in acute myeloid leukaemias (AMLs)

subdivided into the two groups, low and high, according to the level of

CDw131 (b-chain). Flow cytometric analysis of Tie-2 and VEGFR-2

(KDR) in AMLs is subdivided into CDw131 low and CDw131 high

groups. The AMLs with high expression of CDw131 showed a higher

mean fluorescence intensity (MFI) of Tie-2 and a higher percentage of

VEGFR-2-positive cells than AMLs with low CDw131 expression.

Fig 7. Cell cycle status of acute myeloid leukaemia (AML) blasts: total

and sorted populations. (A) Evaluation of the proportion of cycling

blasts in 18 AMLs (total population) divided into the two groups with

low (8 AMLs) and high (10 AMLs) expression of CDw131. The per-

centage of cycling cells was higher in the CDw131 high group with

respect to the CDw131 low group. (B) Percentage of S-phase cells at

level of CDw131 bright and CDw131 dim subpopulation in seven

AMLs. The bright and dim populations were isolated by cell sorting.

The bright populations were more cycling than the corresponding dim

populations in all seven patients analysed.

Expression of IL-3/GM-CSF/IL-5 in AML

ª 2008 The AuthorsJournal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387 383

mutations (four FLT3/ITD and one FLT3 D835), whilst in the

low b-chain group the seven pSTAT5+ AMLs, only one

displayed FLT3 mutations.

Discussion

Acute myeloid leukaemia is a heterogeneous group of

neoplastic disorders with great variability in clinical course

and response to therapy as well as in the genetic and molecular

basis of the pathology. Major advances in the understanding of

leukaemogenesis have been achieved by the characterization

and the study of acquired cytogenetic abnormalities; further-

more, gene mutations also constitute key events in AML

pathogenesis. In this context, mutations or enhanced expres-

sion of some membrane receptors contribute to confer a

proliferative advantage to AML blasts. A significant proportion

(about 40%) of AMLs displays mutations of FLT3 or c-kit

receptors, usually associated with poor cure rates due to

increased relapse risk (Small, 2006; Renneville et al, 2008). In

addition, FLT3 is overexpressed in some AML patients in the

absence of any mutation: the overexpressed FLT3 receptors are

auto-phosphorylated and constitutively signal-like mutated

receptors (Ozeki et al, 2004).

We recently reported that the IL-3Ra chain was over-

expressed in about 45% of AML patients and this overexpres-

sion was associated with increased proliferation of leukaemic

blasts and poor prognosis (Testa et al, 2002). The present

study explored the level of CDw131 expression in AMLs, with

the particular aim of evaluating whether this parameter may be

related to peculiar features and to the autonomous growth of

leukaemic blasts. Our observations indicated that CDw131 is

expressed by the large majority of AMLs and about 23% of

AML samples express this receptor chain at significantly high

levels. One observation suggests that elevated CDw131 expres-

sion may have a role in the proliferation of leukaemic cells. In

fact, we observed that within each leukaemic sample, the

fraction of cycling blasts was preferentially observed in the

fraction of cells expressing the highest CDw131 levels.

Interestingly, a significant and direct correlation between the

level of CDw131 and the level of CD123 or the level of CD116

was observed, thus suggesting that leukaemic cells have a

co-ordinated control of the amount of these receptor chains

expressed on the cell membrane. The comparative analysis of

the immunophenotypic features of CDw131 high and CDw131

low AMLs showed that: (i) CD34 was usually scarcely expressed

in CDw131 high AMLs; (ii) the majority of CDw131 high AMLs

consistently expressed both CD11b and CD14. These observa-

tions indicated that an elevated CDw131 expression on

leukaemic blasts is associated with more differentiated AMLs,

mainly pertaining to M4 and M5 subtypes.

The analysis of FLT3 mutations in AMLs highly expressing

CDw131 showed that about 61% of these AMLs displayed

FLT3 mutations (mainly FLT3-ITD) compared with a fre-

quency of about 35% of FLT3 mutants observed in AMLs

expressing low CDw131 levels. On the other hand, we observed

also that FLT3-ITD+ AMLs displayed an elevated CD123 and

CDw131 expression. These observations suggest that elevated

expression of CDw131 and CD123 is associated with FLT3-

ITD and that these two genes may represent molecular targets

for FLT3-ITD. Interestingly, in a previous study aiming to

evaluate the nuclear morphology of AMLs with mutated FLT3,

Kussick et al (2004) reported preliminary observations sug-

gesting that AMLs with FLT3-ITD strongly expressed CD123.

The mechanism through which FLT3-ITD may stimulate

CDw131 and CD123 expression remains to be determined.

The observation that AMLs with FLT3-ITD strongly express

CD123 may also have some implications for the development

of new therapies for the treatment of this category of AML

patients, who notoriously have a poor prognosis. Our obser-

vations suggest that these AMLs are potential candidates for

experimental treatment with diphtheria toxin-IL3 fusion

protein (Frankel et al, 2008).

Constitutive activation of STAT5 has been demonstrated in

>50% of AML cases (Birkenkamp et al, 2001; Spiekermann

et al, 2003) and may be attributed to activating mutations in

upstream kinases such as FLT3 (Hayakawa et al, 2000;

Fig 8. Analysis of constitutive extracellular signal-regulated kinase

(ERK1/2) and STAT5 phosphorylation in acute myeloid leukaemias

(AMLs) subdivided according to the level of CDw131 expression. Sixteen

AML samples were evaluated by western blotting for phosphor-STAT5

(pSTAT5), total STAT5 (STAT5 TOT), phosphor-ERK (pERK), total

ERK (ERK-TOT) and actin content. Samples corresponding to AMLs

with high CDw131 expression are indicated by an asterisk.

R. Riccioni et al

ª 2008 The Authors384 Journal Compilation ª 2008 Blackwell Publishing Ltd, British Journal of Haematology, 144, 376–387

Spiekermann et al, 2003; Yao et al, 2005; Rocnik et al, 2006),

Kinase Inducing Tyrosine (KIT) (Growney et al, 2005) or

JAK2 (Walters et al, 2006) or alternatively be due to autocrine

growth factor production (Birkenkamp et al, 2001). Finally, a

recent study showed that in AMLs with constitutive STAT5

activation, STAT5 is required for leukaemic cell proliferation

and for maintenance of leukaemic stem/progenitor cells

(Schepers et al, 2007). In the present study we reported the

frequent constitutive STAT5 activation in high CDw131 AMLs

(70%) compared with a lower frequency observed in low

CDw131 AMLs (38%). The high frequency of constitutive

STAT5 activation observed in high CDw131 AMLs was related

to the high frequency of FLT3-ITD observed in these AMLs.

We previously reported the frequent constitutive STAT5

activation in AMLs overexpressing IL-3Ra (Testa et al,

2002). This finding may be now easily explained by the

present observations showing that FLT3-ITD+ AMLs fre-

quently overexpress IL-3Ra.

An additional interesting finding of the present study

was the observation that CDw131 high AMLs frequently

co-expressed membrane endothelial markers such as Tie-2,

VEGF-R1 and VEGF-R2. These findings indicate that these

AMLs may represent a subtype of AMLs, as recently described

(Riccioni et al, 2007) and corresponding to myeloid cells

expressing endothelial markers. The normal counterpart of

these cells represent a population of haematopoietic proangio-

genic myeloid cells required for tumour vessel formation

(Yang et al, 2004; De Palma et al, 2005; Ahn & Brown, 2008).

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