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Increased expression of vascular endothelial growth factor in bone marrow of multiple myeloma patients
European Journal of Internal Medicine 14 (2003) 98–100 www.elsevier.com / locate / ejim
Original article
Increased expression of vascular endothelial growth factor in bone marrow of multiple myeloma patients ´ a , *, Grzegorz Mazur a , Pawel« Surowiak b , Piotr Dzie¸giel b , Tomasz Wrobel Lidia Usnarska-Zubkiewicz a , Kazimierz Kuliczkowski a a
b
Department of Hematology, Wrocl«aw Medical University, Pasteura 4, 50 -367 Wrocl«aw, Poland ´ 60 a, 50 -367 Wrocl«aw, Poland Department of Histology and Embriology, Wrocl«aw Medical University, Chal«ubinskiego Received 2 April 2002; received in revised form 29 October 2002; accepted 4 November 2002
Abstract Background: Angiogenesis (neovascularization) is a multistep process in which new blood vessels grow from existing vessels. Angiogenesis is associated with the growth, dissemination, and metastasis of solid tumors. There is increasing evidence that neovascularization may be important in hematological malignancies. Several studies suggest that vascular endothelial growth factor (VEGF) is one of the most important cytokines responsible for the development, maintenance, and progression of multiple myeloma (MM) by promoting bone marrow angiogenesis. A high serum concentration of VEGF has been reported in MM patients. The aim of this study was to evaluate the expression of VEGF in the bone marrow of MM patients. Methods: Eighteen paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MM were evaluated. In addition, 10 bone marrow core biopsy specimens from adult patients without evidence of malignancy were used as controls. Bone marrow sections were stained immunohistochemically for VEGF. Results: Our data show that multiple myeloma is associated with an increased expression of VEGF in the bone marrow. Conclusions: Our observation supports previous studies suggesting that angiogenesis may play a role in the pathophysiology of hematopoietic malignancies. The clinical significance of this phenomenon needs further investigation. However, this study provides rationale for the use of angiogenesis inhibitors in MM therapy. 2003 Elsevier Science B.V. All rights reserved. Keywords: Angiogenesis; VEGF; Multiple myeloma
1. Introduction Angiogenesis (neovascularization) is the multistep formation of new blood vessels from a existing vessels. It involves extracellular matrix remodeling, endothelial cell migration and proliferation, capillary differentiation, and anastomosis formation. Angiogenesis plays a very important role in physiological vascularization during the normal menstrual cycle, and it occurs in pathophysiological conditions such as wound healing, proliferative re*Corresponding author. E-mail addresses: tomasz [email protected] ] [email protected] (P. Dzie¸giel).
(T.
´ Wrobel),
tinopathy, rheumathoid arthritis, and solid tumors [4]. There is increasing evidence that neovascularization may be important in hematological malignancies [5,8]. Multiple myeloma (MM) is a B-lymphoid cell malignancy characterized by a proliferation of plasma cells derived from a single plasma cell clone, mainly within the bone marrow. Plasma cells produce monoclonal immunoglobulin present in serum or in urine. Increased angiogenesis correlates with plasma cell growth in patients with MM and is associated with other B-cell malignancies such as acute lymphoblastic leukemia and non-Hodgkin’s lymphomas [1]. Several positive and negative regulatory cytokines have been reported to be involved in the angiogenic process.
0953-6205 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0953-6205(03)00027-X
´ et al. / European Journal of Internal Medicine 14 (2003) 98–100 T. Wrobel
99
Vascular endothelial growth factor (VEGF), the most potent direct-acting angiogenic protein known, is a endothelial cell-specific mitogen and angiogenic factor that also increases vascular permeability. VEGF overproduction has been identified as a major factor underlying pathological angiogenesis [7]. The purpose of this study was to determine the expression of VEGF in the bone marrow of patients with MM.
Table 1 Evaluation of the reaction results using the ImmunoReactiveScore (IRS) scale
2. Materials and methods
Table 2 The results of VEGF expression
Eighteen paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MM were evaluated. The diagnosis of MM was made according to South West Oncology Group (SWOG) criteria. In addition, 10 bone marrow core biopsy specimens from adult patients without evidence of malignancy were used as controls. In the control patients, bone marrow biopsies were taken for the evaluation of cytopenias or tumor staging. The samples studied were fixed in 10% buffered formalin and then embedded in paraffin. The preparations were stained with hematoxylin and eosin and evaluated histopathologically. Deparaffinized sections were incubated with mouse monoclonal antibodies against VEGF (clone G153-694; Pharmingen, USA) in dilution with 2 mg antibodies / ml. In the next step, streptavidin–biotinylated peroxidase (LSAB2, Dako, Denmark) complex was used and the activity of the latter was estimated using 3,39-diaminobenzidine (DAB, Dako). In each case the negative control was included with primary negative control (Dako) and then with LSAB2 and DAB [6,11]. The intensity of the immunocytochemical procedure was evaluated using the ImmunoReactive Score (IRS) according to Remmele and Stenger [10] (Table 1). The applied scale took into account both the intensity of the color reaction and the percentage of cells that exhibited a positive reaction. The final result represented the product of the two parameters, and its value ranged from 0 to 12. Statistical analysis was done using the Mann–Whitney U-test. Differences were considered statistically significant at P,0.05.
% Positive cells 0 1 2 3 4
Intensity of the reaction
No positive cells ,10% positive cells 10–50% positive cells 51–80% positive cells .81% positive cells
0 1 2 3
No positive reaction Faint color reaction Moderate color reaction Intense color reaction
MM patients
Control
P
Mean % VEGF (1) cells / range
47% / 5–70%
5% / 0–9%
,0.001
Mean intensity of reaction / range
2/ 1–3
1 / 0–1
,0.001
Mean IRS / range
5/ 1–9
1/ 0–1
,0.001
IRS—The ImmunoReactive Score.
0–1). The differences in VEGF expression measured by the IRS scale in MM and control marrows were highly significant (P,0.001). There was no correlation between VEGF expression and the class of monoclonal immunoglobulin. The results of VEGF expression are summarized in Table 2. Clinical data are presented in Table 3.
4. Discussion Multiple myeloma is characterized by the clonal proliferation of malignant plasma cells in the bone marrow associated with bone loss, renal impairment, and paraproteinemia. VEGF plays an important role in angiogenesis by acting as a potent inducer of vascular permeability, as well as by serving as a specific endothelial cell mitogen [7]. The importance of proangiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully understood in hematopoietic neoplasm. Prominent bone marrow neovascularization occurs in MM. It correlates positively with high plasma cell labeling index
3. Results The patients with MM consisted of eight women and 10 men. The control group of patients consisted of five women and five men. The mean age of the MM patients was 64 years (range 48–70 years), while that of the control group was 62 years (range 45–68 years). All patients had active disease in stages II and III, according to Durie and Salmon [3]. The mean percentage of VEGF-positive cells in the myeloma group and in the control group was 47% (range 5–70%) and 5% (range 0–9%), respectively. The mean intensity of the reaction in MM bone marrows was 2 (range 1–3) while in the control group it was 1 (range
Table 3 Clinical data Age (years)
Sex (M / F)
Monoclonal protein
Stage according to Durie and Salmon
MM patients
64 (48–70)
10 / 8
IgGk-9 IgGl-4 IgAk-4 IgAl-1 IgMk-1
IIA-6 IIIA-7 IIIB-5
Normal controls
62 (45–68)
5/5
–
–
100
´ et al. / European Journal of Internal Medicine 14 (2003) 98–100 T. Wrobel
and disease activity and confers a poor prognosis. Plasma levels of various angiogenic cytokines, such as basic fibroblast growth factor (bFGF) and VEGF, are elevated in patients with active myeloma [2,13,14]. On the other hand, plasma levels of VEGF in other B-cell malignancies are not always a reliable marker of angiogenesis [1]. In this study, we demonstrate increased VEGF expression in bone marrow in active MM. These data raise the possibility that VEGF may play a role in the growth of MM. It may occur through either a paracrine or an autocrine mechanism. In MM, VEGF is expressed and secreted by tumor cells as well as by bone marrow stromal cells [2]. There are some reports suggesting that, in MM, VEGF is not only responsible for increased microvessel density in bone marrow, but also shows a direct effect upon plasma cells [9]. Our data show that MM is associated with an increased expression of VEGF in the bone marrow. This observation supports previous studies suggesting that angiogenesis may play a role in the pathophysiology of hematopoietic malignancies. Although antiangiogenic therapy with thalidomide has been reported to have marked activity in MM, the microvascular density of bone marrow did not change significantly in patients with a response [12]. The clinical significance of this phenomenon needs further investigation. However, this study provides rationale for the use of angiogenesis inhibitors in MM therapy.
Acknowledgements This study was supported by grant KBN No. 4P05B07718.
References [1] Belgore FM, Lip G, Bareford D et al. Plasma levels of vascular endothelial growth factor (VEGF) in haematological cancers. Br J Haematol 2000;110:496–7.
[2] Dankbar B, Padro T, Leo R et al. Vascular endothelial growth factor and interleukin-6 in paracrine tumor–stromal cell interactions in mutiple myeloma. Blood 2000;95:2630–6. [3] Durie BGM, Salmon SE. A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer 1975;36:842–54. [4] Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27–31. [5] Hussong JW, Rodgers GM, Shami PJ. Evidence of increased angiogenesis in patients with acute myeloid leukemia. Blood 2000;95:309–13. [6] Hsu SM, Raine L, Fanger H. Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577–82. [7] McMahon G. VEGF receptor signaling in tumor angiogenesis. Oncologist 2000;5:3–10. [8] Perez-Atayde AR, Sallan SE, Tedrow U et al. Spectrum of tumor angiogenesis in bone marrow of children with acute lymphoblastic leukemia. Am J Pathol 1997;150:815–21. [9] Podar K, Tai Y, Davies FE et al. Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration. Blood 2001;98:428–35. [10] Remmele W, Stenger HE. Vorschlag zur einheitlichen definition eines immunreaktiven score (IRS) fuer den immunohistochemischen oestrogenrezeptor–nachweis (ER–ICA) im mammakarzinomgewebe. Pathologe 1987;8:138–40. [11] Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39:741–6. [12] Singhal S, Mehta J, Desikan R et al. Antitumor activity of thalidomide in refractory multiple myeloma. New Engl J Med 1999;341:1565–71. [13] Vacca A, Ribatti D, Presta M et al. Bone marrow neovascularisation, plasma cell angiogenic potential, and matrix metalloproteinase—2 secretion parallel progression of human multiple myeloma. Blood 1999;13:469–72. [14] Vacca A, Ribatti D, Roncali L et al. Bone marrow angiogenesis and progression in multiple myeloma. Br J Haematol 1994;87:503–8.
Original article
Increased expression of vascular endothelial growth factor in bone marrow of multiple myeloma patients ´ a , *, Grzegorz Mazur a , Pawel« Surowiak b , Piotr Dzie¸giel b , Tomasz Wrobel Lidia Usnarska-Zubkiewicz a , Kazimierz Kuliczkowski a a
b
Department of Hematology, Wrocl«aw Medical University, Pasteura 4, 50 -367 Wrocl«aw, Poland ´ 60 a, 50 -367 Wrocl«aw, Poland Department of Histology and Embriology, Wrocl«aw Medical University, Chal«ubinskiego Received 2 April 2002; received in revised form 29 October 2002; accepted 4 November 2002
Abstract Background: Angiogenesis (neovascularization) is a multistep process in which new blood vessels grow from existing vessels. Angiogenesis is associated with the growth, dissemination, and metastasis of solid tumors. There is increasing evidence that neovascularization may be important in hematological malignancies. Several studies suggest that vascular endothelial growth factor (VEGF) is one of the most important cytokines responsible for the development, maintenance, and progression of multiple myeloma (MM) by promoting bone marrow angiogenesis. A high serum concentration of VEGF has been reported in MM patients. The aim of this study was to evaluate the expression of VEGF in the bone marrow of MM patients. Methods: Eighteen paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MM were evaluated. In addition, 10 bone marrow core biopsy specimens from adult patients without evidence of malignancy were used as controls. Bone marrow sections were stained immunohistochemically for VEGF. Results: Our data show that multiple myeloma is associated with an increased expression of VEGF in the bone marrow. Conclusions: Our observation supports previous studies suggesting that angiogenesis may play a role in the pathophysiology of hematopoietic malignancies. The clinical significance of this phenomenon needs further investigation. However, this study provides rationale for the use of angiogenesis inhibitors in MM therapy. 2003 Elsevier Science B.V. All rights reserved. Keywords: Angiogenesis; VEGF; Multiple myeloma
1. Introduction Angiogenesis (neovascularization) is the multistep formation of new blood vessels from a existing vessels. It involves extracellular matrix remodeling, endothelial cell migration and proliferation, capillary differentiation, and anastomosis formation. Angiogenesis plays a very important role in physiological vascularization during the normal menstrual cycle, and it occurs in pathophysiological conditions such as wound healing, proliferative re*Corresponding author. E-mail addresses: tomasz [email protected] ] [email protected] (P. Dzie¸giel).
(T.
´ Wrobel),
tinopathy, rheumathoid arthritis, and solid tumors [4]. There is increasing evidence that neovascularization may be important in hematological malignancies [5,8]. Multiple myeloma (MM) is a B-lymphoid cell malignancy characterized by a proliferation of plasma cells derived from a single plasma cell clone, mainly within the bone marrow. Plasma cells produce monoclonal immunoglobulin present in serum or in urine. Increased angiogenesis correlates with plasma cell growth in patients with MM and is associated with other B-cell malignancies such as acute lymphoblastic leukemia and non-Hodgkin’s lymphomas [1]. Several positive and negative regulatory cytokines have been reported to be involved in the angiogenic process.
0953-6205 / 03 / $ – see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0953-6205(03)00027-X
´ et al. / European Journal of Internal Medicine 14 (2003) 98–100 T. Wrobel
99
Vascular endothelial growth factor (VEGF), the most potent direct-acting angiogenic protein known, is a endothelial cell-specific mitogen and angiogenic factor that also increases vascular permeability. VEGF overproduction has been identified as a major factor underlying pathological angiogenesis [7]. The purpose of this study was to determine the expression of VEGF in the bone marrow of patients with MM.
Table 1 Evaluation of the reaction results using the ImmunoReactiveScore (IRS) scale
2. Materials and methods
Table 2 The results of VEGF expression
Eighteen paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MM were evaluated. The diagnosis of MM was made according to South West Oncology Group (SWOG) criteria. In addition, 10 bone marrow core biopsy specimens from adult patients without evidence of malignancy were used as controls. In the control patients, bone marrow biopsies were taken for the evaluation of cytopenias or tumor staging. The samples studied were fixed in 10% buffered formalin and then embedded in paraffin. The preparations were stained with hematoxylin and eosin and evaluated histopathologically. Deparaffinized sections were incubated with mouse monoclonal antibodies against VEGF (clone G153-694; Pharmingen, USA) in dilution with 2 mg antibodies / ml. In the next step, streptavidin–biotinylated peroxidase (LSAB2, Dako, Denmark) complex was used and the activity of the latter was estimated using 3,39-diaminobenzidine (DAB, Dako). In each case the negative control was included with primary negative control (Dako) and then with LSAB2 and DAB [6,11]. The intensity of the immunocytochemical procedure was evaluated using the ImmunoReactive Score (IRS) according to Remmele and Stenger [10] (Table 1). The applied scale took into account both the intensity of the color reaction and the percentage of cells that exhibited a positive reaction. The final result represented the product of the two parameters, and its value ranged from 0 to 12. Statistical analysis was done using the Mann–Whitney U-test. Differences were considered statistically significant at P,0.05.
% Positive cells 0 1 2 3 4
Intensity of the reaction
No positive cells ,10% positive cells 10–50% positive cells 51–80% positive cells .81% positive cells
0 1 2 3
No positive reaction Faint color reaction Moderate color reaction Intense color reaction
MM patients
Control
P
Mean % VEGF (1) cells / range
47% / 5–70%
5% / 0–9%
,0.001
Mean intensity of reaction / range
2/ 1–3
1 / 0–1
,0.001
Mean IRS / range
5/ 1–9
1/ 0–1
,0.001
IRS—The ImmunoReactive Score.
0–1). The differences in VEGF expression measured by the IRS scale in MM and control marrows were highly significant (P,0.001). There was no correlation between VEGF expression and the class of monoclonal immunoglobulin. The results of VEGF expression are summarized in Table 2. Clinical data are presented in Table 3.
4. Discussion Multiple myeloma is characterized by the clonal proliferation of malignant plasma cells in the bone marrow associated with bone loss, renal impairment, and paraproteinemia. VEGF plays an important role in angiogenesis by acting as a potent inducer of vascular permeability, as well as by serving as a specific endothelial cell mitogen [7]. The importance of proangiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully understood in hematopoietic neoplasm. Prominent bone marrow neovascularization occurs in MM. It correlates positively with high plasma cell labeling index
3. Results The patients with MM consisted of eight women and 10 men. The control group of patients consisted of five women and five men. The mean age of the MM patients was 64 years (range 48–70 years), while that of the control group was 62 years (range 45–68 years). All patients had active disease in stages II and III, according to Durie and Salmon [3]. The mean percentage of VEGF-positive cells in the myeloma group and in the control group was 47% (range 5–70%) and 5% (range 0–9%), respectively. The mean intensity of the reaction in MM bone marrows was 2 (range 1–3) while in the control group it was 1 (range
Table 3 Clinical data Age (years)
Sex (M / F)
Monoclonal protein
Stage according to Durie and Salmon
MM patients
64 (48–70)
10 / 8
IgGk-9 IgGl-4 IgAk-4 IgAl-1 IgMk-1
IIA-6 IIIA-7 IIIB-5
Normal controls
62 (45–68)
5/5
–
–
100
´ et al. / European Journal of Internal Medicine 14 (2003) 98–100 T. Wrobel
and disease activity and confers a poor prognosis. Plasma levels of various angiogenic cytokines, such as basic fibroblast growth factor (bFGF) and VEGF, are elevated in patients with active myeloma [2,13,14]. On the other hand, plasma levels of VEGF in other B-cell malignancies are not always a reliable marker of angiogenesis [1]. In this study, we demonstrate increased VEGF expression in bone marrow in active MM. These data raise the possibility that VEGF may play a role in the growth of MM. It may occur through either a paracrine or an autocrine mechanism. In MM, VEGF is expressed and secreted by tumor cells as well as by bone marrow stromal cells [2]. There are some reports suggesting that, in MM, VEGF is not only responsible for increased microvessel density in bone marrow, but also shows a direct effect upon plasma cells [9]. Our data show that MM is associated with an increased expression of VEGF in the bone marrow. This observation supports previous studies suggesting that angiogenesis may play a role in the pathophysiology of hematopoietic malignancies. Although antiangiogenic therapy with thalidomide has been reported to have marked activity in MM, the microvascular density of bone marrow did not change significantly in patients with a response [12]. The clinical significance of this phenomenon needs further investigation. However, this study provides rationale for the use of angiogenesis inhibitors in MM therapy.
Acknowledgements This study was supported by grant KBN No. 4P05B07718.
References [1] Belgore FM, Lip G, Bareford D et al. Plasma levels of vascular endothelial growth factor (VEGF) in haematological cancers. Br J Haematol 2000;110:496–7.
[2] Dankbar B, Padro T, Leo R et al. Vascular endothelial growth factor and interleukin-6 in paracrine tumor–stromal cell interactions in mutiple myeloma. Blood 2000;95:2630–6. [3] Durie BGM, Salmon SE. A clinical staging system for multiple myeloma. Correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer 1975;36:842–54. [4] Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27–31. [5] Hussong JW, Rodgers GM, Shami PJ. Evidence of increased angiogenesis in patients with acute myeloid leukemia. Blood 2000;95:309–13. [6] Hsu SM, Raine L, Fanger H. Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577–82. [7] McMahon G. VEGF receptor signaling in tumor angiogenesis. Oncologist 2000;5:3–10. [8] Perez-Atayde AR, Sallan SE, Tedrow U et al. Spectrum of tumor angiogenesis in bone marrow of children with acute lymphoblastic leukemia. Am J Pathol 1997;150:815–21. [9] Podar K, Tai Y, Davies FE et al. Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration. Blood 2001;98:428–35. [10] Remmele W, Stenger HE. Vorschlag zur einheitlichen definition eines immunreaktiven score (IRS) fuer den immunohistochemischen oestrogenrezeptor–nachweis (ER–ICA) im mammakarzinomgewebe. Pathologe 1987;8:138–40. [11] Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991;39:741–6. [12] Singhal S, Mehta J, Desikan R et al. Antitumor activity of thalidomide in refractory multiple myeloma. New Engl J Med 1999;341:1565–71. [13] Vacca A, Ribatti D, Presta M et al. Bone marrow neovascularisation, plasma cell angiogenic potential, and matrix metalloproteinase—2 secretion parallel progression of human multiple myeloma. Blood 1999;13:469–72. [14] Vacca A, Ribatti D, Roncali L et al. Bone marrow angiogenesis and progression in multiple myeloma. Br J Haematol 1994;87:503–8.