Histologic correlation of VEGF and COX-2

expression with tumor size in squamous cell

carcinoma of the larynx and hypopharynx
April 30, 2017
by Rodrigo Gonzalez Bonhin, MD; Guilherme Machado de Carvalho, MD; Alexandre Caixeta Guimaraes, MD;
Vanessa Brito Campoy Rocha, MD; Carlos Takahiro Chone, MD, PhD; Agrcio Nubiato Crespo, MD, PhD;
Eliane Maria Ingrid Amstalden, MD, PhD

Abstract
The prognosis and survival of patients with laryngeal and hypopharyngeal squamous cell carcinoma
(SCC) are related to the clinical staging. Tumor growth and metastasis are closely related to
angiogenesis. Vascular endothelial growth factor (VEGF) and cyclooxygenase 2 (COX-2) are
intimately related to the angiogenic process in which the blood supply required for neoplastic
evolution is ensured. Considering the contributions of VEGF and COX-2 to the formation, growth,
and progression of primary tumors, as well as their metastasis, it has been thought that the
expression of these two factors might be related to the size and/or aggressiveness of laryngeal and
hypopharyngeal cancer. To test that theory, we conducted a retrospective study to evaluate the
expression of VEGF and COX-2 markers in archived specimens of SCC of the larynx and
hypopharynx to correlate their expression with tumor size. These specimens had been obtained from
35 patients-31 men and 4 women, aged 37 to 75 years (mean: 57)-who had been treated for
laryngeal or hypopharyngeal SCC at our tertiary care university hospital over a period of 15 years.
Immunohistochemical analyses were performed with avidin-biotin-peroxidase staining for VEGF and
COX-2 antibodies. The degree of VEGF and COX-2 expression was based on the German scale of
immunoreactivity. Specimens were separated into groups based on the intensity of expression
(none/low and moderate/strong) and tumor size (TNM categories T1/T2 and T3/T4). A total of 7
patients exhibited moderate or strong VEGF expression (3 from the T1/T2 group and 4 from the
T3/T4 group), and 17 patients demonstrated moderate or strong COX-2 expression (8 from the
T1/T2 group and 9 from the T3/T4 group). We found no statistically significant relationship between
tumor size and either VEGF or COX-2 expression. Our study demonstrated that despite the intrinsic
connection between and VEGF and COX-2, neither appears to be related to the size of the SCCs of
the larynx or hypopharynx. Other factors must be involved in the pathogenesis and progression of
this disease, and further studies are needed to identify them.

Introduction
Squamous cell carcinoma (SCC) is the most common malignant tumor of the larynx and
hypopharynx, accounting for about 95% of all cases.1-4 It usually affects men aged 50 to 80
years.5 Among the many risk factors associated with SCC of the larynx or hypopharynx, cigarette
smoking is the most common.5
The prognosis and survival of patients with laryngeal or hypopharyngeal carcinoma depend on the
size of the lesion, the level of invasion and tumor extension to surrounding structures, the number of
metastatic regional lymph nodes, and the development of distant metastasis, since death is often
attributed to uncontrolled regional disease associated with local recurrence of the primary tumor. 1-
4
Clinical staging by TNM category is fundamental in guiding treatment.5

Tumor growth and metastasis are closely related to angiogenesis-that is, the formation of new
capillary blood vessels from the preexisting vasculature. This process is essential to providing
nutrients, oxygen, and growth factors that support cell function and survival. 1,2 A complex interaction
among endothelial cells, extracellular protein matrices, and soluble plasma factors occurs in
angiogenesis.

Endothelial cells leave their quiescent state when stimulated by VEGF and initiate the following
steps: basement membrane dissolution, endothelial cell migration and proliferation, capillary tube
formation, and the maturation and survival of newly formed vessels. As a result, the blood supply
required for neoplastic evolution is ensured.6-8

COX-2 promotes cell migration and dissemination by angiogenesis. In addition, it increases the
synthesis of VEGF.8,9 Elevated levels of COX-2 are related to the activation of oncogenes and
increased activity of growth factors and cytokines. Oncogenes such as Ras are connected to the
augmentation of COX-2 in breast cancer cells. The elevation of COX-2 levels has been analyzed in
several types of malignant tumors, including pancreatic, lung, breast, gastric, colonic, prostatic, and
head and neck.6

COX-2 synthesizes prostaglandin E2 (PGE2), which operates in PGE2 and PGE4 endothelial cell
receptors. In a cAMP-dependent pathway, COX-2 promotes migration and spreading of these cells
through angiogenesis.

Considering the important roles that VEGF and COX-2 play in angiogenesis and their contribution to
the formation, growth, progression, and metastasis of tumors, it has been theorized that the
expression of these two factors might be related to greater size and/or aggressiveness of laryngeal
and hypopharyngeal cancer tumors.2

In this article, we describe our study to evaluate expression of VEGF and COX-2 markers in
laryngeal and hypopharyngeal SCC and to correlate these data with tumor size.

Patients and methods

We conducted a retrospective study of a cross-sectional historical cohort to evaluate the expression
of VEGF and COX-2 markers in SCC of the larynx or hypopharynx and to correlate their expression
with tumor size. We used resected laryngeal and hypopharyngeal SCC specimens obtained from 35
patients-31 men and 4 women, aged 37 to 75 years (mean: 57)-who had been treated for laryngeal
or hypopharyngeal SCC at our tertiary care university hospital over a period of 15 years. The
excisional biopsies and surgical specimens were obtained from the archived collection of the
Department of Pathology at the University of Campinas Teaching Hospital.

There were four types of cancer among the 35 specimens: 6 cases of well-differentiated glottic SCC,
10 cases of moderately or poorly differentiated glottic SCC, 9 cases of well-differentiated nonglottic
SCC of the larynx or hypopharynx, and 10 cases of moderately or poorly differentiated nonglottic
SCC of the larynx or hypopharynx. For all specimens, the paraffin blocks contained enough material
for new cuts. Clinical data and tumor staging were obtained from medical records.

Immunohistochemical tests were performed with avidin-biotin-peroxidase staining for VEGF (IgG1,
1:100) and COX-2 (IgG1, 1:300); in both cases, pretreatment with a citrate buffer was performed.

Pathology slides were analyzed randomly by an investigator who had no knowledge of the previous
diagnosis or clinical status of the corresponding patient; specimens were compared with positive and
negative controls. Photographs (Coolpix 995 camera; Nikon; Tokyo) of highly reactive areas (hot
spots) were scanned at high magnification (x400). Thereafter, images were sent for histologic
analysis on Image Lab software (Bio-Rad; Hercules, Calif.).

The intensity of expression of VEGF and COX-2 was based on the German scale of
immunoreactivity (GSI), which is determined by multiplying the coefficient of the immunomarker
intensity of expression and the coefficient of the percentage of positive cells, considering a sample
space of at least 500 cells (figure). The possible number of GSI values ranges from 0 to 12.

Figure. Elements of the GSI calculation.

Specimens were classified according groups based on the intensity of expression (none/low and
moderate/strong) and tumor size (TNM categories T1/T2 and T3/T4). Tumors in the T1/T2 group (n =
20) were small tumors restricted to their site of origin, exhibiting no invasion of extralaryngeal sites.
The T3/T4 tumors (n = 15) were larger, invasive tumors that did invade extralaryngeal structures.

Statistical analysis. Statistical analysis was performed with the Statistical Package for the Social
Sciences software. Comparisons were made with the chi-square test and the Fisher exact test.

Ethical considerations. The study protocol was approved by our Institutional Ethics Committee.

Results
A total of 7 patients exhibited moderate or strong VEGF expression (3 from the T1/T2 group and 4
from the T3/T4 group), and 17 patients demonstrated moderate or strong COX-2 expression (8 from
the T1/T2 group and 9 from the T3/T4 group). There was no statistically significant correlation
between tumor size and VEGF expression (p = 0.430) or COX-2 expression (p = 0.315) (table).

Table. VEGF and COX-2 expression by T category

 T1/T2 T3/T4 Total

VEGF

None/low 17 11 28

Moderate/strong 3 4 7

Total 20 15 35

COX-2

None/low 12 6 18

Moderate/strong 8 9 17

Total 20 15 35

Discussion
VEGF. In our study, moderate or strong expression of VEGF in epithelial cells of SCC was observed
in only 7 of the 35 specimens (20%). Previous studies of tumors of the head and neck-which
included not only SCC of the larynx, but those of the oral cavity and pharynx, as well-found an
increase of VEGF, which was associated with angiogenesis, neoplastic progression, and a worse
prognosis.6-10 Most of these analyses took into account only the positivity of the marker, regardless
of its intensity.

In addition, a meta-analysis of Kyzas et al found a trend toward a correlation of VEGF positivity with
higher clinical stage (III/IV) and poor histologic differentiation. 11 They wrote that VEGF correlated with
poor survival in patients with SCC of the head and neck. Likewise, Vlachtsis et al found that VEGF
and another molecular biomarker, epidermal growth factor receptor (EGFR), exhibited prognostic
significance in laryngeal cancer.12 Apart from their role in carcinogenesis, both of these markers
appear to play an important role in tumor relapse.

While these studies found that VEGF positivity was a predictor of a poor prognosis, none of them
found that it was related specifically to tumor size.
In another investigation, Srivastava et al tested the use of VEGF as a biomarker. They measured
serum VEGF-A in patients with head and neck SCC and studied its relation to response to
chemoradiotherapy.13 They found that serum VEGF-A levels were significantly higher in patients
who did not respond to treatment, although there was no significant difference in response between
those with T1/T2 and T3/T4 tumors. This finding is in accord with our results.

COX-2. Moderate or strong expression of COX-2 was seen in 17 of the 35 specimens (48.6%).
Several studies have shown that COX-2 participates in the production of PGE2, which induces the
tumor extension associated with the overexpression of this enzyme. 7,14,15 In addition, a recent study
from China found that COX-2 expression, histologic grade, and recurrence were all independent
prognostic factors for laryngeal SCC; high levels of COX-2 expression indicated poor
survival.16 However, unlike these studies, ours found no significant correlation between the
expression of COX-2 and tumor extension.

New treatments. The relationship among VEGF, COX-2, and SCC of the head and neck is
particularly important in terms of the development of new treatment modalities. Studies that have
documented the expression of COX-2 in head and neck SCC have explored the potential role of
selective COX-2 inhibitors.17

Studies of SCC have also examined the use of monoclonal antibodies as a branch of treatment.
Overexpression of EGFR is a common characteristic of head and neck SCC. 18 Cetuximab, a
chimeric anti-EGFR monoclonal antibody that has been approved for multiple indications, might be
found to have a role in SCC with more ongoing studies.19,20

Another promising treatment is celecoxib. This COX-2 inhibitor was tested by Kim et al in vitro
against hamster head and neck carcinoma cells. 21 Their experiment found that a decrease in the
expression of VEGF and COX-2 as associated with an increase in survival. Reductions in
proliferation and in vitro invasion were seen, as well. Celecoxib retarded early-stage carcinogenesis.
However, despite a decrease in neovascularization, it did not inhibit tumor growth.

In conclusion, our study demonstrated that VEGF and COX-2 do not appear to be implicated in the
extension of SCCs of the larynx or hypopharynx. Other factors should be implied in the pathogenesis
and progression of these tumors, and further studies are needed to identify them.

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From the Department of Otolaryngology-Head and Neck Surgery (Dr. Bonhin, Dr. de Carvalho, Dr.
Guimares, Dr. Rocha, Dr. Chone, and Dr. Crespo) and the Department of Pathology (Dr.
Amstalden), Faculty of Medical Sciences, University of Campinas, Campinas, So Paulo, Brazil.
Corresponding author: Vanessa Brito Campoy Rocha, MD, Department of Otolaryngology-Head and
Neck Surgery, Faculty of Medical Sciences, University of Campinas, Rua Tesslia Vieira de
Carvalho, 126 - CEP: 13083-887, Campinas, So Paulo, Brazil. E-mail: vanessacampoy@gmail.com
Ear Nose Throat J. 2017 April-May;96(4-5):176-182