Histomorphometric analysis of angiogenesis in oral submucous fibrosis and oral squamous cell carcinoma associated with oral submucous fibrosis

Aim

The aim of this study will be to appraise the molecular basis of neoangiogenesis in terms of expression of CD105 in the extracellular matrix of OSMF, and OSCC with OSMF cases.

Objectives

Variables measures

Study design

In this project, a total of 90 cases will be divided into three groups as follows: i) Group I, 30 cases with normal oral mucosa; ii) Group II, 30 cases with oral submucous fibrosis; and iii) Group III, 30 cases with oral squamous cell carcinoma with oral submucous fibrosis. Appropriate details from the STROBE checklist have been included in this study protocol.²⁸

Inclusion criteria

Overall, 30 samples with normal oral mucosa retrieved during minor surgical procedures like removal of pericoronal flap, disimpaction of third molar will be used as controls. On the basis of appropriate clinical symptoms of OSMF like, inability to endure hot and spicy food beverages as well as difficulty in mouth opening, 30 clinicopathologically diagnosed cases of OSMF will be selected. On examination, the clinical signs show conspicuous fibrotic bands with rubbery blanching of the oral mucous membrane. The 30 samples of OSCC with OSMF that have undergone surgical treatment and have been histopathologically and clinically diagnosed will be included in the study.

Exclusion criteria

Cases who had undergone presurgical radiotherapy and/or chemotherapy, history of second primary or local and distant recurrence and patients with any other systemic diseases and with coexisting malignancies were excluded from the study.

Sources of the data

We have received approval from The Institutional Ethical Committee [DMIMS (DU)/IEC/2022/761, dated 14/02/2022] of Datta Meghe Institute of Higher Education and Research, (DU), Sawangi(M), Wardha, Maharashtra, India. This project will be undertaken at the Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital.

The study will comprise 30 normal oral mucosa samples (group I) and 30 clinicopathologically diagnosed cases of OSMF (group II).¹⁵ The included cases of OSMF will be histologically evaluated. OED in OSMF will be recorded on the basis of its presence or absence. Furthermore, cases will be categorized into two groups, low-risk epithelial dysplasia (LRED) and high-risk epithelial dysplasia (HRED).¹⁶ On the basis of the association of OSMF, 30 cases of histopathologically diagnosed, surgically treated OSCC cases of various histopathological grades will be retrieved from the archives of the department (group III). Three oral pathologists independently performed histopathological grading of all OSCC cases using Broders grading system in a blinded manner. The archival tissue section of 4 μm will be obtained for groups I, II, and III. Hematoxylin and eosin stains will be used for routine staining procedures and immunohistochemistry for the expression of CD105.

Immunostaining

Paraffin blocks with tumor tissue and normal tissue will be selected. For de-paraffinization, sections will be placed in the xylene solution. Sections will be rehydrated by subjecting them to descending concentrations of alcohol. In order to wash sections, tap water will be used. The washing time for sections in distilled water should be 60 seconds. After washing all the sections, they will be transferred to a Coplin jar containing the retrieval buffer solution. The solution that is used for antigen retrieval is composed of 30 ml of retrieval solution in 1,500 ml of distilled water for 15-20 minutes in the pressure cooker. Cooling will be done at room temperature.

Sections will be dipped once in distilled water. Sections will be washed with Tris buffer solution for at least 5 minutes at room temperature. This step will be repeated three times. For peroxidase blocking, a mixture of 3-5% hydrogen peroxide and methanol will be used for 30 minutes. Tris buffer solution will be used for washing the sections three times for five minutes each. CD105 will be applied at room temperature for 1 hour. Once again washing of sections will be done in Tris buffer solution three times for five minutes each. Envision technique will be performed by utilizing a labeled polymer for 30 minutes at room temperature. To wash the sections, Tris buffer solution will be used three times for five minutes.

The application of the 3,3′-diaminobenzidine (DAB) substrate will be done for 15 to 20 minutes. The working DAB solution is comprised of the following, 1 ml of DAB buffer and 25 ml of DAB concentrate. Washing of sections will be done by Tris buffer, for 15-20 minutes. Sections will be cleaned in distilled water. For counterstaining, Mayer’s hematoxylin will be used, which is done for five minutes. Again, the washing of sections will be done under tap water. Sections are then dried, following which they are mounted in DPX and examined under a microscope.

Evaluation of immunopositive tissue sections

CD105 immunopositive tissue sections will be observed under a light microscope (Leica) at ×100 magnification and further by ×400 magnification.

Morphometric analysis of tumor vessels (CD105 immunopositive cells)

To determine the MVD within and surrounding the tumor, the tissue sections immunostained with the CD105 antibody (Diluted 1:30, Monoclonal Mouse Anti-Human CD105, Endoglin, Clone SN6 h; Product code: M3527, Dako, North America Inc.) will be inspected under a light microscope (Leica) at ×400 magnification, as recommended by Weidner et al.¹⁷ The scanning of tissue sections will be carried out at ×100 to choose two fields with the highest MVD (“hot spots”) and further microvessels will be counted at ×400 magnification.

The identification of every independently stained microvessel in each particular field will be done. Furthermore, the outline of the recognized microvessel will be traced by an image analysis system to determine the MVD, TVA and MVA within and surrounding the tumor tissue sections.

Study design

Retrospective cohort study.

Statistical analysis

Using the single proportion formula and the 2% prevalence of OSMF and OSCC cases in the Oral Pathology and Microbiology Outpatient Department, the sample size was determined as follows¹⁸:

Where,

“Z_α/2²: The level of significance at 5%, i.e., 95% confidence interval = 1.96.

p: sample showing positive E-cadherin expression focally in small group cells in the basal layer of epithelium = 35% = 0.35.

E: error of margin = 10% = 0.10.

$\mathrm{n}={1.96}^2\times 0.35\times \left(1-0.35\right)/{0.10}^2$

$\mathrm{n}=87.39$

$\mathrm{n}=90$”

Expected outcome

The present study will determine the expression of CD105 by immunohistochemistry in OSMF and OSCC associated with OSMF.

We postulate the progressively increased vascularity with the disease progression from LRED to HRED and further its transformation to invasive squamous cell carcinoma. This increased vascularity will be evident by enhanced MVD, TVA and MVA, which is expressed by CD105 immunoexpression. This observation emphasizes the significance of neoangiogenesis in cases of OSMF with epithelial dysplasia and its further progression to OSCC.

Dissemination

This study will be published in an indexed journal.

Study status

Not yet started.

Vascularity/angiogenesis in OSMF

Neoangiogenesis is an essential and fundamental process in development and progression, as well as in the formation of granulation tissue and healing of the wound. Moreover, it is also a crucial stride in the conversion of benign to aggressive, malignant tumors. In oral precancerous disorders the cells undergo alterations during the progression of carcinogenic process. One of the initial and possibly most considerable characteristics expressed by pre-cancerous cells is their capability to generate a neovascular response that is angiogenesis. Therefore, it could be assumed that the expression of neoangiogenic activity may possibly be representative of malignant potential that is attained prematurely in the carcinogenic process.

The abnormal alterations of the microvessels in 27 patients of OSMF were investigated by immunohistochemistry. Microvessel quantity and microvessel area were deliberated by an image analysis system. They observed enhanced microvessel quantity in the primary stage of OSMF and reduced MVA in the intermediate and late stages. From these study results they recommended that hyperplasia of microvessels takes place in the early stages of OSMF.¹⁹ They reported that the typical tissue reaction consequential to hypoxia does not seem to work in this disease. The enhanced vascular dilatation predominantly present in tissue is indicative of tissue adaptability to compensate for hypoxia due to extensive fibrosis.²⁰

The microvessel and mast cell density in patients of various stages of OSMF and normal mucosal tissue was investigated by immunohistochemistry and further associated with the disease progression. The molecular markers, anti-mast cell tryptase and anti-factor VIII related von Willebrand Factor for endothelial cells were used. They observed significantly enhanced microvessel and mast cell density in various stages of OSMF cases. They also observed a positive association between microvessel and mast cell density. They concluded that enhanced microvessel and mast cell density plays a greater role in the pathophysiology of OSMF.²¹

The submucosal vasculature in the early and advanced stages of OSMF and WDSCC was assessed by morphometric analysis. Murgod et al.,²² investigated 30 histopathologically confirmed cases of OSMF from early and advanced clinical stages and WDSCC for assessment of microvascular density and microvascular luminal diameter. They observed significantly enhanced expression in the context of microvascular density and microvascular luminal diameter. The microvascular density and microvascular luminal diameter were considerably better in primary stages of OSMF and WDSCC as compared to normal mucosa, and also in an advanced stage of OSMF as well as in WDSCC in comparison to primary stages of OSMF. The enhanced vascularity from normal to precancerous and cancer tissue highlights the significance of neoangiogenesis in tumor development and progression.²²

Immunoexpression of CD105, E-cadherin, and VEGF in different stages of OSMF were investigated through semi-quantitative analysis in a previous study.²³ They observed upregulation of CD105, E-cadherin, and VEGF expression gradually increasing from without epithelial dysplasia tissue toward advanced grades of dysplasia. They observed that the direct correlation between lesser expression of the E-cadherin molecule and enhanced CD105 expression suggests neoangiogenic attributes of OSMF with an increase in dysplastic grades. This could be used to assess the malignant potential of OSMF.²³

Angiogenesis in OSCC

CD105 expression in 51 patients of OSCC was investigated by immunohistochemistry. MVD was evaluated by measuring the number of CD105-immunostained blood vessels. They revealed that CD105 immunoexpression is enhanced in OSCC as compared to healthy normal oral mucosal tissue. They concluded that “CD105 has a considerable function in the growth and progression of OSCC, which may be reasonably explicit as compared to other endothelial markers”.²⁴

In a previous study, MVD in 27 HNSCC patients was evaluated by immunohistochemistry against CD34 and CD105 antibodies. They observed significantly higher MVD in the T3-T4 stage (advanced clinical stage) of tumors. In the case of tumors with positive lymph nodes, MVD was considerably elevated. On the correlation of MVD with survival data it was observed that the cases with an elevated MVD had a considerably reduced disease-free survival, whereas MVD expressed by CD34 had no correlation with a status of survival. They concluded that “increased expression of CD105 is considered as the single self-determining tumor recurrence marker”.²⁵

CD105 expression and MVD in 19 surgically operated specimens of OSCC were evaluated by immunohistochemistry. The MVD was determined by the “hot spot method”. They observed higher expression of MVD at the central and invading front of OSCC. Furthermore, MVD was higher in the peritumoral region as compared to the intratumoral region and the difference was statistically significant. Thus, they concluded that “enhanced expression of CD105 in the tumor is considered as an imperative predictive marker for the outcome in OSCC”.²⁶

Expression of VEGF and CD105 in 54 cases of OSCC were investigated by immunohistochemistry. They observed increased VEGF expression in OSCC specimens in contrast to premalignant and normal healthy oral tissues. Furthermore, VEGF expression is decreased in poorly differentiated oral squamous cell carcinoma (PDOSCC) as compared to moderately differentiated oral squamous cell carcinoma (MDOSCC) and well differentiated oral squamous cell carcinoma (WDOSCC), and MVA was elevated in OSCC in comparison to potentially malignant lesions and healthy tissues of the oral mucosa. They concluded that “VEGF and CD105 may be considered as reliable markers of tumor angiogenesis and progression in OSCC”.²⁷

Limitations

The study planned to be conducted is an in vitro, cross-sectional study. Therefore, a long term prospective follow up study with a larger sample size is recommended.

Interpretation

It will be possible to see the progressively increased vascularity evident by CD105 immunoexpression with the disease progression from LRED to HRED and further its transformation to invasive squamous cell carcinoma.

Generalizability

A review of the literature reveals an association between CD105 expression and epithelial dysplasia in OSMF. Furthermore, on the basis of neoangiogenesis evaluated by CD105 immunoexpression with MVD, MVA, and TVA, the progression of epithelial dysplasia towards malignancy might be discernible. The management of immunotherapy for different patients might be considered based on the CD105 immunoexpression status.