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NEOPLASIA
From the Departments of Leukemia, Hematopathology, and
Bioimmunotherapy, University of Texas, M. D. Anderson
Cancer Center, Houston, TX.
Angiogenesis has been associated with the growth, dissemination,
and metastasis of solid tumors. The aims of this study were to evaluate
the vascularity and the levels of angiogenic factors in patients with
acute and chronic leukemias and myelodysplastic syndromes (MDS). The
numbers of blood vessels were measured in 145 bone marrow biopsies and
the levels of vascular endothelial growth factor (VEGF), basic
fibroblast growth factor (bFGF), tumor necrosis growth factor- Angiogenesis is the formation of new blood vessels
from an existing vasculature.1 It involves degradation of
extracellular matrix proteins and activation, proliferation, and
migration of endothelial cells and pericytes in a multistep
process.2-4 In addition to its physiologic role in
vascularization during ovulation, placentation, and
embryogenesis,1 angiogenesis has been associated with the
growth, dissemination, and metastasis of solid tumors.5-7 Several positive and negative regulatory molecules have been reported to be involved in the angiogenic process.8 The 2 most
potent and specific positive regulators are vascular endothelial growth factor (VEGF)8,9 and basic fibroblast growth factor
(bFGF).8,10-13 Other cytokines such as tumor necrosis
factor- Little is known about angiogenesis and angiogenesis-related molecules
in leukemia. The normal vascular bed in bone marrow forms a sinusoidal
network supporting the hematopoietic cells, similar to cellular support
in other organs such as kidney and spleen.28 Perez-Atayde
and colleagues29 studied 61 bone marrow biopsies (BMBs) of
40 children with untreated acute lymphoid leukemia (ALL) and 10 control
biopsies.29 They found a significantly higher bone marrow
microvessel density in ALL as well as a higher level of urinary bFGF.
Increased vascularity was also reported in 20 bone marrow samples from
patients with acute myeloid leukemia (AML).30 Expression
of VEGF in leukemic cells of patients with AML was found by Fiedler and
colleagues31 and Hussong and coworkers.30 We
have reported that intracellular levels of VEGF in AML32 and chronic lymphocytic leukemia (CLL)33 are of
prognostic significance. These data suggest that angiogenesis may have
a role in the pathophysiology of leukemias and that antiangiogenesis
therapy could have an anticancer effect.34 In this study,
we expanded on these observations by evaluating vascularity in BMBs of
patients with acute leukemias, chronic leukemias, and myelodysplastic
syndromes (MDS). We measured the number of blood vessels in BMBs and
the plasma levels of VEGF, bFGF, HGF, TNF- Blood vessels in the bone marrow
Immunohistochemical preparation.
All blood vessels were highlighted by staining endothelial cells with
anti-factor VIII (FVIII)-related antigen antibody using a standard
immunoperoxidase technique described
previously.35-37 Factor VIII-related antigen
antibodies were purchased from Dako Corporation (Santa Barbara, CA) and
used at a dilution of 1:400.
Measurement of bone marrow microvessel density.
Microvessel density was assessed blindly. All BMBs were evaluated for
cellularity by light microscopy with a 10 × power ocular lens. Five
cellular representative areas were chosen randomly and examined by
20 × power objective lens. Pictures of the 5 fields were digitized.
Individual microvessels (stained in brown) were counted in each field,
and the vascular area was measured using National Institutes of Health
shared image analysis software. The relationship between the total area
of blood vessels selected (expressed in squared pixels) and the total
picture taken by 20 × power objective lens (313 956 squared pixels)
was calculated and expressed as a percentage. The average number of
blood vessels and average area of the blood vessels were obtained for
the 5 fields. Neither vessel lumens nor red blood cells in vessel
lumens were used to define a blood vessel in the absence of FVIII
staining. Megakaryocytes were stained with FVIII but were easily
distinguishable and not counted.
Measurement of angiogenic factors
Plasma and serum collection.
Plasma and serum samples from 417 patients with newly diagnosed or
relapsed AML, ALL, MDS, CLL, chronic myeloid leukemia (CML), and
chronic myelomonocytic leukemia (CMML) were collected and stored
according to approved protocols. Consent forms were obtained according
to institutional guidelines. These samples were used to measure various
angiogenic factors and compared with 11 healthy individuals used
as controls.
Enzyme-linked immunosorbent assay.
The enzyme-linked immunosorbent assays (ELISAs) for VEGF, bFGF, HGF,
and TNF- Statistical analysis
Increased vascularity in ALL, AML, CML, and MDS The 129 BMBs from patients with leukemia or MDS were evaluated and compared with 16 control marrows. Twenty-three patients had CLL, 20 had ALL, 24 had CML, 30 had AML, and 32 had MDS. Of the patients with MDS, 1 had refractory anemia (RA), 3 had RA with ringed sideroblasts (RARS), 9 had RA with excess blasts (RAEB), 5 had RA with excess blasts in transformation (RAEBt), and 14 had CMML. Blood vessels were well visualized using FVIII staining (Figure 1) and easily distinguishable from megakaryocytes.
Table 1 shows the peripheral blood and
bone marrow characteristics of the leukemia patients and the control
group. A good correlation between vascular area size and number of
blood vessels was found in control marrows and in marrows of leukemia
patients (R = 0.84 and 0.77, respectively;
P < .001 for both).
We found a significant increase in vascularity in CML, AML, ALL, and
MDS patients but not in CLL patients as compared with the controls
(P < .05). The relative vascular area was
marginally increased in MDS (Table 2;
Figure 2). We did not find a significant difference between ALL and AML. When CMML patients were considered separately from MDS patients, their vascularity was significantly higher than that of the control group (0.04) but was not different from
that of the other MDS (RA, RARS, RAEB, and RAEBt) patients.
Differences between plasma and serum levels of VEGF and FGF Initially we tested the differences in VEGF and bFGF levels between plasma and serum in 67 patients. The median platelet count in this group of patients was 173 × 109/L (range, 9-890). VEGF levels were significantly higher in the serum as compared with plasma (P < .001, Kruskal-Wallis test). The median VEGF in plasma was 49.9 pg/mL (range, 24.1-2767.2 pg/mL); it was 163.5 pg/mL (range, 27.6-2461.9 pg/mL) in serum. There was no significant difference (P = .13) in the levels of bFGF between plasma and serum, 8.5 pg/mL (range, 4.4-465 pg/mL) and 7.36 pg/mL (range, 3.7-487 pg/mL), respectively. Overall VEGF levels in plasma and serum correlated with platelet counts (Spearman R = 0.68, P < .001, and 0.5, P < .001, respectively). We found a similar correlation with total white blood cells (WBC). The bFGF levels in plasma and serum also correlated with platelets (R = 0.5, P < .001, and R = 0.32, P = 0.01, respectively), but there was no correlation between bFGF plasma or serum and WBC (P = .14 and P = .49, respectively). High levels of VEGF have been reported in platelets, and it is possible that during the clotting process and the separation of the serum, VEGF is released from the platelets and WBC leading to the detection of high levels.38,39 High levels of bFGF have been reported in platelets, as demonstrated above, without significant effects on the levels of bFGF during clotting. This may suggest that the mechanisms responsible for releasing the VEGF from platelets during serum separation are different from those for bFGF. The other possibility is that the VEGF levels in serum are affected by its release from the WBC, whereas bFGF is not released from the cells. This discrepancy between serum and plasma levels of VEGF has been reported by other investigators,38,39 who recommended the use of plasma rather than serum for analyzing these angiogenic factors. This is an important issue in leukemias and MDS because of the significant variation in the number of platelets among leukemia patients. Measurements of all angiogenic factors in this study were performed using plasma rather than serum.Elevated levels of angiogenic factors in leukemia and MDS Levels of VEGF, bFGF, HGF, TGF- , and TNF- in plasma samples
collected from patients with various leukemias and MDS were evaluated
before therapy or during relapsed disease. These levels in each disease
are shown in Table 3. Except for TGF-,
levels of these factors were significantly higher in patients with
leukemia and MDS. TGF- was not detectable in any of the normal
samples despite the high sensitivity of the assay (25 pg/mL). Rare
samples of leukemias showed expression of TGF-, but overall there
was no significant increase in TNF- in leukemia or MDS
(Table 3).
There was a significant difference in the levels of VEGF
(Figure 4) and bFGF (Figure
5) among groups (P < .001).
CML patients had the highest levels of VEGF, and CLL patients had the
highest levels of bFGF. Except for ALL patients, all groups displayed a
significantly higher level of VEGF compared with healthy controls, with
CML and CMML patients showing the highest levels of VEGF (Table 3).
There was no significant difference in VEGF levels between AML and MDS patients, but there was a significant difference between AML and CMML patients (P = .1). The bFGF levels were also significantly higher in all diseases compared with healthy controls (Table 3 and Figure 5), but the highest levels were detected in CLL (Table 3). There was a direct correlation between VEGF and bFGF plasma levels in CLL, CML (P < .001 for both), and AML (P = .007) patients. No correlation was found between the 2 angiogenic factors in ALL (P = .44), MDS (P = .17), or CMML (P = .32). The level of HGF was also increased in leukemia and MDS (Figure
6) as compared with normal samples
(P < .0001, Kruskal-Wallis test). However, the highest
levels were detected in CMML (1444 pg/mL; range, 448-8657.4) (Table 3).
The level of TNF-
To test the reproducibility of ELISA, 44 random samples were assayed in
duplicate and a Spearman R correlation of 0.98 was demonstrated. To test the stability of the angiogenic factors in
plasma, we used 63 random samples after thawing and refreezing and
tested for the levels of VEGF. The Spearman R correlation was 0.97. All values obtained in this study were in the linear ranges
of the ELISA, reported by the manufacturer to be 31.2 to 2000 pg/mL for
VEGF, 5 to 640 pg/mL for bFGF, 15.6 to 1000 pg/mL for TNF-
Angiogenesis has a major role in tumor growth, dissemination, and metastasis in solid tumors.5-7 Clearly, angiogenic factors and angiogenesis play a significant role in the course and disease process of some leukemias. The reported increased vascularity in pediatric ALL,29 AML,30 and MDS40; the prognostic importance of VEGF in AML32; and the detection of angiogenic factor receptors in leukemia cell lines41 suggest that angiogenic factors may have a direct effect on marrow vascularity as well as on leukemic cells. In our study we observed a significant increase in the number of blood vessels in CML, AML, ALL, and MDS, and a borderline increase in the relative vascular area in ALL, MDS, and AML. When we compared bone marrow cellularity with vascularity, there was no correlation, and vascularity appeared to be independent of cellularity. Increased vascularity cannot be explained by the relative increase in cellularity in marrow alone (median cellularity in controls = 20% versus 85% in ALL, 80% in AML, and 90% in CML) (Figure 1). Furthermore, there was no increase in vascularity in CLL bone marrow, despite cellularity. This suggests that vascularity in hematologic malignancies is an active and controlled process. Increased vascularity was associated with a significant increase in
angiogenic factors, including VEGF, bFGF, TNF- Patients with CMML showed VEGF levels similar to CML, which may reflect the proliferative nature of the CMML despite its frequent classification as a subgroup of MDS. The clinical significance of marrow vascularity and plasma levels of angiogenic factors individually or combined in leukemia and MDS needs further investigation and may suggest novel therapeutic approaches in these diseases. Several new antiangiogenic agents are now available, which may have role in treating leukemia and MDS. In summary, our data suggest that angiogenic factors play a significant role in the leukemic process. Understanding their roles may help in designing new therapeutic strategies for leukemias and MDS.
Submitted October 19, 1999; accepted May 15, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Maher Albitar, Department of Hematopathology, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 72, Houston, TX 77030-4095; e-mail: malbitar{at}mdanderson.org.
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S. Pillozzi, M. F. Brizzi, P. A. Bernabei, B. Bartolozzi, R. Caporale, V. Basile, V. Boddi, L. Pegoraro, A. Becchetti, and A. Arcangeli VEGFR-1 (FLT-1), {beta}1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome Blood, August 15, 2007; 110(4): 1238 - 1250. [Abstract] [Full Text] [PDF]
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T. A. Navas, M. Mohindru, M. Estes, J. Y. Ma, L. Sokol, P. Pahanish, S. Parmar, E. Haghnazari, L. Zhou, R. Collins, et al. Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors Blood, December 15, 2006; 108(13): 4170 - 4177. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(TGF-
82°C. Patient
samples were added to separate microplates each containing a specific
monoclonal antibody. The mixtures were incubated at room temperature
for 2 hours. The plates were washed 3 times to remove any unbound
substances. Enzyme-linked polyclonal antibodies specific for each
protein were added to the wells, and the mixtures 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 450-nm wavelength for the VEGF, bFGF, HGF, and TNF-
