VEGF-C Expression in Squamous Cell Carcinoma and Adenocarcinoma of the Esophagus

Abstract

Vascular endothelial growth factor C (VEGF-C) is the only factor known to cause lymphangiogenesis. In esophageal cancer the histologic tumor type and lymph node metastasis are independent predictors of recurrence and poor outcome. To evaluate the rule of VEGF-C expression in esophageal cancer, we investigated 113 specimens, 59 squamous cell and 54 adenocarcinomas of the esophagus.
The expression of VEGF-C was evaluated using immunohistochemistry (IHC) on 59 paraffin-embedded archival specimens from patients with squamous cell esophageal carcinomas and 54 paraffin-embedded archival specimens of patients with esophageal adenocarcinomas arising in Barrett's mucosa. All patients had a complete tumor resection. A complete and updated follow-up was available for all patients.
The expression of VEGF-C was significantly different between the two histological types of esophageal tumors. Patients with squamous cell carcinoma and lymph node metastases had a significantly higher VEGF-C expression (P < 0.01). In patients with adenocarcinoma of the esophagus there was no correlation between VEGF-C expression and clinicopathological parameters. High VEGF-C expression tended to be correlated with poor survival in squamous cell cancer but not in adenocarcinoma of the esophagus.
The present study indicates that VEGF-C may play a role in tumor progression via lymphangiogenesis in squamous cell carcinoma of the esophagus. This seems not to be true for the adenocarcinoma of the esophagus. These data could help with the understanding of the different onset and characteristics of lymph node metastasis in squamous cell carcinoma and adenocarcinoma of the esophagus.

Full text

VEGF-C Expression in Squamous Cell Carcinoma
and Adenocarcinoma of the Esophagus
Christian Mo
¨
bius, MD,
1
Jose
´
Freire, MD,
2
Ingrid Becker, MD,
3
Marcus Feith, MD,
4
Bjo
¨
rn L. D. M. Bru
¨
cher, MD, FACS,
4
Michael Hennig, MD,
5
J. Ru
¨
diger Siewert, MD,
4
Hubert J. Stein, MD
6
1
Second Department of Surgery, University of Leipzig, Germany
2
Second Department of Surgery, Santa Maria Hospital, Lisbon Medical School, Portugal
3
Pathology Department, Klinikum rechts der Isar, Technische Universita
¨
tMu
¨
nchen, Germany
4
Department of Surgery, Klinikum rechts der Isar, Technische Universita
¨
tMu
¨
nchen, Germany
5
Institute of Medical Statistics and Epidemiology, Klinikum rechts der Isar, Technische Universita
¨
t
Munchen, Germany
6
Department of Surgery, Salzburger Landesklinik, University Hospital Salzburg, Austria
Abstract
Background: Vascular endothelial growth factor C (VEGF-C) is the only factor known to cause
lymphangiogenesis. In esophageal cancer the histologic tumor type and lymph node metastasis
are independent predictors of recurrence and poor outcome. To evaluate the rule of VEGF-C
expression in esophageal cancer, we investigated 113 specimens, 59 squamous cell and 54
adenocarcinomas of the esophagus.
Methods: The expression of VEGF-C was evaluated using immunohistochemistry (IHC) on 59
paraffin-embedded archival specimens from patients with squamous cell esophageal carcinomas
and 54 paraffin-embedded archival specimens of patients with esophageal adenocarcinomas
arising in Barrett’s mucosa. All patients had a complete tumor resection. A complete and updated
follow-up was available for all patients.
Results: The expression of VEGF-C was significantly different between the two histological types
of esophageal tumors. Patients with squamous cell carcinoma and lymph node metastases had a
significantly higher VEGF-C expression (P < 0.01). In patients with adenocarcinoma of the
esophagus there was no correlation between VEGF-C expression and clinicopathological
parameters. High VEGF-C expression tended to be correlated with poor survival in squamous cell
cancer but not in adenocarcinoma of the esophagus.
Conclusions: The present study indicates that VEGF-C may play a role in tumor progression via
lymphangiogenesis in squamous cell carcinoma of the esophagus. This seems not to be true for
the adenocarcinoma of the esophagus. These data could help with the understanding of the
different onset and characteristics of lymph node metastasis in squamous cell carcinoma and
adenocarcinoma of the esophagus.
D
uring the last two decades, surgical therapies of
esophageal cancer in the Western world have
changed significantly. Advances in perioperative man-
agement and standardization of the surgical technique
have resulted in a substantial reduction in the number of
postoperative deaths after esophagectomy in experi-
Correspondence to: Hubert J. Stein, MD, Mu
¨
llner Haupstraße 48,
A–5020 Salzburg, Austria, e-mail: h.j.stein@salk.at
 2007 by the Socie
´
te
´
Internationale de Chirurgie World J Surg (2007)
Published Online: 5 March 2007 DOI: 10.1007/s00268-006-0373-1

enced centers. Nevertheless the overall prognosis of
patients with esophageal cancer remains poor due to local
recurrence and the development of distant metastasis.
1
In the past, adenocarcinoma and squamous cell carci-
noma of the esophagus have been treated as a single
entity, but recently our group presented the histologic tu-
mor type in esophageal cancer to be an independent
prognostic parameter for survival.
1
One of the main fac-
tors determining the prognosis of patients with tumors of
the esophagus is the involvement by tumor of the regional
lymph nodes.
2
But there is a remarkable difference be-
tween the two tumor types that can be involved. Of par-
ticular interest is, that, in contrast to squamous cell
carcinoma, lymphatic spread appears to start later in pa-
tients with esophageal adenocarcinoma.
3
It is also known,
that there is a low rate of lymph node microinvolvement in
patients with adenocarcinoma of the distal esophagus as
compared to patients with squamous cell carcinoma.
4
The molecular mechanism of the proliferation of
lymphatic vessels and the tumor microinvolvement in
lymph node metastasis in esophageal cancer is still not
understood.
Vascular endothelial growth factor (VEGF), now
termed VEGF-A, belongs to the platelet-derived growth
factor family and is the most potent inducer of angio-
genesis and vessel permeability.
5
In total, five forms,
VEGF A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, plus the
placental growth factor (PIGF), have been characterized,
each with its own gene loci.
8
VEGF-C is a ligand at the
VEGF receptor VEGF R3. This is a tyrosine kinase
receptor that is expressed predominantly in the endo-
thelium of lymphatic vessels. It has been shown in
experimental studies that VEGF-C is a lymphoangiogenic
factor that can selectively induce hyperplasia of the lym-
phatic vasculature.
6
In recent investigations, VEGF-C has
been detected in several different cancers, and its levels
in some studies seem to correlate with nodal metastasis
and patient survival.
7,8
In this study, we examined VEGF-C expression in
squamous cell carcinoma and adenocarcinoma of
esophageal cancer to determine whether it is associated
with lymph node metastases or survival in these two dif-
ferent types of esophageal cancer.
MATERIALS AND METHODS
Patients and Tumor Samples
Fifty-nine paraffin-embedded archival specimens from
patients with squamous cell carcinoma and 54 paraffin-
embedded archival specimens of patients with adeno-
carcinoma of the esophagus were studied by immuno-
histochemistry (IHC). All patients had been operated on at
the same institution between 1990 and January of 2001.
None of the patients had neoadjuvant or adjuvant treat-
ment, and all tumors were completely resected. Because
of the selection criteria, only a sample of patients from our
institution was included in the present study. Based on
initial review of the hematoxylin-eosin stained slides of all
surgical specimen sections, two representative paraffin
blocks from each case were selected. In all blocks se-
lected, invasive edge and viable tumor were present.
Histological classification was done according to the
criteria of the TNM and American Joint Committee on
Cancer (AJCC)
9
and were comparable in distribution for
the different stages. Follow-up was complete on all pa-
tients.
Immunohistochemistry
Antibodies against VEGF-C were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). The anti-
body is an affinity-purified goat polyclonal antibody raised
against a peptide corresponding to amino acids 136–155
mapping at the COOH terminus of the VEGF-C of human
origin.
For the immunohistochemical study, paraffin sections
2 lm thick were deparaffinnized and heat treated with
citrate buffer pH 6.0 for 7 min as an epitope retrieval
protocol. Endogenous peroxidase was blocked with
3% hydrogen peroxide for 7 min at room temperature, and
tissue nonspecific binding sites were blocked with skim-
med milk powder at 4% applied for 30 min. Sections were
then incubated with the antibody for 1 h (dilution 1:50 for
VEGF-C) and mixed with skimmed milk powder at
2% again to reduce unspecific staining. Then they were
reacted with biotinylated secondary antibody for 30 min,
avidin-biotin-peroxidase complex (Dako LSAB2 system,
DAKO Co, Carpinteria, CA, USA) was added, and color
was developed using 3-3¢-diaminobenzidine. Counter-
staining was done with hematoxylin. All steps were per-
formed at room temperature. Fetal lung tissue was used as
positive control, and omitting the primary antibody from the
procedure on the protocol was used as negative control.
Data Presentation and Statistical Analysis
Statistical calculations were performed using SAS
statistical software. The tumor stage was divided in two
categories, early cancer (pT1–2) and advanced cancer
(pT3–4) and nodal stage was classified as N0 (lymph
Mo
¨
bius et al.: VEGF-C in Esophageal Squamous Cell Carcinoma and Adenocarcinoma

node negative) and N1 (lymph node positive). VEGF-C
expression was analyzed by scoring the extent and
intensity of staining. The slides were evaluated under a
transmission light microscope by two separate investi-
gators (J.F. and I.B.) in a blind manner in terms of the
patient’s background. For VEGF-C assessment, staining
intensity was scored as 0 (negative), 1 (weak), 2 (med-
ium), or 3 (strong). Extent of staining was scored as
0(0%), 1 (1% –25%), 2 (26%–50%), 3 (51%–75%), or
4 (76%–100%), according to the percentages of the po-
sitive staining areas in relation to the whole carcinoma
area. The product of the intensity and extent score was
used as the final staining score (0–12) for VEGF-C. We
used contingency tables and the Fisher exact test to as-
sess the statistical strength of independent association
between selected covariates (T, N, stage, etc.) and pa-
tient survival. For survival analysis, Kaplan-Meier survival
curves were plotted; differences were tested by log rank
test. All statistical evaluations were done at 95% confi-
dence intervals.
RESULTS
Cohort Results
Overall, 59 patients with squamous cell carcinomas
and 54 patients with adenocarcinoma of the distal
esophagus (Barrett’s carcinomas) were evaluated for
VFGF-C expression. The mean age for the patients with
squamous cell carcinomas was 56.5 years (range: 34–79
years) and the male/female sex ratio was 49:10). The
mean age for the adenocarcinomas patients was 63.7
years (range: 38–82 years), and the male/female sex
ratio was 49:5). The median follow-up of patients with
squamous cell carcinomas was 12.8 months (range: 1.1–
48.9 months), and the median follow-up of patients with
adenocarcinoma was 47.5 months (range: 0–111
months). The clinicopathological data are presented in
Table 1.
Correlation between Clinicopathological Factors
and VEGF-C Expression in Squamous Cell
Carcinoma
Immunohistochemical analyses of esophageal cancer
were conducted using an anti-VEGF-C andibody (Fig. 1).
There was minimal staining in the normal stroma adjacent
to the tumor of VEGF-C. High expression of VEGF-C was
localized in the cytoplasm of cancer cells and predomi-
nately at the invasive edge of the tumor. The expression
of VEGF-C in patient with no positive lymph nodes
(n = 29) was significantly lower than in patients with po-
sitive nodes (n = 30) (P < 0.01) (Fig. 2a). The patients
with higher VEGF-C expression tend to have a lower
survival rate than patients with low VEGF-C expression,
but this difference was not significant (Fig. 2b). There
Table 1.
Clinicopathological categories of the 59 squamous cell
carcinoma of the esophagus and the 54 adenocarcinoma of the
distal esophagus
Squamous
cell carcinoma
Adenocarcinoma
Histological grade
G1 and 2 19 29
G3 and 4 40 25
Depth of invasion
T1 and 2 19 19
T3 and 4 40 35
Lymph node status
N0 29 30
N+ 30 24
Stage
I1217
II–IV 47 37
Figure 1. IHC for VEGF-C on tumor cells (a) in squamous cell
carcinoma of the esophagus and IHC for VEGF-C on tumour
cells (b) in adenocarcinomas of the distal esophagus are shown.
Mo
¨
bius et al.: VEGF-C in Esophageal Squamous Cell Carcinoma and Adenocarcinoma

was no further correlation between clinicopathological
factors and VEGF-C expression in squamous cell carci-
noma.
Correlation between Clinicopathological Factors
and VEGF-C Expression in Adenocarcinoma of
the Distal Esophagus
In patient with adenocarcinoma of the esophagus, the
VEGF-C expression did not have any correlation with
clinicopathological data (Figs. 3a and 3b).
DISCUSSION
VEGF-C is the only known factor that causes lym-
phangiogenesis. It stimulates lymphatic endothelial cells
via the VEGF-R3.
10
The stimulation of lymphatic vessels
has been investigated by experiments using the avian
chorioallantoic membrane assay and in VEGF-C trans-
genic mice. It has been shown that the hyperplasia of
subcutaneous lymphatic channels was induced in
transgenic mice where a vector of VEGF-C was intro-
duced.
11
3029N =
N+N0
12
10
8
6
4
2
0
months
60483624120
cum. survival
1,0
,9
,8
,7
,6
,5
VEGF C < 4
VEGF C > 4
log rank
p= 0,058
A
B
Figure 2. a. The lymph node negative sqamous cell carcinoma tumors showed a significant higher VEGF C expression than the
lymph node negative tumors (P < 0.01). b. The survival in patients with low VEGF C expression (VEGF C expression < 4) and high
VEGF C expression (VEGF C expression > 4).
2430N =
N+N0
VEGF C expression in adenocarcinoma
14
12
10
8
6
4
2
0
months
60483624120
cum. survival
1,0
,8
,6
,4
,2
0,0
VEGF C < 3
VEGF C > 3
log rank p
= 0,83
A
B
Figure 3. a. The VEGF C expression in lymph node negative and lymph node positive adenocarcinoma of the esophagus. In
patients with adenocarcinoma of the esophagus there was no correlation between VEGF C expression and lymph node status. b. The
survival in patients with low VEGF C expression (VEGF C expression < 3) and high VEGF C expression (VEGF C expression > 3.
Mo
¨
bius et al.: VEGF-C in Esophageal Squamous Cell Carcinoma and Adenocarcinoma

Recent reports of clinical investigations suggest that
VEGF-C plays a role in the lymphatic spread of several
cancers. A close correlation between lymph node status
and VEGF-C expression could be shown for lung cancer
and gastric cancer.
7,8
For esophageal cancer, there have been two studies
that investigate the VEGF-C expression only in squa-
mous cell carcinoma: Kitadai et al. found a close corre-
lation between VEGF-C expression in squamous cell
carcinoma and depth of tumor invasion, tumor stage,
venous invasion, lymphatic invasion, and lymph node
metastasis.
12
In contrast, Noguchi et al. found no corre-
lation between VEGF-C expression in squamous cell
carcinoma and clinicopathological features except the
grade of differentiation, which was closely related to
lymph node metastasis.
13
To better understand lymphatic spread in esophageal
cancer, it is important to compare the two major histologic
types of esophageal cancer. The present study, in which
we investigated the VEGF-C expression in squamous cell
carcinoma and adenocarcinoma of the esophagus,
showed a strong correlation between VEGF-C expression
and lymph node metastasis in squamous cell carcinoma,
but not in adenocarcinoma. These findings might help to
demonstrate that squamous cell carcinoma and adeno-
carcinoma of the esophagus are two different tumor
entities, referring to the onset of lymph node metastases
and therefore of therapeutic strategies in terms of the
extent of surgery.
2
Our results indicate the possibility that
VEGF-C produced in squamous cell carcinoma cells
could modulate lymphatic metastasis. This modulation
might be through its receptor, VEGF-R3, but this needs
further investigation. To our knowledge, this is the first
report that compares a possible way for lymphangio-
genesis to occur in squamous cell carcinoma of esoph-
ageal cancer and adenocarcinoma of the esophagus. The
results may help to understand why limited surgery in
early stages of adenocarcinoma of the esophagus is
possible and why radical lymphadenectomy in squamous
cell carcinomas is necessary, even in its early stages.
In conclusion, the present study gives evidence that
VEGF-C might play a role in lymph node metastasis in
squamous cell carcinoma but not in adenocarcinoma of
the esophagus. Recognizing that VEGF-C is the most
important mediator of lymphatic spread in cancer, it is
possible that the different expression in squamous cell
carcinoma and adenocarcinoma of the esophagus might
indicate different ways of metastasizing, with possible
consequences for the clinical treatment.
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