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Table of Contents   
ORIGINAL RESEARCH  
Year : 2019  |  Volume : 30  |  Issue : 4  |  Page : 539-543
Immunohistochemical analysis of osteopontin expression in oral squamous cell carcinoma


1 Department of Oral Pathology, Tamil Nadu Government Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Oral Pathology, Government Dental College, Thrissur, Calicut, Kerala, India
3 Department of Dentistry, Faculty of Dental Sciences, Institute of Medical Sciences, BHU, Varanasi, Uttar Pradesh, India
4 Private Practitioner, Mysore, Karnataka, India

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Date of Submission28-Aug-2017
Date of Decision16-Apr-2018
Date of Acceptance25-Jun-2018
Date of Web Publication18-Nov-2019
 

   Abstract 

Background: Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity. Osteopontin (OPN) has been proved as a biomarker in varying malignant tumors. Only limited studies detail the role of OPN in OSCC. Aims: This study aims to demonstrate the expression of OPN in OSCC and to correlate the expression of OPN with the histologic grades of OSCC. Settings and Design: This is a retrospective immunohistochemical study in Dravidian population (linguistically Malayalam). Materials and Methods: Thirty diagnosed cases of OSCC were subjected to immunohistochemistry using OPN antibody for detection of OPN expression. Ten normal oral mucosal specimens were also stained as controls. Statistical Analysis Used: Chi-square test and ANOVA followed by Bonferroni test. Results: OPN expression was significantly higher in OSCC patients than in controls. In normal oral mucosal specimens, none of them showed OPN immunoreactivity. A significant difference was observed between total scores and intensities of normal and varying grades of OSCC. A significant difference was also observed between the percentage of positive cells for OPN expression of normal and varying grades of OSCC. However, no significant difference was observed between the percentage of positive cells for OPN expression of well-, moderate-, poorly-differentiated carcinomas. Correlation of OPN expression with lymph node status, site, and sex was found to be statistically insignificant. Conclusion: Insights gained from this study may lead to research targeted at the treatment of OSCC.

Keywords: Moderately differentiated squamous-cell carcinoma, oral squamous cell carcinoma, osteopontin, poorly differentiated squamous-cell carcinoma, well-differentiated squamous cell carcinoma

How to cite this article:
Jeyasivanesan DL, Mohamed SP, Pandiar D, Basheer S. Immunohistochemical analysis of osteopontin expression in oral squamous cell carcinoma. Indian J Dent Res 2019;30:539-43

How to cite this URL:
Jeyasivanesan DL, Mohamed SP, Pandiar D, Basheer S. Immunohistochemical analysis of osteopontin expression in oral squamous cell carcinoma. Indian J Dent Res [serial online] 2019 [cited 2019 Dec 9];30:539-43. Available from: http://www.ijdr.in/text.asp?2019/30/4/539/271063

   Introduction Top


About 92.8% of all oral malignancies are squamous cell carcinoma (SCC).[1] In India, oral SCC (OSCC) is the most common malignancy (40%–50% of all malignancies).[2] Osteopontin (OPN) has been proved as a candidate biomarker in various other malignant tumors.[3]

OPN was first described by Senger as a phosphoprotein secreted by transformed, malignant epithelial cells.[4] It interacts with αvb3 integrin, and this interaction is associated with various events involved in tumorigenesis.[5] OPN expression was found to be further related to prognosis or survival.[6] Only limited numbers of studies detail its role in OSCC.[7],[8],[9]


   Materials and Methods Top


The study sample included 30 formalin fixed paraffin embedded tissue blocks retrieved from the archives of the Department of Oral Pathology, Government Dental College, Calicut. Clinical parameters such as age, sex, site, tumor node metastasis (TNM) stage of all the cases were obtained from the archival records. Ten biopsy specimens of oral mucosa of healthy individuals undergoing impacted third molar removal or preprosthetic surgery were taken. All the healthy controls signed consent forms; approval of the Institutional Ethical Committee for the use of human subjects was obtained. All the healthy individuals were nonsmokers and demonstrated normal mucosa at the site of specimen removal. Of the 30 SCC cases, ten were well differentiated, ten were moderately differentiated and ten were poorly differentiated SCC. All the 40 blocks were considered for immunohistochemical staining with OPN. A similar immunohistochemical procedure was performed as described elsewhere.[10] Five μm thick sections were cut from all the blocks for immunostaining. The OPN immunohistochemical kit consisted of mouse monoclonal Anti-OPN as the primary antibody (Leica Microsystems; Novocastra) and the Super Sensitive Polymer-HRP/DAB system as the secondary antibody and detection kit (BioGenex Laboratories, USA).

Scoring and statistical analysis

Immunolocalization of OPN was examined in normal oral mucosal specimens, different histological grades of OSCCs. The results were assessed in a blinded fashion by two pathologists. Three high power fields within the tumor were selected, and expression of OPN in 100 cells/field was evaluated. The expression of OPN was semiquantitatively assessed as staining intensity compared with that of positive control (a) and percentage of positive cells with respect to the total number of epithelial cells (b). We established a score corresponding to the sum of a + b with maximum score being 6.

Staining intensity-(a):

  • 0 = Negative
  • 1 = Weak
  • 2 = Intermediate
  • 3 = Strong (when compared with that of positive control).


Percentage of positive cells-(b):

  • 0 = 0% positive cells
  • 1 ≤ 25% positive cells
  • 2 = 26%–50% positive cells
  • 3 ≥ 50% positive cells.



   Results Top


OPN expression was detected in perinuclear and other cytoplasm regions of the cancer cells. Positive staining was also detected in small numbers of muscle bundles, a few endothelial cells, and fibrinous exudates. In normal oral mucosal specimens (n = 10), 0% showed OPN immunoreactivity. In well-differentiated SCC, out of ten cases, six were positive (60%) (i.e., six cases had a total score above 3). In moderately differentiated carcinomas, 90% were OPN positive and in poorly differentiated SCCs 90% were OPN positive. The OPN expression (mean total score) in OSCC patients ranged from 4 to 5.3. In contrast, the OPN expression in controls was 0.0 (mean total score). Thus, OPN expression was significantly higher in OSCC patients than in control.

Total score

The mean value for OPN total score was 0 in the normal mucosal specimens. The mean values for well-, moderate-, poorly-differentiated carcinomas were 4, 4.6, and 5.3, respectively [Table 1]. A significant difference was observed between total scores of normal and varying grades of OSCC.
Table 1: Mean total score of osteopontin in varying grades of oral squamous cell carcinoma

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Intensity

The mean value for OPN intensity was 0 in the normal mucosal specimens. The mean values for well-, moderate-, and poorly-differentiated carcinomas were 1.5, 2, and 2.5, respectively [Table 2]. A significant difference was observed between intensities of normal and varying grades of OSCC.
Table 2: Mean intensity of osteopontin in varying grades of oral squamous cell carcinoma

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Percentage of positive cells

The mean value of the percentage of positive cells for OPN expression was 0 in the normal mucosal specimens. The mean values for well-, moderate-, and poorly-differentiated carcinomas were 2.5, 2.6, and 2.8, respectively [Table 3]. A significant difference was observed between the percentage of positive cells for OPN expression of normal and varying grades of OSCC. However, no significant difference was observed between the percentage of positive cells for OPN expression of well-, moderate-, poorly-differentiated carcinomas.
Table 3: Mean percentage of positive cells in varying grades of oral squamous cell carcinoma

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Nodal status

To assess whether the OPN expression correlated with nodal status, we compared OSCC cases with the presence or absence of palpable lymph nodes. Sixteen cases were positive for lymph nodes and 14 cases were negative for lymph nodes. OPN expression was high (mean total score 4.92) in cases positive for lymph node when compared to cases negative for lymph node (mean total score 4.37); however, the difference was not statistically significant [Table 4].
Table 4: Correlation between osteopontin expression and lymph node involvement

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Tumor node metastasis staging

To assess whether the OPN expression is associated with the disease stage, we compared OSCC cases after stratification based on the TNM status. Regarding the tumor stage, there were seven patients with Stage I, three with Stage II, and 20 with Stage III. Analysis of the OPN expression (total score) according to the TNM status revealed that the Stage III (mean total score – 4.75) showed a higher level of OPN expression than Stage II (mean – 4) and Stage I (mean – 3.85). However, the difference was not statistically significant [Table 5].
Table 5: Correlation between osteopontin expression and tumor node metastasis staging

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Site

Our oral squamous cell carcinoma cases were from the following different sites

Fifteen cases occurred in buccal mucosa, eight cases in tongue, and seven cases in alveolus (malignancies arising from the attached masticatory mucosa of the edentulous alveolar ridge). We also examined whether there is a difference in OPN expression among various sites. Alveolar SCC showed the highest OPN expression (mean total score – 5.71) and tongue showed the lowest level of OPN expression (mean total score – 4.1), however, the difference was not statistically significant.

Gender

In our sample, 19 were male and 21 were female. Although OPN levels in male patients (mean total score – 3.73) tended to be higher than those in female patients (mean total score – 3.23) the difference was not statistically significant [Table 6].
Table 6: Correlation between osteopontin expression and gender

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In well-differentiated SCC (WDSCC), tumor cells are found to express OPN in low amounts compared to higher grades thus showing a weak immunostaining for OPN [Figure 1]. An increased mean OPN expression is seen in increasing grades of OSCC thus poorly differentiated SCC (PDSCC) shows strong immunostaining for OPN [Figure 2] and moderately differentiated SCC (MDSCC) shows moderate (stronger than WDSCC but weaker than PDSCC) immunostaining for OPN [Figure 3].
Figure 1: Well Differentiated squamous cell carcinoma – Osteopontin immunostaining (Original)

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Figure 2: Poorly differentiated squamous cell carcinoma – Osteopontin immunostaining (Original)

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Figure 3: Moderately differentiated squamous cell carcinoma – Osteopontin immunostaining (Original)

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   Discussion Top


Oral cancer is the eighth-most common type of cancer in the world.[11] Despite modern intervention, the 5-year survival rate for this disease has improved only marginally over the past decade, and recurrent disease is observed in 50% of the patients. Survival curves of oral cancer patients have plateaued over the past two decades and remain among the worst of all cancer sites.[12] Therefore, recent studies in the field have focused on the development of biomarkers for early detection, disease monitoring and determining the prognosis of patients with oral cancer.

OPN, a phosphoprotein, has been shown to play important roles in tumorigenesis, tumor invasion, metastasis, and prognosis in several human cancers such as breast, gastric, brain, ovary, lung, and prostate carcinomas.[4],[13],[14],[15] It has also been suggested that OPN might be a candidate biomarker for head and neck malignancies.[16] Moreover, it has been proven that OPN is an early invasion marker in OSCC.[3] In the present study, we examined OPN expression in a number of oral cancer patients and tried to elicit its prognostic and diagnostic significance as a biomarker.

In the present study, we found OPN expression is significantly higher in OSCC patients than in normal controls. We found that all the normal epithelium samples were negative for OPN expression and the intensity of OPN expression in tumor cells of OSCC was significantly higher when compared with the normal epithelium. This is in accordance with Staibano et al.[17] and Devoll et al.[18] who reported that OPN was not detected in the normal oral mucosa. It is also in accordance with other studies by Matsuzaki et al.[3] in OSCC, who found that OPN expression was negative or very weak in the basal cell layer of normal epithelium, and by Chambers et al.[19] in lung cancer, who found uniformly negative or very low OPN expression in normal section.

Researchers have hypothesized that strong OPN expression detected in their studies [4],[6] is mediated by its binding with CD44 and integrin receptors. The binding of OPN to these receptors can activate c-Src and downstream signaling elements of epidermal growth factor receptor, which can affect the carcinogenesis of different cancers. OPN can induce MMP-2 expression through activating PI3-kinase, which can modulate cell migration, tumor progression, tumor metastasis, and protect cells from apoptosis.

Literature has shown OPN expression to be increasing from lower to higher histological grades in various carcinomas.[3],[20] Similar results have recently been found in OSCC.[7],[8],[9] We also observed an increased mean OPN expression in increasing grades of OSCC in both total score and intensity. This OPN expression showed a statistically significant difference between WDSCC and PDSCC but not between MDSCC and WDSCC and between MDSCC and PDSCC.

We suggest that as tumor cells in moderately and poorly differentiated SCC have undergone anaplastic transformation and as OPN is found to be secreted by transformed epithelial cells, the tumor cells in higher grades of OSCC express high levels of OPN. However, in the WDSCC, tumor cells are terminally differentiated and have functional similarity with normal epithelium, therefore, they express OPN in low amounts compared to higher grades. It is also found that overexpression of OPN may exacerbate the malignant phenotype by increasing cell transformation, migration, and invasion and is associated with oncogenesis and dissemination of various cancers. Furthermore, overexpression of OPN markedly increased the proliferation and invasion ability of squamous carcinoma cell line.[21]

Matsuzaki et al.[3] in their observation found no significant correlation between OPN expression and lymphatic status and metastasis in 46 cases of OSCC. We also observed no significant correlation between OPN expression and prognostic parameters such as lymphatic status and metastasis. In contrary, Chien et al.[22] found OPN immunostaining to significantly correlate with positive nodal status and advanced TNM stage. Their study was conducted by using plasma OPN assay and in 94 samples. This discrepancy in the studies may have been caused by the difference in the sample size and the different methods of analysis.

We found an increased expression of OPN in males compared to females. Even though not statistically significant, this observation is consistent with the study by Chien et al.[22] Of the site, it was alveolus showing highest expression of OPN, but it was not statistically significant.

Matsuzaki et al. in their study have noted that OPN expression was distinctly predominant in the region where basement membrane is destroyed rather than at dysplastic epithelium and because of that they had stressed that OPN can be considered as an early invasive biomarker than a prognostic marker.[3] However, we have not observed these features in all the 30 cases that have been studied and also the staining pattern was found to be almost uniform all over the dysplastic epithelium.

Our data demonstrated that tumor cells of OSCC express OPN and is found to be elevated in increasing grades of OSCC. Our findings suggest that the elevated levels seen in OSCC patients may contribute to the biology of the tumor and to its progression.


   Conclusion Top


Insights gained from this study may lead to research targeted at the treatment of OSCC. The prospective studies with larger sample size correlating with patient survival rate are warranted to assess the relationship of OPN as a tumor marker.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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3.
Matsuzaki H, Shima K, Muramatsu T, Ro Y, Hashimoto S, Shibahara T, et al. Osteopontin as biomarker in early invasion by squamous cell carcinoma in tongue. J Oral Pathol Med 2007;36:30-4.  Back to cited text no. 3
    
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Chiu YW, Tu HF, Wang IK, Wu CH, Chang KW, Liu TY, et al. The implication of osteopontin (OPN) expression and genetic polymorphisms of OPN promoter in oral carcinogenesis. Oral Oncol 2010;46:302-6.  Back to cited text no. 6
    
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El-Tanani MK, Campbell FC, Kurisetty V, Jin D, McCann M, Rudland PS, et al. The regulation and role of osteopontin in malignant transformation and cancer. Cytokine Growth Factor Rev 2006;17:463-74.  Back to cited text no. 14
    
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Le QT, Sutphin PD, Raychaudhuri S, Yu SC, Terris DJ, Lin HS, et al. Identification of osteopontin as a prognostic plasma marker for head and neck squamous cell carcinomas. Clin Cancer Res 2003;9:59-67.  Back to cited text no. 15
    
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Muramatsu T, Shima K, Ohta K, Kizaki H, Ro Y, Kohno Y, et al. Inhibition of osteopontin expression and function in oral cancer cell lines by antisense oligonucleotides. Cancer Lett 2005;217:87-95.  Back to cited text no. 16
    
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Staibano S, Merolla F, Testa D, Iovine R, Mascolo M, Guarino V, et al. OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome. Br J Cancer 2007;97:1545-51.  Back to cited text no. 17
    
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Devoll RE, Li W, Woods KV, Pinero GJ, Butler WT, Farach-Carson MC, et al. Osteopontin (OPN) distribution in premalignant 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.  Back to cited text no. 18
    
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Chambers AF, Wilson SM, Kerkvliet N, O'Malley FP, Harris JF, Casson AG, et al. Osteopontin expression in lung cancer. Lung Cancer 1996;15:311-23.  Back to cited text no. 19
    
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Korita PV, Wakai T, Shirai Y, Matsuda Y, Sakata J, Cui X, et al. Overexpression of osteopontin independently correlates with vascular invasion and poor prognosis in patients with hepatocellular carcinoma. Hum Pathol 2008;39:1777-83.  Back to cited text no. 20
    
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Celetti A, Testa D, Staibano S, Merolla F, Guarino V, Castellone MD, et al. Overexpression of the cytokine osteopontin identifies aggressive laryngeal squamous cell carcinomas and enhances carcinoma cell proliferation and invasiveness. Clin Cancer Res 2005;11:8019-27.  Back to cited text no. 21
    
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Chien CY, Su CY, Chuang HC, Fang FM, Huang HY, Chen CH, et al. Comprehensive study on the prognostic role of osteopontin expression in oral squamous cell carcinoma. Oral Oncol 2009;45:798-802.  Back to cited text no. 22
    

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Correspondence Address:
Dr. Dhana Lakshmi Jeyasivanesan
Department of Oral Pathology, Tamil Nadu Government Dental College and Hospital, Chennai – 600 003, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_474_17

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