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NEOPLASIA
From the Department of Pathology, the Department of
Hematology/Oncology, and the Bone Marrow Transplant Program, The
Arizona Cancer Center, University of Arizona, Tucson, AZ.
Vascular endothelial growth factor (VEGF) is a potent angiogenic
peptide with biologic effects that include regulation of hematopoietic
stem cell development, extracellular matrix remodeling, and
inflammatory cytokine generation. To delineate the potential role of
VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and
receptor expression and its functional significance in MDS bone marrow
(BM) were evaluated. In BM clot sections from normal donors,
low-intensity cytoplasmic VEGF expression was detected infrequently in
isolated myeloid elements. However, monocytoid precursors in chronic
myelomonocytic leukemia (CMML) expressed VEGF in an intense
cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR
receptors, or both. In situ hybridization confirmed the presence of
VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia
(AML) and other MDS subtypes, intense co-expression of VEGF and one or
both receptors was detected in myeloblasts and immature myeloid
elements, whereas erythroid precursors and lymphoid cells lacked VEGF
and receptor expression. Foci of abnormal localized immature myeloid
precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting
autocrine cytokine interaction. Antibody neutralization of VEGF
inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML
and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony
formation in 12 patients. Neutralization of VEGF activity suppressed
the generation of tumor necrosis factor- The ineffective hematopoiesis and heightened risk
of leukemia transformation that distinguish the myelodysplastic
syndromes (MDS) derive from features inherent in the malignant clone
and its interaction with the micro-environment.1-4
Excessive production of growth-inhibitory cytokines such as tumor
necrosis factor (TNF)- Although cellular elaboration of angiogenic peptides represents an
important feature of the malignant phenotype in solid tumors and
multiple myeloma,9-11 the pattern of expression and
relevance in other hematologic malignancies remains undefined. Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with
diverse biologic activities that include the regulation of embryonic
stem cell development, extracellular matrix remodeling, and local
generation of inflammatory cytokines.12-19 VEGF exerts its
biologic effects by interaction with either of 2 high-affinity tyrosine
kinase receptors, the 160-kd c-fms-like tyrosine kinase (Flt-1 or
VEGFR-1) and the 180- to 210-kd fetal liver kinase-1 (KDR, Flk-1, or
VEGFR-2).20,21 Cellular expression of these VEGF receptors
is not restricted to proliferating endothelial cells but is also
demonstrable in macrophages, megakaryocytes, and primitive
hematopoietic stem cells.11,16,22,23 Indeed, homozygous
gene knockout studies have shown that expression of the KDR receptor is
essential for hematopoietic development.15 VEGF exerts
both growth-enhancing and suppressive effects on the in vitro formation
of hematopoietic progenitor that is lineage- and maturation-dependent.
Recombinant human (rhu-)VEGF promotes the expansion of
granulocyte-macrophage progenitors but inhibits the formation of
erythroid bursts and multipotent progenitors.24 Prolonged
systemic administration of rhu-VEGF in murine models results in intense
medullary hyperplasia of immature myeloid elements and arrest of
erythroid maturation.25
Recent investigations suggest that cellular expression of VEGF and
other angiogenic peptides may contribute to the pathobiology of
hematopoietic malignancies. We reported that hematopoietic cell lines
of diverse histogenic origin produce and secrete VEGF and commonly
co-express one or both VEGF receptors.11 Similarly, Fiedler et al26 reported the overexpression and secretion
of VEGF in 72% of acute myelogenous leukemia (AML) specimens and the
corresponding expression of Flt-1 or KDR gene message in 52% and 19%
of patients, respectively. Pruneri et al27 recently reported that bone marrow microvessel density is increased in MDS and
AML and that it directly correlates with myeloblast percentage. To
delineate the role of VEGF in MDS, we investigated the pattern of VEGF
and receptor expression in bone marrow clot sections and evaluated the
relation to cytokine elaboration and the effects of VEGF neutralization
and stimulation on leukemia and committed progenitor formation.
Blood and marrow specimens
Cell lines
Cytokines and reagents Recombinant human VEGF was purchased from R & D Systems (Minneapolis, MN). The recombinant human monoclonal VEGF-neutralizing antibody A.4.6.1,29 which recognizes all isoforms of human VEGF, was kindly provided by Dr Napeoleone Ferrara (Genentech, San Francisco, CA). Recombinant human erythropoietin was provided as a gift from Ortho Biotech (Raritan, NJ; manufactured by Amgen, Thousand Oaks, CA).Liquid suspension cultures Short-term suspension cultures were performed using 1 × 106 bone marrow MNC incubated in media consisting of IMDM and 10% FBS, in the presence or absence of either the VEGF neutralizing recombinant human antibody A.4.6.1 or rhu-VEGF (R & D Systems) at the concentrations indicated. Supernatants were harvested after a 24-hour incubation and cryopreserved at 20°C for subsequent
quantitation of cytokine concentration.
Enzyme-linked immunosorbent assays Cytokine concentrations in patient plasma and cell culture supernatants were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits for human interleukin-1 (IL-1; sensitivity, 0.125 pg/mL), tumor necrosis factor- (TNF-;
sensitivity, 0.5 pg/mL), VEGF (sensitivity, 31.2 pg/mL), and
granulocyte-macrophage colony stimulating factor (GM-CSF; sensitivity,
1.0 pg/mL). All kits were obtained from R & D Systems, and procedures
followed the manufacturer's protocol. Analyses were performed in
triplicate with calibrations performed in duplicate using recombinant
cytokine standards in the appropriate diluent. The color of the
chromogenic reaction was evaluated spectrophotometrically at 490 nm for
IL-1 and TNF- and at 450 nm for VEGF using a plate reader
(Biotech Laboratories, Laguna Hills, CA). The VEGF ELISA recognizes 2 secreted isoforms of VEGF (121 kd and 165 kd), but it lacks reactivity with the bound forms of the cytokine (189 kd and 206 kd).
Progenitor and leukemia colony-forming assays The blast colony-forming capacity of leukemia progenitors (CFU-L) was evaluated using a modification of methods previously described.30 Briefly, 1 × 105 patient BM-MNCs were plated in 0.2 mL methylcellulose (0.8%) and 10% FBS with IMDM in 96-well microtiter. Microwells were plated in triplicate in the presence or absence of varied concentrations of the A.4.6.1 antibody or rhu-VEGF and incubated in a moist atmosphere with 5% CO2. Aggregates of more than 20 cells were counted with an inverted microscope after a 7-day culture, and colony recovery was compared to growth in control plates. CFU-L assays using KG1 cells were initiated at a cell density of 5 × 105 cells (n = 8). Additional aliquots of BM-MNC (2 × 105/mL) were plated in cytokine-defined medium containing 30% FBS, 3 U/mL erythropoietin, 100 U/mL GM-CSF, 100 U/mL IL-3, 5 ng/mL stem cell factor (R & D Systems), and various concentrations of the A.4.6.1 antibody or rhu-VEGF for 14 days. In addition, the growth of bone marrow colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM); burst-forming unit-erythroid (BFU-E); and colony-forming unit granulocyte-macrophage (CFU-GM) was assessed. As previously described,31 hematopoietic colonies (more than 40 cells/colony) and clusters (3-40 cells) were scored after 14 days' incubation using an inverted microscope, and results were expressed as mean colony number per 1 × 105 plated and percentage of colony growth in control cultures.Immunohistochemistry Immunohistochemical analysis was carried out on formalin-fixed, paraffin-embedded bone marrow clot sections as previously described.32 Slides were stained for expression of VEGF, Flt-1/VEGFR-1 (Santa Cruz Biotechnology, Santa Cruz, CA) and KDR/VEGFR-2 (Santa Cruz Biotechnology; Sigma). The anti-VEGF antibody used in these studies has been demonstrated to be specific for VEGF and does not cross-react with other known VEGF/placental growth factor family members. Its specificity has been established through the use of VEGF-specific blocking peptides. Both the KDR and Flt-1 antibodies are also specific and do not cross-react with each other or with other protein tyrosine kinase membrane receptors. All reactions were performed using an automated immunostainer (GenII; Ventana Medical Systems, Tucson, AZ). Detection of bound antibody was assessed through the use of immunoperoxidase methodologies with diaminobenzidine serving as the color substrate or by alkaline phosphatase methodologies using a biotinylated goat-anti-rabbit antibody (DAKO-Patts, Santa Barbara, CA) in conjunction with alkaline phosphatase-conjugated streptavidin followed by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as the color substrate. The Ventana Medical Systems antibody diluent was used as a negative control. Nuclei were counterstained with methyl green or hematoxylin, and sections were evaluated by light microscopy. Endogenous peroxidase was inhibited with methyl alcohol containing 0.01% H2O2.The degree of expression in tumor cells was judged at 400 × magnification as 4+ (very intensely positive), 3+ (moderately intensely positive), 2+ (moderate), 1+ (faint), or 0 (completely negative) throughout the sample. Samples were judged by 2 independent reviewers. In situ hybridization Formalin-fixed, paraffin-embedded BM clots or cores were sectioned 3 µm thick and placed on glass slides with a "sausage" control section containing human placenta, liver, spleen, colon, and pancreas. Slides were baked for 1 hour at 60°C and then deparaffinized in 2 changes of xylene for 10 minutes each and 2 changes of 100% ethanol for 2 minutes each, followed by a graded series of alcohols (95%, 80%, 70%) and, finally, 2 changes of diethylpyrocarbamate-treated water. They were then placed in APK wash (Ventana Medical Systems). All further steps were carried out using an automated in situ hybridization instrument (GenII; Ventana Medical Systems). The details of this procedure have been previously published.32-34 A 24-mer VEGF-specific oligonucleotide probe was designed based on published sequences and was synthesized with 6 biotins at the 3' end (Research Genetics, Huntsville, AL). Before addition to the probe, slides were treated with Protease 1 (Ventana Medical Systems) for 4 minutes. Two hundred microliters of probe diluted to a concentration of 1 ng/µL in a hybridization solution composed of 35% formamide, 5 × Denhardt, 10% dextran sulfate, 100 µg/mL salmon sperm DNA, and 4 × SSC was manually added to each slide. For a negative control, the hybridization solution alone was applied. The slides were then denatured at 65°C for 4 minutes, followed by hybridization at 45°C for 60 minutes. After hybridization, 3 stringency washes 1 × SSC, 0.5 × SSC, and 0.1 ×
SSCwere performed for 4 minutes each at 50°C to remove unbound
probe. Detection was carried out at 40°C by incubating the slides in
streptavidin-alkaline phosphatase for 1 hour (Boehringer-Mannheim
Biochemicals, Indianapolis, IN) followed by overnight incubation in
NBT/BCIP substrate. The slides were counterstained off the instrument
with contrast red (Kierkegaard & Perry Laboratories, Gaithersburg, MD).
Hybridization with a d(T)30 oligonucleotide probe confirmed
the integrity of the mRNA in each sample. To ensure that the
oligonucleotide probe recognized mRNA and not genomic DNA, a subset of
slides was incubated in either DNAse or RNAse before the addition of
the probe. The degree of expression was assessed in a manner identical
to that for the immunohistochemistry samples.
Statistical analysis Statistical analyses were performed using the Student t test (2-tailed for equal variances). P < .05 was considered significant.
Detection of VEGF and VEGF-receptor expression Cellular expression of VEGF and its receptors Flt-1/VEGFR-1 and KDR/VEGFR-2 were evaluated by immunohistochemical staining in bone marrow clot sections from 46 patients with MDS and 17 patients with relapsed (n = 7) or secondary (n = 10) AML. Results were compared to the pattern of protein expression in normal bone marrow specimens (n = 9). Morphologic subtypes of MDS classified according to the French-American British (FAB) criteria35 and the corresponding frequencies of protein detection are summarized in Table 1. In normal bone marrow, the detection of VEGF expression in hematopoietic elements was uncommon (Figure 1). Using immunohistochemistry, VEGF protein expression was not observed in erythroblasts, lymphocytes, or plasma cells. A faint Flt-1 signal was observed in monocytes-histiocytes and in rare myeloid elements, whereas a strong KDR signal was observed in scattered histiocytes.
VEGF expression in MDS and AML specimens was detected in a diffuse
cytoplasmic pattern in myeloid and monocyte precursors with varied
signal intensity (Table 1). In bone marrow specimens from 8 of 10 patients with chronic myelomonocytic leukemia (CMML), cytoplasmic
expression of VEGF, ranging from faint (+1) to intensely positive (+4),
was detected in myelomonocytic cells, whereas no signal was detected in
erythroblasts and plasma cells (Figure 2;
Tables 2 and 3).
Concordant membranous expression of the Flt-1/VEGFR-1 receptor
was observed in monocyte and myeloid precursors in all CMML specimens
(Table 1). Using 2 separate antibodies, a similar pattern of cellular
expression, albeit of lower intensity, was detected for the KDR/VEGFR-2
receptor in 33% (3 of 10) of the CMML specimens examined. To confirm
that the high-intensity VEGF protein signal detected in CMML resulted
from overexpression of the VEGF gene transcript, VEGF mRNA expression
was assessed by in situ hybridization using a VEGF-specific
oligonucleotide probe (Figure 3). Indeed,
we found that the cellular expression of VEGF mRNA was restricted to
neoplastic myelomonocytic cells in a pattern analogous to that observed
for the VEGF protein.
A similar pattern of cellular expression was detected in morphologic
subtypes of MDS other than CMML and in AML specimens. Of particular
importance, foci of myeloid precursors clustered in the central marrow
space, abnormal localized immature precursors (ALIP), consistently
displayed intense expression of VEGF, myeloperoxidase, and the
Flt-1/VEGFR-1 receptor (Figure 4).
Overall, the expression of VEGF was observed in myeloid and monocyte
precursors from 76% (35 of 46) of patients with MDS and in myeloblasts
from 82% (14 of 17) of patients with AMLs. Co-expression of the Flt-1
receptor was demonstrated in 69% (32 of 46) of patients with MDS and
76% (13 of 17) of patients with AML. Membranous expression of the KDR
receptor in blasts and myelomonocytic precursors was detected in 19%
(9 of 46) of patients with MDS, but it was not found in any of the
patients with AML. If KDR was detected in patients with MDS, it was
always found in conjunction with Flt-1 receptor expression. Neither the
intensity of cellular VEGF expression nor the distribution or frequency
of receptor expression varied among relapsed or secondary forms of AML.
Leukemia progenitor formation To determine whether VEGF serves as a trophic stimulant of leukemia progenitor self-renewal in MDS, we evaluated the effects of VEGF neutralization and stimulation on leukemia colony formation (CFU-L) in bone marrow specimens from a group of 15 patients with CMML (n = 4) or RAEB-t (n = 11) with which there was sufficient viably frozen material to perform functional assays. BM-MNCs were plated in cytokine-deficient medium with and without 0.1 to 50 µg/mL of the A.4.6.1 antibody or 1 to 100 ng/mL rhu-VEGF, and leukemia colony formation was assessed after a 7-day incubation. Antibody neutralization of VEGF activity inhibited CFU-L formation in a concentration-dependent fashion in 47% (7 of 15) of the patient specimens, with the magnitude of inhibition ranging from 20% to 100% compared to control cultures (mean, 68% inhibition). Incubation with rhu-VEGF yielded concentration-dependent stimulation of CFU-L formation in 8 patients, including each of the antibody-inhibitable specimens, with maximal stimulation ranging from 1.5-fold to 5.6-fold of control colony growth (mean, 2.44-fold). No significant changes were observed in myeloid cluster growth in response to VEGF neutralization or stimulation. Table 2 illustrates maximal changes in mean leukemia colony number according to culture condition in individual patient specimens.Immunocytochemical stains of cytospin preparations of the KG1 AML cell
line showed that these cells co-express VEGF protein and the
Flt-1/VEGFR-1 and KDR/VEGFR-2 receptors. To determine whether VEGF
receptor ligation triggers a proliferative growth response in a
purified leukemia cell population, CFU-L formation was assessed in
methylcellulose cultures containing 10% FBS after a 24-hour incubation
of KG-1 cells with and without rhu-VEGF supplementation of 50 µg/mL
or IL-1 100 U/mL. Cultures were grown in quadruplicate and were regrown
on 3 separate occasions. VEGF exposure before culture initiation
stimulated leukemia colony formation 2.4-fold compared to control
cultures ELISA quantitation of VEGF, TNF- , and IL-1 by ELISA in specimens from patients with MDS
(n = 24) were evaluated, and the results were compared to those from
a group of healthy allogeneic BM donors (n = 15). Mean plasma
concentrations of IL-1 (3.42 ± 2.94 vs 1.61 ± 0.79 pg/mL;
P = .013) and TNF- (3.14 ± 2.22 vs 1.35 ± 0.83
pg/mL; P = .004) were significantly higher in MDS BM
plasma than in the plasma of healthy donors. However, concentrations of
immunoreactive VEGF were significantly lower (13.8 ± 21.6 vs
58.6 ± 9.7 pg/mL; P < .001) in MDS BM plasma than in
the plasma of healthy allogeneic BM donors, and they were often below
the limits of assay detection. To determine whether the lower
concentrations of VEGF detected in MDS BM plasma reflected excessive
local receptor binding of the peptide or impaired secretion by myeloid
precursors, we evaluated VEGF elaboration from BM-MNC in 24-hour
suspension cultures. Among the 12 MDS patients studied, we demonstrated
elaboration of VEGF from BM-MNC, with corresponding cytokine
concentrations in 24-hour culture supernatants ranging from 7.5 to 34 pg/mL. Mean VEGF concentrations in culture supernatants increased with
BM myeloblast percentage (RA/RARS, 9.2 ± 7.5 pg/mL; RAEB/RAEB-t,
17.4 ± 21.3 pg/mL) and monocyte count (CMML, 22.3 ± 2.8
pg/mL).
To determine whether VEGF elaboration from BM precursors impacted
local generations of TNF-
Effect of VEGF neutralization on committed progenitor formation To determine whether neutralization of VEGF activity improved the formation of committed hematopoietic progenitors in MDS, BM-MNC from 13 patients (RA/RARS, 6; RAEB, 3; RAEB-t, 4) and 2 healthy donors were plated in cytokine-defined medium with and without 0.1 to 50 µg/mL A.4.6.1 antibody, and colony formation was scored after a 14-day incubation. Antibody neutralization of VEGF improved the mean recovery of CFU-GEMM approximately 3-fold (range, 1.3- to 7.5-fold) in 38% (5 of 13) of patient samples, whereas BFU-E was increased 3.7-fold in 46% (6 of 13) of samples (range, 1.5- to 10.7-fold). There was no consistent improvement in mean CFU-GM colony recovery (1.17-fold; range, 0.9- to 1.9-fold) (Table 3). Significant changes in progenitor recovery were not observed with VEGF neutralization in bone marrows from normal donors.
Although it is well established that the growth of solid tumors is
dependent on the formation of neovasculature, the role of angiogenic
factors in hematopoietic malignancies has only recently been
investigated. Fiedler et al26 demonstrated the expression of VEGF gene message in 72% of AML specimens; Aguayo et
al36 reported that myeloblast VEGF protein content is an
independent prognostic variable inversely correlated with disease-free
and overall survival. Pruneri et al27 and Hussong et
al37 described increased microvessel density in bone marrow
trephine biopsies from patients with AML and MDS, the magnitude of
which directly correlated with myeloblast percentage. The current
studies demonstrate that cellular expression and elaboration of VEGF by
malignant monocytes and myeloblasts in MDS and AML may have potent
biologic effects that contribute to both leukemia progenitor
self-renewal and ineffective hematopoiesis in these diseases. Cellular
expression of VEGF was restricted to monocyte and myeloid precursors,
and was associated with membranous co-expression of either the
Flt-1/VEGFR-1 or the KDR/VEGFR-2 receptor, or both, in 69% (32 of 46)
and 19% (9 of 46) of patients with MDS, respectively (Table 1). Our
findings of VEGF and FLT-1/VEGFR-1 expression in AML are consistent
with the molecular studies of Fiedler et al.26 Although
KDR/VEGFR-2 was detected infrequently by these investigators, we did
not detect its expression in a group of 17 patients with AML. Possible
explanations for this discrepancy may lie in the patient selection or
in the method for detection. Fiedler et al26 used RT-PCR to
detect KDR/VEGFR-2 messages whereas we used immunohistochemistry to
examine the protein. Although solution-phase RT-PCR is an extremely
sensitive technique, it lacks the specificity to distinguish expression in individual cells. It is possible that contamination by endothelial cells or macrophages may have contributed to the KDR signal in the
study by Fiedler et al.26 Although we did not use antigen retrieval methodologies for immunohistochemical detection of VEGF and
its receptors, 2 MDS cases that were negative by IHC demonstrated a
significant inhibition of CFU-L and a decrease in both TNF- Antibody neutralization of VEGF activity suppressed leukemia progenitor formation in 46% of patients, whereas rhu-VEGF promoted the growth of leukemia colonies in 56% of CMML and RAEB-t patient specimens. These findings suggest that VEGF serves as a trophic factor that supports leukemia progenitor self-renewal by either autocrine interaction or paracrine induction of myeloid growth factors from stromal elements. Indeed, rhu-VEGF stimulated the in vitro growth of KG1 AML cells, indicating that VEGF has direct trophic effects in receptor-competent leukemias. We cannot exclude indirect paracrine stimulation of leukemic cells by myeloid growth factors elaborated by VEGF-receptor competent cells in the patient specimens studied. We11 and others26 previously reported that VEGF is a potent stimulant of M-CSF, G-CSF, IL-6, stem cell factor, and GM-CSF production from human umbilical cord endothelial cells. Indeed, our findings of VEGF stimulation of GM-CSF elaboration from bone marrow stroma and the clonogenic response observed in selected VEGF-receptor-naive cases support this notion (Table 2). These findings indicate that the production of VEGF by malignant myeloid precursors serves as both an autocrine growth stimulus and a diffusible paracrine signal mediating the local generation of growth factors that foster leukemia survival and self-renewal. Additionally, these data may explain the adverse prognostic relevance of myeloblast VEGF content in AML described by Aguayo et al.36 Although plasma concentrations of VEGF are elevated in patients with
solid tumors, bone marrow plasma concentrations from MDS patients were
significantly lower than corresponding levels from normal allogeneic
donors, whereas corresponding concentrations of TNF- Cellular expression of VEGF and its receptors in normal marrow was
detected in megakaryocytes and tissue macrophages, but rarely in
myeloid cells. Tordjman et al43 recently described the
expression of VEGF165 mRNA in the more primitive cell
population of CD34+/CD38 Investigations by Broxmeyer et al24 have shown that
rhu-VEGF exerts growth-enhancing and -suppressive effects on in vitro colony formation of hematopoietic progenitors mediated by direct cellular interaction and indirect effects mediated by other molecules. VEGF stimulated the proliferation of mature subsets of CFU-GM but
inhibited formation of the more primitive progenitors, CFU-GEMM and
BFU-E. In single-cell assays, VEGF had no effect on primitive progenitor formation from CD34+ cells but retained
enhancing effects on myeloid progenitors. In our studies,
neutralization of VEGF activity with the A.4.6.1 antibody suppressed
the generation of TNF- Perhaps the most compelling observation from these investigations is the uniform co-expression of VEGF and one or both of its receptors by myeloblasts clustered in the central marrow space, ALIP.48 This pattern of dislocation of myeloid precursors from a para-trabecular locale represents an adverse prognostic feature in MDS associated with imminent risk for leukemia transformation. Our findings suggest that such foci of myeloblasts emerge as a result of autocrine or paracrine VEGF stimulation, or both, supplanting physiologic sources of cytokine production. Alternatively, such foci may represent sites of localization of proliferating myeloblasts adjacent to medullary neovasculature. Autocrine VEGF stimulation may promote the homotypic adhesion of myeloid precursors through the up-regulation or activation of cell adhesion molecules, analogous to its effects on endothelial cell targets.49 Additional studies are warranted to delineate the relation between ALIP-bone marrow microvessel distribution and integrin expression-activation status. Nevertheless, these observations suggest that VEGF is a potentially important peptide signal reinforcing leukemia cell survival and contributing to ineffective hematopoiesis in MDS. These studies provide a biologic rationale for the clinical investigation of anti-angiogenic agents in patients with MDS.
Submitted April 6, 2000; accepted November 9, 2000.
Supported in part by National Cancer Institute grant CA-32102, National Institute of Environmental Health Sciences grant ESO6694, and the Jeffrey Anderson Memorial Leukemia Research Fund.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William T. Bellamy, Department of Pathology, University of Arizona, 1501 N Campbell Avenue, Tucson, AZ 85724; e-mail: wbellamy{at}u.arizona.edu.
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A. F. List, K. J. Kopecky, C. L. Willman, D. R. Head, D. L. Persons, M. L. Slovak, R. Dorr, C. Karanes, H. E. Hynes, J. H. Doroshow, et al. Benefit of cyclosporine modulation of drug resistance in patients with poor-risk acute myeloid leukemia: a Southwest Oncology Group study Blood, December 1, 2001; 98(12): 3212 - 3220. [Abstract] [Full Text] [PDF]
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A. Raza, P. Meyer, D. Dutt, F. Zorat, L. Lisak, F. Nascimben, M. du Randt, C. Kaspar, C. Goldberg, J. Loew, et al. Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes Blood, August 15, 2001; 98(4): 958 - 965. [Abstract] [Full Text] [PDF]
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M. H. Mangi, W. T. Bellamy, T. M. Grogan, and A. F. List Misleading information about ALIP and VEGF in myelodysplasia Blood, August 15, 2001; 98(4): 1272 - 1273. [Full Text] [PDF]
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R. J. Klasa, A. F. List, and B. D. Cheson Rational Approaches to Design of Therapeutics Targeting Molecular Markers Hematology, January 1, 2001; 2001(1): 443 - 462. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
.05).
hematopoietic
progenitors. Although primitive stem cells capable of hematopoietic and
endothelial cell differentiation express the KDR/VEGFR-2
receptor,