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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 768-770
BRIEF REPORT
Clinical relevance of intracellular vascular endothelial growth
factor levels in B-cell chronic lymphocytic leukemia
Alvaro Aguayo,
Susan O'Brien,
Michael Keating,
Taghi Manshouri,
Cristi Gidel,
Bart Barlogie,
Miloslav Beran,
Charles Koller,
Hagop Kantarjian, and
Maher Albitar
From the Departments of Leukemia and Hematopathology, University of
Texas MD Anderson Cancer Center, Houston, TX, and the
Myeloma and Transplantation Research Center, University of Arkansas for
Medical Sciences, Little Rock, AR.
 |
Abstract |
Strong evidence exists for an association between high vascular
endothelial growth factor (VEGF) levels and poor prognoses in patients
with solid tumors and acute leukemia. Using Western blot analysis and
solid-phase radioimmunoassay, we measured cellular VEGF levels in
B-cell chronic lymphocytic leukemia (CLL) samples from 225 patients and
correlated these levels with disease characteristics and prognoses. The
median VEGF level in CLL samples was 7.26 times the median level
detected in normal peripheral blood mononuclear cells. Patients with
lower levels of VEGF protein showed a trend toward shorter survival
(P = .07). However, in a subgroup of CLL patients with good
prognoses or early-stage disease (Rai stages 0-II, Binet stages A,B;
 2-M  2.8 mg/dL), lower levels of VEGF were associated with
shorter survival times. For the entire group of patients, no
correlation was found between VEGF levels and 2-M levels or Rai and
Binet stage. Most samples from patients with CLL expressed the 43-kd
VEGF isoform in addition to the commonly expressed 45-kd isoform.
It remains to be seen whether the expression of the 43-kd isoform
is responsible for this reversed correlation with outcome.
(Blood. 2000;96:768-770)
© 2000 by The American Society of Hematology.
| |
Introduction |
Vascular endothelial growth factor (VEGF) is a
potent mitogen for microvascular and macrovascular endothelial cells
derived from arteries, veins, and lymphatics, and overexpression has
been correlated with increased angiogenesis.1-4 Because of
this characteristic, VEGF has been associated with tumor growth,
invasion, and metastasis in solid tumors.5-7 The gene for
human VEGF has been localized to chromosome 6p21.3.8 It is
a single gene containing 8 exons and 7 introns.9
Alternative splicing produces VEGF isoforms with sequences of 121, 165, 189, and 206 amino acids.9 The 165-amino acid isoform
(VEGF165) appears to be the most abundantly expressed
isoform in most cells.10 VEGF isoforms 121 and 165, which
have no or low heparin-binding activity, are secreted by tumor
cells.11 VEGF mRNA is markedly up-regulated in many cancer cells and in tumor-infiltrating lymphocytes.10 VEGF was
originally cloned from HL60 leukemia cells12 and, more
recently, was found to be expressed in other leukemic cell
lines,13-15 but the role of angiogenesis and
angiogenesis-regulatory molecules has not been investigated extensively
in patients with leukemia. Perez-Atayde et al16 found that
children with acute lymphoid leukemia (ALL) had increased angiogenesis
in the bone marrow and high urinary basic fibroblast growth factor.
Fiedler et al14 found VEGF transcription in a high
percentage of patients with acute myeloid leukemia (AML). In a series
of 99 patients with newly diagnosed AML, we found that low levels of
intracellular VEGF protein were associated with higher rates of
complete response (CR) rate and survival (P < .05).15 Salven et al reported that high level
of serum-VEGF in patients with non-Hodgkin lymphoma correlated with
shorter survival times and other poor prognostic features. We studied intracellular VEGF levels in peripheral blood samples from patients with CLL using Western blot and solid-phase radioimmunoassay (RIA).
| |
Study design |
Peripheral blood samples were obtained from 225 patients
diagnosed with B-cell CLL. Diagnosis was based on morphology, flow cytometry analysis (CD5+, CD19+, CD23+), and molecular evaluation. Mononuclear cells from the peripheral blood of 31 normal persons were
used as controls. In 8 patients CD19 and CD3 cells were isolated using
magnetic beads and MACS columns (Miltenyi Biotec, Auburn, CA). The
sorted cells were confirmed to be more than 90% pure B and T cells
using CD20 and CD7, respectively.
Protein was extracted and analyzed by Western blot, as previously
described.17 Quantification of VEGF protein was determined using solid-phase RIA, as previously
detailed.17
| |
Results and discussion |
The clinical characteristics of the 225 patients with CLL are
summarized in Table 1. All analyzed samples
contained at least 70% lymphocytes.
Overexpression of cellular VEGF in CLL samples was
demonstrated on Western blots, whereas normal peripheral blood showed
no detectable VEGF protein (Figure 1A).
Most of the CLL patients expressed mainly the 43-kd VEGF isoform,
though low-level expression of the 45-kd isoform was also seen (Figure
1A). The patterns of expression in most CLL patients were different
from those seen in patients with chronic myeloid leukemia (CML) or
acute myeloid leukemia (AML). Some samples expressed trace amounts of
the 57-kd and 38-kd isoforms. Solid-phase-RIA was used for
quantification. The median number of counts per minute (cpm) in 31 samples of peripheral blood mononuclear cells obtained from normal
persons was assigned a value of 1, and levels for the various samples were expressed relative to the control median. By this method, the
median value of VEGF for the CLL patients was 7.26 cpm (range, 2.45-17 cpm), indicating significantly higher levels of cellular VEGF in CLL
patients than in the normal group (P < .001, Kruskal-Wallis test). All analyzed samples contained more than 70% CD19+/CD5+ cells.
To test whether the presence of residual normal cells in the analyzed
samples would affect the overall values of VEGF, we sorted CD19+ and
CD3+ positive cells from the monocytes and compared VEGF levels of
these 3 subpopulations to those obtained from unfractionated samples.
Using the Kruskal-Wallis test, we found no significant difference in
values obtained from an unfractionated sample and the CD19+ cell
population. In contrast, VEGF values of unfractionated cells were
significantly different from those obtained from CD3+ cells and
monocytes (P = .04 and .02, respectively).
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| Fig 1.
Expression of VEGF protein in CLL and
correlation with survival.
(A) Western blot analysis showed that the patterns of expression in
most samples from patients with CLL were different from those seen in
plasma and cells from patients with CML and AML.
The VEGF protein expressed in CLL samples was mainly the 43-kd and
45-kd isoforms. Some samples expressed trace amounts of the 57-kd and
38-kd isoforms. (B) Patients with VEGF levels above the median had
slightly better survival times (P = .07) when all patients with
CLL were compared. (C) Higher levels of VEGF reflected better survival
times in the subgroup of patients with low 2-M (good prognosis
group). (D) High levels of VEGF correlated with better survival times
in the subgroup of patients with early disease by Rai stages 0 to II.
|
|
VEGF levels correlated directly with absolute lymphocyte counts (R =  .08; P = .05) and inversely with white blood cell counts (R = .12; P = .06). Although a correlation was not found
between VEGF and other prognostic covariates, including Rai or Binet
stages, a trend for a direct correlation was noted for VEGF and
2-microglobulin (R = .01; P = .15). Analysis of
survival revealed better survival times in patients with cellular VEGF
levels above the median7.26 than below the median, though
this difference did not reach statistical significance (P =
.07) (Figure 1B).
The effect of VEGF level on survival was evaluated after dividing
patients into groups with high 2-M (poor prognosis) and low
2-M (good prognosis) concentrations. VEGF levels in patients with
high 2-M levels did not correlate with survival (P = .51). However, low VEGF levels (less than 7.26) correlated with worse outcomes in patients with low levels of 2-M (P = .04)
(Figure 1C).
This was confirmed using Rai and Binet stages; low levels of VEGF
correlated with shorter survival times in patients with Rai stages 0 to
II disease (Figure 1D) and Binet stages A and B disease (P =
.006 for both). No effect of VEGF was noticed for the subgroup of
patients with more advanced disease, Rai stage III or IV (P = .69) and Binet stage C (P = .46). Most (192 of 225) of our
patients were previously untreated. Analysis of the previously
untreated patients showed survival patterns similar to those of the
entire group. On comparing VEGF levels with duration of disease, as
measured from the time of diagnosis to the time of sample collection
and analysis, we found reversed correlation (R = .119; P
= .07). This suggests that VEGF protein expression decreases as CLL progresses.
The exact role of VEGF protein in the growth and proliferation of the B
cells of CLL is unclear. Our demonstration of an inverse relationship
between VEGF expression and survival time, particularly in the subgroup
of patients with early disease, was not expected. Although further
study and evaluation of more homogeneously treated patients is
necessary, the current data suggest that VEGF levels may help identify
patients with early-stage disease who are at high risk for disease
progression and death.
Most studies have found a correlation between high VEGF levels and poor
survival times in patients with solid tumors.18-22 Occasional studies, including studies of pancreatic tumor,
squamous cell head and neck carcinoma, and breast cancer, showed
no correlation between VEGF expression and
survival.7,19,20 We found a correlation between high levels
of VEGF and poor prognosis in the bone marrow of patients with
AML,15 but the inverse correlation between VEGF and
survival in CLL is interesting and requires further investigation. The
possibility that different isoforms of VEGF may have different functions must be explored. We are investigating whether the
43-kd isoform is secreted. Of interest, we found no significant
increase in vascularity in the bone marrow of patients with
CLL.23 In conclusion, VEGF levels in patients with B-cell
CLL are high, and they help predict disease behavior in a
subgroup of patients with early-stage disease for whom prognostic
factors are scarce.
| |
Footnotes |
Submitted May 19, 1999; accepted March 1, 2000.
Reprints: Maher Albitar, Department of Hematopathology, The
University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard,
Box 72, Houston, TX 77030-4095.
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.
| |
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Top
Abstract
Introduction