Annals of Diagnostic Pathology 17 (2013) 421–424

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Annals of Diagnostic Pathology

Osteopontin: a marker for invasive oral squamous cell carcinoma but not for
potentially malignant epithelial dysplasias☆,☆☆
Samapika Routray MDS a,⁎, Supriya M. Kheur MDS b, Mohit Kheur MDS c
a
Department of Oral Pathology & Microbiology, Gitam Dental College & Hospital, Gandhinagar Campus, Rushikonda, Vishakapatanam 530045, India
b
Department of Oral Pathology & Microbiology, D.Y. Patil Vidyapeeth's Dr. D.Y. Patil Dental College and Hospital, Maheshnagar, Pimpri, Pune, Maharastra, India
c
Department of Prosthodontics, M.A. Rangoonwala Dental College Pune, Maharastra, India

a r t i c l e i n f o a b s t r a c t

Keywords: This study aimed to evaluate and correlate osteopontin (OPN) expression in oral squamous cell carcinoma
Osteopontin (OSCC) and potentially malignant disorders including oral leukoplakia and oral submucous fibrosis (OSMF).
Leukoplakia Expression of OPN was investigated in 140 samples including OSCC, oral leukoplakia, and OSMF with or
Oral submucous fibrosis without dysplasia and normal oral mucosa. By using immunohistochemistry. Both intercellular and
Oral squamous cell carcinoma intracellular staining of the keratinocytes was considered to be positive, and intensity grading was assessed.
Statistical analysis was done using analysis of variance. OPN positivity was detected in 85% cases of OSCC,
55% cases of oral leukoplakia, 35% cases of OSMF, and 60% cases of normal mucosa. These study highlights
OPN as a biomarker for malignancy in the form of invasion but not to study progression from dysplasia to
malignant transformation.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction
The incidence of head and neck cancer in the developing world
is on the rise, where it is known to be the third most common cause
of death among the spectrum of cancers [1,2]. In India alone, oral
cancers account for 9.4% of all cancers [3]. Oral squamous cell car-
cinoma (OSCC) is an archetype of cancer occurring when there is a
complex and multistep process. This change is sometimes preceded
by clinically visible lesions, known as “precancerous lesions” [4].
In OSCC, surgery is generally the treatment of choice. Given the
morbidity associated with oral surgery, there is a need to identify
simple markers that estimate the transition potential of oral precancer
to cancer [5]. For decades, numerous investigators have diligently
searched for markers to collaborate malignancy with premalignancy
[6]. One of the first markers to be carefully investigated was the
expression of cytokeratins, but unfortunately, they varied greatly
within dysplastic epithelium, as does the phenotypic expression
(morphology) reflecting genetic alterations [7]. Osteopontin (OPN), a
secreted plasma marker with an established role in tumor transfor-
☆ Key message: As a biomarker osteopontin has already established its efficacy in
cancers such as prostrate, breast, and others, particularly in its role in identifying
metastasis. In oral squamous cell carcinoma also, literature coincides with osteopontin's
role in identifying metastasis and mostly at invasive front, but its role in distinguishing
dysplasia to malignancy is still at a very questionable stage.
☆☆ Conflict of interest: None.
⁎ Corresponding author. Tel.: +91 7702144800.
E-mail addresses: drroutray.samapika@gmail.com (S. Routray),
drskheur@gmail.com (S.M. Kheur).
mation and invasion in head and neck cancer, is being considered as
one of the most promising biomarker for various cancers in the recent
times [8].
Osteopontin, a major sialoprotein of the extracellular matrix, binds
calcium and functions in early-stage mineralization in bone and
dentin. It is an early component of type-1 immunity in activated T
lymphocytes and macrophages, mediating angiogenesis and inhibit-
ing apoptosis in soft tissues. Osteopontin is involved in cell adhesion
and cell migration, as it has the Arg-Gly-Asp (RGD) sequence that
binds certain integrins, including integrin αvβ1, integrin αvβ3, and
integrin αvβ5 and influences cell migration via interaction with CD44
and activation of the epidermal growth factor pathway [9].
The role of OPN in cancer biology has been the major focus of
research for last decade [10]. Although the expression of OPN has
already been studied in OSCC, only a few cases have been inves-
tigated in OSCC cell lines [11] and OSCC of tongue [12]. No studies
have yet analyzed or investigated the relationship between OPN
expression and premalignant lesions and conditions, using immuno-
histochemistry. In the present study, an effort has been made to
standardize the findings of OPN expression in normal tissues and
study its expression in premalignant and malignant lesions in a group
of Indian patients.
2. Materials and methods
One hundred twenty cases of hematoxylin and eosin–stained
slides and their matched formalin-fixed, paraffin-embedded tissue
blocks were retrieved from the surgical pathology archives of Dr D.Y.
Patil Dental College and Hospital, Pune.

1092-9134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.anndiagpath.2013.03.005
422 S. Routray et al. / Annals of Diagnostic Pathology 17 (2013) 421–424

The cases were chosen randomly as follows: Table 1

Biopsy specimens from each group of patient were analyzed for OPN immunolocaliza-
1. Forty cases of leukoplakia showing mild to severe dysplasia, tion according to their histopathologic status and the percentage of OPN-positive cases
2. Forty cases of oral submucous fibrosis (OSMF), and determined
3. Forty cases of OSCC. Study Histologic No. of No. of Total positive % of positive
group grading males females cases cases
The cases included in the control group were of normal epithe-
lium (NE), taken from healthy volunteers. It was obtained during Leukoplakia Mild dysplasia 20 0 14 70
Moderate dysplasia 8 0 0 –
routine dental procedures. The procedural details were explained
Severe dysplasia 8 8 8 80
to the patient, and informed consent was obtained from all the Carcinoma in situ 2 0 0 –
patients. The entire study protocol was approved by the institutional OSMF Atrophied epithelium 34 6 14 35
ethics committee. OSCC Well differentiated 6 8 14 100
All the hematoxylin and eosin–stained slides were reexamined by Moderately 8 10 16 72
differentiated
2 pathologists independently, who confirmed the given diagnosis.
Poorly differentiated 2 0 2 100
Early invasive 4 2 2 33.33
2.1. Immunohistochemistry Normal 4 16 12 60

Blocks of formalin-fixed, paraffin-embedded tissue were cut at

5 μm. Tissues were mounted on positively charged poly-L-lysine–
coated slides. The sections were deparaffinized with xylene and
rehydrated in changes of absolute alcohol for 15 and 1 minutes,
respectively. Antigen retrieval was carried out in Decloacking
chamber (Biocare, USA) with 10-mM Citra solution at 125°C for 30
seconds followed by 90°C for 10 seconds. After the run, the slides were
taken out and allowed to cool down at room temperature.
2.2. For immunostaining
The slides were transferred to i6000 Biogenex (Fremont, CA, USA)
automated immunohistochemistry staining system and washed with
phosphate buffered saline (PBS) solution. Endogenous peroxidase
activity was quenched by immersion of slides in menthol with 3%
hydrogen peroxide for 10 minutes. The sections were then covered
with appropriate volume of Power Block solution (Biogenex, USA), for
universal protein blocking. Then, sections were treated with biotiny-
lated diluted primary antibody (mouse monoclonal antibody OP3N,
Human Osteopontin, 1:100 dilution; Novocastra, UK). After a gentle
wash with PBS, the slides were then covered with appropriate volume
of poly-horseradishperoxidase reagent solution and incubated for 30
minutes at room temperature. After another wash with PBS, the
sections were then covered with appropriate volume of Substrate
Chromogen solution in Tris-HCl with hydrogen peroxide. The sections
were incubated for 10 minutes and rinsed well. Sections were
counterstained in Harris's hematoxylin for 1 minute, dehydrated,
cleared in xylene, and then mounted in dibutyl phthalate xylene. For
negative control, PBS solution was used instead of primary antibody.
2.3. Assessment of staining intensity
The intensity of staining of the epithelium was assessed as nega-
tive (0), mild (1), moderate (2), and intense (3) cytoplasmic staining
with reference to the method used by Grizzle et al [13].
2.4. Statistical analysis
The frequency of OPN-positive cases were compared using analysis
of variance on ranks. In the study group, the percentage of positive
staining was compared using t test.
3. Results
ranged from 22 to 60 years. Ten of them were severely dysplastic,
8 showed moderate dysplasia, and 20 cases showed mild dysplasia.
Two of these cases were diagnosed as carcinoma in situ.
3.2. Immunostaining
Each slide was evaluated by 2 different oral pathologists to
eliminate any interobserver bias. For OPN expression quantification,
the numbers of positive keratinocytes were counted under high
scanner view. Cases were assessed along with its histopathologic
grading, and accordingly, its positivity is enumerated in Table 1. The
frequency of grading and intensity of OPN expression according to
each group are summarized in Table 2.
3.2.1. OPN as marker of dysplasia
A comparative analysis of various grades of dysplasia in leukopla-
kia showed no consistency of OPN positivity. Results showed 55% of
cases positive for OPN in mild dysplasia cases. There was no OPN
staining in moderately dysplastic cases, whereas severely dysplastic
leukoplakia showed an 80% positivity for OPN. In case of OSMF where
the epithelium is atrophic with minimal dysplastic changes, 35% of
cases were positive for OPN staining. There was no statistically
significant difference between OPN staining in NE, leukoplakia, and
OSMF cases. Thus, we concluded that OPN staining was not consistent
with dysplasia seen in leukoplakia and OSMF cases.
3.2.2. OPN as a marker of OSCC
All OSCC patient slides were positive for OPN in varying intensity.
The invasive tumor front in all cases showed an increase in intensity of
staining with OPN as compared with the rest of the tumor tissue.
Intensity grading increases significantly with loss in degree of dif-
ferentiation of tumor cells. Thus, we found that there is a statistically
significant difference between NE (0.6 ± 0.51), leukoplakia (0.7 ±
0.73), and OSMF (0.45 ± 0.68) with OSCC (1.45 ± 1.05).
4. Discussion
As the current trend prevails, tumor markers are receiving more
attention in early detection as well as predicting prognosis of the
Table 2
Total score and percent positivity with various grades of intensity distribution observed
in leukoplakia, OSMF, OSCC, and normal cases

Study Grade I Intensity Total Negative Positive Total percent

3.1. Clinical findings group score cases cases positivity
Grade II Grade III
In the 40 previously diagnosed OSCC cases, the age of the patient Leukoplakia 16 6 – 28 18 22 55
ranged from 25 to 68 years. Twenty cases were diagnosed as well- OSMF 10 4 – 14 26 14 35
OSCC 14 6 8 62 6 34 85
differentiated OSCC; 18, as moderately differentiated; and 2 cases, as
Normal 12 – – 12 8 12 60
poorly differentiated OSCC. Regarding the leukoplakia cases, the age
 S. Routray et al. / Annals of Diagnostic Pathology 17 (2013) 421–424
Fig. 1. Photomicrograph of OSCC section with epithelial cell islands showing grade 2
intensity under low-power view.
lesion [14]. Expression of OPN, an extracellular matrix marker, has
been examined in relation to the carcinogenesis and metastasis of
various malignant tumors such as the lung, esophageal, breast, and
salivary gland, and the results have suggested that OPN is a candidate
biomarker of malignant tumors [12].
In the present study, our main objective was to evaluate the role
of OPN in progression as well as determining prognosis of oral
cancer and precancerous lesions, which are mainly tobacco and
areca nut induced in the Southern Asian countries. Semiquantitative
evaluation of immunopositive reaction of OPN and scoring for each
case revealed that OPN expression was significantly higher in car-
cinoma cells than in normal. The intensity of staining varied in the
islands and nests of epithelial cells in each case (Fig. 1) and espe-
cially in areas showing keratin pearls (Fig. 2). Osteopontin showed a
definite increased expression at the early invasion front than the
neighboring epithelial dysplasia (Fig. 3). In areas where the base-
ment membrane of squamous epithelium was unclear, OPN expres-
sion increased. As there was significant difference between OSCC
and NE (P b .05, significant), it correlated with increase in OPN
expression with malignant transformation. These results were con-
sistent with the study by Muramatsu et al [15], showing that
Fig. 2. Photomicrograph of OSCC section with keratin pearl formation showing grade
2 intensity and a high inflammatory component adjacent to the area under low-
power view.
423
Fig. 3. Photomicrograph of OSCC section showing invasive front with grade 3 intensity
under high-power view.
proliferation and invasion were reduced by inhibition of OPN in
BSC-OF (derived from oral basaloid SCC) cells. In the study by
Matsuzaki et al [12], they showed scores that were significantly
higher in carcinoma nests than in neighboring normal epithelium or
epithelial dysplasia. In cases of early invasive carcinoma, in
particular, expression of OPN showed a remarkable increase at the
invasion front compared with the noninvaded regions. So results
made it evident that OPN is more an early invasion marker than a
prognostic marker in OSCC. As suggested by Zhu et al [16], under
hypoxia, OPN expression is up-regulated via a Ras-activated
enhancer so OPN expression is significantly and consistently high
solid carcinomas. In our study, OSCC perfectly complies with the
high expression of OPN.
In the present study, for NE cases, 12 (60%) were mildly positive,
and 8 (40%) were negative for OPN expression. The results were not
consistent with other studies with NE specimens using the same
Fig. 4. Photomicrograph of oral leukoplakia section showing dysplastic epithelium with
grade 1 intensity under high-power view.
424 S. Routray et al. / Annals of Diagnostic Pathology 17 (2013) 421–424
Osteopontin values in all the groups
1.6
1.4
Mean values
1.2
1 5. Conclusion
0.8
0.6
0.4
0.2
0 premalignant cases did show positive intensity, however, OPN
OSCC LEUKOPLAKIA OSMF Control
Graph 1. Comparision of OPN intensity between leukoplakia, OSMF, OSCC, and normal
(control) groups.
monoclonal antibody [11,12], but the strong intensity in our positive
control guaranteed the accuracy of our method. In the study done by
Devoll et al [11], OPN expression was not detected in epithelium
obtained from normal oral mucosa or gingiva. Matsuzaki et al [12]
found OPN expression to be negative or very weak in the basal cell
layer of normal epithelium. Conversely, Coppola et al [17] saw high
OPN staining but identified in only a few cases of the corresponding
normal tissues (7/113).
In leukoplakia samples, mild dysplasia cases showed 70% posi-
tivity, but moderate dysplasia and carcinoma in situ cases did not
show any positivity, whereas severe dysplasia cases again showed
80% positivity (Fig. 4). When correlated with OSCC intensity grading,
the premalignant lesion and condition showed significant difference.
The sections of both leukoplakia and OSMF cases did show highly
positive staining for muscle tissues. In the study, we also correlated
the OPN intensity of premalignancy with NE samples. The average
total score for OPN expression in the leukoplakia cases was 0.7 ± 0.73,
whereas in NE (control), it was 0.6 ± 0.51. There was no significant
difference found between the groups(P N .05, nonsignificant). The
average total score for OPN expression in the OSMF cases was 0.45 ±
0.686, whereas in NE (control), it was 0.6 ± 0.51. There was no
significant difference found between the groups(P N .05, nonsignifi-
cant). The entire comparison with the mean values are represented
in graphical form (Graph 1).
In the study done by Devoll et al [11], OPN was detected in a
significant percentage of premalignant and malignant oral lesions but
not normal oral epithelium. Localization in a high percentage of
hyperplasias (75%) suggested that OPN expression may be associated
with benign changes not directly related to malignancy. Fifty-two
percent of dysplasias and 54% of carcinomas in situ showed OPN
expression representing an early event in tumor progression. Our
results did show positivity in leukoplakia and OSMF cases but not in
accordance to the intensity assessed in OSCC cases. The premalignant
study groups when compared with the normal cases also did not
show any significant difference suggestive of nearly same intensity
grading (85% +OSCC, 55% +leukoplakia, 35% +OSMF, 60% +normal
epithelium). The same result for OPN was also stated by Ogbureke
et al [18] in their study using dysplastic tissue samples.
As suggested in earlier studies, secretion of OPN into the local
tumor microenvironment promotes tumor cell adhesion and migra-
tion by binding cell surface receptors, such as the αvβ3 integrin and
CD44 receptor. Through its capacity to bind several extracellular
matrix proteins such as fibronectin; osteocalcin; and collagen types I,
II, III, IV, and V, OPN may modulate certain cell–extracellular matrix
interactions required for tumor cell infiltration and metastasis.
Osteopontin may also impact tumor cell phenotype by activating
intracellular signaling pathways involving phospholipase C and tyro-
sine kinase through autocrine/paracrine mechanisms, but as all these
activities are not relevant in the epithelium, probably reasoning the
mild OPN expression in premalignant lesion and condition. Because
of less positivity observed, we concluded that its use as a biomarker
for malignant transformation is very limited and has no role to study
progression from dysplasia to malignant transformation.
Osteopontin expression was observed in 55% positive Leukoplakia
cases, whereas in the study, only 35% positive OPN expression was
observed in OSMF cases with various intensity grading. Although
intensity in premalignancy was not in accordance with the histologic
grading of the same cases. Intensity grading assessed was same as that
seen in the normal epithelium cases. So we would like to conclude
that although our results indicate that OPN expression cannot be used
as a relevant diagnostic and prognostic marker for oral mucosal
dysplasia and malignant transformation, OPN certainly has a role as a
prognostic marker in OSCC.
Acknowledgment
I am thankful to Dr Sanjay Navani for availing me the technical
support in his establishment, Lab surg path, needed to carry out the
thesis work. Dr NK Paniker (HOD, Department of Pathology), Dr Alka
Kale (Dean, K.L.E.I.D.S Belgaum), and Dr Meena Kulkarni for your
unwavering support and intellectual guidance and Mrs SS Garad
(biostatistician) for the analysis of the results.
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