|Year : 2021 | Volume
| Issue : 3 | Page : 267-273
Expression of “periostin” in leukoplakia and oral submucous fibrosis: An immunohistochemical study
Sandhya Sundar, Pratibha Ramani, Herald J Sherlin, Gifrina Jayaraj
Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu, India
|Date of Submission||31-Jul-2020|
|Date of Decision||30-Jan-2021|
|Date of Acceptance||04-Feb-2021|
|Date of Web Publication||18-Jun-2021|
Dr. Pratibha Ramani
Department of Oral and Maxillofacial Pathology and Microbiology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil Nadu.
Source of Support: None, Conflict of Interest: None
Aim: Periostin is an extracellular matrix protein secreted in the tumor microenvironment. It is frequently overexpressed in various human cancers, including oral squamous cell carcinoma (OSCC). The role of periostin in carcinogenesis of OSCC is yet to be established. The present study was aimed at determining “Periostin” expression in leukoplakia and the oral submucous fibrosis (OSMF) to relate with its role in malignant transformation of these disorders to OSCC. Materials and Methods: A total of 63 cases were categorized into three groups, namely Group I: OSCC (n = 21), Group II: Leukoplakia (n = 21), and Group III: Oral submucous fibrosis (n = 21); these groups were subjected to immunohistochemical analysis by using the periostin antibody. Kruskal–Wallis test and Mann–Whitney U tests were used to assess the significant differences in the periostin expression between groups (SPSS software version 23.0). Results: Results indicated that there was significant difference in the periostin levels between the groups (P < 0.005), indicating a fairly strong relationship between periostin expression among groups. A significant difference between the different grades of leukoplakia was observed, whereas there were no significant differences between subgroups of the OSCC and OSMF. Conclusion: The present study justifies periostin as a key stromal element in tumor progression of OSCC. The potential role of periostin in malignant transformation of oral potentially malignant disorders (OPMD) has been suggested.
Keywords: Leukoplakia, Malignant Transformation, Oral Squamous Cell Carcinoma, Oral Submucous Fibrosis, Periostin
|How to cite this article:|
Sundar S, Ramani P, Sherlin HJ, Jayaraj G. Expression of “periostin” in leukoplakia and oral submucous fibrosis: An immunohistochemical study. J Int Oral Health 2021;13:267-73
|How to cite this URL:|
Sundar S, Ramani P, Sherlin HJ, Jayaraj G. Expression of “periostin” in leukoplakia and oral submucous fibrosis: An immunohistochemical study. J Int Oral Health [serial online] 2021 [cited 2021 Oct 26];13:267-73. Available from: https://www.jioh.org/text.asp?2021/13/3/267/318447
| Introduction|| |
OSCC is a highly aggressive neoplasm with increased propensity for early lymph node metastasis and frequent recurrences. Worldwide, its five-year survival rate is around 50%, with prognosis worsening with advanced disease and late presentation. Despite real advances in diagnosis and management, nearly half of all patients diagnosed with an OSCC die as a direct consequence of the disease.
OPMD are chronic conditions with a statistically increased risk of progression to oral cancer. Overall, the worldwide prevalence of OPMD was estimated to be 4.47%, with the most commonly affected population being Asians followed by South Americans and Caribbean. Since these disorders precede most cases of oral cancer in Asia, it provides an opportunity for early intervention. Understanding the mechanisms of malignant transformation leads to early diagnosis of OSCC arising in the background of these PMDs.
OSMF is a high-risk potentially malignant disorder, with a malignant transformation rate estimated to be 7% to 13%., Oral leukoplakia (OLE) is the next frequent PMD with a prevalence of 4.11% associated with tobacco and alcohol consumption. Simultaneous occurrence of OLE and OSMF carries a higher risk for OSCC and experiences early malignant transformation than those with either OLE or OSMF alone. Both the earlier mentioned PMDs come under the high-risk category of the WHO classification of PMD. Novel biomarkers to indicate the potential transformation of these disorders to OSCC is highly needed, which would greatly improve their evaluation and distinguish lesions that may progress to cancer.
Periostin is a 90-kDa secreted extracellular matrix protein belonging to the group of Transforming growth factor beta (TGF-β) inducible proteins. It is frequently overexpressed in various human cancers, including OSCC. Although the role of periostin in the invasion and metastasis of OSCC has been well established, its part in malignant transformation has not been explored. Thus, this study aims at evaluating periostin expression in leukoplakia and OSMF cases to predict their malignant transformation to OSCC.
| Materials and Methods|| |
The present study was performed on patients histopathologically diagnosed with different grades of OSCC, OLE, and OSMF. Formalin-fixed, paraffin-embedded tissue blocks of all the cases were retrieved from the department of Oral and Maxillofacial Pathology between the years 2017 and 2018. A pilot study was performed to determine the potential expression of periostin in OSCC, OLE, and OSMF cases. Based on the results, Z-tests were done for all the three groups and a maximum sample size of 21 per group was obtained. The software used to calculate the sample size was G Power (version 188.8.131.52). Thus, the study material consisted of a total of 63 samples categorized into three groups,
Group I: Oral Squamous Cell Carcinoma (n = 21)
Well-differentiated OSCC (n = 7)
Moderately differentiated OSCC (n = 6)
Poorly differentiated OSCC (n = 4)
Early OSCC (n = 4)
Group II: Oral submucous fibrosis (n = 21)
Early and Moderately Advanced OSMF (n = 7)
Advanced OSMF (n = 14)
Group III: Leukoplakia (n = 21)
Hyperkeratosis with mild dysplasia (n = 8)
Hyperkeratosis with moderate dysplasia (n = 10)
Hyperkeratosis with severe dysplasia (n = 3)
For immunohistochemical study, 3 mm sections were cut from formalin-fixed, paraffin embedded blocks (Leica semiautomated microtome) and mounted on APES-coated slides. The sections were de-paraffinized in xylene, dehydrated in alcohol, and rinsed with distilled water. Antigen retrieval was performed by using Citrate buffer (pH 6.0) for 10min in a pressure cooker. Endogenous peroxidase was blocked (for 5min), and incubation with Periostin Mouse Monoclonal antibody was done overnight.
Detection was performed by using DAKO envision kit [DAKO Denmark A/S]. The sections were then counterstained with Mayer’s hematoxylin, dehydrated and mounted. Normal Human Fallopian tube More Details tissue was used as positive control, and negative controls were achieved by performing the staining procedures with the omission of the primary antibody.
Evaluation of immunohistochemical sections
The presence of the brown-colored end product at the site of target antigen was indicative of positive reactivity. Immunohistochemical staining of periostin was assessed by the evaluation of the stain intensity and percentage of periostin positivity, and the expression of periostin was determined according to the method used by Zhang et al.
The intensity of staining was observed and assessed from three fields. A scale from 0 to 2 was used. The grading was based on the intensity of the stain and the area of positive staining. An individual observer analyzed the immunohistochemically stained sections. The areas after assessing were then graded as 0, 1, 2, and 3 based on the staining intensity. Grading of stain-positive cells was as follows: “0” = No positive cells, “1” = 1–25% stain-positive cells, “2” = 26–50% stain-positive cells, and "3” = 51–100% stain-positive cells. The total score of each specimen was obtained by multiplying the percentage and intensity scores. Those cases with mean scores of <4 were considered to have low expression of periostin, and those with >4 were considered to have high expression of periostin.
All the parameters were tabulated and assessed for statistical significance by using SPSS software (version 23.0). The differences in the expression of periostin between groups were statistically analyzed by using the Kruskal–Wallis test and Mann–Whitney U test. P < 0.05 was considered to be statistically significant.
| Results|| |
A total of 63 cases, comprising 21 cases of OSCC, 21 cases of OSMF, and 21 cases of hyperkeratosis with epithelial dysplasia (clinically leukoplakia), were evaluated for periostin expression. Among the 21 cases of OSCC, 19 cases (90.5%) showed positive periostin expression; out of these, five cases (23.81%) showed mild expression, four cases (19.05%) showed moderate expression, and 10 cases (47.62%) showed intense expression of periostin.
As far as the distribution of periostin was concerned, it was predominantly seen in the connective tissue stroma, around the tumor islands, fibroblasts, and in areas of desmoplasia [Figure 1][Figure 2][Figure 3]. Overall, 31.58% of OSCC cases showed aberrant periostin expression by epithelial (carcinoma) cells. The expression of periostin was not significantly different between the subgroups of OSCC (P = 0.324) [Table 1].
|Figure 1: Photomicrograph of well-differentiated squamous cell carcinoma case showing positive periostin expression surrounding the tumor islands: 10x|
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|Figure 2: Photomicrograph of case of well-differentiated squamous cell carcinoma showing periostin expression by carcinoma cells (epithelial cells) surrounding the tumor islands: 40x|
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|Figure 3: Photomicrograph of case of moderately differentiated squamous cell carcinoma showing periostin expression surrounding the tumor islands: 10x|
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|Table 1: Kruskal–Wallis test to compare mean periostin expression values between subgroups in the OSCC group|
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Ovearll, 52.38% of the cases of hyperkeratosis with epithelial dysplasia (clinically leukoplakia) showed an overall positivity for periostin expression; 87.5% of the cases of hyperkeratosis showed mild epithelial dysplasia; 40% of the cases of hyperkeratosis with moderate epithelial dysplasia cases showed positive expression of periostin; and all the cases of hyperkeratosis with severe epithelial dysplasia were devoid of periostin expression. In all the studied samples of hyperkeratosis with epithelial dysplasia, periostin expression was restricted to the connective tissue stroma with only one case (4.8%) showing epithelial cell expression. The expression of periostin was significantly higher in hyperkeratosis cases with mild dysplasia compared with the moderate and severe dysplasia subgroups (P = 0.055) [Table 2].
|Table 2: Kruskal-Wallis test to compare mean periostin expression values between subgroups in leukoplakia group|
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Overall, 71.43% of the samples of OSMF showed an overall positivity for periostin expression. Considering gradewise distribution, 57.14% cases of early and moderately advanced OSMF (n = 7) and 78.57% cases of advanced OSMF (n = 14) showed periostin expression. The periostin expression in the positive cases were restricted to areas of fibrosis and hyalinization [Figure 4], with two cases (9.5%) showing expression by epithelial cells. The expression of periostin was not significantly different between the subgroups of OSMF (P = 0.961) [Table 3].
|Figure 4: Photomicrograph of advanced case of OSMF showing periostin expression in the areas of severe fibrosis: 40x|
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|Table 3: Mann-Whitney test to compare mean periostin expression values between subgroups in OSMF group: The expression of periostin was not significantly different between the subgroups of OSMF (P = 0.961). The periostin expression in the positive cases was restricted to areas of fibrosis and hyalinization, with two cases (9.5%) showing expression by epithelial cells|
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The differences in the expression of periostin between the cases of OSCC, OSMF, and hyperkeratosis with epithelial dysplasia (clinically leukoplakia) were compared by using the Kruskal-Wallis test. The results of the Kruskal-Wallis test indicated that there was a significant difference in the periostin level between the groups (P = 0.005) [Table 4], indicating a fairly strong relationship between periostin expression among groups [Figure 1]. Prominent differences between the test groups were assessed by a pairwise comparison of groups by using Mann-Whitney U test. The results indicated that the periostin expression was significantly higher in OSCC compared with leukoplakia (P = 0.003) [Table 5] and OSMF (P = 0.014) [Table 6].
|Table 4: Kruskal-Wallis test compare mean periostin expression values between groups: The differences in expression of periostin between the cases of OSCC, OSMF, and hyperkeratosis with epithelial dysplasia (clinically leukoplakia) were compared. There was a significant difference in the periostin level between the groups (P = 0.005), indicating a fairly strong relationship between periostin expression among groups|
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|Table 5: Mann-Whitney Test to compare mean periostin expression values between OSCC and leukoplakia groups: Prominent differences between the test groups were assessed by a pairwise comparison of groups by using Mann-Whitney U test. The results indicated that periostin expression was significantly higher in OSCC compared with leukoplakia (P = 0.003).|
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|Table 6: Mann-Whitney test to compare mean periostin expression values between OSCC and OSMF groups: Prominent differences between the test groups were assessed by a pairwise comparison of groups by using Mann-Whitney U test. The results indicated that periostin expression was significantly higher in OSCC compared with OSMF (P = 0.014)|
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| Discussion|| |
In the present study, 90.5% of OSCC cases showed upregulated periostin expression, which is analogous with the results shown in previous studies.,,, The tumor cells in the advancing front breach the basement membrane to help the epithelial component invade the stromal components. These infiltrating tumor cells need to induce stromal periostin to support their colonization, anchorage-dependent growth, and epithelial mesenchymal transition (EMT) programs (as they are the cell adhesion molecules). Periostin expression was observed in 25% cases of early squamous cell carcinoma, 85.71% cases well-differentiated squamous cell carcinoma, 100% cases moderately differentiated carcinoma, and 100% of cases in poorly differentiated carcinoma. This heterogeneity of periostin in different degrees of tumor differentiation could be attributed to varied production of TGF-β and other stimulators that are essential for periostin secretion. This also correlates the periostin expression with poor prognosis in OSCC, as studies have correlated poorly differentiated tumors with nodal metastasis in OSCC.
Periostin staining was localized primarily in the stroma, with the extracellular matrix and fibroblast surrounding the tumor islands and this was in accordance with the studies by., Qin et al. have identified stromal cells as the principal source of Periostin protein (POSTN), with high expression in cancer-associated fibroblasts (CAFs) and TGF β as the key inducer of its production. This stromal-derived periostin may modulate the tumor microenvironment, which is required for the maintenance of cancer stem cells, and be responsible (through integrin-mediated adhesion) for the formation of podosome structure to facilitate cancer cell migration.
Although the robust staining of periostin within tumor cells was not observed, 31.58% of OSCC tumors were positive for epithelial periostin as comparable to those reported by Choi et al. (58%). Five out of six cases (83.33%) showing epithelial periostin expression were highly invasive cancers with a type III/IV histological pattern of invasion. These findings suggest that epithelial expression of periostin may be associated with a more aggressive tumor phenotype in OSCC, which is supported by other studies where the OSCC cells engineered to overexpress periostin, showed enhanced tumor growth, invasion, and spontaneous metastasis to lymph nodes.
Overall, 52.38% of the cases of hyperkeratosis with epithelial dysplasia (clinically leukoplakia) showed positive periostin expression. Studies by Costea et al. have revealed the stromal regulation of epithelial morphogenesis, especially that of fibroblasts. Dysplasia represents the progressive loss of the stromal control over epithelial morphogenesis. Once the dysplastic epithelium accumulates further mutations resulting in invasion, the stroma responds by acting as a co-conspirator. Consistent with this, the periostin is overexpressed in oral cancer cases compared with those cases of hyperkeratosis with epithelial dysplasia or OSMF.
The progressive reduction in the periostin expression could be visualized in different grades of hyperkeratosis with dysplasia cases from mild, moderate to severe dysplasia. Since OLE is associated with tobacco and the mechanical stress produced by these agents, increased periostin expression could have been seen in mild dysplasia. This periostin produced by resident fibroblasts as part of the reactive and inflammatory process induces epithelial cell proliferation and activates the NF-κB in these cells. As more mutations and dysplastic changes are acquired as in the case of severe dysplasia, the protective role of periostin may be lost unless the tumor cells fall into connective tissue to induce periostin secretion from differentiated myofibroblasts.
In the current study, 57.14% of cases of early and moderately advanced OSMF and 78.57% of cases of advanced OSMF showed periostin expression. Periostin interacts with other ECM molecules in stroma to regulate collagen fibrillogenesis and cross-linked structures, and these could be expressed in areas of fibrosis. Increased fibrosis of the connective tissue induces hypoxia, and subsequent overexpression of HIF-1 α causes activation of many hypoxia-inducible genes (such as VEGF) that play a role in malignant transformation and progression of OSF.The progressive fibrosis seen in advanced OSMF may predispose to atrophy of the epithelium and subsequent malignant changes.
The epithelial periostin expression was observed in one case of moderately advanced OSMF with mild dysplasia and another case of advanced OSMF with severe dysplasia. Periostin probably exerts its protumorigenic effect not only through its effect on ECM fibrillogenesis but also by binding to the integrins and consequent activation of intracellular pathways, enhancing the invasiveness. We, therefore, hypothesize that periostin, if present in cases of OSMF, might contribute in explaining the process of malignant transformation.
Therefore, our present study confirms the essential role of stromal and epithelial-derived periostin in promoting tumor growth and invasion in OSCC. Periostin expression in cases of hyperkeratosis with mild and moderate dysplasia could mark a normal reactive process, whereas the absence of periostin expression in severe dysplasia indicates a loss of control of stroma over the epithelial morphogenesis due to progressively acquired mutations of carcinomatous cells. Thus, periostin is not a conceivable marker to predict the malignant transformation of leukoplakia (hyperkeratosis with epithelial dysplasia). As far as OSMF is concerned, the potential role of periostin in the pathological process and malignant transformation of OSMF has been proposed. However, further studies with large sample sizes will ascertain the role of periostin as a diagnostic marker in malignant transformation of OSMF.
| Conclusion|| |
The increased expression of periostin derived from stromal and carcinoma cells can be a marker of aggressive behavior, as it correlates with poor prognosis. It might help us to identify the subset of patients who require more aggressive treatment methods to attain a better survival rate.
Periostin might help intervene in the early process of carcinogenesis, counsel patients for prompt follow-up, and provide an improved quality of life to the patients, thus proving to be a predictive marker of malignant transformation of OSMF.
Financial support and sponsorship
Conflict of interest
Sandhya S. was involved in study conception, data collection, data analysis, and article writing. Pratibha Ramani was involved in study conception, data analysis, data interpretation, and article writing. All the authors approved the final version of the article for publication.
Ethical policy and Institutional Review board statement
Approval from the institutional review board was obtained (SRB/SDMDS11/18/OMP/02).
Patient declaration of consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/ have given his/ her consent for his/ her images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Data availability statement
The data set used in the current study is available on request.
| References|| |
Scully C, Bagan J. Oral squamous cell carcinoma: Overview of current understanding of aetiopathogenesis and clinical implications. Oral Dis 2009;15:388-99.
Mello FW, Miguel AFP, Dutra KL, Porporatti AL, Warnakulasuriya S, Guerra ENS, et al
. Prevalence of oral potentially malignant disorders: A systematic review and meta-analysis. J Oral Pathol Med 2018;47:633-40.
Rajendran R. Oral submucous fibrosis: Etiology, pathogenesis, and future research. Bull World Health Organ 1994;72:985-96.
Yang PY, Chen YT, Wang YH, Su NY, Yu HC, Chang YC. Malignant transformation of oral submucous fibrosis in Taiwan: A nationwide population-based retrospective cohort study. J Oral Pathol Med 2017;46:1040-5.
Warnakulasuriya S, Ariyawardana A. Malignant transformation of oral leukoplakia: A systematic review of observational studies. J Oral Pathol Med 2016;45:155-66.
Speight PM, Khurram SA, Kujan O. Oral potentially malignant disorders: Risk of progression to malignancy. Oral Surg Oral Med Oral Pathol Oral Radiol 2018;125:612-27.
Takeshita S, Kikuno R, Tezuka K, Amann E. Osteoblast-specific factor 2: Cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem J 1993;294 (Pt 1):271-8.
Conway SJ, Izuhara K, Kudo Y, Litvin J, Markwald R, Ouyang G, et al
. The role of periostin in tissue remodeling across health and disease. Cell Mol Life Sci 2014;71:1279-88.
Siriwardena BS, Kudo Y, Ogawa I, Kitagawa M, Kitajima S, Hatano H, et al
. Periostin is frequently overexpressed and enhances invasion and angiogenesis in oral cancer. Br J Cancer 2006;95:1396-403.
Zhang ZR, Chen LY, Qi HY, Sun SH. Expression and clinical significance of periostin in oral lichen planus. Exp Ther Med 2018;15:5141-7.
Kudo Y, Ogawa I, Kitajima S, Kitagawa M, Kawai H, Gaffney PM, et al
. Periostin promotes invasion and anchorage-independent growth in the metastatic process of head and neck cancer. Cancer Res 2006;66:6928-35.
Kudo Y, Iizuka S, Yoshida M, Nguyen PT, Siriwardena SB, Tsunematsu T, et al
. Periostin directly and indirectly promotes tumor lymphangiogenesis of head and neck cancer. PLoS One 2012;7:e44488.
Deraz EM, Kudo Y, Yoshida M, Obayashi M, Tsunematsu T, Tani H, et al
. MMP-10/stromelysin-2 promotes invasion of head and neck cancer. PLoS One 2011;6:e25438.
Qin X, Yan M, Zhang J, Wang X, Shen Z, Lv Z, et al
. Tgfβ3-mediated induction of periostin facilitates head and neck cancer growth and is associated with metastasis. Sci Rep 2016;6: 20587.
Choi P, Jordan CD, Mendez E, Houck J, Yueh B, Farwell DG, et al
. Examination of oral cancer biomarkers by tissue microarray analysis. Arch Otolaryngol Head Neck Surg 2008;134:539-46.
Jakobsson PA, Eneroth CM, Killander D, Moberger G, Mårtensson B. Histologic classification and grading of malignancy in carcinoma of the larynx. Acta Radiol Ther Phys Biol 1973;12:1-8.
Costea DE, Kulasekara K, Neppelberg E, Johannessen AC, Vintermyr OK. Species-specific fibroblasts required for triggering invasiveness of partially transformed oral keratinocytes. Am J Pathol 2006;168:1889-97.
Izuhara K, Nunomura S, Nanri Y, Ogawa M, Ono J, Mitamura Y, et al
. Periostin in inflammation and allergy. Cell Mol Life Sci 2017;74:4293-303.
Ekanayaka RP, Tilakaratne WM. Oral submucous fibrosis: Review on mechanisms of malignant transformation. Oral Surg Oral Med Oral Pathol Oral Radiol 2016;122:192-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]