Histopathology 2018, 72, 330–338. DOI: 10.1111/his.

13381

PTEN allelic loss is an important mechanism in the late

stage of development of oral leucoplakia into oral squamous
cell carcinoma
Ligia A N Miyahara,1 Fl

avia S C Pontes,2 Rommel M R Burbano,2 Nicolau Conte Neto,2
Douglas M Guimar~aes,2 Felipe P Fonseca3 & H
elder A R Pontes1,2
1
Oral Diagnosis Department, Semiology and Oral Pathology Areas, Piracicaba Dental School, University of Campinas
(UNICAMP), S~ao Paulo, Brazil, 2Jo~ao de Barros Barreto University Hospital (HUJBB), Federal University of Para
(UFPA), Para, Brazil, and 3Department of Oral Surgery and Pathology, School of Dentistry, Federal University of
Minas Gerais (UFMG), Minas Gerais, Brazil

Date of submission 14 July 2017

Accepted for publication 30 August 2017
Published online Article Accepted 31 August 2017

Miyahara L A N, Pontes F S C, Burbano R M R, Conte Neto N, Guimar~

aes D M, Fonseca F P & Pontes H A R
(2018) Histopathology 72, 330–338. https://doi.org/10.1111/his.13381
PTEN allelic loss is an important mechanism in the late stage of development of oral
leucoplakia into oral squamous cell carcinoma

Aim: The aim of this study was to analyse allelic loss
of the phosphatase and tensin homologue deleted on
chromosome 10 (PTEN) gene and its protein
immuno-expression in dysplastic oral lesions and oral
squamous cell carcinomas (OSCCs).
Methods and results: Samples were collected from
153 patients [20 ranulas used as a control (C); 30
leucoplakias with mild dysplasia (MD); 30 leu-
coplakias with moderate to severe dysplasia (MSD);
73 oral squamous cell carcinoma (OSCC)]. PTEN pro-
tein expression was investigated using immunohisto-
chemistry, and PTEN allelic loss was analysed by
fluorescence in-situ hybridisation (FISH). Differences
among groups were evaluated using the v2 test.
Keywords: gene deletion, immunohistochemistry, leucoplakia, oral cancer, PTEN phosphatase
PTEN expression was higher in MSD (P = 0.002) and
OSCC (P = 0.0259) compared with the C group; addi-
tionally, a higher expression was observed in MSD
(P = 0.0035) and OSCC (P = 0.049) than MD.
Regarding FISH analysis, a higher hemizygous (single
copy) loss was observed in OSCC than in C
(P = 0.0467) and in OSCC than in MD (P = 0.0175),
as well as a higher homozygous deletion in OSCC
compared with C (P = 0.0159) and OSCC than MD
(P = 0.0145).
Conclusion: The results of this work suggest that
PTEN allelic loss is an important mechanism in the
late stage of the development of oral potentially
malignant lesions into oral cancer.

Introduction the oral cavity. It is a significant problem in many

Oral squamous cell carcinoma (OSCC) is one of the
most common types of cancer in the world,1 account-
ing for more than 95% of all malignant neoplasms in
Address for correspondence: H
elder Ant^ onio Rebelo Pontes DDS,
PhD, Jos
e Malcher Street, no. 1913, Apartment 801, 66060-230
S~
ao Braz, Bel
em, Par
a, Brazil. e-mails: harp@ufpa.br or harp1@
uol.com.br
© 2017 John Wiley & Sons Ltd.
parts of the world, particularly in underdeveloped
countries, owing to its high incidence and unsatisfac-
tory 5-year survival rate and because treatment can
result in severe functional and cosmetic defects.2 Oral
carcinogenesis is a multistep process that involves the
activation of proto-oncogenes and inactivation of
tumour suppressor genes. However, the underlying
molecular mechanisms remain to be understood fully.
A proportion of OSCCs develop from oral potentially

malignant lesions, such as oral leucoplakia, oral sub-
mucous fibrosis and oral erythroplakia.
The presence and degree of epithelial dysplasia are
used to assess the risk of leucoplakia progression to
malignancy; however, histological grading has lim-
ited value in predicting the individual risk of each
case. Furthermore, the criteria reproducibility of
microscopic grading of dysplastic lesions is very low,
and poses significant difficulties among patholo-
gists.3–5 Therefore, the understanding of the molecu-
lar mechanisms that govern the evolution of oral
potentially malignant lesions into OSCC is highly rel-
evant for clinical practice, leading potentially to
the identification of genes and proteins suitable for
therapeutic targeting and predictors of prognostic
determination.5–7
Akt/protein kinase B is a major downstream target
of growth factor receptor tyrosine kinase that signals
via phosphatidylinositol-3 kinase (PI3K), and is
activated frequently in different human malignan-
cies.6,8–10 Phospho-Akt (p-Akt) controls a variety of
critical cellular pathways during the carcinogenic
process, including those leading to apoptosis inhibi-
tion and increased cell proliferation, as well as
enhanced tumour cell invasion, angiogenesis and cell
metabolism, through glucose metabolism.6,11 Phos-
phatase and tensin homologue deleted on chromo-
some 10 (PTEN) is a tumour suppressor gene, and its
major substrate is phosphatidylinositol (3,4,5)-tripho-
sphate (PIP-3), which dephosphorylates PIP3 to
regenerate PIP2 and which antagonises PI3K func-
tion. Consequently, PTEN down-regulates Akt activity
by dephosphorylating and regulating PI3K signalling
negatively.12 Conversely, loss of PTEN expression
results in increased Akt activity and continued cell
survival and proliferation.
Recent studies have proposed that activation of Akt
has been shown to be associated with advanced
stages and poor prognosis in OSCC;7,13–15 further-
more, studies using OSCC cell lines have shown that
induced PTEN overexpression down-regulated Akt
phosphorylation significantly and induced apopto-
sis.16 Regarding oral potentially malignant lesions,
several studies have shown that p-Akt may play an
important role in the conversion of these lesions to its
malignant counterpart.6,7,15,17
The expression of PTEN in potentially malignant
oral lesions remains to be investigated, and its impor-
tance to the oral carcinogenesis process deserves to
be understood more clearly. Therefore, the aim of this
study was to determine the expression and the allelic
loss of PTEN in oral leucoplakia with different degrees
of epithelial dysplasia and in OSCC.
© 2017 John Wiley & Sons Ltd, Histopathology, 72, 330–338.
PTEN in oral lesions 331
Materials and methods
TISSUE SAMPLES
Samples were collected from the Service of Oral
Pathology of the Jo~ ao de Barros Barreto University
Hospital (Bel
em, Brazil). The local ethics committee
approved this study (process no. 1 426 757).
The specimens were obtained from 153 patients [20
samples of morphologically normal epithelium obtained
from ranulas with inflammatory infiltrated located dis-
tant from the overlying epithelium to be used as a control
group (group C); 30 oral leucoplakias with mild dysplasia
(MD); 30 leucoplakias with moderate to severe dysplasia
(MSD); and 73 OSCC]. Histological sections (5 lm) of all
samples were stained routinely with haematoxylin and
eosin (H&E) and analysed under light microscopy. Two
independent oral pathologists without prior knowledge of
the clinical data assessed the stained sections to classify
the histological grades of oral dysplasia according to a
previously proposed binary system criteria of classifica-
tion.3 In cases of disagreement, pathologists discussed the
findings and achieved a final agreement.
IMMUNOHISTOCHEMISTRY
Immunohistochemical reactions were performed in
3 lm sections that were dewaxed with xylene and rehy-
drated in ethanol series. Antigen retrieval was performed
by immersing the slides in an ethylenediamine tetraace-
tic acid (EDTA) solution (pH 8.0) for 15 min in a micro-
wave oven. Peroxidase activity was blocked with a 6%
hydrogen peroxide and methanol (1:1) solution in two
baths of 15 min each at room temperature. After wash-
ing in Tris buffer (pH 7.4), the slides were incubated
with primary antibody (polyclonal anti-human PTEN;
Invitrogen, Carlsbad, CA, USA) in a dilution of 1:100
overnight for 18 h at 4°C. The slides were subsequently
exposed to the avidin–biotin complex [LSAB-
Kit + horseradish peroxidase (HRP); Dako Cytomation,
Glostrp, Denmark] and to the 3,30 -diaminobenzidine
chromogen (DAB+; Dako Cytomation). The sections
were counterstained with Mayer’s haematoxylin, dehy-
drated in ethanol, cleared in xylene and mounted. Pros-
tate cancer tissue was used as a positive control; the
negative control was obtained by omitting the primary
specific antibody during the reaction. The results were
considered positive when brown-coloured nuclear and
cytoplasmic stainings were observed, characterising the
presence of DAB in the immunohistochemical reaction.
The scoring system used has been described previ-
ously in the literature.18 Briefly, the analysis was
based on both the intensity and distribution of stain-
ing. The distribution of stained cells was organised as
332 L A N Miyahara et al.

follows: 0 (0% of cells stained), 1 (1–50% of cells Table 1. Clinicopathological profile of OSCC samples
stained) and 2 (51–100% of cells stained). The inten-
sity of staining was rated as follows: 0 (no staining), Characteristics Data (%)
1 (mild staining), 2 (moderate staining) and 3 (strong Age (years)
staining). The final record for each case was defined
Average 61.13
by the sum of the values obtained as follows: FR0,
FR2, FR3, FR4 and FR5. Finally, FR0 and FR2 were Range 33–87
considered negative staining, while FR3, FR4 and
Sex
FR5 were considered positive staining.
Male/female 44/29 (60.27%/39.73%)

FLUORESCENCE IN-SITU HYBRIDISATION Tobacco consumption

For FISH analysis, 5 lm histological sections of 10% Smoker/non-smoker 51/22 (69.86%/30.14%)

formalin-fixed tissues were dewaxed in xylene and TNM classification
rehydrated in ethanol series. The denaturation process
was carried out on a hot plate at 95°C for 10 min. Five T classification
ll of PTEN probe were applied on the samples accord- T1 11 (15.07%)
ing, to the manufacturer’s instructions (Cat. no.
T2 17 (23.29%)
07J74001, Vysis LSI PTEN SpectrumOrange/CEP 10
SpectrumGreen Probes; Abbott Molecular Inc., Des T3 21 (28.77%)
Plaines, IL, USA). The samples were incubated in a
T4 24 (32.87%)
humid chamber protected from light at 37°C for
approximately 16 h. Sections were stained with 40 ,6- N classification
diamidino-2-phenylindole, dihydrochloride (DAPI), N0/pN+ 35/38 (47.95%/52.05%)
and the slides were incubated in a humid chamber pro-
tected from light for 10 min at 8°C. N1 16
Analysis was performed using an Olympus BX 41 N2 19
fluorescence microscope using the Case Data Manager
5.0 program, where 100 cells/case were counted ran- N3 3
domly regarding the probe signals in each allele of M classification
the gene investigated. Hemizygous (single-copy) PTEN
deletion was assigned when >50% of nuclei exhibited M0 73 (100%)
clonal loss of PTEN and adjacent probes. Homozygous M1 0 (0%)
PTEN deletion was defined by a simultaneous lack of
Staging
both PTEN locus signals in 30% of scored nuclei.19
I 8 (10.96%)

STATISTICAL ANALYSIS II 11 (15.07%)

To determine any significant difference regarding the III 20 (27.40%)

allelic loss of PTEN and its immuno-expression among IV 34 (46.57%)
the groups investigated, a v2 test was applied. Graph-
Pad Prism version 5.0 (GraphPad Software, San Anatomical location
Diego, CA, USA) was used for statistical analysis, and Floor of the mouth 27 (36.99%)
statistical significance was set at P < 0.05 with 95%
confidence intervals. Tongue 23 (31.50%)

Alveolar ridge 14 (19.18%)

Results Palate 5 (6.85%)

Jugal mucosa 3 (4.11%)

The clinicopathological characteristics of 60 patients
with leucoplakia and 73 patients with OSCC are sum- Retromolar region 1 (1.37%)
marised in Tables 1 and 2.

© 2017 John Wiley & Sons Ltd, Histopathology, 72, 330–338.


Table 2. Clinicopathological profile of leucoplakia samples
Characteristics Data (%)
Age (years)
Average 55.63
Range 14–84
Sex
Male/female 28/32 (46.67%/53.33%)
Tobacco consumption
Smoker/non-smoker 25/33 (41.67%/55%)
Not specified 2 (3.33%)
Anatomical location
Tongue 32 (53.33%)
Floor of the mouth 10 (16.67%)
Others (jugal mucosa, retromolar 18 (30%)
region, palate, attached gingiva,
alveolar ridge
This study comprised 73 patients (44 male and 29
female) with OSCC. An average age of 61.13 years
was observed, with a range of 33–87 years. Fifty-one
patients (69.86%) were smokers and 22 (30.14%)
were non-smokers. Tumours were localised mainly in
the floor of the mouth (36.99%), tongue (31.50%),
and alveolar ridge (19.18%). Tumours classified as
T4 represented the majority of the cases (32.87%),
followed by T3 (28.77%), T2 (23.29%) and T1
(15.07%). A large proportion of the patients had
regional or locoregional involvement (52.05%), and
35 of 73 (47.95%) were free of lymph node involve-
ment. Distant metastases were not observed in these
samples. Following the AJCC criteria, patients were
categorised as stage IV tumours in 46.57% of the
cases, stage III in 27.40%, stage II in 15.07% and
stage I in 10.96%.
Regarding oral leucoplakia, 60 cases were retrieved
(30 with MD and 30 with MSD). Of these samples,
28 (46.67%) were male and 32 (53.33%) female. An
average age of 55.63 years was observed, with a
range of 14–84 years. The proportion of smokers/
non-smokers was 25/33 (ratio 1:1.3). The majority
of the cases affected the tongue (53.33%), followed
by the floor of the mouth (16.67%).
IMMUNOHISTOCHEMICAL STAINING
PTEN immunoreaction showed, predominantly, a
cytoplasmatic staining. However, nuclear staining
© 2017 John Wiley & Sons Ltd, Histopathology, 72, 330–338.
PTEN in oral lesions 333
was sometimes observed. In normal oral mucosa and
dysplastic tissues, the immunoreaction was found pre-
dominantly in the parabasal and basal layers,
whereas OSCC showed a diffuse distribution in neo-
plastic islands (Figure 1).
In the C group, 35% (seven cases) were positive for
PTEN immuno-expression, whereas 65% (13 cases)
were negative. In the oral leucoplakia group, MD
showed that 43.33% (13 cases) were positive and
56.67% (17 cases) were negative, whereas MSD
showed positivity in 80% of the cases (24 cases) and
20% (six cases) demonstrated negativity. Regarding
OSCC samples, 63.01% (46 cases) were positive and
36.99% (27 cases) were negative.
PTEN expression was higher in MSD (P = 0.002)
and OSCC (P = 0.0259) compared with the C group.
Additionally, a higher expression was observed in
MSD (P = 0.0035) and OSCC (P = 0.049) than MD
(Table 3).
FLUORESCENCE IN-SITU HYBRIDISATION
ANALYSIS
All samples of normal epithelium did not demonstrate
allelic loss. In MD, only 3.33% (one of 30) showed
homozygous deletion; in MSD, 3.33% (one of 30)
showed a PTEN hemizygous (single copy) deletion,
whereas 10% (three of 30) showed homozygous dele-
tion. In OSCC samples, single-copy loss was observed
in 13.70% (10 of 73) of the cases, and bi-allelic loss
occurred in 20.55% (15 of 73) (Figure 2). Statisti-
cally, a higher hemizygous loss was observed in OSCC
than C (P = 0.0467) and OSCC than MD
(P = 0.0175), as well as a higher homozygous dele-
tion in OSCC than C (P = 0.0159) and OSCC than
MD (P = 0.0145) (Tables 4 and 5).
Discussion
Despite advances in therapeutic approaches, the mor-
bidity and mortality associated with OSCC have not
improved significantly in the last 30 years, with the
5-year overall survival rate varying between 30%
and 50%.13 OSCC may be preceded by a potentially
malignant lesion, and the presence of epithelial dys-
plasia seems to be the most important prognostic
indicator of its potential for malignant transforma-
tion;20 however, the incidence of malignant change
varies in different studies, possibly as a result of differ-
ences in ethnic and environmental factors.6 More-
over, the low reproducibility of current histological
criteria for grading epithelial dysplasia is largely
recognised and remains to be improved. These facts
334 L A N Miyahara et al.

A B

C D

Figure 1. Immunoexpression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in normal, dysplastic and malignant
epithelium (streptavidin–biotin). A, Normal oral epithelium; B, epithelium with mild dysplasia; C, epithelium with moderate to severe dys-
plasia; D, oral squamous cell carcinoma.

Table 3. v2 test; P-value combination among groups at pathways that govern the progression of oral carcino-
immunohistochemistry genesis.6,21,22
PTEN is a tumour suppressor that opposes PI3K
C MD MSD OSCC function by dephosphorylating PIP3 to regenerate
C – NS 0.002 0.0259 PIP2. It is believed that down-regulation of PTEN
leads to an accumulation of PIP3, leading to
MD NS – 0.0035 0.049
increased activation of PI3K and pAkt. pAkt partici-
MSD NS NS – NS pates in the regulation of the cell cycle, proliferation,
apoptosis, cell adhesion, migration, invasion and
OSCC NS NS NS –
metastasis during cancer progression by regulating
C, control group; MD, mild dysplasia; MSD, moderate/severe dys- various downstream substrates.
plasia; OSCC, oral squamous cell carcinoma; NS, not significant. Therefore, in this study, we investigated the allelic
loss of PTEN and its immuno-expression in dysplastic
oral lesions and in OSCC. We employed the binary
highlight the necessity of identifying molecular mark- system to graduate dysplasia because this system has
ers that could predict oral leucoplakia aggressiveness been proved to be a reliable tool, reducing variability
and clinical course more clearly. For this reason, it is and enhancing the reproducibility of epithelial dyspla-
crucial to understand the deregulated molecular sia.3,23 Our results revealed that PTEN expression
© 2017 John Wiley & Sons Ltd, Histopathology, 72, 330–338.
 PTEN in oral lesions 335

A B

C D

Figure 2. Vysis LSI phosphatase and tensin homologue deleted on chromosome 10 (PTEN) SpectrumOrange/CEP 10 SpectrumGreen Probes
hybridise to band 10q23 (SpectrumOrange LSI PTEN) and to the centromere, band region 10p11.1–q11.1 (SpectrumGreen CEP 10) of
human chromosome 10. Fluorescence in-situ hybridization (FISH) image of PTEN in normal, dysplastic and malignant epithelium. A, Normal
oral epithelium; B, epithelium with mild dysplasia; C, epithelium with moderate to severe dysplasia; D, oral squamous cell carcinoma.

Table 4. v2 test; P-value combination among groups at Table 5. v2 test; P-value combination among groups at
hemizygous deletion homozygous deletion

C MD MSD OSCC C MD MSD OSCC

C – NS NS 0.0467 C – NS NS 0.0159

MD NS – NS 0.0175 MD NS – NS 0.0145

MSD NS NS – NS MSD NS NS – NS

OSCC NS NS NS – OSCC NS NS NS –

C, control group; MD, mild dysplasia; MSD, moderate/severe dys- C, control group; MD, mild dysplasia; MSD, moderate/severe dys-
plasia; OSCC, oral squamous cell carcinoma; NS, not significant. plasia; OSCC, oral squamous cell carcinoma; NS, not significant.

was higher in MSD (P = 0.002) and in OSCC
(P = 0.0259) when both were compared with the C
group; moreover, a higher expression was observed
in MSD (P = 0.0035) and OSCC (P = 0.049) than
© 2017 John Wiley & Sons Ltd, Histopathology, 72, 330–338.
MD. In addition, a higher hemizygous loss was
observed in OSCC than C (P = 0.0467) and OSCC
than MD (P = 0.0175), as well as a higher homozy-
gous deletion in OSCC when compared with C
336 L A N Miyahara et al.
(P = 0.0159) and in OSCC when compared with MD
(P = 0.0145). To the best of our knowledge, this is
the first study analysing the expression and PTEN
allelic loss in oral leucoplakias and in OSCC together.
The molecular mechanism underlying malignant
transformation of leucoplakias has not yet been elu-
cidated. It is now accepted widely that there are
divergent molecules and pathways that affect the
process of transformation from leucoplakia to oral
squamous cell carcinoma sequentially.24,25 Recently,
the tumour microenvironment was shown to carry
out an important role in oral leucoplakia and oral
cancer progression.26,27 In addition, several factors
including age, sex, smoking and oral subsite, as
well as dysplasia grade, have been suggested as
predictors of the risk of malignant transformation of
oral leucoplakia.26–28 These lines of evidence sug-
gest that carcinogenic transformation of a leu-
coplakia is multifactorial and is patient-specific.29
Therefore, it is possible that inter-individual and
interpopulation differences in risk could be
explained partially by different distributions of
genetic variants that may cause variation in the
ability to metabolise carcinogens and/or effective
repair of the damage caused by them.30 The above
observations contribute to understanding of 20% of
negative expression of PTEN in MSD group and
37% of negative expression in OSSC group. We
must also keep in mind that some oral cancers can
develop from lesions that lack dysplastic
changes.31,32 On this basis we can explain, in some
measure, the proportion of cases in the C group
with expression-positive PTEN.
Recently, Chaves et al. studied p-Akt function in
oral epithelial dysplasia and in OSCC, observing that
p-Akt immunostaining was significantly higher in the
malignancy group than in dysplasias and controls.15
Other studies investigated p-Akt in the process of
malignant transformation of oral epithelium, suggest-
ing that activation of the PI3K-AKT signal pathway
is involved in the malignant phenotype acquisition
process from its early stages.6,7,33 In addition, Akt
expression was shown to be a prognostic determinant
for patients affected by OSCC.13,34 In this regard, in
our work the increase in PTEN expression observed
in the evolution of dysplastic lesions can be under-
stood as an attempt to control the increased expres-
sion of Akt described in several studies of oral
dysplastic lesions.6,7,15,33 In addition, a decrease in
PTEN expression was observed in OSCC. Reduced
PTEN protein expression in OSCC has also been
observed in previous studies,21,35,36 suggesting a
PTEN functional inactivation in this malignancy.
Here we also investigated the loss of PTEN copy
number in oral dysplastic lesions. Our results
showed that the loss of PTEN copy number in oral
leucoplakias can represent an important mechanism
for the development of oral potentially malignant
lesions into progression to oral cancer when the loss
of PTEN copy number occurred in 34.25% of our
sample (10 hemizygous deletion and 15 homozygous
deletion). As PTEN is a tumour suppressor gene, for
its complete inactivation there is a need for both
copies to be deleted. In the cases of OSCC in which
there was hemizygous deletion, evaluation of the
mutation or hypermethylation of the PTEN promoter
region in the remaining alleles may elucidate
whether the loss of PTEN expression in OSCC sam-
ples would be occurring through genetic or epige-
netic events. Some authors have already addressed
this issue, demonstrating that mutation in PTEN is
a rare event in HNSCC,36–41 whereas others have
shown that hypermethylation of the PTEN promoter
region is frequent in this tumour,36,42 which is an
important mechanism of epigenetic silencing that
may contribute to cancer.
Some papers highlight that homozygous deletion is
unlikely to play a key role in the PTEN inactivation
process in OSCC.43,44 However, in this study bi-allelic
loss occurred in 20.55% of the OSCC group. This
homozygous loss of PTEN alleles was statistically
higher in OSCC samples when compared with lesions
that presented in MD and the C group. An important
increase in loss of both alleles was observed in OSSC
when compared with MSD, although there was no
statistically significant difference.
In conclusion, taking into consideration the fact
that longitudinal studies are more indicated to attri-
bute predictive value for malignant transformation,
this cross-sectional study permits only speculation, to
some extent, that PTEN bi-allelic loss can be an
important mechanism in the late stage of develop-
ment of oral potentially malignant lesions into oral
cancer. Moreover, we observed an increase in the
expression of PTEN in the dysplastic lesions, suggest-
ing an attempt of PTEN to control the increased
expression of Akt. After acquisition of the malignant
phenotype, a decrease in PTEN expression was
observed, which may be explained by the increased
deletion in this group.
Conflicts of interest
The authors state that they have no potential con-
flicts of interest.
© 2017 John Wiley & Sons Ltd, Histopathology, 72, 330–338.

Acknowledgements
This work was supported by the Coordination for the
Improvement of Higher Education Personnel (CAPES/
PROEX).
Authors contribution
L. A. N. Miyahara, H. A. R. Pontes, F. P. Fonseca, R. M.
R. Burbano: study concepts. L. A. N. Miyahara, D. M.
Guimaraes, F. S. C. Pontes: data acquisition. F. S. C.
Pontes, D. M. Guimaraes: data analysis and interpreta-
tion. N. Conte Neto: statistical analysis. L. A. N. Miya-
hara, N. Conte Neto: capture of images. L. A. N.
Miyahara, H. A. R. Pontes, F. S. C. Pontes: manuscript
preparation. R. M. R. Burbano, F. P. Fonseca, D. M. Gui-
maraes: manuscript editing. L. A. N. Miyahara, H. A. R.
Pontes, F. S. C. Pontes, D. M. Guimaraes, F. P. Fonseca,
N. Conte Neto, R. M. R. Burbano: manuscript review.
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