mia (ALL) and 15 chronic lymphocytic leukemia (CLL) before therapy and 14 ... indicate that angiogenesis in B-cell lymphocytic leukemias likely to represent an ...
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χχ χχ χχ x Haema 2006; 9(0): 301-305 Review article
B-cells from lymphocytic leukemias are capable of synthesis of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor (b-FGF) S. Aref 1, O. Salama1, S. Shamaa2, T. Goda2, H. Mourkos1
Hematology Unit clinical pathology Department, Oncology Department, Internal Medicine Department, Mansoura Faculty of Medicine, Mansoura University, Mansoura, Egypt
Abstract. Angiogenesis is a crucial event in the survival and progression of solid as well as hemopoietic
malignancies. To determine whether angiogenesis in lymphoid leukemia is an intrinsic property of leukemic cells, Peripheral blood mononuclear B cell lineage were separated from 25 acute lymphoblastic leukemia (ALL) and 15 chronic lymphocytic leukemia (CLL) before therapy and 14 ALL, 9 CLL during remission and cultured for 24 hs. Culture supernatans were assayed for the secretion of VEGF and b-FGF using enzyme linked immunosorbent assay. These data demonstrated that leukemic B-cells isolated from CLL and ALL patients at diagnosis spontaneously secrete both VEGF and b-FGF (55.1;7.0-295, 68.2,5.0-400.0 respectively) in CLL and (70.4; 20.4-130 ;99.1,37.4-172.0 respectively) in ALL. The pretreatment b-FGF and VEGF levels were significantly higher as compared to that in remission both in ALL and CLL(P97% clonal B-cells. These purified B cells were then used immediately for laboratory studies.
Cell preparation and culture Purified B cells were cultured in RPMI containing 10% FCS at density of 1x106 cells per well in 24 well plates for 24 hs prior to collection of cell free supernatants. We assessed the levels of VEGF and b-FGF in the culture subernatant. We used PBMC from healthy donors as controls. Enzyme-linked immunosorbent assay. The culture medium (200 µL) harvested from CLL and ALL B-cells cultured under normoxic conditions was tested for the presence of soluble VEGF protein and bFGF by a commercial human VEGF immunoassay kit (Quantikine) according to the manufacturer’s protocol (R&D Systems, Abingdon, UK). Briefly, culture supernatant were added to separate microplates each containing a specific mono-
B-cells from lymphocytic leukemias are capable of synthesis of vascular endothelial growth factor(VEGF) and basic fibroblast growth factor (b-FGF)
clonal antibody. The mixtures were incubated at room temperature for 2 hours. The plates were washed 8 times to remove any unbound substances. Enzyme-linked polyclonal antibodies specific for each protein were added to the wells, and the mixture were incubated at room temperature for 2 hours followed by another washing to remove any unbound antibody or enzyme reagent. A substrate solution was added to the wells, and a blue color developed. The intensity of the blue was proportionate to the amount of cytokine bound in the initial step. The color development was stopped and the intensity of the color was measured and compared with a standard curve. Reading was done at wavelength as stated by manufacturer.
Statistical analysis Statistical analysis was done by using Excel program and SPSS version 10 (statistical package of social science). First part of the data was to test the distribution of the data. The data was of non parametric style ; therefore Mann-Whitny
U test was used to do statistical difference between 2 groups. Significance was considered when P value < 0.05.
Results I-CLL and ALL B-cells secrete VEGF and b-FGF in culture medium We determined the secretion VEGF and b-FGF from clonal B-cells isolated from CLL patients (n= 15), and ALL (n=25) at diagnosis. The patient group included in this study were from Rai stages II, III, IV, and ALL FAB L1, L2. We assessed the levels of VEGF and b-FGF in the medium of CLL-B cells, that have been freshly isolated and incubated without in vivo stimulation for 24 hs. Table 2. shows the data obtained from these experiments. These data demonstrate that B-CLL and ALL B-cells spontaneously secrete VEGF and b-FGF which are significantly higher as compared to the levels at remission and in normal control (P0.05 for all) (Table 2).
Table 1. Descriptive data of the investigated patients and control group.
Splenomegaly (%) Hepatomegaly (%) Lymphadenopathy(%)
36.0 7.0 36.0 7..0 68.0 0.0
B-CLL Diagnosis (n=15) Remission(n=9) 47(40-64) 8/7 II 4 II 4 III 8 III 5 IV 3 86.0 44.0 73.0 33.0 60.0 55.0
Follow up period (month) Hemoglobin (G/dl) median (range TLC (Χ10 9/L) median (range) Platelets (Χ109/L) median(range)
6-9 9.4(9-12.7) 11.5(11 -13.2)
7-16 6.7(8.2-11.6) 10.9(10.5-12.7)
13.4(12.8-15.0)
34.0(29-83) 8.6(5.5-12.0)
132(70-180) 20(18-40)
7.5(4.5-9.0)
66(20-89) 180(170-280)
118(40- 130) 180 (110-270)
210 (185-350)
PB blast cells (%) median (range) PB lymphocytes (%) median (range) BM Lymphocytes (%) Lymphoblast (%)
90±15% 0.0
-
-
-
78.0(48-95%) 18(23-50)
23(18-32)
75(64-98%) 0.5(0-.1.0)
85.0(40- 100) 31(18- 55)
-
Age (years) median (range) Sex (male/female) FAB/Rai stages
B-ALL Diagnosis(n=25) Remission (n=14) 39(20-44) 16/9 L1 9 7 L2 16 7
Control (n=13) 26(18-61) 9/4 -
PB: peripheral blood, TLC: total leucocytic count
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S. Aref et al
II-Levels of VEGF and b-FGF levels in culture medium at diagnosis in B - CLL and B-ALL subdivision. The median level of VEGF and b-FGF were significantly higher in advanced Rai stage IV, followed by III, then II and the difference was statistically significant. (P< 0.01 for both). Also the levels of VEGF and b-FGF in CLL patients were significantly higher at diagnosis as compared to their levels in remission (P0.05 CLL Rai stages II III IV
304
32.0(7.0-36) 144.0(48.0-198.0) 186.1(50.0-295.0) P
