Oral Diseases (2012) doi:10.1111/odi.

12010
© 2012 John Wiley & Sons A/S
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ORIGINAL ARTICLE

Expression of proliferative markers in ameloblastomas

and malignant odontogenic tumors
D Otero1, SQC Lourenço1, I Ruiz-Ávila2, M Bravo3, T Sousa4, PAS de Faria5, MA González-Moles6
1
Pathology Graduate Program, Fluminense Federal University (UFF), Niterói, Brazil; 2Clinical University Hospital San Cecilio of
Granada, University of Granada, Granada, Spain; 3Preventive and Public Health in Dentistry Department, University of Granada,
Granada, Spain; 4Oncology Graduate Program, National Cancer Institute, Rio de Janeiro, Brazil; 5Pathology Division, National
Cancer Institute, Rio de Janeiro, Brazil; 6Department of Oral Medicine, School of Dentistry, University of Granada, Granada, Spain

OBJECTIVE: To compare the proliferative activity in Introduction

ameloblastoma and malignant odontogenic tumors, as
assessed by Ki-67 immunostaining and determine Ameloblastoma is the second most common odontogenic
whether expression of substance P (SP) and NK-1 tumor, accounting for approximately 1% of all oral tumors
receptor (NK-1R) is related to cell proliferation in these (Barnes et al, 2005; Costa et al, 2012). The solid multicy-
tumors. stic ameloblastoma (AS/M) is considered the most com-
MATERIALS AND METHODS: Immunohistochemistry mon variant and displays greater clinical aggressiveness
was used to evaluate protein expression in 44 benign (Barnes et al, 2005). The most important feature of AS/M
and malignant odontogenic tumors from 39 patients. is its ability to produce significant destruction of the jaw-
Immunohistochemistry was performed with anti-SP, bones before generating clinical manifestations such as
anti-NK-1R, and anti-Ki-67 monoclonal antibodies, and deformity or facial asymmetry (Black et al, 2010). This
the clinical and pathological data of the patients with insidious behavior of ameloblastoma probably indicates
odontogenic tumor were evaluated. that it is a slow-growing tumor that is present for a long
RESULTS: Expression of Ki-67 in malignant odontogenic time, a situation which could allow the development of
tumors was significantly higher than in ameloblastomas adaptive mechanisms by the tumor to enable its intraos-
(P < 0.001), and the expression level was associated with seous growth (Black et al, 2010). Several studies (Ong’uti
higher expression of NK-1R. Among the ameloblasto- et al, 1997; Piattelli et al, 1998; Sandra et al, 2001;
mas, there was significantly higher expression of Ki-67 Hirayama et al, 2004; Bologna-Molina et al, 2008, 2009;
in peripheral ameloblastic-like cells (3.3 ± 4.1) than in Migaldi et al, 2008; Gomes et al, 2010) reported a low
stellate reticulum-like cells (2.6 ± 3.7) (P = 0.04). In the cellular proliferation index in ameloblastoma, although
majority of tissue locations of the malignant tumors, exhibiting great infiltrative potential and a high recurrence
expression of SP and NK-1R was positively correlated rate. So far, it is not possible to predict which ameloblas-
with higher expression of Ki-67. toma will develop a recurrent or aggressive behavior
CONCLUSION: These findings show that the expres- (Barnes et al, 2005). Malignant odontogenic tumors can
sion level of Ki-67 in ameloblastomas was positively cor- arise from a benign odontogenic tumor or as a primary
related with the rate of growth of odontogenic tumors. neoplasm. These malignant lesions are exceedingly rare
Overexpression of NK-1R complex in malignant odonto- and occur more frequently in the elderly. They cause
genic tumors could be part of the trigger stimulus that extensive jawbone destruction with involvement of local
results in higher proliferative activity of the tumor. lymph nodes and distant metastases (Barnes et al, 2005).
Oral Diseases (2012) doi:10.1111/odi.12010 Two studies reported that the growth and proliferative
activity of ameloblastoma can be correlated with activation
Keywords: Kodontogenic tumors; substance P; receptor NK-1; of the mitogen-activated protein kinase (MAPK) signaling
Ki-67; ameloblastoma; malignant odontogenic tumor pathway through upregulation of certain proteins such as
Ras and urokinase plasminogen activator (uPA) (Kumam-
oto et al, 2004; Zhong et al, 2010). Our group recently
showed that another type of odontogenic tumor also with
a locally aggressive behavior, keratocystic odontogenic
tumor (KOT), exhibited overexpression of substance P
Correspondence: MA González-Moles, Facultad de Odontología, Paseo (SP) and its receptor (NK-1R) in association with an
de Cartuja s/n, 18071, Granada, Spain. Tel: +34 958243804, Fax: +34
958240908, E-mail: [email protected] increased rate of proliferation in the epithelium (high
Received 21 May 2012; revised 16 July 2012; accepted 24 July 2012 Ki-67 expression), and with prognostic implications
 Proliferative markers in odontogenic tumors
D Otero et al

2
(González-Moles et al, 2008). SP is a neuropeptide that hydrated, and incubated in 0.5% (v/v) H2O2 in methanol for
exerts multiple physiological functions through its binding 20 min to block endogenous peroxidase activity. Slides
to NK-1 receptor (Esteban et al, 2006; Muñoz and Cove- were then washed with Tris-buffered saline (TBS) and
ñas, 2010). Recently, it was suggested that overexpression steamed for 15 min at 100°C in 10 mM sodium citrate buf-
of SP and/or NK-1R may be related to malignant tumor fer (pH 6.0) for antigen retrieval. Subsequently, they were
growth through activation of MAPK signaling, as has incubated for 60 min with 1:500 diluted antisubstance P
been demonstrated both in tumor tissue and in malignant (Sigma, St Louis, MO, USA) and anti-NK-1R (Sigma–
cell lines (Muñoz and Coveñas, 2010). Thus, the aims of Aldrich, Madrid, Spain) monoclonal antibodies, followed
this study were as follows: (i) to compare the proliferative by washing in phosphate-buffered saline (PBS). The color
activity, based on Ki-67 expression, of primary ameloblas- was developed using diaminobenzidine as the chromogen.
toma, recurrent ameloblastoma, and malignant odontogenic Slides were washed with TBS after each step. Slides were
tumors; and (ii) to analyze whether SP and NK-1R expres- counterstained with Mayer’s hematoxylin. Formalin-fixed,
sion is associated with the proliferative activity in ame- paraffin-embedded oral squamous cell carcinoma (for
loblastomas and malignant odontogenic tumors. Ki-67) and glioma cells (for SP and NK-1R) served as posi-
tive controls. As a negative control, the primary antibody
was replaced with PBS. Positive immunoexpression for SP
Materials and methods
and NK-1R was defined by brown staining in the nucleus,
Forty-four tissue samples from 39 patients were studied, cytoplasm, and plasma membrane of the odontogenic epi-
of which 32 were ameloblastomas including AS/M thelial cells in all neoplasms. The stroma was also evaluated
(n = 30), peripheral ameloblastoma (n = 1), and desmo- for SP expression. Ki-67 expression was considered positive
plastic ameloblastoma (n = 1); and 12 were malignant when brown staining was present in the cell nucleus of epi-
odontogenic tumors, including ameloblastic carcinoma thelial cells. Quantification of immunostaining was
(n = 6), ghost cell odontogenic carcinoma (n = 1), performed through digital image analysis. Six fields
primary intraosseous squamous cell carcinoma derived (50 9 objective), selected from areas, whenever possible,
from a keratocystic odontogenic tumor (n = 1), and malig- containing ameloblast-like cells, stellate reticulum-like cells,
nant odontogenic tumors that were not classifiable (n = 4). and stroma (only for SP), were evaluated per slide. In all
The specimens were selected from the records of the cases and for all markers (SP, NK-1R, and Ki-67), total and
National Cancer Institute, Department of Oral Pathology positive cell counts were made, and the percentage of posi-
of the University of São Paulo (Bauru), Anatomic Pathol- tive cells was calculated. Image analysis was performed
ogy Service of the Antonio Pedro University Hospital with ImageJ 1.45s software (Wayne Rasband, National
(Federal Fluminense University) and a private pathology Institutes of Health, USA).
laboratory (all of them in Brazil), and from the records of
the Jaen Hospital Complex, Spain. Clinical data were Statistical analysis
gathered from medical records of all patients, including SPSS Windows v.15.0 (SPSS Inc., Chicago, IL, USA)
age, gender, and site of the odontogenic neoplasm. Also was used for the descriptive analysis. For the calculation
recorded was whether the lesion was a primary tumor or a of P-values, SUDAAN v.7.5 (Research Triangle Institute,
recurrence neoplasm. The microscopic features used for RTP, NC, USA) was applied. The statistical methods used
diagnosing the malignant odontogenic tumors that were are reported in the table footnotes.
not classifiable by the World Health Organization in 2005
(Barnes et al, 2005) were as follows: basilar hyperplasia,
increased mitotic index, nuclear pleomorphism, perineural
Results
invasion, or other histologic evidence of malignancy pres- The group of patients with ameloblastoma comprised 12
ence of sheets, islands, or trabeculae of epithelium, the females (41.4%) and 17 males (58.6%), ranging in age
absence of stellate reticulum-like structures, and round-to- from 9 to 80 years (mean 42.0 years, standard deviation
spindled epithelial cells with little or no differentiation 18.3). The group of patients with malignant odontogenic
toward the columnar cell morphology of ameloblastoma. tumors comprised 3 women (30%) and 7 men (70%), aged
The research protocol was approved by the Research 27–76 years (mean 48.4 years, standard deviation 17.8).
Ethics Committee of Federal Fluminense University Table 1 shows the clinical and pathological characteristics
(Niterói-RJ) and the National Cancer Institute (RJ). of the tumors. Table 2 shows the immunoexpression data
for SP, NK-1R, and Ki-67 in ameloblastomas and in
Immunohistochemistry analysis malignant odontogenic tumors (Figure 1). It is important
Immunohistochemistry was performed on formalin-fixed to highlight the higher Ki-67 expression observed in
paraffin-embedded sections (4 lm). For Ki-67 detection malignant odontogenic tumors than in ameloblastomas.
(monoclonal anti-Ki-67, clone Mib-1; diluted 1:200, Dako, Moreover, among the ameloblastomas, there was signifi-
Carpinteria, CA, USA), the immunohistochemical study cantly higher expression of Ki-67 in peripheral ameloblas-
was performed automatically, using Autostainer Link equip- tic-like cells (3.3 ± 4.1) than in stellate reticulum-like
ment (Dako, Carpinteria, CA, USA) and EnVisionTM FLEX cells located in more central areas of the follicles
reagents (K8002; Dako), as reported previously by our (2.6 ± 3.7) (P = 0.04; SUDAAN) (data not shown in
group (González-Moles et al, 2010). Manufacturer’s table). Comparisons of Ki-67 expression between primary
instructions were rigorously followed. For detection of SP (2.6 ± 3.7) and recurrent (4.1 ± 4.2) ameloblastomas, and
and NK-1R, the slides were deparaffinized in xylene, between primary (20.2 ± 15.8) and recurrent (5.6 ± 4.2)

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 Proliferative markers in odontogenic tumors
D Otero et al

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Table 1 Clinical and pathological description of the tumors (n = 44a) Table 2 Immunoexpression findings for SP, NK-1R, and Ki-67 in amelo-
blastoma and malignant odontogenic tumors, considering the tumor as
All tumors Ameloblastoma Malignant the unit of analysis (n = 44)
Variable (n = 44) (n = 32) (n = 12) P-value
Ameloblastoma Malignant
Localization, n (%) Variable (n = 32) (n = 12) P-valuea
Mandible 28 (63.6%) 23 (71.9%) 5 (41.7%) 0.119b
Maxilla 16 (36.4%) 9 (28.1%) 7 (58.3%) Substance P
Type, n (%) Nucleus (PA + SRb) 41.8 ± 27.2 40.6 ± 24.1 0.893
Ameloblastoma 32 (72.7%) 32 (100%) – Cytoplasm (PA + SRb) 51.9 ± 23.8 66.7 ± 24.0 0.064
Ameloblastic 6 (13.6%) – 6 (49.9%) Membrane (PA + SRb) 61.4 ± 22.6 71.5 ± 15.5 0.097
carcinoma Lymphocytes 72.8 ± 20.5 57.0 ± 11.8 0.002
Ghost cell 1 (2.3%) – 1 (8.4%) Stroma 90.1 ± 17.4 81.9 ± 32.9 0.431
odontogenic NK-1R
carcinoma Nucleus (PA + SRb) 5.9 ± 9.7 17.0 ± 16.9 0.026
Carcinoma 1 (2.3%) – 1 (8.4%) Cytoplasm (PA + SRb) 34.8 ± 27.3 56.6 ± 35.2 0.059
derived from Membrane (PA + SRb) 43.8 ± 26.9 58.9 ± 31.5 0.147
keratocysts Lymphocytes 29.6 ± 36.3 32.1 ± 29.2 0.804
Malignant 4 (9.1%) – 4 (33.3%) Stroma 8.9 ± 16.4 11.1 ± 21.7 0.742
odontogenic Ki-67
tumor not Nucleus (PA + SRb) 2.9 ± 3.7 8.0 ± 8.4 0.042
classifiable
a
Primary/recurrence, n (%) Comparison of means by the DESCRIPT procedure in SUDAAN to
Primary 19 (43.2%) 17 (53.1%) 2 (16.7%) 0.029b account for clustering (multiple tumors per patient).
b
Recurrence 25 (56.8%) 15 (46.9%) 10 (83.3%) PA, peripheral ameloblast-like cells; SR, stellate reticulum-like cells.
a
Corresponding to 39 patients.
b
Chi-square analysis, corrected for multiple tumors by using the CROS-
STAB procedure in SUDAAN. by peripheral expansion of the follicles and that this pat-
tern of proliferation possibly indicates some degree of cen-
tral maturational activity, as was also pointed out by
malignant odontogenic tumors, did not show any signifi- Sandra et al, 2001;. Two studies (Piattelli et al, 1998;
cant differences (P = 0.72 and P = 0.07, respectively) Hirayama et al, 2004), although not ours, reported that
(data not shown in table). Table 3 displays the results of recurrent ameloblastoma has a significantly higher prolifer-
statistical comparisons between the expression of SP or ative rate than primary tumors.
NK-1R and the expression of Ki-67. In the majority of tis- The results published on ameloblastic carcinomas dem-
sue locations of the malignant tumors, expression of SP onstrate that they have a higher proliferation rate com-
and NK-1R is positively correlated with higher expression pared with ameloblastomas (Han et al, 2003; Bologna-
of Ki-67. Table 4 shows the correlation between SP and Molina et al, 2009; Yoon et al, 2011). Our results for
NK-1R expression in tumors of the series. In 9 tumors, ameloblastic carcinoma and other malignant odontogenic
we observed at least one field with positive SP nuclear tumors are in agreement with those previous findings. The
expression and negative NK-1R membrane expression (8 malignant odontogenic tumors in our study showed a
ameloblastomas, 14 fields; 1 malignant tumor, 1 field). higher proliferative index than the ameloblastomas. Thus,
Five of these fields showed more than 20% positive SP we were interested in investigating molecular mechanisms
nuclei (20.1–42.1%). that could be involved in this increased proliferation asso-
ciated with malignancy. Two studies (Kumamoto et al,
2004; Zhong et al, 2010) reported activation of the
Discussion
MAPK signaling pathway as a result of upregulation of
Our results, which are in line with the few published stud- uPA and mutation of the Ras protein, which resulted in
ies by other authors (Ong’uti et al, 1997; Nagao et al, increased proliferative activity. Our group recently
1999; Sandra et al, 2001; Han et al, 2003; Meer et al, described SP/NK-1R overexpression associated with
2003; Slater, 2004; Yoon et al, 2011), indicate that ame- increased Ki-67 expression in KOT, another odontogenic
loblastomas have a low cellular proliferation index. In the tumor with local aggressiveness and a tendency to recur-
present study, only 3.3% of peripheral ameloblastic-like rence (González-Moles et al, 2008). SP, a member of the
cells and 2.6% of stellate reticulum-like cells expressed tachykinin family of neuropeptides, exerts its action by
the proliferation marker Ki-67. This low proliferative binding to the NK-1 receptor. SP has a wide range of
activity could explain the slow development of the tumor functions, including regulation of neurogenic inflammation
in areas of the jaw with low resistance to growth such as and the immune response, and participation in psychologi-
the marrow spaces and the emergence of adaptive mecha- cal stress pathways (Esteban et al, 2006; Muñoz and Cov-
nisms to host cortical defensive measures. An interesting eñas, 2010). It is known that SP stimulates mitogenesis by
finding of this and previous studies (Ong’uti et al, 1997; activating NK-1R in several normal and tumor cell types
Gomes et al, 2010; Tekkeşİn et al, 2012) is that prolifera- (Luo et al, 1996; Palma et al, 2000; Muñoz et al, 2005,
tion was observed to be located predominantly in periph- 2007, 2008, 2010b). Through these effects, activation of
eral cells, whereas the stellate reticulum-like cells the MAPK pathway could occur, including activation of
presented fewer cells positive for Ki-67 expression. This the extracellular signal-regulated kinases 1 and 2 (ERK1/
suggests that the growth of ameloblastomas is produced 2) (Luo et al, 1996). Activation of NK-1 receptors by SP

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D Otero et al

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(a) (b)

(c) (d)

Figure 1 Immunostaining of Ki-67, SP, and

NK-1R in ameloblastomas and malignant
odontogenic tumors. (a) Ki-67 expression in a
solid/multicystic type of ameloblastoma shows
staining in peripheral cells and stellate
reticulum-like cells (immunohistochemical
technique; 9200). (b) Ki-67 expression in
tumoral epithelial cells of a malignant
odontogenic tumor (immunohistochemical
technique; 9200). (c) Substance P expression
(e) (f) in the nucleus, cytoplasm, and plasma
membrane of epithelial cells in a solid/
multicystic type of ameloblastoma
(immunohistochemical technique; 9500). (d)
Nuclear, cytoplasmic, and membrane expression
of SP in malignant odontogenic tumor
(immunohistochemical technique; 9500). (e)
Low expression of NK-1R in a solid/multicystic
type of ameloblastoma (immunohistochemical
technique; 9500). (f) Intense expression of
NK-1R in the cytoplasm and membrane of
epithelial cells in a malignant odontogenic
tumor (immunohistochemical technique; 9500)

Table 3 Pearson correlations between substance P or NK-1R immunoex- Table 4 Pearson correlations between substance P and NK-1R immuno-
pression and Ki-67 immunoexpression (n = 264 fieldsa) expression (n = 264 fieldsa)

Ameloblastoma Malignant Ameloblastoma Malignant

Variable (n = 192 fields) (n = 72 fields) Localization (n = 192 fields) (n = 72 fields)

Substance P Nucleus (PA + SRb) 0.05 0.58*

Nucleus (PA + SRb) 0.00 0.41* Cytoplasm (PA + SRb) 0.43* 0.49*
Cytoplasm (PA + SRb) 0.10 0.40* Membrane (PA + SRb) 0.31* 0.27
Membrane (PA + SRb) 0.07 0.30* Lymphocytes 0.08 0.15
Lymphocytes 0.12 0.24* Stroma 0.08 0.16*
Stroma 0.17 0.50*
NK-1R a
Corresponding to 44 tumors (6 fields/tumor 9 44 tumors = 264).
Nucleus (PA + SRb) 0.06 0.03 b
PA, peripheral ameloblast-like cells; SR, stellate reticulum-like cells.
Cytoplasm (PA + SRb) 0.02 0.27* *P < 0.05, with the REGRESS procedure in SUDAAN to account for
Membrane (PA + SRb) 0.08 0.23* clustering (6 fields per tumor).
Lymphocytes 0.01 0.28*
Stroma 0.01 0.09
a
Corresponding to 44 tumors (6 fields/tumor 9 44 tumors = 264). malignant odontogenic lesions compared with ameloblas-
b
PA, peripheral ameloblast-like cells; SR, stellate reticulum-like cells. tomas [statistically significant in the nucleus (P = 0.026)
*P < 0.05, with the REGRESS procedure in SUDAAN to account for and with a tendency to significance in the cytoplasm
clustering (6 fields per tumor). (P = 0.059)], suggesting that, as happens in other oral
lesions and malignancies, including lichen planus and oral
stimulates the formation of a scaffolding complex com- squamous cell carcinoma (Eistetter et al, 1992; Esteban
prising internalized receptor, b-arrestin, src, and ERK1/2. et al, 2006; Muñoz et al, 2008, 2010b; Brener et al,
Once activated, ERK1/2 is translocated into the nucleus 2009; González-Moles et al, 2009), upregulation of NK-
where it aids in inducing proliferation and protecting the 1R could be an oncogenic event in malignancies of odon-
cell from apoptosis (Eistetter et al, 1992). Our results togenic origin. This idea is supported by the significant
showed overexpression of NK-1R in tumor cells of association between the expression of NK-1R and the

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D Otero et al

5
proliferative activity of the malignant odontogenic tumors Black CC, Addante RR, Mohila CA (2010). Intraosseous amelo-
in our series. Overexpression of NK-1R could be an blastoma. Oral Surg Oral Med Oral Pathol Oral Radiol En-
effective oncogenic mechanism because only nanomolar dod 110: 585–592.
amounts of SP are necessary to stimulate NK-1R (Luo Bologna-Molina R, Mosqueda-Taylor A, Lopez-Corella E et al
(2008). Syndecan-1 (CD138) and Ki-67 expression in different
et al, 1996). The subject has potential therapeutic interest,
subtypes of ameloblastomas. Oral Oncol 44: 805–811.
given that preliminary studies by our group have Bologna-Molina R, Mosqueda-Taylor A, Lopez-Corella E et al
demonstrated that NK-1R antagonists (e.g. L-733,060, (2009). Comparative expression of syndecan-1 and Ki-67 in
L-732,138, aprepitant), after binding to NK-1R, exert a peripheral and desmoplastic ameloblastomas and ameloblastic
dose-dependent antiproliferative effect that induces apop- carcinoma. Pathol Int 59: 229–233.
tosis in a large number of human tumor cell lines (e.g. Brener S, González-Moles MA, Tostes D et al (2009). A role
melanoma, glioma, neuroblastoma, retinoblastoma, larynx, for the substance P⁄NK-1 receptor complex in cell prolifera-
gastric, and colon carcinoma cell lines) (Esteban et al, tion in oral squamous cell carcinoma. Anticancer Res 29:
2006). Other antitumor actions linked to the NK-1R antag- 2323–2329.
onists include inhibition of angiogenesis and blocking the Costa DO, Maurício AS, Faria PA, Silva LE, Mosqueda-Taylor
A, Lourenço SQ (2012). Odontogenic tumors: a retrospective
migration of tumor cells. These observations suggest that
study of four Brazilian diagnostic pathology centers. Med Oral
NK-1R antagonists might be candidate drugs for antican- Pathol Oral Cir Bucal 17: e389–e394.
cer treatments (Esteban et al, 2006; Muñoz et al, 2010a). Eistetter HR, Mills A, Brewster R, Alouani S, Rambosson C,
Another finding of interest was the SP expression Kawashima E (1992). Functional characterization of neuroki-
observed in the nucleus of epithelial cells in some fields nin-1 receptors on human U373MG astrocytoma cells. Glia 6:
of tumors in the absence of membrane NK-1R expression, 89–95.
indicating perhaps that SP could act as a transcription fac- Esteban F, Muñoz M, González-Moles MA, Rosso M (2006). A
tor that is constitutively activated, driving the expression role for substance P in cáncer promotion and progression: a
of genes involved in proliferation and invasion, as occurs mechanism to counteract intracellular death signals following
with other transcription factors known to be involved in oncogene activation or DNA damage. Cancer Metastasis Rev
25: 137–145.
the development of cancer such as signal transducers
Gomes CC, Duarte AP, Diniz MG, Gomez RS (2010). Current
and activators of transcription (STAT) and Nuclear factor- concepts of ameloblastoma pathogenesis. J Oral Pathol Med
kappaB (NFkB). 39: 585–591.
In conclusion, according to the present findings, the low González-Moles MA, Mosqueda-Taylor A, Esteban F et al
proliferative activity of ameloblastomas could enable these (2008). Cell proliferation associated with actions of the sub-
tumors to develop slowly while establishing adaptive mech- stance P/NK-1 receptor complex in keratocystic odontogenic
anisms to persist in and invade the jawbone. Malignant tumours. Oral Oncol 44: 1127–1133.
odontogenic tumors, on the other hand, present a higher rate González-Moles MA, Esteban F, Ruiz-Avila I et al (2009). A
of growth, which could be associated, in part, with onco- role for the substance P ⁄ NK-1 receptor complex in cell pro-
genic mechanisms linked to overexpression of NK-1R. Our liferation and apoptosis in oral lichen planus. Oral Dis 15:
162–169.
results indicate that this could be a molecular mechanism
González-Moles MA, Ruiz-Avila I, Gil-Montoya JA, Esteban F,
associated with the malignant transformation of odonto- Bravo M (2010). Analysis of Ki-67 expression in oral squa-
genic epithelium, although its absolute demonstration mous cell carcinoma: why Ki-67 is not a prognostic indicator.
requires studies designed specifically to test this hypothesis. Oral Oncol 46: 535–530.
Han B, Li L, Wang H (2003). Expression of Ki-67 antigen in
ameloblastoma and its clinical significance. Hua Xi Kou Qiand
Conflict of Interest Xue Za Zhi 21: 153–154.
Hirayama T, Hamada T, Hasui K, Semba I, Murata F, Sugihara
None declared.
K (2004). Immunohistochemical analysis of cell proliferation
and suppression of ameloblastoma with special reference to
plexiform and follicular ameloblastoma. Acta Histochem Cyto-
Acknowledgements chem 37: 391–398.
We thank Dolores Gomez and Dra. Vanessa Soares Lara for their Kumamoto H, Takahashi N, Ooya K (2004). k-Ras gene status
assistance with part of this research. The first author was sup- and expression of Ras/mitogen-activated protein kinase
ported by CAPES and FAPERJ. (MAPK) signaling molecules in ameloblastoma. J Oral Pathol
Med 33: 360–367.
Luo W, Sharif TR, Sharif M (1996). Substance P-induced mito-
Author contributions genesis in human astrocytoma cells correlates with activation
of the mitogen-activated protein kinase signaling pathway.
Daniela and Thais did the experiments. Dra. Isabel evaluated the Cancer Res 56: 4983–4991.
results, Dr.Bravo did the statistical analysis, Dra. Simone and Meer S, Galpin JS, Altini M, Coleman H, Ali H (2003). Prolifer-
Dr. Paulo did the histopathologic diagnosis. Dr. Miguel Angel ating cell nuclear antigen and Ki67 immunoreactivity in ame-
evaluated the results and wrote the article. loblastomas. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 95: 213–221.
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