[version 1; peer review: 1 approved, 1 not approved]
No competing interests were disclosed.
The practice of betel nut chewing and its various custom-made preparations like pan masala, gutkha, khaini, or kharra is extensive in Southeast Asian countries including India. The chewing of these betel nut preparations has been acknowledged as one of the primary risk factors leading to oral submucous fibrosis (OSMF). It is a persistent progressive disease frequently affecting the oral and oropharyngeal mucosa and at times the superior third of mucosa of the esophagus.
Commonly oral precancerous disorders are clinically identifiable diseases that further progress to oral cancer. In general, the malignant transformation of it begins with single-cell atypia subject to genetic mutation and/or various carcinogenic factors like tobacco, areca nut, betel quid, virus, and alcohol. The malignant potential of betel nut without tobacco has been recognized and classified as a group one human carcinogen as per a cancer monograph on betel-quid and areca nut chewing and some areca nut derived nitrosoamines by II International Agency for Research on Cancer.
Prediction of the clinical behavior of oral potentially malignant disorders (OPMDs) is complex because histological characterization may not always provide prognostic judgment.
To understand the etiopathogenesis, progression of the disease, and malignant transformation in OSMF various immunohistochemical biomarkers have been studied. The association of clinical grades/stages of OSMF with molecular biomarkers is a crucial area of research. Nevertheless, the association of biomolecular expression with grades of oral epithelial dysplasia (OED) has been overlooked in cases of OSMF. OED exhibits a significant role to suggest a malignant transformation of OSMF. However due to atrophic epithelium, the evaluation of OED in OSMF is challenging. In other OPMDs, the principles advocated for OED grading are mainly based on alterations in the proliferating epithelium. Taking into consideration the characteristic “atrophic epithelium” of OSMF, these criteria will not be suitable for the assessment of OED in OSMF. In view of this, the application of biomarkers may be useful for the OED grading in OSMF.
Neoangiogenesis is considered to be an imperative characteristic in epithelial dysplastic lesions. It facilitates the requirement of nutrition for the growth and development of dysplastic cells. There are numerous angiogenic factors that are formed by the cells undergoing dysplastic changes, as well as tissue stromal cells like fibroblasts, macrophages, mast cells, and tumor cells. Further progression of dysplastic cells to invasive tumor cells and their metastasis is governed by these angiogenic factors.
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.
To evaluate CD105 expression in OSMF cases To evaluate CD105 expression in OSCC associated with OSMF cases To compare CD105 expression in OSMF and OSCC associated with OSMF cases
Clinicopathological and histopathological features of OSCC with OSMF cases. Quantitative measurement of CD105 expression (micro vessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA)) in OSMF and OSCC with OSMF.
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.
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.
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.
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).
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.
CD105 immunopositive tissue sections will be observed under a light microscope (Leica) at ×100 magnification and further by ×400 magnification.
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
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.
Retrospective cohort study.
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 follows
Where,
“Z
α/2
2: The level of significance at 5%,
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.
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.
This study will be published in an indexed journal.
Not yet started.
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.
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.
The submucosal vasculature in the early and advanced stages of OSMF and WDSCC was assessed by morphometric analysis. Murgod
Immunoexpression of CD105, E-cadherin, and VEGF in different stages of OSMF were investigated through semi-quantitative analysis in a previous study.
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”.
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”.
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”.
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”.
The study planned to be conducted is an
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.
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.
No data are associated with this article.
Zenodo: STROBE checklist for ‘Histomorphometric analysis of angiogenesis in oral submucous fibrosis and oral squamous cell carcinoma associated with oral submucous fibrosis’.
Data are available under the terms of the
We acknowledge the support of laboratory technicians from the Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe), Wardha. We also acknowledge Dr. Padmashri Kalmegh for her support in the plagiarism check of the manuscript.
The practice of betel nut chewing, which is common in Southeast Asian countries, has been identified as a significant risk factor for the development of oral submucous fibrosis (OSMF). OSMF is a progressive disease that affects the oral and oropharyngeal mucosa, as well as the esophagus. The disease is characterized by the loss of epithelial rete ridges, atrophy of the epithelium, increased fibrosis in connective tissue, reduced vascularity, and hyalinization, which leads to stiffness of the oral mucous membrane and limited mouth opening. OSMF is also associated with a high risk of malignant transformation to oral cancer, with oral epithelial dysplasia playing a significant role in assessing this potential transformation.
The study aims to evaluate the expression of CD105, a marker of angiogenesis, in OSMF and oral squamous cell carcinoma (OSCC) associated with OSMF. The objectives include evaluating CD105 expression in OSMF cases, in OSCC cases associated with OSMF, and comparing CD105 expression in OSMF and OSCC associated with OSMF. The study will also assess clinicopathological and histopathological features of OSCC with OSMF cases, as well as quantitatively measure CD105 expression in OSMF and OSCC with OSMF.
The study design involves a total of 90 cases divided into three groups: normal oral mucosa, OSMF, and OSCC with OSMF. The inclusion criteria specify the selection of samples with normal oral mucosa, clinically diagnosed cases of OSMF, and histopathologically diagnosed cases of OSCC with OSMF. Cases that have undergone presurgical radiotherapy and/or chemotherapy, as well as those with systemic diseases or coexisting malignancies, were excluded from the study.
Immunohistochemistry will be performed to evaluate CD105 expression in the extracellular matrix of OSMF and OSCC with OSMF cases. This will involve the use of paraffin blocks containing tumor tissue and normal tissue, de-paraffinization, and immunostaining with the CD105 antibody.
Morphometric analysis of tumor vessels, measured by microvessel density (MVD), total microvessel area (TVA), and mean microvessel area (MVA), will be conducted to assess the level of CD105 expression in OSMF and OSCC with OSMF. Statistical analysis will be performed to determine the expected outcome of the study, which aims to identify the expression of CD105 by immunohistochemistry in OSMF and OSCC associated with OSMF.
In conclusion, the study aims to provide insights into the association between CD105 expression and epithelial dysplasia in OSMF, as well as the potential use of CD105 immunoexpression as a marker for assessing malignant potential and guiding immunotherapy for patients with OSMF and OSCC associated with OSMF.
Major Points
1. The exclusion of moderate dysplasia and applying binary dysplasia classification to the malignant transformation of OSF raises concerns about the validity of the findings. Moderate dysplasia represents an important intermediate stage in the progression to malignancy and cannot be disregarded without justification. By excluding this category, the study may overlook critical insights into the dynamics of neoangiogenesis during the early stages of carcinogenesis in OSMF. In the context of OSMF, excluding moderate dysplasia seems questionable, as dysplasia may promote fibrosis and vice versa.
2. A study evaluated the expression of TGF-β1 protein in 30 cases of oral squamous cell carcinoma (OSCC) to understand its role in oral carcinogenesis. Results showed increased TGF-β1 expression in different oral epithelial dysplasia and OSCC stages compared to normal mucosa. TGF-β1 expression was markedly enhanced in both OSCC cells and stromal cells. The study concluded that enhanced TGF-β1 expression could be responsible for transforming oral premalignant lesions to OSCC, aggressive tumor growth, metastasis, and resistance to treatment [refer 2] .....this TGF-β1 released from dysplastic epithelium can promote fibrosis. It has also been shown in a study exploring the Relationship between Fibrosis Thickness and Epithelial Dysplasia in Oral Submucous Fibrosis. that Increased fibrosis thickness correlated with a higher incidence of epithelial dysplasia. Advancement of fibrosis heightens the risk of epithelial dysplasia development in OSMF patients. Prevention of fibrosis progression may reduce the risk of dysplasia and oral squamous cell carcinoma [refer3] Thus, a two-way loop exists between fibrosis and epithelial dysplasia in the malignant transformation of Oral submucous fibrosis, this evident in very high rate of malignant transformation rate.
3. Moreover, the assumption that the epithelium in OSMF is always in the atrophic stage is unsubstantiated and may introduce bias into the analysis [refer 4]. Anura et al explores the role of cellular competitiveness in oral submucous fibrosis (OSF) conditions, using computer-assisted neighbourhood analysis in quantitative immunohistochemistry (IHC) framework. It found that different states of OSF condition may be associated with different forms of competitiveness within epithelial neighboring cells, which may shape the present and future of the pre-malignant condition. The findings suggest that atrophic epithelium is associated with stress-driven competitive environments, while cells with high c-Myc+ give rise to hyperplastic epithelium. They stated that both hyperplastic and atropic epitltlium may co-exist in the same tissue. The heterogeneous nature of OSMF suggests that epithelial changes can vary widely among cases, making it essential to consider multiple phenotypic presentations rather than imposing a predetermined assumption.
4. Coexisting oral leukoplakia and erythroplakia further complicate the interpretation of findings, and have a higher malignant transformation rate in the background of OSMF. These conditions have distinct pathophysiological characteristics that may confound the assessment of neoangiogenesis, specifically in OSMF and OSCC with OSMF. The study's conclusions may lack specificity and clinical relevance without appropriate stratification or control for these variables.Also, the proposal of a grading system without considering the potential harm of biopsies promoting fibrosis and malignant transformation raises ethical concerns.
5. In summary, the article's methodological limitations, flawed interpretation of results, and inadequate consideration of confounding variables, ethical concerns undermine its scientific rigor and relevance. As such, it cannot be accepted for publication in its current form.
Is the study design appropriate for the research question?
No
Is the rationale for, and objectives of, the study clearly described?
No
Are sufficient details of the methods provided to allow replication by others?
No
Are the datasets clearly presented in a useable and accessible format?
No
Reviewer Expertise:
Oral Cancer, Epigenetics, Oral Potentially Malignant Disorders, Salivary Gland tumors
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
The manuscript is well written, all the morphometric analysis and details have been explained.
the manuscript is properly readable, plagiarism is not noted,
ethical policies have been followed.
The study design is properly framed.
The article is fulfilling all aspects and can be considered for publication.
The source of data is appropriate.
Is the study design appropriate for the research question?
Yes
Is the rationale for, and objectives of, the study clearly described?
Yes
Are sufficient details of the methods provided to allow replication by others?
Yes
Are the datasets clearly presented in a useable and accessible format?
Yes
Reviewer Expertise:
MOLECULAR BIOLOGY, NEO -ANGIOGENESIS, TUMOR MARKERS
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.