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

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Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
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

Important Notice

Original article / research
Year : 2009 | Month : December | Volume : 3 | Issue : 6 | Page : 1891 - 1899

The Immunohistochemical Evaluation Of The Expression Of Bcl-2 In Different Histological Grades Of Squamous Cell Carcinoma

SURI C

* (MDS) – Oral & Maxillofacial Pathology Sri Sai College of Dental Surgery Vikarabad

Correspondence Address :
Dr. Charu Suri,Address: Dept of Oral & Maxillofacial Pathology,Sri Sai College of Dental Surgery,R.R District,Vikarabad – 01 Phone No.: 9989233003,EmailId:charusuri87&yahoo.com

Abstract

The bcl-2 proto-oncogene was first discovered in B-cell lymphomas and is the prototype of cell death regulatory genes. It’s gene product, the bcl-2 protein, directly or indirectly inhibits the release of cytochrome C, thus preventing the activation of caspases and hence, inhibiting apoptosis. This leads to cellular immortalization, contributing to formation of the tumour and facilitating the permanent acquisition of mutations.
Studies show increased expression of the bcl-2 protein in about 50% of human cancers. Keeping this in view, our study was undertaken to evaluate the expression, to quantify and to determine the intensity and the pattern of bcl-2 in various histological grades of Oral Squamous Cell Carcinoma.
Method:The present study investigated the immunohistochemical expression of bcl-2 in 38 cases, of which 32 were histologically diagnosed as Oral Squamous Cell Carcinomas and 5 normal lymph nodes along with 1 normal oral mucosa were included as controls.
Each specimen was sectioned at 3 micron thickness, immunostained with the bcl-2 antibody and viewed under a light microscope.
Results:All the 38 cases showed bcl-2 immunopositivity. The number of bcl-2 positive cells was more in poorly differentiated SCC than in well differentiated SCC. The intensity of bcl-2 expression was more in moderately differentiated SCC, while in poorly differentiated SCC, an equal number of cases showed light and dark intensity. When the distribution pattern of bcl-2 expression was assessed, the tumour islands devoid of central keratinization showed bcl-2 expression in all the tumour cells.
Summary and Conclusion:In OSCCs, the number of cells expressing bcl-2 increased from well differentiated to poorly differentiated, showing an inverse relationship with the degree of differentiation. Further correlative studies using markers for other members of the bcl-2 family are necessary to elucidate the specific molecular defects critical to the biology of this tumour, which will have an impact on it’s diagnosis and treatment.

Keywords

Bcl-2, Squamous cell carcinoma (SCC), apoptosis

Introduction
The prevalence of tumours of the head and neck region is rapidly increasing and in particular, Squamous Cell Carcinoma (SCC) represents the most frequent malignant tumour of the oral cavity. It has long been evident that cancer has a multifactorial aetiology and is a multistep process involving initiation, promotion and tumour progression.

Carcinogenesis involves the activation of oncogenes and inactivation of tumour suppressor genes. The three major antiproliferative pathways recognized are: inhibition of cell growth, induction of cell differentiation and programmed cell death (apoptosis).

Normally, the elimination of genetically damaged cells by apoptosis precludes the development of tumours and also plays a role in the effective response to cancer therapy, including chemo and radiotherapy (1).On the contrary, the imbalance of the apoptotic pathway contributes to the immortalization of replicating cells, thus favouring the accumulation of sequential genetic damage that normally would induce cell death (2).

The bcl-2 proto-oncogene was first discovered in B-cell lymphomas which exhibited the t(14:18) q (32:31) chromosomal aberration and is the prototype of cell death regulatory genes (3). It’s gene product, the bcl-2 protein, blocks a distal step in an evolutionary conserved pathway of apoptosis. It’s abnormal expression, usually in terms of overexpression in genetically modified cells such as tumour cells, contributes to the expansion of the damaged cell clone by preventing cell turnover due to programmed cell death, leading to cellular immortalization.

Aims And Objectives
Our study aims to explore the bcl-2 protein immunoreactivity in Oral Squamous Cell carcinoma in an attempt to elucidate its influence on the biological behaviour of this neoplasm.

By promoting cell survival, bcl-2 facilitates the permanent acquisition of mutations and malignant transformation (4).Moreover, increased bcl-2 expression in cancer cells possibly reflects tumour cell resistance to apoptosis and may have implications for their responsiveness to treatments(5).

Material and Methods

The material for the study included 38 formalin fixed paraffin embedded tissue blocks. Among these, 10, 12 and 10 blocks of each histologically diagnosed well moderate and poorly differentiated oral squamous cell carcinomas respectively and 5 of normal lymph nodes and 1 of normal mucosa as controls, were considered for immunohistochemical staining for bcl-2.
The antibodies and reagents used for the immunohistochemical technique were obtained from DAKO cytomation (DENMARK) and SIGMA ALDRICH companies (USA).

3 micron thick sections were taken onto poly-L-lysine adhesive coated micro slides and incubated for 1 hour at 48 degree centigrade. The sections were then dewaxed through 3 changes of xylene, hydrated through descending grades of alcohol (100%, 90%, 70%) and brought to water. The micro sections were then dipped in freshly prepared 3% H2O2 in methanol for 20 minutes to block endogenous peroxidase activity, followed by a wash in PBS
For antigen retrieval, the micro sections were immersed in preheated 0.01 millimolar sodium citrate buffer and boiled for 8 minutes in a 5 litre domestic stainless steel pressure cooker on an electric heater. This increases the immunoreactivity of the antigen in tissues by enabling the antigen blocked by the cross-linking of the formalin fixative to be uncovered for binding to the relevant antibody. The slides were allowed to cool down in citrate buffer, were then washed in distilled water for 5 minutes and were then washed thoroughly in 3 changes of Phosphate Buffered Saline (PBS)

The sections were then treated with 10% goat serum at room temperature for 30 minutes to block the non–specific antigen sites, followed by incubation with primary antibody (bcl-2) prediluted at 370C (1:50) dilution for 60 minutes in a humid chamber. For reagent control, no primary antibody was added.

The sections were then incubated with biotinylated secondary antibody (Fc conjugated goat anti-mouse IgG) diluted to 1:200, at room temperature for 30 minutes, followed by incubation with the streptavidin-peroxidase conjugate diluted at a concentration of 1:200, for 30 minutes.

For visualization, the sections were incubated with Diaminobenzidine tetra hydrochloride (DAB) at a concentration of 1:50 for 5 minutes. The sections were then lightly counterstained using Meyer’s hematoxylin for 95 seconds and were then gently washed in running tap water for 2 minutes. The sections were then dehydrated through ascending grades of alcohol, were mounted with DPX and were covered with coverslips.

Evaluation
Assessment of the antigen expressed cells was performed by using a light microscope at 10X and 40X magnifications.

The criteria used to define the bcl-2 antigen positive cells were
• Brown staining in lymphocytes (intrinsic positive areas)
• Brown staining within the cytoplasm and the nucleus of the tumour cells.
In each case, three fields were selected randomly and 100 cells were counted per field under 40X magnification. The intensity of staining was graded as light /dark by comparing with the internal positive control, which were lymphocytes. In the different grades of squamous cell carcinoma, the pattern of staining was considered as periphery when the expression was seen only in the peripheral cells of the islands, while it was graded as entire when all the tumour cells within the islands expressed bcl-2 reactivity (Table/Fig 1).

Statistical Analysis
The data was analyzed using t-test for the number of bcl-2 positive cells and the by using the Kendall’s tau–b test for staining intensity and location to eliminate the interobserver bias. Comparisons within the different grades of OSCCs were subjected to the Fischer test for the number of positive cells and to the Chi Square test for staining intensity and location. For the intragroup comparison, Tukey HSD was applied for the number of bcl-2 positive cells, Fischer Exact test and Chi Square test were applied for staining intensity and the Fischer Exact test was applied for staining location.

Results

Results And Observation
In the present study, the control sections of the lymph nodes showed bcl-2 expression in the mantle zone lymphocytes and most interfollicular lymphocytes, whereas only occasional germinal centre lymphocytes expressed bcl-2. In the normal oral mucosa, bcl-2 expression was observed in a few cells of the basal layer. The suprabasal spinous layers were devoid of the bcl-2 antigen expression.

It was observed that all the 32 squamous cell carcinoma cases tested for the expression of bcl-2, showed immunopositivity. The immunohistochemical staining of the tumour cells showed granular cytoplasm and the nuclear envelope staining was positive. The sites of expression (periphery/entire island) in tumour islands/sheets were noted and the intensity of the expression of bcl-2 was also graded as light or dark when compared to the internal positive control lymphocytes. To eliminate subjective bias, two observers independently evaluated the expression of bcl-2 and to minimize the interobserver bias, t- test was applied and the p value was found to be non-significant Thus, the mean number of bcl-2 expressing cells counted by observer 1 was then subjected for further statistical analysis.

When the cells were evaluated for bcl-2 expression in well, moderately and poorly differentiated OSCCs, the mean number of cells were found to be 187.600, 223.750 and 264.700, respectively. Fischer test was applied on the results to compare the number of cells expressing bcl-2 in different grades of OSCCs and the p value was found to be very highly significant (p= 0.001) (Table/Fig 2).

When bcl-2 expression was compared between the different grades of OSCCs, it was found that;
• Well vs moderately differentiated - mean difference of 36.150 cells was observed;
• Well vs poorly differentiated – mean difference of 77.100 cells was observed;
• Moderately vs poorly differentiated – mean difference of 40.950 cells was
observed

These inter comparison results when subjected to the Tukey HSD test, showed p values which were very highly significant (p=0.001) in all the inter comparisons (Table/Fig 3).

After ruling out the interobserver bias (Table/Fig 4) for the staining intensity of bcl-2 positive cells, it was observed that only 2 out of 10 cases of the well differentiated OSCCs showed a dark intensity of expression as compared to 10 out of 12 cases of the moderately differentiated and 5 out of 10 of the poorly differentiated OSCC cases, i.e. out of 32 samples , 17 showed dark staining and 15 showed light staining The results obtained were compared using the Chi Square “χ2” test and the value obtained was 8.843, with the p value being significant (p= 0.012) (Table/Fig 5).

On comparing the intensity of the expression between individual grades
• 2 of the 10 well differentiated cases showed dark staining as compared to 10 out of the 12 moderately differentiated cases. The “χ2 “value was found to be 8.824 and the p value as 0.003, the difference being highly significant (Table/Fig 6) (Table 6a).
• Likewise, on comparing well differentiated (2 of 10 cases) vs poorly differentiated cases (5 of 10 cases) using the Fischer Exact test, the p value obtained was found to be 0.35, the difference being non-significant (Table/Fig 6) (Table 6 b).
• Similarly, on comparing moderately differentiated (10 of 12 cases) vs poorly differentiated cases (5 of 10 cases), the p value obtained by using the Fischer Exact test was found to be 0.172, which showed no significant difference (Table/Fig 6) (Table 6 c).

When compared for the location of the positive cells (periphery/entire island) in tumour island/sheets after interobserver bias was ruled out (Table/Fig 7) , it was observed that 9 out of 10 cases of well differentiated OSCCs showed staining in entire tumour islands as compared to 11 out of 12 cases of moderately differentiated and 1 out of 10 cases of poorly differentiated OSCCs. The results obtained were compared using the Chi Square “χ2” test. The “χ2 “value obtained was 27.594, with the p value being very highly significant (p= 0.001) (Table/Fig 8).

On comparing the location of the positive cells between individual grades, it was seen that 9 of the 10 well differentiated cases showed staining in the entire island as compared to 11 out of 12 of moderately differentiated cases. The p value obtained with the Fischer Exact test was 1, the difference being non- significant (Table/Fig 8).

Observations were also noted for the expression of bcl-2 in the connective tissue stroma and positive staining was observed in a few of the endothelial cells lining the blood vessels and secretory ducts of the minor salivary glands

Discussion

Programmed cell death or apoptosis is a critical step in cell differentiation and turnover and in tissue homeostasis. Perhaps the best studied regulatory proteins are the bcl-2 family of molecules. Recent advances on cancer biology have shown that the process of carcinogenesis may involve not only increased cell proliferation, but also decreased cell death or increased cell survival. Mutations of any of the genes encoding anti-apoptotic proteins or any changes in the levels of their expression can lead to increased cell survival.

In this immunohistochemical study, Bcl-2 expression was observed in 32 cases of squamous cell carcinoma, which was also seen by Ravi et al (1999) in their study on squamous cell carcinoma (6). The expression of bcl-2 was in the form of granular cytoplasmic staining with an accentuation around the nuclear membrane. This is due to the cellular localization of bcl-2 in the outer membrane of the mitochondria, endoplasmic reticulum and the nuclear membrane.

In this study, the number of bcl-2 positive cells was more in the poorly differentiated squamous cell carcinoma (mean 264.700) than in well differentiated squamous cell carcinoma (mean 187.600). An increase of bcl-2 expression in poorly differentiated squamous cell carcinoma was also seen by Yu chen et al (2000) and Muzio et al (2003) (7),(8). This observation of an increase in the number of bcl-2 positive cells with a decrease in the differentiation, probably reflects that bcl-2 is expressed in keratinocytes that have an increased capacity for survival.

However, in earlier studies by Loro et al (1999), bcl-2 oncoprotein expression was suppressed in poorly differentiated squamous cell carcinoma. This variation in the expression pattern which was detectable by the bcl-2 antibody, could be because of elective caspase activation, resulting in the proteolytic degradation of bcl-2 in tumour cells (9).

Studies done on squamous cell carcinoma of the oesophagus and the oral cavity by Jordan et al (1996), showed a higher percentage of immunoreactivity in poorly differentiated than in well differentiated carcinomas (10). However, this uniform immunoreactivity was not observed in carcinomas of the lung in studies by Francesso Pezella et al (1993) (11). This variation suggests that bcl-2 expression may be organ specific and may depend on the biological behaviour of individual tumours.

The activity of the bcl-2 family is partly regulated by the formation of homo and heterodimers and the relative concentration of the pro-apoptotic and anti-apoptotic members which would decide the outcome of a cell challenged by an apoptotic stimulus. In this study, the intensity of bcl-2 expression was dark in fewer cases of well differentiated carcinomas (2 cases) as compared to moderately differentiated squamous cell carcinomas (9 cases). In poorly differentiated SCC, an equal number of cases (5 each) showed light and dark intensities. The increased number of cases showing a dark intensity of expression in moderately differentiated carcinomas could be because of the increased activity of the bcl-2 protein during the early stages of tumour progression. However, as there is a functional interaction between different members of bcl-2 and also, because of the binding of this molecule to other cellular proteins, their intrinsic activity is modulated during the later stages of tumorigenesis.

Terminal differentiation is a form of programmed cell death which occurs in the oral epithelium where the basal cells proliferate, mature and differentiate to form keratin squames. When the distribution pattern of bcl-2 expression was assessed, it was observed that the tumour islands which were devoid of central keratinization showed bcl-2 expression in all the tumour cells. However, those islands which had keratinized neoplastic cells showed diminished or no immunoreactivity in the centre with staining which was restricted to only the peripheral cells. These observations are similar to those of Teni et al (2002) (12).However, Loro et al (1999) observed that the tumour islands devoid of any keratin in the central parts of the islands showed a progressive loss of bcl-2 immunoreactivity(13). which suggests that the central cells are differentiated and further, have the potential to terminally differentiate. In poorly differentiated tumours, immunoreactivity for bcl-2 was seen throughout the tumour cell population. This overexpression possibly reflects the resistance of these tumour cells to apoptosis and increased cell survival.

Bcl-2 immunoreactivity in the normal oral mucosa is confined to the basal cell layer and possibly is involved in the preservation of an adequate reservoir of proliferating stem cells, whereas the other apoptotic proteins like bax predominate in the superficial layers.13In all the cases examined for the overlying dysplastic epithelium, bcl-2 expression was observed even in the superficial layers, suggesting an alteration in the normal expression of bcl-2. Non-neoplastic epithelium adjacent to OSCCs observed in a few cases, showed expression of bcl-2 in a few basal cells as was also seen in normal epithelium which was taken as control.

Bcl-2 protein upregulation in tumour microvascular endothelial cells can be induced by vascular endothelial growth factor (VEGF) which enhances the availability of oxygen and nutrients for tumour cells. Bcl-2 expression was also observed in the endothelial cells lining the blood vessels. Nor JE et al (2001) observed bcl-2 expression in endothelial cells and suggested that these cells that overexpress bcl-2 secrete increased amounts of chemokine IL-8 which functions as an endogenous inducer of tumour angiogenesis (14).

Bcl-2 expression was also observed in a few duct cells of the minor salivary glands, which is consistent with the findings of Qi Long et al (1993) who examined the bcl-2 expression in embryonic non-haematopoietic tissues, wherein bcl-2 expression was seen in all the duct cells of all exocrine glands including salivary, pancreas and sweat glands. The expression seen in few of the duct cells, possibly suggests that these cells are undifferentiated reserve cells (15).

The findings from our study show that inhibition of apoptosis is a frequent event in squamous cell carcinomas. The bcl-2 family of proteins appears to be involved in regulating the terminal differentiation of keratinocytes. The downregulation of bcl-2 expression was concomitant with terminal differentiation and an increased over expression of this protein protects the tumour cells from undergoing apoptosis, thus facilitating their survival.

However, additional studies by using a larger sample size and by assessing the family of bcl-2 proteins may provide useful information for a more accurate tumour classification based on a biological basis.

Conclusion

Summary And Conclusion
In OSCCs, the number of cells expressing bcl-2 increased from well differentiated to poorly differentiated, showing an inverse relationship with the degree of differentiation of the tumour. As the onco-protein bcl-2 regulates programmed cell death by allowing the tumour cells to escape apoptosis, it promotes their survival, thereby facilitating the acquisition of further mutations.

Further correlative studies using markers for other members of the bcl-2 family are necessary to elucidate the specific molecular defects critical to the biology of this tumour, which will have an impact on diagnosis and treatment.

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