Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Mohan Z Mani

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Dr Mohan Z Mani,
Professor & Head,
Department of Dermatolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Research Protocol
Year : 2023 | Month : October | Volume : 17 | Issue : 10 | Page : EK01 - EK04 Full Version

Analysis of BRAF V600E in Precancerous and Cancerous Lesions of Colorectum by Immunohistochemistry: A Research Protocol


Published: October 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/60304.18447
Shreya Giri Goswami, Arvind Bhake, Sunita Vagha

1. Junior Resident, Department of Pathology, Jawaharlal Nehru Medical College, DMIMS (DU), Wardha, Maharashtra, India. 2. Professor and Director, Department of Pathology, Jawaharlal Nehru Medical College, DMIMS (DU), Wardha, Maharashtra, India. 3. Head, Department of Pathology, Jawaharlal Nehru Medical College, DMIMS (DU), Wardha, Maharashtra, India.

Correspondence Address :
Dr. Shreya Giri Goswami,
Department of Pathology, Datta Meghe Institute of Medical Sciences, DMIMS (DU), Wardha-442004, Maharashtra, India.
E-mail: shreya18g@gmail.com

Abstract

Introduction: Colorectal cancers have been extensively studied in recent years to understand their molecular abnormalities and their impact on treatment outcomes, as they continue to be a major global health burden. The Adenomatous Polyposis Coli (APC) β-catenin pathway and the Mitogen-activated Protein Kinase (MAPKs) pathway are the topic of extensive research in colorectal pathology.

Need of the study: The mutation in B-type Rapidly Accelerated Fibrosarcoma (BRAF) kinase is known to be associated with the pathogenesis and progression of precancerous lesions to cancerous lesions. Precancerous lesions carry a high risk of developing Adenocarcinoma. Therefore, the expression of BRAF can serve as a useful predictive and prognostic biomarker for disease outcomes and establish correlations with other clinicopathological parameters.

Aim: The present study aims to assess the immunoexpression of BRAF V600E in precancerous and cancerous lesions of the colorectum, as well as the relationship between BRAF expression, histological grading, and the Tumour, Lymph Nodes, and Metastasis (TNM) staging of colorectal cancers.

Materials and Methods: This will be an ambispective observational study (both prospective and retrospective) conducted at the Department of Pathology, Rural Hospital, Wardha, Maharashtra, India, over a two-year period (Retrospective data: August 2021 to July 2022, Prospective data: August 2022 to July 2023). A total of 25 cases each of precancerous and cancerous colorectal lesions will be included. Immunohistochemical expression of BRAF V600E will be performed on tissue sections from each case, and scoring will be conducted. The relationship between BRAF expression and clinicopathological parameters (such as tumour site, tumour size, number of positive lymph nodes, and perineural invasion), histological grade, and TNM stage will be assessed. The results will be analysed using Statistical Package for the Social Sciences (SPSS) software, version 27.0. The Chi-square test will be used to investigate the relationship and association between BRAF V600E expression and clinicopathological parameters. Furthermore, Pearson’s correlation coefficient method will be utilised to determine the correlation between two parameters. A significance level of 95% (p-value <0.05) will be considered.

Keywords

Cancer, Immunoexpression, Mutation, Polyp, Prognosis

Cancers of the colorectum have remained a focal area in oncology due to the need to understand the pathogenetic mechanisms, molecular basis of carcinogenesis, and available treatment options. Until a decade ago, colorectal cancers were primarily assessed for their prognosis and predictive outcomes based on histological types, grade, and disease stage. Extensive studies have been conducted on the clinical situations and syndromes associated with precursor lesions, providing insights into the pathogenesis of colorectal cancers. With advancements in molecular genetics, conventional histological typing has been scrutinised, leading to the identification of various molecular pathogenetic mechanisms that can be targeted for colorectal cancer treatment (1).

Colorectal cancer is known to arise from precancerous lesions with a high risk of harbouring cancers such as adenovillous polyps, villous polyps, and other hereditary polyposis conditions. Conversely, hereditary non-polyposis cancers are also observed. The transition from known precancerous to cancerous lesions is well-documented in the literature, highlighting certain molecular defects in the cells (2),(3). Modern-day pathology, with the utilisation of defined molecular techniques, has implicated numerous candidate genes and pathways in the development of colorectal cancer. The APC β-catenin pathway and MAP kinase pathway are among the most important ones (4).

Literature reports studies that focus on the detection of mutations in components of the APC β-catenin pathway, which is a part of the Wingless-related Integration Site (WNT) pathway (5),(6). Precancerous lesions have been found to exhibit Kristen Rat Sarcoma viral oncogene homolog (KRAS) mutations with a prevalence of approximately 10%, whereas cancerous lesions show a higher frequency of such mutations (1). Loss of function of the Tumour Protein 53 (TP53) is also associated with more aggressive forms of cancer and poorer outcomes in patients with colorectal lesions (1),(7). Microsatellite instability, resulting from DNA mismatch repair deficiency, has also been linked to the development of both precancerous and cancerous colorectal lesions. Molecular changes in the mismatch repair pathway impact cell survival and proliferation in colorectal cancer development. Genes such as MutL Homolog 1 (MHL1), MutS Homolog 2 (MSH2), Transforming Growth Factor Beta Receptor 2 (TGFBR2), Bcl-2-associated X protein (BAX), BRAF, Transcription Factor 4 (TCF4), and Insulin-like Growth Factor 2 Receptor (IGF2R) within the mismatch repair pathway have been extensively reported to be associated with sporadic and familial colorectal cancers (2),(8). Hypermethylation in the absence of mutation, particularly in a subset of microsatellite-stable colon cancers, has also been demonstrated (2),(4). Among the aforementioned molecular defects, BRAF is a single gene defect that has been extensively studied in the past 10 years for both precancerous and cancerous lesions of colorectal cancer (1),(2),(3),(4),(5). However, such studies in the Indian population are scarce.

The BRAF V600E mutation is known to be associated with the pathogenesis of adenocarcinoma of the colon and the progression of colorectal lesions to cancerous ones (9),(10). Studies in the literature have found correlations between BRAF immunexpression and various clinicopathological features, such as familial vs sporadic colonic cancers, young vs old age, tumour grade, tumour size, nodal stage, TNM stage, and metastasis (11),(12). Some studies have performed BRAF immunexpression to suggest prognosis and treatment options, especially in the form of antibody therapies (12),(13),(14),(15),(16).

Based on the available literature, the BRAF mutation status has been recognised for its utility as a prognostic and predictive biomarker. The present protocol is necessary to assist clinicians in planning targeted therapies for colorectal cancers, as there is a limited number of studies on mutant BRAF in combined precancerous and cancerous lesions of the colon in the Indian population. Therefore, the aim of this study is to investigate the immunexpression of BRAF V600E in precancerous and cancerous lesions of the colorectum, as well as the clinicopathological features of colorectal cancers. The following objectives have been identified for the study:

1. To examine the immunexpression of BRAF V600E in precancerous and cancerous lesions of the colorectum.
2. To compare the frequency of immunexpression of BRAF V600E in precancerous and cancerous lesions (adenocarcinoma of the colorectum).
3. To study and correlate the immunexpression of BRAF V600E with the histological grade of colorectal cancer and lymphovascular invasion.
4. To study and correlate the immunexpression of BRAF V600E with the nodal tumour stage of colorectal cancer.
5. To correlate the immunexpression of BRAF V600E with the TNM staging of colorectal cancers.
6. To study and correlate the immunexpression of BRAF V600E with the metastasis of colorectal cancers.

Based on the results of previously published studies on the immunexpression of BRAF in colorectal cancers, it is hypothesised that BRAF plays an important role in the molecular pathogenesis of colorectal cancers and that high BRAF protein expression is associated with disease aggressiveness.

Review of Literature

Colorectal cancer is one of the most common cancers and a leading cause of death worldwide. Numerous studies have been conducted to assess and correlate BRAF with the molecular mutation status in colorectal cancers.

Kanik P et al., conducted a study on 82 colorectal cancer patients to investigate BRAF and KRAS protein expression using immunohistochemistry on paraffin-embedded tissue blocks and its correlation with various clinicopathological parameters and prognosis (1). They found that 63% of the tumours were BRAF positive, while KRAS positivity was seen in 34% of the tumours. Patients with positive lymph node status, positive perineural invasion, and preoperative serum Carcinoembryonic Antigen (CEA) levels >5.0 ng/mL showed higher BRAF immunopositivity. No significant correlation was observed between KRAS and clinicopathological parameters. Additionally, a higher positive BRAF expression was observed in advanced-stage patients compared to early-stage patients. However, the study did not find any impact on the survival of colorectal cancer patients. The findings of the study established BRAF as a useful indicator of disease aggressiveness in colorectal cancer.

Shetty O et al., conducted a study to assess the frequency of BRAF mutation patterns in colorectal cancer patients at a tertiary care centre using paraffin-embedded tissue sections (8). They analysed a total of 298 cases for BRAF gene mutation and found that three cases (1%) showed BRAF V600E mutation, which was associated with metastatic characteristics. The study revealed that the right side of the colon was predominantly affected by BRAF gene mutations. However, the study found a low frequency of BRAF alterations, which had no significant impact on patient survival.

Gonzalez-Colunga KJ et al., conducted a retrospective study on 135 cases of colon cancer (11). The objective of the study was to evaluate the diagnostic performance of BRAF and its association with histopathological characteristics in colon cancer. Out of 135 cases, 9.6% (13) had BRAF mutations, showing intense and diffuse staining on immunohistochemistry. The study revealed that tumours with BRAF mutations tended to be larger in size. The study concluded that BRAF V600E demonstrated excellent diagnostic capabilities, making it a viable alternative for molecular examinations.

Wasti H et al., conducted a study to establish a correlation between the expression of KRAS and BRAF V600E and the histological grades observed in tissue sections of colorectal cancer (12). They analysed a total of 51 cases of colorectal cancer, with 72.5% being male and 27.4% being female patients. The majority of the tumours (72.5%) were localised to the left side of the colon. KRAS expression was observed in 80.39% of the cases, whereas 39.2% of the cases showed BRAF V600E expression. The study found no significant correlation between KRAS and BRAF expression. KRAS overexpression showed a positive correlation with histological tumour grades and its variants, while BRAF V600E showed no positive correlation with histological variants and tumour grades. Additionally, the study’s findings indicated the presence of BRAF V600E mutation in the normal mucosa surrounding the tumour, suggesting that BRAF mutation may serve as an early event in the development of colorectal cancer. The study highlighted the importance of incorporating BRAF V600E as a standard biomarker in the routine diagnosis of colorectal cancer.

Material and Methods

The study will be conducted as an ambispective observational study, involving both prospective and retrospective data collection. It will be carried out in the Department of Pathology at a rural hospital in Wardha, Maharashtra, India, over a period of two years (retrospective data: August 2021 to July 2022, prospective data: August 2022 to July 2023). Ethical clearance for the study has been obtained with the approval number DMIMS (DU)/IEC/2022/1059. Written consent will be obtained from all subjects participating in the study.

Sample size calculation: The sample size calculation was performed using the incidence rate from a previous study (17). The formula used was Cochrane’s formula:

n=(Zα/2)2×p×(1-p)/E2,

where, Zα/2=Level of significance at 5% (95% Confidence interval)=1.96
p=Incidence of colorectal cancer=4.9%=0.049 (GLOBOCON 2020) (17)
E=Error of margin=10%=0.10
n=1.962×0.049×(1-0.049)/0.102=17.90

Therefore, the initial sample size of seventeen cases was increased to 25 cases each for the groups of precancerous and cancerous colorectal lesions.

Inclusion criteria: The inclusion criteria for the study are cases diagnosed with precancerous polyps/lesions and adenocarcinoma of the colorectum based on biopsy or surgical specimens.

Exclusion criteria: The exclusion criteria include colorectal biopsies with inflammatory histomorphology and glandular dysplasia, as well as cases with biopsy and blocks that do not have representative histomorphology of precancerous and cancerous lesions. Cases with deficient clinical data and details will also be excluded.

Study Procedure

Methodology and parameters studied: The study will collect patient information, including name, age, gender, registration number, unit, department, In-Patient/Out-Patient Department (IPD/OPD), as well as clinical findings such as symptoms, examination results, and provisional diagnosis. Basic laboratory work-up, including complete blood count, biochemistry, and tumour marker analysis, will also be conducted using the BRAF V600E marker from Abcam. Radiological investigations, such as abdominal sonography, barium studies, Computed Tomography (CT), and Magnetic Resonance Imaging (MRI), will be noted from the patient’s case sheets. The study will collect biopsy and surgically resected specimens, as well as specimens of colectomy for suspected precancerous and cancerous lesions of the colorectum.

Laboratory methods: Grossing of biopsy, polypectomy, and colectomy specimens will be done using different techniques (18). The sections for the specimens will be processed by an automated histokinette to make paraffin blocks using a microtome. After proper processing, Haematoxylin and Eosin (H&E) staining of paraffin sections will be carried out using standard methods. The histological grading of colorectal adenocarcinoma will be performed based on its degree of differentiation as given by WHO guidelines (19). Staging of colorectal carcinoma will be done according to the American Joint Committee on Cancer (AJCC) guidelines and Dukes classification (Table/Fig 1),(Table/Fig 2) (20),(21).

Immunohistochemical assessment: The paraffin-embedded sections of biopsy and surgically resected specimens diagnosed as precancerous or cancerous lesions of the colorectum (adenocarcinoma) will be selected for immunohistochemistry. Only representative blocks of the lesion will be selected for immunohistochemistry. Immunohistochemistry will be carried out using the biotin-avidin peroxidase complex method with monoclonal antibodies against BRAF V600E (22). The sections will be rinsed in Tris-Buffered Saline (TBS) and then incubated in a 10% casein solution for 10 minutes. They will be further incubated with a properly diluted primary antibody for 60 minutes. After washing with TBS, the sections will be incubated with an optimally biotinylated secondary antibody for 30 minutes. Following another wash with TBS, the sections will be incubated with freshly prepared labeled streptavidin or streptavidin biotin complex for 30 minutes. After another wash with TBS, the sections will be exposed to a DAB substrate solution. They will then be washed in running water, counterstained with haematoxylin, dehydrated, cleared, and mounted (22). Scoring of immunoexpression of BRAF V600E in cases of colorectal cancer will be done based on a semi-quantitative scoring (Table/Fig 3) system (13).

Primary outcome: The expected primary outcome of the study will be the assessment of immunopositivity of BRAF in precancerous and cancerous lesions of the colorectum.

Secondary outcome: The secondary outcome, through its results, will help determine the frequency of immunoexpression of BRAF and its correlation with histological grade, lymphovascular invasion, and TNM staging of colorectal cancer. It may also provide information on the relationship between BRAF immunoexpression and metastatic colorectal cancer and nodal stage.

Statistical Analysis

The software used will be SPSS software, version 27.0. The statistical analysis of the association and relationship between the immunoexpression of BRAF V600E and clinicopathological parameters will be conducted using the chi-square test. The correlation between two parameters will be calculated using Pearson’s correlation coefficient method. A significance level or confidence interval of 95% (p-value <0.05) will be considered statistically significant.

References

1.
Kanik P, Gajjar K, Ghosh N. Immunohistochemical localisation of KRAS and BRAF and its clinical utility in patients with colorectal cancer. Colorec Cancer [Internet]. 2018 [cited 2022 Jun 27];04(1):01-07. [crossref]
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DOI and Others

Doi: 10.7860/JCDR/2023/60304.18447

Date of Submission: Sep 20, 2022
Date of Peer Review: Nov 26, 2022
Date of Acceptance: Jun 14, 2023
Date of Publishing: Oct 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Oct 01, 2022
• Manual Googling: May 17, 2023
• iThenticate Software: Jun 10, 2023 (8%)

ETYMOLOGY: Author Origin

EMENDATIONS: 8

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