Expression of VEGF and its receptors by myeloma cells - Nature

Leukemia (2003) 17, 2025–2031 & 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu

Expression of VEGF and its receptors by myeloma cells S Kumar1, TE Witzig1, M Timm1, J Haug1, L Wellik1, R Fonseca1, PR Greipp1 and SV Rajkumar1 1

Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA

Angiogenesis or new vessel formation is an essential component in the growth and progression of neoplasms and there is growing evidence of its importance in hematological malignancies including multiple myeloma (MM). Vascular endothelial growth factor (VEGF) is believed to play a role in tumor angiogenesis. We studied the expression of VEGF and its receptors (VEGFR1 or Flt-1 and VEGFR2 or Flk-1/KDR) by myeloma cell lines and plasma cells isolated from patients, using different methods. VEGF expression by the plasma cells was demonstrated by immunohistochemistry in 18 of 20 patients with MM. Enzyme-linked immunosorbent assay demonstrated VEGF secretion in all six different myeloma cell lines studied. Five patient marrow samples and seven different myeloma cell lines were then studied for VEGF mRNA expression by reverse-transcriptase polymerase chain reaction (RTPCR), which was positive in all. We further evaluated the expression of both VEGFR1 and VEGFR2 in different myeloma cell lines and five sorted myeloma bone marrow samples by RTPCR. All the myeloma cell lines expressed VEGFR1 and three of the cell lines expressed VEGFR2. VEGFR1 expression was detected in all and VEGFR2 in all but one of the sorted marrow samples. Increased expression of VEGF by the myeloma cells taken in the context of the suspected prognostic value of marrow angiogenesis suggests a pathogenetic role for this cytokine and presence of its receptors on myeloma cells points toward an autocrine mechanism. Demonstration of the presence of VEGFR2 in our study provides a potential biological explanation for the preclinical activity observed with VEGFR2 inhibitors. Leukemia (2003) 17, 2025–2031. doi:10.1038/sj.leu.2403084 Keywords: multiple myeloma; angiogenesis; vascular endothelial growth fector; VEGFR1; Flk-1

and is being increasingly recognized.4 Increasing levels of tumor angiogenesis has been associated with poor prognosis for a variety of hematological malignancies as well as solid tumors. As in solid tumors, new vessel formation (occurring in the bone marrow) seems to play an important role in the pathophysiology of myeloma,5–8 leukemias9,10 and in myelofibrosis.11 Increased bone marrow microvessel density in patients with myeloma appears to be a powerful prognostic indicator.7 Various cytokines have been invoked as being responsible for driving the process of neovascularization seen in the setting of solid tumors and hematological malignancies.12 Vascular endothelial growth factor (VEGF) appears to have a particularly important role in the biology of MM.13,14 Studies have shown that myeloma cells are capable of secreting VEGF contributing to new vessel formation in the bone marrow in myeloma. In turn, the microvascular endothelial cells as well as the marrow stromal cells are stimulated by the VEGF resulting in increased secretion of interleukin (IL)-6, which has been shown to be a potent growth factor for the malignant plasma cells.15,16 An autocrine pathway of myeloma cell stimulation by VEGF may also exist in addition to this paracrine pathway. We examined the expression of VEGF and their receptors by different methods on the plasma cells to examine this hypothesis further.

Introduction

Bone marrow samples were obtained from patients with MM and plasma cells were isolated from the patient samples using MACS separation columns using antibodies against CD138 as described by the manufacturer (Miltenyi Biotec, Bergisch Gladbach, Germany). These cells were further analyzed by Wright–Giemsa stain and a purity of over 96% was confirmed.

Multiple myeloma (MM) is characterized by a clonal proliferation of abnormal plasma cells in the bone marrow resulting in a variety of different clinical manifestations like osteolytic bone lesions leading to pathologic fractures, anemia, life-threatening hypercalcemia and renal failure. It accounts for more than 10% of all hematological malignancies and nearly 1% of all malignancies. It is estimated that there will be 14 600 new patients with a diagnosis of myeloma in the United States alone during the year 2002 and there will be 10 800 estimated deaths due to myeloma in the same period.1 The median survival from diagnosis among patients treated with conventional chemotherapy is 3–4 years.2 Introduction of high-dose chemotherapy with stem cell rescue has led to improved survival, but these patients eventually relapse from their disease and it remains an incurable disease with the currently available therapeutic modalities.3 Angiogenesis or new blood vessel formation from existing blood vessels (in contrast to vasculogenesis or de novo formation of blood vessels) occurs physiologically during normal growth, tissue healing and regeneration. The role of abnormal, increased angiogenesis in the development and spread of tumors had been suspected for nearly two decades Correspondence: Dr SV Rajkumar, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA; Fax: þ 1 507 266 4972 Received 30 December 2002; accepted 16 June 2003

Methods and materials

Isolation of patient plasma cells

Myeloma cell lines and cell culture Eight different myeloma cell lines were used for the study (ARH9, OCI-My5,17 KAS 6/1,18 RPMI 8226,19 KP-6,18 ANBL6,20 U266B1,21 OPM2). All cell lines were maintained at 371C in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine. Three of these cell lines, KP6, ANBL-6 and KAS 6/1, were also supplemented with IL-6 at 5 ng/ml.

Enzyme-linked immunosorbent assay Intracellular and secreted levels of VEGF were estimated by enzyme-linked immunosorbent assay (ELISA) using Quantikines human VEGF assay (R and D systems, Minneapolis, MN, USA). For intracellular levels of VEGF, 5 106 cells from each cell line were washed in RPMI 2, snap frozen in liquid nitrogen, and then thawed at 371C. The snap freezing and

Vascular endothelial growth factor in myeloma S Kumar et al

2026 thawing was repeated two more times. Cellular debris was then spun down and supernatant saved for analysis. Secreted levels were estimated using cell culture supernatants taken on day 7 from each cell line. Briefly, 200 ml of cell culture supernatant, cell lysate or standard (recombinant human VEGF165) was added to the microplate wells with the recommended diluent and incubated at room temperature for 2 h. The wells were washed and 200 ml of VEGF conjugate (polyclonal antibody against VEGF conjugated to horseradish peroxidase) was added and further incubation carried out at room temperature for 2 h. The wells were washed and 200 ml of hydrogen peroxide and tetramethylbenzidine was added and incubated for 20 min at RT. After stopping the reaction with 2 N sulfuric acid, the optical density was read at 450 nm using a microtiter plate reader.

Reverse-transcriptase polymerase chain reaction Total RNA was isolated from cell lines and patient samples using an RNeasys Kit as described by the manufacturer (Qiagen, Inc., Hilden, Germany). To generate cDNA and for amplification, 100 ng of RNA was used in a one-step reverse-transcriptase polymerase chain reaction (RT-PCR) kit as described by the manufacturer (Qiagen, Inc., Hilden, Germany). Nested PCR was performed to detect VEGFR1 and VEGFR2, and one round of PCR to detect VEGF. We chose an RT-nested-PCR approach to improve the sensitivity for detection of the VEGF receptors, since previous studies using RT single-step PCR have failed to demonstrate its presence consistently. Amplification of the housekeeping gene b-actin was used to verify mRNA isolation and RT-PCR techniques. Primer sequences were as follows: VEGF 50 -GAA GTG GTG AAG TTC ATG GAT GTC-30 (forward) and 50 -CGA TCG TTC TGT ATC AGT CTT TCC-30 (reverse); VEGFR1 (outer) 50 -GAG AAT TCA CTA TGG AAG ATC TGA TTT CTT ACA GT-30 (fwd) and 50 -GAG CAT GCG GTA AAA TAC ACA TGT GCT TCT AG-30 (rev); VEGFR2 (outer) 50 -CAA CAA AGT CGG GAG AGG AG-30 (fwd)12 and 50 -ATG ACG ATG GAC AAG TAG CC-30 (rev);22 VEGFR1 (inner) 50 -CTG AGA ACA ACG TGG TGA AGA TT-30 (fwd) and 50 -CTG ACA TCA TCA GAG CTT CCT GA-30 (rev); VEGFR2 (inner) 50 -CAC TGA GCA GGA GAG CGT GT-30 (fwd) and 50 -CTC ACT CTG CGG ATA GTG AGG TT-30 (rev); b-actin 50 -CGC TGC GCT GGT CGT CGA CA30 (fwd) and 50 -GTC ACG CAC GAT TTC CCG CT-30 (rev).23 PCR-amplified products were electrophoresed in 1.0% agarose gel and stained with ethidium bromide. Expression was verified using ABI Prism 377 DNA Sequencer (Perkin-Elmer, Lincoln, NE, USA). Precautions were taken to avoid contamination and negative controls were run with each step. Preparation of samples and PCR reactions were carried out in different areas. Quantitative RT-PCR for VEGF transcripts was carried out with the RNA Amplification Kit-SYBR Green I (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions using the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). Quantifications were made with a DNA external standard generated by PCR. The VEGF estimations were normalized using the housekeeping gene GAPDH. Primers were designed using Oligo 6.24 software (Molecular Biology Insights, Inc., Cascade, CO, USA) as follows: forward: 50 -AGG AGG GCA GAA TCA TCA CGA-30 ; reverse: 50 -TGA GGT TTG ATC CGC ATA ATC-30 .

Bone marrow immunohistochemistry Bone marrow biopsy samples were obtained from patients with diagnosis of MM. VEGF immunohistochemical staining of the Leukemia

bone marrow was performed using a labeled streptavidin–biotin peroxidase method, on a Ventana ES automated immunohistochemistry stainer (Ventana Medical Systems, Tucson, AZ, USA) using buffers and detection reagents supplied by the manufacturer. After deparaffinization, the primary antibody (VEGF mouse monoclonal antibody, Santacruz Biotechnology, Santa Cruz, CA, USA, diluted 1:500) was incubated with tissue sections for 24 min. The AEC (amino ethyl carbazole) detection kit (Ventana Medical Systems) was used for antigen visualization; sections were counterstained with a light hematoxylin and then cover slipped with Kaiseri’s glycerol jelly (Mayo Medical Laboratories, Rochester, MN, USA). VEGF expression was graded as follows: , no staining; þ , weak staining (0–30% plasma cells positive); þ þ , weak to moderate staining (30–60% plasma cells positive); þ þ þ , strong staining (460% plasma cells positive). All patients had given written informed consent for research use of bone marrow and serum specimens. Approval for the study was obtained from the Mayo Institutional Review Board in accordance with federal regulations and the Declaration of Helsinki.

Functional studies Ability of VEGF to activate intracellular downstream kinases was examined in three different myeloma cell lines. Phosphorylation of the mitogen-activated protein kinase (MAPK) and members of the signal transducers and activators of transcription (Stat) family members Stat1, Stat3 and Stat5 was evaluated after stimulation with differing concentrations of VEGF. Human MM cell lines JJN3, 8226 and OPM2 were maintained in RPMI 1640 containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ ml streptomycin. Cells were starved 16 h in RMPI 1640 and then stimulated with 10, 20 or 50 ng/ml VEGF121 and VEGF165. Cells were washed with phosphate-buffered saline, lysed and centrifuged. Cell lysates (50 mg/lane) were separated by SDSPAGE prior to transfer to nitrocellulose. The blots were probed with phospho-STAT1, phospho-STAT3 Tyr705, phospho-STAT3 Ser727, phospho-STAT5 and phospho-p44/42 MAPK (Cell Signaling Technology), followed by incubation for 1 h with horseradish peroxidase-conjugated secondary antibodies and visualization with the ECL detection kit (Amersham).

Apoptosis studies Ability of VEGF to protect from dexamethasone-induced apoptosis was examined using the myeloma cell line MY5. A constant number of cells were exposed to dexamethasone (1.5 mM) or VEGF 121 or VEGF 165 at concentrations of 0, 4, 20 and 50 ng/ml or both. The presence of apoptotic cells was detected by flow cytometry, using 7-AAD as a marker of apoptosis.

Results

VEGF expression by RT-PCR The presence of VEGF expression by patient plasma cells and the myeloma cell lines was evaluated using RT-PCR. VEGF expression was evident in all the five patient samples studied. Seven different myeloma cell lines (ARH9, OCI-My5, KAS6/1, RPMI 8226, KP6, ANBL-6, U266) were also studied for VEGF


2027 expression by RT-PCR. All cell lines demonstrated the presence of VEGF mRNA (Figure 1). Two different isoforms of VEGF (VEGF and VEGF) were observed in all samples studied. VEGF mRNA expression was quantitated by RT-PCR in eight patient samples and the results were as shown in Figure 2. Melting curve analysis of the VEGF PCR products was performed demonstrating a clear peak at 831C (Figure 3b). Further demonstration of the specificity was performed by electrophoresing the PCR products on a 2% agarose gel (Figure 3a).

VEGF secretion by the plasma cells

Figure 1 VEGF mRNA expression detected by RT-PCR in sorted myeloma cells from five patients and in different cell lines.

VEGF protein secretion by different myeloma cell lines was examined by ELISA on the cell culture supernatants. Rat serum and plasma, human plasma and RPMI medium were used as controls. Six cell lines (ANBL-6, KP6, KAS6/1, OCI-My5, RPMI 8226, OPM2) were studied. The cells were seeded into six-well plates at a concentration of 200 000 cells per well in RPMI 1640 with 10% FBS. Culture supernatants were removed after 96 h and the VEGF measured as previously described. VEGF secretion ranged from 217 to 3050 pg/ml (Figure 4) compared to undetectable levels in the normal human plasma.

VEGF expression by immunohistochemistry

Figure 2 Quantitative RT-PCR for VEGF mRNA. Values are expressed as a ratio of VEGF to GAPDH expression.

The expression of VEGF by myeloma cells was studied by immunohistochemistry in bone marrow biopsy samples from 20 patients with MM. Plasma cells in the bone marrow demonstrated variable expression of VEGF as shown in the representative pictures (Figure 5a–d). Of the 20 samples, 18 were positive for VEGF expression and the level of expression based on the intensity, and a relatively high level of expression was seen in 13 patients (grade þ þ þ ). Five patients had weak to moderate grade of staining and it was negative in the remaining two. We

Figure 3 Gel demonstrating VEGF amplicon following quantitative PCR carried out on purified myeloma cells from five patient samples (a). Also included are the melting curves from the five patient samples (b). Leukemia


2028 also noticed some degree of staining by the megakaryocytes in the marrow as well.

The expression patterns for VEGF and their receptors in the different cell lines are summarized in Table 1.

Expression of VEGF receptors

Evaluation of VEGF effect on myeloma cells

We further evaluated the expression of both VEGF receptors, VEGFR1 and VEGFR2, in five sorted myeloma bone marrow samples and in seven different myeloma cell lines (ARH9, OCIMy5, KAS 6/1, 8226, KP-6, ANBL-6, U266) by nested RT-PCR. All the myeloma cell lines expressed VEGFR1 and three of the cell lines (KP6, KAS 6/1, ANBL6) expressed VEGFR2. VEGFR1 expression was present in plasma cells from all five patients, whereas strong expression of VEGFR2 was seen in three patient samples, weak in one and none in the fifth patient (Figures 6 and 7). The RT-PCR results were confirmed by sequence analysis.

We were able to demonstrate STAT3 activation by both VEGF 121 and VEGF 165 in the JJN3 myeloma cell line (Figure 8). The

Figure 4 VEGF secretion as determined by ELISA on the supernatants of different myeloma cell lines. VEGF values are expressed in pg/ml. Intracellular levels were determined by ELISA on cell lysates.

Figure 6 Detection of VEGFR1 receptors using RT-PCR in sorted myeloma cells from five patients and in different cell lines.

Figure 5 (a) Immunostain of bone marrow demonstrating lack of staining by myeloma cells for VEGF. Areas of intense staining represent erythroid precursors ( 400). (b, c) Bone marrow in MM with plasma cells demonstrating variable degree of staining for VEGF ( 400). (d) Higher power view clearly demonstrating the cytoplasmic VEGF staining of plasma cells. Arrow demonstrates a stained plasma cell with the characteristic perinuclear Hof. Leukemia


2029 phosphorylation of the other members of the STAT family was not observed in any of the cell lines studied. In addition, MAPK activation was demonstrated in the 8226 cell line

after stimulation with VEGF121, but not with VEGF165. MAPK phosphorylation was not seen in the other cell lines studied. Exposure of My5 cells to dexamethasone consistently led to significant apoptosis of the cells. The addition of VEGF 121 and VEGF 165 at various concentrations did not have any effect on the cell viability. Addition of VEGF 121 and VEGF 165 at the same concentrations along with dexamethasone did not result in any decrease in the apoptotic effect of dexamethasone in these cells.

Discussion

Figure 7 Detection of flk1/KDR receptors using RT-PCR in sorted myeloma cells from five patients and in different cell lines. Table 1

ARH9 MY5 KAS-6/1 8226 KP6 ANBL-6 U266

Figure 8

VEGF and VEGF receptors in myeloma cell lines VEGF

FLT-1

KDR

+ + + + + + +

+ + + + + + +

+ + +

In MM, emerging evidence point to the presence of a maze of autocrine and paracrine interactions in the bone marrow between stroma and the malignant plasma cells mediated by various cytokines and growth factors. A variety of cytokines (IL-1b, TNF-a, TGF-b, IL-6, bFGF, VEGF) have been implicated in this complex cascade of interactions. Ongoing studies continue to unravel the interactions between these players in the pathogenesis and progression of MM. VEGF is a heparin binding glycoprotein that is intimately associated with the induction of angiogenesis and endothelial cell proliferation and is an important regulator of vasculogenesis. In addition to its vasculotrophic effects, it also induces vasodilatation, vascular permeability and cellular migration.24 In addition to the widely studied VEGF-A, this family of cytokines includes VEGF-B, C, D, E and placental growth factor. Although several splice variants of VEGF-A have been found, the two most commonly encountered are VEGF121 and VEGF165.24 VEGF can be secreted by a variety of cells and its secretion is often stimulated by different cytokines, hypoxia and several activated oncogenes.25 VEGF activity is mediated mainly through two receptors, VEGFR1 and VEGFR2.26,27 Although the affinity of VEGFR1 for its ligand is much stronger than that of VEGFR2, the latter seems to be responsible for most of the angiogenic effect of VEGF. A third receptor (VEGFR3 has been identified and appears to be restricted to lymphatic epithelium.

STAT3 and ERK activation in myeloma cell lines following VEGF stimulation. Leukemia


2030 Bone marrow angiogenesis plays an important role in the pathogenesis and progression of MM as with other hematological malignancies as well as solid tumors. In the setting of myeloma, the degree of bone marrow angiogenesis as estimated by the microvessel density, has been shown to be a powerful prognostic factor for survival.7,8 In a study of 400 patients, the degree of bone marrow angiogenesis was found to increase progressively across the spectrum of plasma cell proliferative disorders.28 Secretion of VEGF by the myeloma cells as shown in our study as well as by others is likely to be playing a role in the initiation and maintenance of the abnormal angiogenesis. The proliferative vascular endothelium is responsive to VEGF stimulation and has been shown to secrete IL-6 on stimulation.15 IL-6 is a key cytokine regulator of myeloma cell growth29 and is an essential growth factor for myeloma cells. In the bone marrow, the stromal cells are capable of secreting IL-6 in addition to the vascular endothelial cells. VEGF has been shown to be capable of stimulating IL-6 secretion by the stromal cells15 and is probably responsible for the increased secretion of IL-6 by stromal cells noted when cocultured with myeloma cells.30 IL-6 secretion by the bone marrow stromal cells can also be induced by other cytokines like IL-1b and TNF-a.31,32 IL-6 in turn has been shown to be capable of stimulating VEGF secretion by myeloma cells.15 Thus enough evidence exists to hypothesize the presence of a paracrine feedback loop of cytokines that stimulate, maintain and possibly lead to progression of the myeloma cell mass. Using multiple methods we have confirmed the increased expression and secretion of VEGF by the myeloma cells using sorted samples from patients with myeloma as well as several established myeloma cell lines. These results confirm work carried out by Bellamy et al,12 who showed expression of VEGF mRNA and secretion of the corresponding protein by different human hematopoietic tumor cell lines, representing different hematologic malignancies. VEGFR1 receptor expression was found in five of the 12 cell lines studied, suggesting the possibility of an autocrine pathway. Stimulation of vascular endothelial cells by VEGF led to an increase in the mRNA for several hematopoietic growth factors and IL-6. Myeloma cells in the bone marrow were shown to express VEGF, and normal bone marrow myeloid and monocytic cells surrounding the myeloma cells had elevated expression of both the VEGFR1 and VEGFR2 high-affinity VEGF receptors. Dankbar et al15 studied the paracrine interactions between the myeloma cells and the marrow stromal cells. They demonstrated VEGF expression and secretion by myeloma cell lines and plasma cells isolated from the marrow of patients with MM. However, VEGF receptors VEGFR1 and VEGFR2 expression when evaluated by immunohistochemistry or RT-PCR was found to be weak or absent on the myeloma cells. They found abundant expression of these receptors by marrow stromal cells and also demonstrated recombinant human VEGF-induced, time- and dose-dependent increase in IL-6 secretion by stromal and microvascular endothelial cells. IL-6 is a potent growth factor for myeloma cells, and these findings demonstrate the existence of a paracrine pathway. Podar et al16 studied the cellular pathways of VEGF stimulation in the myeloma cells and demonstrated stimulation of tumor cell proliferation by VEGF by a protein kinase C-independent Raf-1-MEK-extracellular signal-regulated protein kinase (ERK) pathway. Most of the previous studies have failed to demonstrate the presence of VEGFR-2 on these cells,16 although the presence of VEGFR1 has been consistently demonstrated. By using a more sensitive RT-nested-PCR method we have been able to Leukemia

demonstrate its presence on myeloma cells from patient samples as well as on myeloma cell lines. Secretion of VEGF by myeloma cells and the simultaneous presence of both VEGFR1 and VEGFR2 receptors on its surface raise the possibility of an autocrine pattern of stimulation in addition to the paracrine mechanisms discussed previously. The demonstration of VEGFR2 on the myeloma cells is especially important since this seems to be the functionally more active receptor. In this particular context, demonstration of its presence strengthens the biological plausibility for the previously demonstrated effects of VEGF on myeloma cells.16 We do want to point out that, while demonstration of VEGF required only an RT-PCR approach, a nested approach was needed for demonstrating the VEGF receptors pointing to a low level of expression. However, the level of expression need not correlate with that of activity for cytokine receptors. We were able to demonstrate activation of the MAPK and STAT pathways in some of the cell lines following stimulation with VEGF. But the results of the functional studies fail to demonstrate a significant stimulatory or antiapoptotic effect for VEGF. Further studies are needed to confirm or disprove an autocrine role for secreted VEGF in the disease biology. The role of VEGF and its receptors in the biology of myeloma as evidenced by our data and other data presents a great opportunity for using inhibitors of VEGF or its receptors for therapy of myeloma. Several novel molecules like SU6668 and PTK787 are currently undergoing evaluation utilizing this strategy. Demonstration of the presence of VEGFR2 in our study provides biological explanation for the preclinical activity observed with VEGFR2 inhibitors. Results of these clinical trials and in vitro studies with these agents will shed more light on the role of this cytokine and its receptors in the biology of myeloma.

Acknowledgements This work is supported in part by the Goldman Philanthropic Partnerships, IL, USA and Grants CA 93842, CA 85818 and CA62242, National Cancer Institute, Bethesda, MD, USA. Dr Rajkumar is a Leukemia and Lymphoma Society of America Translational Research Awardee and is also supported by the Multiple Myeloma Research Foundation.

References 1 Jemal A, Thomas A, Murray T, Thun M. Cancer statistics. CA Cancer J Clin 2002; 52: 23–47. 2 Kyle RA. Long-term survival in multiple myeloma. N Engl J Med 1983; 308: 314–316. 3 Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 1996; 335: 91–97. 4 Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995; 333: 1757–1763. 5 Sezer O, Niemoller K, Eucker J, Jakob C, Kaufmann O, Zavrski I et al. Bone marrow microvessel density is a prognostic factor for survival in patients with multiple myeloma. Ann Hematol 2000; 79: 574–577. 6 Rajkumar SV, Fonseca R, Witzig TE, Gertz MA, Greipp PR. Bone marrow angiogenesis in patients achieving complete response after stem cell transplantation for multiple myeloma. Leukemia 1999; 13: 469–472. 7 Rajkumar SV, Leong T, Roche PC, Fonseca R, Dispensieri A, Lacy MQ et al. Prognostic value of bone marrow angiogenesis in multiple myeloma. Clin Cancer Res 2000; 6: 3111–3116.


2031 8 Vacca A, Ribatti D, Roncali L, Ranieri G, Serio G, Silvestris F et al. Bone marrow angiogenesis and progression in multiple myeloma. Br J Haematol 1994; 87: 503–508. 9 Schneider P, Jerome MV, Paysant J, Soria PC, Vannier JP. The role of angiogenesis in leukemia proliferation. Am J Pathol 1999; 155: 1007–1008. 10 Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas D et al. Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood 2000; 96: 2240–2245. 11 Mesa RA, Hanson CA, Rajkumar SV, Schroeder G, Tefferi A. Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia. Blood 2000; 96: 3374– 3380. 12 Bellamy WT, Richter L, Frutiger Y, Grogan TM. Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. Cancer Res 1999; 59: 728–733. 13 Di Raimondo F, Azzaro MP, Palumbo G, Bagnato S, Giustolisi G, Floridia P et al. Angiogenic factors in multiple myeloma: higher levels in bone marrow than in peripheral blood. Haematologica 2000; 85: 800–805. 14 Sezer O, Jakob C, Eucker J, Niemoller K, Gatz F, Wernecke K et al . Serum levels of the angiogenic cytokines basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in multiple myeloma. Eur J Haematol 2001; 66: 83–88. 15 Dankbar B, Padro T, Leo R, Feldmann B, Kropff M, Mesters RM et al. Vascular endothelial growth factor and interleukin-6 in paracrine tumor–stromal cell interactions in multiple myeloma. Blood 2000; 95: 2630–2636. 16 Podar K, Tai YT, Davies FE, Lentzsch S, Sattler M, Hideshima T et al. Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration. Blood 2001; 98: 428–435. 17 Hitzler JK, Martinez-Valdez H, Bergsagel DB, Minden MD, Messner HA. Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease. Blood 1991; 78: 1996–2004. 18 Westendorf JJ, Ahmann GJ, Greipp PR, Witzig TE, Lust JA, Jelinek DF. Establishment and characterization of three myeloma cell lines that demonstrate variable cytokine responses and abilities to produce autocrine interleukin-6. Leukemia 1996; 10: 866–876. 19 Matsuoka Y, Moore GE, Yagi Y, Pressman D. Production of free light chains of immunoglobulin by a hematopoietic cell line derived from a patient with multiple myeloma. Proc Soc Exp Biol Med 1967; 125: 1246–1250. 20 Jelinek DF, Ahmann GJ, Greipp PR, Jalal SM, Westendorf JJ, Katzmann JA et al. Coexistence of aneuploid subclones within a myeloma cell line that exhibits clonal immunoglobulin gene

21

22

23

24 25

26 27

28

29 30

31 32

rearrangement: clinical implications. Cancer Res 1993; 53: 5320– 5327. Nilsson K, Bennich H, Johansson SG, Ponten J. Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient. Clin Exp Immunol 1970; 7: 477–489. Brown LF, Berse B, Jackman RW, Tognazzi K, Manseau EJ, Dvorak HF et al. Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am J Pathol 1993; 143: 1255–1262. Fiedler W, Graeven U, Ergun S, Verago S, Kilic N, Stockschlader M et al . Vascular endothelial growth factor, a possible paracrine growth factor in human acute myeloid leukemia. Blood 1997; 89: 1870–1875. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13: 9–22. Okada F, Rak JW, Croix BS, Lieubeau B, Kaya M, Roncari L et al. Impact of oncogenes in tumor angiogenesis: mutant K-ras upregulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells. Proc Natl Acad Sci USA 1998; 95: 3609–3614. de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992; 255: 989–991. Terman BI, Dougher-Vermazen M, Carrion ME, Dimirov D, Armellino DC, Gospodarowicz D et al. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 1992; 187: 1579–1586. Rajkumar SV, Mesa RA, Fonseca R, Schroeder G, Plevak MF, Dispenzieri A et al. Bone marrow angiogenesis in 400 patients with monoclonal gammopathy of undetermined significance, multiple myeloma, and primary amyloidosis. Clin Cancer Res 2002; 8: 2210–2216. Michio K, Toshio H, Tadashi M, Tetsuya T, Yasuhiro H, Koji I et al. Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 1988; 332: 83–85. Gupta D, Treon SP, Shima Y, Hideshima T, Podar K, Tai YT et al. Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications. Leukemia 2001; 15: 1950–1961. Shalaby MR, Waage A, Espevik T. Cytokine regulation of interleukin 6 production by human endothelial cells. Cell Immunol 1989; 121: 372–382. Thomas X, Anglaret B, Magaud JP, Epstein J, Archimbaud E. Interdependence between cytokines and cell adhesion molecules to induce interleukin-6 production by stromal cells in myeloma. Leukemia Lymphoma 1998; 32: 107–119.

Leukemia