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

Users Online : 124953

AbstractMaterial and MethodsResultsDiscussionConclusionReferencesDOI and Others
Article in PDF How to Cite Citation Manager Readers' Comments (0) Audio Visual Article Statistics Link to PUBMED Print this Article Send to a Friend
Advertisers Access Statistics Resources

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 Dermatolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018

Prof. Somashekhar Nimbalkar

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

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

Dr. Kalyani R

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

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

Dr. Saumya Navit

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

Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
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,
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,
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
(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)
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.
On April 2011

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
On Jan 2020

Important Notice

Original article / research
Year : 2023 | Month : November | Volume : 17 | Issue : 11 | Page : EC01 - EC06 Full Version

Combined Cyclin D2 and Protein Convertase 2 Genes in Differentiating Various Follicular-patterned Lesions and Neoplasms of the Thyroid: A Cross-sectional Study

Published: November 1, 2023 | DOI:
Prasanna Venkadesa Perumal, Neelaiah Siddaraju, Sunil Kumar Saxena, Soundravally Rajendiran, Ramachandra V Bhat

1. Laboratory Technician, Department of Microbiology, Jawaharlal Institute of Medical Education and Research (JIPMER), Puducherry, India. 2. Senior Professor, Department of Pathology, Jawaharlal Institute of Medical Education and Research (JIPMER), Puducherry, India. 3. Professor, Department of Otorhinolaryngology, Jawaharlal Institute of Medical Education and Research (JIPMER), Puducherry, India. 4. Professor, Department of Biochemistry, Jawaharlal Institute of Medical Education and Research (JIPMER), Puducherry, India. 5. Dean and Professor, Department of Pathology, Indira Gandhi Medical College and Research Institute (IGMC and RI), Puducherry, India.

Correspondence Address :
Dr. Neelaiah Siddaraju,
Senior Professor, Department of Pathology, Jawaharlal Institute of Medical Education and Research (JIPMER), Puducherry-605006, India.


Introduction: A pretherapeutic distinction between benign and malignant thyroid nodules is critical for the clinical management of patients presenting with thyroid nodules. However, certain follicular-patterned lesions, such as adenomatous nodules, follicular neoplasms comprising Follicular Adenoma (FA) and Follicular Carcinoma (FC), as well as the Follicular Variant of Papillary Thyroid Carcinoma (FVPTC), can pose a significant dilemma during pre-therapeutic Fine Needle Aspiration Cytology (FNAC) evaluation. The present (pilot) study explores the possible utility of messenger Ribonucleic Acid (mRNA) and the protein expression of the two relatively less-explored genes, Cyclin D2 (CCND2) and Protein Convertase 2 (PCSK2), in distinguishing various follicular-patterned thyroid lesions and neoplasms by testing these molecular markers initially on histopathological sections.

Aim: To assess the RNA and protein expressions of CCND2 and PCSK2 genes in differentiating follicular-patterned thyroid neoplasms.

Materials and Methods: This was a cross-sectional analytical study conducted over 6 years, from August 2014 to August 2020, at the Department of Pathology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India. Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) along with Immunohistochemistry (IHC) was performed on a total of 75 tissue samples from follicular-patterned thyroid lesions and neoplasms, including 19 Follicular Hyperplasias (FHs), 10 Nodular Goitres (NGs), 17 FAs, 8 FCs, and 12 FVPTCs, along with nine Conventional Papillary Thyroid Carcinomas (CPTCs). After confirming the RNA and protein expression levels in each of these lesions, Immunoreactive Scoring (IRS) was performed to assess their IHC expression. The Kruskal-Wallis and Analysis of Variance (ANOVA) tests were used to analyse the mRNA expression data, and Pearson analysis was conducted to correlate the IHC data between the study groups. A p-value of <0.05 was considered statistically significant.

Results: Among the 75 thyroid lesions studied, both NG and FA showed a relatively higher cyclinD2 mRNA expression, with fold changes of 1.21 and 1.46, respectively. This was also reflected in IHC, with moderate nuclear expression observed in these cases. The PCSK2 mRNA expression was similar to that of CCND2, with the only difference noted between FH and FA. On IHC, eight out of 75 cases had positive PCSK2 expression, including five FHs, two FVPTCs, and one FA, while the remaining 67 cases were negative.

Conclusion: The CCND2 and PCSK2 genes assessed in the present study, regarding their mRNA and protein expressions, were not found to be of any practical value in distinguishing benign and malignant follicular lesions or neoplasms of the thyroid.


Adenoma, Carcinoma, Follicular pattern, Follicular variant, Immunohistochemistry, Messenger ribonucleic acid expression, Papillary carcinoma

A variety of thyroid disorders are common worldwide. Thyroid Function Tests (TFT), imaging studies, and FNAC are the common investigative modalities employed in thyroid work-up. The role of FNAC in establishing the neoplastic and non neoplastic nature of a thyroid nodule is well-established. The Bethesda System of Reporting Thyroid Cytopathology (TBSRTC) is the universally accepted pattern for reporting thyroid FNAC. Although FNAC is highly effective in detecting malignancies such as conventional papillary carcinoma, medullary carcinoma, anaplastic carcinoma, and high-grade Non-Hodgkin’s Lymphoma (NHL), it cannot distinguish between a FA and FC, which are categorised as ‘follicular neoplasm’ or ‘suspicious of follicular neoplasm’ under TBSRTC. Furthermore, lesions like FH/adenomatous nodules and FVPTC often cause significant diagnostic difficulty, creating a major hurdle in deciding the nature of treatment or surgery (1),(2),(3).

Distinguishing various follicular-patterned thyroid lesions is not a problem confined to cytology alone. A significant diagnostic dilemma is encountered even in certain instances of histopathology. IHC markers like galectin-3, CBP/p300 Interacting Transactivator with Glu/Asp Rich Carboxy-Terminal Domain-1 (CITED), Hector Battifora Mesothelial Cell-1 (HBME-1), and cytokeratin-19 (CK-19), which were considered ‘promising’ in earlier studies, were subsequently shown to be of limited practical value (4),(5),(6). Notably, the revised American Thyroid Association (ATA) in 2015 recommended the incorporation of ‘molecular testing’ in the pre-therapeutic follow-up of doubtful thyroid follicular lesions, a recommendation that has been accepted by TBSRTC-2017. Currently, molecular assays like Affirma gene classifier and ThyroSeq® are being applied to FNAC samples. However, the currently available molecular tests are not 100% effective in identifying the precise nature of doubtful thyroid nodules, which is why the search for further markers is ongoing (7). There has been significant emphasis on certain less-explored genes like CCND2 and PCSK2 (8),(9),(10),(11).

The CCND2 is one of the members of the D-type cyclin family, the others being CCND1 and cyclin D3. The D-type cyclins exhibit a similar amino acid sequence, and their major function is in cell cycle regulation, especially in G1/S transition. They are also involved in cellular differentiation and oncogenic transformation. CCND2 was the first to be discovered among them and has been extensively studied, while CCND1 is relatively less explored. CCND2, located on chromosome 12q13, is usually expressed in B-lymphocytes (12). It has been shown to increase phosphorylation of the retinoblastoma (Rb) protein and promote cell cycle progression, cell proliferation, and tumourigenesis (13). Studies have documented its overexpression in gastric (12) and thyroid cancers (11).

The PCSK2 gene encodes a member of the subtilisin-like proPCSK family. A total of nine mammalian PCSK proteins have been discovered so far and have been classified based on their cleavage preferences. PCSK2 is located on chromosome number 20b and p11.2, consisting of 12 exons. The PCSK2 protein, synthesised as an inactive form (72kDa) in the endoplasmic reticulum, is transported to the Golgi complex, where it transforms into mature secretory granules (64kDa) (14). The protein functions as a mediator of prohormones and pro-peptides in neuroendocrine cells (15). Studies have highlighted its role as an IHC marker for the detection of a variety of metastatic neuroendocrine tumours (13).

Molecular methods to diagnose and distinguish follicular-patterned thyroid neoplasms have not been widely used in India. The present study assessed the utility of mRNA and protein expression of two important but less emphasised genes, PCSK2 and CCND2, on tissue samples using RT-qPCR and IHC techniques to address the long-standing diagnostic dilemma of differentiating follicular thyroid tumours.

Material and Methods

This cross-sectional analytical study was conducted over a six-year period, from August 2014 to August 2020, at the Department of Pathology, JIPMER, Puducherry, India, in collaboration with the Departments of Biochemistry, JIPMER, Puducherry, and the Department of Pathology, Indira Gandhi Medical College and Research Institute (IGMC&RI), Puducherry, India. The study commenced after obtaining approval from the Research Monitoring and Ethics Committees (IEC NO: JIP/IEC/SC/2015/19/783) of JIPMER.

Inclusion criteria: The study included all well-circumscribed or encapsulated lesions with histopathological diagnosis of adenomatoid nodule/FH, FA, FC, FVPTC, which had preoperative FNAC interpretations/diagnosis of NG, adenomatous nodule/FH, Follicular Lesion of Undetermined Significance (FLUS), Follicular Neoplasm/Suspicious for FN (FN/SFN), or FVPTC.

Exclusion criteria: Cases without a preoperative FNAC interpretation/diagnosis were excluded from the study.

Sample size calculation: The sample size was estimated based on the area under the curve of 0.725 in ROC with a negative-positive ratio of 0.666 (derived from our previous hospital records) and with an alpha error of 0.05 and a beta error of 0.20 using Medcalc 15.6.1 Software.

Study Procedure

Relevant clinical and laboratory results, including imaging findings, were gathered for all 75 cases included in the study. All cases had preoperative FNAC diagnosis, and thyroidectomy was performed based on the FNAC diagnosis or strong clinical suspicion of malignancy. Histopathological diagnosis was considered the gold standard. Strict precautionary measures were followed during the collection of tissue samples from the pathological lesions for molecular studies. Normal thyroid tissue collected from the surrounding pathological lesions served as the control. Fresh tissue from the lesional area of the thyroidectomy specimen was transferred into RNA later solution and stored at -80°C. RNA was isolated from the frozen tissue specimens using the RNA extraction kit (Roche Life Science). The extracted RNA was converted into cDNA using the cDNA synthesis kit (Roche Life Sciences). RT-qPCR was conducted to study the mRNA expression of the candidate genes using the Universal Probe Library Assay kits supplied by Roche Life Sciences. RT-qPCR was performed using the UPL probe numbers 49 (CCND2) and 21 (PCSK2) supplied by Roche Life Sciences. For the CCND2 gene, the Forward Primer-CCGCAGTGCTCCTACTTCAA and the Reverse Primer-GCCAAGAAACGGTCCAGGTA were used. For the PCSK2 gene, the Forward Primer-CGTGCAGGACCCTGAGAAAA and the Reverse Primer-GTCTCCTCTTCTGGTTGCGT were used. The β-actin (probe Number-9) served as the housekeeping gene (positive control). IHC for both PCSK2 and CCND2 proteins was performed in all 75 cases. The antibody kits for PCSK2 and CCND2 were obtained from ORIGENE Technologies (US) and Biorbyt Ltd (UK), respectively. Antibody dilution was done according to the manufacturer’s protocol. The working condition of both antibodies was tested using the control tissues recommended by the manufacturers. For each tissue sample, 3 μm thick sections were prepared from the routinely processed and paraffin-embedded tissue blocks, which were placed on poly-L-lysine coated slides for immunostaining. The slides were treated with 3% hydrogen peroxide in methanol for 20 minutes to block endogenous peroxidase. Antigen retrieval was done using the pressure cooker method with citrate buffer (pH 6.4). Primary antibody incubation was performed for 2 hours, followed by polymer horseradish peroxidase incubation for 40 minutes. Diaminobenzidine (DAB) was used as the chromogen. The IHC slides were examined, and an IRS was performed by the Pathologist (NS) (16). The scoring pattern is elaborated in (Table/Fig 1).

Statistical Analysis

For multiple comparisons between the study groups, the Kruskal-Wallis test was used for unpaired data, while a one-way ANOVA was used for paired data. Pearson’s correlation analysis was performed to assess the correlation of IHC expression in each study group. All statistical tests were conducted using GraphPad Instat Version 3.06, and a p-value of <0.05 was considered statistically significant.


The patients’ age ranged from 19 years to 75 years, with a median of 56 years. There were 60 women and 15 men, resulting in an M:F ratio of 1:4. The preoperative cytological diagnosis and their histopathological correlation with the mean age of the patients are provided in (Table/Fig 2).

mRNA expression: The PCSK2 mRNA expression showed no significant change in any of the study groups, except for the FH group, which exhibited a significantly elevated level compared to FA and CPTC (p-value <0.001) (Table/Fig 3). Among the 75 cases, eight showed PCSK2 gene mRNA expression, including five FHs and one case each of FVPTC, FA, and NG, while the remaining 67 cases had a similar expression pattern (Table/Fig 4). Relative PCSK2 gene expression did not yield significant results for differentiating between FC and FA (p-value 0.17). The CCND2 mRNA expression levels remained unchanged in the FC group compared to the FA, FH, and NG groups (Table/Fig 5). The CCND2 gene expression did not show significant results for differentiating between FC and FA (p-value 0.44) (Table/Fig 6). The expression pattern of CCND2 was similar in all study groups, except the FC group, which exhibited a lesser fold change. Therefore, there was no statistical evidence to support a significant difference among the various study groups.

IHC expression: Among the 75 study cases, only one case each of FA and NG exhibited positive expression (moderate) of CCND2 (Table/Fig 7). None of the malignant cases expressed the marker. A high percentage of FAs (94.2%) and NGs (91.6%) were negative for goitreCCND2, indicating its poor concordance even among non malignant thyroid lesions (Table/Fig 7). None of the FH cases showed CCND2 positivity, suggesting that the marker was not specific for either benign neoplastic or non neoplastic lesions. The majority of cases exhibited a negative staining pattern for CCND2, with focal nuclear positivity observed in one case each of FA and NG (Table/Fig 8). Immunohistochemically, PCSK2 expression was observed only in a few cases of FVPTC, FA, and FH, while the remaining study groups were all negative (100%) (Table/Fig 9). Out of 12 FVPTCs, only 2 were PCSK2 positive, with one showing moderate expression and the other showing strong expressions. Among 19 FHs, 5 expressed the marker, with three exhibiting moderate expressions and the remaining two showing mild and strong expressions, respectively (Table/Fig 10). Pearson correlation analysis for PCSK2 IHC expression showed no correlation between FA vs. FVPTC (p-value 0.771) and FH vs. FVPTC (p-value 0.585), with R-values of 0.07 and 0.13, respectively. The correlation between FA and FH showed a moderate correlation with an R-value of 0.47 and a p-value of 0.052 (Table/Fig 11).

The majority of cases exhibited negative protein expression for CCND2 on IHC (73 negatives and 2 positives), so correlation analysis was not performed. Overall, both CCND2 and PCSK2 were not deemed suitable for routine application in differentiating various follicular-patterned neoplasms/lesions based on their immunohistochemical results.


The follicular-patterned thyroid lesions pose significant diagnostic challenges, both in cytology and histopathology, resulting in a “grey zone” of uncertainty. As a result, there is a continuous search for reliable Immunohistochemical (IHC) markers and molecular assays. In the present study, we examined 75 cases with histopathological confirmation, of which 66 were follicular-patterned lesions including NG, FH, FA, FC, and FVPTC. The significant difference in the number of non neoplastic and neoplastic follicular-patterned lesions between cytological and histopathological interpretations highlights the gravity of the problem faced by cytopathologists. In the present study, authors aimed to evaluate the effectiveness of CCND2 and PCSK2 genes, along with their corresponding proteins, in distinguishing among various follicular-patterned lesions and neoplasms.

The CCND2 and PCSK2 genes encode proteins belonging to the Cyclin-D and subtilisin-like proPCSK families, respectively. According to the literature, CCND2 is frequently downregulated in cancer conditions, although upregulation has been reported in malignancies such as breast and prostate cancers (17). Takano Y et al., investigated the role of CCND2 and CCND1 genes in gastric and breast cancers using IHC and Western blotting methods (12). They found that CCND2 overexpression was significantly associated with cancer invasion and lymph node metastasis. Overexpression of CCND2 was also observed in ovarian granulosa cell tumours (18). Sarkar S et al., studied CCND2 expression in colorectal cancer and found that positive expression was associated with tumour progression (19). In another significant study, three candidate genes including CCND2, PCSK2, and UbcH10 were analysed in 84 follicular neoplasms. The mRNA expression of all three genes was compared, and it was found that only UbcH10 yielded significant results. CCND2 and PCSK2 expressions were more frequently observed in benign thyroid tumours (20).

However, an analysis of CCND2 gene expression in follicular tumours by Prabakaran I et al., did not reveal any significant difference between benign and malignant follicular tumours (21). They did notice relatively higher expression of the CCND2 gene in normal and benign thyroid tissues compared to malignant follicular tumours. Krause K et al., studied the utility of multiple genes for differentiating between benign and malignant thyroid tumours (22). PCSK2 was one of the genes included in their study; however, according to their results, PCSK2 alone was not able to differentiate between benign and malignant thyroid neoplasms. The possibility of an altered mechanism of pro-protein processing involved in the tumour progression of thyroid neoplasms has remained elusive for most researchers. Nejjari M et al., showed that PCSK2 dysfunction causes malignant cell migration and metastasis in colonic cancer (23). According to the study by Weber F et al., PCSK2 was found to be effective in differentiating follicular thyroid tumours, although it failed to provide similar results in the present study (17). Weber F et al., observed a significant downregulation of both CCND2 and PCSK2 mRNA in FC compared to FA, with PCSK2 showing a fold difference downregulation of 263 times (17). The CCND2 and PCSK2 mRNA results of the present study with some of the important studies documented in the literature has been presented in (Table/Fig 12) (11),(21).

Cerutti J et al., conducted a study on multiple gene expression patterns using Serial Analysis of Gene Expression (SAGE) analysis. In their study, six of the genes analysed were unable to distinguish between FA and FC (24). PCSK2 was one of the genes included in the SAGE analysis, and it showed mRNA expression of 30 to 69% in FC and 30 to 40% in FA. Therefore, PCSK2 expression may be rarely observed in thyroid follicular-patterned neoplasms. While some studies have demonstrated significant downregulation of CCND2 and PCSK2 in FC [17,22], our present study did not find any difference in their expression between FC and FA. Both Weber F et al., and Krause K et al., used Beta-actin as the common housekeeping gene in their studies (17),(22). Similarly, the present study also employed Beta-actin as the common housekeeping gene, but authors were unable to demonstrate the diagnostic utility of CCND2 and PCSK2 gene assays in distinguishing follicular-patterned neoplasms of the thyroid.

There has been significant literature on the role of IHC in differentiating benign and malignant thyroid nodules [4-6]. Various IHC panels, including markers such as galectin-3, HBME-1, CITED-1, CK-19, Thyroid Peroxidase (TPO), and Thyroglobulin (TG), have been extensively studied (4). While these markers have shown high sensitivity (>80%) and specificity (>80%), some of them are also known to be expressed in a considerable number of benign thyroid nodules and even in normal thyroid tissue (4),(5),(6). More recently, markers such as anti-BRAFV600E (VE1), Trophoblast Cell-surface Antigen-2 (TROP-2), CD56, and Ki67 (a proliferative marker) have been found to be useful as IHC markers for distinguishing benign and malignant thyroid nodules, as well as various follicular-patterned lesions including Non Invasive Follicular Tumour with Papillary (NIFTP)-like nuclear features [5,6].

Limited literature is available on the IHC assessment of PCSK2 and CCND2 in thyroid neoplasms (17). CCND2 has been well-studied as an IHC marker in gastric carcinomas, where it exhibits cytoplasmic, nuclear, or combined cytoplasmic and nuclear expression in certain normal tissues like the salivary gland and a variety of malignancies including Diffuse Large B-Cell Lymphoma (DLBCL), melanoma, prostatic and thyroid carcinomas (25). Cytoplasmic expression of CCND2 in gastric carcinoma has been associated with poor prognosis (12). PCSK2, which displays cytoplasmic expression, has been found to be useful in detecting neuroendocrine tumours of midgut, pulmonary, pheochromocytoma, and paraganglioma origin at metastatic sites (13).

Regarding their role in thyroid neoplasms, Weber F et al., performed IHC for CCND2 and PCSK2, both of which showed negative expression in FC, while FA had positive expression. They claimed that the results obtained with the combined PCSK2 and CCND2 markers were superior to other studies documented in the literature. None of their FC cases showed positive expression for PCSK2. Our PCSK2 findings are fairly similar to those of Weber F et al., with both studies showing negative PCSK2 expression in FC (17). However, the same is not true for PCSK2 expression in our FA cases, which were CyclinD2all negative, in contrast to the Weber F et al., series where all FAs were PCSK2 positive (17). In the present study, authors recorded positive PCSK2 expression in five FHs and a single FA. All these immunohistochemically positive PCSK2 cases also had a correspondingly increased PCSK2-mRNA level. Thus, both markers were not found to be of any practical benefit.


The authors could have arrived at more meaningful conclusions by assessing CCND2 and PCSK2 IHC as part of an immunopanel with other immunohistochemical markers that have already been tested with reasonably good sensitivity and specificity, as highlighted above. This is a limitation of the present study. However, the insignificant CCND2 and PCSK2 IHC results documented in the present study make them obviously inferior to the other markers highlighted in the literature.


The preoperative distinction of various follicular-patterned thyroid nodules is diagnostically challenging for cytopathologists, although it is critical for patient management. These lesions often pose significant diagnostic dilemmas even upon histopathological examination. While sensitive molecular assays like Thyroseq and affirma gene classifier are currently being used on preoperative cytological samples, they are not completely specific, leading to a continued search for reliable molecular markers. In the present study, authors assessed the CCND2 and PCSK2 genes, based on their proven utility in certain malignancies, to distinguish between benign and malignant thyroid follicular lesions/neoplasms. However, authors did not find them to be of any practical value at both the mRNA and protein levels.


Han K, Ha J, Kong JS, Kim JS, Myung JK, Koh JS, et al. Cytological features that differentiate follicular neoplasm from mimicking lesions. J Pathol Transl Med. 2018;52(2):110-20. [crossref][PubMed]
Balakrishnan K, Sumathi V, Elangovan RS, Hemalatha G. Follicular neoplasms cytohistomorphological aspects: A case study of 50 cases. Int J Sci study. 2016;4(4):225-30.
Faquin WC. Diagnosis and reporting of follicular-patterned thyroid lesions by fine needle aspiration. Head Neck Pathol. 2009;3(1):82-85. [crossref][PubMed]
Saleh H, Jin B, Barnwell J, Alzohaili O. Utility of immunohistochemical markers in differentiating benign from malignant follicular-derived thyroid nodules. Diagn Pathol. 2010;5:9. [crossref][PubMed]
Chuang H-W, Wang J-S, Tsai J-W, Hsu C-T, Lin K-J. Immunohistochemistry helps to distinguish non-invasive follicular thyroid neoplasm with papillary-like nuclear features/non-invasive encapsulated follicular variant of papillary thyroid carcinoma with other follicular thyroid lesions. Medicina (Kaunas). 2021;57(11):1246. [crossref][PubMed]
Dwivedi SS, Khandeparkar SGS, Joshi AR, Kulkarni MM, Bhayekan P, Jadhav A, et al. Study of immunohistochemical markers (CK-19, CD-56, Ki-67, p53) in differentiating benign and malignant solitary thyroid nodules with special reference to papillary thyroid carcinomas. J Clin Diagn Res. 2016;10(12):EC14-EC19. [crossref][PubMed]
Tamhane S, Gharib H. Thyroid nodule update on diagnosis and management. Clin Diabetes Endocrinol. 2016;2:17. [crossref][PubMed]
Jaaks P, Bernasconi M. The proprotein convertase furin in tumour progression: The PC furin in tumour progression. Int J Cancer. 2017;141(4):654-63. [crossref][PubMed]
Shen Y, Dong S, Liu J, Zhang L, Zhang J, Zhou H, et al. Identification of potential biomarkers for thyroid cancer using bioinformatics strategy: A study based on GEO datasets. Biomed Res Int. 2020;2020:9710421. [crossref][PubMed]
Leone V, Angelo D, Rubio I, Freitas PM, Fedrico A, Colamaio M, et al. MiR-1 is a tumor suppressor in thyroid carcinogenesis targeting CCND2, CXCR4, and SDF-1 alpha. J Clin Endocrinol Metab. 2011;96(9):E1388-98. [crossref][PubMed]
Yuan S, Liu Z, Yu S, Wang X, Shi J. CCND2 and miR-206 as potential biomarkers in the clinical diagnosis of thyroid carcinoma by fine-needle aspiration cytology. World J Surg Oncol. 2023;21(1):22.[crossref][PubMed]
Takano Y, Kato Y, van Diest PJ, Masuda M, Mitomi H, Okayasu I. CyclinD2 overexpression and lack of p27 correlate positively and cyclin E inversely with a poor prognosis in gastric cancer cases. Am J Pathol. 2000;156(2):585-94. [crossref][PubMed]
Remes SM, Leijon H, Vesterinen T, Louhimo J, Pulkkinen V, Ezer S, et al. PCSK2 expression in neuroendocrine tumors points to a midgut, pulmonary or pheochromocytoma-paraganglioma origin. APMIS. 2020;128(11):563-72. [crossref][PubMed]
Benjannet S, Rondeau N, Paquet L, Boudreault A, Lazure C, Chrétien M, et al. Comparative biosynthesis, covalent post-translational modifications, and efficiency of pro-segment cleavage of the prohormone convertases PC1 andPC2: Glycosylation, sulphation, and identification of the intracellular site of prosegment cleavage of PC1 and PC2. Biochem J. 1993;294(3):735-43. [crossref][PubMed]
Gyamera-Acheampong C, Mbikay M. Proprotein convertase subtilisin/kexin type4 in mammalian fertility: A review. Hum Reprod Update. 2009;15(2):237-47. [crossref][PubMed]
Fedchenko N, Reifenrath J. Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue- A review. Diagn Pathol. 2014;9:221. [crossref][PubMed]
Weber F, Shen L, Aldred MA, Morrison CD, Frilling A, Saji M, et al. Genetic classification of benign and malignant thyroid follicular neoplasia based on a three-gene combination. J Clin Endocrinol Metab. 2005;90(5):2512-21. [crossref][PubMed]
Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, et al. Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature. 1996;384(6608):470-74. [crossref][PubMed]
Sarkar S, Dey BK, Dutta A. MiR-322/424 and 503 are induced during muscle differentiation and promote cell cycle quiescence and differentiation by down-regulation of Cdc25A. Mol Biol Cell. 2010;21(13):2099-2366. [crossref][PubMed]
Guerriero E, Ferraro A, Desiderio D, Pallante P, Berlingieri MT, Iaccarino A, et al.UbcH10 expression on thyroid fine-needle aspirates. Cancer Cytopathol. 2010;25;118(3):157-65. [crossref][PubMed]
Prabakaran I, Grau J, Lewis R, Fraker D, Guvakova M. Rap2AI up regulated in invasive cells dissected from follicular thyroid cancer. J Thyroid Res. 2011;2011:979840. [crossref][PubMed]
Krause K, Eszlinger M, Gimm O, Karger S, Engelhardt C, Dralle H, et al. TFF3-based candidate gene discrimination of benign and malignant thyroid tumors in a region with borderline iodine deficiency. J Clin Endocrinol Metab. 2008;93(4):1390-93. [crossref][PubMed]
Nejjari M, Berthet V, Rigot V, Laforest S, Jacquier M-F, Seidah NG, et al. Inhibition of proprotein convertases enhances cell migration and metastases development of human colon carcinoma cells in a rat model. Am J Pathol. 2004;164(6):1925-33. [crossref][PubMed]
Cerutti J, Delcelo R, Amadei M, Nakabashi C, Maciel R, Peterson B, et al. A preoperative diagnostic test that distinguishes benign from malignant thyroid carcinoma based on gene expression. J Clin Invest. 2004;113(8):1234-42. [crossref][PubMed]
Hans CP, Weisenburger DD, Greiner T, Chan WC, Aoun P, Cochran GT, et al. Expression of PKC-beta or cyclin D2 predicts for inferior survival in diffuse large B-cell lymphoma. Modern Pathol. 2005;18(10):1377-84.[crossref][PubMed]

DOI and Others

DOI: 10.7860/JCDR/2023/65064.18650

Date of Submission: Apr 27, 2023
Date of Peer Review: Jun 16, 2023
Date of Acceptance: Aug 14, 2023
Date of Publishing: Nov 01, 2023

• 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. Yes

• Plagiarism X-checker: Apr 29, 2023
• Manual Googling: Jul 13, 2023
• iThenticate Software: Aug 12, 2023 (8%)

ETYMOLOGY: Author Origin


JCDR is now Monthly and more widely Indexed .
  • Emerging Sources Citation Index (Web of Science, thomsonreuters)
  • Index Copernicus ICV 2017: 134.54
  • Academic Search Complete Database
  • Directory of Open Access Journals (DOAJ)
  • Embase
  • EBSCOhost
  • Google Scholar
  • HINARI Access to Research in Health Programme
  • Indian Science Abstracts (ISA)
  • Journal seek Database
  • Google
  • Popline (reproductive health literature)