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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3401-3413
A Strong Expression of CD44-6v Correlates With Shorter Survival of
Patients With Acute Myeloid Leukemia
By
S. Legras,
U. Günthert,
R. Stauder,
F. Curt,
S. Oliferenko,
H.C. Kluin-Nelemans,
J.P. Marie,
S. Proctor,
C. Jasmin, and
F. Smadja-Joffe
From INSERM U268 Hôpital Paul-Brousse, Villejuif, France; Basel
Institute for Immunology, Basel, Switzerland; University Hospital
Innsbruck, Innsbruck, Austria; U168 Villejuif, France; Leiden
University Medical Center, Leiden, The Netherlands; Hôpital
Hôtel-Dieu, Paris, France; and The Royal Victoria Infirmary,
Newcastle upon Tyne, UK.
 |
ABSTRACT |
CD44 is a ubiquitous cell-surface glycoprotein that displays many
variant isoforms (CD44v) generated by alternative splicing of exons 2v
to 10v. The expression of variant isoforms is highly restricted and
correlated with specific processes, such as leukocyte activation and
malignant transformation. We have herein studied CD44v expression in
acute myeloid leukemia (AML) and, for comparison, in normal
myelopoiesis. Protein expression of total CD44 and of CD44-3v, -6v, and
-9v isoforms has been measured using specific monoclonal antibodies and
flow cytometry. The composition of variant exon transcripts has been
analyzed by semi-quantitative reverse transcriptase-polymerase chain
reaction followed by Southern hybridization with exon-specific probes.
Our data show that (1) CD44-6v isoforms are expressed on 12.0% ± 2.5% of normal CD34+ cells; this expression is sharply
upregulated through monopoiesis and, inversely, downregulated during
granulopoiesis. Also, CD44-3v and CD44-9v isoforms are detected on 10%
and 14% of normal monocytes, respectively. (2) Sixty-nine from a total
of 95 AML patients display a variable proportion (range, 5% to 80%)
of CD44-6v+ leukemic cells. (3) A shorter overall
survival characterizes the group of AML patients displaying more than
20% of CD44-6v+ leukemic cells (8 months v 18 months, P < .02). These data suggest, for the first time,
that the protein expression of CD44-6v containing isoforms may serve as
a new prognostic factor in AML.
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INTRODUCTION |
THE CD44 ANTIGEN is a highly glycosylated
transmembrane protein encoded by a unique 20-exon gene on chromosome 11 in humans.1 It displays multiple isoforms. The so-called
standard isoform CD44s (90 kD) is the most common one and
is encoded by 10 standard exons (1s to 10s). A substantial number of
so-called variant isoforms, CD44v (110 to 250 kD), have also been
described.2 They are generated by the alternative splicing
of nine variant exons (2v to 10v) in multiple combinations, and
characterized by additional sequences inserted into the extracellular
domain of the molecule.3
CD44 is thought to play an important role in myelopoiesis because
anti-CD44 monoclonal antibodies (MoAbs) fully inhibit in vitro
long-term hematopoiesis on pre-established stroma.4,5 In
addition, it has been shown that CD44 mediates the adhesion of
CD34+ hematopoietic progenitors (HPC) to
hyaluronan,6-8 its best-known ligand,9-11 which
is present in the hematopoietic extracellular matrix.7,12-15
The standard CD44 isoform is expressed on all types of mature blood
cells,11,16,17 on the majority of mononuclear bone marrow
(BM) precursors,18,19 and on all CD34+
HPC.20,21 The level of its expression varies according to hematopoietic cell lineage and stage of differentiation. For example, it is high on monocytic cells, intermediate on polymorphonuclear cells
(PMN) and on CD34+ HPC, and low on erythroid cells and
platelets. The variant isoforms CD44-6v and CD44-9v have been detected
on monocytes, macrophages, lymphocytes, and dendritic
cells.2,22-27 Inflammation, mitogens, and
inflammatory cytokines have been shown to upregulate CD44-3v, 6v, and
CD44-9v expression in monocytes and lymphocytes.22-26,28-30 As far as myeloid cells are concerned, only CD44-10v has been shown on
a few human BM myeloid precursors.19 The expression of
CD44-6v has been also recently reported on HPC in the
rat.31
Several CD44 variant isoforms are overexpressed in malignant
hematopoietic cells. For example, CD44-6v or CD44-9v isoforms are
upregulated in non-Hodgkin's lymphoma (NHL) and
myeloma,26,27,29,32 and CD44-10v expression is enhanced in
chronic myeloid leukemia (CML) and acute myeloid leukemia
(AML).19,33 Moreover, the expression of CD44-6v and CD44-9v
correlates with an unfavorable clinical evolution in lymphoma and
myeloma,27,32 suggesting a role of these CD44 variant
isoforms in hematopoietic malignancies.
In the present study our aim has been to evaluate the role of CD44
variant isoforms in the pathophysiology and clinical evolution of AML.
AML is a heterogenous disease, characterized by the accumulation of
leukemic cells in BM and peripheral blood (PB). These cells display
cytological and antigenic features of immature granulocytic and/or monocytic cells. On the basis of these features, AML has been classified into distinct subtypes: myeloblastic (M1/M2), promyelocytic (M3), myelomonocytic (M4), and monoblastic
(M5).34,35
We have analyzed the expression of 3v-, 6v-, and 9v-containing CD44
isoforms on circulating leukemic cells using flow cytometry; we have
determined the exon composition of these isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR); and we have correlated the results with clinical characteristics. For comparison we
have also analyzed the CD44v expression pattern in normal myeloid cells
at various stages of maturation. We show that in normal subjects the
expression of CD44-6v isoforms is restricted to CD34+ cells
and to monocytic cells. We also show that CD44-6v is expressed on AML
leukemic cells and, most importantly, that its expression level is
correlated with the overall survival of AML patients treated by
conventional chemotherapy.
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MATERIALS AND METHODS |
AML Patients and Clinical Data
Leukemic cells from 95 patients with AML were collected at diagnosis
(82 patients) or at relapse (13 patients) from the following hospitals:
Leiden University Hospital (Leiden, The Netherlands; 45 samples),
Hotel-Dieu (Paris, France; 21 samples), Henri Mondor (Creteil, France;
13 samples), The Royal Victoria Infirmary (Newcastle, UK; 9 samples),
Hôpital Paul-Brousse (Villejuif, France; 3 samples), Hôpital St Louis (Paris, France; 2 samples), and Institut
Gustave-Roussy (Villejuif, France; 2 samples). The main clinical
features of these patients are summarized in Table 1. All patients
displayed more than 60% blasts in PB. The patients' treatments are
indicated in Table 1. They include (1)
conventional induction chemotherapy,36 ie, administration
of anthracycline plus cytarabine, which may be followed by allograft,
and (2) palliative chemotherapy. Two M3 patients also received
all-trans retinoic acid. The patients' survival was
followed-up for at least 2 years after complete remission.
Isolation of Leukemic Cells
Low-density mononuclear cells from PB were separated by density
gradient centrifugation on Ficoll-hypaque density 1.077 g/mL (Pharmacia
LKB, Uppsala, Sweden) and resuspended in RPMI 1640 plus 10% fetal calf
serum (FCS). Preliminary experiments showed that cell freezing and
thawing did not change the antigenic profile of these cells. Therefore,
AML low-density mononuclear cells were cryopreserved in 25% FCS plus
10% dimethylsulfoxide in RPMI 1640 culture medium, and stored in
liquid nitrogen until use. Four hours before CD44 analysis, cells were
thawed at room temperature in RPMI 1640 culture medium containing 50%
FCS, then washed twice in RPMI 1640 plus 2% FCS. B and T lymphocyes
were removed from all samples by specific immunoadsorption on Dynabeads
(Dynal, Oslo, Norway) coated with MoAbs to CD2 and CD19, according to the manufacturer's recommendations. Monocytes were removed from myeloblastic and promyelocytic AML (M1 to M3), using Dynabeads coated
with MoAb to CD14. Cell suspensions containing more than 95% of AML
blasts were thus obtained. The absence of CD2+ and
CD19+ cells (and of CD14+ cells in M1 to M3
AML) was verified by flow cytometry (Fig
1).
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| Fig 1.
Specific gating of AML blasts. Cell suspensions
containing more than 95% of AML blasts were prepared by removing
CD2+ CD19+ cells (lymphocytes) using
specific immunoadsorption on Dynabeads coated with specific MoAbs as
described in Materials and Methods. In the example shown here, which is
from an M1 AML patient, CD14+ monocytes have also been
removed. AML blasts, which are characterized by large forward and side
scattering values37 (1), are gated in the
CD2neg, CD19neg (2) and CD14neg (3)
windows.
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Isolation of Normal Myeloid Cells
BM and circulating myeloid cells from distinct lineages and at
different stages of maturation are intermingled. Therefore, for both
RT-PCR and fluorescence-activated cell sorter (FACS) analysis,
populations enriched with CD34+ cells and granulocytic
precursors were isolated from BM; because circulating mature monocytes
and PMN could be easily identified on the basis of specific
cell-surface antigen expression (CD14 and CD15, respectively) and
light-scattering properties,18 they were directly labeled
and analyzed by flow cytometry, as detailed below; they were isolated
only for RT-PCR studies. Monocytic precursors were too scarce for being
isolated as a pure population for RT-PCR and therefore they were only
analyzed by flow cytometry (see section "Flow Cytometric
Analysis").
Normal Immature Hematopoietic Cells (CD34+ cells)
and CD34neg Granulocytic Precursors (GP)
Samples from normal BM were obtained following informed consent from
donors receiving hip surgical intervention (Dr J. de Thomasson,
Clinique Chirurgicale de Choisy, Paris, France) or BM cell donors for
allograft (Dr J.L. Pico, Institut Gustave Roussy, Villejuif, France).
Normal donors comprised three females and two males with age ranging
from 42 to 75 years, except for one 1-year-old child. In the case of
donors with hip surgical intervention, hematopoietic cells were
extracted from normal BM by grounding the trabeculae with a potter and
vortexing the samples several times in phosphate-buffered saline (PBS).
Light-density mononuclear cells were separated by density gradient
centrifugation on Ficoll-hypaque density 1.077 g/mL (Pharmacia LKB),
resuspended in RPMI 1640 plus 10% FCS and processed for the isolation
of two distinct cell populations, respectively, enriched in
CD34neg granulocytic precursors and CD34+
cells. Mature monocytes were removed by adhesion on plastic for 2 hours
at 37°C and after two washes with PBS. The most differentiated precursors and stromal cells were discarded by adhesion on soy bean
agglutinin CELLector flasks (Applied Immune Sciences Inc, Menlo Park,
CA).
CD34+ cells were recovered by adhesion on ICH3 anti-CD34
MoAb-coated CELLector flasks (Applied Immune Sciences Inc), according to the manufacturer's instructions. Flow cytometric analysis indicated that all samples contained more than 95% CD34+ cells. The
nonadherent CD34neg cell population was highly enriched in
immature granulocytic cells by incubation with Dynabeads coated with
MoAbs to CD2, CD19, CD14, and CD71 for depleting lymphocytes, monocytic
cells, and erythroblastic cells. Microscopic observation of
May-Grünwald Giemsa-stained cytosmears indicated that this cell
population comprised 3% ± 1% myeloblasts, 21% ± 2%
promyelocytes, 34% ± 4% myelocytes, and 35% ± 3%
metamyelocytes. It was designated as CD34neg granulocytic
precursors (CD34neg GP). We confirmed by flow cytometry
that the purification procedure did not alter the expression level of
CD44.
Normal PMN and Monocytes
Samples of heparinized PB were obtained from five informed and
consenting adult donors. To recover PMN, the blood sample was first
diluted with PBS containing 3% gelatin (Plasmion; Rhone-Poulenc, Lyon,
France), with a ratio of 4:1 (vol/vol); the red blood cells were
pelleted by low-speed centrifugation (50g for 1 minute, at 20°C), and the nucleated cells, recovered in the upper phase, were
layered on Ficoll-hypaque density 1.077 g/mL (Pharmacia LKB) and
centrifuged at 500g for 30 minutes, at 20°C. The pellet
comprised more than 98% granulocytes. For isolating monocytes,
light-density mononuclear cells were recovered by centrifugation on
Ficoll-hypaque as described above and suspended in RPMI 1640 medium
plus 10% FCS (5 × 106 cells/mL). Monocytes were
collected by adhesion for 2 hours on plastic (T-flask Costar,
Cambridge, MA), at 37°C with 5% CO2 in air. At least
90% of plastic adherent cells were shown to express the
monocyte-specific antigen CD14.
MoAbs
The MoAb F10-44-2 (IgG2a),22 which is directed at an
epitope in the CD44 common region, cannot discriminate between CD44s and CD44v-containing isoforms. Its binding reflects the total amount of
CD44 on the cell surface. It was conjugated to phycoerythrin (PE) and
used at 5 µg/mL; this MoAb was provided by Serotec (Oxford, UK).
MoAbs directed at CD44 variant epitopes were specific for 3v (BBA11,
IgG2b), 6v (VFF-18, IgG1), and 9v (FW11-24, IgG1). BBA11 was obtained
from R&D SystemsEurope (Abingdon, UK) and used at 1 µg/mL. VFF-18 was
purchased from Bender MedSystems (Vienna, Austria) and used at 10 µg/mL, and FW11-24 was a hybridoma supernatant,22 used
undiluted. Fluorescein isothiocyanate (FITC)-conjugated MoAb to CD14
(IgG2b, 5 µg/mL) provided by Coulter (Coulter Immunology, Hialeah,
FL) was used to identify monocytic cells. FITC-conjugated anti-CD15
MoAb (IgM, 1 µg/mL; Becton Dickinson, San Jose, CA) was used to
characterize cells from the granulocytic lineage. Anti-CD34 MoAb
(HPCA-2, IgG1, 1 µg/mL) conjugated to FITC was obtained from Becton
Dickinson. Antibodies were used at saturating concentrations. Murine
IgG1 and IgG2a conjugated with either FITC or PE were from Immunotech
(Marseille, France). PE-conjugated goat F(ab )2 anti-mouse
IgG (H + L) (PE-GAM) was purchased from Southern Biotechnologies
Associates (Birmingham, AL) and used in a 1:50 dilution.
Immunofluorescent Staining and Flow Cytometry of Normal and
Leukemic Cells
Gating of normal and leukemic cells.
PMN and mature monocytes were stained directly in the heparinized blood
samples, after red blood cells lysis using 0.8% ammonium chloride, and
they were identified by specific electronic gating18 and on
the basis of specific cell-surface antigen expression (CD15 and CD14,
respectively). CD34+ cells were isolated as described and
identified by staining with an FITC-conjugated MoAb directed to CD34,
as well as by specific scatter features. Monocytic precursors were
identified in the CD34neg BM population by staining with an
FITC-conjugated MoAb to CD14, and by specific scatter
features.18 Granulocytic precursors were isolated in the
CD34neg BM population as described above, and identified by
staining with an FITC-conjugated MoAb to CD15, and by specific scatter features.18 Suspensions containing more than 95% of AML
blasts were prepared. As described above, these blasts were
characterized both by specific scatter parameters37 and by
a CD2neg, CD19neg phenotype, as shown in Fig 1.
In addition, in myeloblastic (M1/M2) and promyelocytic (M3) AML,
leukemic blasts were also characterized by a CD14neg
phenotype (Fig 1).
Immunofluorescent staining and flow cytometry.
For labeling, cells were suspended in RPMI 1640 containing 0.2% bovine
serum albumin (BSA) and 0.02% NaN3 (label medium) at 106 cells/mL; incubated at 4°C for 30 minutes with 5 µg/mL PE-conjugated MoAb F10-44-2, which labeled both CD44s and CD44v
(total CD44); and washed twice with label medium. CD44v expression was
analyzed after successive incubation with 5% normal human serum (AB
type), unconjugated MoAb, 5% goat serum, and GAM-PE. The MoAb binding was measured by flow cytometry relative to isotype-matched control antibodies, using a Profile II flow cytometer (Coulter Immunology) equipped with a 15-mW argon laser and calibrated using a panel of
fluorescent beads (Immunobrite; Coulter, Hialeah, FL). Under these
conditions, the fluorescence intensity of total CD44 on HL60 and MO7e
cells, respectively, used as negative and positive controls for variant
expression, was constant. Adjustment of the crossover fluorescence was
obtained by compensation of the two single-stained samples to limit
superposition of the fluorochrome emission spectra. Each measurement
was performed on 5,000 cells.
The staining intensity of total CD44 and CD44 variant isoforms was
measured as the relative mean fluorescence intensity (MFI) compared
with isotypic controls. The relative MFI of CD44v was much lower than
the one of total CD44 (maximum of 6). For this reason, the amount of
CD44v isoforms was also evaluated by the percentage of labeled cells
over the background (cells incubated with isotype-matched control
antibodies). It was considered as negative ( ) when less than 5% of
the cells were labeled, positive (+) for 5% to less than 20% of
labeled cells (similar to normal CD34+ cells, see Results),
and highly positive (++) when more than 20% of the cells were
CD44v+ (similar to normal monocytes, see Results).
CD34+CD44-6v+ and
CD34+CD44-6vneg cells were sorted on a
FACSvantage (Becton Dickinson) equipped with an INNOVA70-4 Argon ion
laser (Coherent Radiation, Palo Alto, CA) tunned at 488 nm and
operating at 500 mW. Highly diffusive and/or too large objects
were rejected. Positivity or negativity for the CD44-6v among the
CD34+ cells was determined using control cells labeled with
FITC-conjugated anti-CD34 MoAb (FITC-HPCA2) and PE-conjugated IgG1.
Compensation was set up with single-stained samples.
Clonogenic Features of
CD34+CD44-6v+ and
CD34+CD44-6vneg Cells
CD34+CD44-6v+ and
CD34+CD44-6vneg cells were cultured, in
triplicate, in 35-mm non-tissue culture grade Petri dishes (Falcon;
reference 1008; Becton Dickinson, Plymouth, UK) containing 1 mL of
complete methylcellulose medium purchased from StemCell Technologies
Inc (Vancouver, British Columbia, Canada) (0.8% methylcellulose in Iscove's modified Dulbecco's medium [IMDM], 30% FCS, 1% deionized BSA, 10 4 mol/L 2-mercaptoethanol). Colony-stimulating
factors were provided as 5% of phytohemagglutinin-human
leukocyte-conditioned medium (Hemostim H2400; StemCell Technologies
Inc, Vancouver, Canada). Human erythropoietin purified from human urine
was added at 3 U/mL (StemCell Technologies Inc). Plates were
incubated at 37°C in a fully humidified atmosphere containing 5%
CO2 in air. Colony-forming units monocytic (CFU-M),
granulocytic (CFU-G), granulo/monocytic (CFU-GM), and
granulocytic/erythroid/monocytic/megakaryocytic (CFU-GEMM), and
burst-forming units-erythroid (BFU-E) were scored at day 16 following
the standard criteria. Their number was expressed as the percentage of
total colonies obtained.
Preparation of RNA and cDNA/Reverse-Transcriptase Polymerase Chain
Reaction (RT-PCR)
Variant exon expression was analyzed at the RNA level on enriched cell
populations, prepared as described above. Both normal myeloid cells
(five samples per cell type) and AML leukemic cells from 70 patients
(from all French-American-British [FAB] types) were analyzed. Total
cellular RNA was prepared and semi-quantitative RT-PCR performed as
described previously.27 Briefly, total cellular RNA was
prepared using 105 purified cells, followed by cDNA
synthesis. cDNA amounts were equilibrated semi-quantitatively for the
housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) as a
marker transcript. Equilibrated amounts of cDNA were taken for
CD44-specific PCR amplification using primers homologous to CD44s
regions close to the variant sequences. The cDNA from CD44s lacking all
variant exons thus yielded a product of 67 bp. The exon-specific probes
were labeled with 33P-dATP (Megaprime labeling kit;
Amersham, Zürich, Switzerland), and blots were hybridized and
washed under stringent conditions (0.2 × standard sodium
citrate [SSC], 0.1% sodium dodecyl sulfate, 65°C). Blots were
exposed to Kodak BioMax MR films (Eastman Kodak Co, Rochester, NY) at
room temperature for 12 to 48 hours. Staining intensity was scored
semiquantitatively on a scale of to ++++ (, negative; ±,
faint, not clearly above background; +, weakly positive; ++,
moderately positive; +++ and ++++, strongly positive).
Statistical Analysis
The mean MFI value ± 1 SE from the distinct cell populations were
compared by the Kruskal-Wallis test. The comparisons two by two were
performed by Student's t-test depending on the number of
cases. The statistical significance between the presence of CD44
isoforms and the clinical parameters was determined by Fisher's exact
test or chi-squared test depending on the number of cases. The
probability of overall survival was computed according to the
Kaplan-Meier method.38 Statistical comparisons between
curves were based on log-rank tests. Relationship between prognostic factors was determined using Cox's proportional hazard model for covariate analysis of censored data.39 All survival
analyses were performed by using the statistical package BMDP (BMDP
Statistical Software, Los Angeles, CA). The correlation between the
expression of CD44v isoforms and the following clinical parameters were
investigated: overall survival rate (alive v deceased), rate
and duration of complete remission, and rate of relapse. In addition,
correlation of CD44v expression and AML subtype, age, number of
circulating leukemic blasts, and percentage of CD34+
leukemic cells were also analyzed. Survival rate, age, and white blood
cell number per microliter (WBC/µL) were expressed as median.
| |
RESULTS |
Flow Cytometric Analysis of CD44 Isoform Expression
Normal myeloid cells.
The amount of total CD44 was measured using PE-conjugated F10-44-2
MoAb. This MoAb, which is directed to an epitope in the constant domain
of the molecule, binds both standard and variant CD44 isoforms. The
highest expression of CD44 was observed on monocytic cells, including
monocytic precursors (MP, CD34neg CD14+ cells)
and mature monocytes (relative MFI = 200 ± 25 and 80 ± 1, respectively). CD34+ cells and PMN displayed also a
significant amount of CD44 (relative MFI of 26 ± 3 and 26 ± 1,
respectively), whereas maturing granulocytic precursors (GP,
CD34neg CD15+ cells) expressed total CD44 only
weakly (relative MFI = 4 ± 1) (Fig
2). The GP population comprised 3% ± 1%
myeloblasts, 21% ± 2% promyelocytes, 34% ± 4% myelocytes, and
35% ± 3% metamyelocytes.
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| Fig 2.
Scatter plot of total CD44 expression on normal myeloid
cells and on AML leukemic cells. Normal Myeloid Cells:
CD34+ cells (very immature hematopoietic cells); GP,
granulocytic precursors (CD34, CD15+ BM
cells); PMN, polymorphonuclear cells; MP, monocytic precursors (CD34, CD14+ BM cells); Mono, mature
monocytes (circulating CD14+ cells). AML Leukemic Cells:
from AML patients with the following FAB types.34,35 M1/M2,
myeloblastic AML; M3, promyelocytic AML; M4, myelomonocytic AML; M5,
monoblastic AML. Mature monocytes and PMN from PB were identified
according to their forward angle and side scatter.37
CD34+ cells and MP were double-stained with (1) an
FITC-conjugated MoAb directed to either CD34, which is specific for
very immature hematopoietic cells, or CD14, a monocyte-specific antigen
and (2) a PE-conjugated anti-CD44 MoAb F10-44-2, which binds to an epitope located in the constant part of the CD44 molecule. Negative controls were cells labeled with FITC and PE-conjugated isotype-matched control antibodies. The MFI was determined by flow cytometry, relative
to cells labeled with PE-conjugated IgG2a (negative controls), as
described in Materials and Methods. Each symbol refers to the MFI value
per patient, and the horizontal bars indicate the mean MFI value in
each category of patients. The normal BM cells and the leukemic cell
populations were isolated as described in Materials and Methods. The
gating of leukemic cells is shown in Fig 1.
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The expression of variant CD44 proteins (CD44v) was investigated using
variant-specific MoAbs (described in Materials and Methods).
A small proportion of CD34+ cells (12.0% ± 2.5%) was
found to display CD44-6v isoforms (relative MFI of 2 ± 0, Fig 3A
and B, and Fig
4). We sorted
CD34+CD44-6v+ and
CD34+CD44-6vneg cells and analyzed their
clonogenic features, using a methylcellulose assay: the
CD34+CD44-6v+ cell population comprised more
CFU-M (44% ± 7% v 24% ± 5%, P < .05, Student's t-test) and less BFU-E (10% ± 2% v
31% ± 8%, P < .05, Student's t-test), than
the CD34+CD44-6vneg cells. CFU-G were in equal
number in CD34+CD44-6v+ and
CD34+CD44-6vneg cell populations (Table
2).
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| Fig 3.
Cell-surface expression of CD44-3v, -6v, and -9v
containing isoforms on normal myeloid cells. (A) Expression of CD44-6v
on CD34+ cells. CD34+ cells were isolated
from normal BM by specific immunoadsorption, as described in Materials
and Methods. Histogram 1 defines the morphologic gate. Cells were
double-stained with the anti-6v MoAb VFF18 plus GAM-PE and
FITC-conjugated HPCA-2 MoAb directed to CD34 (histogram 2). Quadrant
limits defining positivity and negativity have been set up using cells
labeled with PE-conjugated and FITC-conjugated control antibodies
(histograms 3 and 4), as described in Materials and Methods. Adjustment
of the crossover fluorescence was obtained by compensation of the two
single-stained samples to limit superposition of the fluorochrome
emission spectra. (B) CD34+ cells and monocytic cells.
CD34+ cells, very immature hematopoietic cells; MP,
monocytic precursors (CD34neg, CD14+ BM
cells); Mature monocytes, circulating CD14+ cells. Mature
monocytes from PB were identified according to their forward angle and
side scatter.37 CD34+ cells and MP were
double-stained with (1) an FITC-conjugated MoAb directed to CD34, which
defines very immature hematopoietic cells, and CD14, a
monocyte-specific antigen, respectively and (2) MoAbs directed at
CD44-3v (BBA11), -6v (VFF-18), and -9v (11-24), plus PE-conjugated goat
anti-mouse IgG. Negative controls (gray lines) were cells labeled with
FITC and PE-conjugated isotype-matched control antibodies. Flow
cytometric analysis was performed as described in Materials and
Methods. Four independent experiments gave similar results. HPC were
negative for CD44-3v and CD44-9v proteins.
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| Fig 4.
Scatter plot of CD44v protein expression on AML leukemic
cells and normal myeloid cells. Normal myeloid cells:
CD34+ cells (very immature BM hematopoietic cells). GP,
granulocytic precursors (CD34, CD15+ BM
cells); PMN, polymorphonuclear cells; MP, monocytic precursors (CD34neg, CD14+ BM cells); Monocytes: mature
monocytes (circulating CD14+ cells). AML leukemic cells
from AML patients with the following FAB types.34,35 M1/M2,
myeloblastic AML; M3, promyelocytic AML; M4, myelomonocytic AML; M5,
monoblastic AML. CD34+ cells, GP, and MP were identified
by double-staining with (1) an FITC-conjugated MoAb directed to CD34,
CD15 for GP and CD14 for MP, respectively, (2) unconjugated MoAbs
directed to variant epitopes 3v, 6v, and 9v plus PE-goat anti-mouse
(PE-GAM). Leukemic cells were identified by specific electronic
gating.37 Flow cytometric analysis was performed as
described in Materials and Methods. The staining intensity of variant
CD44 isoforms was evaluated by measuring the percentage of labeled
cells over the background (cells incubated with isotype-matched control
antibodies). Each symbol ( ) refers to the percent CD44v cells per
patient. Expression of CD44v was considered as negative () when less
than 5% of the cells were labeled, low (+) when 5% to less than
20% of cells were labeled, and high (++) when more than 20% of
the cells were CD44v+.
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CD44-6v was also detected on about half of the monocytic cells
(relative MFI: 5 ± 1, Figs 3B and 4). Considering that the level of
CD44-6v expression was significantly higher on monocytic cells than on
CD34+ cells, we defined as strongly positive (++) the level
of CD44-6v on normal monocytic cells and as positive (+) the one on
normal CD34+ cells (Fig 4).
In addition, CD44-9v was found on 25% of monocytic precursors and 14%
of mature monocytes (relative MFI: 3 ± 2), whereas CD44-3v was only
detected on 10% of mature monocytes (Figs 3B and 4). No significant
expression of CD44v isoforms could be evidenced on GP or on PMN (<5%
of positive cells, Fig 4).
AML leukemic cells.
Flow cytometric analysis of CD44 isoform expression was performed in
AML cells from PB. In 10 patients, analysis was performed on both blood
and BM blasts: a similar CD44 expression pattern was observed
(comparison not shown, data from blood are shown, Figs 2 and 4). As
detailed in Materials and Methods, the labeling was performed on cell
suspension containing more than 95% AML blasts, after removal of PMN,
lymphocytes, and monocytes, the latter for myeloblastic (M1/M2) and
(M3) promyelocytic AML types. In addition, the AML blasts were
specifically gated as described.37
Total CD44 was expressed on AML leukemic cells (Fig 2). The mean MFI
value was similar in M1/M2, M4, and M5 AML (32 ± 4, 38 ± 6, and
48 ± 3, respectively). It was higher in M3 AML (98 ± 7).
CD44-6v expressing leukemic cells were detected in 69 of 95 patients
(Fig 4). The proportion of leukemic cells labeled above the background
(cells labeled with isotype-matched control antibodies) ranged between
5% and 80% in 34 of 51 M1/M2 cases (median value, 20%), in 7 of 8 M3
patients (median value, 23%) and in 24 of 30 M4/M5 patients (median
value, 43%). More than 20% of CD44-6v expressing leukemic cells (mean
relative MFI value of 3 ± 1, 4 ± 1, and 5 ± 1, respectively) were displayed by 15 M1/M2, 6 M3, and 16 M4/M5 patients as well as on 2 of 6 patients with undefined or M7 AML type. By reference to the high level of CD44-6v expression on normal monocytes, as described above, these patients were designated as
CD44-6v++. The intensity of CD44-6v expression positively
correlated with the level of total CD44 (P < .01,
chi-squared test, n = 82, samples collected at relapse were not
included).
A weak expression of CD44-3v was observed in 6 of 51 M1/M2 patients, 4 M3, and 9 M4/M5 cases. Six M5 patients were strongly CD44-3v+ (Fig 4). Only a low expression of CD44-9v was
evidenced in 11 M1/M2, 4 M3, and 6 M4/M5 patients.
Correlation of CD44 Isoform Expression With the Clinical Outcome of
Patients
To determine whether CD44-6v isoform expression influences the clinical
outcome of AML patients, the rate of first complete remission was
compared in CD44-6v++ and CD44-6v+/neg groups
of patients undergoing induction chemotherapy. Because treatment may
modify CD44-6v expression,40 only patients with de novo AML
have been included in this statistical study (n = 63, Table 1): the
remission rate was 69% for CD44-6v++ group (n = 23) and
58% for CD44-6v+/neg cases (n = 40); the difference was
not statistically significant (P = .33, chi-squared test).
The median overall survival of CD44-6v++ and
CD44-6v+/neg cases has been compared using the
Kaplan-Meier method.38 To analyze a homogenous group of
patients, this statistical analysis has been restricted to patients
treated by conventional induction chemotherapy and with a well-defined
FAB AML type. Consequently, the two M3 patients treated with
all-trans retinoic acid, as well as the three patients with
undefined FAB AML type, were excluded from the analysis (Table 1).
First, M1 to M5 FAB types have been analyzed together
(n = 48, Fig 5A). The median overall
survival of CD44-6v++ patients was 8 months, compared with
18 months for CD446v+/neg cases. The difference was
statistically significant, with P < .02 (Kaplan-Meier method38). Moreover, the disease-free
survival (DFS), measured for patients achieving a first complete
remission, was shorter in the CD44-6v++ group of patients
than in the CD44-6v+/neg group (6 months v 17 months, P < .06, n = 29, Kaplan-Meier
method,38 Fig 5D).
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| Fig 5.
Correlation of CD44-6v isoform expression with survival
of AML patients. Survival curves of AML patients according to the expression of CD44-6v on leukemic cells at diagnosis, have been computed using the Kaplan-Meier method.38 Statistical
comparisons between curves were based on log-rank tests. (A, B, and C)
Overall survival curves of AML patients treated by conventional
chemotherapy36 (anthracyclin plus cytosin arabinosine). (D
and E) Disease-free survival curves of AML patients induced in complete
remission by conventional chemotherapy. M1/M2, myeloblastic AML; M4,
myelomonocytic AML; M5, monoblastic AML. Significant numbers have been
obtained by grouping M4 and M5 patients for overall survival, but not
for disease-free survival analysis.
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|
Second, M1/M2 AML (n = 28) have been separately analyzed. A
strong CD44-6v expression was clearly associated with a shorter overall
survival. Indeed, this latter was 5.6 months in the group of
CD44-6v++ patients, and 14.6 months in the group of
CD44-6v+/neg patients (P < .04, Kaplan-Meier
method,38 Fig 5B). Moreover, the DFS was 3 months and 16 months for CD44-6v++ and CD44-6v+/neg groups,
respectively (P = .15, n = 18, Kaplan-Meier
method,38 Fig 5E).
Statistical significance was not achieved in the group of
myelomonocytic plus monoblastic AML (M4/M5, P = .48,
n = 16, Kaplan-Meier method,38 Fig 5C).
The expression of CD44-6v was not correlated with the patients' age
(P = .3, n = 82, Student's t-test), or with the
number of circulating blasts (P = .4, n = 82, Student's
t-test), which are both important prognostic
factors in AML. An eventual correlation with karyotypic abnormalities
could not be investigated because karyotype analyses were available
only in a minority of cases.
Finally, there was no correlation between patients' survival and the
expression of total CD44 (P = .6, n = 48, Kaplan-Meier method38).
Determination of the CD44v Exon Composition in AML Leukemic
Cells and in Normal Myeloid Cells
Semi-quantitative RT-PCR analysis has been performed, followed by
Southern blotting and detection with exon-specific probes, as described
by Stauder et al.27 Using HPRT controls, equal amounts of
cDNAs (Fig 6)have been analyzed per RT-PCR reaction, and the composition of CD44
isoforms has been determined for each sample.
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| Fig 6.
Analysis of CD44v exon composition in normal myeloid and
AML leukemic cells. RNA and cDNA were prepared as described in
Materials and Methods. Using primers for HPRT, the amounts of cDNA were equilibrated to this internal standard. A CD44-specific PCR was performed and the PCR products were blotted and hybridized with 33P-labeled exon-specific probes indicated at each blot.
Represented are the autoradiographs and the photos taken from the CD44
and HPRT-PCR reaction products as indicated. Negative controls () were performed by omitting the cDNA. (1) Normal myeloid cells: CD34+ cells, blot A, lanes 1, 2, 3, CD34
granulocytic precursors GP (blot A, lane 4), PMN (blot A, lane 5), and
monocytes (blot A, lanes 6 and 7). RT-PCR has been performed on five
distinct samples of PMN, monocytes, GP, and CD34+ cells.
Similar results have been obtained (data not shown). (2) Leukemic cells
from AML patients with the following FAB types: M1 (blot A, lanes 8 through 14), M2 (blot A, lanes 15 through 18), M3 (blot B, lanes 1 through 4), M4 (blot B, lanes 5 through 11) and M5 (blot B, lanes 12 through 16). The sample shown on blot B lane 17 is from a patient with
acute lymphoid leukemia. (3) Control experiment: Lymphocytes (lane 1) or
monocytes (lane 3) were isolated from a patient with a myeloblastic AML
(M1) and mixed in a ratio 5:95, with HL60 cells that express variant
exons only very weakly (CD44vneg/low). The presence of 5%
of lymphocytes from the AML patient did not result in the expression of
any detectable 6v signal (lane 2), and only a faint expression of
CD44-6v in the directly spliced version resulted from the presence
of 5% of monocytes from the AML patient (lane 4).
{/ANNT;4224n;;0n;0n}3
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Normal myeloid cells.
Exon 6v was expressed in CD34+ cells (Fig 6A, lanes 1 through 3), PMN (lane 5), and monocytes (lanes 6 and 7). Only
granulocytic precursors (GP) did not express this variant exon (lane
4). In CD34+ cells, 6v was found in a directly spliced
version (ie, the variant exon flanked directly by the framework of CD44
standard, size 196 bp). In these cells, 6v was also combined with other
variant exons, such as 7v to 10v (724 bp, lane 1), 10v (400 bp, lane
2), and 7v to 9v (520 bp, lane 3); a single exon 6v-containing
combination was observed in each CD34+ cell sample. In
contrast, 6v was contained in a great variety of high-molecular-weight
isoforms in each sample of PMNs (lane 5) and monocytes (lanes 6 and 7).
The large 6v-containing isoforms were most abundant in samples of PMN
and monocytes shown in lanes 5 and 6.
Exon 3v was expressed in the directly spliced version in
CD34+ cells and GP (size 193 bp, lanes 1 to 4). In
contrast, a great variety of high-molecular-weight transcripts
containing exon 3v in combination with exons 8v, 9v, and 10v were
abundantly produced in monocytes and PMN (lanes 5 to 7). In
CD34+ cells and GP, a few transcripts containing 8v + 9v
(262 bp, lanes 1 to 4) and 8v + 9v + 10v (466 bp, lanes 1, 3 and 4)
were also synthesized.
AML leukemic cells.
CD44-6v transcripts were found to be expressed in all AML patients
(except in one patient, who displayed only a weak CD44-6v protein
expression; Fig 6A, lane 14). In a few patients (18 of 70), CD44-6v
transcripts were only in the directly spliced version (size 196 bp), as
shown in Fig 6A, lanes 8 and 16, and Fig 6B, lanes 8 and 11. In half of
the AML patients (39 of 70), 6v transcript was also combined with other
CD44 variant exons in a variety of large-size transcripts, as shown in
Fig 6A, lanes 9, 10, 12, 15, and 18 and Fig 6B, lanes 1 to 3, 5 to 7, 9, 10, 13 through 16). These large transcripts combined in particular
6v + 7v (325 bp), 6v + 8v to 10v (595 bp), and 6v to 10v (724 bp). They
were very abundant in 15 of 70 patients (scored in semi-quantitative
score: ++ to ++++, Table 3).
Strong signals for 3v, 8v, and 10v transcripts were also obtained in
all AML patients, both in the directly spliced version (193 bp, 187 bp,
and 271 bp, respectively) and in multiple combinations with other
exons. For example, 3v was also found to be combined with 2v (322 bp),
and 10v was frequently associated in an 8v + 9v + 10v complex (466 bp).
Exon 9v was always associated with 8v (262 bp). 4v and 5v were rarely
expressed.
We asked whether the strong variant mRNA signals may be produced by
contaminating lymphocytes or monocytes, which could have been
incompletely removed by the immunoabsorption procedure. To answer this
question, semi-quantitative RT-PCR analysis was performed on a cell
mixture containing monocytes and lymphocytes from a patient with a
myeloblastic AML (M1 type), mixed in a ratio 5:95, with HL60 cells,
that express variant exons only very weakly (CD44vneg/low).
As shown in Fig 6C, the presence of 5% of lymphocytes from the AML
patient did not result in the expression of any detectable 6v signal
(lane 2), and the presence of 5% of monocytes from the AML patient
lead only to a faint expression of CD44-6v in the directly spliced
version (lane 4). This faint signal contrasted with the abundance of 6v
transcripts (both directly spliced and combined with other exons) in
the AML blast sample from this patient (Fig 6A, lane 12). This mixing
experiment indicates that the bulk of the 6v transcripts detected by
semi-quantitative RT-PCR analysis in AML blast samples were synthesized
by the leukemic cells themselves.
To summarize, large CD44 transcripts combining 3v, 6v, and 8v to 10v
are much more abundant in AML leukemic cells and in normal monocytes
than in CD34+ cells and in GP. However, contrasting with
the prognostic importance of 6v protein expression in AML, the
composition of 6v-containing transcripts did not correlate with the
survival rate of AML patients.
| |
DISCUSSION |
In this study we report on the expression of CD44 variant isoforms in
normal myeloid cells and in AML patients, and its correlation to
clinical data. Using antibodies to CD44-3v, -6v, and -9v, flow cytometry, and RT-PCR analysis of variant-specific transcripts, we have
shown that CD44-6v is expressed on 12% of normal CD34+ HPC
and on 50% of monocytic cells, whereas it could not be detected on
granulocytic cells. We also demonstrated that AML leukemic cells
display CD44-6v and, to a lesser extent, CD44-3v and -9v isoforms.
Furthermore, we give evidence that a strong expression of CD44-6v
correlates with a shorter survival of AML patients treated with
conventional chemotherapy.
Because of the high reproducibility of CD44-6v isoform expression
through more than seven experiments, it seems relevant to attest that
this variant isoform is displayed by about 12% of normal human
CD34+ cells (Fig 3A). Cell-sorting experiments indicate
that the CD34+ CD44-6v+ cell population is
enriched in progenitors committed to the monocytic lineage (Table 2).
In addition, CD44-6v is strongly expressed on monocytic precursors
(CD34neg CD14+ cells). This suggests that
monocytic differentiation may be associated with an upregulation of
CD44-6v expression, and that this isoform may be particularly involved
in monopoiesis. Inversely, CD34+ CD44-6v+ cells
are depleted from BFU-E, suggesting that CD44-6v is not much expressed
on erythroid progenitors. Finally, granulocytic committed progenitors
are found in equal number in CD34+CD44-6v+ and
CD34+CD44-6vneg cell populations. Their
differentiation into granulocytic precursors (CD34neg
CD15+ cells) is probably associated with a downregulation
of CD44-6v because granulocytic precursors do not display CD44-6v.
In PMN, 6v-containing transcripts are abundant, although CD44-6v
protein cannot be detected by FACS analysis. Activation of PMN during
the in vitro isolation procedure may lead to the synthesis of
6v-specific transcripts, but it may be too short for final protein
expression on the membrane. Arguing in favor of this hypothesis, we
have observed significant changes in the level of CD10 and CD11b
antigens, which have been shown to be surface markers for neutrophil
activation.41,42
In AML, CD44-6v is more frequently and more strongly expressed than
CD44-3v and CD44-9v, with 39 CD44-6v++ patients, versus 8 CD44-3v++ and 2 CD44-9v++ cases (Fig 4). In
contrast to this low frequency in 3v and 9v protein expression, a great
amount of 3v and 9v specific transcripts were detected in nearly all
AML leukemic cell samples (Fig 6). Similarly, 6v protein could not be
detected on the blasts from up to 26 AML patients, despite a
significant amount of 6v specific transcripts (Fig 6, except in one
case, 6A lane 14). As a hypothesis, the accessibility of variant
epitopes to the specific MoAbs may be prevented by a particular
conformation of the CD44v proteins, or by changes in the glycosylation
pattern of the molecules, due in particular to the insertion of variant
sequences.43 Using another MoAb to CD44-6v isoform, the
FW11-9,22 similar data have been obtained, indicating that
the present results are not restricted to the particular MoAb (VFF18)
used here.
The overall survival of the AML patients is clearly related to the
protein expression level of CD44-6v containing isoforms with a value of
8 months in the CD44-6v++ group compared with 18 months in
the CD44-6v+/neg group (P < .02, n = 48, Fig
5A). The level of this expression is unlikely to influence the
achievement of the first complete remission, since the rate of first
complete remission did not significantly differ in the
CD44-6v++ and CD44-6v+/neg groups of patients
(P = .33, see Results). On the contrary, the level of 6v
containing CD44 molecules may accelerate the relapse, because the
disease-free survival of patients achieving a first complete remission
was significantly shorter in the CD44-6v++ group than in
the CD44-6v+/neg group (Fig 5D). In this regard it would be
of interest to determine whether CD44-6v has a role in stimulating the
proliferation of residual leukemic cells.
Because CD44-6v may be differently involved in the evolution of the
different FAB types of AML, we have separately analyzed the influence
of its expression in M1/M2 (myeloblastic) and M4/M5 (myelomonocytic and
monoblastic) leukemic cells.
We have found that a strong CD44-6v protein expression is also
correlated with a shorter overall survival in M1/M2 (Fig 5B), suggesting a role for CD44-6v in the pathology of these AML.
Interestingly, CD44-6v expression is strikingly higher in myeloblastic
leukemic cells than in normal granulocytic precursors (Fig 4). This
suggests that CD44-6v is overexpressed in M1/M2 AML compared with the
normal cellular counterpart, and that this dysregulation in CD44-6v
gene expression may play a part in the pathophysiology of this type of
AML. The overexpression of 6v specific transcripts in M1/M2 blasts
compared with normal granulocytic precursors (Fig 6A) also supports the
hypothesis of a CD44-6v gene dysregulation in these AML.
Total CD44 is also overexpressed in M1/M2 AML compared with the normal
granulocytic precursors (Fig 2). However, its overexpression is not
correlated with the clinical evolution (P = .6, n = 48), underlining that the various isoforms of CD44 constitute a family of
molecules endowed with specific functions.2
No correlation between CD44-6v expression and overall survival has been
found in M4/M5 AML (Fig 5C). Because this analysis has been performed
on only 16 patients, and although the P value is very far from
the statistical significance (P = .48), further studies
dealing with more cases are necessary for better evaluation of the
involvement of CD44-6v in M4/M5 AML. It is remarkable that the CD44-6v
expression pattern is broadly similar in M5 leukemic cells and in
normal monocytic precursors that are their putative normal cell
counterparts. Both cell types strongly express CD44-6v containing
proteins (Figs 3B and 4), and high-molecular-weight 6v-containing
transcripts (Fig 6), indicating a comparable composition of CD44-6v
containing isoforms. A leukemic amplification of a CD44-6v+
subset of monoblastic cells may account for the very high frequency of
CD44-6v+ blasts in several M5 AML patients.
Although the clinical evolution of patients with promyelocytic AML (M3)
is generally favorable,44 the outcome of the eight M3 AML
patients from our cohort has not been better than that of other FAB
types. Indeed, four M3 patients rapidly died because of AML, and
complete remission has been achieved in only three of them (the
clinical evolution of one patient has not been documented; Table 1).
Accordingly, these M3 cases are likely to represent a selection of
poor-prognosis patients, all the more since most of them display more
than 10,000 WBC/µL, a feature associated with an unfavorable clinical
evolution in this type of AML. Because of the low number of M3 cases,
no survival analysis has been performed. However, it is noticeable that
4 of 5 CD44-6v++ cases died, whereas 2 CD44-6v+/neg patients are still in complete remission.
How CD44-6v overexpression participates in the leukemic process is
still elusive because the specific functions of CD44-6v containing
isoforms are unknown so far. CD44-6v overexpression may participate in
the release of BM leukemic cells into the circulation. Indeed, it is
noteworthy that the exon 6v-encoded peptidic sequence displays several
potential sites of glycosylations.2 Since glycosylation of
CD44 has been shown to negatively regulate its binding to
hyaluronan,45,46 an overexpression of CD44-6v containing isoforms may diminish the CD44-mediated adhesion of early myeloid cells
to hyaluronan6-8 and, thereby, to BM stroma. Interestingly, it is known that adhesion of immature myeloid cells to the BM stroma,
which involves CD44,4,5,47 is crucial for regulating their
proliferation and their differentiation along the granulocytic and the
monocytic lineages. Therefore, by decreasing the myeloid cell-stroma
interaction, overexpression of 6v-containing CD44 molecules may impair
stroma-dependent control of both proliferation (as suggested above) and
differentiation of myeloid cells, thus perhaps contributing to the
freezing of myelopoiesis in AML. Overexpression of CD44-6v isoforms may
also confer affinity to a specific ligand distinct from hyaluronan and
favor an extra-hematopoietic localization of the leukemic cells, as
observed in certain AML patients.36
In conclusion, this study is the first report that the protein
expression of CD44-6v containing isoforms on leukemic cells may
influence the survival of AML patients. Accordingly, it suggests that
CD44-6v may serve as a new prognostic factor in AML, and that analysis
of its expression may contribute to improve the management of new
therapeutical approaches aimed at AML patients with adverse prognostic
factors.48
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FOOTNOTES |
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Abstract
Introduction
Abstract