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Potential autocrine function of vascular
endothelial growth factor in head and neck
cancer via vascular endothelial growth factor
receptor-2
Panayiotis A Kyzas, Dimitrios Stefanou, Anna Batistatou and Niki J Agnantis
Department of Pathology, University of Ioannina, Medical School, Ioannina, Greece
Vascular endothelial growth factor is a peptide with well-defined actions on the vasculature and fundamental
role in tumor angiogenesis. Its action in vascular endothelium is exerted in a paracrine manner. The
immunohistochemical expression of this protein by cancer cells in head and neck squamous cell carcinoma
was correlated with increased tumor aggressiveness and poor survival in previous studies. In the past years, an
increasing amount of studies demonstrated potential autocrine action of vascular endothelial growth factor in
various neoplasms. However, the existence and the impact of such autocrine action in head and neck cancer
have not been demonstrated yet. In this retrospective study, we evaluated the expression of vascular
endothelial growth factor and its receptors in neoplastic cells, in a cohort of patients with head and neck
squamous cell carcinoma, and compared this expression with tumor aggressiveness, clinicopathologic
parameters and outcome. High expression of vascular endothelial growth factor was strongly correlated with
high expression of vascular endothelial growth factor receptor-2 (but not vascular endothelial growth factor
receptor-1) on the cancer cells (Po0.001). The co-overexpression of both the protein and vascular endothelial
growth factor receptor-2 was associated with higher tumor proliferation rate (Po0.001). The above co-
overexpression also correlated with worse survival (log rank Po0.05) in patients with oral–larynx squamous
cell carcinoma. Our results suggest that an autocrine vascular endothelial growth factor loop, mediated via
vascular endothelial growth factor receptor-2, probably exists in head and neck squamous cell carcinoma.
These observations support the hypothesis that the use of vascular endothelial growth factor receptor-2
inhibitors as adjuvant antiangiogenic therapy might have beneficial effects for these patients, by disrupting
both paracrine (endothelial-dependent) and autocrine actions of vascular endothelial growth factor.
Modern Pathology (2005) 18, 485–494, advance online publication, 8 October 2004; doi:10.1038/modpathol.3800295
Keywords:
VEGF; VEGFR-2; VEGFR-1; angiogenesis inhibitors
It is well established that the local growth, and
metastatic spread of solid neoplasms is supported
by tumor angiogenesis,
1,2
which refers to a combina-
tion of angiogenesis and vasculogenesis. In this
process, the main blood supply to a tumor derives
from the growth and extension of pre-existing blood
vessels (angiogenesis), but circulating endothelial
cell precursors may contribute to the growing
vascular tree as well (vasculogenesis).
3,4
In head
and neck squamous cell carcinoma, the quantifica-
tion of tumor angiogenesis by intratumoral micro-
vessel counting correlates with poor clinical
outcome,
5,6
but results of various studies are
conflicting and inconclusive in regard of the size
of the estimated risk.
7,8
A number of putative angiogenic factors such as
basic fibroblast growth factor, platelet-derived
growth factor, interleukins, and vascular endothelial
growth factor (VEGF), are expressed in head and
neck squamous cell carcinoma.
4
VEGF is considered
a leading candidate for tumor angiogenesis in head
and neck squamous cell carcinoma, exhibiting its
effect in the vasculature in a paracrine pattern.
1,2
This protein binds with high affinity to the tyrosine
kinase receptors VEGFR-1 (also known as flt-1) and
VEGFR-2 (also known as Flk-1/KDR), which are
expressed in endothelial cells during development,
wound healing, and in tumor vasculature.
9
Studies
Received 9 July 2004; revised and accepted 18 August 2004;
published online 8 October 2004
Correspondence: Dr PA Kyzas, DDS, Department of Pathology,
University of Ioannina, Medical School, 45 110 Ioannina Greece.
E-mail: md03791@cc.uoi.gr
Modern Pathology (2005) 18, 485–494
&
2005 USCAP, Inc All rights reserved 0893-3952/05
$30.00
www.modernpathology.org
examining the association between VEGF immuno-
histochemical expression and patients’ survival in
head and neck cancer are more consensus than those
examining microvessel density, pointing towards a
correlation with poorer overall survival.
10–16
Also,
many studies failed to show association of VEGF
expression with higher microvessel density.
15,16
These aspects raise the possibility that VEGF, which
is produced by neoplastic cells, might serve not only
for vessel sprouting, but have other functions as
well.
Recently, VEGFR-1 and VEGFR-2 expression by
tumor cells in melanoma, Kaposi sarcoma and breast
cancer, as well as in melanoma, choriocarcinoma
and leukemia cell lines was reported.
17–24
There are
also studies describing the existence of VEGF
receptors in cancer cell lines and in limited number
of tissue samples in head and neck squamous cell
carcinoma.
25,26
These observations suggest that
VEGF can function as an autocrine growth factor.
This hypothesis was experimentally confirmed both
for solid tumors (such as prostate adenocarcinoma)
24
and also for hematological malignancies (such as
acute leukemia).
19
It is yet unconfirmed whether
VEGF exhibits autocrine function in head and neck
cancer, and what is the impact of this action, if any.
In the present study, we performed immuno-
histochemical evaluation of VEGF, VEGFR1 and
VEGFR2 expression in 81 primary tumor samples
from patients with head and neck squamous cell
carcinoma. Our aim was to investigate the presence
of co-overexpression of the protein and its receptors;
and the possible correlation of this co-overexpres-
sion with tumor aggressiveness, clinicopathologic
parameters and overall survival of this cohort of
patients.
Materials and methods
Tissues and Clinical Parameters
A total of 81 paraffin-embedded archival tissue
blocks for patients with head and neck squamous
cell carcinoma specimens were obtained from the
Department of Pathology of the General University
Hospital of Ioannina. Of these samples, 68 were also
included in our previous study.
27
Specimens con-
sisted of surgical material obtained from the primary
tumor. These patients with head and neck squamous
cell carcinoma were treated in the Ear Nose Throat
(ENT) Department (Director: Professor A Skevas) of
the same hospital, between the years 1998 and 2004.
All hematoxylin–eosin-stained sections were re-
viewed, the quality of the material was checked,
and the best section from each specimen was
selected. We selected a section showing central
and peripheral areas of the tumor (intratumor and
peritumor areas), avoiding areas with necrosis.
Patient’s records were reviewed, and clinicopatho-
logical characteristics as well as follow-up data
were noted. TNM staging was established by IUCC
system,
28
and grading was performed according
to WHO.
29
Immunohistochemistry
We performed immunostaining on formalin-fixed,
paraffin-embedded tissue sections using EnVision
System for monostaining, and additionally, the
alkaline phosphatase-labeled procedure for double
staining. Seven adjacent 4-mm-thick sections from
each paraffin-embedded block were cut. The first
four were used for staining with VEGF, Ki-67,
VEGFR-1 and VEGFR-2, respectively, and the rest
three were used for double staining for VEGF/Ki-67,
VEGF/VEGFR-2 and VEGF/VEGFR-1, respectively.
Monostaining
For usual staining we used the EnVision System
(DAKO) and the monoclonal antibodies VEGF
(VEGF Ab-3 JH121, Neomarkers, USA, dilution
1:50), Ki 67 (clone MIB1, DAKO, dilution 1:50),
VEGFR-1 (RP 077, DBS, USA, dilution 1:1000) and
VEGFR-2 (RP 076, DBS, USA, dilution 1:2000).
Sections were at first deparaffinized in xylene and
rehydrated through graded concentrations of alco-
hol. Sections were heated in a microwave oven for 2
circles of 5 min each, in high pH buffer (DAKO), for
antigen retrieval. Endogenous peroxidase’s activity
was blocked with H
2
O
2
solution in methanol
(0.01 M), for 30 min. Nonspecific binding was
blocked by incubating the sections for 30 min with
serum block (DAKO). After washing with PBS for
5 min, all of the primary antibodies were incubated
over night at 41C. Then sections were washed for
10 min with PBS. Envision HRP system (DAKO)
was used for visualization of the expression of
the antibodies. Diaminobenzidinetetrahydrochlo-
ride (DAB) was used as a chromogen. All sections
were counterstained with hematoxylin.
Double Staining
The first antibody (Ki-67, VEGFR-2 and VEGFR-1,
respectively) was applied as described above. After
the visualization of the expression of the antibodies
with DAB, sections were washed in PBS for 5 min.
Serum block was applied for 5 min and afterwards,
the second antibody (VEGF) was incubated over-
night at 41C. After washing with PBS for 5 min,
secondary antibody (Multilink, Biogenex) was ap-
plied for 20 min, followed by 10 min washing with
buffer. Alkaline-phosphatase link (Biogenex) was
applied for 20 min, and the expression of the second
antibody was visualized with Fast-Red (Biogenex),
diluted in naphthol phosphate. One drop of levi-
mazole was added to reduce unspecific background
staining. All sections were then counterstained with
hematoxylin.
VEGF and VEGFR-2 expression in squamous cancer cells
PA Kyzas et al
486
Modern Pathology (2005) 18, 485–494
Antibody Expression
Two observers (DS and PK) without knowledge of
the clinical data performed evaluation of the stain-
ing, in the sections with the monostaining. Double
staining was used only for the qualitative evaluation
of the coexpression pattern. Cell counts were
performed only in the sections with monostaining.
The percentage of the tumor cells that exhibited a
positive immunoreactivity was determined using a
40 objective lens. At least 1000 neoplastic cells
were counted in each case. For VEGF, a cutoff of
20% cytoplasmic staining in the tumor cells was
used to distinguish low from high VEGF-expressing
tumors, in order to be in consensus with the
majority of the studies performed so far.
12–14
For
Ki-67, nuclear staining was evaluated and a cutoff of
20% was used to separate tumors with low vs high
mitotic rate, as described previously.
30,31
VEGF receptors are mainly expressed in tumor
vasculature;
9
but their expression in cancer cells
was also described.
25,26
Our aim was to evaluate
their expression on the tumor cells, in a quantitative
way. The intensity of the membranous-cytoplasmic
immunohistochemical staining was evaluated as
follows: , negative, þ , focal expression o5% of
cancer cells, and þþ, diffuse expression 45% of
cancer cells. Samples with þþ staining were
classified as ‘high expression group’, and those with
and þ were assigned as ‘low expression group’.
Survival and Recurrence analysis
The cohort of our patients was divided into two
groups regarding the location of the primary tumor.
It is well known that squamous cell carcinoma of the
lower lip, is more curable than those of the oral
cavity or the larynx,
32
and the major problem for this
malignancy is local recurrence after surgical re-
moval of the primary tumor. On the other hand,
squamous cell carcinoma of the oral cavity and
larynx are more aggressive. We performed survival
analysis in the subgroup of patients with oral–
larynx squamous cell carcinoma, and recurrence
analysis in the subgroup of patients with squamous
cell carcinoma of the lower lip. We also examined
whether there was difference in survival rates when
the subgroup of patients with oral–larynx squamous
cell carcinoma, was further separated to patients
with oral squamous cell carcinoma, and patients
with larynx squamous cell carcinoma.
Statistical Analysis
Analyses were conducted in the SPSS software
version 11.0. (SPSS, Inc., Chicago, IL, USA). The
expression of the antibodies was considered
as dichotomous variable using the cutoff value
described above. For comparison between variables
we used w
2
-test. Analyses for survival and recurrence
were done using the Kaplan–Meier method and
different subgroups were compared using the log-
rank test. Cox regression model was used to
determine the importance of different factors. All
differences were considered positive if Po0.05.
P-values are two-tailed.
Results
Clinicopathological Data
A total of 81 patients with head and neck squamous
carcinoma were included in this retrospective study.
The median age of the patients was 65 (range 33–98)
years. Of the patients, 63 (78%) were male and 18
(22%) were female. In all, 50 of the tumors were
located in the lower lip, 22 in the oral cavity and
nine in larynx. A total of 21 patients (26%)
presented with lymph node metastases at the time
of diagnosis. Only one of them suffered from lower
lip squamous cell carcinoma. Of the patients, 56
(69%) had cancer classified as lower (I and II)
clinical stage, and 25 (31%) as high (III and IV). Nine
patients (11%) had histological high-grade tumors,
51 (63%) had tumors with median differentiation
and 21 (26%) of the tumors were well differentiated.
Available follow-up data existed for 28 of the 31
patients with oral–larynx squamous cell carcinoma
and for 33 of the 50 patients with lower lip
squamous cell carcinoma. Patients were followed
up until death or recurrence, respectively, or for at
least 24 months. Of the 28 patients with oral–larynx
squamous cell carcinoma, 17 were dead at the end
of the follow-up period, and 10 of the 33 patients
with lip squamous cell carcinoma showed local
recurrence.
VEGF Expression
High VEGF staining in the cytoplasm of the tumor
cells was observed in 27 of the 81 tumor samples
(33%). The intensity of immunostaining was often
strong, but the staining pattern was heterogeneous
(Figure 1a). Some of the inflammatory cells infiltrat-
ing the tumors, especially macrophages, were found
to stain strongly for VEGF. Weak staining was
occasionally observed in vascular endothelium as
well.
The correlation of VEGF expression with the
expression of its receptors and Ki-67 is summarized
in Table 1. High VEGF expression was strongly
associated with increased expression of VEGFR-2
(Po0.001), and Ki-67 (Po0.001), but not VEGFR-1
(P ¼ 0.13). Of the tumors, 23 presented co-over-
expression of both VEGF and VEGFR-2; and all but
one of these tumors also presented with high Ki-67
expression (Po0.001). Overexpression of both VEGF
and VEGFR-1 was observed only in 12 tumor
samples; and for these tumors, the correlation with
VEGF and VEGFR-2 expression in squamous cancer cells
PA Kyzas et al
487
Modern Pathology (2005) 18, 485–494
Ki-67 overexpression was not formally significant
(P ¼ 0.055).
There was no statistically significant correlation
between VEGF expression and age, sex, histological
grade and lymph node status. On the other hand,
high VEGF expression was correlated with a higher
clinical stage (P ¼ 0.04). High VEGF expression
was very rare in tumors located in the lower lip;
whereas tumors located in the oral cavity and larynx
more often expressed high VEGF immunostaining
(P ¼ 0.03). There was no difference in VEGF expres-
sion between tumors located in the oral cavity from
those located in larynx (Table 2).
VEGFR-2 Expression
Intracytoplasmic and membrane staining in
tumor endothelium was observed in all of the
samples, but the intense of the staining was
heterogeneous and often weak (Figure 1b). Surpri-
singly, intense cytoplasmic and membrane stain-
ing was present in tumor cells in 46 out of
the 81 samples (57%) (Figure 1c, d), and in some
cases staining was very strong. As mentioned
above, there was a strong association of VEGFR-2
overexpression with high VEGF and Ki-67
expression, and an impressive correlation of the
co-overexpression of VEGF and VEGFR-2 with
Ki-67.
Association of VEGFR-2 expression with clinico-
pathologic parameters followed the same pattern as
VEGF expression; that is absence of any significant
association of its expression with age, sex, histo-
logical grade and lymph node status, whereas a
strong correlation was observed for higher clinical
stage (P ¼ 0.02) and for tumors located in the oral
cavity and larynx (P ¼ 0.001).
Figure 1 (a) Diffuse cytoplasmic VEGF immunoreactivity in cancer cells in a laryngeal carcinoma (magnification 400). (b) VEGFR-2
expression in vascular endothelium in squamous cell carcinoma of the floor of the mouth (magnification 200). (c and d) Membranous-
cytoplasmic expression of VEGFR-2 in cancer cells in squamous cell carcinoma of the tongue (magnification 1000 and 200,
respectively). (e) VEGFR-1 expression in vascular endothelium in squamous cell carcinoma of the floor of the mouth (magnification
400). (f) Membranous-cytoplasmic expression of VEGFR-1 in cancer cells in squamous cell carcinoma of the tongue (magnification
200). (g) and (h) Intense nuclear immunostaining with proliferation marker Ki-67 in squamous cell carcinoma of the larynx
(magnification 1000 and 400, respectively).
VEGF and VEGFR-2 expression in squamous cancer cells
PA Kyzas et al
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Modern Pathology (2005) 18, 485–494
VEGFR-1 Expression
The cell-staining pattern was the same as VEGFR-2
(intracytoplasmic and membranous), with hetero-
geneous expression in tumor vasculature (Figure
1e). The overexpression of VEGFR-1 by tumor cells
was not as frequent as for VEGFR-2; it was observed
in 27 of the tumor samples (33%) (Figure 1f).
Figure 1 Continued.
Table 1 Correlation between the expression of the four (VEGF, VEGFR-1, VEGFR-2 and Ki-67) antibodies used for immunohisto-
chemistry
VEGF VEGFR-1 VEGFR-2 Ki-67
High Low P-value High Low P-value High Low P-value High Low P-value
VEGFR-1 High 12 15 NS
Low 15 39
VEGFR-2 High 23 23 o0.001 25 21 o0.001
Low 4 31 2 33
Ki-67 High 23 21 o0.001 19 25 NS 38 6 o0.001
Low 4 33 8 29 8 29
VEGF/ Yes 12 11 NS 22 1 o0.001
VEGFR-2 No 15 43 22 36
NS: nonsignificant.
VEGF and VEGFR-2 expression in squamous cancer cells
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Modern Pathology (2005) 18, 485–494
No association was observed between VEGFR-1
expression and age, sex, histological grade, lymph
node status and clinical stage, whereas tumors
located in the oral cavity and larynx, presented
more often with VEGFR-1 overexpression (P ¼ 0.02).
Ki-67 (MIB-1) Expression
Intense nuclear staining of the proliferation marker
Ki-67 was observed in 44 of the tumor samples
(54%) (Figure 1g, h), and its presence was associated
with a more aggressive tumor profile, including
higher clinical stage (P ¼ 0.01) and the presence of
lymph node metastasis at the time of diagnosis
(P ¼ 0.004).
Double Immunostaining
The statistically addressed co-overexpression of
VEGF/VEGFR-2, and VEGF/Ki-67 in tumor samples
was confirmed by double immunostaining (Figure
2). The double staining pattern was quite similar for
VEGF/VEGFR-2 and VEGF/Ki-67 expression respec-
tively (Figure 2h–j). We observed cancer cells that
were spontaneously expressing VEGF, VEGFR-2 and
Ki-67 (Figure 2a, b). Also, cells in the same tumor
area were expressing either VEGF or VEGFR-2
(Figure 2l). For some samples, expression of both
VEGF and Ki-67 was more than 75% of the tumor
cells (Figure 2h, k). Immunoreactivity for VEGFR-1
was located in different and independent tumor
areas than VEGF and no agreement with VEGF/Ki-
67 double staining pattern was observed.
Survival and Recurrence Analysis
VEGF expression, VEGFR-2 overexpression and
VEGF/VEGFR-2 co-overexpression were correlated
with worse survival in the group of patients with
oral and larynx squamous cell carcinoma (in all
cases log rank Po0.05). There was also a trend
towards an association of the presence of lymph
nodes at the time of diagnosis with worse survival
(P ¼ 0.08). No association was observed between
clinical stage, histological grade, Ki-67 expression
and VEGFR-1 expression with overall survival. The
small number of patients did not allow us to run Cox
regression model to determine the importance of the
three different potential prognostic variables.
33
Kaplan–Meier analyses showed the same results
for the above factors, when the group was further
separated in patients with oral and larynx squamous
cell carcinoma respectively (log rank Po0.05, for all
three factors).
As we found in a previous study,
34
VEGF over-
expression was the only independent prognostic
factor for recurrence in patients with squamous cell
carcinoma of the lower lip (log rank Po0.001). No
one from the currently examined markers (Ki-67,
VEGFR-2 and VEGFR-1) was correlated with recur-
rence in these patients.
Table 2 Correlation between the expression of VEGF and VEGFR-2 and the clinicopathological characteristics of the patients
VEGF VEGFR-2
High Low P-value High Low P-value
Location Oral 10 12 0.03 16 6 0.001
Larynx 5 4 8 1
Lower lip 12 38 22 28
Histological grade Low 3 6 NS 5 4 NS
Medium 16 35 27 23
High 8 13 14 7
Survival at 24 months (oral, larynx) Dead 12 5 0.03 14 3 0.04
Alive 5 8 4 9
NS: nonsignificant.
Figure 2 (a) Double immunostaining with VEGF (red) and Ki-67 (brown) in squamous cell carcinoma of the tongue. The same cancer cell
expresses the two markers (magnification 1000). (b) The same tumor sample as in (a), in an adjacent section. VEGFR-2 (brown) and
VEGF (red) are expressed in the same cancer cell (magnification 1000). (c) Expression of both VEGF and VEGFR-2 in the same cells in
squamous cell carcinoma of the floor of the mouth. There are also cells expressing either VEGF or VEGFR-2 (magnification 200). (d–f)
Double immunostaining with VEGF (red) and Ki-67 (brown) in squamous cell carcinoma of the larynx. The same cancer cells express
both the two markers (magnification 1000, 1000 and 400, respectively). (g–k) Double immunostaining with VEGF (red) and Ki-67
(brown) in squamous cell carcinoma of the tongue (g–j) and larynx (k). We can see overexpression of both of the markers in a high
percentage of the tumor cells. (l) Expression of both VEGF and VEGFR-2 in the same cells in squamous cell carcinoma of the lower lip.
Tumor cells in the same tumor area express either VEGF or VEGFR-2 (magnification 400).
VEGF and VEGFR-2 expression in squamous cancer cells
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VEGF and VEGFR-2 expression in squamous cancer cells
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Discussion
Among all the known angiogenic growth factors,
VEGF plays a paramount and indispensable role in
regulating the multistep process of tumor angiogen-
esis.
1–4
This protein induces the proliferation,
differentiation, and migration of vascular endothe-
lial cells,
4,35
increases the permeability of the
capillaries,
1,35
and also enhances the survival of
endothelial cells by preventing their apoptosis.
36
However, it is quite possible that its action is not
limited in the paracrine, endothelial dependent,
angiogenic pathway, but also extends towards a
potential autocrine action between cancer cells in
various malignancies.
19–24
VEGF activities are mediated via two tyrosine
kinase receptors VEGFR-1 (also known as flt-1) and
VEGFR-2 (also known as Flk-1/KDR). Although
VEGF binds with VEGFR-1 with higher affinity, it
is well known that mitogenic and proliferating
actions of VEGF in endothelial cells are mediated
via VEGFR-2.
9
Activation of VEGFR-1 by VEGF in
endothelial cells lacking VEGFR-2 does not induce
cell proliferation.
9
VEGFR-1 plays an important role
in modulating the activities of VEGFR-2
37,38
and the
circulating levels of VEGF, in regulating extracellu-
lar matrix proteolytic activity,
39
and in controlling
the release of nitric oxide by endothelial cells.
9
These aspects are mainly due to the different signal
transduction cascades induced by VEGFR-1 and
VEGFR-2; and specifically, for cell proliferation, to
the activation of mitogen-activated protein kinase
(MAPK) only by VEGFR-2.
36
It is therefore concei-
vable that the potential autocrine action of VEGF is
mediated via VEGFR-2.
The most interesting finding in our study is the
co-overexpression of VEGF and (VEGFR-2) in head
and neck squamous cell carcinoma cells. We have
also demonstrated a strong association of VEGF/
VEGFR-2 overexpression with a higher cell prolif-
eration rate, as determined by Ki-67 overexpression.
These aspects are clear hints towards the existence
of a VEGF autocrine loop, in squamous carcinoma.
Patients with oral and larynx squamous cell carci-
noma exhibited worse survival rates when there was
co-overexpression of VEGF and VEGFR-2. Also, this
co-overexpression was correlated with higher clin-
ical stage. Tumors located in the lower lip demon-
strated VEGF/VEGFR-2 overexpression very rarely,
and this might offer a logical explanation for their
decreased aggressiveness. In our previous work,
34
we found that VEGF overexpression was the only
independent prognostic factor for recurrence in
patients with squamous cell carcinoma of the lower
lip (log rank Po0.001), and these results were
confirmed by the current study. No one from the
currently examined markers (Ki-67, VEGFR-2 and
VEGFR-1) was correlated with recurrence in these
patients. Only four of these patients had co-over-
expression of VEGF and VEGFR-2, and no asso-
ciation with recurrence was observed for this
co-overexpression. However, this can be spurious
due to the very limited number of the patients with
VEGF/VEGFR-2 co-overexpression.
The above observations indicate that a potential
autocrine function of VEGF may increase VEGF-
related tumor aggressiveness. Previous reports
25,26
demonstrated the existence of VEGF receptors in
head and neck squamous cell carcinoma cells, along
with a predominant overexpression of VEGFR-2.
This is in agreement with our results. Double
staining confirmed the statistically addressed asso-
ciations and revealed the colocalization of VEGF
and VEGFR-2 in cancer cells, and occasionally the
expression of all three of VEGF/VEGFR-2/Ki-67, in
the same cell. Also, the expression of either VEGF or
VEGFR-2 in cancer cells located in the same tumor
area, suggests the existence of a paracrine (endothe-
lial-independent) loop between cancer cells. Similar
paracrine action between cancer cells and stromal
cells that infiltrate the tumor was previously
described.
26
Some limitations of our study should be dis-
cussed. First, the sample size is limited and does not
allow the performance of multivariate analysis.
33
Such an analysis would have confirmed observa-
tions of the univariate analysis for the predictive
ability of VEGF and VEGFR-2 co-overexpression on
the patients’ outcome and on the aggressive beha-
vior of the tumors. Second, the co-overexpression of
the protein and its receptor was not observed in all
of the tumor samples; and thus, a potential autocrine
action of VEGF might not be a general property of
head and neck squamous cell carcinoma.
VEGF/VEGFR-2 autocrine pathway was proposed
and confirmed in vitro for other malignancies.
19,24
On the other hand, it is reported that VEGF
stimulates DNA synthesis in choriocarcinoma cell
lines, which express both VEGFR-1 and VEGFR-2.
23
VEGF autocrine activity was also described in
benign neoplasms, such as cardiac myxomas, which
also express both receptors.
40
We observed VEGFR-1
expression in neoplastic cells in our samples, but its
expression was correlated neither with VEGF nor
with Ki-67 expression. Thus, autocrine VEGF path-
ways are not yet completely understood, and
probably there are differences in the manifestation
between various neoplasms. However, it is quite
possible that VEGF autocrine action is an enhancer
of uncontrolled tumor proliferation and along
with tumor angiogenesis regulates tumor metastatic
potential, increasing tumor aggressiveness.
Understanding autocrine faction of VEGF and
cross talks between autocrine and paracrine path-
ways will help to the development of new ther-
apeutic strategies, and to the critical evaluation of
the usefulness of the currently used antiangiogenic
agents. A fundamental approach to inhibit angio-
genesis during tumorigenesis is the disruption of
VEGF/VEGFR-2 pathway.
41
This could suppress
tumor growth by limiting their blood supply, by
changing the morphology, wall structure and
VEGF and VEGFR-2 expression in squamous cancer cells
PA Kyzas et al
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Modern Pathology (2005) 18, 485–494
function of tumor vasculature to a more normal
fashion,
42
and thus improving drug penetration in
tumors, and also by blocking VEGF autocrine path-
way and thus reducing uncontrolled neoplastic cell
proliferation.
Currently, a lot of VEGFR-2 inhibitors are under-
going preclinical and clinical evaluation.
41
The most
studied is SU5416 (semaxanib), which also presents
inhibitory activity towards VEGFR-1. Studies in
mice with transgenic prostate adenocarcinoma
(TRAMP model)
43
revealed a significant decrease
in intratumor microvessel density, an increment in
cancer cell’s apoptotic index, and pronounced
regions of cell death. However, these observations
were not confirmed by a recently performed phase II
trial.
44
Although initial results from clinical trials in
acute leukemia
45
showed a favorable outcome for
patients taking SU5416, results from phase II studies
for melanoma,
46
multiple myeloma,
47
and renal
cancer
48
diminish original enthusiasm. No clinical
trial has been performed so far regarding the efficacy
of VEGFR-2 inhibitors as adjuvant chemotherapy in
patients with head and neck cancer. Results of
clinical trials are necessary for the design of future
kinase inhibitors, and observations like the above
described, for possible autocrine VEGF/VEGFR-2
pathway in head and neck cancer, provides an
additional reason (besides the antiangiogenic effect)
of the potential usefulness of these agents. The field
of the antiangiogenic therapy remains open to
debate concerning the efficacy and tolerability of
the antigenic agents, and the spectra of malignan-
cies, which might have the maximal benefit from
this therapeutic strategy.
In conclusion, this study demonstrates that an
autocrine VEGF loop, mediated via VEGFR-2,
probably exists in head and neck squamous cell
carcinoma. This aspect, combined with the well-
known angiogenic effects of VEGF, increases
VEGF-related tumor aggressiveness. The above
observations, in association with the results of
previous reports, may lay the foundation for a
different therapeutic approach, in which patients
with VEGF-producing, VEGR-2-expressing head and
neck cancer, may receive adjuvant antiangiogenic
therapy, based on VEGFR-2 inhibitors, alone or in
combination with currently used chemotherapy.
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