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ORIGINAL ARTICLE
Year : 2020  |  Volume : 9  |  Issue : 1  |  Page : 46-51

Quantitative analysis of microvessels density in different grades of oral squamous cell carcinoma


1 Department of Oral Pathology and Microbiology, SIBAR Institute of Dental Sciences, Tekallapadu, Guntur, Andhra Pradesh, India
2 Department of Oral pathology, Government Dental College and Hospital, Vijayawada, Andhra Pradesh, India

Date of Submission30-Apr-2019
Date of Acceptance14-Nov-2019
Date of Web Publication14-May-2020

Correspondence Address:
Dr. Jyothsna Mandapati
Department of Oral pathology,Government Dental College and Hospital, Vijayawada, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JDRNTRUHS.JDRNTRUHS_56_19

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  Abstract 


Background: Oral cancer is the sixth most common cancer in the world today. Angiogenesis plays a vital role in neoplasm growth and metastasis. It is modulated by both angiogenic stimulators and angiogenic inhibitors. Cluster of differentiation 31 (CD31) is expressed on endothelial cells and has been implicated in the adhesion and migration of endothelial cells during angiogenesis.
Aims and Objectives: The aim and objectives of this study are to analyze the expression of CD31 in different histological grades of oral squamous cell carcinoma (OSCC), to determine the microvessel area (MVA) using a CD31 marker.
Materials and Methods: The retrospective study was undertaken on 10 cases each of different grades of squamous cell carcinoma. Immunostaining using CD31 was used to demonstrate blood vessels. MVA was calculated using measurement tools in image analysis software and compared between the groups. One-way analysis of variance was used for comparing the parameter for multiple groups. Pairwise comparison of different grades of squamous cell carcinoma with the mean vascular area by Tukey's multiple analysis was done.
Results: The mean vascular area decreased from well-differentiated squamous cell carcinoma to moderately differentiated carcinoma and was statistically significant (P ≤ 0.05) and increased from moderately differentiated squamous cell carcinoma to poorly differentiated squamous cell carcinoma and showed no statistical significance (P ≥ 0.05).
Conclusion: Our study suggests that CD31 can be used as a prognostic indicator in OSCC.

Keywords: Angiogenesis, CD31, microvessel area, oral squamous cell carcinoma


How to cite this article:
Alivelu D, Kattappagari KK, Mandapati J, Poosarla C, Gontu SR, Reddy Baddam VR. Quantitative analysis of microvessels density in different grades of oral squamous cell carcinoma. J NTR Univ Health Sci 2020;9:46-51

How to cite this URL:
Alivelu D, Kattappagari KK, Mandapati J, Poosarla C, Gontu SR, Reddy Baddam VR. Quantitative analysis of microvessels density in different grades of oral squamous cell carcinoma. J NTR Univ Health Sci [serial online] 2020 [cited 2020 May 30];9:46-51. Available from: http://www.jdrntruhs.org/text.asp?2020/9/1/46/284316




  Introduction Top


Oral cancer is the sixth most common cancer in the world today. Oral epithelial cell malignancy of oral squamous cell carcinoma (OSCC) is a malignant tumor of epithelial cells exhibiting squamous differentiation characterized by the formation of keratin and the presence of intercellular bridges. The South Central Asian countries such as Sri Lanka, Bangladesh, India, and Pakistan are under high risk due to high exposure to risk factors. In India, the incidence rate of oral cancer is 12.6 per 100,000 population. They represent 94% of all oral malignancies.[1] Oral cancer is a multifactorial disease, caused by extrinsic factors including tobacco, alcohol, oncogenic viruses (human papillomavirus, Epstein–Barr virus), and radiation and intrinsic factors such as malnutrition and iron deficiency anemia. Tobacco habit and excessive alcohol consumption have been estimated to account for about 90% of oral cancers.[2]

The pathogenesis of oral cancer is a complex process, due to deregulation of growth and differentiation of the affected tissue. Angiogenesis is one of the factors that play an important role in tumor growth and metastasis. Angiogenesis is the ability of preexisting vasculature to form new microvessels.[3],[4] Angiogenesis is a multistep process, modulated by both angiogenic stimulators, such as vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FGF), interleukins, and tumor necrosis factor (TNF), and angiogenic inhibitors such as thrombospondin-1, angiostatin, and endostatin. Any shift in the net balance between angiogenic stimulators and inhibitors has a profound effect on tumor growth and metastasis.[3] Angiogenic factors are produced by tumor cells that directly activate the endothelial cells to sprout and grow toward the developing tumor.[3],[5]

Angiogenesis can be used as a prognostic marker, by measuring tumor microvessel density, or angiogenic factor expression, which may predict the risk of tumor development and metastasis. Since the end purpose of angiogenesis in tumorigenesis is to supply the required nutrients to the rapidly growing tumor, the sum total of the vessel area in supporting tumor connective tissue is likely to be more than the adjacent normal tissue. Hence, the mean vessel area may also be one of the parameters to assess neoplastic activity including tumor progression. A cluster of differentiation 31 (CD31) or platelet endothelial cell adhesion molecule-1 (PECAM-1) is a transmembrane glycoprotein of the immunoglobulin super family, expressed on endothelial cells. It has been implicated in the adhesion and migration of endothelial cells during angiogenesis and may emerge as a possible therapeutic target in oral carcinomas.[6] This study attempts to correlate the expression of CD31 in different grades of OSCC, to determine the microvessel area (MVA) per unit area using a CD31 marker in different histological grades of OSCC.


  Materials and Methods Top


This study was performed using 30 formalin-fixed paraffin-embedded tissue blocks of each category, which were diagnosed as histopathologically well-differentiated, moderately differentiated, and poorly differentiated OSCC. All these cases were retrieved from the archives of Department of Oral Pathology and Microbiology, Sibar Institute of Dental Sciences, Guntur. The ethical approval for this study was obtained from the ethical committee of SIBAR institute of Dental Sciences, Guntur (Ref No: 81/IEC/SIBAR/2016), and was performed based on the criteria proposed by the World Health Organization in 2005.

Thirty cases of OSCC were categorized into three groups as follows:

  • Group I: Well-differentiated squamous cell carcinomas (n = 10)
  • Group II: Moderately differentiated squamous cell carcinomas (n = 10)
  • Group III: Poorly differentiated squamous cell carcinomas (n = 10).


Serial sections having thickness of 4 μm were prepared. All these sectioned were subjected to hematoxylin and eosin for reconfirmation of histopathological diagnosis. Histopathologically confirmed cases were subjected to immunohistochemical analysis using anti-CD31 antibody.

For immunohistochemical stain, sections were fixed on micro slides which are coated with poly-l-lysine. The tissue was deparaffinized using xylene with each dip of 10 min. The rehydration of tissue was performed by three dips for 5 min, each in 90%, 80%, and 70% alcohol. Thereafter, the tissue was placed in a distilled water bath and was not allowed to dry. Tissue sections were dipped in buffer solution prepared by mixing 1.21 g of Tris ethylenediaminetetraacetic acid (EDTA) buffer with 0.37 g of EDTA and incubated at 850 W for 5 min, 600 W for 10 min, and 400 W for 5 min in a microwave oven. The slides were allowed to cool and placed in a water buffer prepared by adding 950 mL of distilled water with 50 mL of buffer. Excessive buffer from the slides was removed using tissue paper. Then the tissue specimen was covered with 50 μL of hydrogen peroxide, incubated for 5 min and gently washed twice with phosphate-buffered saline. The tissue was covered with primary antibody–antimonoclonal anti-CD31 antibody and incubated for 1 h at room temperature and gently washed twice with phosphate-buffered saline. The secondary link antibody was added. Specimens were incubated for 30 min at room temperature and given three gentle rinses with phosphate-buffered saline. The substrate chromogen solution prepared by mixing 1 mL of substrate buffer and a drop of diaminobenzidine was incubated for 5 min at room temperature and later gently rinsed with phosphate-buffered saline. Then the slides were counterstained in a bath of hematoxylin for 2–5 min and washed under tap water for 5 min. Positive CD 31 expression was as a light brown stain in the blood vessels. All stained blood vessels areas demonstrating positivity for CD31 were identified at a magnification of 20×, and the number of positivity stained blood vessels was counted on 10 representative areas of the section, in minimum of 100 microvessels per field [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]. The CD31 positivity was marked and the score was entered in Excel sheet.
Figure 1: 20× photomicrograph of hematoxylin and eosin of well-differentiated squamous cell carcinoma

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Figure 2: 20× photomicrograph of immune positivity of microvessels of well-differentiated squamous cell carcinoma

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Figure 3: 20× photomicrograph of hematoxylin and eosin of moderately differentiated squamous cell carcinoma

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Figure 4: 20× photomicrograph of immune positivity of microvessels of moderately differentiated squamous cell carcinoma

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Figure 5: 20 × photomicrograph of hematoxylin and eosin of poorly differentiated squamous cell carcinoma

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Figure 6: 20 × photomicrograph of immune positivity of microvessels of moderately differentiated squamous cell carcinoma

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Statistical analysis

The collected data were entered in Excel spread sheet, and statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) software version 20.0 (SPSS Inc., Chicago IL, USA). The mean of microvessels was determined using one-way analysis of variance (ANOVA). P value of 0.05 or less was considered for statistical significance. Pairwise comparison of three different grades of OSCC was performed using “Tukey's multiple analysis.”


  Results Top


A total 30 cases of OSCC were divided into three groups such as well-differentiated, moderately differentiated, and poorly differentiated squamous cell carcinoma. All the groups were subjected to immunohistochemical analysis using anti-CD31 antibody. The number of immunopositive cells was evaluated in each case and a score was given accordingly.

Comparison of the mean vascular area in different grades of squamous cell carcinoma was done. The mean vascular area was 145.47 ± 93 in Group I (well-differentiated squamous cell carcinoma), 137.88 ± 71 in Group III (poorly differentiated squamous cell carcinoma), and 62.35 ± 37 in Group II (moderately differentiated squamous cell carcinoma) using statistical test one-way ANOVA. Comparison of the mean vascular area in different grades of squamous cell carcinoma showed statistical significance (P ≥ 0.0370) [Table 1] and [Graph 1].
TABLE 1: Comparison of three groups with respect to mean vascular area by one-way anova

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Pairwise comparison of the three grades of squamous cell carcinoma with respect to the mean vascular area was done using Tukey's multiple analysis. A high statistical significant was noticed on comparing Group I and Group III (P = 0.0410); however, a comparison of Group I and Group III, and Group II and Group III showed no statistical significance (P = 0.9790) [Table 2].
TABLE 2: Pairwise Comparisons of Three Groups with Respect to Mean Vascular Area by Tukey'S Multiple Post Hoc Procedures

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The mean vascular area in three different groups with respect to score was performed using Kolmogorov–Smirnov test: Group I (P = 0.6600), Group II (P = 0.2100), and Group III (P = 0.9900). There was no statistical significance in Group I, Group II, and Group III [Table 3].
TABLE 3: Normality of mean vascular area in three groups by kolmogorov-smirnov test

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  Discussion Top


Oral cancer is the sixth most common cancer in the general population representing a major health problem in many parts of the globe.[7] It is estimated that 62% of oral cancers occur in developing countries.[8] In India, oral cancer represents the most common cancer which constitutes a major health problem.[9] OSCC is a malignant epithelial neoplasm which accounts for 95% of the malignant tumors of the oral cavity. More than 60% of the patients with OSCC present with advanced stages of the disease.[10] The environmental factors combined with alteration in the genetic factors such as oncogenes and tumor suppressor genes are the major etiological factors for head and neck carcinomas.[4]The causative factors in the development of OSCC are tobacco in the form of chewing or smoking tobacco, consumption of alcohol, diet, and viral infections.[11] Carcinogenesis is a multistep process which leads to the accumulation of genetic alterations that occur even before the development of malignant phenotype. Various changes in DNA can progress from normal keratinocytes to dysplastic cells, then progress to potentially malignant cells, and finally leads to malignant keratinocytes, which has the capacity for proliferation in uncontrolled fashion than normal keratinocytes.[12] As a tumor progresses, cells undergo further mutations leading to increased heterogeneity of the tumor cell population. The tumor cells will get detached because of the lack of adherence property and invade the neighboring tissues. These individual tumor cells can enter the blood vessels and lymphatic channels from which they are transported to the distant sites leading to metastasis.[13] Invasion and dissemination of squamous cell carcinoma require active cell migration through the extracellular matrix with the remodeling of intercellular adhesions.[14] Thus, tumor angiogenesis could be a novel second target for anticancer therapy. There are numerous factors that contribute to angiogenesis such as fibroblastic growth factor (FGF), VEGF, TGF, angiogenin, and angiotropin.[15] In this study, the immunohistochemical expression of anti-CD 31 was assessed among the different grades of OSCC. In this study, a decreased expression of CD 31 was observed with increased grade of OSCC which was in accordance with the studies conducted by Pazouki et al., Zhang et al., Maruthi et al., and Jyothsna et al.[16],[17],[18],[19] A strong expression of MVA was observed in well-differentiated squamous cell carcinoma when compared with moderately differentiated squamous cell carcinoma and poorly differentiated squamous cell carcinoma. The microvessel expression indicates that for better prognosis, the MVA is a very important factor in the behavior of malignant tumors.

The mean values of CD-31 immunopositive microvessels in this study are higher in well-differentiated squamous cell carcinoma, when compared with moderately and poorly differentiated squamous cell carcinoma. Moderately differentiated squamous cell carcinoma cases showed moderate expression of CD-31. In this study, angiogenesis was measured using CD31 and it was observed that the mean vascular area decreased from well- to moderately differentiated squamous cell carcinoma. But the mean vascular area increased from moderately to poorly differentiated squamous cell carcinoma. These observations are in accordance with the studies done by Wadhwan et al. in 2015 and Bouguezzi et al. in 2016.[20],[21] In this study, expression of CD-31 advises that angiogenic marker. CD31 can be useful prognostic factor in different grades of OSCC. However, further studies on the distribution in the expression of CD31 are required on large sample size using different angiogenic parameters including mean vessel density, mean vessel area, total vessel area, and vessel perimeter for a better understanding of the role of angiogenesis on tumor progression and prognosis.


  Conclusion Top


The results of this study suggest the number of microvessel from well-differentiated squamous cell carcinoma to moderately and poorly differentiated squamous cell carcinoma. It implies CD31 expression in newly formed sprouting vessels during neoangiogenesis. The MVA was increasing from moderately differentiated squamous cell carcinoma to poorly differentiated squamous cell carcinoma, which might be due to increased requirement of blood supply which may simply be met by an increase in vessel area. CD31 may be a useful prognostic indicator in OSCC. But further investigations into angiogenic factors induced by tumor cells might provide a better understanding of angiogenesis in different grades of squamous cell carcinoma and help predict their response to antiangiogenic agents.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Zhang YY, Sun CF, Peng J. Expression of CD31, CD34 and CD105 in squamous cell carcinoma of the tongue and their relationships with lymph node metastasis. Hua Xi Kou Qiang Yi Xue Za Zhi 2008;26:201-5.  Back to cited text no. 17
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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