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,
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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.
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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

Original article / research
Year : 2023 | Month : November | Volume : 17 | Issue : 11 | Page : EC18 - EC22 Full Version

Immunoexpression of p53 and p16 in Low and High-grade Serous Ovarian Cancer: A Cross-sectional Study


Published: November 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/65393.18696
Ranajit Mandal, Rajib Kumar Mondal, Sarbari Kar Rakshit, Anup Kumar Roy, Rathin Hazra

1. Senior Resident, Department of Pathology, Nil Ratan Sircar Medical College, Kolkata, West Bengal, India. 2. Associate Professor, Department of Pathology, Nil Ratan Sircar Medical College, Kolkata, West Bengal, India. 3. Assistant Profesor, Department of Pathology, Nil Ratan Sircar Medical College, Kolkata, West Bengal, India. 4. Profesor, Department of Pathology, Nil Ratan Sircar Medical College, Kolkata, West Bengal, India. 5. Associate Professor, Department of Pathology, Diamond Harbour Government Medical College, Diamond Harbour, West Bengal, India.

Correspondence Address :
Rathin Hazra,
Vill-Hasanpur, Ward-13, Joynagar Mojilpur Municipality, PO: Ramakantanagar, PS: Joynagar, District South 24 Parganas,Joynagar-743395, West Bengal, India.
E-mail: hazra_rathin@rediffmail.com

Abstract

Introduction: The most common malignancy of the ovary is serous carcinoma, which can be classified as either Low-grade Serous Ovarian Carcinoma (LGSOC) or High-grade Serous Ovarian Carcinoma (HGSOC) and originates from the surface epithelium. However, the overall prognosis for both cancers is very poor. Immunohistochemical analysis of p53 and p16 expression is commonly used to detect mutations. Diffuse and strong mutations (mutant type) are almost always observed in cases of HGSOC, while focal expression (wild type) suggests the absence of mutations in HGSOC. LGSOCs are characterised by a low number or absence of genetic mutations.

Aim: To investigate the association between p53 and p16 expression in different grades and stages of Serous Ovarian Carcinoma (SOC).

Materials and Methods: This observational cross-sectional descriptive study was conducted on 62 patients diagnosed with ovarian serous carcinoma. The study focused on examining the expressions of p53 and p16 using Immunohistochemistry (IHC) in the Department of Pathology, Nil Ratan Sircar Medical College and Hospital, Kolkata, West Bengal, India, over a period of one and a half years (February 2021 to July 2022). The study parameters included clinical features, histological findings, staging, The Federation of Gynaecology and Obstetrics (FIGO), grading, p53 and p16 expression by IHC in all cases, and the association between p53 and p16 expression with the grade and stage of the cancer. Statistical analysis was performed using Analysis of Variance (ANOVA) and Statistical Package for Social Sciences (SPSS) software. A p-value of less than 0.05 was considered significant.

Results: A total of 62 cases were included in the study, with 55 cases (88.70%) classified as LGSOC and 7 cases (11.29%) as HGSOC. The mean age for LGSOC was 53.5 years, while for HGSOC it was 54 years. Among the HGSOC cases (n=55), 45 cases (81.80%) showed diffuse positive results (mutant type) for p53. In contrast, there was no diffuse p53 expression in LGSOC cases (n=7), with 5 cases (71.40%) showing focal positivity (wild type). The p-value for comparing p53 expression in both cases was significant (<0.00001). As for p16 expression, among the HGSOC cases (n=55), 31 cases (56.40%) showed diffuse positivity (mutant type), while among the LGSOC cases (n=7), most of the cases, 5 cases (71.40%), showed focal positivity (wild type). The p-value for comparing p16 expression in both cases was significant (<0.003794).

Conclusion: In conclusion, p53 along with p16 are good markers for grading SOC, and p53 is highly effective in differentiating HGSOC from LGSOC based on the positivity pattern (diffuse and strong positive for high-grade/mutant type, and focal positive for low-grade cancers). Thus, p53 has become an attractive target for the development of molecule-targeted therapies for this disease.

Keywords

High-grade serous ovarian carcinoma, Low-grade serous ovarian carcinoma, Serous ovarian carcinoma

The most dangerous gynaecological malignancy is epithelial ovarian cancer. Worldwide, there are 140,000 annual associated deaths due to this disease. A total of 90% of ovarian cancers develop from the surface epithelium, among which serous lesions comprise 60-80%. This cancer has a poor prognosis, and the survival period is also very short (1). The objectives of the present study were to diagnose cases of low-grade and high-grade serous ovarian cancer through histopathological examination and to study the immunoexpression of p53 and p16 in the diagnosed cases.

Due to a long, relatively asymptomatic latency period, poor awareness, and inadequate diagnostic infrastructure, most patients present late in the advanced stages of the disease, resulting in poor overall survival (1). For many years, scientific efforts have been made to identify prognostic factors based on molecular markers. The classification of surface epithelial tumours is based on the type of tumour cell (mucinous, serous, endometrioid, clear cell) and further subclassified as benign, borderline, or malignant carcinoma. These epithelial ovarian cancers are a heterogeneous group of diseases, and their determination is relevant for prognostication and treatment prediction.

Histologically, serous carcinoma has two grades: low-grade and high-grade. Low-grade serous carcinomas are distinguished from HGSOCs by a lesser degree of nuclear pleomorphism (less than threefold variation in nuclear size). In difficult cases, low-grade serous carcinomas show a lower mitotic rate than HGSCs (12 or fewer MF per 10 HPF) (2). Regarding p53 immunostaining, LGSOC shows a normal pattern compared to the abnormal staining (so-called all-or-nothing patterns) present in 95% of HGSCs (2). HGSOCs are the predominant type of SOCs, comprising 80%-90% of ovarian cancer cases. They are very aggressive in nature, with most cases being diagnosed in advanced stages and having low overall survival rates. On the other hand, LGSOCs commonly exhibit an indolent growth pattern, with better prognosis and Progression-Free Survival (PFS) rates. While important diagnostic information can be obtained from the morphology and histological grades of SOCs, IHC plays a crucial role in differential diagnosis and the evaluation of molecular features, further aiding in the characterisation of morphology and clinical behaviour (3).

The 53-KDa protein is a nuclear protein encoded by the tumour suppressor tp53 gene. It plays a role in maintaining genome integrity and promotes cellular apoptosis during Deoxyribonucleic Acid (DNA) damage. Most HGSOCs commonly exhibit tp53 gene mutations. Increased tp53 mutations are associated with strong and diffuse nuclear expression (mutant type) or complete absence of expression (null type). Focal expression (wild type) suggests the absence of mutations in HGSOC. In the case of LGSOCs, genetic mutations are low or p53 gene mutations are almost absent in these tumours (3).

Detection of tp53 mutations has significant importance in clinical practice. Nucleotide sequencing is the most reliable technique for detecting gene mutations, but it is labour-intensive and time-consuming. Therefore, immunohistochemical analysis of p53 expression is currently preferred as a surrogate for mutational analysis (4). Yemelyanova A et al., analysed p53 expression as a surrogate marker for mutational analysis in ovarian carcinoma (4).

The CDKN2A gene is a tumour suppressor gene that encodes p16. It functions to slow down cell progression from the G1 to S phase and regulates the cell cycle. Approximately 60%-80% of HGSOCs show diffuse p16 staining. Therefore, p16, along with p53, should be used as IHC markers in the differential diagnosis of SOCs and to distinguish between low-grade and high-grade serous ovarian cancer (3).

Material and Methods

It was an observational, cross-sectional descriptive study conducted in the Department of Pathology, Nil Ratan Sircar Medical College and Hospital, Kolkata, West Bengal, India, for one and a half years (February 2021 to July 2022). Special permission was obtained from the Institutional Ethical Committee (IEC number: NMC/855, dated 26.02.2021) for the present study. The study included all female patients who presented with ovarian Space Occupying Lesions (SOL) and underwent oophorectomy, salpingo-oophorectomy, and Total Abdominal Hysterectomy with Bilateral Salpingo-oophorectomy (TAH BSO).
I
nclusion criteria:
1. Low-grade serous ovarian cancer {Tumour with mild to moderate cytological atypia and a lower mitotic count, usually up to 12 mitoses per 10 High Power Fields (HPFs)}.
2. High-grade serous ovarian cancer (Tumour with marked cytological atypia, presence of multinucleated cells, and a higher mitotic count, more than 12 mitoses per 10 HPFs).
Exclusion criteria:
• All other serous ovarian tumours except LGSOC and HGSOC.
• Metastasis to the ovary.
Control: For p53 positive control, a diagnosed case of HGSOC was taken. For p16 positive control, a diagnosed case of Human Papilloma Virus (HPV) positive cervical carcinoma was taken. For negative controls of p53 and p16, a case of colloid goiter was taken.

Study Procedure

The biological material was processed using a common histopathological technique with 10% formalin fixation. In each case, the standard protocol for surgical grossing of the TAH BSO specimens was followed. After a detailed examination of the gross specimen, multiple representative tissue samples were taken from the tumour and ovarian capsule. The samples were processed according to the standard protocol, embedded in paraffin, and cut into tissue blocks. The tissue blocks were then stained with Haematoxylin and Eosin (H&E). The H&E stained slides were examined to determine the tumour histology, grade, lymphovascular invasion, and capsular invasion. The tumour was staged according to the International Federation of Gynaecology and Obstetrics (FIGO) cancer staging system (1).

Processing for Immunohistochemistry (IHC): For IHC analysis of p16 and p53, 4.0 μm paraffin sections were taken on poly-L-lysine coated slides. The sections were deparaffinised in xylene and then 19hydrated in descending grades of ethanol. Antigen retrieval was performed by heating the sections at 95ÂşC in Tris-Ethylene Diamine Tetra Acetic Acid (EDTA) buffer (pH 9.0) for three cycles of 5 minutes each. The sections were then incubated with power block (Biogenex, USA) for 10 minutes to reduce non specific antibody binding. Primary antibodies against human p16 (Dako Antihuman p16, ready to use) and human p53 (Dako Anti-human p53, ready to use) were used. The sections were incubated with the primary antibodies for one hour at 4ÂşC. After three washes with Trisphosphate Buffer Solution (TBS), a secondary antibody was added and incubated for 30 minutes. Following another three washes with TBS, the sections were treated with 3,3’-diaminobenzidine substrate (DAB tetrahydrochloride) for 10 minutes. The sections were then counterstained with Haematoxylin, dehydrated with ethanol and xylene, and permanently mounted with DPX. A positive control was taken from p53-positive breast carcinoma, and negative control sections were processed by omitting the primary antibody. The results were compiled and subjected to statistical analysis. Slides showing non contibutory staining were not included in the statistical analysis. The immunohistochemical results were reported as the percentage of labeled cells for each case to obtain the Positivity Index (PI). The intensity of the reaction was assessed as none, weak, intermediate, or strong (5).

Assessment of IHC staining (2): The IHC staining was assessed using the following semi-quantitative criteria: Nuclear p53 protein expression was analysed in each case, and the percentages of cells with nuclear staining were estimated as mentioned below.

Nuclear p53 staining:

• Negative: if less than 1% of the nuclei showed staining.
• Focal positive: if 1% to 69% of the nuclei showed staining.
• Diffuse positive: if 70% or more of the nuclei showed staining.

Cytoplasmic and nuclear p16 staining:

• Negative: if less than 10% of the cells showed staining, or if the staining was absent or of low intensity.
• Focal positive: if 10% to less than 89% of the cells showed staining.
• Diffuse positive: if 90% or more of the cells showed staining.

Laboratory investigations and parameters studied: In the present study, detailed history and clinical findings of the patients were collected with their consent. After TAH and BSO operation, careful macroscopic examination was performed, and representative sections were taken for grading the SOC as low-grade or high-grade using a binocular microscope. The FIGO staging (1) was determined through microscopic examination. Immunohistochemical examination was conducted on each case to assess the expression of p53 and p16 in low-grade and high-grade serous carcinomas. Finally, the p-value was calculated to determine the association of these immunomarkers with the different grades and stages of carcinoma.

Statistical Analysis

The images were acquired using a Nikon Eclipse E600 microscope and Lucia 5 software program. Statistical analysis was conducted using ANOVA and Pearson tests with SPSS 10 software. The data was tabulated in Microsoft Excel and analysed with appropriate statistical methods, including the chi-square test. A p-value of less than 0.05 was considered statistically significant.

Results

In the present study, all 62 collected specimens (100%) were TAH and BSO. Among them, 55 cases (88.70%) were HGSOC (Table/Fig 1), and 7 cases (11.30%) were LGSOC (Table/Fig 2). The mean age for HGSOC was 54 years, while the mean age for LGSOC was 53.5 years. The majority of HGSOC cases, 34 (61.80%), occurred between 56-65 years (Table/Fig 3). Most of the SOC cases, 49 (79.00%), occurred at postmenopausal age. Among the low-grade cases (n=7), 3 cases (42.8%) were associated with endometriosis. All high-grade and low-grade cases had a common clinical presentation of abdominal distension and fatigue. Among other symptoms of high-grade cases, ascites was present in 41 cases (74.5%), low back pain in 41 cases (74.5%), and weight loss in 31 cases (56.4%). Most low-grade cases experienced constipation and pelvic discomfort.

In all SOC cases (n=62), 45 cases (72.60%) showed diffuse p53 expression, 9 cases (14.50%) showed focal positivity, and 8 cases (12.90%) were negative. For p16 expression, 33 cases (53.20%) showed diffuse expression, 14 cases (22.60%) showed focal positivity, and 15 cases (24.20%) were negative. Among the 55 HGSOC cases, 45 cases (81.80%) showed diffuse p53 expression (Table/Fig 4), 6 cases (10.90%) showed negative expression, and 4 cases (7.30%) showed focal positivity. Among the 7 LGSOC cases, there was no diffuse p53 expression, 5 cases (71.40%) showed focal positivity (Table/Fig 5), and 2 cases (28.60%) showed negative expression (Table/Fig 6).

Among the 55 HGSOC cases, 31 cases (56.40%) showed diffuse p16 expression (Table/Fig 7), 15 cases (27.30%) showed negative expression, and 9 cases (16.30%) showed focal positive expression. Among the 7 LGSOC cases, 2 cases (28.6%) showed diffuse p16 expression, while 5 cases (71.40%) showed focal positivity, and there was no negative expression (Table/Fig 8). Out of 62 cases, the highest number, 30 cases (48.4%), were Stage III according to FIGO staging, followed by FIGO I with 21 cases (33.90%) and FIGO II with 11 cases (17.70%). No cases of Stage IV were found in the present study. Diffuse expression of p53 in FIGO I, II, and III was observed in 9 cases (42.90%), 9 cases (81.80%), and 27 cases (90.00%), respectively. No cases of Stage IV were found in our study (Table/Fig 9). The focal expression of p53 (wild p53) was highest in 7 cases (33.33%) under FIGO I stage. Diffuse p16 immunoexpression was highest in 11 cases (100%) under FIGO Stage II, while focal p16 expression was highest in 14 cases (66.70%) under FIGO Stage I (Table/Fig 10).

Discussion

Ovarian serous cancer, whether low-grade or high-grade, is a lethal malignancy in adult females. HGSOC is the
predominant variety of ovarian surface epithelial cancer and is known for its aggressive nature. Only 30% of these patients survive for a maximum of five years (1). A binary pathway for the development of this cancer has been proposed based on the tumour grade. HGSOC is believed to directly arise from the ovarian surface epithelium, the fimbrial end of the fallopian tube, or the epithelium of cortical inclusion cysts. These tumours do not yet have a definitive precursor lesion and are strongly associated with mutations in the p53 tumour suppressor gene. On the other hand, LGSOC seems to evolve from benign cyst adenoma through the stage of a borderline serous tumour in a sequential step-wise fashion. LGSOC has more mutations in the BRAF and KRAS genes compared to the p53 gene, which is more commonly mutated in HGSOC. Therefore, different genes and pathways seem to be involved in the development of different grades of ovarian serous carcinoma (1).

Comparing to HGSOC, LGSOCs have an indolent growth pattern. Although they are not very responsive to chemotherapy, their prognosis is better than HGSOC. They have longer disease PFS rates. While valuable research and publications have been done worldwide regarding p53 and p16 expression in low-grade and high-grade serous ovarian cancer, research in India is quite limited (1). There is no published record of any such study done in West Bengal. Consequently, the therapeutic implications, such as the development of targeted chemotherapeutics and immunotherapies, as well as their clinical trials, relevance, economic feasibility, and practical feasibility, remain largely unexplored.

The present study was conducted on 62 cases of serous ovarian cancer, with 55 cases (88.70%) being high-grade and seven cases (11.30%) being low-grade. The received specimens mainly consisted of total abdominal hysterectomy with bilateral salpingo-oophorectomy in all the cases. There were no salpingo-oophorectomy or oophorectomy specimens, indicating that the clinicians and surgeons took precautions to avoid the chance of recurrence, poor prognosis, or further spread. Overall, the mean age for the present study population was 53.5 years. The lowest and highest ages for the cases were 40 and 68 years, respectively. This signifies that the incidence of serous ovarian cancer increases with age. Elderly patients with serous carcinomas are more likely to experience early recurrence, recalcitrance, and platinum resistance than younger patients (1).

Grossly, serous carcinoma presents as a large solid cystic mass with an irregular outer surface. The cut surface is also multicystic to solid and contains haemorrhagic areas. Sometimes, a gritty sensation can be felt during grossing due to calcified psammoma bodies. Microscopically, HGSOC cases are composed of solid areas with occasional papillary projections, severe cellular atypia, tumour giant cells, a high mitotic count, and plenty of psammoma bodies in a background of haemorrhage and necrosis (Table/Fig 1) (1). On the other hand, LGSOC cases show multicystic masses with fine papillary projections, mild to moderate cellular atypia, low mitotic figures, and occasional psammoma bodies (Table/Fig 2) (1). The majority of HGSOC cases (61.80%) occurred between 56-65 years, while the majority of LGSOC cases (42.80%) occurred between 56-65 years (Table/Fig 3). The mean age for low-grade and high-grade cases was 53.5 years and 54 years, respectively, similar to the findings of Jha R and Karki S, (mean age 51 years) and Deng F et al., (mean age 56 years) [6,7]. They stated that compared with younger patients, elderly patients with SOC are commonly characterised by high tumour grade, poor performance status, and undertreatment. In our study, the majority of SOC cases (79.00%) occurred at postmenopausal age. The Chi-square statistic was 0.2753, and the p-value was 0.599793. This result was not significant at p<0.05, similar to the findings of Qasim YA et al., (p-value was 0.6234) (8).

All of the high-grade and low-grade cases in the present study had a common clinical presentation, which included abdominal distension and fatigue. Among other symptoms, ascites, low back pain, and weight loss were present in 41 cases (74.50%), 41 cases (74.50%), and 31 cases (56.40%) of high-grade cases, respectively. Most of the low-grade cases experienced constipation and pelvic discomfort, similar to the findings of Hunn J and Rodriguez GC, (n=54 cases, HGSOC- 47, LGOC-07 cases). Among high-grade cases, ascites was present in 30 cases (63.82%), and weight loss in 25 cases (53.19%) (9).

In the case of high-grade and high-stage cancer, tumour growth involves the capsule and can spread through lymphovascular invasion and peritoneal spread. The inflammatory reactions due to metastatic deposits cause the accumulation of serous fluid, which is manifested by ascites. Endometriosis was the predominant cause of LGSOC in the present study, supported by the findings of Hjerpe E et al., (10). However, no definite cause was found in most of the high-grade cases. The exact cause or biomolecular pathway is still under continuous research.

Among all the SOC cases (n=62), 45 cases (72.60%) showed diffuse p53 expression, and 33 cases (53.20%) showed diffuse p16 expression. Therefore, p53 associated with p16 are commonly used as IHC markers in the differential diagnosis of SOC. Among all the HGSOC cases (n=55), 45 cases (81.80%) showed diffuse p53 expression (mutant type) (Table/Fig 4), 6 cases (10.90%) were negative (null type), and 4 cases (07.30%) showed focal positivity. Therefore, diffuse and null type (51 cases) made up a total of 92.7% of p53 expression, similar to the findings of Marinas¸ MC et al., (n=20, 18 HGSOC cases showed diffuse positivity) and Amaral JD et al., (n=50, 44 HGSOC cases showed diffuse positivity) [1,11]. They concluded that diffuse p53 mutations were present in nearly 100% of high-grade serous ovarian cancer. However, Köbel M et al., found that p53 was completely absent in 153 (30.30%) cases, focally expressed in 62 (12.00%) cases, and overexpressed in 287 (57.70%) cases of 502 HGSOC cases. They also found an association between the complete absence of p53 and an unfavourable outcome (12).

In the case of LGSOC (n=07), there was no diffuse p53 expression, while five cases (71.40%) showed focal positivity (wild type) (Table/Fig 5), and two cases (28.60%) had negative expression. The p-value for the comparison of p53 expression in high-grade and low-grade cases was <0.00001, indicating a significant difference. This finding is similar to the study by Cole A et al., where all LGSOC cases (100%) showed wild type mutation (13).

Out of the 62 cases, diffuse expression of p53 was noticed in 9 (42.90%), 9 (81.80%), and 27 (90.00%) cases under FIGO stages I, II, and III, respectively. No cases of Stage IV were found. The focal expression of p53 (wild type) was highest in 07 cases (33.33%) under FIGO stage I. The Chi-square statistic was 17.0701, and the p-value was significant (0.001873). This finding is similar to the studies by Kmet LM et al., (n=51, diffuse expression in 21 (90.00%) under Stage III cancer) and Arik D and Kulacoglu S, (n=28, diffuse expression in 17 (87.00%) under Stage III cancer). They found that p53 abnormalities were positively associated with increasing tumour grade and stage [14,15].

Among all the HGSOC cases (n=55), 31 cases (56.40%) showed diffuse p16 expression, 15 cases (27.30%) were negative, and 09 cases (16.30%) showed focal positivity. This finding is similar to the study by Rambau PF et al., where p16 block expression was most frequent in 4334 HGSOC cases (56.00%) out of 6525 cases (16). Phillips V et al., also demonstrated that p16 was highly expressed in 38 cases (49.35%) of HGSOC (n=77 cases) (17).

Among the LGSOC cases (n=7), 02 cases (28.60%) showed diffuse p16 expression, while 05 cases (71.40%) showed focal positivity. There were no cases with negative p16 expression. The Chi-square statistic for the comparison of p16 expression in high-grade and low-grade cases was 11.1486, and the p-value was significant (<0.003794). Most low-grade cases exhibit an indolent
growth pattern and take more time to spread. Therefore, they are often identified at a lower stage. In the present study, most of the LGSOC cases were found at FIGO Stage I. On the contrary, high-grade cases are rapid growers and spread quickly, often presenting with non specific and vague symptoms. This is why the maximum number of HGSOC cases was found at FIGO Stage III in the present study.

Out of the 62 cases, diffuse p16 immunoexpression washighest at FIGO Stage II (n=11), which was 100%. Focal p16 expression was highest in 14 cases (66.70%) under FIGO Stage I (n=21). The chi-square statistic was 43.3338, and the p-value was significant (<0.00001). This finding is similar to the study by Shandiz FH et al., where diffuse p16 immunoexpression was highest at FIGO Stage II (n=30 cases, 63.82%), and focal p16 expression was highest under FIGO Stage I (n=12 cases, 25.53%) (18).

Out of the 62 cases, the maximum number of cases (30, 48.40%) were found at Stage III according to FIGO staging, followed by FIGO I with 21 cases (33.90%) and FIGO II with 11 cases (17.70%). This finding is similar to the study by Seidman JD et al., where out of 113 cases, FIGO I had 30 cases (26.54%), FIGO II had 33 cases (29.20%), and FIGO III had 50 cases (44.24%) (19). No cases of Stage IV were found in the present study.

Limitation(s)

The present study included only a small study population of 62 patients who attended a tertiary care hospital. Since, the present study is single-centered and conducted in Kolkata, West Bengal, India the results cannot be generalised to all regions.

Conclusion

So, authors concluded from the study that p53 is highly effective in differentiating HGSOC from LGSOC based on the pattern of positivity. Extensive diffuse and strong nuclear positivity in highgrade cases signifies increased tp53 gene mutations (mutant type). Zero diffuse positivity in low-grade confirms that tp53 gene mutation is never present in low-grade cases. p16 can also differentiate between high-grade (56.40% diffuse expression) and low-grade (28.60% diffuse expression) SOC. However, p53 has been shown to be a robust and more effective immunomarker than p16 for differentiating these SOCs. All investigations and procedures were conducted in a government hospital, and the cost was covered by the hospital.

References

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DOI and Others

DOI: 10.7860/JCDR/2023/65393.18696

Date of Submission: May 22, 2023
Date of Peer Review: Jul 26, 2023
Date of Acceptance: Aug 24, 2023
Date of Publishing: Nov 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for the present 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. Yes

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: May 15, 2023
• Manual Googling: Aug 22, 2023
• iThenticate Software: Sep 09, 2023 (14%)

ETYMOLOGY: Author Origin

EMENDATIONS: 7

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