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COX-2 expression correlates with VEGF-C and
lymph node metastases in patients with head
and neck squamous cell carcinoma
Panayiotis A Kyzas, Dimitrios Stefanou and Niki J Agnantis
Department of Pathology, University of Ioannina, Medical School, Ioannina, Greece
Recent observations suggest an implication of the cyclooxygenase-2 (COX-2) in tumor lymphangiogenesis
through an upregulation of vascular endothelial growth factor-C expression. It is unknown whether this
mechanism also acts in squamous cell carcinoma of the head and neck region. We performed a retrospective
study of 70 patients with head and neck squamous cell carcinoma in order to investigate whether COX-2
immunohistochemical expression correlates with vascular endothelial growth factor-C expression. We also
examined the association of the expression of these molecules with clinicopathologic parameters (especially
lymph node status) and outcome for these patients. We performed immunostaining on formalin-fixed, paraffin-
embedded tissue sections by the routine streptavidin–biotin peroxidase labeled procedure. Increased
cyclooxygenase-2 expression was observed in 30 of the 68 tumor samples (44%), while high vascular
endothelial growth factor-C expression occurred in 26 of the 68 tumor samples (38%). High expression of the
two proteins was correlated with the presence of lymph node metastasis at the time of diagnosis, and the
observed association was even stronger when there was overexpression for both the antibodies (Po0.001).
High expression of vascular endothelial growth factor-C, but not of COX-2 was correlated with increased
mortality in patients with oral–larynx squamous cell carcinoma. When multivariate Cox regression model was
applied, the presence of lymph node metastasis at the time of diagnosis, combined with overexpression of both
the antibodies, was the only independent prognostic factor for mortality of these patients. Our results suggest
that a lymphangiogenic pathway, in which COX-2 overexpression stimulates vascular endothelial growth factor-
C upregulation, probably exists in head and neck squamous cell carcinoma. Also, the predictive ability
for mortality of regional lymph node metastasis can be improved with the combined evaluation of the
immunohistochemical expression of these two proteins.
Modern Pathology (2005) 18, 153–160, advance online publication, 23 July 2004; doi:10.1038/modpathol.3800244
Keywords:
cyclooxygenase-2; head and neck cancer; lymphangiogenesis; metastasis; survival; vascular
endothelial growth factor-C
Clinical and pathological observations have shown
that lymph node metastases at the time of diagnosis,
is the most reliable prognostic factor for mortality or
recurrence in patients with head and neck squa-
mous cell carcinoma.
1
However, to date, the
mechanisms promoting the entrance of the cancer
cells into the lymphatics and their dissemination to
the cervical lymph nodes are poorly understood.
Lymphangiogenesis, the formation of new lym-
phatic vessels, has recently become a new research
frontier in understanding tumor aggressiveness,
progression and metastases. Today, intratumor lym-
phatic vessels can be directly identified with the use
of new markers such as podoplanin, lymphatic
vessel endothelial hyaluronan receptor (LYVE-1),
desmoplakin, vascular endothelial growth factor
receptor-3 (VEGFR-3) and homeobox gene prox-1.
2
Two members of the vascular endothelial growth
factor (VEGF) family, VEGF-C and VEGF-D are
considered as major lymphangiogenic factors. Pre-
vious reports suggest that the upregulation of VEGF-
C strongly correlates with regional lymph node
metastases in thyroid, prostate, stomach, breast,
lung and oral carcinomas.
3,4
Cyclooxygenase (COX) is the key enzyme in
prostaglandin’s metabolism and so far, two isoforms
of this enzyme have been characterized: COX-1 and
COX-2. COX-1 is constitutively expressed in most
tissues, and plays a role in regulating normal
physiological function and inflammation.
5
COX-2
has been reported to be expressed intensively in
Received 18 May 2004; revised and accepted 28 June 2004;
published online 23 July 2004
Correspondence: Associate Professor D Stefanou, Department of
Pathology, University of Ioannina, Medical School, 45 110
Ioannina Greece.
E-mail: dstefan@cc.uoi.gr
Modern Pathology (2005) 18, 153–160
&
2005 USCAP, Inc All rights reserved 0893-3952/05
$30.00
www.modernpathology.org
various malignancies
6–8
and it is suggested that
COX-2 overexpression correlates with tumor aggres-
siveness and poor prognosis.
9
Thus, inhibition of
COX-2 activity may have a therapeutic value. COX-2
is also proposed to be a regulator of tumor
angiogenesis in head and neck squamous cell
carcinoma.
10,11
Recently, COX-2 was shown to be
an upregulator of VEGF-C in lung adenocarcino-
ma,
12
suggesting a role as a promoter of lymphan-
giogenesis.
In this retrospective study, we examined whether
COX-2 immunohistochemical expression correlates
with VEGF-C expression or with clinicopathologic
parameters and especially lymph node involvement
in a cohort of patients with head and neck squamous
cell carcinoma.
Materials and methods
Tissues and Clinical Parameters
A total of 70 paraffin-embedded archival tissue
blocks concerning head and neck squamous cell
carcinoma specimens were obtained from the De-
partment of Pathology of the General University
Hospital of Ioannina. Specimens were, with regard
to 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 1998–2002. All
hematoxylin–eosin-stained sections were reviewed,
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. Patients’ records were
reviewed, and clinicopathological characteristics as
well as follow-up data were noted. TNM staging was
established by IUCC system, and grading was
performed according to WHO.
Immunohistochemistry
We performed immunostaining on formalin-fixed,
paraffin-embedded tissue sections (one slide for
each antibody per case) using the streptavidin–
biotin peroxidase-labeled procedure. We used the
ImmunoCruz Staining System (Santa Cruz Biotech-
nology) and the goat polyclonal antibodies VEGF-C
(N-19: sc-7133, Santa Cruz Biotechnology, dilution
1:300), and COX-2 (M-19: sc-1747, Santa Cruz
Biotechnology, dilution 1:100). Briefly, two adjacent
4-mm-thick sections from each paraffin-embedded
block were cut. The sections were deparaffinized in
xylene and rehydrated through graded concentra-
tions of alcohol. Sections were heated in a micro-
wave oven for two circles of 5 min each, in High pH
Buffer (DAKO), for antigen retrieval. Endogenous
peroxidase activity was blocked with H
2
O
2
solution
in methanol (0.01 M), for 30 min. Nonspecific bind-
ing was blocked by incubating the sections for
30 min with serum block (Santa Cruz Biotechno-
logy). After washing with phosphate-buffered saline
(PBS) for 5 min, primary antibodies were incubated
overnight at 41C. Then sections were washed for
10 min with PBS. Biotinylated secondary antibody
was applied for 30 min, and sections were washed
for 10 min in PBS. Sections were then incubated for
30 min with horseradish peroxidase (HRP)–Strepta-
vidin complex. HRP-substrate was used for visuali-
zation of the expression of the antibodies and
diaminobezidinetetrahydrochloride (DAB) was used
as a chromogen. All sections were counterstained
with hematoxylin.
Antibody Expression
Two observers (DS and PK) without knowledge of
the clinical data performed evaluation of the stain-
ing. The percentage of the tumor cells that exhibited
a positive cytoplasmic immunoreactivity was deter-
mined using a 40 objective lens. At least 1000
neoplastic cells were counted in each case. The
intensity of the immunohistochemical staining was
evaluated as follows: , negative, þ , focal expres-
sion o5% of cancer cells, þþ , focal expression in
5–20% of cancer cells; and þþþ , diffuse expres-
sion 420% of cancer cells. Samples with þþ and
þþþ staining for COX-2 and VEGF-C, respectively,
were classified as ‘high expression group’, and those
with and þ were assigned as ‘low expression
group’, as described previously.
12
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,
13
and the major problem for
this malignancy is local recurrence after surgical
removal of the primary tumor. On the other hand,
squamous cell carcinoma of the oral cavity and
larynx are more aggressive. To date, the most reliable
prognostic factor for survival of patients with oral or
laryngeal squamous cell carcinoma is the presence
of lymph node metastases at the time of diagnosis,
but the accuracy of the prediction is far from per-
fect and certainly it can be improved.
1
Thus, 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 those with larynx squamous cell
carcinoma.
COX-2 and VEGF-C expression in squamous cancer
PA Kyzas et al
154
Modern Pathology (2005) 18, 153–160
Statistical Analysis
Analyses were conducted in the SPSS software
version 11.0. (SPSS, Inc., Chicago, IL, USA). VEGF-C
and COX-2 expression were considered dichoto-
mous variables using the cutoff value described
above. For comparison between variables we used w
2
test. Analyses for survival and recurrence were
conducted 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
Clinicopathologic Data
The clinicopathologic characteristics of the patients
enrolled in the study are listed in Table 1. For two
patients, clinical information was inadequate, and
therefore they were excluded from further analysis.
Thus, a total of 68 patients were included in the
analysis for the correlations of the antibodies
expression with the clinicopathologic parameters.
The median age of the patients was 67 (range 33–98)
years. Of the patients, 53 were male and 15 were
female. In total, 38 of the tumors were located in the
lower lip, 21 in the oral cavity and nine in the
larynx. A total of 20 patients (29%) presented with
lymph node metastases at the time of diagnosis.
Only one of them suffered from lower lip squamous
cell carcinoma.
Available follow-up data existed for 28 of the 31
patients with oral–larynx squamous cell carcinoma
and for 33 of the 39 patients with lower lip
squamous cell carcinoma. Patients were followed
up until death or recurrence or for at least 24
months. Of the 28 patients with oral–larynx squa-
mous, 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.
COX-2 and VEGF-C Expression
Increased, diffuse cytoplasmic staining for COX-2
was observed in 30 out of the 68 tumor samples
(44%—‘High expression group’). The intensity of
immunostaining was often strong, but the staining
pattern was heterogeneous. Interestingly, intense
staining was observed in acinic epithelial cells and
ducts of normal minor salivary glands infiltrated by
tumor cells (Figure 1). The same staining pattern
was observed for VEGF-C, for which high expression
was observed in 26 out of the 68 tumor samples
(38%) (Figure 2).
The correlation of the antibodies expression with
clinicopathologic parameters is summarized in
Table 1 Correlation of clinicopathologic characteristics of the patients included in the study with immunohistochemistry results
COX-2 expression VEGF-C expression
High Low P-value High Low P-value
Gender
Female 15 (24%) 6 9 NS 3 12 NS
Male 53 (76%) 24 29 23 30
Histological Grade
Poorly 12 (18%) 9 3 8 4 NS
Moderate 24 (34%) 10 14 NS (P ¼ 0.55) 8 16
Well 32 (48%) 11 21 10 22
Stage
a
I 28 (42%) 8 20 0.035 2 26 o0.001
II 16 (24%) 8 8 9 7
III 9 (12%) 3 6 4 5
IV 15 (22%) 11 4 11 4
N Status
N negative (N0, Nx) 48 (72%) 16 32 0.006 11 37 o0.001
N positive (N1, N2, N3) 20 (28%) 14 6 15 5
Location
Oral 21 (31%) 12 9 0.038 13 8 0.003
Lower lip 38 (57%) 12 26 8 30
Larynx 9 (12%) 6 3 5 4
COX-2 expression
High 22 8 o0.001
Low 4 36
a
According to IUCC.
COX-2 and VEGF-C expression in squamous cancer
PA Kyzas et al
155
Modern Pathology (2005) 18, 153–160
Table 1. Increased COX-2 expression was strongly
associated with the presence of lymph node metas-
tases at the time of diagnosis (P ¼ 0.006). There was
also a correlation of positive COX-2 expression with
higher clinical stage (P ¼ 0.035). High COX-2 ex-
pression was rare in tumors located in the lower lip,
whereas tumors located in the oral cavity and larynx
more often expressed high COX-2 immunostaining
(P ¼ 0.038). No correlation was observed between
COX-2 expression and histological grade, although a
trend towards an association of high COX-2 expres-
sion with lower grade of differentiation could not be
ruled out (P ¼ 0.055). A very strong correlation was
observed between COX-2 and VEGF-C expression:
Out of the 30 tumor samples, which were positive
for COX-2 expression, 22 also demonstrated high
VEGF-C expression (Po0.001).
Positive VEGF-C expression was also strongly
correlated with lymph node status (Po0.001) and
advanced clinical stage (Po0.001). VEGF-C expres-
sion was positive more often in squamous cell
carcinoma of the oral cavity and larynx (P ¼ 0.003).
No statistically significant correlation was observed
between VEGF-C expression and histological grade.
We considered the subgroup of the 22 tumors with
high expression both for COX-2 and VEGF-C. A
comparison between this group of patients and the
group of the rest of the patients showed a strong
correlation of the overexpression of both the anti-
bodies only with the presence of lymph node
metastases at the time of diagnosis (Po0.001).
Survival and Recurrence Analysis
VEGF-C expression was the only prognostic factor
for outcome in the subgroup of patients with oral
and larynx squamous cell carcinoma (log rank
P ¼ 0.035) (Figure 3). The mean survival time for
VEGF-C positive expression was 21 months, while
the mean survival time for VEGF-C negative expres-
sion was 42 months. Neither COX-2-positive ex-
pression nor overexpression of both antibodies had
any significant association with survival. Clinical
stage and lymph node status did not show any
significant association with survival, although a
trend towards correlation of the presence of lymph
nodes at the time of diagnosis with worse survival
could not be excluded (log rank P ¼ 0.08). We
performed analysis for survival between the group
of patients with lymph node metastases at the time
of diagnosis, for which staining for both the
antibodies was high (Node þ /COX-2 þ /VEGF-C þ ),
and the rest of the patients. This group of patients
Figure 1 Diffuse cytoplasmic COX-2 immunoreactivity: in ductal epithelial cells of normal minor salivary glands (a, b magnification
400), in carcinoma cells in squamous cell carcinoma of the floor of the mouth, of the tongue and the larynx (c, e, f magnification
1000), and in both minor salivary glands and carcinoma cells in squamous cell carcinoma of the floor of the mouth (d magnification
400).
COX-2 and VEGF-C expression in squamous cancer
PA Kyzas et al
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Modern Pathology (2005) 18, 153–160
had significantly higher risk of death than the others
(Figure 4, log rank P ¼ 0.0026). Multivariate analysis
with Cox regression model for the two, above
described, prognostic factors, revealed that the
combination of positive lymph node status with
overexpression of both COX-2 and VEGF-C was the
only independent prognostic factor of outcome for
these patients (P ¼ 0.006).
We also performed the above analyses separately
for patients with oral or larynx squamous cell
carcinoma. VEGF-C overexpression was correlated
with worse survival both in the patients with oral
(log rank P ¼ 0.02) or larynx (log rank P ¼ 0.04)
squamous cell carcinoma. Neither COX-2-positive
expression nor overexpression of both the anti-
bodies had any significant association with survival
in both of the subgroups. Patients with oral
Figure 2 High VEGF-C immunoreactivity in carcinoma cells in a
squamous cell carcinoma of (a) the tongue (magnification 1000)
and (b) larynx (magnification 1000).
Figure 3 Kaplan–Meier curve for the overall survival of the
patients with oral–larynx squamous cell carcinoma, according to
the expression (VEGF-C). The two curves are significantly
different (log rank P ¼ 0.035).
Figure 4 Kaplan–Meier curve for the overall survival of the
patients with oral–larynx squamous cell carcinoma, according to
lymph node status, the expression of COX-2 and the expression of
(VEGF-C). COX-2 þ and VEGF-C þ are referred to as ‘high
expression groups’ (see Materials and methods for more). The
two curves are significantly different (log rank P ¼ 0.0026).
COX-2 and VEGF-C expression in squamous cancer
PA Kyzas et al
157
Modern Pathology (2005) 18, 153–160
squamous cell carcinoma with lymph node metas-
tases and overexpression of COX-2 and VEGF-C
(Node þ /COX-2 þ /VEGF-C þ ), presented worse sur-
vival than the rest of the patients with oral
squamous cell carcinoma (log rank P ¼ 0.03). The
same pattern was observed in patients with laryn-
geal squamous cell carcinoma (log rank P ¼ 0.02).
Neither COX-2 nor VEGF-C overexpression
showed any significant association with local
recurrence for patients with squamous cell carcino-
ma of the lower lip. The lack of correlation remained
when patients with overexpression of both the
antibodies were considered as a different subgroup.
Discussion
Previous reports revealed the implication of COX-2
in squamous epithelial carcinogenesis and ad-
dressed the contribution of this enzyme to the
growth and progression of head and neck squamous
cell carcinoma, by various pathways.
10,11,13,14
Its role
is mediated by a number of molecules such as
VEGF-A, CD44 and matrix metalloproteinases,
15
and
results in the induction of tumor angiogenesis,
increased invasiveness, apoptosis and immune
surveillance inhibition and increased cell prolifera-
tion. Also, increased COX-2 expression was asso-
ciated with poor prognosis in a number of
malignancies,
16–18
and head and neck squamous cell
carcinoma, although the small number of patients
enrolled in the studies for head and neck squamous
cell carcinoma does not permit the extraction of
valid conclusions.
10,11,13,14
VEGF-C is a member of the VEGF family and along
with VEGF-D is considered to be responsible for
tumor lymphangiogenesis and lymph node meta-
stases.
19
This function is mediated via VEGF
receptor-3 (VEGFR-3), which is strictly expressed
only in the lymphatic vessels and stimulated by the
above molecules. Recent studies suggest a direct
correlation of VEGF-C expression with the presence
of lymph node metastases in head and neck
squamous cell carcinoma,
20
and with lymphatic
vessel density (LVD) in this malignancy.
21
Also,
other reports implied the prognostic significance of
the positive expression of this protein for the
clinical outcome.
22–24
To our knowledge, this is the first study examin-
ing the correlation of COX-2 with VEGF-C in
patients with head and neck squamous cell carci-
noma. The implication of COX-2 to lymphangiogen-
esis is possible, since COX-2 is a pleiotropic enzyme
that mediates many cellular functions.
25
An upre-
gulation of VEGF-C by COX-2 through a HER-2/Neu-
depentent pathway was recently described for lung
adenocarcinoma.
12
In this cascade, COX-2 catalyzes
the synthesis of prostaglandin E2 (PGE2) that binds
to EP1 prostaglandin receptor. The activation of this
receptor increases the tyrosine phosphorylation of
the human epidermal growth factor receptor HER/
Neu-2. Signaling is mediated through mitogen-
activated protein kinase (MAPK) p38 and leads to
a stimulation of the transcription factor NF-kB, and
finally to an increased transcription of VEGF-C gene
and thus, an upregulation of this protein expres-
sion.
26
It is also proposed that the mechanisms by
which COX-2 and its derived prostaglandins regu-
late angiogenesis and lymphangiogenesis are quite
distinct, since COX-2 function in angiogenesis is
mediated mainly through EP2.
26
However, interac-
tions and crosstalks between these pathways cannot
be excluded. It is unknown whether the same
pathway acts in squamous cell carcinoma and what
are the possible crossacts (if any) with the COX-2
mediated angiogenesis procedure, for the signifi-
cance of which in head and neck squamous cell
carcinoma, enough evidence exists.
10,11,27
In this study the most impressive finding was the
strong association of the spontaneous overexpres-
sion of COX-2 and VEGF-C with the presence of
lymph node metastases at the time of diagnosis.
These results confirm the previous observations for
each protein independently, and also suggest that a
lymphangiogenic pathway, in which COX-2 over-
expression stimulates VEGF-C upregulation and
thus the formation of new lymphatic vessels, which
is the first step for spreading and establishment of
tumor cells to the regional lymph nodes, probably
exists in head and neck squamous cell carcinoma.
Also, in our cohort of patients, lymph node
metastasis was not correlated with increased mor-
tality. However, patients with lymph node metas-
tases had a significantly higher risk of death when
the staining was positive for both of the antibodies,
than the rest of the study population. In our study,
all of the eight node-positive patients with oral–
larynx squamous cell carcinoma, with COX-2 and
VEGF-C overexpression died before 36 months.
Therefore, the accuracy of the predictive ability for
mortality of regional lymph node metastases can be
improved with the combined evaluation of lymph
node status along with the examination of the
immunohistochemical expression of COX-2 and
VEGF-C.
The lack of any association of both COX-2 and
VEGF-C with local recurrence in squamous cell
carcinoma of the lower lip is a challenging finding.
In our previous work,
28
we found that the immuno-
histochemical expression of VEGF-A was the only
independent prognostic factor for recurrence for this
cohort of patients. VEGF-A upregulation can be
promoted not only by COX-2 pathway but also by
intratumoral hypoxic conditions mediated via hy-
poxia-induced factor (HIF).
29,30
On the other hand,
as our results show, COX-2 and VEGF-C-positive
expression correlate with a higher clinical stage and
the presence of lymph node metastases, while the
majority of these patients presented with an early
stage of the disease. In fact, only one patient had
lymph node metastases at the time of diagnosis.
These aspects suggest that lymphangiogenesis may
COX-2 and VEGF-C expression in squamous cancer
PA Kyzas et al
158
Modern Pathology (2005) 18, 153–160
not be an early step in squamous carcinoma
progression.
In conclusion, this study suggests that COX-2
correlates with VEGF-C and both of them with
the presence of lymph node metastases in head
and neck squamous cell carcinoma. The expression
of these proteins, combined with the lymph
node status of the patient, produces a promising
prognostic marker for mortality of these patients.
However, the complex phenomenon of tumor lymp-
hangiogenesis in head and neck squamous cell
carcinoma needs further investigation. COX-2 and
VEGF-C expression should be examined in the
context of other proposed lymphangiogenic mole-
cules such as VEGF-D and the expression of VEGFR-
3 should also be under consideration.
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