Yonago Acta medica 2013;56:29–37

Original Article

Lymphatic Vessel Density and Vascular Endothelial Growth Factor Expression in
Squamous Cell Carcinomas of Lip and Oral Cavity: A Clinicopathological Analysis
with Immunohistochemistry Using Antibodies to D2-40, VEGF-C and VEGF-D
Soh Watanabe,* Masako Kato,† Isamu Kotani,* Kazuo Ryoke* and Kazuhiko Hayashi†*Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8504, Japan and †Division of Molecular Pathology, Department of Pathology, School of Medicine, Tottori University Faculty of Medicine, Yonago, 683-8503, Japan

ABSTRACT
Background Squamous cell carcinoma (SCC) of
the oral region often metastasizes to the cervical lymph
nodes. To investigate whether the risk of cervical lymph
node metastasis are predictable through lymphatic vessel density (LVD) and vascular endothelial growth factor (VEGF) expression, we assessed the relationshipbetween LVD and clinicopathological parameters, andVEGF expression in SCC of the oral region.
Methods The subjects were 109 patients with SCC ofthe oral region including the lip. Clinicopathological parameters examined for the association with LVD in a peritumoral hot spot were lymph node metastasis, histologicalgrade and disease stage. The association with VEGF
expression was similarly studied. LVD was detected byimmunohistochemistry using D2-40.
Results LVD was significantly higher in lip cancer
than in other oral tumors (P < 0.0001), while there wereno significant differences of LVD among other cancersof the oral cavity. LVD tended to decrease with diseaseprogression, increase of tumor size and increase of metastatic lymph node size. Eighty-four of 109 tumors werepositive for VEGF-C or D. VEGF-C-positive tumor lesions were also positive for VEGF-D. Significantly higher levels of VEGF-C and D expressions were associatedwith large size of lymph node metastases (P = 0.02).
Conclusion SCC of the oral region including the
lip that produces VEGF-C and D is significantly more
likely to cause cervical lymph node metastasis. LVD in aperitumoral hot spot does not directly indicate the risk ofcervical lymph node metastasis, but instead may reflect
lymphangiogenesis due to VEGF together with loss oflymphatic vessels through tumor growth and progression.

The oral region has a role in various essential and complex functions to breath, masticate, swallow and vocalize, which are important for sustaining life. The oral region extends from the lips to the palatoglossal fold, classified into the lips, buccal mucosa, gingiva, floor of themouth, tongue and palate. Although it contains various
anatomically distinct tissues, squamous cell carcinoma(SCC) accounts for more than 90% of all malignant tumors originating in the oral region.1 Unlike SCC of theskin and other organs, SCC of the oral region is likely tometastasize to the cervical lymph nodes at an early period, so cervical lymph node metastasis is considered to
be a prognostic factor for patients with oral cancer.2, 3
Regarding the relationship between oral SCC andcervical lymph node involvement, an association withhistological grade and tumor size was suggested,4 whileassociation with cervical metastasis of histological grade
but no primary tumor location or size5 was suggested.
Lymphatic vessels in the tongue have many connections
and are linked across the midline. This anatomy of thelymphatics suggests that tumors in the oral region caneasily metastasize to the opposite side from the primarytumor or can cause bilateral metastases.1
In order to establish lymph node metastasis, it is alsoessential for tumor lymphangiogenesis to be induced.
Previously, SCC of the oral cavity, larynx/pharynx
and esophagus, and squamous cancer of the lungs andgastrointestinal tract were investigated regarding therelationship between tumors and lymphangiogenesis bymeans of assessment of lymphatic vessel density (LVD)
and expression of vascular endothelial growth factors(VEGFs).6–11 However, there is no detailed study of
the associations of site-specific LVD in oral SCC with
VEGF expression or clinicopathological findings. In thepresent study, we performed histopathological assessment of LVD in over 100 patients with oral SCC, andexamined the association between LVD and clinicopathological findings. Our results showed that, contrary
to the previous reports, LVD in peritumoral tissue “hotspots” tended to decrease with an increase of tumor sizeand stage progression. On the other hand, the expression of VEGFs in SCC was significantly associated with

Key words
lip; lymphatic vessel density; oral cavity;
squamous cell carcinoma; vascular endothelial growth factor
Corresponding author: Masako Kato, MDmakato@med.tottori-u.ac.jp
Received 2012 December 07
Accepted 2012 December 25
Abbreviations: HE, hematoxylin and eosin; LVD, lymphatic vessel
density; SCC, squamous cell carcinoma; VEGF, vascular endothelial growth factor; VEGFR-3, VEGF receptor-3

29

S. Watanabe et al.
Table 1. Clinicopathological characteristics of 109 patients with SCC of lip and oral cavity
Characteristics
GenderMale
FemaleAge (yr)
Range
Mean ± SD
T1
Primary tumor size
T2
T3
T4
Size of regional lymph node metastasis N0 (no metastasis)
N1 (≤ 3 cm)
N2 (between N1 and N3)
N3 (> 6 cm)
Clinical stage (UICC)
Stage I
Stage II
Stage III
Stage IV
Histological differentiation
Well differentiatedModerately differentiatedPoorly differentiatedPatientsn (%)

LVD(mean ± SD)

63 (57.5)
46 (42.2)
23–93
65 ± 1320 (18.3)
43 (39.4)
9 (8.3)37 (34.0)
65 (59.6)
18 (16.5)
24 (22.0)
2 (1.8)18 (16.5)
28 (25.7)
16 (14.7)
47 (43.1)
44 (40.4)
46 (42.2)
19 (17.4)

19.0 ± 11.9
21.3 ± 12.2

0.345

20.0 ± 11.9
23.4 ± 11.0
21.6 ± 13.9
21.7 ± 12.3
15.9 ± 8.9
21.3 ± 12.6
20.1 ± 13.4
17.0 ± 8.6
12.0 ± 2.8
22.4 ± 9.7
22.8 ± 15.5
21.9 ± 14.1
16.7 ± 8.7
21.3 ± 14.1
19.0 ± 10.4
19.5 ± 10.2

0.928
0.253

P-value

0.608

0.971

0.708

LVD, lymphatic vessel density; SCC, squamous cell carcinoma; UICC, Union Internationalis Contra Cancrum.

1). The subjects were also classified into 4 groups basedon tumor size: T1 in 20 patients, T2 in 43 patients, T3
in 9 patients and T4 in 37 patients (Table 1). Histologically, the tumor was classified as well differentiated in
44 patients, moderately differentiated in 46 patients andpoorly differentiated in 19 patients. With regard to thepresence or absence of cervical lymph node metastasis,
there was no cervical lymph node metastasis (N0) in 65
subjects, while 18 had a solitary lymph node metastasis
measuring 3 cm or less in maximum diameter (N1) on
the same side as the primary tumor, 2 had a lymph nodemetastasis measuring 6 cm or more in maximum diameter (N3) and 24 had a lymph node metastasis between
N1 and N3 (N2). We performed histopathological assessment of LVD using the pathological specimens fromeach group. Furthermore, to assess the factors present at
an early stage that could lead to lymphatic metastasis,

lymphatic metastasis. This suggests that growth of lymphatics may be promoted by the production of VEGFs intumor tissue, but this is not directly reflected by LVD in
peritumoral hot spots. Instead, with regard to the mechanism of cervical lymph node metastasis by oral SCC,
factors other than LVD in peritumoral hot spots may be
involved.
SUBJECTS AND METHODS
Subjects and clinicopathological features

The subjects were 109 patients who underwent biopsy
or surgical resection of tumors in the oral region at theDivision of Oral and Maxillofacial Biopathological
Surgery (Tottori University Hospital, Japan) between
January 1991 and 2005. They did not receive radiation
therapy nor chemotherapy before biopsy or surgicalresection. They included 63 men and 46 women aged
between 23 and 93 years, with a mean of 65 ± 13 years.
Table 1 shows their clinicopathological features. Regarding the histology of the tumor, the final diagnosis
was SCC based on examination of biopsy or resected
specimens in all patients. Oral SCC was classified into
6 types according to the location of the primary tumor:
gingiva in 45 patients, tongue in 41 patients, buccal mucosa in 8 patients, floor of the mouth in 7 patients, palate in 5 patients and lip in 3 patients (Table 2). Clinicaltumor staging according to the TNM classification was
as follows: stage I in 18 patients, stage II in 28 patients,
stage III in 16 patients and stage IV in 47 patients (Table

Table 2. LVD among the primary sites of 109 patientswith SCC
Primary site

Patientn (%)

LVD(mean ± SD)

TongueGingiva
Buccal mucosa
Oral floor
PalateLip

41 (37.6)
45 (41.3)
8 (7.3)7 (6.4)5 (4.6)3 (2.8)20.2 ± 8.7
17.8 ± 10.4
25.9 ± 19.4
15.3 ± 8.1
14.6 ± 7.2
54.3 ± 6.0***

LVD, lymphatic vessel density; SCC, squamous cell carcinoma.
***P < 0.0001.

30

D2-40/VEGFs expression of lip/oral SCC
Table 3. Antibodies used and dilutions
Antibody

Clone

Dilution

Antigen retrieval

Source

D2-40
VEGF-CVEGF-D

D2-40
Polyclonal
78923

1:100
1:400
1:100

Microwave heating
Microwave heating
Microwave heating

DAKO (Glostrup, Denmark)R&D Systems (Mineapolis, MN)
R&D Systems (Mineapolis, MN)

VEGF, vascular endothelial growth factor.

we also examined the association between the abovementioned clinicopathological parameters and LVD instage-I and II patients. The present study was performed
with approval from the ethics committee of the Tottori
University Faculty of Medicine (approval number: 1499).

opsy or surgery. One of each set of serial sections was
subjected to hematoxylin-eosin (HE) staining and wasused to evaluate histopathological findings according to
the TMN classification of the Union Internationalis Contra Cancrum. The other serial sections were subjected toimmunohistochemical analysis.

Histopathological examination
Immunohistochemistry

Serial paraffin sections measuring 4 µm in thickness
were prepared from tumor tissue blocks obtained at bi-

Immunohistochemical analysis was performed by theavidin-biotinylated peroxidase complex method. Paraffin sections were deparaffinized, and then subjected
to microwave heating for 10 min in 0.01 M sodium citrate buffer (pH 6.0). Next, the sections were treated inmethanol containing 0.3% hydrogen peroxide solution to
inhibit endogenous peroxidase, incubated with a blocking reagent to prevent nonspecific reactions, and thenincubated at 4 ˚C overnight with various primary antibodies (Table 3) at various dilutions. The sections were
subsequently washed with phosphate-buffered saline (pH7.4) and then incubated for 30 min with the biotinylated
secondary antibody at room temperature, followed byreaction for 1 h with avidin-biotinylated peroxidase complex solution at room temperature. Color was developedwith 3-3'-diaminobenzidine-tetrahydrochloride, andafter nuclear staining, dehydration and lucidification,
specimens were served for microscopic examination.
The primary antibodies used were listed in Table 3. Sections incubated in phosphate-buffered saline without any
primary antibody were used as the negative control.

1A

LVD measurement

Sections immunostained with D2-40 were used to measure the lymphatic vessel count. Clinicopathologicalfindings were evaluated by 2 independent examiners (SWand MK) in a blinded fashion, and mean values were calculated. Lymphatic vessel measurement was performed
according to a modification of Weidner’s method (1991).12
In brief, a region of the peritumoral stroma that was appropriate for determining the lymphatic vessel count was
defined as a hot spot (Fig. 1), and was used to measure
the number of lymphatic vessels positive for D2-40 ineach of 2 arbitrary fields under an objective lens with amagnification power of 20 (field area: 0.949 mm2). Thenthe total number of lymphatic vessels in the 2 fields wascalculated. The total lymphatic count of the 2 fields in the

1B
Fig. 1. HE staining and D2-40 immunostaining of resected SCC
specimens of the tongue.
A: Well differentiated SCC showing solid proliferation.
B: There are D2-40-positive lymphatic vessels in the peritumoral
stroma. The cytoplasm of some tumor cells at the margin of
the cancer is also positive for D2-40. The circled area was defined as the hot spot. Lymphatic vessels were counted in 2 hotspot fields and totaled.
HE, hematoxylin and eosin; SCC, squamous cell carcinoma.

31

S. Watanabe et al.

Association between clinicopathological findings
and LVD

hot spot was compared among groups stratified for age,sex, primary tumor site, clinical stage, tumor size, grade
and the presence or absence of lymphatic metastasis.

In relation to tumor size, LVD was 23.4 ± 11.0 for T1,
21.6 ± 13.9 for T2, 21.7 ± 12.3 for T3 and 15.9 ± 8.9 forT4 disease (Table 1). Regarding the association between
LVD and tumor size, the lymphatic vessel count tended
to decrease with an increase of tumor size, but there
was no significant difference of LVD among each tumor
size category. LVD was 21.3 ± 12.6 in patients without
lymph node metastasis (N0 group), 20.1 ± 13.4 in the N1
group, 17.0 ± 8.6 in the N2 group and 12.0 ± 2.8 in theN3 group. There were no significant differences of LVD
among these 4 groups, although LVD tended to decrease
with an increase of lymph node metastasis size. According to the clinical tumor stage, LVD was 22.4 ± 9.7 instage I, 22.8 ± 15.5 in stage II, 21.9 ± 14.1 in stage III
and 16.7 ± 8.7 in stage IV. The lymphatic vessel counttended to decrease with stage progression, but multiple
regression analysis revealed no significant differenceof LVD between each stage. Regarding the histologicalgrade of SCC, LVD was 21.3 ± 14.1 in well-differentiated SCC, 19.0 ± 10.4 in moderately differentiated SCC
and 19.5 ± 10.2 in poorly differentiated SCC. There wasno significant difference of LVD among the different
grades of SCC. No significant differences of LVD were
found in relation to sex and age.
Assessment of the association between LVD andclinicopathological findings in stage-I and II patients revealed no significant correlations between LVD and age(P = 0.712), sex (P = 0.873), tumor size (P = 0.772) ortumor histological grade (P = 0.057) by multiple regression analysis.

Examination of VEGF-C and D expressions

Staining for VEGF-C or D was performed using serial
paraffin sections. For both VEGF-C and VEGF-D, morethan 50% positive tumors cells were judged as positive
staining. We examined the relationship between the results of VEGF-C or D staining and clinicopathologicalfindings or LVD.
Statistical Analysis

Relationships between clinicopathological parametersand LVD were assessed by multivariate analysis using a
multiple linear regression model. Associations between
the expression of VEGF-C or D and clinicopathological parameters or LVD were investigated by multivariate
logistic regression analysis. For statistical analysis of therelationship between the primary site and LVD, analysis ofvariance was used, followed by multiple comparison with
the Dunnett’s method and Scheff’s method. SPSS Statistics software version 19.0 (IBM, Tokyo, Japan) was used
for all analyses, and statistical significance was accepted at
P < 0.05.
RESULTS
Site-specific LVD in oral SCC

The LVD measured in the 2 hot spot fields was 20.2 ± 8.7
for tongue cancer, 17.8 ± 10.4 for gingival cancer, 25.9
± 19.4 for cancer of the buccal mucosa, 15.3 ± 8.1 fororal floor cancer, 14.6 ± 7.2 for palatal cancer and 54.3 ±6.0 for lip cancer (Table 2). HE- and D2-40-stained sections of representative examples of tongue, gingiva, lip,
palate and oral floor cancers are shown in Figs. 2A to J.
In the peritumoral stromal tissue, D2-40-poisitive lymphatic vessels of various sizes were observed (Figs. 2B,
D, F, H and J). Blood vessel components apart from thelymphatic endothelium and the negative controls were
negative for D2-40 staining. In addition, the marginsof the tumor tissue and in infiltrating areas were D2-40
positive (Figs. 2B and F). Site-specific LVD showed asignificant difference between lip cancer and cancers of
the gingiva, tongue, buccal mucosa, oral floor and palate(post-hoc test, P < 0.0001). The LVD of lip cancer was
significantly higher than that for cancers of the tongue,
gingiva, buccal mucosa, oral floor and palate. However,no significant differences of LVD were noted amongcancers of the tongue, gingiva, buccal mucosa, oral floor
and palate.

Relationship between VEGF-C or D immunostaining and LVD or clinicopathological findings

Immunostaining was positive for VEGF-C and D in thecytoplasm of tumor cells (Figs. 3B, E and F). Tumor
tissue with more than 50% positive cells is judged to
be positive for VEGF staining. Based on this criterion,
we determined 84 of the 109 tumors to be positive for
VEGF-C (Fig. 3B and E) and 25 of them, negative (Fig.
3G). In the case of VEGF-D, we determined 84 of the
109 tumors to be positive for VEGF-D, which were also
positive for VEGF-C (Figs. 3E and F). In other words,
the expression patterns of VEGF-C and D were identical.
When we investigated the relationship between LVD
and staining for VEGF-C or D, LVD was 19.7 ± 11.9 in
VEGF-C-positive patients and 21.2 ± 12.2 in VEGFC-negative patients. Regarding the association between
LVD and either positive or negative staining for VEGFC or D, logistic regression analysis revealed no statistically significant associations. When VEGF-C/VEGF32

D2-40/VEGFs expression of lip/oral SCC

ACEGI: HE staining

D2-40/VEGFs expression of lip/oral SCC

BDFHJ: D2-40 immunostaining

A & B Cancer of the tongue

2A

2B

C & D Cancer of the gingiva
E & F Cancer of the lip
G & H Cancer of the palate

2C

2D

2E

2F

I & J Cancer of the oral floor

Fig. 2. HE staining and D2-40
immunostaining of SCC tissues in
representative patients.
ACEGI: Well differentiated SCC
showing cord-like proliferationand invasion. Bar = 100 µm.

2G

2H

2I

2J

BDFHJ: There are D2-40-positive
lymphatics in the peritumoral
stroma. These lymphatic vessels
are clearly distinguishable from
D2-40-negative blood vessels.
HE, hematoxylin and eosin; SCC,
squamous cell carcinoma.

33

S. Watanabe et al.

A–H: Tongue cancer
A & B Specimen 1

VEGF-Cimmunostaining

HE staining

3A

3B

C–F Specimen 2

D2-40
immunostaining

HE staining

3C

3D

VEGF-Dimmunostaining

VEGF-Cimmunostaining

3E

3F

G & H Specimen 3

D2-40
immunostaining

VEGF-Cimmunostaining

3G

3H

Fig. 3. HE staining, D2-40 immunostaining, VEGF-C immunostaining and VEGF-D immunostaining of SCC.
A: Well-differentiated SCC showing cord-like proliferation and invasion.
B: The cytoplasm of tumor cells is positive for VEGF-C staining. Non-neoplastic squamous cells in the tongue mucosa are negative for
VEGF-C.
C: Small cords of SCC cells proliferated and invaded the stromal tissues of the tongue.
D: Several D2-40-postivie lymphatics are observed in the stroma in the vicinity of the tumor tissues. Blood vessels are negative for D240.
E: The majority of the tumor cell nests are strongly positive for VEGF-C.
F: The majority of tumor cell nests are positive for VEGF-D. The VEGF-D staining pattern corresponds to that of VEGF-C.
G: A few tumor cells are positive for VEGF-C, but many tumor cells are negative.
H: Many D2-40-positive lymphatic vessels can be seen in the stroma near the tumor tissues. Tumor cells in the infiltrating lesion (leftupper region) are also strongly positive for D2-40.
The same magnification was used for A to H. Bar = 100 µm. HE, hematoxylin and eosin; SCC, squamous cell carcinoma; VEGF, vascular endothelial growth factor.

34

D2-40/VEGFs expression of lip/oral SCC

D positivity and D2-40 staining were compared, LVD
was low in some VEGF-C- and D-positive tumors (Figs.
3D–F), while LVD was high in some VEGF-C- and Dnegative tumors (Figs. 3G and H).
Regarding the relationship between clinicopathological findings and VEGF staining, the VEGF-C-positive
rate increased significantly with an increase in the sizeof lymph node metastases (logistic regression analysis, P= 0.02). Similarly, the VEGF-D-positive rate increased
significantly with an increase in the size of lymph nodemetastases (P = 0.02). However, no significant association was observed between VEGF expression and theclinicopathological factors such as tumor size and clinical stage.

metastasis. Other factor like the distance from the cervical lymph nodes might also influence the biological behavior of lip cancer. Furthermore, the lip is a prominentsite where tumors are easily detected, which contributes
to early diagnosis and treatment. This might also explain why the prognosis of lip cancer is better than thatof cancer of other oral regions. In general, oral cancerof the tongue and floor of the mouth metastasize to thecervical lymph nodes relatively often. However, in thepresent investigation of site-specific LVD in peritumoral
hot spots, we found no significant differences amongsites other than the lip, such as the gingiva, tongue, buccal mucosa, oral floor and palate. Therefore, based on
the mechanism of cervical lymph node metastasis which
occurs more easily in cancer of the tongue and oral floor
than in cancer of the gingiva and palate, we found that
the LVD of hot spots does not directly reflect susceptibility to cervical lymph node metastasis.
Regarding the relationship between LVD and oral
SCC, lymphangiogenesis was associated with metastasis-free survival in tongue cancer patients.13 In addition,
the presence of lymph node metastasis was significantly
associated with a higher intratumoral lymphatic density,a higher incidence of tumor relapse and shorter levels of
5-year overall survival and disease-free survival.9 Moreover, LVD was correlated with tumor grade and nodal
status in oral SCC patients.17 In the present study of 109
patients with lip and oral SCC, we assessed all correlations between LVD and clinicopathological parameters
including tumor size, presence or absence of lymph nodemetastases, histological grade and clinical tumor stage;
however, multivariate analysis revealed no significant associations between LVD and clinicopathological parameters. In addition, we examined the associations between
LVD and these clinicopathological parameters among
stage-I and II patients without lymph node metastasis,and found no significant associations between LVD and
any of these factors. Rather, our results showed lymphatic vessel count decrease along with tumor size increase,which tended to decrease with the advance of diseasestage or lymph node metastasis. In an investigation of
adenocarcinoma of the rectum examined on the association between LVD and clinicopathological factors,18 theprognosis was not necessarily correlated with the LVD
at the peripheral zone of the tumor or inside tumor tissue, but actually with lymphangiogenesis in the regional
lymph nodes. In patients with oral cancer, the primarytumor induces sentinel lymph node lymphagiogenesis
prior to the onset of lymph node metastasis.19 In otherwords, lymphagiogenesis in the regional lymph nodes,
rather than peritumoral lymphatic hyperplasia, is an important prognostic factor.

DISCUSSION
SCC of the oral region is likely to cause lymph node metastases, which influences the prognosis. For this reason,
several studies have investigated the association between
lymphatic vessels and clinicopathological findings inpatients with oral SCC.9,13,14 However, these studies
mainly focused on SCC of the tongue and the numberof patients investigated was limited. In the present study,
we investigated the relationship between histopathologically assessed LVD and clinicopathological findings in
109 patients with SCC of various parts of the oral cavity,
including the lip; i.e., the gingiva, tongue, buccal mucosa, floor of the mouth and palate. No study has everexamined more than 100 patients with SCC originatingin the oral region, particularly with stratification according to the site of the primary tumor.
With regard to LVD in oral SCC, the tumor sitespecific LVD was 28.3 ± 12.5 for tongue cancer and 16.5
± 5.66 for cancer at other sites.13 The peritumoral LVDof oral cancer ranged widely from 1 to 43.9 Moreover,
the LVD of oral SCC was 12.89 ± 9.31.14 In the present study, we totalled the lymphatics in two 0.949-mm2
fields. When converted to a per-unit value, our LVD values were slightly lower than or similar to those reported
above.
As we observed in the present investigation of sitespecific LVD, lip cancer showed a significantly higher
LVD level than cancer arising at other sites.15, 16 Studies
on lip cancer were not many, especially with regard to
LVD, and no study reported higher LVD in the peritumoral “hot spot” of lip cancer than in other types of oral
cancer. This result may be related to an anatomical feature of the lip, high vascularity, which imparts the characteristic red color. Despite the high LVD observed inlip cancer at the hot spot, the prognosis of this cancer isquite good, with a low frequency of cervical lymph node35

S. Watanabe et al.
REFERENCES
1 Johnson N, Franceschi S, Ferlay J, Ramadas K, Schmid S,
MacDonald DG et al. Squamous cellcarcinoma. In: Barnes L,
Eveson JW, Reichart P, Sidransky D, eds. World Health Organization Classification of Tumours. Pathology & Genetics
Head and Neck Tumours. Lyon: IARC Press; 2005. p. 168-75.
2 Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. Oral Oncol. 2009;45:309-16. PMID:
18804401.
3 Kreppel M, Scheer M, Drebber U, Ritter L, Zöller JE. Impact
of podoplanin expression in oral squamous cell carcinoma:
clinical and histopathologic correlations. Virchows Arch.
2010;456:473-82. PMID: 20393745.
4 Martínez-Gimeno C, Rodríguez EM, Vila CN, Varela CL.
Squamous cell carcinoma of the oral cavity: a clinicopathologic scoring system for evaluating risk of cervical lymph nodemetastasis. Laryngoscope. 1995;105:728-33. PMID: 7603278.
5 Umeda M, Yokoo S, Take Y, Omori A, Nakanishi K, Shimada
K. Lymp node metastasis in squamous cell carcinoma of theoral cavity: correlation between histologic features and theprevalence of metastasis. Head Neck. 1992;14:263-72. PMID:
1517076.
6 Beasley NJP, Prevo R, Banerji S, Leek RD, Moore J, vanTrappen P, et al. Intratumoral lymphangiogenesis and lymph
node metastasis in head and neck cancer. Cancer Res. 2002;
62:1315-20. PMID:11888898.
7 Yuan P, Temam S, El-Naggar A, Zhou X, Liu DD, Lee JJ, etal. Overexpression of podoplanin in oral cancer and its association with poor clinical outcome. Cancer. 2006;107:563-9.
PMID: 16804930.
8 Adachi Y, Nakamura H, Kitamura Y, Taniguchi Y, Araki
K, Shomori K, et al. Lymphatic vessel density in pulmonary
adenocarcinoma immunohistochemically evaluated with
anti-podoplanin or anti-D2-40 antibody is correlated withlymphatic invasion or lymph node metastases. Pathol Int.
2007;57:171-7. PMID: 17316411.
9 Zhao D, Pan J, Li XQ, Wang XY, Tang C, Xuan M. Intratumoral lymphangiogenesis in oral squamous cell carcinoma
and its clinicopathological significance. J Oral Pathol Med.
2008;37:616-25. PMID: 19012622.
10 Raica M, Cimpean AM, Ribatti D. The role of podoplanin intumor progression and metastasis. Anticancer Res. 2008;28:
2997-3006. PMID: 19031946.
11 Garcia-Carracedo D, Rodrigo JP, Astudillo A, Nieto CS,
Gonzalez MV. Prognostic significance of lymphangiogenesis in pharyngolaryngeal carcinoma patients. BMC Cancer.
2010;10:416-25. PMID: 20698954.
12 Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N Engl J Med. 1991;324:1-8. PMID: 1701519.
13 Miyahara M, Tanuma J, Sugihara K, Semba I. Tumor lymphangiogenesis correlates with lymph node metastasis andclinicopathologic parameters in oral squamous cell carcinoma. Cancer. 2007;110:1287-94. PMID: 17674352.
14 Oriveira-Neto HH, Gleber-Netto FO, Ferreira de Sousa S,
França CM, Ferreira Aguiar MC, Silva TA, et al. A comparative study of microvessel density in squamous cell carcinomaof the oral cavity and lip. Oral Surg Oral Med Oral Pathol
Oral Radiol. 2012;113:391-8. PMID:22669145.
15 Vartanian JG, Carvalho AL, de Araújo Filho MJ, Hattori M
Jr, Magrin J, Kowalski LP. Predictive factors and distribution of lymph node metastasis in lip cancer patients and their

VEGF-C and D promote lymphatic vessel growth
and are involved in lymphangiogenesis.20 Both VEGFs
are related to LVD, and bind to VEGF receptor-3 (VEGFR-3) on the surface of lymphatic endothelial cells to
induce the proliferation and growth of new lymphatic
vessels.21 Expression of both VEGFs has been reported
in oral cancer.22 In the present study, immunostaining oforal SCC specimens showed a high positivity for VEGFC and D (84/109 both; 77%). Moreover, when the staining patterns were compared, VEGF-C and D showedalmost identical patterns. Significant associations between VEGF-C expression and LVD were reported.14,17
We observed no significant association of VEGF-C andD positivity with LVD, but significantly more VEGFC and D positivity among patients with larger lymph
node metastases. It is likely that lymphangiogenesis occurs due to the production of VEGF-C by tumor cells,
but conversely LVD in peritumoral hot spots tends todecrease with an increase of tumor size and stage progression. The production of VEGF-C by tumor cells
also promotes lymphangiogenesis, but lymphatic vessels at the tumor periphery are damaged due to tumor
progression and this may lead to a decrease of the LVD.
Furthermore, there is a possibility that lymphangiogenesis also occurs in remote sentinel lymph nodes due to
VEGF production. In relation to VEGF-C and D expressions in tumor tissue, it is thought that lymphagiogenesis
in the regional lymph node affects the prognosis. These
reports support our present finding that lymph node metastasis was more frequent in VEGF-C and D-positive
patients.
In conclusion, we examined the association between
clinicopathological parameters and LVD or the expression of VEGF-C or D in SCC of the oral region including the lip. Our site-specific investigation revealed asignificant increase of the LVD in lip cancer, whereas
no significant difference of LVD was observed in cancers arising at other sites. In addition, in terms of therelationship between clinicopathological parameters and
LVD, LVD tended to decrease with increasing diseasestage and size of lymph node metastasis. Furthermore,patients with larger lymph node metastases were significantly more likely to be positive for VEGF-C and
D. Peritumoral LVD decreased with tumor progression,
while the expression of VEGF showed a strong correlation with lymph node metastasis, suggesting that further
study is needed to determine the mechanisms of cervicallymph node metastasis in patients with oral SCC.
The authors declare no conflict of interest.

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D2-40/VEGFs expression of lip/oral SCC
implications on the treatment of the neck. Oral Oncol. 2004;
40:223-7. PMID: 14693248.
16 Batista AC, Costa NL, Oton-Leite AF, Mendonça EF, Alencar
RCG, Silva TA. Distinctive clinical and microscopic featuresof squamous cell carcinoma of oral cavity and lip. Oral Surg
Oral Med Oral Pathol Oral Radiol Endod. 2010;109:e74-9.
PMID: 20219590.
17 Sedivy R, Beck-Mannagetta J, Haverkampf C, Battistutti W,
Hönigschnabl S. Expression of vascular endothelial growth
factor-C correlates with the lymphatic microvessel density andthe nodal status in oral squamous cell cancer. J Oral PatholMed. 2003;32:455-60. PMID: 12901726.
18 Jakob C, Aust DE, Liebscher B, Baretton GB, Datta K, Muders MH. Lymphangiogenesis in regional lymph nodes is anindependent prognostic marker in rectal cancer patients after
neoadjuvant treatment. PLoS One. 2011;6:e27402. PMID:

22087309.
19 Ishii H, Chikamatsu K, Sakakura K, Miyata M, Furuya N,
Masuyama K. Primary tumor induces sentinel lymph node
lymphangiogenesis in oral squamous cell carcinoma. Oral
Oncol. 2010;46:373-8. PMID: 20308006.
20 Clarijs R, Ruiter DJ, de Waal RMW. Lymphangiogenesis inmalignant tumours: does it occur? J Pathol. 2001;193:143-6.
PMID: 11180158.
21 Su JL, Yen CJ, Chen PS, Chuang SE, Hong CC, Kuo IH, etal. The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer. 2007;96:541-5. PMID: 17164762.
22 Shintani S, Li C, Ishikawa T, Mihara M, Nakashiro K, Hamakawa H. Expression of vascular endothelial growth factor
A, B, C, and D in oral squamous cell carcinoma. Oral Oncol.
2004;40:13-20. PMID: 14662410.

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