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

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On Sep 2018




Prof. Somashekhar Nimbalkar

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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."



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Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
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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.
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Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




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




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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 : January | Volume : 17 | Issue : 1 | Page : EC15 - EC20 Full Version

Evaluation of Nuclear Morphometry and Ki-67 Proliferative Marker in Astrocytomas: An Ambispective Study


Published: January 1, 2023 | DOI: https://doi.org/10.7860/JCDR/2023/58640.17378
Palak Nanda, Sapna Patel, Apoorva Cherukuri

1. Tutor Cum Postgraduate Student, Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India. 2. Associate Professor, Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India. 3. Student, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India.

Correspondence Address :
Dr. Sapna Patel,
House No-#3, Vth Main Road, Behind Dhobhi Ghat, Yadavagiri, Mysuru, Karnataka, India.
E-mail: drsapnaharish@gmail.com

Abstract

Introduction: Astrocytomas are the commonest primary Central Nervous System (CNS) tumours. Its diagnosis is based on histopathological criteria defined by the World Health Organisation (WHO) 2016 that grades astrocytoma into four grades. The subjective nature of WHO grading has prompted for more objective methods to evaluate nuclear features. Furthermore, Ki-67, a marker of cellular proliferation is a useful diagnostic tool that also helps in prognostic evaluation and to plan adjuvant therapy in astrocytomas.

Aim: To evaluate the nuclear morphometry and Ki-67 proliferative marker in astrocytomas and to assess the relationship of WHO grade with proliferative activity using Ki-67 immunostaining.

Materials and Methods: This ambispective study was conducted in the Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India, with a total duration of four years (October 2017 to September 2021). From 35 astrocytoma cases, Haematoxylin and Eosin (H&E) stained slides were retrieved and reviewed appropriately by two pathologists and graded using the WHO criteria. Nuclear morphometric analysis was performed on the H&E slides using Olympus BX-41 research microscope. The parameters evaluated were mean nuclear length, mean nuclear diameter, mean nuclear perimeter, mean nuclear area, Mean Nuclear Roundness Factor (MNRF) and mean nuclear ellipse form. The cases were then stained with Ki-67 antibody. The relationships between WHO grade of astrocytoma and nuclear morphometry and WHO grade with proliferative index was analysed.

Results: According to WHO grading, 4 (11.4%) cases were grade 1, 12 (34.3%) grade 2, six (17.1%) grade 3 and 13 (37.1%) grade 4. Significant correlation was seen between WHO grading and mean nuclear length, diameter, perimeter, area and Ki-67 with p-value <0.001.

Conclusion: Astrocytoma is an extremely heterogeneous disease with unpredictable outcome. The widely used WHO grading is subjective, while nuclear morphometry, using computer assisted image analysis, can ensure more objective assessment. The Ki-67 index could provide valuable information and may compliment the other parameters.

Keywords

Gliomas, Histopathological grading, Proliferative index

Primary malignant Central Nervous System (CNS) tumour constitutes 2% of all malignancies, with relatively higher mortality and morbidity (1),(2). They are broadly classified as gliomas or non gliomas. Astrocytoma, a tumour of star shaped glial cells called astrocytes is the most common type of glioma (3). The WHO grading system is being widely used and is based on histologic determinants like hypercellularity, mitosis, endothelial proliferation and necrosis. Grade 1 corresponds to pilocytic astrocytoma, grade 2 to diffuse astrocytoma, grade 3 to anaplastic astrocytoma and grade 4 as Glioblastoma Multiforme (GBM). The latest 2016 World Health Organisation (WHO) classification of astrocytoma has incorporated molecular characteristics and now the tumour is differentiated into Isocitrate Dehydrogenase (IDH)-mutant and IDH-wild type (3),(4).

Histopathological examination although considered gold standard, however is not so reliable due to tumour heterogeneity, sampling error, poor reproducibility and the subjective nature of reporting (3). Over the years, many newer diagnostic modalities have surfaced, like nuclear morphometry (5). Nuclear morphometry aids in highly accurate measurements of the different nuclear facets of a tumour cell. Computer assisted image analysis has helped to calculate mean major axis, minor axis, nuclear area and nuclear perimeter, which relate to overall size of the nucleus and roundness of nucleus, which corresponds to nuclear shape (6),(7). Ki-67, a non histone nuclear protein which was first discovered by Gerdes J et al., is expressed in the proliferative phase of the cell cycle and is present in all phases except G0 phase (8),(9). Ki-67 Labelling Index refers to the percentage of cells that are positive for Ki-67 immunostaining (10).

The aim of the present study was to objectively assess the cases of astrocytomas and compare the results with conventional histologic examination. The objectives were to study nuclear morphometry of astrocytomas using computer assisted image analysis, correlate with histological grading and finally evaluate the relationship of histologic grade with proliferative activity using Ki-67 immunostaining.

Material and Methods

This ambispective study was conducted in the Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India, with a total duration of four years (October 2017 to September 2021). Ethical clearance from the Institutional Ethical Committee was obtained (ISS/MC/PG/5189/2019-20).

Inclusion and Exclsuion criteria: Thirty five cases included astrocytic tumours of varying grades. Mixed glioneuronal tumours and cases diagnosed as mixed oligoastrocytomas were excluded from the study.

Study Procedure

For retrospective cases, Haematoxylin and Eosin (H&E) stained slides were retrieved for morphometry and paraffin embedded blocks for Ki-67 Immunohistochemistry (IHC). For prospective cases, surgically removed brain tumour tissues were fixed in formalin, paraffin embedded and stained with H&E followed by immunostaining for Ki-67. All the cases were reviewed appropriately by two pathologists and graded using the WHO criteria published in 2016 (4). Kappa statistics was used to measure the agreement in diagnosis amongst both pathologists. Finally, a consensus diagnosis was made by both the pathologists. For computer assisted nuclear morphometric analysis, Olympus BX-41 research microscope connected with Jenoptix (Germany) progress Charge-Coupled Device (CCD) camera and progress capture pro imaging software was utilised. At 400X magnification, 100 nuclei were studied in each case. The software calculated the quantitative nuclear facets like nuclear length, nuclear diameter, nuclear perimeter and nuclear area. Other two parameters were calculated and included, Mean Nuclear Roundness Factor (MNRF)=perimeter 2/4 area and nuclear form ellipse (MNFe)=longest diameter/shortest diameter. Ki-67 IHC was performed on 3 μm thick sections on poly-lysine coated slides. Monoclonal antibody Ki-67 (Novocastra, code no. Ki-67-MMI-R7-C) was used by standard streptavidin-biotin technique using novostain universal detection kit (Novocastra, code no. RTU-Ki-67-MM1). Sections from reactive lymph node was used as positive control whereas sections treated with tris-buffer solution instead of primary antibody was used as negative control. Brown granular nuclear reactivity indicated positivity. LI was derived, which is the percentage of positively stained nuclei amongst 1000 cells noted under 400X magnification.

For optimal LI calculation, highly cellular area with evenly distributed cells having clear morphology and good staining was selected. Regions devoid of necrosis and endothelial proliferation were preferable.

Statistical Analysis

Kappa statistics was used to analyse the agreement and interobserver variability in diagnosis and grading of astrocytomas amongst the two pathologists. Statistical Package for Social Sciences (SPSS) version 13.0 was used to find the mean value of all parameters for each grade of astrocytoma, and Pearson’s correlation coefficient was calculated. The p-value <0.05 was considered statistically significant.

Results

After appropriate reviewing of H&E slides by two pathologists, 29 cases were in concordance to each other, whereas disagreement was seen in six cases. Kappa statistics was used to find out this agreement amongst both pathologists in WHO grading and was 74.7%, implying substantial agreement (fair agreement, κ=0.00 to 0.20; moderate agreement, κ=0.21 to 0.45; substantial agreement, κ=0.46 to 0.75; near perfect agreement, κ=0.76 to 0.99; perfect agreement, κ=1.00). Finally, both pathologists arrived to a consensus and there were four grade I cases (Table/Fig 1)a, 12 grade II cases (Table/Fig 1)b, six grade III (Table/Fig 1)c and 13 grade IV cases (Table/Fig 1)d.

Among these 35 cases, 12 were retrospective and 23 prospective cases with a male predominance and M:F ratio of 3:2. The age group ranged from 14-70 years with a mean age of 40.6 years. Age range for grade 1 tumour was 14-48 years, for grade 2 was 34-70 years, grade 3 was 18-60 years and for grade 4 was 17-68 years.

The correlation test was applied between WHO grade and nuclear morphometry among all 35 cases (Table/Fig 2) and the mean was calculated for each parameter in all cases followed by calculation of p-values and Pearson’s coefficient (Table/Fig 3).

Pearson’s correlation was used to evaluate the relationship between WHO grade and nuclear morphometry and positive correlation was found between WHO grade and nuclear length, diameter, perimeter and area. Since, p-value of <0.05 is considered statistically significant, all the mean values of nuclear size morphometry in this study were statistically significant. However, nuclear roundness factor and nuclear form ellipse were not statistically significant (Table/Fig 3). The photographs of nuclear morphometric analysis of each grade of tumour using research microscope are demonstrated in (Table/Fig 4).

Ki-67 immunostaining: The Ki-67 LI was calculated based on the number of nuclei showing brown granular positivity for every 1000 nuclei counted in each case as shown in (Table/Fig 2),(Table/Fig 5). The mean values of Ki-67 for the lower three grades were similar, whereas it was significantly higher for GBM (Table/Fig 6). The Ki-67 results were correlated to consensus WHO grade using Pearson’s correlation coefficient (p≤0.001). The coefficient was 0.643, which implies it positively correlated with WHO grade.

Discussion

Astrocytomas are heterogenous in their presentation. In this study, the age ranged from 14-70 years. Grade 1 astrocytoma is commonly seen in the paediatric age group and the youngest one in this study was 14 years and was histologically diagnosed with pilocytic astrocytoma (Table/Fig 1)a (11). The variation in the age was also supported by Sukheeja D et al., (12). This study showed a greater incidence of grade 2 and grade 4 tumours with lower prevalence of grade 1 and grade 3 tumours. A similar trend was seen by Ralte AM et al., (13). Other studies like of Johannessen AL and Torp SH, and Akaishi K et al., showed grade 3 and 4 to be more common (14),(15). The studies conducted by Ralte AM et al., Johannessen AL and Torp SH, and Akaishi K et al., showed mean age of occurrence being directly proportional to the grade of astrocytoma, whereas this study saw the highest mean age for grade 2 tumour (Table/Fig 7) (13),(14),(15).

Nuclear Morphometry

Even with advancement in WHO grading of CNS tumours, disconcordance among pathologists remain to certain extent.

Reason for this could be pathologists experience and the unclear criteria put forth by WHO. As grade of the tumour increases, cellularity and mitosis also increase. However, there is no defined cut-off for these criteria, which leads to subjective error in grading (4).

Aldape K et al., in their study found that in 457 astrocytoma cases, 23% had variable diagnosis, and 16% among them lead to patient mismanagement (16). In the current study, there was disagreement in 6 (17%) out of 35 cases. Thus, histology alone cannot be sufficiently considered reliable to predict patient outcome. Newer methodologies like molecular genetics and Deoxyribonucleic Acid (DNA) ploidy are not always affordable by all patients, since they are expensive. Hence, nuclear morphometry is a simpler and cheaper tool to objectively differentiate between benign and a malignant tumour (6). Till now, very few studies have been conducted on computer assisted morphometric analysis of CNS tumours. The variables used in a similar study conducted by Boruah D and Deb P, included major axis, minor axis, nuclear area, nuclear perimeter and nuclear roundness, very similar to variables used in this study, with addition of nuclear form ellipse (6). The initial four parameters of this study assessed the nuclear size and the last two derived variables correspond to the nuclear shape of the tumour cells.

The study done by Boruah D and Deb P, on nuclear morphometry on gliomas showed negative correlation of roundness factor with WHO grade (6). In the present study, there was a negative correlation for nuclear form ellipse. Both of these parameters pertain to nuclear shape. A technical error for this insignificant correlation may include missing out the details of nuclear shape irregularity while manually contouring on the research computer. A previous study done by Ikeguchi M et al., has investigated the interobserver variation in morphometry (17). Other sources of error could be due to error in calibration and standardisation of the software (18).

The WHO grading is very subjective in which poor sampling, fixation and sectioning can lead to a false diagnosis (3). Such limitations can be overcome with the evaluation of increased abnormality in nuclear size and shape. Tosi P et al., in their study stated that morphometric parameters like nuclear area are independent to tissue processing issues (19). The best technique that could supplement grading are stereological factors like nuclear length, diameter, perimeter and area that predict the nuclear size.

Extensive work has been done on nuclear morphometry in tumours of other systems such as breast carcinoma, renal cell carcinoma, thyroid carcinoma (19),(20),(21),(22). A couple of studies have been devoted to conducting morphometric analysis on cytological smears (23). Similar parameters have been used in the domain of morphometry in the study of these organ systems (20),(21),(22).

Ikeguchi M et al., studied computerised nuclear morphometry use in evaluating colorectal adenocarcinoma and their metastatic potential (17). They inferred that the mean nuclear area increased as the case progressed from non neoplastic to an adenoma to colorectal adenocarcinoma. They also concluded that the capability of the tumour cells to metastasize into lymphovascular space and to distant organ depended on the nuclear area. Hence, the study stated that nuclear morphometry can be used as a screening program to assess the metastatic potential of the carcinoma.

Similar study conducted by Pienta KJ and Coffey DS, on breast carcinoma saw a steep increase in nuclear area among those cases that were node-metastasis positive (20). Computer assisted image analysis in these studies support the fact that an aggressive tumour that gets graded into a higher category would expectedly have bigger nuclear area.

Ki-67 Labelling

The Ki-67 is a monoclonal antibody that targets protein expressed in a proliferating cell, except in the resting phase (G0 phase) of cell cycle (24). It has already been used for breast tumours, lymphomas and other intracranial malignancies such as gliomas in general and meningioma (25),(26),(27),(28). The antibody can be utilised as a tool to supplement the diagnosis of astrocytoma.

In the present study, the mean LI of grade 1, grade 2 and grade 3 astrocytomas were within proximity to each other and were low. A similar observation was made by Sengupta S et al., (29). There are a few studies which had differing results. The study conducted by Johannessen AL and Torp SH, showed a significant leap of the mean LI from grade 2 to grade 3 astrocytomas (14). Ralte AM et al., also showed a similar finding (13). The present study showed overlapping of Ki-67 LI amongst some of the grades of astrocytomas. A study conducted by Moskowitz SI et al., on Ki-67 proliferation on newly detected grade 4 GBM found it to range from 0-76.4% (30). In a study, Raghavan R et al., described that the presence of well differentiated regions in higher grades of astrocytoma is responsible for giving an overlapping result (31).

A few studies like the ones conducted by Thotakura M et al., have shown Ki-67 correlation with histologic grade, much like the present study (32). Significant correlation of the marker to astrocytoma grade was observed in Sengupta S et al., study, identical to the results of this study (29). The issues that may occur with Ki-67 immunostaining range from type of products (manufacturing company), characteristic of the secondary antibody, fixative used and time for fixation and expertise in counting of Ki-67 positive cells (32).

Another issue encountered with calculation of the proliferative index is that an amount as huge as 1000 cells needs to be evaluated for Ki-67 positivity under a microscope. It is not impossible that sometimes pathologists do guess work to save time (33), and when its wrong, it alters the course of treatment. Therefore, newer automated computer assisted techniques are emerging for Ki-67 calculation (33).

Pleomorphic Xanthoastrocytoma (PXA) is a low-grade tumour with favourable prognosis (4). The tumour exhibits moderate cellularity and high level of pleomorphism, however with no areas of necrosis (34). Mitosis is also rare (35). The nucleus is multilobulated and bizarre. A case like this may be misdiagnosed as GBM, if it shows any doubtful undifferentiated areas. It was difficult for the two pathologists to accurately differentiate both entities in cases of small biopsy which may not have been representative of the lesion. In the present study, there were two cases of PXA, both of which were subjected to interobserver variability prior to arriving at a consensus diagnosis. The benign nature of this tumour was supported by low Ki-67 LI. Similar finding was noted in a case reported by Ng WH et al., where an elderly female presented with an aggressive case of PXA with an initial over diagnosis of glioblastoma (36). Despite having areas of necrosis on radiographic and histological examination, the LI of Ki-67 was surprisingly low.

Despite WHO grading being gold standard in diagnosis of astrocytomas, the present study demonstrated discordance in six out of 35 cases with significantly positive correlation for nuclear morphometric analysis of nuclear size and either statistically insignificant or a negative Pearson’s coefficient for nuclear shape. Hence, nuclear morphometry which is relatively an easy tool can enhance the accuracy of diagnosis. In addition, Ki-67 LI showed overlapping results amongst some of the grades of astrocytomas. Hence, combined studies including WHO grading, nuclear morphometry and Ki- 67 LI in astrocytomas may help in arriving at correct diagnosis with better patient care.

Limitation(s)

In the present study, the mean values of all parameters in grade 2 tumours were lower than that of grade 1. This could be attributed to the fact that out of four grade 1 tumours, two were PXA. This tumour presented with large, hyperchromatic, multinucleated cells. Thus, its morphometric values were quite higher and have put the mean of grade 1 parameters slightly at a higher level than grade 2 tumours. The result might have altered if equal number of cases were assessed for each grade of astrocytomas.

Conclusion

Astrocytomas are the most common glial tumour with varied characteristics. The WHO grading being the gold standard for diagnosis, accuracy is not guaranteed at all the times and nuclear morphometry is a relatively easy tool that can enhance the accuracy of diagnosis. It is mostly helpful as a supplementary tool on stereotactic small biopsies, which may not be representative of the tumour. In addition, Ki-67 is an immunomarker that is detected at any stage of the cell cycle except the quiescent phase. In the present study, grade 1 to grade 3 showed low Ki-67 labelling indices, whereas it was significantly increased for GBM with a positive correlation to WHO grade. As noted in the present study, both morphometry and IHC themselves are not sufficient to accurately diagnose astrocytoma, instead they make excellent supplementary techniques that complements the gold standard, histopathology.

Acknowledgement

The authors are extremely grateful to the laboratory technical staffs of the hospital for their contribution in this study. Authors are also thankful to the Head of the Department for immeasurable support and for providing great facility and opportunity.

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DOI and Others

DOI: 10.7860/JCDR/2023/58640.17378

Date of Submission: Jun 23, 2022
Date of Peer Review: Aug 20, 2022
Date of Acceptance: Dec 21, 2022
Date of Publishing: Jan 01, 2023

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

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• iThenticate Software: Nov 22, 2022 (22%)

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