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Indian Pediatr 2013;50:
659-662 |
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Serum Vascular Endothelial Growth Factor-A
Levels During Induction Therapy in Children with Acute
Lymphoblastic Leukemia
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Manas Kalra, Veronique Dinand, *Sangeeta Choudhary, Anupam Sachdeva
and Satya Prakash Yadav
From Pediatric Hematology Oncology and BMT, Department of Pediatrics,
Institute of Child Health; and *Department of Research,
Sir Ganga Ram Hospital, New Delhi, India.
Correspondence to: Dr Satya P Yadav, Pediatric Hematology Oncology
and BMT Unit, Department of Pediatrics, Institute of Child Health, Sir
Ganga Ram Hospital, Delhi 110 060, India.
Email: [email protected]
Received: August 18, 2012;
Initial Review: September 10, 2012;
Accepted: November 22, 2012.
PII: S097475591200746
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Objective: To evaluate serum vascular endothelial growth factor
(VEGF) levels in children with acute lymphoblastic leukemia (ALL) during
the induction phase of chemotherapy.
Design: Prospective sudy.
Setting: Hospital-based study over 18 months.
Patients: 30 children with ALL and 17 healthy
age- and sex-matched controls.
Intervention: Serum concentration of VEGF-A–165
isoform (s-VEGF) was measured by enzyme-linked immunoabsorbant assay at
diagnosis and at the end of induction chemotherapy.
Main outcome measures: s-VEGF was compared with
markers of tumor burden. Kinetics of s-VEGF was assessed in response to
induction chemotherapy.
Results: Median VEGF was significantly lower in
untreated patients than in controls (17.0 vs. 42.6 pg/mL, P=0.004).
s-VEGF levels were fairly correlated with WBC count (r=-0.56, P=0.004)
and LDH (r=-0.52, P=0.02) at diagnosis. All patients but one were
in morphologic remission at the end of induction. Median s-VEGF
concentration on day 29/33 was significantly higher than on day 1 (44.2
pg/mL, P=0.009).
Conclusion: Untreated children with ALL have
significantly lower s-VEGF concentration than controls. At the end of
the induction therapy, s-VEGF increased to levels similar to controls.
The role of ligand-receptor interaction between VEGF and VEGF receptors
on leukemia cells needs to be further delineated.
Key words: Angiogenesis, hematological malignancy, VEGF.
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Vascular Endothelial Growth Factor (VEGF) is an
essential regulator of physiologic and pathologic angiogenesis. VEGF
over-expression is associated with tumor growth, invasion and metastasis
in malignancies, especially solid tumors [1]. VEGF is also thought to
play some role in etiopathogenesis of leukemia and multiple myeloma, as
dysregulation of VEGF expression and signaling pathways are seen in
these hematologic malignancies [2]. Direct and indirect inhibition of
VEGF and its receptors may provide a potent novel targeted therapy to
overcome resistance to chemotherapy [3].
Data on VEGF in acute lymphoblastic leukemia (ALL)
patients are limited and studies show conflicting results. VEGF 165
is the principal isoform of VEGF-A, the prototype member of the VEGF
family. We prospectively evaluated the levels of serum VEGF165
(s-VEGF) in children with ALL during the induction phase of
chemotherapy, in order to assess whether s-VEGF correlates with risk
stratification and with treatment response.
Methods
This prospective study was performed on newly
diagnosed children 1-18 years of age with ALL in the Pediatric
Hematology and Oncology Unit of Sir Ganga Ram Hospital between September
2008 and February 2010. A sex-and age-matched control population was
taken. The diagnosis of ALL was based on bone marrow morphology and
flowcytometry. Demographic data of cases and controls were recorded.
Written informed consent was taken from parents/guardians of cases and
controls. Ethical committee clearance was obtained from the institution
before starting the study.
High-risk ALL was defined as white blood cell (WBC)
>1,00,000/mm 3, translocation
t(9;22) or Bcr-Abl recombination, t(4;11) or MLL gene rearrangement,
prednisolone poor response (peripheral blasts >1000/ mm3
on day 8 or absence of morphological remission at the end of induction
(bone marrow blasts >5%). Patients with none of the above were
classified as standard risk group. UKALL-XI protocol (29 day-induction)
was used for standard risk patients and BFM-95 protocol (33
day-induction) for high-risk patients. Complete remission was defined by
the presence of <5% blasts in the bone marrow and cerebrospinal fluid
clear of malignant cells at the end of induction therapy.
Blood samples were obtained on Day 1 and Day 29/33 in
ALL cases and once in controls. Serum was separated and stored at -70ºC
to avoid loss of bioactive human VEGF. The Assay Designs Human VEGF
Enzyme Immunometric Assay kit was used to assess serum concentration of
VEGF 165.
Mann-Whitney U-test or Kruskal-Wallis test were
applied to compare initial s-VEGF level in patients and controls, and
between various patient groups. Correlation of s-VEGF level with other
continuous variables at diagnosis was done by Spearman’s correlation
coefficient. Comparison of s-VEGF on day 1 and on day 29/33 was done by
Wilcoxon signed-rank test.
Results
The study group comprised of 30 children with ALL (Table
I and Web Table I). The control group had 17
healthy children (9 males and 8 females) with a median age of 5 years
(range 2–12 years). The median s-VEGF value was significantly lower in
patients than in controls (Table II). At the time
of ALL diagnosis, median s-VEGF did not differ significantly between
sex, age groups, immunophenotypes and ploidy on bone marrow cytogenetics.
TABLE I Characteristics of 30 ALL Patients at Diagnosis
Characteristic |
Value |
Age, median (range) |
6 years (1–14) |
Male sex |
20 (66.7) |
ALL immunophenotyping |
Pre-B CALLA positive ALL |
26 (86.7) |
Pre-B Ph1 positive ALL |
1 (3.3) |
T-cell ALL |
3 (10.0) |
WBC |
2,600 (700 – 314,000) |
<20,000/mm3 |
21 (70.0) |
20,000 - 100,000/mm3 |
7 (2.3) |
>100,000/mm3 |
2 (6.7) |
LDH (n=26) > 2 ULN |
15 (53.8) |
CNS disease |
1 (3.3) |
Cytogenetics (n=26) |
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Hyperdiploidy >51 |
7 (26.9) |
46,XX / 46,XY |
15 (57.7) |
Unsuccessful culture |
4 (15.4) |
TEL-AML1 (n=21) |
FISH Positive |
3 (14.3) |
Bcr-Abl fusion* (n=20)
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1 (5.0) |
Protocol |
UKALL-XI |
27 (90.0) |
BFM-95 |
3 (10.0) |
Risk group |
Standard risk |
27 (90.0) |
High risk |
3 (10.0) |
Notes. *LDH normal value 91-180 IU/ml. ULN: upper limit of
normal; *by FISH (qPCR). |
TABLE II Serum VEGF in ALL Cases and Controls
VEGF (pg/mL) |
Cases at diagnosis (N=30) |
Controls (N=17) |
Cases at end of induction (N=30) |
P-value |
Median (IQR) |
17.0 (3.0 – 47.2) |
42.6 (32.0 – 74.7) |
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0.004 |
Median (IQR) |
17.0 (3.0 – 47.2) |
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44.2 (16.7 – 102.3) |
0.009 |
There was a fair inverse correlation of s-VEGF levels
with absolute WBC count at diagnosis (Spearman’s correlation coefficient
-0.56, P=0.004) and LDH (r=-0.52, P=0.02). There was no
correlation between s-VEGF and hemoglobin or platelet count. Patients
with high-risk and intermediate risk ALL showed lower s-VEGF levels than
those with standard-risk (8.6 pg/mL [interquartile range-IQR 0.2-48.2]
vs. 24.8 [IQR 10.8-53.5]), but the difference was not
statistically significant (P=0.2).
All patients but one were in morphologic remission at
the end of induction. Median s-VEGF at the end of induction therapy was
significantly higher than at diagnosis (Web Table I) and
was not significantly different from s-VEGF in controls (P=0.91).
The patient that did not attain remission was a 5-year old boy with
pre-B phenotype, low WBC, and normal cytogenetics. His s-VEGF on day 1
and day 29 were 10.7 and 59 pg/mL, respectively.
Discussion
Our results show an inverse correlation between
s-VEGF concentration at the time of diagnosis and disease burden, as
suggested by increased WBC counts and LDH. We also found lower levels of
s-VEGF at the time of diagnosis of ALL as compared to the end of
induction therapy. With the attainment of remission, s-VEGF rose to
levels almost similar to those of healthy controls. Our results
corroborate the study by Yetgin, et al. [4] wherein the median
level of s-VEGF at the time of diagnosis was significantly lower than
those of the control group and of the patients in remission, with no
relation between Hb, WBC, absolute blast percentage and s-VEGF.
Likewise, Aref, et al. [5] found significantly lower s-VEGF
levels in pre-B ALL newly diagnosed patients at diagnosis than in
controls, levels which increased to near controls in remission.
Considering the fact that VEGF is expressed in
normal hematopoietic cells, we hypothesize that in children suffering
from ALL, the proportion of hematopoietic cells is decreased in
comparison with tumor cells and consequently, the level of VEGF may be
lower. However, during remission, the renewal of normal hematopoiesis
may explain the rise of s-VEGF near normal levels, as evidenced in these
reports and in our study [4,5].
Another hypothesis that may explain our findings
relates to the interaction between VEGF receptors (VEGFR) and VEGF [6].
In CLL and non-Hodgkin lymphomas, receptors present in soluble form in
the plasma bind the ligand before it can bind to the actual cell-bound
receptor and thus decrease the activity of angiogenic factors such as
VEGF [7,8]. Both VEGF and VEGFR-1 are expressed on leukemia cells,
including ALL cells [9]. Recent data from children enrolled in anti-angiogenic
Children’s Oncology Group (COG) clinical trials showed a significant
increase of plasma VEGF and decrease of soluble VEGFR-2 after treatment
with tyrosine kinase inhibitors targeting VEGF receptors [10]. These
results lead us to hypothesize that low s-VEGF levels in our untreated
children with ALL may reflect a higher VEGFR expression on leukemia
cells that would bind s-VEGF, thus decreasing unbound s-VEGF
proportionately to tumor burden via ligand-receptor interaction. Further
studies on VEGFR-1 and VEGFR-2 expression are required to confirm such
hypothesis.
A few clinical studies on VEGF in pediatric
hematological malignancies report results conflicting with ours. Yang,
et al. [11] found significantly higher plasma VEGF concentrations
in children with ALL before treatment than in normal controls. A COG
study showed that standard-risk ALL pediatric patients [12] with low
VEGF-A levels at diagnosis and at the end of induction had significantly
superior event-free survival (EFS). Furthermore, patients who had an
increase in VEGF-A during induction had poorer EFS [12]. A similar
association of high s-VEGF level and poor overall and relapse-free
survival were described in childhood ALL 1,
[11, 13] and
AML patients [14].
The possible explanation for the above observations
and ours is a clearance of abnormal cell population from the bone marrow
as the treatment progresses and reestablishment of normal hematopoiesis.
Hematopoiesis is greatly disturbed in the bone marrow microenvironment
when there is an excessive malignant proliferation of blasts and this
probably hinders the normal marrow angiogenesis, leading to low levels
of angiogenic peptides. With the action of multiagent chemotherapy, this
abnormal cell population gives way to normal angiogenesis and
hematopoietic activities in the bone marrow.
Our study has certain limitations. The most important
one is the small number of patients included in the study, which may be
the cause for non-statistically significant differences after risk
stratification. Furthermore, the cohort consisted mostly of patients who
attained remission at the end of induction therapy, so the possible
relationship between resistant disease and VEGF levels could not be
attained.
Further investigations on the role of angiogenesis
and angiogenic factors in childhood ALL will provide additional
information in understanding the complex interaction between
angiogenesis and the biology of leukemic cells. This understanding can
eventually lay the pathways for clinically helpful targeted molecules to
further improve the outcome of pediatric leukemias.
Contributors: All authors have contributed,
designed and approved the study.
Funding: None; Competing interests; None
stated.
What is Already Known?
• Vascular endothelium growth factor (VEGF)
plays an important role in tumor angiogenesis in solid tumors
but its role in leukemia is not well known.
What This Study Adds?
• Serum VEGF levels are low at diagnosis and
increase to normal levels after end of induction chemotherapy in
children with acute lymphoblastic leukemia.
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