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

1Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN, USA

Angiogenesis or new vessel formation is an essential compo- and is being increasingly recognized.4 Increasing levels of tumor nent in the growth and progression of neoplasms and there is angiogenesis has been associated with poor prognosis for a growing evidence of its importance in hematological malig- nancies including multiple myeloma (MM). Vascular endothelial variety of hematological malignancies as well as solid tumors. growth factor (VEGF) is believed to play a role in tumor As in solid tumors, new vessel formation (occurring in the bone angiogenesis. We studied the expression of VEGF and its marrow) seems to play an important role in the pathophysiology receptors (VEGFR1 or Flt-1 and VEGFR2 or Flk-1/KDR) by of myeloma,5–8 leukemias9,10 and in myelofibrosis.11 Increased myeloma cell lines and plasma cells isolated from patients, bone marrow microvessel density in patients with myeloma using different methods. VEGF expression by the plasma cells appears to be a powerful prognostic indicator.7 was demonstrated by immunohistochemistry in 18 of 20 patients with MM. Enzyme-linked immunosorbent assay de- Various cytokines have been invoked as being responsible for monstrated VEGF secretion in all six different myeloma cell driving the process of neovascularization seen in the setting 12 lines studied. Five patient marrow samples and seven different of solid tumors and hematological malignancies. Vascular myeloma cell lines were then studied for VEGF mRNA expres- endothelial growth factor (VEGF) appears to have a particularly sion by reverse-transcriptase polymerase chain reaction (RT- important role in the biology of MM.13,14 Studies have shown PCR), which was positive in all. We further evaluated the that myeloma cells are capable of secreting VEGF contributing expression of both VEGFR1 and VEGFR2 in different myeloma cell lines and five sorted myeloma bone marrow samples by RT- to new vessel formation in the bone marrow in myeloma. In PCR. All the myeloma cell lines expressed VEGFR1 and three of turn, the microvascular endothelial cells as well as the marrow the cell lines expressed VEGFR2. VEGFR1 expression was stromal cells are stimulated by the VEGF resulting in increased detected in all and VEGFR2 in all but one of the sorted marrow secretion of interleukin (IL)-6, which has been shown to be a samples. Increased expression of VEGF by the myeloma cells potent growth factor for the malignant plasma cells.15,16 An taken in the context of the suspected prognostic value of autocrine pathway of myeloma cell stimulation by VEGF may marrow angiogenesis suggests a pathogenetic role for this cytokine and presence of its receptors on myeloma cells points also exist in addition to this paracrine pathway. We examined toward an autocrine mechanism. Demonstration of the pre- the expression of VEGF and their receptors by different methods sence of VEGFR2 in our study provides a potential biological on the plasma cells to examine this hypothesis further. explanation for the preclinical activity observed with VEGFR2 inhibitors. Leukemia (2003) 17, 2025–2031. doi:10.1038/sj.leu.2403084 Methods and materials Keywords: multiple myeloma; angiogenesis; vascular endothelial growth fector; VEGFR1; Flk-1 Isolation of patient plasma cells

Introduction Bone marrow samples were obtained from patients with MM and plasma cells were isolated from the patient samples using Multiple myeloma (MM) is characterized by a clonal prolifera- MACS separation columns using antibodies against CD138 tion of abnormal plasma cells in the bone marrow resulting in a as described by the manufacturer (Miltenyi Biotec, Bergisch variety of different clinical manifestations like osteolytic bone Gladbach, Germany). These cells were further analyzed by lesions leading to pathologic fractures, anemia, life-threatening Wright–Giemsa stain and a purity of over 96% was confirmed. 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 Myeloma cell lines and cell culture patients with a diagnosis of myeloma in the United States alone during the year 2002 and there will be 10 800 estimated deaths Eight different myeloma cell lines were used for the study 17 18 19 18 due to myeloma in the same period.1 The median survival from (ARH9, OCI-My5, KAS 6/1, RPMI 8226, KP-6, ANBL- 20 21 diagnosis among patients treated with conventional chemother- 6, U266B1, OPM2). All cell lines were maintained at 371C apy is 3–4 years.2 Introduction of high-dose chemotherapy with in RPMI 1640 media supplemented with 10% heat-inactivated stem cell rescue has led to improved survival, but these patients fetal bovine serum (FBS), penicillin, streptomycin and eventually relapse from their disease and it remains an incurable L-glutamine. Three of these cell lines, KP6, ANBL-6 and KAS disease with the currently available therapeutic modalities.3 6/1, were also supplemented with IL-6 at 5 ng/ml. Angiogenesis or new blood vessel formation from existing blood vessels (in contrast to vasculogenesis or de novo formation of blood vessels) occurs physiologically during Enzyme-linked immunosorbent assay normal growth, tissue healing and regeneration. The role of abnormal, increased angiogenesis in the development and Intracellular and secreted levels of VEGF were estimated by spread of tumors had been suspected for nearly two decades enzyme-linked immunosorbent assay (ELISA) using Quanti- kines human VEGF assay (R and D systems, Minneapolis, 6 Correspondence: Dr SV Rajkumar, Mayo Clinic, 200 First Street MN, USA). For intracellular levels of VEGF, 5 Â 10 cells from SW, Rochester, MN 55905, USA; Fax: þ 1 507 266 4972 each cell line were washed in RPMI Â 2, snap frozen in liquid Received 30 December 2002; accepted 16 June 2003 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 bone marrow was performed using a labeled streptavidin–biotin spun down and supernatant saved for analysis. Secreted levels peroxidase method, on a Ventana ES automated immuno- were estimated using cell culture supernatants taken on day 7 histochemistry stainer (Ventana Medical Systems, Tucson, AZ, from each cell line. Briefly, 200 ml of cell culture supernatant, USA) using buffers and detection reagents supplied by the cell lysate or standard (recombinant human VEGF165) was added manufacturer. After deparaffinization, the primary antibody to the microplate wells with the recommended diluent and (VEGF mouse monoclonal antibody, Santacruz Biotechnology, incubated at room temperature for 2 h. The wells were washed Santa Cruz, CA, USA, diluted 1:500) was incubated with tissue and 200 ml of VEGF conjugate (polyclonal antibody against sections for 24 min. The AEC (amino ethyl carbazole) detection VEGF conjugated to horseradish peroxidase) was added and kit (Ventana Medical Systems) was used for antigen visualiza- further incubation carried out at room temperature for 2 h. The tion; sections were counterstained with a light hematoxylin and wells were washed and 200 ml of hydrogen peroxide and then cover slipped with Kaiseri’s glycerol jelly (Mayo Medical tetramethylbenzidine was added and incubated for 20 min at Laboratories, Rochester, MN, USA). VEGF expression was RT. After stopping the reaction with 2 N sulfuric acid, the optical graded as follows: À, no staining; þ , weak staining (0–30% density was read at 450 nm using a microtiter plate reader. plasma cells positive); þþ, weak to moderate staining (30–60% plasma cells positive); þþþ, strong staining (460% plasma cells positive). Reverse-transcriptase polymerase chain reaction All patients had given written informed consent for research use of bone marrow and serum specimens. Approval for the Total RNA was isolated from cell lines and patient samples using s study was obtained from the Mayo Institutional Review Board in an RNeasy Kit as described by the manufacturer (Qiagen, Inc., accordance with federal regulations and the Declaration of Hilden, Germany). To generate cDNA and for amplification, Helsinki. 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 Functional studies performed to detect VEGFR1 and VEGFR2, and one round of PCR to detect VEGF. We chose an RT-nested-PCR approach to Ability of VEGF to activate intracellular downstream kinases was improve the sensitivity for detection of the VEGF receptors, since examined in three different myeloma cell lines. Phosphorylation previous studies using RT single-step PCR have failed to of the mitogen-activated protein kinase (MAPK) and members of demonstrate its presence consistently. Amplification of the the signal transducers and activators of transcription (Stat) family housekeeping gene b-actin was used to verify mRNA isolation members Stat1, Stat3 and Stat5 was evaluated after stimulation and RT-PCR techniques. Primer sequences were as follows: with differing concentrations of VEGF. Human MM cell lines VEGF 50-GAA GTG GTG AAG TTC ATG GAT GTC-30 (forward) JJN3, 8226 and OPM2 were maintained in RPMI 1640 contain- and 50-CGA TCG TTC TGT ATC AGT CTT TCC-30 (reverse); ing 10% FBS, 2 mML-glutamine, 100 U/ml penicillin and 100 mg/ VEGFR1 (outer) 50-GAG AAT TCA CTA TGG AAG ATC TGA TTT ml streptomycin. Cells were starved 16 h in RMPI 1640 and then CTT ACA GT-30 (fwd) and 50-GAG CAT GCG GTA AAA TAC stimulated with 10, 20 or 50 ng/ml VEGF and VEGF . Cells ACA TGT GCT TCT AG-30 (rev); VEGFR2 (outer) 50-CAA CAA 121 165 were washed with phosphate-buffered saline, lysed and AGT CGG GAG AGG AG-30 (fwd)12 and 50-ATG ACG ATG GAC centrifuged. Cell lysates (50 mg/lane) were separated by SDS- AAG TAG CC-30 (rev);22 VEGFR1 (inner) 50-CTG AGA ACA ACG PAGE prior to transfer to nitrocellulose. The blots were probed TGG TGA AGA TT-30 (fwd) and 50-CTG ACA TCA TCA GAG with phospho-STAT1, phospho-STAT3 Tyr705, phospho-STAT3 CTT CCT GA-30 (rev); VEGFR2 (inner) 50-CAC TGA GCA GGA Ser727, phospho-STAT5 and phospho-p44/42 MAPK (Cell GAG CGT GT-30 (fwd) and 50-CTC ACT CTG CGG ATA GTG Signaling Technology), followed by incubation for 1 h with AGG TT-30 (rev); b-actin 50-CGC TGC GCT GGT CGT CGA CA- horseradish peroxidase-conjugated secondary antibodies and 30 (fwd) and 50-GTC ACG CAC GAT TTC CCG CT-30 (rev).23 visualization with the ECL detection kit (Amersham). 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, Apoptosis studies USA). Precautions were taken to avoid contamination and negative controls were run with each step. Preparation of Ability of VEGF to protect from dexamethasone-induced samples and PCR reactions were carried out in different areas. apoptosis was examined using the myeloma cell line MY5. A Quantitative RT-PCR for VEGF transcripts was carried out constant number of cells were exposed to dexamethasone with the RNA Amplification Kit-SYBR Green I (Molecular (1.5 mM) or VEGF 121 or VEGF 165 at concentrations of 0, 4, 20 Probes, Eugene, OR, USA) according to the manufacturer’s and 50 ng/ml or both. The presence of apoptotic cells was instructions using the LightCycler (Roche Diagnostics GmbH, detected by flow cytometry, using 7-AAD as a marker of Mannheim, Germany). Quantifications were made with a DNA apoptosis. 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, Results Inc., Cascade, CO, USA) as follows: forward: 50-AGG AGG GCA GAA TCA TCA CGA-30; reverse: 50-TGA GGT TTG ATC VEGF expression by RT-PCR CGC ATA ATC-30. The presence of VEGF expression by patient plasma cells and Bone marrow immunohistochemistry the myeloma cell lines was evaluated using RT-PCR. VEGF expression was evident in all the five patient samples studied. Bone marrow biopsy samples were obtained from patients with Seven different myeloma cell lines (ARH9, OCI-My5, KAS6/1, diagnosis of MM. VEGF immunohistochemical staining of the RPMI 8226, KP6, ANBL-6, U266) were also studied for VEGF

Leukemia Vascular endothelial growth factor in myeloma S Kumar et al 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 electro- phoresing the PCR products on a 2% agarose gel (Figure 3a).

VEGF secretion by the plasma cells

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 Figure 1 VEGF mRNA expression detected by RT-PCR in sorted plates at a concentration of 200 000 cells per well in RPMI 1640 myeloma cells from five patients and in different cell lines. 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

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 demon- strated variable expression of VEGF as shown in the representa- tive 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 Figure 2 Quantitative RT-PCR for VEGF mRNA. Values are patients (grade þþþ). Five patients had weak to moderate expressed as a ratio of VEGF to GAPDH expression. 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 Vascular endothelial growth factor in myeloma S Kumar et al 2028 also noticed some degree of staining by the megakaryocytes in The expression patterns for VEGF and their receptors in the the marrow as well. 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, We were able to demonstrate STAT3 activation by both VEGF VEGFR1 and VEGFR2, in five sorted myeloma bone marrow 121 and VEGF 165 in the JJN3 myeloma cell line (Figure 8). The samples and in seven different myeloma cell lines (ARH9, OCI- My5, 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.

Figure 4 VEGF secretion as determined by ELISA on the super- natants of different myeloma cell lines. VEGF values are expressed in Figure 6 Detection of VEGFR1 receptors using RT-PCR in sorted pg/ml. Intracellular levels were determined by ELISA on cell lysates. 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 ( Â 400). (b, c) Bone marrow in MM with plasma cells demonstrating variable degree of staining for VEGF ( Â 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 Vascular endothelial growth factor in myeloma S Kumar et al 2029 phosphorylation of the other members of the STAT family after stimulation with VEGF121, but not with VEGF165. was not observed in any of the cell lines studied. In addition, MAPK phosphorylation was not seen in the other cell lines MAPK activation was demonstrated in the 8226 cell line 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

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 Figure 7 Detection of flk1/KDR receptors using RT-PCR in sorted vasculotrophic effects, it also induces vasodilatation, vascular myeloma cells from five patients and in different cell lines. 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 Table 1 VEGF and VEGF receptors in myeloma cell lines of VEGF-A have been found, the two most commonly 24 encountered are VEGF121 and VEGF165. VEGF can be secreted VEGF FLT-1 KDR by a variety of cells and its secretion is often stimulated by 25 ARH9 + + À different cytokines, hypoxia and several activated oncogenes. MY5 + + À VEGF activity is mediated mainly through two receptors, KAS-6/1 + + + VEGFR1 and VEGFR2.26,27 Although the affinity of VEGFR1 8226 + + À for its ligand is much stronger than that of VEGFR2, the latter KP6 + + + seems to be responsible for most of the angiogenic effect of ANBL-6 + + + VEGF. A third receptor (VEGFR3 has been identified and U266 + + À appears to be restricted to lymphatic epithelium.

Figure 8 STAT3 and ERK activation in myeloma cell lines following VEGF stimulation.

Leukemia Vascular endothelial growth factor in myeloma S Kumar et al 2030 Bone marrow angiogenesis plays an important role in the demonstrate its presence on myeloma cells from patient samples pathogenesis and progression of MM as with other hemato- as well as on myeloma cell lines. Secretion of VEGF by logical malignancies as well as solid tumors. In the setting of myeloma cells and the simultaneous presence of both VEGFR1 myeloma, the degree of bone marrow angiogenesis as estimated and VEGFR2 receptors on its surface raise the possibility of an by the microvessel density, has been shown to be a powerful autocrine pattern of stimulation in addition to the paracrine prognostic factor for survival.7,8 In a study of 400 patients, the mechanisms discussed previously. The demonstration of degree of bone marrow angiogenesis was found to increase VEGFR2 on the myeloma cells is especially important since progressively across the spectrum of plasma cell proliferative this seems to be the functionally more active receptor. In this disorders.28 particular context, demonstration of its presence strengthens the Secretion of VEGF by the myeloma cells as shown in our biological plausibility for the previously demonstrated effects of study as well as by others is likely to be playing a role in the VEGF on myeloma cells.16 We do want to point out that, while initiation and maintenance of the abnormal angiogenesis. The demonstration of VEGF required only an RT-PCR approach, a proliferative vascular endothelium is responsive to VEGF nested approach was needed for demonstrating the VEGF stimulation and has been shown to secrete IL-6 on stimulation.15 receptors pointing to a low level of expression. However, the IL-6 is a key cytokine regulator of myeloma cell growth29 and is level of expression need not correlate with that of activity for an essential growth factor for myeloma cells. In the bone cytokine receptors. We were able to demonstrate activation of marrow, the stromal cells are capable of secreting IL-6 in the MAPK and STAT pathways in some of the cell lines addition to the vascular endothelial cells. VEGF has been shown following stimulation with VEGF. But the results of the to be capable of stimulating IL-6 secretion by the stromal cells15 functional studies fail to demonstrate a significant stimulatory and is probably responsible for the increased secretion of IL-6 by or antiapoptotic effect for VEGF. Further studies are needed to stromal cells noted when cocultured with myeloma cells.30 IL-6 confirm or disprove an autocrine role for secreted VEGF in the secretion by the bone marrow stromal cells can also be induced disease biology. by other cytokines like IL-1b and TNF-a.31,32 IL-6 in turn has The role of VEGF and its receptors in the biology of myeloma been shown to be capable of stimulating VEGF secretion by as evidenced by our data and other data presents a great myeloma cells.15 Thus enough evidence exists to hypothesize opportunity for using inhibitors of VEGF or its receptors for the presence of a paracrine feedback loop of cytokines that therapy of myeloma. Several novel molecules like SU6668 and stimulate, maintain and possibly lead to progression of the PTK787 are currently undergoing evaluation utilizing this myeloma cell mass. strategy. Demonstration of the presence of VEGFR2 in our study Using multiple methods we have confirmed the increased provides biological explanation for the preclinical activity expression and secretion of VEGF by the myeloma cells using observed with VEGFR2 inhibitors. Results of these clinical trials sorted samples from patients with myeloma as well as several and in vitro studies with these agents will shed more light on the established myeloma cell lines. These results confirm work role of this cytokine and its receptors in the biology of myeloma. 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 Acknowledgements hematologic malignancies. VEGFR1 receptor expression was found in five of the 12 cell lines studied, suggesting the This work is supported in part by the Goldman Philanthropic possibility of an autocrine pathway. Stimulation of vascular Partnerships, IL, USA and Grants CA 93842, CA 85818 and endothelial cells by VEGF led to an increase in the mRNA for CA62242, National Cancer Institute, Bethesda, MD, USA. Dr several hematopoietic growth factors and IL-6. Myeloma cells in Rajkumar is a Leukemia and Lymphoma Society of America the bone marrow were shown to express VEGF, and normal Translational Research Awardee and is also supported by the bone marrow myeloid and monocytic cells surrounding the Multiple Myeloma Research Foundation. 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 References marrow stromal cells. They demonstrated VEGF expression and secretion by myeloma cell lines and plasma cells isolated from 1 Jemal A, Thomas A, Murray T, Thun M. Cancer statistics. CA the marrow of patients with MM. However, VEGF receptors Cancer J Clin 2002; 52: 23–47. VEGFR1 and VEGFR2 expression when evaluated by immuno- 2 Kyle RA. Long-term survival in multiple myeloma. N Engl J Med histochemistry or RT-PCR was found to be weak or absent on the 1983; 308: 314–316. 3 Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF myeloma cells. They found abundant expression of these et al. A prospective, randomized trial of autologous bone marrow receptors by marrow stromal cells and also demonstrated transplantation and chemotherapy in multiple myeloma. Inter- recombinant human VEGF-induced, time- and dose-dependent groupe Francais du Myelome. N Engl J Med 1996; 335: 91–97. increase in IL-6 secretion by stromal and microvascular 4 Folkman J. Seminars in Medicine of the Beth Israel Hospital, endothelial cells. IL-6 is a potent growth factor for myeloma Boston. Clinical applications of research on angiogenesis. N Engl J cells, and these findings demonstrate the existence of a Med 1995; 333: 1757–1763. 16 5 Sezer O, Niemoller K, Eucker J, Jakob C, Kaufmann O, Zavrski I paracrine pathway. Podar et al studied the cellular pathways et al. Bone marrow microvessel density is a prognostic factor for of VEGF stimulation in the myeloma cells and demonstrated survival in patients with multiple myeloma. Ann Hematol 2000; stimulation of tumor cell proliferation by VEGF by a protein 79: 574–577. kinase C-independent Raf-1-MEK-extracellular signal-regulated 6 Rajkumar SV, Fonseca R, Witzig TE, Gertz MA, Greipp PR. Bone protein kinase (ERK) pathway. marrow angiogenesis in patients achieving complete response after Most of the previous studies have failed to demonstrate the stem cell transplantation for multiple myeloma. Leukemia 1999; 16 13: 469–472. presence of VEGFR-2 on these cells, although the presence of 7 Rajkumar SV, Leong T, Roche PC, Fonseca R, Dispensieri A, Lacy VEGFR1 has been consistently demonstrated. By using a more MQ et al. Prognostic value of bone marrow angiogenesis in sensitive RT-nested-PCR method we have been able to multiple myeloma. Clin Cancer Res 2000; 6: 3111–3116.

Leukemia Vascular endothelial growth factor in myeloma S Kumar et al 2031 8 Vacca A, Ribatti D, Roncali L, Ranieri G, Serio G, Silvestris F et al. rearrangement: clinical implications. Cancer Res 1993; 53: 5320– Bone marrow angiogenesis and progression in multiple myeloma. 5327. Br J Haematol 1994; 87: 503–508. 21 Nilsson K, Bennich H, Johansson SG, Ponten J. Established 9 Schneider P, Jerome MV, Paysant J, Soria PC, Vannier JP. The role immunoglobulin producing myeloma (IgE) and lymphoblastoid of angiogenesis in leukemia proliferation. Am J Pathol 1999; 155: (IgG) cell lines from an IgE myeloma patient. Clin Exp Immunol 1007–1008. 1970; 7: 477–489. 10 Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas D 22 Brown LF, Berse B, Jackman RW, Tognazzi K, Manseau EJ, Dvorak et al. Angiogenesis in acute and chronic leukemias and HF et al. Increased expression of vascular permeability factor myelodysplastic syndromes. Blood 2000; 96: 2240–2245. (vascular endothelial growth factor) and its receptors in kidney and 11 Mesa RA, Hanson CA, Rajkumar SV, Schroeder G, Tefferi A. bladder carcinomas. Am J Pathol 1993; 143: 1255–1262. Evaluation and clinical correlations of bone marrow angiogenesis 23 Fiedler W, Graeven U, Ergun S, Verago S, Kilic N, Stockschlader M in myelofibrosis with myeloid metaplasia. Blood 2000; 96: 3374– et al . Vascular endothelial growth factor, a possible paracrine 3380. growth factor in human acute myeloid leukemia. Blood 1997; 89: 12 Bellamy WT, Richter L, Frutiger Y, Grogan TM. Expression of 1870–1875. vascular endothelial growth factor and its receptors in hemato- 24 Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular poietic malignancies. Cancer Res 1999; 59: 728–733. endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13 Di Raimondo F, Azzaro MP, Palumbo G, Bagnato S, Giustolisi G, 13: 9–22. Floridia P et al. Angiogenic factors in multiple myeloma: higher 25 Okada F, Rak JW, Croix BS, Lieubeau B, Kaya M, Roncari L et al. levels in bone marrow than in peripheral blood. Haematologica Impact of oncogenes in tumor angiogenesis: mutant K-ras up- 2000; 85: 800–805. regulation of vascular endothelial growth factor/vascular perme- 14 Sezer O, Jakob C, Eucker J, Niemoller K, Gatz F, Wernecke K et al . ability factor is necessary, but not sufficient for tumorigenicity of Serum levels of the angiogenic cytokines basic fibroblast growth human colorectal carcinoma cells. Proc Natl Acad Sci USA 1998; factor (bFGF), vascular endothelial growth factor (VEGF) and 95: 3609–3614. hepatocyte growth factor (HGF) in multiple myeloma. Eur J 26 de Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams Haematol 2001; 66: 83–88. LT. The fms-like tyrosine kinase, a receptor for vascular endothelial 15 Dankbar B, Padro T, Leo R, Feldmann B, Kropff M, Mesters RM growth factor. Science 1992; 255: 989–991. et al. Vascular endothelial growth factor and interleukin-6 in 27 Terman BI, Dougher-Vermazen M, Carrion ME, Dimirov D, paracrine tumor–stromal cell interactions in multiple myeloma. Armellino DC, Gospodarowicz D et al. Identification of the KDR Blood 2000; 95: 2630–2636. tyrosine kinase as a receptor for vascular endothelial cell growth 16 Podar K, Tai YT, Davies FE, Lentzsch S, Sattler M, Hideshima T factor. Biochem Biophys Res Commun 1992; 187: 1579–1586. et al. Vascular endothelial growth factor triggers signaling 28 Rajkumar SV, Mesa RA, Fonseca R, Schroeder G, Plevak MF, cascades mediating multiple myeloma cell growth and migration. Dispenzieri A et al. Bone marrow angiogenesis in 400 patients Blood 2001; 98: 428–435. with monoclonal gammopathy of undetermined significance, 17 Hitzler JK, Martinez-Valdez H, Bergsagel DB, Minden MD, multiple myeloma, and primary amyloidosis. Clin Cancer Res Messner HA. Role of interleukin-6 in the proliferation of human 2002; 8: 2210–2216. multiple myeloma cell lines OCI-My 1 to 7 established from 29 Michio K, Toshio H, Tadashi M, Tetsuya T, Yasuhiro H, Koji I et al. patients with advanced stage of the disease. Blood 1991; 78: Autocrine generation and requirement of BSF-2/IL-6 for human 1996–2004. multiple myelomas. Nature 1988; 332: 83–85. 18 Westendorf JJ, Ahmann GJ, Greipp PR, Witzig TE, Lust JA, Jelinek 30 Gupta D, Treon SP, Shima Y, Hideshima T, Podar K, Tai YT et al. DF. Establishment and characterization of three myeloma cell lines Adherence of multiple myeloma cells to bone marrow stromal that demonstrate variable cytokine responses and abilities to cells upregulates vascular endothelial growth factor secretion: produce autocrine interleukin-6. Leukemia 1996; 10: 866–876. therapeutic applications. Leukemia 2001; 15: 1950–1961. 19 Matsuoka Y, Moore GE, Yagi Y, Pressman D. Production of free 31 Shalaby MR, Waage A, Espevik T. Cytokine regulation of light chains of immunoglobulin by a hematopoietic cell line interleukin 6 production by human endothelial cells. Cell Immunol derived from a patient with multiple myeloma. Proc Soc Exp Biol 1989; 121: 372–382. Med 1967; 125: 1246–1250. 32 Thomas X, Anglaret B, Magaud JP, Epstein J, Archimbaud E. 20 Jelinek DF, Ahmann GJ, Greipp PR, Jalal SM, Westendorf JJ, Interdependence between cytokines and cell adhesion molecules Katzmann JA et al. Coexistence of aneuploid subclones within a to induce interleukin-6 production by stromal cells in myeloma. myeloma cell line that exhibits clonal immunoglobulin gene Leukemia Lymphoma 1998; 32: 107–119.

Leukemia