ORIGINAL ARTICLE
Cyclooxygenase-2 expression and clinical parameters in laryngeal squamous cell
carcinoma, vocal fold nodule, and laryngeal atypical hyperplasia
Cağdas
Sayar, MD,1 Hamide Sayar, MD,2 Süleyman Özdemir, MD,3* Tahsin Selçuk, MD,4 Orhan G€orgülü, MD,5 Yücel Akbas
, MD,6
5
Mustafa Kemal Olgun, MD
1
Kahramanmaras State Hospital, Department of Otolaryngology – Head and Neck Surgery, Kahramanmaras, Turkey, 2Sutcu Imam University School of Medicine,
Department of Pathology, Kahramanmaras, Turkey, 3Cukurova University School of Medicine, Department of Otolaryngology – Head and Neck Surgery, Adana, Turkey,
4
Karaman State Hospital, Department of Otolaryngology – Head and Neck Surgery, Karaman, Turkey, 5Adana Numune Education and Research Hospital, Department of
Otolaryngology – Head and Neck Surgery, Adana, Turkey, 6Adana Galeria Ear, Nose, and Throat Hospital, Department of Otolaryngology – Head and Neck Surgery, Adana,
Turkey.
Accepted 2 November 2011
Published online 13 January 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.22911
ABSTRACT: Background. The diagnostic role of cyclooxygenase-2
(COX-2) expression in laryngeal atypical hyperplasia, vocal fold nodule,
and laryngeal squamous cell carcinoma was examined.
Methods. Specimens obtained from patients diagnosed with vocal fold
nodule (n ¼ 35), atypical hyperplasia (n ¼ 35), laryngeal squamous cell
carcinoma (n ¼ 35), and clinical parameters were evaluated retrospectively.
Results. Although no staining was observed in patients with vocal fold
nodules, staining was noted in laryngeal atypical hyperplasia and
squamous cell carcinoma. The percentage of COX-2 staining was the
highest in the carcinoma group.
Laryngeal cancers constitute approximately 1.2% of all
cancers and 25% of head and neck cancers, most of
which are squamous cell carcinomas. Every year in the
world, more than 151,000 people will be diagnosed with
laryngeal cancers, and more than 82,000 people will die
from the disease.1
Vocal fold nodules, benign small lesions of the larynx,
is a commonly used term for a variety of vocal fold
lesions that is bilateral epithelial swellings of the anterior
third of the true vocal fold. It is assumed that voice overuse, misuse, or abuse increases this mechanical stress
leading to localized mechanical trauma.2,3
Epithelial hyperplastic laryngeal lesions caused by pathogenic factors injuring the mucosa may contain reactive
and/or preneoplastic histologic changes. The grading system devised and tested in Ljubljana comprises simple
hyperplasia (benign spinous layer augmentation), abnormal
hyperplasia (benign basal and parabasal layer augmentation), atypical hyperplasia (risky for malignancy), and carcinoma in situ (actually malignant, without invasion).
Cyclooxygenase (COX) enzyme catalyzes prostaglandin
synthesis from arachidonic acid. COX, discovered
Conclusion. It was determined that COX-2 staining was significantly
associated with laryngeal squamous cell carcinoma. It should be noted
that overexpression of COX-2, a potentially important factor in the
evolution of carcinogenesis in precancerous lesions, might be an
C 2012 Wiley Periodicals, Inc.
indicator of the development of carcinoma. V
Head Neck 35: 52–56, 2013
KEY WORDS: larynx, squamous cell carcinoma, vocal fold nodule,
laryngeal atypical hyperplasia, cyclooxygenase-2
approximately 20 years ago, has 2 isoforms. These isoforms, known as COX-1 and COX-2, have diverse properties. COX-1 is mainly present in normal tissues and
mediates the production of prostaglandins, which are important for the control of normal physiologic functions.4
COX-2 is induced by growth factors, tumor promoters,
oncogenes, and carcinogens and synthesized as an early
response to these inducers. COX-2 induction and its association with laryngeal squamous cell carcinoma occur via
genes, growth factors, inflammation, and angiogenesis.
COX-2 has been suggested as a target for the inhibition of
the growth of head and neck squamous cell carcinomas.5,6
Increased COX-2 expression has been described in a
wide variety of human premalignant and malignant
conditions.
To our knowledge, up to now, no investigation was
done about the COX-2 staining in benign lesions
(nodules). For this reason, in this study, we aimed to
evaluate the immunohistochemical expression of COX-2
in vocal fold nodules, atypical hyperplasia, and laryngeal
cancers and compared them with each other.
MATERIALS AND METHODS
Patient selection and study design
*Corresponding author: S. Özdemir, Cukurova University School of Medicine,
Department of Otolaryngology – Head and Neck Surgery, Adana, Turkey. E-mail:
[email protected]
52
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JANUARY 2013
Our study included 105 patients who did not receive
previous radiotherapy or chemotherapy between 2005 and
CYCLOOXYGENASE-2 EXPRESSION
FIGURE 1. Age groups. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
2008 in the Department of Otolaryngology at Adana
Numune Training and Research Hospital. Patients’ medical records were evaluated retrospectively. The study protocol was approved by the ethics committees of all the
institutions involved.
All the patients in our study had been treated according
to a standard procedure. Specimens were obtained by
excision of laryngeal nodule, cordectomy, stripping, and
laryngectomy.
Diagnostic confirmation and histologic assessment
The specimens were histologically confirmed as vocal
fold nodules (n ¼ 35; 17 men; 18 women), atypical
hyperplasia (n ¼ 35; 29 men; 6 women), and laryngeal
squamous cell carcinoma (n ¼ 35; 35 men).
Atypical hyperplasia was classified according to the
Ljubljana classification. Classification of the tumors was
performed according to clinical, radiological, and histopathological examinations. The modified classification
system based on the general principles of the American
Joint Committee on Cancer Staging and End Results
Reporting was used for classification in the laryngeal
squamous cell carcinoma group.
Immunohistochemical staining
Tissue specimens were fixed in 10% buffered formaldehyde and embedded in paraffin according to routine methods. Slides from all tumor, atypical hyperplasia, and vocal
fold nodule blocks were reviewed by a pathologist (H. S.)
to select representative areas of the lesion for further
processing and immunohistochemistry. The criteria for tumor block selection were viable tumor tissue without necrosis and the presence of an invasive front. Only blocks
estimated to be composed of at least 60% tumor cells
were included. The specimens were sectioned (4 lm) and
mounted onto polylysine-coated glass slides. Sections
were dewaxed in xylene, deparaffinized, and rehydrated
using graded ethanols. After microwave pretreatment in
citrate buffer (pH 6.0) for antigen retrieval, slides were
immersed in 0.3% hydrogen peroxide for 20 minutes to
block endogenous peroxidase activity. Then the sections
were washed with phosphate-buffered saline, followed by
blocking avidin and biotin using the avidin-biotin blocking kit (Lab Vision).
Slides were incubated for 1 hour with a monoclonal
antibody to human COX-2, (Dako Cytomation EnVision
Clone CX-294, Code M3617, 1:100 dilution).Then the
sections were treated with biotinylated goat antipolyvalent
(Lab Vision). Streptavidin-peroxidase conjugate was
applied at room temperature. After subsequent staining
with chromogen AEC (DakoCytomation), counterstained
with Mayer’s hematoxylin, and dehydrated and mounted,
a pathologist (H. S.), blinded to the clinical outcomes,
examined each sample core to score the tissue sections
for tumor staining intensity.
To determine percentage labeling indices, for each section, at least 500 and usually more than 1000 carcinoma
cells were analyzed in randomly selected fields of tumor
tissue using high-power (40 objective and 10 ocular)
magnification. A mean percentage of cytoplasmic-positive
tumor cells was determined and a level of immunoreaction was graded into 3 categories as follows: 0% to 10%
of cells stained scored as 0; 11% to 30% as 1þ; 31% to
60% as 2þ; and 61% to 100% as 3þ positive cells.
Statistical analysis
SPSS 12.0 for Windows (SPSS, Chicago, IL) was used
for statistical analysis of the data. Descriptive statistics
results were expressed as the mean, median, SD, minimum,
and maximum for numeric variables, and as frequency
tables for categorical variables. Between-group comparisons were performed using the Kruskall–Wallis test for
numeric variables and using the chi-square test for categorical variables. The alpha significance level was set at
p < .05. Subgroup analysis of numeric variables was performed by the Mann–Whitney U test. The alpha significance level was evaluated after Bonferroni correction. The
relationship between ordinal and numeric variables was
assessed by Spearman correlation analysis.
RESULTS
Squamous cell carcinoma, atypical hyperplasia, and
vocal fold nodules were grouped by age as follows: <40,
41 to 60, and >61 years (Figure 1).
The percentage of COX-2 staining epithelial/tumor cells
and several clinical parameters (age, sex, T classification,
smoking status [packs/year], tumor localization) were
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SAYAR ET AL.
FIGURE 2. Cyclooxygenase-2 (COX-2) staining degrees of some cases. (A) A case with no COX-2 staining (AEC Chromogen 200). (B) A case with
1þ COX-2 staining (AEC Chromogen 200). (C) A case with 2þ COX-2 staining (AEC Chromogen 200). (D) A case with 3þ COX-2 staining (AEC
Chromogen 200). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
analyzed. Scoring was performed based on the extent of
COX-2 staining (Figure 2).
In vocal fold nodules, there was no COX-2 staining.
That means vocal fold nodules were grade 0.
A statistically significant difference was found for the
percentage of COX-2 staining in the study groups (p <
.001). Although no staining was noted in vocal fold nodules, staining was noted in 45.7% (20.0% 1þ, 20.0% 2þ,
and 5.7% 3þ) of atypical hyperplasia and 91.4% (5.7%
1þ, 31.4% 2þ, and 54.3% 3þ) of squamous cell carcinoma
(Figure 2).
There was a statistically significant difference among
the groups in terms of smoking (p < .001) as follows:
40.0% of patients with vocal fold nodules, 42.9% of
patients with atypical hyperplasia, and 94.3% of patients
with squamous cell carcinoma were smokers (Table 1).
TABLE 2. The relationship between smoking and COX-2 staining in the
groups.
TABLE 1. Smoking status of the groups.
Smoking
No
Yes
Total
No. of patients
%
No. of patients
%
No. of patients
Smoking
Vocal
fold
nodule
Atypical
hyperplasia
Squamous
cell
carcinoma
Total
21
60.0
14
40.0
35
20
57.1
15
42.9
35
2
5.7
33
94.3
35
43
41.0
62
59.0
105
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Staining
No staining
Staining
Total
No. of patients
%
No. of patients
%
No. of patients
Abbreviation: COX-2, cyclooxygenase-2.
Chi-square test, P value <.001.
Chi-square test, P value <.001.
54
COX-2 staining was noted in 54.8% of the smokers.
There was a statistically significant difference between
the percentage of COX-2 staining of smokers and nonsmokers (p < .001; Table 2). The percentage of COX-2
staining was increased as smoking rates were increased.
There was a statistically significant difference between
the groups with respect to the duration of smoking (p ¼
.001). The duration of smoking in patients with vocal
fold nodules (18.4 6 16.4 packs/year) was significantly
lower than in the patients with atypical hyperplasia (40.3
6 22.2 packs/year; p ¼ .001) and squamous cell carcinoma (34.8 6 13.9 packs/year; p ¼ .001). There was no
statistically significant difference between the duration of
smoking in patients with atypical hyperplasia and those
with squamous cell carcinoma (p ¼ .589).
JANUARY 2013
No
Yes
Total
38
88.4
5
11.6
43
28
45.2
34
54.8
62
66
62.9
39
37.1
105
CYCLOOXYGENASE-2 EXPRESSION
TABLE 3. Clinical characteristics of carcinoma patients.
Clinical characteristic
Location
Glottic laryngeal cancer
Supraglottic laryngeal cancer
Transglottic laryngeal cancer
T classification
T1
T2
T3
T4
No. of patients
%
8
22
5
22.9
62.9
14.3
7
8
13
7
20.0
22.9
37.1
20.0
The T classification and tumor localization of the carcinoma group are presented in Table 3. The intensity of
COX-2 staining increased from the nodule group toward
atypical hyperplasia and carcinoma groups. The increase
was the highest in the carcinoma group (Table 4).
There was no statistically significant relationship
between the percentage of COX-2 staining and age (p ¼
.679) and duration of smoking (p ¼ .085) T classification
(p ¼ .898) in the carcinoma group.
DISCUSSION
COX-2 is induced by smoking, radiotherapy, ras and
src oncogenes, hypoxine, interleukin-1, benzopyrene,
ultraviolet light, epidermal growth factor-b, and tumor necrosis factor-a. COX-2 is also synthesized after stimulation of serum and tumor inducers, mitogens, and endotoxins.4,7,8 COX-2 seems to act in tumorigenesis by
synthesizing prostaglandins, especially PGE-2, which
facilitates tumor progression by a number of mechanisms
including affecting cell proliferation, reducing apoptosis,
suppressing the immune response, and favoring invasion
by their action on matrix metalloproteinases.9 COX-2
contributes to the development and progression of head
and neck squamous cell carcinomas in several other ways
such as interactions with the genes, growth factors,
inflammation, and angiogenesis.7,10 Increased levels of
COX-2 have been demonstrated at the mRNA and protein
levels in head and neck squamous cell carcinomas.11
COX-2 is predominantly released in the gastrointestinal
system, genitourinary system, lung, skin, and head and
neck tumors.4,7 Mestre et al11 found that COX-2 protein
was noted in all 6 cases of head and neck squamous cell
carcinomas. Chan et al7 reported that COX-2 is expressed
in 100% of tumors from a mixed group of 10 patients
with head and neck tumors. Ranelletti et al12 reported
COX-2 overexpression in 31% of patients (19 of 61). In
yet another study, COX-2 was positive in 88% of head
and neck squamous cell carcinoma samples.9 In the present study, COX-2 was positive in 91.4% of the samples
in the carcinoma group. Differences in results between
the studies may partly be explained by different kinds of
patient materials and the lack of a uniform measurement
method for COX-2 expression.
Smoking is a well-known risk factor for the development of laryngeal cancers. Procarcinogens in tobacco
stimulate COX-2 transcription.13 Stimulation of COX-2
leads to chronic inflammation, which is a risk factor for
squamous cell carcinogenesis.14 In our study, the percentage of COX-2 staining was a statistically significant difference between smokers and nonsmokers. The percentage of COX-2 staining was increased as smoking rates
were increased.
It was reported that COX-2 overexpression is associated
with poor survival.12,15 However, Beyazit et al16 did not
find a significant relationship between COX-2 release and
survival. Ranelletti et al12 compared survival analysis of
patients who were COX-2–negative and –positive and
found a poor clinical prognosis in patients who were
COX-2-negative. These findings show that COX-2 negativity is associated with a more aggressive behavior in laryngeal squamous cell carcinomas. But, we did not investigate the relationship between COX-2 release and
survival in the carcinoma group.
Because COX-2 is an inducible molecule and may not
be detected in normal mucosa in the absence of any
inducing factor, in normal tissue, the presence of p53
may inhibit COX-2 release. Plescia et al15 found that
COX-2 was positive in all tumors and negative in normal
tissue surrounding the tumor. We observed that normal
squamous epithelium adjacent to a tumor stained weakly
for COX-2 protein. COX-2 was expressed in the atypical
hyperplasia areas, whereas it was apparently absent in the
vocal fold nodule.
COX-2 was previously investigated as a potential target
for the inhibition of growth of head and neck squamous
cell carcinomas.5
It has been reported that the COX-2 overexpression in
patients with glottic cancers is associated with increased
risk of mortality.17 Atula et al9 did not find a relationship
between the intensity of COX-2 staining and sex, age, tumor grade, size (T classification), and metastases (N classification). Similarly, no statistically significant relationship was found among the percentage of staining and age,
duration of smoking, and T classification in our study.
COX-2 is upregulated through enzymatic reactions after
radiotherapy.8 None of the patients received previous
radiotherapy in the current study.
It has been reported that the combined use of retinoids
with COX-2 selective inhibitors may be more effective in
the prevention of upper respiratory and gastrointestinal
system cancers compared to the use of single agents
alone.11
TABLE 4. Intensity of COX-2 staining in the groups.
No. of patients (%) by group
Staining
intensity
Vocal fold
nodule
Atypical
hyperplasia
Squamous cell
carcinoma
0
35 (100.0)
19 (54.3)
3 (8.6)
57 (54.3)
1þ
0 (0.0)
7 (20.0)
2 (5.7)
9 (8.6)
2þ
0 (0.0)
7 (20.0)
11 (31.4)
18 (17.1)
3þ
0 (0.0)
2 (5.7)
19 (54.3)
21 (20.0)
Total
Abbreviation: COX-2, cyclooxygenase-2.
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SAYAR ET AL.
COX-2 inhibitors reduce immunosuppression mediating
tumor formation.15 Inhibition of COX-2 activity suppresses
the growth of squamous cells releasing high amounts of
COX-2.18 Therefore, COX-2 inhibitors have a potential
role in the prevention and treatment of head and neck squamous cell carcinomas.19
In conclusion, this study demonstrates that COX-2 is
markedly upregulated in laryngeal squamous cell carcinoma. We noted a significant difference between the
study groups regarding COX-2 staining positivity, indicating a potential role for cancer development. Also, we
found that there was a statistically significant difference
in the percentage of COX-2 staining between smokers
and nonsmokers. In addition to tobacco cessation, COX-2
inhibitors may have a potential role in the prevention and
treatment of the laryngeal carcinoma. This marker should
be included in a prospective clinicopathologic study with
long-term patient follow-up to confirm their significance
as indicators for an increased risk for the development of
laryngeal carcinoma from atypical hyperplasia.
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