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

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On Sep 2018




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Prof. Somashekhar Nimbalkar
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Chairman, Research Group, Charutar Arogya Mandal, Karamsad
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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."



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Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
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Dr. Saumya Navit

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Professor and Head
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Saraswati Dental College
Lucknow
On Sep 2018




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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.


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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.
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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 : 2022 | Month : March | Volume : 16 | Issue : 3 | Page : XC06 - XC10 Full Version

Association of IHC p16INK4a expression and ELISA Plasma p16INK4a Protein in Squamous Cell Carcinoma of Uterine Cervix: A Concept of Liquid Biopsy


Published: March 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/51540.16127
Kalyani Raju, CV Raghuveer, SR Sheela, B Sharath

1. Professor and Head, Department of Pathology, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka, India. 2. Pro-Vice Chancellor, Department of Pathology, Yenopoya University, Mangaluru, Karnataka, India. 3. Professor and Head, Department of Obstetrics and Gynaecology, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka, India. 4. Associate Professor, Department of Cell Biology and Molecular Genetics, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and Research, Kolar, Karnataka, India.

Correspondence Address :
Kalyani Raju,
Professor and Head, Department of Pathology, Sri Devaraj Urs Medical College, Sri Devaraj Urs Academy of Higher Education and Research, Tamaka, Kolar, Karnataka, India.
E-mail: drkalyanir@rediffmail.com

Abstract

Introduction: Cervical cancer is the 3rd most common cancer among women worldwide. p16 biomarker is the surrogate marker in cervical cancer and can be detected by Immunohistochemistry (IHC).

Aim: To evaluate the association of IHC p16INK4a expression in tissue sections and Enzyme-linked Immunosorbent Assay (ELISA) p16INK4a protein in plasma of paired samples in Squamous Cell Carcinoma (SCC) of uterine cervix.

Materials and Methods: This laboratory-based observational, pilot study was conducted at tertiary care centre of Sri Devaraj Urs Medical College in South India from June 2020 to May 2021. Total 17 new cases of SCC of cervix diagnosed by histopathology were considered for the study. The cases were staged as per International Federation of Gynaecology and Obstetrics (FIGO) staging system and classified by histopathology as keratinising and non keratinising types. IHC p16INK4a evaluation was done on tissue sections and classified as block positivity, ambiguity and negative. ELISA p16INK4a estimation was done using plasma from Dipotassium Ethylenediaminetetraacetic Acid (K2-EDTA) blood sample of same cases. Statistical analysis was done using Statistical Package for the Social Sciences (SPSS) version 22.0 software.

Results: The age ranged from 30-80 years with mean of 54.3±12.0 years. Plasma ELISA p16INK4a level ranged from 3.4-19.6 ng/mL with a mean of 7.2±2.35 ng/mL in SCC of cervix. The plasma p16INK4a ELISA levels of 5.1 to 6.2 ng/mL, 6.0 to 6.6 ng/mL and 5.5 to 9.7 ng/mL predicts negative, ambiguity and block positivity of IHC p16INK4a expression respectively in corresponding tissue biopsy. Plasma ELISA p16INK4a levels were maximum in Non Keratinising Squamous Cell Carcinoma (NKSCC) followed by Well Differentiated Squamous Cell Carcinoma (WDSCC), Moderately Differentiated Squamous Cell Carcinoma (MDSCC) and Poorly Differentiated Squamous Cell Carcinoma (PDSCC).

Conclusion: The plasma ELISA p16INK4a levels and IHC p16INK4a expression were maximum in higher disease stage compared to lower stage. This was a pilot study to evaluate the association between tissue IHC p16 expression and plasma ELISA p16 levels. Further study has to be done on larger study population with standardised procedure to prove the hypothesis.

Keywords

Cervical cancer, Enzyme-linked immunosorbent assay, Immunohistochemistry marker, p16 biomarker, p16 protein, Squamous cell carcinoma of cervix

Cervical cancer is the 3rd most common cancer among women worldwide with 11.7% of global prevalence. It ranks 5th in cancer related deaths in women. The annual estimated global incidence is 500,000 and India contributes approximately 100,000 (1),(2),(3). Cervical cancer is the second most common cancer in developing countries among females (1). Human Papilloma Virus (HPV) is proved aetiological factor (1),(3).

p16 is a cyclin dependent kinase inhibitor. In cervical cancer, the High Risk-HPV (HR-HPV) E7 protein inactivates pRb protein resulting in increased synthesis and accumulation of p16 protein in tissues and cells which can be demonstrated by IHC or immunocytochemistry techniques respectively. p16 shows marked positivity (90%) in High grade Squamous Intraepithelial Lesion (HSIL) and invasive SCC of cervix (4),(5),(6). Lower Anogenital Squamous Terminology (LAST) criteria in 2012 classifies p16 immunoreactivity as block positive, ambiguous and negative considering p16 biomarker expression in the nucleus with or without cytoplasmic staining (7),(8).

Liquid biopsy is a technique where the liquid sample as blood, body fluids and urine are used to isolate Circulating Tumour Cells (CTC), circulating tumour Deoxyribonucleic Acid (ctDNA), Ribonucleic Acid (RNA), exosomes and proteins which are shed by tumour cells (9),(10). Studies have reported that estimation of p16 protein by ELISA on lysed samples of cervical cells had positive association with HISL and SCC of uterine cervix (4),(5).

The aim of this study was to evaluate the association of IHC p16 expression with Enzyme-linked Immunosorbent Assay (ELISA) plasma p16 protein levels in Squamous Cell Carcinoma (SCC) of cervix cases and derive the significance of plasma p16 protein levels in cervical cancer. A part of this study of IHC p16 expression correlating the clinical features is already published (11).

Material and Methods

This laboratory-based observational, pilot study was conducted in Department of Pathology in co-ordination with Department of Obstetrics and Gynaecology at Sri Devaraj Urs Medical College (Tertiary Health Care Centre) Kolar, Karnataka, India, from June 2020 to May 2021. Ethical clearance for the study has been obtained from Institutional Ethics Committee (SDUMC/KLR/IEC/45/2020-21 dated:09-05-2020).

Sample size calculation: Sample size was calculated using the formula:

Z2×pq/d2

Considering the prevalence (p) of p16 expression as 95% in cervical cancer (12), an absolute error (d) of 5% and confidence level of 95%. The estimated sample size for the study was 60. Considering 10% non responsive case, total sample size of 70 was considered.

Inclusion and Exclusion criteria: Total 70 new cases diagnosed as SCC of cervix clinically and confirmed by histopathology were included in the study. Patients with Cervical Intraepithelial Neoplasia (CIN), postchemotherapy cases, postradiotherapy cases, relapse cases, Cervical Glandular Intraepithelial Neoplasia (CGIN), adenocarcinoma of cervix, metastatic deposits in the cervix and any other primary malignancy in the patient were excluded from the study.

Procedure

Following informed consent, in clinically suspected cases of cervical cancer, cervical biopsy was taken and fixed in 10% buffered formalin for routine histopathological diagnosis and IHC p16 biomarker expression. A 6 mL of blood was collected in Dipotassium Ethylenediaminetetraacetic Acid (K2-EDTA) vacutainer and centrifuged at 1500 rpm for 10 minutes. The separated plasma was kept in vials at -80o centigrade for quantitative estimation of p16 protein by ELISA sandwich method.The case details including age, hospital number, biopsy number, presenting complaints, personal history, menstrual history, family history, past history, per abdominal examination findings, per speculum examination findings, per vaginal examination findings and per rectal examination findings were entered in the excel sheet. Following histopathology report confirmation, staging of the disease as per International Federation of Gynaecology and Obstetrics (FIGO) staging was noted (13). The slides were screened by two Pathologists and classified histologically as keratinising and non keratinising types. Keratinising SCC was further analysed (14):

• Poorly Differentiated Squamous Cell Carcinoma (PDSCC)
• Moderately Differentiated Squamous Cell Carcinoma (MDSCC)
• Well Differentiated Squamous Cell Carcinoma (WDSCC)
• Non Keratinising Squamous Cell Carcinoma (NKSCC)
-Large Cell Type (NKLCSCC)
-Small Cell Type (NKSCSCC)

Tissue sections were cut from paraffin blocks and subjected to IHC p16 biomarker (Mouse monoclonal anti-p16INK4 clone G175-405, Biogenex, USA), primary antibody, on all cases with positive and negative control. The procedure followed was as per the manufacturer’s instruction. The p16 expression on tissue sections were classified as block positivity, ambiguous staining and negative as per LAST criteria 2012.

• “Block” pattern staining means strong, continuous, nuclear positivity with or without cytoplasmic staining extending from basal layers upwards for atleast 1/3rd thickness of the epithelium (basal and parabasal layers) which can be further graded as 1/3rd, 2/3rd and more than 2/3rd and laterally over a significant area with diffuse staining of >25% of cells.
• “Ambiguous” staining means strong and basal (strong, diffuse, continuous, involves only lower 1/3rd without upward extension) or weak, diffuse and discontinuous staining, involving atleast 2/3rd of the epithelium or strong, focal and discontinuous located at any level of the epithelium.
• “Negative” staining means total absence or weak or focal and discontinuous or only cytoplasmic staining (Table/Fig 1), (Table/Fig 2), (Table/Fig 3) (8).

The separated plasma of the cases were subjected to p16 protein estimation by quantitative sandwich ELISA method using Human p16 ELISA kit (ImmunoTag, Catalogue No: ITEH01637) and performed as per manufacturer’s instruction. A standard curve was constructed by plotting the average Optical Density (OD) for each standard on the vertical axis (Y) against the concentration on the horizontal axis (X) and curve was drawn through the points on the graph. Using the OD of samples, the concentration of the p16 protein was estimated and expressed as ng/mL. The values were entered in the master chart (excel sheet).

Statistical Analysis

Data was analysed using Statistical Package for the Social Sciences (SPSS) version 22.0 software. Categorical data was represented in the form of frequencies and proportions. Continuous data was represented as mean and standard deviation. Chi-square/Fisher-exact test was used to find the significance of difference between the categorical parameters. Analysis of Variance (ANOVA) test was used to find the significance of difference between the continuous data. The p-value <0.05 was considered as statistically significant. Statistical analysis for association between IHC p16 expression in tissue sections and ELISA p16 protein in plasma was done through sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and diagnostic accuracy at different cut-off values of plasma p16 protein levels.

Results

Total 70 cases of cervical cancer of SCC type were considered for the study. The age ranged from 30-80 years with mean of 54.3±12.0 years. Maximum cases were seen in age group of 40 to 49 years (n=19) followed by 60 to 69 years (n=18). Bleeding per vagina was the most common presentation (n=40) followed by white discharge per vagina (n=30), pain in lower abdomen (n=40), postcoital bleeding (n=4), mass per vagina (n=2) and other constitutional symptoms (n=48). The p-value between age of cases and IHC p16 expression was 0.008. All cases between 30-59 years of age showed block positivity for p16 IHC (i.e., 100%) (Table/Fig 4).

The ELISA plasma p16 levels ranged from 3.4 to 19.6 ng/mL with mean of 7.24±2.35 ng/mL. Plasma p16 levels were maximum in a case of 80 years and minimum in age range of 50-59 years. There was no statistical significant association (p-value=0.294) between stage of the disease and IHC p16 expression. However, all stage I cases (100% cases) showed block positive p16 expression. Regarding ELISA plasma p16 levels in different stages of the disease, maximum levels of p16 was recorded in stage IV of the disease. The p-value was 0.068 (Table/Fig 5). The p-value between IHC p16 expression and ELISA p16 values in stage II, III and IV was 0.975, 0.917 and 0.652, respectively.

There was no statistical significant association (p-value=0.887) between histopathological grade and IHC p16 expression of the disease. Total 33 (86.8%) of the cases of WDSCC and 14 (93.3%) of MDSCC cases showed block positive expression p16 IHC but it was not statistically significant (p-value=0.887). Regarding, ELISA plasma p16 levels in different histological grades of the disease, maximum was recorded in NKLCSCC followed by NKSCSCC/WDSCC and minimum in MDSCC/PDSCC. The p-value was 0.018. The p-value between IHC p16 and ELISA p16 in WDSCC, MDSCC and PDSCC was 0.682, 0.406 and 0.008, respectively (Table/Fig 6).

Among 70 cases, IHC p16 biomarker expression showed block positive, ambiguity and negative in 62 (88.6%), 5 (7.1%) and 3 (4.3%) cases, respectively. Regarding ELISA plasma p16 levels in different groups of IHC p16 expression as per LAST criteria, the levels were maximum in ambiguity group followed by block positivity and then negative cases with p-value of 0.598 (Table/Fig 7). Validity of plasma p16 ELISA in predicting negative expression of IHC p16 by LAST criteria is showed in (Table/Fig 8) with plasma p16 ELISA range of 5.1 to 6.2 ng/mL. Validity of plasma p16 ELISA in predicting ambiguity expression of IHC p16 by LAST criteria is showed in (Table/Fig 9) with plasma p16 ELISA range of 6.0 to 6.6 ng/mL. Validity of plasma p16 ELISA in predicting Block positive expression of IHC p16 by LAST criteria is showed in (Table/Fig 10) with plasma p16 ELISA range of 5.5 to 9.7 ng/mL. (Table/Fig 11) shows the sensitivity, specificity, PPV, NPV and diagnostic accuracy at different cut-off levels of ELISA plasma p16 protein in predicting negative, ambiguity and block positive IHC p16 expression by LAST criteria, respectively.

Discussion

Cervical cancer is the common cancer in women worldwide. The incidence and mortality has decreased by 75% to 80% in developed countries by well-organised screening programs. Papanicolaou test (pap) test for screening cervical cancer has markedly decreased the incidence of cervical cancer. Inspite of well-developed screening procedure, women in developing countries are diagnosed at late stage of the disease resulting in high mortality (14),(15),(16). High Risk Human Papillomavirus (HPV) is the proved aetiological factor and HPV vaccine has been implemented in many countries (1),(3),(17).

The common age range of cervical cancer reported is 27 to 80 years of age with mean age of 54.2 years and maximum cases are noted between 41 to 60 years of age (1). In this study, the age ranged from 30-80 years with mean of 54.3±12.0, maximum cases ranged from 40 to 49 years followed by 60 to 69 years. The most common symptom reported is vaginal bleeding (2). In the present study, bleeding per vagina was the most common presentation followed by white discharge per vagina. Histologically, squamous cell carcinoma has been classified as non keratinising and keratinising variants constituting 68.8% and 26% of cases respectively with 5.2% having no data (1). In the present study, non keratinising and keratinising variants constituted 10% and 90%, respectively.

p16 is a regulator of cell cycle and a tumour suppressor protein. It forms complex with Cyclin Dependent Kinase 4 (CDK4) and CDK6 and activate pRb which results in arrest of cell cycle at G1-S phase of cell cycle. In non HPV cancer, p16 gene can be inactivated due to deletion, mutation, hypermethylation or loss of heterozygosity resulting in increased CDK activity, inactivation of pRb and increase in cell proliferation (4). In HPV related cancers, as in cervical cancer, the HR-HPV infects the tissue and releases E7 protein which inactivates pRb protein resulting in increased synthesis and accumulation of p16 protein in cells due to negative feedback mechanism and results in increased cell proliferation (4),(5),(6).

There is improvement in diagnostic agreement when histomorp-hological diagnosis of cervical biopsy is correlated with IHC p16 expression as an adjuvant biomarker (4),(15). Studies have reported positive association of p16 expression with HSIL and SCC of cervix (4),(5),(6). p16 expression in cervical tissue infection with HR-HPV along with integration of viral genome with host genome (17),(18). The criteria for interpretation of p16 expression across globe is not uniform (7),(19). LAST criteria (2012) define p16 expression as block positive, ambiguous and negative. Lower Anogenital Squamous Terminology (LAST) project is co-sponsored by College of American Pathologists (CAP) and American Society for Colposcopy and Cervical Pathology (ASCCP). LAST gives standard guidelines for utility of p16 biomarker (8),(9). In the present study, IHC p16 expression of block positivity, ambiguity and negative constituted 88.6%, 7.1% and 4.3%, respectively.

Tumour diagnosis is conventionally done by radiological findings and invasive surgical biopsy. In surgical or tissue biopsy a small chunk of tissue is taken from the cancer tissue for histopathological examination and diagnosis. Of late non invasive technique where blood sample, urine and body fluids are used to extract CTC and genetic material for cancer diagnosis and treatment which is called as “Liquid Biopsy” (20),(21),(22). In this technique the liquid sample is used to isolate CTC, circulating tumour DNA (ctDNA), RNA, Exosomes and proteins which are shed by tumour cells into blood circulation, body fluids or urine in most of the cancers depending on the site of the cancer. This technique enables non invasive profiling of solid tumours, the results which can be comparable with that of tissue biopsy (9),(10). Studies have shown that estimation of p16 protein by ELISA on lysed samples of cervical cells had positive correlation with HSIL and SCC of cervix. The levels were low in Low grade Squamous Intraepithelial Lesion (LSIL) cases and normal cervix. The rate of detection of cervical dysplasia by p16 ELISA and HR-HPV DNA were similar (4),(6). In the present study, p16 protein was estimated in the plasma of cases of SCC of cervix by ELISA method which showed a range of 3.4-19.6 ng/mL with a mean of 7.2±2.35 ng/mL.

There was significant association between age distribution among cases and IHC p16 expression with younger age (30 to 59 years) showing 100% block positivity probably because of evolving phase of the disease. However, association between age distribution and plasma ELISA p16 levels was not significant. There was no significant association between stage of the disease with IHC p16 expression and plasma ELISA p16 levels. However, all stage I cases showed block positivity and maximum cases of block positivity were in stage III followed by stage II indicating increased expression at higher stage of the disease. Plasma ELISA p16 levels was maximum in stage IV followed by stage II indicating plasma ELISA. p16 increases with stage of the disease. There was no significant association between histological type and IHC p16 expression. However, there was significant association between plasma ELISA p16 levels and histological type where the levels were maximum in WDSCC, followed by MDSCC and PDSCC.

There was no statistical significant association between IHC p16 expression and plasma ELISA p16 levels. However the plasma ELISA p16 levels were maximum in ambiguity and block positive cases compared to negative cases. In addition this study shows that the plasma p16 ELISA levels of 5.1 to 6.2 ng/mL, 6.0 to 6.6 ng/mL and 5.5 to 9.7 ng/mL predicts negative, ambiguity and block positivity of IHC p16 expression in corresponding tissue biopsy.

Limitation(s)

The limitation of this study was, all the cases were SCC of cervix and Cervical Intraepithetial Neoplasia (CIN) as High grade Cervical Intraepithetial Neoplasia (CIN) or Low grade Cervical Intraepithetial Neoplasia (CIN) was not considered for the study as authors thought of proving the hypothesis in frank cases of SCC of cervix and then consider in CIN. There is limitation in numbers of cases, hence, further studies should be done on larger study population and the procedure has to be standardised to prove the hypothesis of the concept of liquid biopsy. However, as far as author’s knowledge, the concept of this study with liquid biopsy is first of its kind in English literature. This is only a pilot study to find the association between tissue IHC p16 expression and plasma ELISA p16 levels.

Conclusion

Plasma ELISA p16 protein level predicts negative, ambiguity and block positivity of IHC p16 expression respectively in corresponding tissue biopsy. Plasma ELISA p16 levels were maximum in NKSCC and WDSCC, followed by MDSCC and PDSCC. The plasma ELISA p16 levels and IHC p16 expression were maximum in higher disease stage compared to lower stage. This observation suggests the possibility of using the blood sample to diagnose and predict the prognosis of SCC of cervix, the concept of liquid biopsy.

References

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DOI and Others

DOI: 10.7860/JCDR/2022/51540.16127

Date of Submission: Jul 23, 2021
Date of Peer Review: Oct 07, 2021
Date of Acceptance: Dec 21, 2021
Date of Publishing: Mar 01, 2022

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: Jul 26, 2021
• Manual Googling: Dec 20, 2021
• iThenticate Software: Jan 19, 2022 (22%)

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