Annals of Diagnostic Pathology 17 (2013) 421–424

Contents lists available at ScienceDirect

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 mation and invasion in head and neck cancer, is being considered as
one of the most promising biomarker for various cancers in the recent
The incidence of head and neck cancer in the developing world times [8].
is on the rise, where it is known to be the third most common cause Osteopontin, a major sialoprotein of the extracellular matrix, binds
of death among the spectrum of cancers [1,2]. In India alone, oral calcium and functions in early-stage mineralization in bone and
cancers account for 9.4% of all cancers [3]. Oral squamous cell car- dentin. It is an early component of type-1 immunity in activated T
cinoma (OSCC) is an archetype of cancer occurring when there is a lymphocytes and macrophages, mediating angiogenesis and inhibit-
complex and multistep process. This change is sometimes preceded ing apoptosis in soft tissues. Osteopontin is involved in cell adhesion
by clinically visible lesions, known as “precancerous lesions” [4]. and cell migration, as it has the Arg-Gly-Asp (RGD) sequence that
In OSCC, surgery is generally the treatment of choice. Given the binds certain integrins, including integrin αvβ1, integrin αvβ3, and
morbidity associated with oral surgery, there is a need to identify integrin αvβ5 and influences cell migration via interaction with CD44
simple markers that estimate the transition potential of oral precancer and activation of the epidermal growth factor pathway [9].
to cancer [5]. For decades, numerous investigators have diligently The role of OPN in cancer biology has been the major focus of
searched for markers to collaborate malignancy with premalignancy research for last decade [10]. Although the expression of OPN has
[6]. One of the first markers to be carefully investigated was the already been studied in OSCC, only a few cases have been inves-
expression of cytokeratins, but unfortunately, they varied greatly tigated in OSCC cell lines [11] and OSCC of tongue [12]. No studies
within dysplastic epithelium, as does the phenotypic expression have yet analyzed or investigated the relationship between OPN
(morphology) reflecting genetic alterations [7]. Osteopontin (OPN), a expression and premalignant lesions and conditions, using immuno-
secreted plasma marker with an established role in tumor transfor- 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.
☆ 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 2. Materials and methods
role in identifying metastasis and mostly at invasive front, but its role in distinguishing
dysplasia to malignancy is still at a very questionable stage. One hundred twenty cases of hematoxylin and eosin–stained
☆☆ Conflict of interest: None.
⁎ Corresponding author. Tel.: +91 7702144800.
slides and their matched formalin-fixed, paraffin-embedded tissue
E-mail addresses: drroutray.samapika@gmail.com (S. Routray), blocks were retrieved from the surgical pathology archives of Dr D.Y.
drskheur@gmail.com (S.M. Kheur). 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– ranged from 22 to 60 years. Ten of them were severely dysplastic,
coated slides. The sections were deparaffinized with xylene and 8 showed moderate dysplasia, and 20 cases showed mild dysplasia.
rehydrated in changes of absolute alcohol for 15 and 1 minutes, Two of these cases were diagnosed as carcinoma in situ.
respectively. Antigen retrieval was carried out in Decloacking
chamber (Biocare, USA) with 10-mM Citra solution at 125°C for 30 3.2. Immunostaining
seconds followed by 90°C for 10 seconds. After the run, the slides were
taken out and allowed to cool down at room temperature. Each slide was evaluated by 2 different oral pathologists to
eliminate any interobserver bias. For OPN expression quantification,
2.2. For immunostaining the numbers of positive keratinocytes were counted under high
scanner view. Cases were assessed along with its histopathologic
The slides were transferred to i6000 Biogenex (Fremont, CA, USA) grading, and accordingly, its positivity is enumerated in Table 1. The
automated immunohistochemistry staining system and washed with frequency of grading and intensity of OPN expression according to
phosphate buffered saline (PBS) solution. Endogenous peroxidase each group are summarized in Table 2.
activity was quenched by immersion of slides in menthol with 3%
3.2.1. OPN as marker of dysplasia
hydrogen peroxide for 10 minutes. The sections were then covered
A comparative analysis of various grades of dysplasia in leukopla-
with appropriate volume of Power Block solution (Biogenex, USA), for
kia showed no consistency of OPN positivity. Results showed 55% of
universal protein blocking. Then, sections were treated with biotiny-
cases positive for OPN in mild dysplasia cases. There was no OPN
lated diluted primary antibody (mouse monoclonal antibody OP3N,
staining in moderately dysplastic cases, whereas severely dysplastic
Human Osteopontin, 1:100 dilution; Novocastra, UK). After a gentle
leukoplakia showed an 80% positivity for OPN. In case of OSMF where
wash with PBS, the slides were then covered with appropriate volume
the epithelium is atrophic with minimal dysplastic changes, 35% of
of poly-horseradishperoxidase reagent solution and incubated for 30
cases were positive for OPN staining. There was no statistically
minutes at room temperature. After another wash with PBS, the
significant difference between OPN staining in NE, leukoplakia, and
sections were then covered with appropriate volume of Substrate
OSMF cases. Thus, we concluded that OPN staining was not consistent
Chromogen solution in Tris-HCl with hydrogen peroxide. The sections
with dysplasia seen in leukoplakia and OSMF cases.
were incubated for 10 minutes and rinsed well. Sections were
counterstained in Harris's hematoxylin for 1 minute, dehydrated, 3.2.2. OPN as a marker of OSCC
cleared in xylene, and then mounted in dibutyl phthalate xylene. For All OSCC patient slides were positive for OPN in varying intensity.
negative control, PBS solution was used instead of primary antibody. 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.
2.3. Assessment of staining intensity Intensity grading increases significantly with loss in degree of dif-
ferentiation of tumor cells. Thus, we found that there is a statistically
The intensity of staining of the epithelium was assessed as nega- significant difference between NE (0.6 ± 0.51), leukoplakia (0.7 ±
tive (0), mild (1), moderate (2), and intense (3) cytoplasmic staining 0.73), and OSMF (0.45 ± 0.68) with OSCC (1.45 ± 1.05).
with reference to the method used by Grizzle et al [13].
4. Discussion
2.4. Statistical analysis
As the current trend prevails, tumor markers are receiving more
The frequency of OPN-positive cases were compared using analysis
attention in early detection as well as predicting prognosis of the
of variance on ranks. In the study group, the percentage of positive
staining was compared using t test.
Table 2
Total score and percent positivity with various grades of intensity distribution observed
3. Results 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) 421–424 423

Fig. 3. Photomicrograph of OSCC section showing invasive front with grade 3 intensity
under high-power view.
Fig. 1. Photomicrograph of OSCC section with epithelial cell islands showing grade 2
intensity under low-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
lesion [14]. Expression of OPN, an extracellular matrix marker, has higher in carcinoma nests than in neighboring normal epithelium or
been examined in relation to the carcinogenesis and metastasis of epithelial dysplasia. In cases of early invasive carcinoma, in
various malignant tumors such as the lung, esophageal, breast, and particular, expression of OPN showed a remarkable increase at the
salivary gland, and the results have suggested that OPN is a candidate invasion front compared with the noninvaded regions. So results
biomarker of malignant tumors [12]. made it evident that OPN is more an early invasion marker than a
In the present study, our main objective was to evaluate the role prognostic marker in OSCC. As suggested by Zhu et al [16], under
of OPN in progression as well as determining prognosis of oral hypoxia, OPN expression is up-regulated via a Ras-activated
cancer and precancerous lesions, which are mainly tobacco and enhancer so OPN expression is significantly and consistently high
areca nut induced in the Southern Asian countries. Semiquantitative solid carcinomas. In our study, OSCC perfectly complies with the
evaluation of immunopositive reaction of OPN and scoring for each high expression of OPN.
case revealed that OPN expression was significantly higher in car- In the present study, for NE cases, 12 (60%) were mildly positive,
cinoma cells than in normal. The intensity of staining varied in the and 8 (40%) were negative for OPN expression. The results were not
islands and nests of epithelial cells in each case (Fig. 1) and espe- consistent with other studies with NE specimens using the same
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- Fig. 4. Photomicrograph of oral leukoplakia section showing dysplastic epithelium with
power view. 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 mild OPN expression in premalignant lesion and condition. Because
of less positivity observed, we concluded that its use as a biomarker
1.6 for malignant transformation is very limited and has no role to study
1.4 progression from dysplasia to malignant transformation.
Mean values

1.2
1 5. Conclusion
0.8
0.6 Osteopontin expression was observed in 55% positive Leukoplakia
0.4 cases, whereas in the study, only 35% positive OPN expression was
0.2 observed in OSMF cases with various intensity grading. Although
0 premalignant cases did show positive intensity, however, OPN
OSCC LEUKOPLAKIA OSMF Control
intensity in premalignancy was not in accordance with the histologic
Graph 1. Comparision of OPN intensity between leukoplakia, OSMF, OSCC, and normal
grading of the same cases. Intensity grading assessed was same as that
(control) groups. 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
monoclonal antibody [11,12], but the strong intensity in our positive dysplasia and malignant transformation, OPN certainly has a role as a
control guaranteed the accuracy of our method. In the study done by prognostic marker in OSCC.
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 Acknowledgment
layer of normal epithelium. Conversely, Coppola et al [17] saw high
OPN staining but identified in only a few cases of the corresponding I am thankful to Dr Sanjay Navani for availing me the technical
normal tissues (7/113). support in his establishment, Lab surg path, needed to carry out the
In leukoplakia samples, mild dysplasia cases showed 70% posi- thesis work. Dr NK Paniker (HOD, Department of Pathology), Dr Alka
tivity, but moderate dysplasia and carcinoma in situ cases did not Kale (Dean, K.L.E.I.D.S Belgaum), and Dr Meena Kulkarni for your
show any positivity, whereas severe dysplasia cases again showed unwavering support and intellectual guidance and Mrs SS Garad
80% positivity (Fig. 4). When correlated with OSCC intensity grading, (biostatistician) for the analysis of the results.
the premalignant lesion and condition showed significant difference.
The sections of both leukoplakia and OSMF cases did show highly References
positive staining for muscle tissues. In the study, we also correlated
[1] Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of eighteen
the OPN intensity of premalignancy with NE samples. The average major cancers in 1985. Int J Cancer 1993;54:594–606.
total score for OPN expression in the leukoplakia cases was 0.7 ± 0.73, [2] Muir CS, Nectoux J. International patterns of cancer. In: Schottenfeld D, Fraumeni
whereas in NE (control), it was 0.6 ± 0.51. There was no significant JF, editors. Cancer epidemiology and prevention. 2nd ed. New York: Oxford
University Press; 1996. p. 141–67.
difference found between the groups(P N .05, nonsignificant). The [3] Bhattacharjee A, Chakraborty A, Purkaystha P. Prevalence of head and neck
average total score for OPN expression in the OSMF cases was 0.45 ± cancers in the North East—an institutional study. Indian J Otolaryngol Head Neck
0.686, whereas in NE (control), it was 0.6 ± 0.51. There was no Surg 2006;58:15–9.
[4] Kuffer R, Lombardi T. Premalignant lesions of the oral mucosa. A discussion about
significant difference found between the groups(P N .05, nonsignifi- the place of oral intraepithelial neoplasia (OIN). Oral Oncol 2002;38:125–30.
cant). The entire comparison with the mean values are represented [5] Scully C, Burkhardt A. Tissue markers of potentially malignant human oral
in graphical form (Graph 1). epithelial lesions. J Oral Pathol Med 1993;22:246–56.
[6] Lee JS, Lippman SM, Hong WK, et al. Determination of biomarkers for intermediate
In the study done by Devoll et al [11], OPN was detected in a end points in chemoprevention trials. Cancer Res 1992;52:2707–10.
significant percentage of premalignant and malignant oral lesions but [7] Lindberg K, Rheinwald JG. Suprabasal 40kd keratin (k19) expression as an immu-
not normal oral epithelium. Localization in a high percentage of nohistologic marker of premalignancy in oral epithelium. Am J Pathol 1989;134:
89–98.
hyperplasias (75%) suggested that OPN expression may be associated
[8] Jain S, Chakraborty G, Bulbule A, et al. Osteopontin: an emerging therapeutic
with benign changes not directly related to malignancy. Fifty-two target for anticancer therapy. Expert Opin Ther Targets 2007;11:81–90.
percent of dysplasias and 54% of carcinomas in situ showed OPN [9] Sodek J, Ganss B, McKee MD. Osteopontin. Crit Rev Oral Biol Med 2000;11:
expression representing an early event in tumor progression. Our 279–303.
[10] Chakraborty G, Jain S, Behera R, et al. The multifaceted roles of osteopontin in cell
results did show positivity in leukoplakia and OSMF cases but not in signaling, tumor progression and angiogenesis. Curr Mol Med 2006;6:819–30.
accordance to the intensity assessed in OSCC cases. The premalignant [11] Devoll RE, Li W, Woods KV, et al. Osteopontin (OPN) distribution in premalignant
study groups when compared with the normal cases also did not and malignant lesions of oral epithelium and expression in cell lines derived from
squamous cell carcinoma of the oral cavity. J Oral Pathol Med 1999;28:97–101.
show any significant difference suggestive of nearly same intensity [12] Matsuzaki H, Shima K, Muramatsu T, et al. Osteopontin as a biomarker in early
grading (85% +OSCC, 55% +leukoplakia, 35% +OSMF, 60% +normal invasion by squamous cell carcinoma in tongue. J Oral Pathol Med 2007;36:30–4.
epithelium). The same result for OPN was also stated by Ogbureke [13] Grizzle WE, Myers RB, Manne U, Srivastava S. Immunohistochemical evaluation of
biomarkers in prostatic and colorectal neoplasia. In: Hanausek M, Walaszek Z,
et al [18] in their study using dysplastic tissue samples. editors. Methods in molecular medicine, Vol. 14, Tumor marker protocols. Totowa,
As suggested in earlier studies, secretion of OPN into the local NJ: Humana Press Inc; 1997. p. 143–60.
tumor microenvironment promotes tumor cell adhesion and migra- [14] Williams DM, Daniels TE. The present and potential role of cell markers in oral
diagnosis. J Oral Pathol 1987;16:186–8.
tion by binding cell surface receptors, such as the αvβ3 integrin and [15] Muramatsu T, Shima K, Ohta K, et al. Inhibition of osteopontin expression and
CD44 receptor. Through its capacity to bind several extracellular function in oral cancer cell lines by antisense oligonucleotides. Cancer Lett 2005;
matrix proteins such as fibronectin; osteocalcin; and collagen types I, 217:87–95.
[16] Zhu Y, Denhardt DT, Cao H, et al. Hypoxia upregulates osteopontin expression in
II, III, IV, and V, OPN may modulate certain cell–extracellular matrix
NIH-3T3 cells via a Ras activated enhancer. Oncogene 2005;24:6555–63.
interactions required for tumor cell infiltration and metastasis. [17] Coppola D, Szabo M, Boulware D, et al. Correlation of osteopontin protein
Osteopontin may also impact tumor cell phenotype by activating expression and pathologic stage across a wide variety of tumor histologies. Clin
intracellular signaling pathways involving phospholipase C and tyro- Cancer Res 2004;10:184–90.
[18] Ogbureke KUE, Abdelsayed RA, Kushner H, et al. Two members of the SIBLING
sine kinase through autocrine/paracrine mechanisms, but as all these family of proteins, DSPP and BSP, may predict the transition of oral epithelial
activities are not relevant in the epithelium, probably reasoning the dysplasia to oral squamous cell carcinoma. Cancer 2010;4:1710–7.