Int. J. Oral Maxillofac. Surg.

2008; 37: 372–378

doi:10.1016/j.ijom.2007.11.021, available online at http://www.sciencedirect.com

Research Paper

Expression of vascular S. E. S. Faustino1, D. T. Oliveira1,

S. Nonogaki2, G. Landman3,
A. L. Carvalho4, L. P. Kowalski4

endothelial growth factor-C

1
Department of Stomatology, Area of
Pathology, Bauru School of Dentistry -
University of São Paulo, Bauru, Brazil; 2Adolfo
Lutz Institute, Pathology Division, São Paulo,

does not predict occult lymph- Brazil; 3Department of Pathology, A. C.

Camargo Cancer Hospital, São Paulo, Brazil;
4
Department of Head and Neck Surgery and

node metastasis in early oral Otorhinolaryngology, A. C. Camargo Cancer

Hospital, São Paulo, Brazil

squamous cell carcinoma

S. E. S. Faustino, D. T. Oliveira, S. Nonogaki, G. Landman, A. L. Carvalho, L. P.
Kowalski: Expression of vascular endothelial growth factor-C does not predict occult
lymph-node metastasis in early oral squamous cell carcinoma. Int. J. Oral
Maxillofac. Surg. 2008; 37: 372–378. # 2007 International Association of Oral and
Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Abstract. Strong vascular endothelial growth factor-C (VEGF-C) expression has been
correlated to occurrence of lymph-node metastases in patients with oral squamous
cell carcinoma (OSCC). The incidence of occult lymph-node metastasis remains a
decisive factor in the prognosis of patients with early OSCC. The aim of this study
was to evaluate VEGF-C expression as a predictor of occult lymph-node metastasis
in OSCC. Eighty-seven patients with primary OSCC arising in the tongue or floor of
mouth, clinically T1N0M0 or T2N0M0, with (pN+) and without (pN0) occult
lymph-node metastases were analyzed for VEGF-C expression by malignant cells.
Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who
were submitted to elective neck dissection. No statistically significant difference
was found between OSCC with and without occult lymph-node metastasis in
regard to VEGF-C immunoexpression by malignant cells and clinicopathologic
features. Independently of VEGF-C expression, lymph-node metastasis (pN+) was
Keywords: VEGF-C; oral squamous cell carci-
the most significant prognostic factor for overall survival of patients with OSCC noma; occult metastasis.
(p = 0.030). These findings indicate that isolated VEGF-C expression by malignant
cells is not of predictive value for occult lymph-node metastasis in the early stages Accepted for publication 26 November 2007
of OSCC. Available online 4 March 2008

Tumor invasion and metastasis is a critical
event of human cancer frequently asso-
ciated with a fatal outcome. It has been
speculated that the VEGF family, particu-
larly VEGF-C and VEGF-D, may function
as angiogenic/lymphangiogenic factors
that facilitate tumor progression and
metastasis9,15,23,29,30,43.
The role of VEGF-C in angiogenesis/
lymphangiogenesis, disease progression
and patient prognosis has been extensively
studied in various types of human tumor,
including OSCC2,4,19,22,24,28,37,42. Evi-
dence is emerging to correlate the pre-
sence of VEGF-C expression in tumor

0901-5027/040372 + 07 $30.00/0 # 2007 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

cells with an increased likelihood of
lymph-node metastases, and consequently
to suggest it as a prognostic indicator in
oral cancer11,16,17,20,21,25,33,35,36,41,44.
It is well established that positive patho-
logic neck nodes (pN+) have the most
decisive influence on prognosis in patients
with OSCC13. The incidence of occult
lymph-node metastasis (pN+) in patients
with OSCC, clinical stages I and II, ran-
ging from 23.1% to 45%1,7,26,31, continues
to be one of the strongest arguments indi-
cating elective neck dissection of these
tumors. There are also a high percentage
of patients who do not have metastasis in
the pathological exam (pN0), and in such
cases the undesirable cosmetic and func-
tional effects of neck dissection ideally
should be avoided1. Determining whether
or not elective neck dissection will be
beneficial to the patient continues to be
an important clinical dilemma.
Detection of better markers to identify
patients with biologically aggressive
tumors and/or poor prognoses would pro-
vide a much-needed opportunity to target
patients at risk of development of metas-
tasis24, contributing to the proper selection
of patients for elective neck dissection1.
Hence, there is an urgent need to identify
characteristics of the primary tumor that
might predict nodal metastasis36, improv-
ing the patient’s survival10,11. In this
study, isolated VEGF-C expression by
malignant cells as a predictor of occult
lymph-node metastasis was investigated
in early stages of OSCC located in the
tongue and floor of mouth.
Patients and Methods
Patient and tumor samples
This study was based on the analysis of 87
patients (68 males and 19 females) who
underwent surgical treatment for primary
OSCC from 1968 to 2001, at the Head and
Neck Surgery and Otorhinolaryngology
Department of the A.C. Camargo Cancer
Hospital, Brazil. The inclusion criteria
were: (1) primary OSCC located in the
tongue or floor of mouth, clinical stages I
(T1N0M0) and II (T2N0M0), confirmed by
biopsy; (2) patients who did not undergo
radiotherapy, chemotherapy or other treat-
ment prior to surgery; (3) patients without
other simultaneous primary tumors; (4)
tumor tissue available for microscopic ana-
lysis. Clinical data of the patients were
obtained from the medical records and
included gender, age, ethnic group (white
or not white), tobacco and alcohol con-
sumption, tumor location, TNM stage38,39,
treatment (surgery, postoperative adjuvant
VEGF-C expression and occult lymph-node metastasis in oral cancer
radiotherapy), and clinical follow up (local
recurrence, regional recurrence and death).
A formalin-fixed 3-mm section of tumor
tissue was taken from the pathology
archive for hematoxylin & eosin (HE)
staining and immunohistochemistry ana-
lyses of occult lymph-node metastasis and
VEGF-C expression. The histopathologi-
cal malignancy grading of the OSCC was
established by an experienced pathologist
according to BRYNE et al.6 (1989), without
knowledge of the clinical data. The pre-
sence of vascular embolization, and peri-
neural, muscular and salivary gland
infiltrations in the OSCC were reviewed
in the HE stained tumor sections.
VEGF-C immunoexpression in OSCC
The 87 specimens of OSCC previously
fixed in 10% buffered formalin and
embedded in paraffin were cut into 3-
mm-thick sections for the standard strep-
tavidin-biotin–peroxidase complex
method (StreptABComplex/HRP, Duet
Mouse/Rabbit, Dako ref K0492, Glostrup,
Denmark). After antigen retrieval using
10 mM citrate buffer, pH 6.0, in a pressure
cooker for 4 min, endogenous peroxidase
activity was blocked by incubation in 3%
H2O2 for 20 min. The sections were incu-
bated overnight at 4 8C with the primary
antibody VEGF-C (C-20) (Santa Cruz
Biotechnology, sc-1881, Santa Cruz,
CA, USA), dilution 1:100, in phosphate-
buffered saline with bovine serum albu-
min solution to block a non-specific reac-
tion. Then, the tumor sections were
incubated with second antibody biotiny-
lated anti-goat Ig made in rabbit (Vector
BA-5000, Burlingame, CA, USA) diluted
in phosphate-buffered saline 1:500, for
30 min, at 37 8C. The antigen–antibody
reaction was detected using streptavidin-
biotin-based detection kit StreptABCom-
plex/HRP Duet, mouse/rabbit (Dako A/S,
K0492, Denmark) and visualized using
3.3’diaminobenzidine tetrahydrochloride
(DAB/SIGMA, ref D-5637, St. Louis,
MO, USA). Sections were counterstained
with Harris hematoxylin before being
dehydrated and cover slipped. Human pla-
centa was used as positive control. Normal
oral mucosa from the surgical margins was
used as internal control. For a negative
control, the primary antibody was omitted
during the immunohistochemical staining.
Quantitative computer-assisted analysis
of 30 invasive tumor front fields (X400
magnification: 93,992.05 mm2/field) in
each tumor sample was performed to eval-
uate the cytoplasmic immunoreactivity of
VEGF-C expressed by malignant cells. The
images of the invasive tumor front in each
373
section were digitally captured with a cam-
era (Axiocam MR3, Zeiss, Jena, Germany)
attached to a light microscope (Axioskop2
Plus, Zeiss, Jena, Germany) and recorded
by a computer program system (Axiovision
4.5, Zeiss, Jena, Germany). Immunostain-
ing results of VEGF-C expressed by malig-
nant cells were evaluated by two
investigators without prior knowledge of
the tumor’s histopathologic features and
the patient’s clinical status. A combined
score for VEGF-C expression was based
on: (a) intensity of the immunostaining
(0 = negative; 1 = weak; 2 = moderate;
3 = strong; 4 = very strong) and (b) percen-
tage of positive cells (0 = 0% positive cells;
1 = <25% positive cells; 2 = 25–50% posi-
tive cells; 3 = 50–75% positive cells;
4 = >75% positive cells), as described pre-
viously by SOINI et al.40. The final immu-
nostaining score was determined by the
sum of (a) + (b) ranged from 0 to maxi-
mally 8. A final score with value greater
than 6 was considered as strong VEGF-C
expression.
Statistical analysis
All statistical analyses were performed
using SPSS statistical software version
10.0 (SPSS Inc., Chicago, IL, USA). Asso-
ciations between VEGF-C expression and
the variables studied were verified accord-
ing to Chi-square test or Fisher’s exact
test. P values less than 0.05 were consid-
ered statistically significant. The survival
probabilities (overall and disease-free sur-
vivals) were estimated using the Kaplan–
Meier method and to compare survival
curves the log-rank test was used. The
follow-up period was the time between
surgery date and the death or last patient
information date.
Results
Clinical features
The analysis of 87 patients with OSCC
revealed a white male predominance with
frequently tobacco (83%) and/or alcohol
(76%) consumption. The patients’ ages
ranged from 35 to 89 years (mean 59.36
years 10.91 standard deviation [SD]).
The tumor was located in the tongue
(69% of cases) and floor of the mouth
(31% of cases). On the basis of the Union
Internationale Contre le Cancer38,39 criteria
of oral cavity carcinomas, a total of 28 cases
(32%) were at clinical stage I (T1N0M0)
and 59 (68%) clinical stage II (T2N0M0).
All 87 patients underwent surgical treat-
ment of primary tumor resection and 64 of
them were submitted to elective neck dis-
374 Faustino et al.
Fig. 1. Weak cytoplasmic immunostaining for VEGF-C in OSCC (A and B). Strong cytoplasmic immunostaining for VEGF-C in OSCC (C
and D).
section (54 patients: ipsilateral neck
dissection; 9 patients: bilateral neck dis-
section). Only 19 patients received post-
operative adjuvant radiotherapy.
During clinical follow up, local and
regional recurrences were observed in
15 (17%) and 14 (16%) patients respec-
tively, and three patients (3.4%) devel-
oped both. Two patients with OSCC
developed distant metastases (one within
lung and the other liver).
Microscopically, most of the OSCC
cases analysed were well to moderately
differentiated, showing solid cords of neo-
plastic cells as pattern of invasion. Most of
the tumors presented intense keratiniza-
tion, discrete nuclear polymorphism, few
mitotic figures per high-power field (X400
magnification) and intense-to-moderate
lymphocytic infiltration.
According to histopathological malig-
nant grading described by BRYNE et al.6, 69
OSCCs (79%) were classified as more
differentiated tumors (5–12 points) and
18 (21%) as less differentiated tumors
(13–20 points). Surgical margins were
negative in 84 patients (97%), but three
patients (3.4%) had positive surgical mar-
gins (a surgical margin was classified as
positive when there was OSCC or OSCC
in situ at the margin). Fourteen patients
(22%) among the 64 patients submitted to
elective neck dissection presented positive
lymph nodes confirmed by histopatholo-
gical analysis (pN+) at the moment of
primary tumor resection.
Immunostaining for VEGF-C expressed
by malignant cells
Immunohistochemical analysis showed
strong cytoplasmic immunostaining for
VEGF-C in 66 (75.9%) of the 87 tumors
and weak immunostaining in 21 (24%)
(Fig. 1). Some of the inflammatory cells
infiltrating the tumors, especially macro-
phages, were found to stain strongly for
VEGF-C.
No statistically significant correlations
were found regarding clinicopathologic
parameters (including gender, ethnic
group, age, tobacco, alcohol, tumor site,
T stage, local recurrence, regional recur-
rence, lymphatic or blood embolization,
perineural, muscular and salivary gland
infiltrations) and the VEGF-C expression
by malignant cells of the OSCC (Table 1).
There was no statistical correlation
between VEGF-C expression and occult
lymph-node metastases as well as histo-
pathological malignancy grading in the
invasive front of the tumors (Table 2).
The clinical follow up for the 87
patients with OSCC ranged from 5.4 to
272.1 months (mean 82.2  63.1 SD). At
the end of the follow-up period, 33
patients (38%) were alive and disease free,
19 patients (22%) had died of recurrence
(local, regional or distant), 28 patients
(32%) had died from cause other than
tumor, and 7 (8%) were considered lost
to follow up because they reached a clin-
ical outcome in less than 5 years.
Details concerning 5-year and 10-year
overall and disease-free survival rates are
summarized in Table 3. VEGF-C expres-
sion by malignant cells was not a signifi-
cant prognostic factor for patients with
OSCC.
Excluding VEGF-C expression, the
neck lymph-nodal status influenced the
overall survival rates of the 64 patients
submitted to elective neck dissection
(Fig. 2 and Table 4). The 5-year and 10-
year overall survival rates were, respec-
tively, 67% and 58% (pN0 group),
whereas for the 14 patients with occult
lymph-node metastasis they were 50% and
29% (pN+ group) (p = 0.030), as summar-
ized in Table 4.
Discussion
Although the metastatic dissemination of
tumor cells to regional lymph nodes is a
common feature of OSCC arising in the
 VEGF-C expression and occult lymph-node metastasis in oral cancer 375

Table 1. Correlation between clinicopathologic parameters and VEGF-C expression in OSCC has gained importance in recent years as
VEGF-C expression an adjuvant tool, in order to understand the
P mechanism of metastatic spread via lym-
Weak Strong phatic vessels.
Variable Category N % N % Most of the patients with OSCC sub-
mitted to elective neck dissection in our
Gender Male 16 76 52 79 0.802
Female 5 24 14 21 series showed strong cytoplasmic immu-
Ethnic group White 20 95 60 91 0.525 nostaining for VEGF-C, including pN0 and
Not white 1 5 6 9 those with occult lymph node metastasis
Age 59 years 10 48 35 53 0.666 (pN+), as shown in Fig. 1. These results
>59 years 11 52 31 47 confirm the observation that tumor cells of
*
Tobacco Yes 17 85 55 90 0.524 OSCC, in the early stage of development,
No 3 15 6 10 express VEGF-C11,16,17,20,21,25,33,35,36,41,44,
Alcohol* Yes 16 80 50 82 0.844 which may elevate the risk of precocious
No 4 20 11 18 nodal metastasis.
Tumor site Tongue 16 76 44 67 0.411
Floor of mouth 5 24 22 33
VEGF-C expression of OSCC located
T stage T1 10 48 18 27 0.082 in the tongue and floor of mouth was not
T2 11 52 48 73 influenced by other clinicopathological
Local recurrence Yes 2 10 13 20 0.282 parameters (gender, ethnic group, age,
No 19 90 53 80 tobacco and alcohol consumption, tumor
Regional recurrence Yes 4 19 10 15 0.672 site, T stage, local recurrence, regional
No 17 81 56 85 recurrence, lymphatic or blood emboliza-
Lymphatic embolization Yes 5 24 20 30 0.567 tion, perineural, muscular and salivary
No 16 76 46 70 gland infiltration) as described in
Blood embolization Yes 2 10 10 15 0.515
Table 1. In contrast to the results of KISHI-
No 19 90 56 85 11 16 33
MOTO et al. , LI et al. , SEDIVY et al. ,
Perineural infiltration Yes 12 57 32 49 0.489 35 36
No 9 43 34 51 SHINTANI et al. , SIRIWARDENA et al. and
Muscular infiltration Yes 18 86 53 80 0.577 TANIGAKI et al.41, the present results do not
No 3 14 13 20 show a statistically significant association
Salivary gland infiltration Yes 5 24 24 36 0.288 between lymph-node metastasis and
No 16 76 42 64 VEGF-C expression by malignant cells
TOTAL 21 100 66 100 of the OSCC (clinical stage I and II).
*
Excluding patients with lost records. These authors did not investigate, as in
the present study, the occult lymph-node
tongue and floor of mouth, it is not clear sels9,15,23,29,30,43, but little is known about metastasis from OSCC, and most of them
whether the tumor utilizes existing lym- the role of tumor lymphangiogenesis in analyzed tumors at both early and
phatic channels or whether dissemination metastasis. VEGF-C overexpression advanced clinical stages (from I to IV).
requires the formation of new lymphatic seems to induce new lymphatic and blood Occult lymph-node metastases (pN+)
vessels (lymphangiogenesis)4,8,18,27,32,34. vessels in the vicinity of the primary can- were detected in 22% of the 64 patients
In the last decade, VEGF-C and VEGF- cer14,33. In this context, the correlation who were submitted to elective neck dis-
D were identified as lymphangiogenic between VEGF-C and lymphatic vessel section in this series. This percentage is
growth factors and ligands to the receptor density in human OSCC, evaluated by close to those reported by AMARAL et al.1
VEGFR-3 present in the lymphatic ves- immunohistochemical parameters25,33,36, (23%) and OKAMOTO et al.26 (24%), but

Table 2. Correlation between histopathological malignancy grading, neck lymph-nodal status and VEGF-C expression in OSCC
VEGF-C
P
Weak Strong TOTAL
Variable Category N % N % N %
Malignancy grading Less differentiated 3 17 15 83 18 100 0.406
More differentiated 18 26 51 74 69 100
Neck lymph-nodal status* pN+ (ONM) 2 14 12 86 14 100 0.876
pN0 8 16 42 84 50 100
ONM = occult lymph node metastasis.
*
Excluding patients not submitted to elective neck dissection.

Table 3. Five-year and 10-year survival rates of the 87 patients with OSCC according to VEGF-C expression
Overall survival (%) Disease-free survival (%)
Variable Category P P
5-year 10-year 5-year 10-year
VEGF-C Weak 63 53 0.682 65 65 0.975
Strong 62 46 66 66
376 Faustino et al.
Table 4. Five-year and 10-year overall survival rates of the 64 patients with OSCC submitted to
elective neck dissection
Variable Category
Neck lymph-nodal status pN+ (ONM)
pN0
ONM = occult lymph-node metastasis.
lower than those of RUSSOLO et al.31 (34%)
and BYERS et al.7 (45%) in patients with
OSCC.
The immunostaining of VEGF-C in
OSCC is a histopathological parameter that
has been evaluated for others11,17,33,35,41
but generally few microscopic fields were
analyzed and frequently the extension of
the examined area and field magnification
were not specified. The present study
involved an objective and reproducible
evaluation of VEGF-C expression in malig-
nant cells of OSCC using a computer sys-
tem that permitted capture of the tumor area
to be analyzed by investigators. Thirty
invasive front tumor fields were evaluated
(X400 magnification) in each sample; with-
out doubt, a representative area of the most
invasive region of the OSCC. Use of this
objective methodology minimizing the
subjectivity and facilitates comparison of
the results with those of future studies, an
essential step for establishment of the real
influence of VEGF-C expression by malig-
nant cells on the clinical and biological
behavior of the OSCC.
According to the histopathological
malignancy grading, most (79%) of the
87 OSCC cases were classified as well-to-
moderately differentiated (more differen-
tiated tumors), a finding consistent with
other published studies11,35,41. There was
no statistical correlation between malig-
Fig. 2. Cumulative overall survival probability curves for groups with lymph-node metastasis
(pN+) and without lymph-node metastasis (pN0).
Overall survival (%)
P cells does not directly influence the clin-
5-year 10-year
50 29 0.030
67 58
nancy grading and VEGF-C expression
(Table 2). Bryne’s grading system6 was
used, in which the morphological features
are determined from the most invasive
front of the tumors. The invasive tumor
front consists of the most aggressive cells,
which have the ability to invade surround-
ing tissue structures, including vessels,
and thereby metastasize. The characteris-
tics of the invasive tumor front are of
major significance for the prognosis of
oral cancer3,5.
VEGF-C expression in the malignant
cells did not influence the likelihood of
overall and disease-free survival for the
patients with OSCC located in the tongue
and floor of the mouth (Table 3). The only
prognostic factor that reached statistical
significance and influenced cumulative
overall survival rates was the occurrence
of occult lymph-node metastasis (pN+), as
showed in Table 4 and Fig. 2. These results
reinforce previous findings that, in the early
stages of OSCC, the prediction of occult
lymph-node metastasis is, at present, indis-
pensable for improvement in the probabil-
ity of survival1,7,10,11,12,31. The pursuit of
predictive factors should be increased to
reduce the morbidity of elective neck dis-
section in patients who do not have metas-
tasis in the pathological exam (pN0).
In conclusion, although VEGF-C
expression has been occasionally regarded
as a prognostic factor in OSCC, these
results suggest that the isolated immu-
noexpression of VEGF-C in malignant
ical outcome and prognosis of patients
with early OSCC of tongue and floor of
mouth. In spite of this, further studies
should be performed with regard to
VEGF-C, microvessel density and the
occurrence of regional lymph-node metas-
tases because, in the future, understanding
the influence of lymphangiogenic growth
factors and tumour immunomodulation
may yield an array of new therapies to
limit the metastatic spread of oral cancer.
Acknowledgments. The authors thank
FAPESP (Fundação de Amparo à Pesquisa
do Estado de São Paulo, grant #2005/
04577-4) and CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel
Superior) for supporting this study.
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