Immunoexpression of E-cadherin in oral potentially malignant disorders, oral squamous cell carcinoma and its correlation with clinicopathological parameters

Padmashri Kalmegh; Alka Hande; Madhuri Gawande; Swati Patil; Archana Sonone; Aayushi Pakhale

doi:10.12688/f1000research.132516.1

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Study Protocol

Immunoexpression of E-cadherin in oral potentially malignant disorders, oral squamous cell carcinoma and its correlation with clinicopathological parameters

[version 1; peer review: 1 approved with reservations]

Padmashri Kalmegh ¹, Alka Hande¹, Madhuri Gawande¹, Swati Patil¹, Archana Sonone¹, Aayushi Pakhale¹

Padmashri Kalmegh ¹, Alka Hande¹, [...] Madhuri Gawande¹, Swati Patil¹, Archana Sonone¹, Aayushi Pakhale¹

PUBLISHED 22 May 2023

Author details Author details

¹ Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442004, India

Padmashri Kalmegh
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Alka Hande
Roles: Investigation

Madhuri Gawande
Roles: Software

Swati Patil
Roles: Software

Archana Sonone
Roles: Visualization

Aayushi Pakhale
Roles: Project Administration

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Oncology gateway.

This article is included in the Datta Meghe Institute of Higher Education and Research collection.

Abstract

The most common malignancy of the head and neck region is “oral squamous cell carcinoma” (OSCC) because of its low survival rate and the increasing incidence in some geographic areas. The process of invasion and metastasis, which is noted in most cancers, requires loss of cell-to-cell attachment. It is therefore important to identify a marker that would help in the identification of lesions that would acquire the ability to transform into OSCC. Cell-to-cell adhesion and cell motility are monitored by E-cadherin; its loss is associated with OSCC progression. Early detection, proper analysis, and correct handling of oral potentially malignant disorder (OPMD) are beneficial to preventing its malignant progression. Thus, the purpose of the present study is to identify the expression and determine the role of E-cadherin in OPMD and OSCC.

Keywords

Cell adhesion, E-cadherin, Oral potential malignant disorder, Oral squamous cell carcinoma, Malignant transformation, Immunoexpression, Immunohistochemistry

Corresponding author: Padmashri Kalmegh

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2023 Kalmegh P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Kalmegh P, Hande A, Gawande M et al. Immunoexpression of E-cadherin in oral potentially malignant disorders, oral squamous cell carcinoma and its correlation with clinicopathological parameters [version 1; peer review: 1 approved with reservations]. F1000Research 2023, 12:524 (https://doi.org/10.12688/f1000research.132516.1) First published: 22 May 2023, 12:524 (https://doi.org/10.12688/f1000research.132516.1) Latest published: 28 Jul 2023, 12:524 (https://doi.org/10.12688/f1000research.132516.2)

Introduction

Carcinoma is the second highest cause of morbidity after cardiovascular disorders in developed countries.¹ Globally, oral squamous cell carcinoma (OSCC) positions sixth amongst all categories of carcinoma. India has the highest number of OSCC cases which contributes to one-third of the total OSCC cases all over the world. This causes a huge challenge for developing countries.² Per annum, around 75,000-80,000, new cases and 50,000-55,000 deaths from OSCC are reported in India. OSCC is the commonest type of carcinoma in India and it contributes the highest incidence among Asian countries This rise in the prevalence of OSCC is an alarming sign for community health.³^,⁴ According to the American Joint Committee on Cancer, in India, 65-75% of patients are detected in the Stage III-IV of OSCC, which is significantly higher than in western countries. As these cases are detected in the advanced phase, the treatment becomes unmanageable. The five to ten-year survival rate of OSCC patients has increased to nearly 50% over the previous few decades.⁵^,⁶ OSCC has become the major cause of morbidity and mortality in the Indian population due to the habit of chewing smokeless tobacco and areca nuts.⁷

It has been observed that oral potentially malignant disorders (OPMDs) can progress into OSCC.¹^,⁸^,⁹ Early detection, proper analysis, and correct handling of OPMDs are beneficial to prevent their malignant progression.¹ There are numerous cutting-edge diagnostic methods that can predict the progression and assess the malignant transformation of OPMDs. In order to reduce the death rate and enhance the patient’s general health, the management of OPMD is crucial. The World Health Organization (WHO) defined OPMDs as the presence of a lesion or disease that poses a risk of malignancy at the time of initial diagnosis or at a later period. The prevalence of OPMDs, in the general population, is 1-5%.⁸ In the current era, five percent of cases of OPMD are detected under the age of 30 years.⁹

According to WHO, OSCC is classified into well-differentiated, moderately differentiated and poorly differentiated; upon which the management and the life span of the patient depends.¹⁰^,¹¹ It has been observed that aggressive OSCC is preceded by the pre-invasive stage which continues for a long duration. In the preinvasive stage, along with the progression of the condition, disturbances at the molecular level occur which leads to loss of cellular cohesion.

Cellular adhesion can be defined as a process by which cells intermingle and attach to neighboring cells via specific molecules on the cellular surface. For the proper functioning of multicellular organisms, cellular adhesion is necessary. This is regulated by cell adhesion molecules (CAMs), including cellular interactions, cellular migrations, cell cycle and cellular signalling as well as in morphogenesis during the development and regeneration of tissue.¹²^,¹³ CAMs play a crucial role in various pathological conditions including inflammatory disorders, infectious and autoimmune diseases oncogenesis.¹⁴ In carcinomas, transformed cells of the epithelial layer grows abnormally, breaking through the basement membrane which leads to invasion of the underlying mesenchyme. In determining cancer prognosis, the rate of differentiation and the grade of invasiveness of carcinomas has significant value.¹⁴^,¹⁵ Atypical genetic expression is intricately involved in cell proliferation and genetic elements that regulate cellular adhesion and cellular motility are features of tumour start and progression.¹⁶

CAMs are transmembrane Ca²⁺-dependent homophilic adhesion receptors. On the basis of structure, four main families of CAMs are identified: integrin, selectin, immunoglobulin gene (IgG-like superfamily), and cadherin. In addition to serving as a molecular bond, it also has a role in maintaining cell-to-cell connections, controlling cell polarity, morphogenesis, cell recognition, and cell sorting during development.¹⁷ E-cadherin (E-Cadh) is located on the epithelial cell surface in the areas of intercellular contacts known as “adherens junction”, a major molecule in cell-to-cell adhesion.¹⁸^,¹⁹ In addition, this highly conserved molecule has a significant role in tumor development, progression and its malignant cell transformation. The association between decreased expression of E-cadh and invasive properties may not be a general phenomenon, since the invasiveness of cells and dedifferentiation of carcinomas can occur even in the presence of E-Cadh.¹⁹

E-Cadh glycoprotein encoded by CDH 1 gene represents a calcium-dependent intercellular adhesion molecule; its loss is related to invasion and metastasis in various cancer models. Thus, CDH 1 gene is considered a tumor suppressor gene. The cytoplasmic moiety of E-cadh binds to β- and γ-catenin, linked to the cytoskeleton through α-catenin, and the moiety present extracellularly is a calcium-dependent receptor that maintains the homophilic interactions. The first discovered classical cadherins were the E-cadh family, which prompts intercellular adhesion.²⁰ Reduced E-cadh expression is indicative of its function in preventing tumor invasion/metastasis. It has been observed that the probability of metastasis is increased when the activity of tumor differentiation decreased.²¹^–²³

With this premise, we designed the following protocol for the evaluation of E-cadh immunoexpression in OPMDs, OSCC, and its correlation with clinicopathological parameters.

Protocol

Aim

To evaluate E-cadh immunoexpression in OPMDs and OSCC.

Objectives

1. To assess the E-Cadh immuno-expression in OPMDs.
2. To evaluate the E-Cadh immuno-expression in OSCC.
3. To compare and co-relate the E-Cadh expression between the OPMDs, OSCC and normal mucosa.
4. To assess the immuno-expression of E-cadh in OSCC and its correlation with clinicopathological parameters.

Methods

Study design

In this cross-sectional study, a total of 90 samples will be divided into three groups: The groups are as follows:

Group I: 30 samples with OPMD.

Group II: Thirty samples with OSCC.

Group III: Thirty samples with normal oral mucosa (NOM).

Inclusion and exclusion criteria

For the OPMD group, we will select 30 samples of erythroplakia, leukoplakia, oral submucous fibrosis (OSMF), and oral lichen planus. A total of 30 samples of the NOM, which will be used as controls, will be collected from the gingival and vestibular mucosa after the extraction of the impacted teeth. The 30 samples of OSCC that have undergone surgical treatment the most frequently and have been histopathologically and clinically diagnosed will be considered in the eligibility criteria for the study.

Patients having a past history of oral malignancy, recurrent or distant disease and pre-operative chemotherapy, radiation therapy, and the study will not include patients who have undergone surgery, with the exception of biopsy.

In demographic data clinical presentation, habits and their duration, histopathological findings, and operative details will be noted in detail. Following disease-free survival for four to five years, follow-up information will be gathered. Each hematoxylin and eosin-stained tissue section will be closely examined at low power magnification (100×).

Sources of the data

After receiving approval from the institutional ethical committee [DMIMS (DU)/IEC/2022/759] at the Datta Meghe Institute of Medical Sciences, Deemed to be University, Sawangi (M), Wardha, Maharashtra, India, this study will be conducted at the Department of Oral Pathology and Microbiology, Sharad Pawar Dental College and Hospital.

A total sample size of 90 samples will be chosen after clinical and histopathological confirmation. For surgically collected OSCC samples from 2005 to 2018 in this institute, the department’s archives will be searched. A total of 90 cases will be selected randomly; study Group I – OPMD (30 samples), Group II – OSCC (30 samples), and Group III – Normal mucosa (30 samples). OSCC patients will be histopathologically graded using Broder’s grading system.

Statistical analysis

Using the Single Proportion Formula and the 2% prevalence of OPMD and OSCC cases in the Oral Pathology and Microbiology outpatient department, the sample size is determined as follows:

n = {Z_{α / 2}}^{2} \times p \times (1 - p) / E^{2}

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

n = 1.96² × 0.35 × (1-0.35)/0.10²

n = 87.39

n = 90

Formula reference - Cochran W. G. et al. (1977)²⁴

Immunostaining

A paraffin block having a suitable mass of tumor and an acceptable amount of normal tissue will be selected. On Poly-L-Lysine coated slides, sections with thicknesses of 3 μm will be cut and placed. For de-paraffinization, sections are 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 is 60 seconds. After washing all the sections will be transferred to a Coplin jar containing the retrieval buffer solution. The solution which is used for antigen retrieval will be composed of 30 mL of retrieval solution in 1500 mL of distilled water for 15 to 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 five minutes at room temperature. This step is repeated thrice. 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. E-cadh will be applied at room temperature for one hour. Once again washing of sections will be done in Tris buffer solution thrice five minutes each. Visualization will be performed by utilizing a labelled polymer for 30 minutes at room temperature. For washing the sections Tris buffer solution will be used thrice for five minutes.

The application of the DAB (3,3′-diaminobenzidine) substrate will be done for 15 to 20 minutes. The working DAB solution is comprised of the following – one mL of DAB buffer and 25 μL of DAB concentrate. This time, 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 will be done for five minutes. Again, the washing of sections will done under tap water. These will be dried; once the sections are dried, they will be mounted in DPX. After that, examination will be carried out under a microscope.

Expected results

The current study will determine the expression of E-cadh by immunohistochemistry in OPMD and OSCC. There will be variation in the immunoexpression of E-cadh amongst the OPMD and OSCC. Further, there will be a positive correlation of E-cadh immunoexpression with the clinicopathological parameters of OSCC.

Dissemination

Results will be published in an indexed journal.

Study status

The proposed study is under process.

Discussion

E-cadherin

Current research has directed to awareness of the mechanism of cellular adhesion. It observed that a strong intercellular adhesion is necessary for the formation of tight tissue sheets. This is one of the classical properties of the generation of epithelia. In order to fulfill the accomplishment of cell-to-cell adhesion, the composition and function of cells of the epithelium must be strongly maintained. E-cadh is a type I superfamily member and a calcium-dependent transmembrane glycoprotein. It is also known as the invasion or tumor suppressor gene which is important for regulating the structural integrity and organization of the epithelium. It is determined by the CDH-1 gene that is located on chromosome 16q-22.1.²⁵^–²⁷

Gurkiran Kaur et al. (2009) assessed the E-Cadh expression in OSCC by immunohistochemistry (IHC) in 37 samples. They concluded that “E-Cadh expression is reduced in advanced cases of OSCC and inverse relation between loss of cell differentiation, cellular adhesion and E-Cadh”.²⁸

Yuwanati et al. (2011) investigated E-cadh expression in OPMD and OSCC by in vivo study. There was evidence that during the progression of OPMD to OSCC, a significant role was played by E-cadh. In order to compare E-cadh expression in normal healthy mucosa, OPMD and OSCC, 20 cases of each OPMD and OSCC were included in the study. After they studied 40 cases in context to patient’s age, sex, tumor location, TNM classification, and clinical stage, it was concluded that decreased expression of E-cadh may serve as a helpful indicator for the transformation of OPMD into OSCC.²⁹

von Zeidler et al. (2014) observed the function of E-cadh as a significant biomarker in the malignant tranformation of OPMDs. For E-cadh immunostaining, they excised specimens surgically diagnosed with OPMDs and OSCC. They concluded that dysplastic changes in the epithelium increase the risk of malignant transformation, which decreases E-cadh expression and therefore E-cadh can be utilized as a potential biomarker to find lesions with a high risk of developing into cancer.³⁰

Akhtar et al. (2016) investigated the diagnostic and prognostic significance of E-Cadh in OSCC metastasis. This was an –in vivo study, where biopsies and specimens were evaluated for all premalignant lesions as well as OSCC. Since it was an in vivo trial, alterations brought on by therapy and patient follow-up were also examined. Their research determined that E-cadh immunohistochemistry stains can be used to measure the invasiveness and recurrence of OSCC. Also, they noted that the biomarker E-cadh may be used in upcoming studies on early detection, diagnosis, and patient survival.³¹

Kushwaha et al. (2019) evaluated the immunohistochemical and histopathological expression of E-cadh in OSCC. In addition, the evaluation of qualitative and quantitative expressions of E-cadh and its correlation with the number of tumor cells was done. There were 20 samples of well-differentiated OSCC, 20 samples of moderately differentiated OSCC, and 10 samples of normal mucosa. The result was that there was a notable reduction in E-cadh expression as OSCC advanced from well-differentiated to higher histological grades. The authors further concluded that E-cadh was a reliable indicator for evaluating the invasiveness of OSCC.³²

Ilangani Sathish et al. (2020) evaluated E-Cadh expression in OPMD, in a prospective study. Their study was intended to evaluate the relationship amid the E-Cadh expression & OPMD. For this, 50 patients were chosen, of whom 25 samples were sent for histological analysis and the other 15 patients underwent real-time PCR to assess E-Cadh expression. According to their analysis, “high E-Cadh expression in OPMD & also concluded reduction in E-Cadh expression can be employed as tumor marker which might determine the progression of normal and OPMD to OSCC”.³³

Limitations

The study to be conducted is an in-vitro, cross-sectional study, and its application in clinical practice is recommended.

Interpretation

The immunoexpression of E-cadh may grow from NOM to leukoplakia to OSCC.

Generalisability

A review of the literature reveals a correlation between the expression of E-cadh and the development of OPMDs and OSCC cases. In the case of OPMDs, the disease’s development and prognosis might be tracked. The management of immunotherapy for different patients might be considered based on the E-cadh immunoexpression status.

Ethical considerations

Ethical approval was received from the institutional ethical committee at the “Datta Meghe Institute of Medical Sciences, Deemed to be University, Sawangi (M), Wardha, Maharashtra, India”.

Data availability

Underlying data

No data are associated with this article.

Reporting guidelines

Zenodo: STROBE checklist for “Immunoexpression of E-Cadherin in oral potentially malignant disorders, oral squamous cell carcinoma and its correlation with clinicopathological parameters”, https://doi.org/10.5281/zenodo.7715199.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgements

We acknowledge the support of laboratory technicians from Department of Oral and Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College and Hospital, Datta Meghe Institute of Higher Education and Research, Sawangi (Meghe), Wardha.

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Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 22 May 2023

Author details Author details

¹ Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, 442004, India

Padmashri Kalmegh
Roles: Conceptualization, Data Curation, Formal Analysis, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Alka Hande
Roles: Investigation

Madhuri Gawande
Roles: Software

Swati Patil
Roles: Software

Archana Sonone
Roles: Visualization

Aayushi Pakhale
Roles: Project Administration

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (2)

version 2

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Published: 28 Jul 2023, 12:524

https://doi.org/10.12688/f1000research.132516.2

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https://doi.org/10.12688/f1000research.132516.1

© 2023 Kalmegh P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Kalmegh P, Hande A, Gawande M et al. Immunoexpression of E-cadherin in oral potentially malignant disorders, oral squamous cell carcinoma and its correlation with clinicopathological parameters [version 1; peer review: 1 approved with reservations]. F1000Research 2023, 12:524 (https://doi.org/10.12688/f1000research.132516.1)

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Open Peer Review

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Reviewer Report 20 Jul 2023

Reetoja Nag, Department of Biomedical Engineering, Emory University, Atlanta, Georgia, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.145444.r186793

Given the high incidence of OSCC in India, this study is significant in the sense that assessment of E-cadherin expression in OPMDs may help in early detection and prevention of OSCC. However, the authors can take a few points in ... Continue reading

The authors plan on checking the E-cadherin expression in different types of OPMDs (erythroplakia, leukoplakia, oral lichen planus, oral submucous fibrosis).They should take into consideration that there have been studies in which the E-cadherin expression is different for different types of OPMDs and ensure that there is uniform distribution of the different types of OPMDs (within the OPMD section)so that there is minimal chance of bias (Reference: Sridevi, Ugrappa, et al. "Expression of E-cadherin in normal oral mucosa, in oral precancerous lesions and in oral carcinomas." European journal of dentistry 9.03 (2015): 364-372).
The authors should give a short detail about how they are assessing the E-cadherin expression between the groups (Normal, OPMD, OSCC). Are they checking the intensity of the expression or any other feature(s)? Are they doing visual assessment or using any image analysis software for this purpose? If they are using image analysis software, are they taking reference from recently conducted studies or using a different methodology of their own?
If possible, it would be very useful if the authors can show some initial results of their methodology, from publicly available datasets/cases. For example: Some university archives may provide data upon request, or some websites like human protein atlas (https://www.proteinatlas.org/), TCGA (https://www.cancer.gov/ccg/) may contain cases/datasets.

Is the rationale for, and objectives of, the study clearly described?

Partly
Is the study design appropriate for the research question?

Partly
Are sufficient details of the methods provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?

Partly

References

1. Sridevi U, Jain A, Nagalaxmi V, Kumar UV, et al.: Expression of E-cadherin in normal oral mucosa, in oral precancerous lesions and in oral carcinomas.Eur J Dent. 2015; 9 (3): 364-372 PubMed Abstract | Publisher Full Text

Competing Interests: No competing interests were disclosed.

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, however I have significant reservations, as outlined above.

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Author Response 28 Jul 2023

Padmashri Kalmegh, Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Wardha, 442004, India

28 Jul 2023

Author Response

Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method ... Continue reading Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method to assess the intensity of E-cadherin under the heading Assessment of E-cadherin immunoreactivity.

Respond to comment 3.

The results for the methodology from previous studies/researches has been mentioned under discussion subheading.

Following studies has been referred.
Gurkiran Kaur et al. (2009)
Yuwanati et al. (2011)
von Zeidler et al. (2014)
Sridevi et al. (2015).
Akhtar et al. (2016)
Kushwaha et al. (2019)
Ilangani Sathish et al. (2020)

Thank you.
Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method to assess the intensity of E-cadherin under the heading Assessment of E-cadherin immunoreactivity.

Respond to comment 3.

The results for the methodology from previous studies/researches has been mentioned under discussion subheading.

Following studies has been referred.
Gurkiran Kaur et al. (2009)
Yuwanati et al. (2011)
von Zeidler et al. (2014)
Sridevi et al. (2015).
Akhtar et al. (2016)
Kushwaha et al. (2019)
Ilangani Sathish et al. (2020)

Thank you.
Competing Interests: No competing interests were disclosed. Close
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Author Response 28 Jul 2023

Padmashri Kalmegh, Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Wardha, 442004, India

28 Jul 2023

Author Response

Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method ... Continue reading Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method to assess the intensity of E-cadherin under the heading Assessment of E-cadherin immunoreactivity.

Respond to comment 3.

The results for the methodology from previous studies/researches has been mentioned under discussion subheading.

Following studies has been referred.
Gurkiran Kaur et al. (2009)
Yuwanati et al. (2011)
von Zeidler et al. (2014)
Sridevi et al. (2015).
Akhtar et al. (2016)
Kushwaha et al. (2019)
Ilangani Sathish et al. (2020)

Thank you.
Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method to assess the intensity of E-cadherin under the heading Assessment of E-cadherin immunoreactivity.

Respond to comment 3.

The results for the methodology from previous studies/researches has been mentioned under discussion subheading.

Following studies has been referred.
Gurkiran Kaur et al. (2009)
Yuwanati et al. (2011)
von Zeidler et al. (2014)
Sridevi et al. (2015).
Akhtar et al. (2016)
Kushwaha et al. (2019)
Ilangani Sathish et al. (2020)

Thank you.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 22 May 2023

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Version 2 (revision) 28 Jul 23	read	read
Version 1 22 May 23	read

Reetoja Nag, Emory University, Atlanta, USA
Sankar Jagadeeshan, Ben-Gurion University of the Negev, Beer-Sheva, Israel

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Reviewer Report

5 Views

26 Oct 2023 | for Version 2

Sankar Jagadeeshan, Ben-Gurion University of the Negev, Beer-Sheva, Israel

5 Views Cite this report Responses(0)

Not Approved

In this study protocol the authors wanted to study the immunoexpression of E-cadherin in OPMD and OSCC in patients.

The authors failed to provide a clear rationale for why specifically assessing the expression of E-cadherin in OPMD and OSCC, also there are multiple studies that has evaluated the expression of E-cadherin in normal oral mucosa to oral lesions to oral carcinoma. Thus, the novelty of the study protocol is in question.
In the methodology section, the authors mentioned that they will be using 30 samples each of OPMD, OSCC and normal oral mucosa. As OPMD and OSCC has many subclasses, how are the authors going to get a clear inference from the results if they are using only 1 or 2 samples from each subclass. For example, OPMD comprises of oral leukoplakia, erythroplakia, oral submucous fibrosis (OSMF), proliferative verrucous leukoplakia and others, if the authors include one from each subclass and look for the E-cadherin expression then the result will vary and will be biased. The authors need to include atleast 10 samples from each subclass and statistically analyse the variation of subclasses with the main class. Like-wise OSCC, have weakly, moderately and well differentiated tumors, E-cadherin expression in these categories varies and will complicate the analysis as well as results. So the study design has to be changed and should include a comprehensive approach with strong statistical support.
The authors need to mention the antibody (the source, dilution etc) for better reproducibility.
The authors main objective is to analyse the expression of e-cadherin during the progression of oral carcinogenesis. The authors have not provided any information how they are going to rule out the demographic/ patient individual parameters such as habits influence the expression of E-cadherin.
Since the authors, the authors provide this manuscript under study protocol section, the authors need to provide the representative images of the immunostained sections of oral tissue representing low staining intensity, moderately staining intensity, Strong stained. Also, for pattern of E-cadherin expression in membrane, in cytoplasmic, and both membranous and cytoplasmic staining.
How could the authors interpret that the immunoexpression of E-cadherin may grow from NOM to leukoplakia to OSCC, when the study has not been completed and its vice versa as per previous findings on the same objective?
The authors need to clearly mention how this present study protocol is different from previous studies and how the results of this study is unique by its own?
In the discussion part, the authors have mentioned the findings of the previous similar studies but did not mention what are the drawbacks of those studies and how the authors new study protocol overcome the limitations of the previous studies.

Is the rationale for, and objectives of, the study clearly described?

No
Is the study design appropriate for the research question?

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

Competing Interests

No competing interests were disclosed.

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.

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7 Views

02 Aug 2023 | for Version 2

Reetoja Nag, Department of Biomedical Engineering, Emory University, Atlanta, Georgia, USA

7 Views Cite this report Responses(0)

Approved

The authors have answered the comments appropriately

Competing Interests

No competing interests were disclosed.

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.

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20 Views

20 Jul 2023 | for Version 1

Reetoja Nag, Department of Biomedical Engineering, Emory University, Atlanta, Georgia, USA

20 Views Cite this report Responses(1)

Approved With Reservations

The authors plan on checking the E-cadherin expression in different types of OPMDs (erythroplakia, leukoplakia, oral lichen planus, oral submucous fibrosis).They should take into consideration that there have been studies in which the E-cadherin expression is different for different types of OPMDs and ensure that there is uniform distribution of the different types of OPMDs (within the OPMD section)so that there is minimal chance of bias (Reference: Sridevi, Ugrappa, et al. "Expression of E-cadherin in normal oral mucosa, in oral precancerous lesions and in oral carcinomas." European journal of dentistry 9.03 (2015): 364-372).
The authors should give a short detail about how they are assessing the E-cadherin expression between the groups (Normal, OPMD, OSCC). Are they checking the intensity of the expression or any other feature(s)? Are they doing visual assessment or using any image analysis software for this purpose? If they are using image analysis software, are they taking reference from recently conducted studies or using a different methodology of their own?
If possible, it would be very useful if the authors can show some initial results of their methodology, from publicly available datasets/cases. For example: Some university archives may provide data upon request, or some websites like human protein atlas (https://www.proteinatlas.org/), TCGA (https://www.cancer.gov/ccg/) may contain cases/datasets.

Is the rationale for, and objectives of, the study clearly described?

Partly
Is the study design appropriate for the research question?

Partly
Are sufficient details of the methods provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?

Partly

References

Competing Interests

No competing interests were disclosed.

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Responses (1)

Author Response

28 Jul 2023

Padmashri Kalmegh, Department of Oral & Maxillofacial Pathology and Microbiology, Sharad Pawar Dental College & Hospital, Datta Meghe Institute of Higher Education and Research, Wardha, 442004, India

Respond to comment 1.

I have added suggested information and reference under the discussion subheading, cited as 34.

Respond to comment 2.

I have mentioned the method to assess the intensity of E-cadherin under the heading Assessment of E-cadherin immunoreactivity.

Respond to comment 3.

The results for the methodology from previous studies/researches has been mentioned under discussion subheading.

Following studies has been referred.
Gurkiran Kaur et al. (2009)
Yuwanati et al. (2011)
von Zeidler et al. (2014)
Sridevi et al. (2015).
Akhtar et al. (2016)
Kushwaha et al. (2019)
Ilangani Sathish et al. (2020)

Thank you.

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Immunoexpression of E-cadherin in oral potentially malignant disorders, oral squamous cell carcinoma and its correlation with clinicopathological parameters

Abstract

Keywords

Introduction

Protocol

Aim

Objectives

Methods

Study design

Sources of the data

Statistical analysis

Dissemination

Study status

Discussion

E-cadherin

Limitations

Interpretation

Generalisability

Ethical considerations

Data availability

Underlying data

Reporting guidelines

Acknowledgements

References

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