|Year : 2016 | Volume
| Issue : 4 | Page : 281-285
Histomorphometric analysis of vascularity in oral epithelial dysplasia
Sanapala Venkata Naga Sashi Kiran, Sri Lalitha Kaja, Kiran Kumar Kattapagari, Poosarla Chandra Shekar, Chitturi Ravi Teja, Baddam Venkat Ramana Reddy
Department of Oral Pathology and Microbiology, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
|Date of Web Publication||23-Dec-2016|
Sanapala Venkata Naga Sashi Kiran
Department of Oral Pathology and Microbiology, Sibar Institute of Dental Sciences, Takkellapadu, Guntur - 522 509, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Context: Potentially malignant disorders such as leukoplakia and erythroplakia are often associated with dysplastic changes that have an increased risk for malignant transformation. Vascularity could have a role in the transformation of potentially malignant disorders to oral squamous cell carcinoma.
Aims: The present study was aimed to assess the mean vessel area (MVA) of the blood capillaries in different grades of epithelial dysplasia by histomorphometric analysis, which could aid in predicting the malignant transforming potential as well as prognosis of potentially malignant disorders.
Materials and Methods: The study consisted of 30 histologically proven epithelial dysplasia cases categorized into three groups of mild, moderate, and severe epithelial dysplasia based on the World Health Organization (WHO) 2005 criteria. Paraffin-embedded tissue sections of 3-4 microns were stained with Masson's trichrome. Morphometric analysis of MVA was done using image analysis software 6.0.
Statistical Analysis Used: Statistical analysis was done using the software Statistical Package for the Social Sciences (SPSS) 20.0. The mean and standard deviation of the three groups were analyzed using Kruskal-Wallis analysis of variance (ANOVA) test. Pairwise comparison was done using Mann-Whitney U test. P value <0.05 was considered to be statistically significant.
Results: The MVA was highest in severe epithelial dysplasia (7575.16) followed by moderate (4070.4) and mild (4014.29) epithelial dysplasias. A high statistical significance was observed on comparison of the three groups (P 0.0004). On pairwise comparison, a statistical significance was observed between mild and severe epithelial dysplasias (P 0.0032) and moderate and severe epithelial dysplasias (P 0.0002).
Conclusions: The MVA was increased as the grade of dysplasia progressed. This suggests that altered vascularity could play a role in the progression and malignant potential of dysplastic lesions and also in assessing the prognosis.
Keywords: Epithelial dysplasia, histomorphometry, Masson′s trichrome, vascularity
|How to cite this article:|
Kiran SS, Kaja SL, Kattapagari KK, Shekar PC, Teja CR, Reddy BV. Histomorphometric analysis of vascularity in oral epithelial dysplasia. J NTR Univ Health Sci 2016;5:281-5
|How to cite this URL:|
Kiran SS, Kaja SL, Kattapagari KK, Shekar PC, Teja CR, Reddy BV. Histomorphometric analysis of vascularity in oral epithelial dysplasia. J NTR Univ Health Sci [serial online] 2016 [cited 2022 Jan 19];5:281-5. Available from: https://www.jdrntruhs.org/text.asp?2016/5/4/281/196563
| Introduction|| |
Cancer development is characterized by a two-step process with the initial presence of a precursor lesion (potentially malignant disorders), which can subsequently develop into a well-established cancer. Among the precursor lesions, oral leukoplakia is the best known that has a high rate of malignant transformation. Many studies have shown that 16-62% of oral carcinomas are preceded by oral leukoplakia when diagnosed.  Leukoplakia is defined as "a white plaque of questionable risk having excluded (other) known diseases or disorders that carry no increased risk for cancer."  The term epithelial dysplasia denotes the histopathological changes that have an increased risk for development of malignancy. Potentially malignant disorders such as leukoplakia and erythroplakia are associated with dysplastic changes. 
Oral epithelial dysplasia is one of the important histological points of potentially malignant disorders such as leukoplakia, erythroplakia, and erythroleukoplakia. The malignant transformation of potentially malignant disorders into oral squamous cell carcinoma was 6.6-36%. 
Knowing the pathogenesis of oral cancer is a very important aspect in terms of the patient's prognosis and therapeutic point of view. A large volume of literature shows a significant increase in the research work on the role of vascularity during transformation of normal mucosa to epithelial dysplasia and epithelial dysplasia transforming into oral squamous cell carcinoma.  However, potentially malignant disorders with epithelial dysplasia are more prone to the development of cancer than those without dysplasia. Also, the grading of epithelial dysplasia cannot accurately predict the cases that can eventually develop into cancer. Angiogenesis or neovascularization is very important for the development and progression of normal epithelium to dysplasia from dysplasia to squamous cell carcinoma.  Many biological markers have opened new horizons with their ability in prediction of the malignant potential of potentially malignant disorders among which angiogenesis, characterized by an increase in neovascularization could be an important biologic predictor of the malignant potential of the precursor lesions. 
The aim of the present study was to assess the mean vessel area (MVA) of the blood capillaries in different grades of epithelial dysplasia by histomorphometric analysis, which could aid in predicting the malignant transforming potential as well as prognosis of potentially malignant disorders.
| Materials and methods|| |
A total of 30 histopathological sections diagnosed as dysplasia from the period of 2011-2014 were selected for this study. The cases were diagnosed histopathologically as mild epithelial dysplasia, moderate epithelial dysplasia, and severe epithelial dysplasia in Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, South India. We used the World Health Organization (WHO) 2005 criteria [Table 1] for diagnosis of epithelial dysplasia. A total of 30 cases of epithelial dysplasia were divided into three groups.
|Table 1: Criteria for grading of oral epithelial dysplasia according to WHO (2005)|
Click here to view
Tissue sections of 3-4 microns were done and stained with hematoxylin and eosin stain for confirmation and grading of dysplasia. The selected cases were again sectioned and stained with Masson's trichrome to assess the mean vascular area. Photomicrographs were captured using Jenoptik (Olympus (India) Pvt Ltd) camera 3.0 and analyzed using image analysis software (Image-Pro Express version 6, Roper Technologies). The blood capillaries were identified by the endothelial cell lining and five fields for each case were captured under 40X magnification. The mean vascular area of the blood capillaries was assessed using Image-Pro Express 6.0. The mean of each case was assessed and statistical analysis was done by Kruskal-Wallis ANOVA and Mann-Whitney U test. P value <0.05 was considered to be statistically significant [Figure 1] [Figure 2] [Figure 3] [Figure 4] [Figure 5] [Figure 6].
- Group I: Mild epithelial dysplasia (10 cases)
- Group II: Moderate epithelial dysplasia (10 cases)
- Group III: Severe epithelial dysplasia (10 cases)
|Figure 1: 10× view of hematoxylin- and eosin-stained photomicrograph of mild epithelial dysplasia|
Click here to view
|Figure 2: 10× view of Masson's trichrome-stained photomicrograph of mild epithelial dysplasia with endothelial-lined blood vessels|
Click here to view
|Figure 3: 10× view of hematoxylin- and eosin-stained photomicrograph of moderate epithelial dysplasia|
Click here to view
|Figure 4: 10× view of Masson's trichrome-stained photomicrograph of moderate epithelial dysplasia showing endothelial cell-lined blood|
Click here to view
|Figure 5: 10× view of hematoxylin- and eosin-stained photomicrograph of severe epithelial dysplasia|
Click here to view
|Figure 6: 10× view of Masson's trichrome-stained photomicrograph of severe epithelial dysplasia showing endothelial cell-lined blood capillaries|
Click here to view
| Results|| |
Thirty cases of oral epithelial dysplasia were taken for this study, out of which 10 (33.3%) were mild epithelial dysplasia, 10 (33.3%) were moderate epithelial dysplasia, and 10 (33.3%) were severe epithelial dysplasia.
The mean of MVA was done by Kruskal-Wallis ANOVA. Mean and standard deviation of MVA in mild epithelial dysplasia were 4014.29 ± 2023.7, moderate epithelial dysplasia were 4070.4 ± 896.42, and in severe epithelial dysplasia were 7575.16 ± 2741.23. The MVA was highest in severe epithelial dysplasia when compared to moderate and mild epithelial dysplasias. This was statistically significant (P ≤ 0.05) [Table 2] and Graph 1].
|Table 2: Comparison of three groups (mild, moderate, and severe epithelial dysplasias) with respect to MVA by kruskal-wallis anova|
Click here to view
The pairwise comparison of mild epithelial dysplasia, moderate epithelial dysplasia, and severe epithelial dysplasia with respect to MVA was done using Mann-Whitney U-test. On comparison of mild and severe epithelial dysplasias and moderate and severe epithelial dysplasias, a high statistical significance with P value of 0.0032 and 0.0002, respectively, was obtained. But there was no statistical significance on comparison between mild and moderate epithelial dysplasias (P value 0.2899) [Table 3].
|Table 3: Pairwise comparison of three groups (mild, moderate, and severe epithelial dysplasias) with respect to MVA by mann-whitney U test|
Click here to view
| Discussion|| |
The common potentially malignant disorders of oral mucosa such as leukoplakia and erythroplakia have high chances of progression to oral squamous cell carcinoma. To detect the presence of cytological and architectural alterations of epithelial dysplasia, microscopic examination of the haematoxylin- and eosin-stained sections remains the gold standard. The criteria used for diagnosis of epithelial dysplasia through cytological and architectural changes were loss of polarity of basal cells, increased nuclear-cytoplasmic ratio, drop-shaped rete ridges, irregular epithelial stratification, increased mitotic figures, abnormal mitotic figures, presence of mitotic figures in the superficial half of the epithelium, nuclear hyperchromatism, enlarged nuclei, cellular and nuclear pleomorphism loss of intercellular adherence, and keratinization of single cells or cell groups.  The slow cellular proliferation in potentially malignant conditions could obscure the recognition of neoplastic activity and this fails to pursue the aggressive nature of the lesion, often leading to delay in the diagnosis and early intervention that adversely affects the prognosis.  Angiogenesis is essential for the proliferation of tumor cells and the level of expression of angiogenic factors could reflect the aggressiveness of the tumor.  Assessment of MVA could serve as an aid in determining the aggressiveness and prognosis of the dysplastic lesions.
The present study analyzed the MVA in different grades of epithelial dysplasia, with 10 cases in each group. The MVA area was increased as the grade of dysplasia progressed and a high statistical significance was obtained on comparison of mild and moderate epithelial dysplasias with severe epithelial dysplasia. Pujari et al. observed an increase in the MVA from the normal mucosa to leukoplakia and squamous cell carcinoma.  The increase in MVA could be attributed to various cytokines, macrophages, mast cells, and neutrophils stimulated by the altered keratinocytes of the dysplastic epithelium. The dysplastic cells could also block the inhibitors of angiogenesis, thereby promoting angiogenesis. As the grade of dysplasia increases, more number of dysplastic cells have the capability to produce chemical signals that can trigger the process of angiogenesis. Thus, angiogenesis by providing nutrients and oxygen promotes the progression of the tumor and could be used as prognostic marker to assess the aggressiveness of the dysplastic lesions and their transformation to carcinoma.
| Conclusion|| |
As the grade of dysplasia progressed the MVA also increased, suggesting a role for angiogenesis in the progression and malignant potential of the dysplastic lesions. Thus, histomorphometric assessment of MVA could serve as a prognostic factor in determining the malignant potential of the dysplastic lesions. But further studies with larger samples and long-term follow-up are needed to confirm its role.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Reibel J. Prognosis of oral pre-malignant lesions: Significance of clinical, histopathological, and molecular biological characteristics. Crit Rev Oral Biol Med 2003;14:47-62.
Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J Oral Pathol Med 2007;36:575-80.
Lumermen H, Freedman P, Kerpel S. Oral epithelial dysplasia and the development of invasive squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:321-9.
Johnstone S, Logan RM. The role of vascular endothelial growth factor (VEGF) in oral dysplasia and oral squamous cell carcinoma. Oral Oncol 2006;42:337-42.
Regezi JA, Sciubba J, Jordan RC. Oral Pathology, Clinical Pathologic Correlations. 5 th
ed. Philadelphia: Saunders; 2008. p. 48-52.
Pitiyage G, Tilakaratne WM, Tavassoli M, Warnakulasuriya S. Molecular markers in oral epithelial dysplasia: Review. J Oral Pathol Med 2009;38:737-52.
Barnes L, Eveson JW, Reichart P, Sidransky D. World Health Organization Classification of Tumours: Pathology and Genetics of Head and Neck Tumours. Lyon: IARC Press; 2005.
Rhodus NL. Oral cancer: Leukoplakia and squamous cell carcinoma. Dent Clin North Am 2005;49:143-65, ix.
Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc Health Risk Manag 2006;2:213-9.
Pujari RV, Vanaki SS, Puranik RS, Desai RS, Motupalli N, Halawar S. Histomorphometric analysis of vascularity in normal buccal mucosa, leukoplakia, and squamous cell carcinoma of buccal mucosa. J Oral Maxillofac Pathol 2013;17:334-9.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]