J Oral Pathol Med

doi: 10.1111/jop.12385 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

wileyonlinelibrary.com/journal/jop

Immunohistochemical expression of cyclooxygenase-2

(COX-2) in oral nevi and melanoma
Juliana de Souza do Nascimento1, Rom
an Carlos2, Wilson Delgado-Aza~
nero3, Adalberto Mosqueda
4 5
Taylor , Oslei Paes de Almeida , M

ario Jos
e Roma~nach , Bruno Augusto Benevenuto de Andrade6
6

1
School of Dentistry, Fluminense Federal University, Nova Friburgo, Rio de Janeiro, Brazil; 2Pathology Section, Centro Cl
ınico de
Cabeza y Cuello/Hospital Herrera Llerandi, Guatemala City, Guatemala; 3Oral Pathology, Universidad Peruana Cayetano Heredia,
Lima, Peru; 4Departamento de Atenci
on a la Salud, Universidad Aut
onoma Metropolitana, Mexico, Mexico; 5Oral Pathology, School of
Dentistry, State University of Campinas, Piracicaba, S~ao Paulo, Brazil; 6Oral Pathology, Department of Oral Diagnosis and Pathology,
School of Dentistry, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

BACKGROUND: Cyclooxygenase-2 (COX-2) catalyses
the conversion of arachidonic acid to prostaglandin, and
its overexpression has been demonstrated in different
malignant tumors, including cutaneous melanoma. How-
ever, no data about the expression of this protein in oral
melanocytic lesions are available to date. The aim of this
study was to evaluate the immunohistochemical expres-
sion of COX-2 in oral nevi and melanomas, comparing
the results with correspondent cutaneous lesions.
METHODS: COX-2 was evaluated by immunohisto-
chemistry in 49 oral melanocytic lesions, including 36
intramucosal nevi and 13 primary oral melanomas, and in
four cutaneous nevi and eight melanomas.
RESULTS: All cases of oral and cutaneous melanomas
were positive for COX-2. On the other hand, all oral and
cutaneous melanocytic nevi were negative.
CONCLUSION: COX-2 is highly positive in oral melano-
mas and negative in oral nevi and might represent a
useful marker to distinguish melanocytic lesions of the
oral cavity.
J Oral Pathol Med (2015)
stimuli, including growth factors, cytokines, bacterial
lipopolysaccharides, hormones, and tumor promoters (6,
7). COX-2 is also induced in processes of cellular growth
and differentiation and is involved in the regulation and
differentiation of human keratinocytes (4). Some studies
have shown the involvement of COX-2 in the pathogenesis
of different human cancers from breast, stomach, lung, and
skin, including squamous cell carcinoma, basal cell
carcinoma, and malignant melanoma (3, 6, 7).
In cutaneous melanoma, studies revealed that overex-
pression of COX-2 is correlated with its development and
progression, being associated with Clark and Breslow
thickness, and consequently with a poorer prognosis (8,
9). Oral melanoma is extremely rare, accounting for less
than 1% of all melanomas, and currently, little is known
about its pathogenesis, including etiological factors and
molecular mechanisms (10). To date, immunohistochemical
findings of COX-2 in oral nevi and melanoma are not
available in the English language literature. The aim of the
present study was to evaluate the immunohistochemical
expression of COX-2 in oral nevi and melanomas, compar-
ing the results with their correspondent cutaneous lesions.

Keywords: cutaneous melanocytic nevi; cutaneous melanoma;

cyclooxygenase-2; intramucosal melanocytic nevi; oral melanoma
Introduction
Cyclooxygenase-2 (COX-2) is an enzyme that catalyzes the
conversion of arachidonic acid to prostaglandin (1–5). Its
expression might be induced in response to numerous
Correspondence: Bruno Augusto Benevenuto de Andrade, DDS, PhD,
Department of Oral Diagnosis and Pathology, School of Dentistry, Federal
University of Rio de Janeiro (UFRJ). Av. Professor Rodolpho Paulo Rocco,
325, 1° andar, Rio de Janeiro 21941-913, Brazil. Tel: +55 21 39382087,
Fax: +55 21 39382087, E-mail: augustodelima33@hotmail.com
Accepted for publication October 19, 2015
Material and methods
Formalin-fixed, paraffin-embedded tissue blocks and clini-
cal information of 49 oral melanocytic lesions (13 primary
oral melanomas and 36 intramucosal nevi) and 12 cutaneous
melanocytic lesions (four superficial spreading melanomas
and eight compound melanocytic nevi) were obtained from
the charts of four oral pathology laboratories from Latin
America (Guatemala, Mexico, Peru and Brazil). All
melanomas were revised using hematoxylin/eosin prepara-
tions to confirm diagnosis. Primary oral melanomas were
histopathologically classified according to Prasad et al. (11),
and 1, 4 and 8 cases corresponded to levels I (in situ), II and
III, respectively (Fig. 1). Cutaneous melanomas were clas-
sified using Breslow thickness (12) and Clark level (13), and
three cases corresponded to Clark level II and Breslow
 COX-2 in oral nevi and melanoma
de Souza do Nascimento et al.
2
A B
Figure 1 Histopathological aspects of oral melanoma: (A) Oral in situ melanoma with neoplastic melanocytes arranged in a pagetoid pattern. (B). Level III
oral melanoma composed mainly by epithelioid and plasmacytoid neoplastic melanocytes (hematoxylin and eosin staining, (A) 9100, (B) 9400).
thickness between 1 and 2 mm, while one case was Clark
level III and Breslow thickness between 2 and 4 mm.
For immunohistochemical staining, 3-lm-thick sections
mounted on silane-coated glass slides were used. Briefly,
the sections were deparaffinized, rehydrated in graded
ethanol solutions and after antigen retrieval with EDTA/
Tris buffer (pH 9.0) in a pressure cooker; endogenous
peroxidase activity was blocked with 20% H2O2 by five
cycles of 5 min each. Overnight incubation with the primary
antibody COX-2 (Clone: CX-294; Dako Corporation,
Carpinteria, CA, USA) diluted in BSA (bovine serum
albumin-1:100) was followed by the secondary antibody
conjugated with polymer dextran marked with peroxidase
(Dako EnVision Labelled Polymer; Dako, Glostrup, Den-
mark). The reaction was developed with Permanent Red
(Permanent Red Substrate System; Dako) and counter-
stained with Carazzi hematoxylin. Sections of breast
carcinoma were included in all reactions as positive control
for COX-2. Negative controls of reactions were performed
by omitting the primary antibody. Only cytoplasmic staining
was considered as positive. Analysis of cyclooxygenase-2
was expressed as the percentage of positive cells (number of
positive tumor cells/total number of tumor cells) for each
case studied, based in a semiquantitative analysis according
to Lee et al. (7). The fraction of positive cells was estimated
using a four-tiered score (<1% = 0, 1–9% = score 1,
10–50% = score 2, >50% = score 3).
Results
Intramucosal nevi patients included 30 women and six men,
aged 16–67 years, located in hard palate (n = 13), buccal
mucosa (n = 11), gingiva (n = 10), and not specified
(n = 2), while the primary oral melanomas corresponded
to eight women and three men, aged 23–86 years, located in
hard palate (n = 6), hard palate and upper gingiva (n = 3),
and upper gingiva (n = 2). Considering the cutaneous
lesions, compound nevi patients included five women and
three men, with age ranged from 16 to 67 years, located in
upper extremity (n = 3), lower extremity (n = 2), head and
neck (n = 2), and trunk (n = 1), while cutaneous melano-
mas corresponded to one woman and three men, aged 31–
75 years, located in upper extremity (n = 2), lower extrem-
ity (n = 1), and head and neck (n = 1).
All oral melanomas either in situ or invasive showed
immunohistochemical positivity for COX-2. Eight cases of
level III, three cases with level II, and one case with level I
J Oral Pathol Med
showed positivity in more than 50% of malignant cells
(score 3), while only one case with level II demonstrated
positivity in 10–50% of neoplastic cells (score 2). All oral
nevi were negative, but keratinocytes of the granular and
spinous layers of the normal oral epithelium were diffusely
positive, serving as control (Fig. 2).
Similarly, COX-2 protein was significantly expressed in
all cutaneous melanomas and negative in all compound
melanocytic nevi. In cutaneous melanomas, COX-2 showed
higher positivity in cases with Clark level III and Breslow
thickness >2 mm, when compared with melanomas Clark
level II and Breslow thickness <2 mm. One case with Clark
level III and Breslow thickness between 2 and 4 mm
showed score 3 (more than 50% positive cells), while three
cases with Clark level II and Breslow thickness between 1
and 2 mm showed a score of 2. Normal epithelium of the
skin was negative for COX-2, except in sebaceous glands,
which strongly expressed this protein (Fig. 3).
Discussion
Cyclooxygenase-2 (COX-2) is an enzyme constitutively
expressed in organs such as the brain, testis, trachea, and
kidney (4, 5), being involved in the production of
prostaglandins in inflammatory and neoplastic processes
(1–7). Its overexpression has been related to advanced
stage and/or prognosis of different diseases (5, 7–9), and
few studies have considered the role of COX-2 in
melanomas and nevi; the present study is the first
immunohistochemical study of COX-2 in oral melanocytic
lesions (1–9, 11–19).
We found strongly immunopositivity for COX-2 in oral
melanomas, and all intramucosal melanocytic nevi were
negative for this protein. Indeed, cutaneous melanomas and
compound melanocytic nevi showed similar results, as also
observed by Denkert et al. (6) and Bianchini et al. (18).
Kuzbicki et al. (8) demonstrated that the immunohisto-
chemical intensity of COX-2 is associated with Clark level
and Breslow thickness, suggesting this protein as a molec-
ular prognostic marker. We also found a single case of
cutaneous melanoma with Clark level III and Breslow
thickness >2 mm exhibiting high COX-2 positivity, but this
finding was different from those found in oral melanomas,
in which both invasive and in situ lesions showed high
positivity for COX-2. Although melanomas from the skin
and oral cavity are consistently positive for COX-2, its high
positivity in in situ oral melanomas suggests that the role of
 COX-2 in oral nevi and melanoma
de Souza do Nascimento et al.

3
A B

C D

Figure 2 Immunohistochemical expression of COX-2 in oral melanocytic lesions: (A) Intramucosal nevus is negative for COX-2. The spinous layer of
normal oral epithelium served as positive control. (B) Oral in situ melanoma showing positivity for COX-2 (score 3). (C, D) Invasive oral melanoma
demonstrating high expression of COX-2 (score 3) (Envision—Permanent red, (A) 9200, (B) 9100, (C, D) 9200).

A B

C D

Figure 3 Immunohistochemical expression of COX-2 in cutaneous melanocytic lesions: (A, B) Compound melanocytic nevus was negative for COX-2,
which showed positivity only in normal sebaceous glands (A, B). (C) Tumor cells positive for COX-2 in Clark II melanoma of the skin (score 2). (D) High
COX-2 positivity in tumor cells of Clark III melanoma of the skin (score 3) (Envision—Permanent red, A–D, 9200).

COX-2 in the neoplastic progression might be different in
oral cavity when compared with its cutaneous counterpart.
Currently, there is little evidence to claim that oral
melanocytic nevi increases the risk for oral melanomas, but
the development of melanomas has been attributed of being
preceded by pigmented lesions of unknown characteristics
(17). Kuzbicki et al. (8) showed COX-2 positive cells only
occasionally in benign melanocytic lesions, particularly in
dysplastic nevi. It would be interesting to determine COX-2
expression in atypical melanocytic hyperplasias of the oral
cavity, but considering their rarity, multicentric studies are
encouraged for this purpose.
The granular and spinous layers of the normal oral
epithelium as well as sebaceous glands of the normal skin

J Oral Pathol Med

 COX-2 in oral nevi and melanoma
de Souza do Nascimento et al.
4
were strongly positive for COX-2, representing inner
positive controls in the present study. Such observations
agree with those reported by other authors and indicate a
role of COX-2 in the regulation of the differentiation of
keratinocytes (3, 15, 16). In vitro studies demonstrated that
keratinocytes and melanoma cells produce different types of
prostaglandins—keratinocytes produce mainly prostaglan-
din E2 (PGE2) and melanoma cells synthesized both PGE2
and prostaglandin F2a (15). Indeed, the synthesis of COX-2
was absent in normal melanocytes and clearly detected in
melanoma cell lines (16).
Interestingly, our results with oral nevi and melanoma
were similar with those found in a previous study evaluating
fatty acid synthase (FASN), a key enzyme responsible for
synthesis of fatty acids (20). FASN is overexpressed in
many human cancers such as oral melanoma cells because
of their increased energy obtained from lipid metabolism
(20). Lipid bodies are structures surrounded by a phospho-
lipid monolayer with a unique fatty acid composition that
has emerged as dynamic organelles involved in lipid
metabolism in inflammation and cancer (21). Increased
lipid body numbers have been described in tumor cells,
possibly representing sites of compartmentalization of
COX-2 and PGE2 production (21). Considering that, we
may suggest that the overexpression of COX-2 and FASN
in oral melanoma might be associated to the production of
fatty acids needed for tumor growth.
It is well known that oral melanoma has a poor prognosis
(17), and in most types of cancer, high expression of COX-2
is usually related to a poorer prognosis (4). The present
study did not related COX-2 expression levels with oral
melanoma prognosis. Kuzbicki et al. (8) showed that the
immunohistochemical intensity of COX-2 is associated with
Clark level and Breslow thickness, which indicates that
COX-2 expression might represent a prognostic and
predictive marker in human malignant melanoma, possibly
being relevant for malignant transformation of oral
melanocytes (1–19).
In summary, COX-2 was consistently positive in invasive
and in situ oral melanomas, and consistently negative in
intramucosal melanocytic nevi, as also observed in their
cutaneous counterparts. Nevertheless, COX-2 staining
intensity in cutaneous melanomas increases with increasing
depth of invasion, while in oral melanomas, expression
seems to be similar. COX-2 represents a useful diagnostic
marker on differentiating benign and malignant melanocytic
lesions of the oral cavity. Future studies of COX-2
expression in oral atypical melanocytic lesions would be
interesting to better understand the biology and develop-
ment of oral melanomas.
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