Expression of vascular endothelial growth factor and its receptors in multiple myeloma and other hematopoietic malignancies

Expression of Vascular Endothelial Growth Factor Receptors in Multiple Myeloma and Other Hematopoietic Malignancies William Vascular angiogenic

endothelial peptide

regulation

of

growth factor with biologic

hematopoietic

extracellular matrix tokine generation.

stem

remodeling, The importance

tors such as VEGF, while tumors, has not been fully topoietic neoplasms. lines, representing

receptors. malignant

VEGF plasma

tients studied. receptors was

pression

(IL-6).

revealed that in an inhibition

detected

hein

marrow

multiple myof VEGF and its

(IL-l data

and KDR plasma

sections from normal cytoplasmic VEGF ex-

infrequently

of tumor

in pa-

in the normal marsurrounding the tu-

in isolated

and megakaryocytes. using patient-derived

of recombinant human VEGF tion. Neutralization of VEGF generation

cell pro-

of several be involved Bone

with

elevated cells

antibody neutralization of colony growth,

and interleukin-lfi nuclear cells. These

solid hema-

protein production was detected cells from 78% of the myeloma

bone marrow clot donors, low-intensity was

in

tumor diseases,

While expression of the Flt-I not observed in the malignant

cytes, macrophages, ony-forming assays

the

expression known to

diagnosed for expression

cells, both were markedly row myeloid and monocytic mor. In allogeneic

cyfac-

and express at least one of its of human vascular endothelial

interleukin-6

samples from patients eloma were examined

development,

inflammatory of angiogenic

hematopoietic lineages and

cells to VEGF increased the matopoietic growth factors including

cell and

is a potent that include

clearly established elucidated in human

Human multiple

duce and secrete VEGF two receptors. Exposure

myeloma

(VEGF) effects

of VEGF whereas the

stimulated activity

necrosis

myelo-

In vitro colmaterial resulted addition

colony formaalso suppressed

factor-alpha

(TNF-cr)

/3) from bone marrow monoraise the possibility that VEGF

may play a role in the plasms such as multiple

growth of hematopoietic neomyeloma through paracrine

and perhaps Semin Oncol

mechanisms. Copyright

Saunders

autocrine 28551459.

0

2001

by

W6.

Company.

M

ULTIPLE MYELOMA is a B-cell neoplasia in which there is an accumulation of terminally differentiated B cells (plasma cells) primarily in the bone marrow. In most patients the disease follows a course in which the plasma cells are predominantly in a nonproliferative phase in the bone marrow. This may be followed by an active phase in which a small percentage of proliferating tumor cells (usually 6%) may be detected among the predominantly nonproliferating plasma cells. Finally, there is a fulminant phase with an increase in the proliferating plasmablastic compartment Seminars in Oncology, Vol 28, No 6 (December),

200 I : pp 55 I-559

and Its

T. Bellamy

and extramedullary disease.lz2 In the United States, multiple myeloma will be diagnosed in an estimated 14,400 people in the year 2001 and will account for approximately 11,200 cancer deaths, or approximately 18% of all cancer deaths from hematopoietic malignancies.3 Myeloma demonstrates a progressive, usually fatal, course with current treatments generally producing only temporary remissions.+ The 5year survival for patients with myeloma is 25%, with less than 5% alive after 10 years.4 Traditional therapy has centered on various chemotherapy regimens such as melphalan-prednisone and, more recently, vincristinedoxorubicin-dexamethasone (VAD). While such regimens have produced objective responses in untreated patients, their overall efficacy has been limited by the emergence of drug resistance. Antiangiogenic therapies represent a potential new approach to treating cancer. Tumors require neovascularization to grow beyond a few millimeters and antiangiogenic agents function to inhibit tumor growth by starving tumor cells of vital nutrients, as well as potentially preventing hematogenous spread.5 While it is well established that the growth of solid tumors is dependent on neovascularization, the role of angiogenic factors in hematopoietic malignancies has only recently been studied. Investigations in our laboratory and elsewhere indicate that expression of angiogenic factors may contribute to the pathobiology of hematopoietic malignanciese-is Vacca et al were the first to report that patients with multiple myeloma had an increased bone marrow microvascular density as compared to patients with monoclonal gammopathy of unknown significance (MGUS) and

From the Department of Pathology, University of Arkma, Tucson, AZ. Supported in part by funding from the Multiple Myeloma ResearchFoundation. Address reprint requeststo William. T. Bellamy, PhD, Depmtmerit of Pathology, University of Arizona, 1.501N Campbell Awe, Tucson, AZ 85724. Copyright 0 200 1 by W. B Saunders Company 0093-7754/01/2806-0009$35.00/O doi:10.1053/sonc.200i.28606 551

552

WILLIAM

normal bone marrow controls.6 Similar findings have also been reported in B-cell non-Hodgkin’s lymphoma (NHL), myelodysplasia syndrome (MDS), and acute lymphocytic and myelogenous leukemias,s-l5 suggesting the angiogenic process is activated in these malignancies. Pruneri et all4 reported that bone marrow microvessel density was increased in patients with MDS and acute myelogenous leukemia (AML), and was directly correlated with myeloblast percentage. Fiedler et al” reported overexpression and secretion of VEGF in 72% of AML specimens, and corresponding expression of the receptors Fit-1 or KDR in 52% and 19% of cases, respectively. Adding support to the hypothesis that angiogenic factors are playing a role in these tumors are the recent reports that thalidomide has been demonstrated to be effective in patients with refractory myeloma.16J7 Although the mechanism for this effect has yet to be established, thalidomide has been demonstrated to possess antiangiogenic activity.i8 VEGF

AND

ITS RECEPTORS

While tumor angiogenesis is influenced by a number of regulatory factors, one key cytokine in this process is vascular endothelial growth factor. There are currently six members of the VEGF family: VEGF-A through VEGF-E, as well as placental growth factor (Fig 1). Most of the studies related to the angiogenic activity of these molecules have centered on VEGF-A, hereafter referred to simply as VEGF. Several excellent re-

A

I

B

C

D

I

I

VEGFRI

VEGFR2

(FM)

WW

NP-I

E

I

I

VEGFRB

Intracellular

(F&4)

Fig I. VEGF family members and their receptors. There are currently 6 members of the VEGF family, VEGF-A through VEGF-E, shown above, as well as placental growth factor (not shown). VEGF-A specifically binds 2 high-affinity TK receptors, Flt-INEGFRI NP-I. VEGF-A

and KDRIVEGFRZ, is considered to

as well as a third be the predominant

receptor, member

of the VEGF family in its ability to stimulate angiogenesis. VEGF-C functions primarily to stimulate endothelial cells in lymphatics via the Flt-4NEGFR3 receptor but may also stimulate angiogenesis through its interaction with KDR/VEGFRZ.

T. BELLAMY

views of the VEGF family have been recently published and the reader is referred to these for more details.lg-z2 VEGF is a potent angiogenic peptide with diverse biological activities that include regulation of embryonic stem cell development, extracellular matrix remodeling, and the local generation of inflammatory cytokines.19 At least five different transcripts of the VEGFeA gene have been reported. In human cell lines these transcripts encode polypeptides of 121, 145, 165, 189, and 206 amino acids.19 Three of these-the 121, 145, and 165 isoforms-are secreted from the cell, while the 189 and 206 isoforms remain associated with the cell surface through heparin-binding domains. The secreted VEGF,,, isoform has been the predominant transcript identified in most systems. Each of these isoforms are generated by differential splicing of mRNA derived from a single gene containing eight exons,19 and while they have unique physical properties, all are mitogenic to vascular endothelial cells and induce vascular permeabilization.19 VEGF exerts its effects through an interaction with either of two high-affinity tyrosine kinase (TK) receptors, the 180skd c-+-like TK (Flt-l/ VEGFR-1) and the 200-kd fetal liver kinase-1 receptor (KDR/Flk-l/VEGFR-2).z3 Cellular expression of VEGF receptors is not restricted to proliferating endothelial cells, but is also demonstrable in macrophages, megakaryocytes, and primitive hematopoietic stem cells.iiJ4 The specific roles for these two VEGF receptors are not entirely understood. While the KDR/VEGFR2 receptor is generally believed responsible for endothelial cell proliferation, the function of Flt-l/VEGFRl is less clear. The essential requirement of both receptors however, has been definitively established by the lethal nature of either KDRjVEGFR2 or Flt-l/ VEGFRl gene knockouts.zsJ6 A third VEGF receptor has been recently described. Neuropilin1 (NP-1) is an isoform-specific VEGF receptor, binding only VEGF,,,, and serving to enhance KDR-mediated chemotaxis.27 Although the responsible VEGF 7receptor subtype has not been conclusively established, VEGF stimulation of endothelial cells results in the release of various cytokines, including those capable of maintaining hematopoietic tumor growth.11 Thus, even those cells lacking VEGF receptors may nevertheless be affected by angiogenic factors such as VEGF indie rectly through paracrine pathways.

EXPRESSION

OF VEGF

VEGF

553

IN MYELOMA

AS A SURVIVAL

FACTOR

VEGF is a potent mitogenic stimulator of endothelial cells, but it may also exert effects on cells in a manner unrelated to its angiogenic activity. An inverse relationship exists between apoptosis and angiogenesis.28,29 Several studies have demonstrated the ability of VEGF to function as a survival factor for endothelial cells.sOJi Immature blood vessels, such as those present in tumors with active angiogenesis, are dependent on the presence of VEGF, undergoing regression via apoptosis following its removal. 3o Maintenance of vessel integrity may be due to the ability of VEGF to increase the expression of Bcl-2 and other antiapoptotic genes, a mechanism with clear implications in hematopoietic tumors.31-33 In support of such findings, Katoh et al have demonstrated that VEGF reduces apoptotic cell death of normal hematopoietic stem cells and tumor cell lines following exposure to ionizing radiation or chemotherapeutic agentsj2J3 in part, by inducing the expression of the anti-apoptotic gene, Mel-1. Thus, it is possible that VEGF, in addition to its role in stimulating endothelial cells, may play a role in survival or maintenance of hematopoietic stem cells by interfering with apoptotic cell death. Although it is presently unclear what fraction of neovascular growth can be attributed to VEGF as opposed to other polypeptides with angiogenic activity, a central role of VEGF in tumor growth in vivo was demonstrated by Kim et al, who showed that the addition of neutralizing antibodies to VEGF resulted in a marked suppression of tumor growth.34 Levels of VEGF mRNA and protein expression in human tumors often correlate positively with malignant progressiorPJ6 and our own studies have demonstrated a functional role for VEGF in the growth of hematopoietic tumor cells in vitro.11J5 PARACRINE

EFFECTS

OF VEGF

Paracrine control mechanisms are increasingly recognized as being important for tumor growth. While the requirement of tumor-stromal interactions is well established in multiple myeloma, the involvement of angiogenic factors, which act pri+ manly on the stromal elements, is not fully appreciated. In multiple myeloma, the stromal compartment is involved in interactions between the malignant plasma cells and the microenvironment

of the bone marrow by means of cell-cell contact, adhesion molecules, and the release of cytokines?J* In normal bone marrow as well as that of myeloma patients, endothelial cells are found in close contact with hematopoietic cells and have been demonstrated to produce interleukin-6 (IL6), a key growth factor in multiple myeloma.37 IL-6 is well established as a paracrine growth factor in myeloma, both in vitro and in vivo.1,38-40 IL-6 was initially believed to be an autocrine growth factor but is now generally believed to act through a paracrine mechanism in all but a few myeloma cell lines. In normal bone marrow as well as in myeloma patients, the stromal cells appear to be the major producers of cytokines including IL6.39,40 Work performed in our laboratory has demonstrated that exposure of microvascular endothelial cells to VEGF results in increased expression of several growth factor cytokines, including IL-6.11 In support of our earlier findings, Dankbar et al observed that both the 121 and 165 isoforms of VEGF induced both a time- and dose-dependent increase in IL-6 production from microvascular endothelial cells.4l Such findings reveal that VEGF can increase the expression of growth factors with known stimulatory effects in multiple myeloma. One of the functions of IL-6 is to act as a survival factor for myeloma cells by preventing tumor cell apoptosis triggered by dexamethasone, serum starvation, and Fas.42-44 Thus, it is conceivable that endothelial cells may, in response to angiogenic factors such as VEGF, release cytokines capable of sustaining tumor growth. The increase in hematopoietic growth factor mRNA and protein in response to VEGF stimulation thus supports the possibility that VEGF may stimulate the growth of myeloma cells through paracrine mechanisms. The potential effects of VEGF in myeloma are not limited to the neoplastic plasma cell alone. Bone disease in multiple myeloma is the most common presenting clinical symptom, resulting in pathologic fractures and bone pain.lz2 This serious complication is due in part to increased bone resorption secondary to the activation of osteoclasts.q5 Numerous osteoclast activating factors including tumor necrosis factor (TNF), IL-l and IL-6 are released by myeloma cells directly or by the normal marrow cells in response to the myeloma ce11s.4°+49 Several studies have demonstrated that VEGF can directly or indirectly,

WILLIAM

Fig 2.

Potential

T. BELLAMY

roles

ofVEGF

in multiple my&ma. Malignant plasma cells produce VEGF, which stimulates the bone marrow stromal cells to produce growth and survival factors such as IL-6, providing a paracrine mechanism of tumor growth. Increased VEGF levels provide a stimulus to increase bone marrow vascular density via activating angiogenesis. VEGF also stimulates activation of osteoclasts and inhibits maturation of dendritic cells, potentially affecting lytic bone lesions in myeloma, as well as the body’s immune response (see text).

through its stimulatory actions on TNF-a and IL-l@, stimulate the activation of osteoclasts thus contributing to the lytic lesions in myeloma.50-52 There has been considerable interest in immunologic approaches to treating myeloma, particularly the use of dendritic cells in vaccine-based therapies or adoptive T-cell-based approaches. Gabrilovich et al have reported that VEGF inhibits the maturation of dendritic cells, thus potentially diminishing the efficacy of such approaches.53,54 VEGF, in addition to its potent angiogenic activity, thus has the potential to impact several key areas related to myeloma growth and progression through paracrine-based mechanisms (Fig 2). EXPRESSION HEMATOPOIETIC Expression

OF VEGF IN HUMAN TUMORS

in Hematopoietic Tumor Cell Lines

Increasingly, VEGF expression is being found in a variety of hematopoietic neoplasms. We undertook studies to examine the expression of angiogenie growth factors and their receptors in a series of human hematopoietic tumor cell lines representing multiple cell lineages. Our findings revealed the expression of VEGF, basic fibroblast growth factor (bFGF), or both by all of the cell lines in our panel.11 Reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blot analyses revealed transcripts corresponding to the 12 1 and the 165 isoforms of VEGF in all of the cell lines examined to date. In contrast to VEGF,

bFGF was found in approximately 60% of the hematopoietic cell lines. These findings demonstrate that while different angioregulatory molecules may be operating in a tumor, VEGF appears to be expressed in a vast majority of the tumors examined and therefore is likely to be playing an important role. We have also examined the expression of the two VEGF receptors: Flt-l/ VEGFRl and KDR/VEGFR2, as well as the recently described KDR coreceptor, NP- 1. While all three receptors were found expressed among the cell lines, expression of the Flt-l/VEGFRl receptor was by far the more common occurrence (Tables 1 and 2). The finding of VEGF receptor expression in these cell lines suggests the possibility of an autocrine pathway operating in which the tumor cells may stimulate their own growth or survival follotiing VEGF exposure.

Expression in Multi&

Myeloma

Patient

Specimens

Expression of VEGF and its high-affinity receptors Flt-l/VEGFRl and KDR/VEGFRZ was also evaluated in clinical specimens from patients diagnosed with multiple myeloma, myelodysplasia, acute myelocytic leukemia, or NHL, and the results compared to those observed in normal bone marrow. For the myeloma, MDS, and AML studies, immunohistochemical analysis was carried out on formalin-fixed, paraffin-embedded bone marrow clots or cores as previously described.ll The NHL studies used formalin-fixed, paraffin-embed-

EXPRESSION

Table

555

OF VEGF IN MYELOMA

I. Angiogenic Growth Human Hematopoietic

Factor Expression Tumor Cell Lines

in

Cell VEGF

bFGF

VEGF-C

HL-60 KG-I

AML

Disease

POS

Neg

AML

POS

POS

NALM 8226

ALL Multiple my&ma

POS

Neg

ARH-77 U-266

Multiple myeloma Multiple my&ma Multiple my&ma

POS POS

Neg POS

POS

POS

POS

POS

Histiocytic lymphoma Burkitt’s lymphoma (EBV+)

POS Pos

POS

Burkitt’s lymphoma T-cell lymphoma

Pos POS

POS

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

Line

IM-9 u-937 Raj i Ramos Hut78

Abbreviations: virus.

(EBV-)

Neg POS

Pos, positive: Neg, negative; EBV, Epstein-Barr

ded lymph nodes. Specimens were stained for expression of VEGF, Fit-l/VEGFR-1, and KDR/ VEGFR-2. The anti-VEGF antibodies used in these studies have been demonstrated to be specific for VEGF and do not cross-react with other known VEGF/placental growth factor family members. Both the KDR and Flt-1 antibodies are also specific and do not cross-react with each other or with other protein TK membrane receptors. All reactions were performed using an automated immunostainer (Gen”, Ventana Medical Systems [VMS], Tucson, AZ). Detection of bound antibody was assessed using immunoperoxidase methodologies with diaminobenzidine serving as the color substrate or by alkaline phosphatase methodologies using a biotinylated goat-anti-rabbit antibody in conjunction with alkaline phosphataseconjugated streptavidin followed by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as the color substrate. The VMS antibody diluent was used as a negative control. Nuclei were counterstained with methyl green or hematoxylin and sections evaluated by light microscopy. Endogenous peroxidase was inhibited with methyl alcohol containing 0.01% H,O,. The degree of expression in tumor cells was judged at 400x magnification as 4+ (very intensely positive), 3 + (moderately intensely positive), 2-t (moderate), 1 + (faint), or 0 (completely negative) throughout the sample. All samples were judged in a blinded manner by at least two independent reviewers.

In normal bone marrow, VEGF expression was uncommon, and generally restricted to myeloid elements, macrophages, and megakaryocytes, whereas immunohistochemical staining in erythroblasts and lymphocytes was not observed. To date, a total of 42 myeloma patients have been assessed with similar findings observed in all (Table 3). Plasma cell expression of VEGF was observed in the bone marrows from 33 of these patients (78%) in a diffuse cytoplasmic pattern that varied in intensity, ranging from l+ to 3+, among the individual patients. Although a gradient of VEGF expression is usually seen in solid tumors, with the strongest expression being in areas of hypoxia, we did not observe such a finding in the bone marrows of myeloma patients. Expression of VEGF in the malignant plasma cells was confirmed using in situ hybridization to VEGF mRNA. Neither the Flt-1 nor KDR receptors were observed to be expressed by malignant plasma cells; however, both receptors were markedly elevated in the normal marrow myeloid and monocytic cells surrounding the tumor in these patients-a pattern consistent with a paracrine role of VEGF in myeloma. Expression

in Other Hematopoietic

Tumors

In contrast to myeloma where a paracrine mechanism appears to predominate, in samples obtained from MDS patients, we detected concordant overexpression of VEGF and Flt-1 in monocytoid precursors of the chronic myelomonocytic

Table

2. VEGF Receptor Expression in Human Hematopoietic Tumor Cell Lines

Cell Line

Disease

Fit-I

KDR

NP- I

POS

Neg Pos

POS

HL-60 KG- I

AML AML

NALM 8226

ALL Multiple my&ma

ARH-77 U-266

Multiple my&ma Multiple my&ma

POS

IM-9 u-937

Multiple my&ma Histiocytic lymphoma

Pos Pos

Neg Neg Neg Neg

Raji Ramos Hut78

Burkitt’s lymphoma Burkitt’s lymphoma T-cell lymphoma

Pos Pos

Neg Neg

POS

Neg

Neg Pos Neg POS

(EBV+) (EBV-)

* Positive in the presence of bone marrow Abbreviation: ND, not determined.

Neg Neg

stromal

POS POS Neg/Pos* Neg ND Neg 4 Neg Neg Neg

cells.

556

WILLIAM

i

I

,i”

‘I ’

TabkJ.‘“Summary of &%&&hi~~rhchdcal C&t&ion a% VECF d&J Its Receptors z: -: Hemxopoietic Matigaancier ,:c ,:,:‘; : 7 : g; 87‘u, m .: ‘i”,, Diagnosis

NO.

NOtTlld Multiple

VEGF

9 my&ma

o*

in I r,

,/ f! i ,“: : ,~ ,I_

Fit- I

KDR

o*

0”

42

33

0

0

I7

I4

13

0

Low-grade

IO

3

I

I

High-grade

IO

8

8

3

AML

:

NHL

Myelodysplasia RA

8

8

7

2

RARS

6

3

2

0 3

CMML

IO

8

8

RAEB-t

22

I6

I5

*Samples

were

negative

with

occasional

positive

4 cells ob-

served. Abbreviations:

RA, refractive

mia with

ringed sideroblasts;

leukemia:

RAEB-t, refractive

anemia: RARS. refractive CMML,

chronic

ane-

myelomonocytic

anemia with excess blasts in trans-

formation.

leukemia (CMML) specimens. In bone marrow specimens from eight of 10 patients with CMML, cytoplasmic expression of VEGF, ranging from faint (+I) to intensely positive (+4), was detected in myelomonocytic cells, whereas no signal was detected in erythroblasts and plasma cells. Concordant membranous expression of the Flt-l/ VEGFR-1 receptor was observed in monocyte and myeloid precursors in all CMML specimens. Using two separate antibodies, a similar pattern of cellular expression, albeit of lower intensity, was detected for the KDR/VEGFR-2 receptor in 33% (3/10) of the CMML specimens examined. To confirm that the high-intensity VEGF protein sig nal detected in CMML resulted from overexpression of the VEGF gene transcript, VEGF mRNA expression was also assessed by in situ hybridization using a VEGF-specific oligonucleotide probe. Indeed, we found that cellular expression of VEGF mRNA was restricted to neoplastic myelomonocytic cells in a pattern analogous to that observed for the VEGF protein. Among other MDS morphologic subtypes, VEGF and Flt- 1 expression was restricted to myeloblasts and immature myeloid elements and was absent from erythroid precursors. Overall, expression of VEGF was observed in myeloid and monocyte precursors from 76% (35/ 46) of MDS and in myeloblasts from 82% (14/17)

T. BELLAMY

of AML cases. Coexpression of the Flt-1 receptor was demonstrable in 69% (32/46) of MDS and 76% (13/17) of AML cases. Membranous expression of the KDR receptor in blasts and myelomonocytic precursors was detected in 19% (9/ 46) of MDS, but was not found in any of the AML cases. In MDS cases where KDR was detected, it was always found in conjunction with Flt-1 receptor expression. This coexpression of the receptor and ligand consistently identified central foci of abnormal localizing precursors (ALP), an adverse histologic feature predictive of disease progression.15 Our investigations in NHL also suggest that VEGF may contribute to the regulation of tumor growth in these hematologic malignancies through both autocrine and paracrine mechanisms. Biopsy specimens from patients diagnosed with B-cell intermediate-/high-grade diffuse large cell lymphoma or low-grade follicular lymphoma were examined by immunohistochemistry and in situ hybridization for expression of VEGF, Flt-1, and KDR. VEGF expression was found both in the tumor vasculature, as well as in the tumor cells in the majority of lymphoma patient samples studied. In the neoplastic lymphocytes from both lowgrade and high-grade lymphomas VEGF expression was observed in an intense cytoplasmic pattern (4+) with membranous coexpression of the Flt-1 and/or KDR receptors. In situ hybridization, again, confirmed the presence of VEGF mRNA in the neoplastic cells. Expression of VEGF and its receptors was related to tumor grade, with a higher percentage of VEGF-positive cells in the intermediate-/high-grade group (80%) than in low-grade lymphomas (30%). Microvascular density was significantly increased in those patients with highgrade (62 2 17 vessels/O.74 mm’) versus low-grade (34 210 vessels/O.74 mm’) disease (I’ < .OOl). In the studies to date, either an increase in microvessel density or expression of various angiogenie growth factors, as demonstrated above, has suggested a role for angiogenesis in these tumors. However, using patient-derived material, we have demonstrated a functional role for VEGF in heneutralization of matopoietic tumors. Antibody VEGF activity suppressed leukemia progenitor formation in a concentration-dependent manner in 46% of MDS cases, whereas recombinant human (rhu)-VEGF promoted the growth of leukemia colonies in 56% of CMML and refractive anemia

EXPRESSION

OF VEGF IN MYELOMA

with excess blasts in transformation (RAEB-t) patient specimens.15 In addition, altering VEGF levels also had a marked impact on the expression of inflammatory cytokines. VEGF neutralization suppressed the concentration of TNF-ol and IL-l/3 to 44% and 39% of the concentrations observed in supernatants from control bone marrow mononuclear cultures, whereas the addition of rhu-VEGF resulted in increased cytokine concentrations.i5 Such findings suggest that VEGF serves as a trophic factor supporting progenitor self-renewal via either autocrine interaction or the paracrine induction of growth factors from stromal elements. Indeed, rhu-VEGF directly stimulated the in vitro growth of the KG-1 AML cell line, indicating that VEGF has direct trophic effects in receptor-competent tumors. CONCLUSIONS There is increasing evidence that angiogenic growth factors such as VEGF play a role in hematopoietic malignancies including multiple my eloma. Production of VEGF by malignant hematopoietic cells may serve as both an autocrine growth stimulus, and a diffusible paracrine signal mediating the local generation of growth factors that foster tumor survival and self-renewal. Autocrine production of VEGF may contribute to leukemia transformation in MDS, and potentiate progenitor self-renewal in AML, CMML, and NHL, while paracrine mechanisms are likely operating in myeloma. By identifying a role for VEGF in multiple myeloma and other hematopoietic malignancies, new treatment strategies are thus opened. The finding of such a role for VEGF in these tumors does not preclude the probability that other angiogenic growth factors are operating however. Studies have already demonstrated basic fibroblast growth factor expression, as well as matrix metalloproteinases-2 and -9, in patients with myeloma,rs*s5 thus further strengthening the possible applications of antiangiogenic therapies to the clinical setting. Traditionally, treatment of myeloma has been hampered by the development of drug resistance in this generally incurable disease. Kerbel has proposed that the inhibition of angiogenesis may represent one approach to overcome acquired drug resistance.56 While it is too premature to ascertain whether such an approach will be of benefit in myeloma, developing treatment strategies that target both the stromal and

557

tumor compartments, such as combining traditional cytotoxic chemotherapy with antiangiogenie agents, may indeed have an impact on drug resistance and improve the therapeutic response in myeloma. Findings such as those demonstrating expression of angiogenic growth factors and increased vascularity in hematopoietic tumors provide a biologic rationale for the clinical investigation of antiangiogenic agents in patients with myeloma and other hematopoietic malignancies. REFERENCES 1. Bataille R, Harousseau J Med 3361657-1664, 1997 2. Salmon

SE, Cassady

JL: Multiple

JR: Plasma

my&ma.

N

cell neoplasms,

Engl

in DeVita

VT, Hellman S and Rosenberg SA (eds): Cancer: Principles and Practice of Oncology (ed 5). Philadelphia, PA, Lippincott, 1997,

pp 2344-2387

3. Greenlee

RT,

statistics 2001. 4. Anderson

Hill-Harmon

multiple myeloma today. 5. Folkman J: Clinical esis. N Engl J Med 6. Vacca angiogenesis

MB, Murray

T, et al: Cancer

CA Cancer J Clin 51:15-36, 2001 KC, Hamblin TJ, Traynor A: Management Semin Hematol 36:3-8, applications of research

333:1757-1763,

A, Ribatti D, and progression

to1 87:503-508,

of

1999 on angiogen-

1995

Roneaii L, et al: Bone marrow in multiple myeloma. Br J Haema-

1994

7. Vacca A, Di Loreto M, Ribatti D, et al: Bone marrow of patients with active multiple myeloma: Angiogenesis and plasma

cell

adhesion

molecules

CD44. Am J Hematol 8. Ribatti D, Vacca trum

in the

stroma

LFA-1,

VLA-4,

LAM-1

and

50:9-14, 1995 RD, Nice B, et al: Angiogenesis of B-cell

immunohistochemical

non-Hodgkin’s

and ultrastructural

to1 56:45-53, 1996 9. Perez-Atayde AR,

Sallan

spec-

lymphomas. study.

SE, Tedrow

An

Eur J Haema-

U, et al: Spectrum

of tumor angiogenesis in the bone marrow of children acute lymphoblastic leukemia. Am J Pathol 150:815-821, 10. Fiedler thelial human

W,

Graeven

growth factor, acute myeloid

11. Bellamy,

U, Ergun

WT,

Richter

vascular endothelial growth hematopoietic malignancies. 12. Bellamy

WT,

vascular endothelial tors in non-Hodgkin’s 40:227,

S, et al: Vascular

a possible paracrine growth leukemia. Blood 89:1870-1875, L, Frutiger

with 1997 endo-

factor in 1997

Y, et al: Expression

factor (VEGF) and its receptors Cancer Res 59:728-733, 1999

Richter

L, Frutiger

cell growth lymphoma.

Y, et al: Expression

of in of

factor (VEGF) and its recepProc Am Assoc Cancer Res

1999 (abstr)

13. Vacca A, Ribatti D, Presta M, et al: Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human

multiple

14. Pruneri myelodysplastic

myeloma.

15. Bellamy WT, dothelial cell growth abnormal

Blood

G, Bertolini syndromes.

localized

93:3064-3073,

1999

F, Soligo D, et al: Angiogenesis in Br J Cancer 81:1398-1401, 1999

Richter L, Sirjani D, et al: Vascular factor (VEGF) is an autocrine promoter immature

myeloid

precursors

(ALIP)

enof and

558

leukemia progenitor formation in my&dysplastic syndromes. Blood 97:1427-1434, 2001 16. Singhal S, Mehta J, Desikan R, et al: Antitumor activity of thalidomide in refractory multiple myeloma. N Engl J Med 341:1565-1571, 1999 17. Rajkumar SV, Fonseca R, Dispenzieri A, et al: Thalidomide in the treatment of relapsed multiple myeloma. Mayo Clin Proc 75897-901, 2000 18. D’Amato RJ, Loughnan MS, Flynn E, et al: Thalidomide is an inhibitor of angiogenesis. Proc Nat1 Acad Sci USA 91:4082-4085, 1994 19. Dvorak HF, Brown LF, Detmar M, et al: Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Path01 146: 1029-1039,1995 20. Veikkola T, Karkkainen M, Claesson-Welsh L, et al: Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60:203-212, 2000 21. Clauss M: Molecular biology of the VEGF and the VEGF receptor family. Semin Thromb Hemost 26:561-569, 2000 22. Ferrara N: Vascular endothelial growth factor and the regulation of angiogenesis. Recent Prog Horm Res 55:15-35, 2000 23. Terman BI, Dougher-Vermazen M, Carrion ME, et al: Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 187:1579-1586, 1992 24. Ziegler BL, Valtieri M, Porada GA, et al: KDR receptor: A key marker defining hematopoietic stem cells. Science 285: 1553-1558, 1999 25. Fong G-H, Rossant J, Gertsenstein M, et al: Role of the Fltl receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 376:66-70, 1995 26. Shalaby F, Rossant J, Yamaguchi TP, et al: Failure of blood island formation and vasculogenesis in Flk-l-deficient mice. Nature 376~62-66, 1995 27. Soker S, Takashima S, Miao HQ, et al: Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92:735745, 1998 28. Holmgren L, O’Reilly MS, Folkman J: Dormancy of micrometastases: Balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nature Med 1:149-153, 1995 29. Lu C, Tanigawa N: Spontaneous apoptosis is inversely related to intratumoral microvessel density in gastric carcinoma. Cancer Res 57:221-224, 1997 30. Benjamin LE, Golijanin D, Itin A, et al: Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest 103:159-165, 1999 31. Nor JE, Christensen J, Mooney DJ, et al: Vascular endothelial growth factor (VEGF) -mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression. Am J Path01 154:375-384, 1999 32. Katoh 0, Tauchi H, Kawaishi K, et al: Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation. Cancer Res 55~5687.5692, 1995

WILLIAM

T. BELLAMY

33. Katoh 0, Takahashi T, Oguri T, et al: Vascular endothelial growth factor inhibits apoptotic death in hematopoietic cells after exposure to chemotherapeutic drugs by inducing MCL-1 acting as an antiapoptotic factor. Cancer Res 58:55655569, 1998 34. Kim JK, Li B, Winer J, et al: Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in viva. Nature 362841,844, 1993 35. Ohta Y, Endo Y, Tanaka M, et al: Significance of vascular endothelial growth factor messenger RNA expression in primary lung cancer. Clin Cancer Res 2:1411-1416, 1996 36. Brown LF, Berse B, Jackman RW, et al: Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am J Pathol 143:1255-1262, 1993 37. Bataille R, Harousseau JL: Multiple myeloma. N Engl J Med 336:1657-1664, 1997 38. Klein B, Zhang X, Lu 2, et al: Interleukin-6 in human multiple myeloma. Blood 85:863-872, 1995 39. Klein B, Zhang XG, Jourdan M, et al: Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood 73:517-526, 1989 40. Kawano M, Hirano T, Matsuda T, et al: Autocrine generation and requirement of BSF/IL-6 for multiple myelomas. Nature 332:83-85, 1988 41. Dankbar B, Padre T, Leo R, et al: Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma. Blood 95:2630-2636, 2000 42. Kawano MM, Mihara K, Huang N, et al: Differentiation of early plasma cells on bone marrow stromal cells requires interleukin-6 for escaping apoptosis. Blood 85:487-494, 1995 43. Lichtenstein A, Tu Y, Fady C, et al: Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol 162: 248255, 1995 44. Hata H, Matsuzaki H, Takeya M, et al: Expression of Fas/Apo-1 (CD-95) and apoptosis in tumor cells from patients with plasma cell disorders. Blood 86:1939-1945, 1995 45. Bataille R, Chappard D, Marcelli C, et al: Mechanisms of bone destruction in multiple myeloma. J Clin Onco17:19091914, 1989 46. Croucher PI, Apperley JF: Bone disease in myeloma. Br J Haematol 103:902-910, 1998 47. Kawano M, Yamamoto I, Iwato K: Interleukin-1 beta rather than lymphotoxin as a major bone resorbing activity in human multiple myeloma. Blood 73:1646-1649, 1989 48. Torcia M, Lucibello M, Vannier E, et al: Modulation of osteoclast-activating factor activity of multiple myeloma bone marrow cells by different interleukin-1 inhibitors. Exp Hematol 24868874, 1996 49. Klein B, Zhang XG, Jourdan M, et al: Interleukin-6 is the central tumor growth factor in vitro and in viva in multiple myeloma. Eur Cytokine Netw 1:193-201, 1990 50. O’Brien CA, Lin SC, Bellido T, et al: Expression levels of GP-130 in bone marrow stromal cells determine the magnitude of osteoclastogenic signals generated by IL-6 type cytokines. J Cell Biochem 79:532-541, 2000 51. Deckers MM, Karperien M, van der Bent C, et al: Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation. Endocrinology 141: 1667-1674, 2000

EXPRESSION

OF VEGF IN MYELOMA

52. Nakagawa

M,

endothelial growth tic bone resorption Lett 473:161-164,

Kaneda

559

T, Arakawa

factor (VEGF) and survival 2000

T, et al: Vascular

directly enhances osteoclasof mature osteoclasts. FEBS

53. Gabrilovich DI, Chen HL, Girgis KR, et al: Production of vascular endothelial growth factor by human tumors inhibits the functional 2:1096-1103, 54.

maturation 1996

Gabrilovich

DI,

of Ishida

dendritic T,

Oyama

cells. T,

Nature

Med

et al: Vascular

endothelial

growth

factor

cells and dramatically hematopoietic lineages 55. Vacca A, Ribatti

inhibits

the development

human lymphoid tumor cells. Correlation sion. Dev Immunol 7:77-88, 2000 56.

Kerbel

of dendritic

affects the differentiation of multiple in viva. Blood 924150-4166, 1998 D, Ria R, et al: Proteolytic activity of

RS: Inhibitron

of tumor

to circumvent acquired resistance agents. Bioessays 13:31-36, 1991

with

angiogenesis to anti-cancer

tumor

progres-

as a strategy therapeutic