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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 14  |  Issue : 4  |  Page : 378-382

The assessment of expression of midkine in epithelial dysplasia and oral squamous cell carcinoma


1 Department of Oral Pathology, Malla Reddy Institute of Dental Sciences, Hyderabad, Telangana, India
2 Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (Deemed to be University), Wardha, Maharashtra, India

Date of Submission11-Nov-2019
Date of Decision28-Nov-2019
Date of Acceptance15-Dec-2019
Date of Web Publication16-Jul-2020

Correspondence Address:
Dr. Alka Hande
Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi (Meghe), Wardha - 442 001, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_178_19

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  Abstract 


Background: Oral squamous cell carcinoma (OSCC) is categorized by a cancerous tissue growth of the epithelial cells of the oral cavity and is brought about by an array of genomic abnormalities. The survival rate of OSCC has remained nearly unchanged. For this reason, the identification of new molecules that could serve as targets for antineoplastic therapies is of prime importance. Midkine (MK) is considered to be a novel marker for determining prognosis and in early detection of cancer. Our study aims at assessing the expression of MK in oral epithelial dysplasia and OSCC. It is also an attempt to find correlation between the expression of MK and 3-year postoperative survival of patients with OSCC. Materials and Methods: The study included fifty histopathologically diagnosed cases of OSCC of various grades and fifty histopathologically diagnosed cases of epithelial dysplasia from the archives of the department of oral pathology and microbiology. These 100 samples were retrieved from a period of 3 years, i.e. 2009 to 2012. Results: Out of fifty patients with epithelial dysplasia, 47 (94%) patients showed negative expression for MK and only 3 (6%) patients showed positive expression for MK. The percentage of MK expression was PDSCC (100%), MDSCC (93%), and (76%) WDSCC showed positive expression of MK. As the Broder's grading progresses, from well to moderate to poor, the expression of MK also increased. Conclusion: According to our results, it shows that MK should be considered a target for the treatment of OSCC.

Keywords: Epithelial dysplasia, midkine, oral squamous cell carcinoma


How to cite this article:
Alvi S, Hande A, Chaudhary M, Gawande M, Patil S, Sharma P. The assessment of expression of midkine in epithelial dysplasia and oral squamous cell carcinoma. J Datta Meghe Inst Med Sci Univ 2019;14:378-82

How to cite this URL:
Alvi S, Hande A, Chaudhary M, Gawande M, Patil S, Sharma P. The assessment of expression of midkine in epithelial dysplasia and oral squamous cell carcinoma. J Datta Meghe Inst Med Sci Univ [serial online] 2019 [cited 2020 Aug 13];14:378-82. Available from: http://www.journaldmims.com/text.asp?2019/14/4/378/289845




  Introduction Top


Oral squamous cell carcinoma (OSCC) falls into a larger category known as head-and-neck squamous cell carcinomas. OSCC is characterized by an array of genomic abnormalities which may have developed over a period of time.[1],[2] Majority of all OSCCs are preceded by clinical premalignant lesions and conditions such as oral leukoplakia, erythroplakia, oral lichen planus, and oral submucous fibrosis.[3],[4],[5] The 5-year survival rate has remained nearly unchanged for the past two decades.[6] For this reason, recent studies have focused on identifying new molecules that is carcinogenesis-relevant biological markers which could serve as targets for antineoplastic therapies and ultimately reduce the morbidity and mortality associated with OSCC.[7],[8] These new molecules are midkine (MK), Pleotropin, and leptin, out of which MK shows promising results. The expression of MK, a heparin-binding growth factor, is increased during the early differentiation stage in embryonal carcinoma cells and contributes to cell proliferation, angiogenesis, and antiapoptosis, which ultimately leads to tumor progression.[9],[10] MK is also involved in fibrinolytic activities and remodeling of mesenchyme, which is associated with tumor cell migration and metastasis.[11] MK participates in tumorigenesis via increased expression of antiapoptotic proteins Bcl-2, leading to a decrease in cell death.[12] MK also promotes migration and survival of tumor cells by activation of various signaling pathways such as PI3 kinase and mitogen-activated protein (MAP) kinase pathway, followed by suppression of caspases, thus contributing to tumor progression, including angiogenesis, cell proliferation, and antiapoptosis.[13] Therefore, MK is considered to be a novel marker for determining prognosis and in early detection of cancer.

Thus, the present study aims to assessing the expression of MK in oral epithelial dysplasia and OSCC. It is also an attempt to find correlation between the expression of MK and 3 years' postoperative survival of patients with OSCC.


  Materials and Methods Top


Sample

The study included fifty histopathologically diagnosed cases of OSCC of various grades and fifty histopathologically diagnosed cases of epithelial dysplasia from the archives of the department of oral pathology and microbiology. These 100 samples were retrieved from a period of 3 years, i.e. 2009 to 2012. These samples were subjected to immunohistochemistry (IHC) labeled with primary antibody monoclonal mouse anti-human MK (clone A-9; Santa Cruz, CA, USA). The labeling index was calculated that is number of IHC-positive cells × 100/total number of cells observed.[14] Follow-ups were taken after 3-years approximately or more after surgery.

Labeling index for midkine expression

Anti-MK immunoreactivity was confined primarily to the cytoplasm. One hundred cells from five randomly selected representative fields (×400) of each section were counted. All the cells whose cytoplasm stained brown were scored positive regardless of the intensity of staining. It was considered positive when all immunoreactivity levels were more than 10% with anti-MK antibody.

Statistical analysis

Statistical analysis (SPSS Version 20 (Chicago, Illinois, USA)) was carried in the present study to obtain various results which were as follows:

Statistical analysis for the following factors was carried out:

  1. Expression of MK in oral epithelial dysplasia
  2. Expression of MK with Broder's Grading in OSCC
  3. Comparison of MK expression in epithelial dysplasia and OSCC
  4. Correlation of MK expression with the status of life in OSCC.


Ethical clearance

Ethical clearance was obtained from the Institutional Ethical Committee of SPDC, Sawangi (Meghe), Wardha, on 11th March 2019. With ethical clearance no DMIMS(DU)/IEC/2019-20/318.


  Results Top


This immunohistochemical study was designed for the evaluation of MK expression in oral epithelial dysplasia, OSCC, and to correlate its expression with the dead and live status of the patients. Evaluation of expression of immunostaining with MK was performed and labeling indices were calculated.

Out of fifty patients with epithelial dysplasia, 47 (94%) patients showed negative expression for MK and only 3 (6%) patients showed positive expression for MK [Figure 1]. On comparing all the three groups of Broder's grading with the status of life, it was found that 19 out of 25, 9 out of 15, and 1 out of 10 were alive in WDSCC, MDSCC, and PDSCC groups, respectively. 9/10, 6/15, and 6/25 were dead in PDSCC, MDSCC, and WDSCC groups, respectively. There was a significant comparison between status of life and Broder's grading, indicating poor survival of patients with increase in grades of Broder's grading [Figure 2]. There was a significant positive association between MK expression and Broder's grading in OSCC. As the Broder's grading progresses, from well to moderate to poor, the expression of MK also increased [Figure 3]. There was a significant comparison difference in the expression of MK between epithelial dysplasia and OSCC. Only 3 (6%) out of fifty patients with epithelial dysplasia showed positive expression for MK. Whereas, 43 (86%) out of fifty patients with OSCC showed positive expression [Figure 4]. There was a significant correlation of expression of MK with the status of life. As the expression of MK increases, the status of life decreases. Ninety-five percent of dead patients were positive for MK expression, whereas 80% alive patients were negative for MK expression [Figure 5].
Figure 1: Expression of midkine in epithelial dysplasia

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Figure 2: Comparison of Broder's grading with the status of life in oral squamous cell carcinoma group

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Figure 3: Expression of midkine with Broder's grading in oral squamous cell carcinoma

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Figure 4: Comparison of midkine expression in epithelial dysplasia and oral squamous cell carcinoma

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Figure 5: Correlation of midkine expression with status of life in oral squamous cell carcinoma

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Among fifty selected samples of OSCC, 25 were WDSCC, 15 of them were MDSCC, and 10 were PDSCC. On comparing all the three groups of Broder's grading with status of life, it was found that 19 out of 25, 9 out of 15, and 1 out of 10 were alive in WDSCC, MDSCC, and PDSCC groups, respectively. There was a significant correlation between status of life and Broder's grading, indicating poor survival of patients with increase in grades of OSCC [Figure 6], [Figure 7], [Figure 8]. Out of fifty patients with epithelial dysplasia, 43 patients showed negative expression for MK and only three patients showed positive expression for MK, well, moderately, and poorly differentiated squamous cell carcinoma showing positive MK expression, respectively.
Figure 6: Expression of midkine in well differentiated squamous cell carcinoma (×100)

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Figure 7: Expression of midkine in moderately differentiated squamous cell carcinoma (×100)

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Figure 8: Expression of midkine in poorly differentiated squamous cell carcinoma (×100)

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  Discussion Top


This study was done to take into consideration the increased expression of MK in precancerous and cancerous tissue. The present study correlated expression of MK in normal, dysplastic, and malignant tissue. This study revealed that there was an increased expression of MK in malignant tissue compared to that of dysplastic and normal. This is similar to a study conducted by Ye et al., who demonstrated that MK was expressed in colorectal adenomas with moderate and severe-grade dysplasia and in carcinomas, but not in mild-grade dysplasia or in normal tissues.[15] This suggests that the protein is associated with early-stage carcinogenesis. The study also evaluated fifty patients with OSCC, out of which 43 (86%) patients showed positive expression. We found an increase in the expression of MK with an increase in grading of OSCC. The percentage of MK expression was PDSCC (100%), MDSCC (93%), and (76%) WDSCC showed positive expression of MK. There are various reasons which explain the increased expression of MK in severe dysplasia and increasing grades of OSCC; p53 gene controls MK expression indirectly via various transcription factors, which ultimately upregulates the expression of MK cell it plays an important proliferation, survival, apoptosis, and tumor progression.[7],[16] MK expression regulates cell proliferation via PI3/AKT pathway, thereby leading to tumorigenesis. MK expression in epithelial dysplasia and OSCC could also be attributed to activation of various transcription factors such as nuclear factor kappa-B and thyroid transcription factor-1, which causes suppression of apoptosis, cell proliferation, and tumorigenesis.[7],[17] There was a significant correlation of expression of MK with the status of life. As the expression of MK increases, the status of life decreases. Ninety-five percent of dead patients were positive for MK expression. There are various factors which contribute to poor survival in OSCC patients; MK acts on keratinocytes, fibroblasts, tumor cells, and endothelial cells to promote growth, survival, migration, secretion of cytokines, and fibrinolytic activity.[18],[19],[20] The increased MK concentrations in early-stage OSCC were strongly associated with poor survival. Hence, MK concentrations may be a useful marker not only for cancer screening but also for predicting prognosis of OSCC patients.[21],[22] Low-density lipoprotein receptor-related protein and protein tyrosine phosphatase receptor-ζ serve as MK receptors. Through these receptors, MK recognizes glycosaminoglycans on tumor cells which are essential for migration and metastasis of tumor cells. MK exhibits angiogenic action which accelerates tumor growth; this was demonstrated by coexpression of vascular endothelial growth factor (VEGF) with MK which is a potent mitogen for endothelial cells. MK is involved in epithelial–mesenchymal transition of tumor cells by secreting various cytokines and growth factors such as interleukin-8 and bone morphogenetic protein-2. Thus, MK is involved in remodeling of mesenchyme and also associated with tumor cell migration and metastasis.[23] MK participates in tumorigenesis via increased expression of antiapoptotic proteins such as bcl-2, leading to a decrease in cell death. MK promotes migration and survival of tumor cells by activation of PI3 kinase and MAP kinase pathway, followed by suppression of caspases. Phosphorylation of STAT3 by MK stimulates the proliferation of tumor cells.[24] p53, a major tumor suppressor gene, which has an important role in apoptosis regulation, tumor progression, and determining prognosis. Stabilized, mutated, and nonfunctional p53 can be demonstrated by IHC and thus it is considered as an indicator of prognosis and tumor progression.[25] MK expression increases with stabilization and mutation of p53. It is hypothesized by Raynolds et al., and p53 may control MK expression indirectly via transcription factors HIF-1α and NF-κB which upregulates the expression of MK.[26],[27] Thus, MK possesses a strong relation with p53 gene status and MK gene expression is regulated by p53 gene. According to our results, it shows that MK should be considered a target for the treatment of OSCC.[28],[29],[30],[31],[32],[33]

Limitations

Correlation of other markers such as VEGF and Bcl-2 with MK-positive cells in OSCC should be carried out. The use of MK as a potential marker for evaluation of the success of treatment modalities.


  Conclusion Top


We believe that an increase in MK expression in severe epithelial dysplasia suggests that it is not only an early marker of carcinogenesis but also indicative of tumor progression. Furthermore, increase in the expression of this marker with increasing grades of OSCC is indicative of increased biological aggressiveness and thereby contributing to poor 3-year survival.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]



 

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