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

Users Online : 43200

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
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



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




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011
Anuradha

Dear team JCDR, I would like to thank you for the very professional and polite service provided by everyone at JCDR. While i have been in the field of writing and editing for sometime, this has been my first attempt in publishing a scientific paper.Thank you for hand-holding me through the process.


Dr. Anuradha
E-mail: anuradha2nittur@gmail.com
On Jan 2020

Important Notice

Original article / research
Year : 2022 | Month : May | Volume : 16 | Issue : 5 | Page : EC01 - EC06 Full Version

Comparative Analysis of Ki-67 in Different Scoring Patterns and its Association with other Prognostic Markers of Breast Carcinoma


Published: May 1, 2022 | DOI: https://doi.org/10.7860/JCDR/2022/52704.16231
Archana Buch, Mukta Naik, Shirish Chandanwale, Hetal Rathod, Barnali Paul, Charusheela Gore

1. Professor, Department of Pathology, Dr. D.Y Patil Medical College, Hospital and Research Centre, Dr. D.Y Patil Vidyapeeth, Pune, Maharashtra, India. 2. Third Year PG Resident in Pathology, Department of Pathology, Dr. D.Y Patil Medical College, Hospital and Research Centre, Dr. D.Y Patil Vidyapeeth, Pune, Maharashtra, India. 3. Professor, Department of Pathology, Dr. D.Y Patil Medical College, Hospital and Research Centre, Dr. D.Y Patil Vidyapeeth, Pune, Maharashtra, India. 4. Professor, Department of Community Medicine, Dr. D.Y Patil Medical College, Hospital and Research Centre, Dr. D.Y Patil Vidyapeeth, Pune, Maharashtra, India. 5. Third Year PG Resident in Pathology, Department of Pathology, Dr. D.Y Patil Medical College, Hospital and Research Centre, Dr. D.Y Patil Vidyapeeth, Pune, Maharashtra, India. 6. Professor & HOD, Department of Pathology, Dr. D.Y Patil Medical College, Hospital and Research Centre, Dr. D.Y Patil Vidyapeeth, Pune, Maharashtra, India.

Correspondence Address :
Dr. Mukta Naik,
1904/A, Queens, Pride Palms, Opposite Bayer Compound, Kolshet Road, Thane (WEST)-400607, Maharashtra, India.
E-mail: dr.muktanaik23@gmail.com

Abstract

Introduction: Immunohistochemical evaluation of Ki-67 is widely used for estimation of tumour proliferation in breast cancer. Till date, no specific method or a cut-off point for Ki-67 exists.

Aim: To perform a comparative analysis between different scoring patterns and mean Ki-67 value and association of mean Ki-67 with other prognostic markers like tumour size, lymph node status, tumour stage and grade and different molecular subtypes of breast cancer.

Materials and Methods: A cross-sectional study was conducted at Dr. D.Y Patil Medical College, Hospital and Research Centre, Pune, Maharashtra, India, between August 2019 to August 2021. Total of 50 new diagnosed cases of breast cancer were studied for the histologic type, grade and stage of the tumour. Immunohistochemistry for Oestrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2) and Ki-67 was performed. Association of Ki-67 with other prognostic markers like tumour size, lymph node status, tumour stage and grade and different molecular subtypes of breast cancer was evaluated by expressing Ki-67 as a continuous variable (mean±SD) and also by dividing Ki-67 into different scoring patterns (I: ≤14%, >14%, II: ≤15%, 16-30%, >30% and III: <20%, 20-50%, ≥50%). Statistical tests like Kruskal-Wallis test (mean Ki-67 with tumour size, tumour grade and molecular subtypes), Mann-Whitney Rank Sum Test (mean Ki-67 with lymph node status) and Analysis of variance (ANOVA) (mean Ki-67 with staging) respectively.

Results: Out of 50 patients, 40 (80%) were older than 50-year-old. Twenty six (52%) cases affected the left breast. A total of 49 (98%) were diagnosed as Invasive Ductal Carcinoma (IDC). Among them 26 (52%) cases were of grade III and 25 (50%) cases were of Luminal A. Mean Ki-67 and molecular subtypes of breast cancer had statistically significant association (p= 0.002). No association was found between mean Ki-67 and tumour size (p=0.608), lymph node status (p=0.506) stage (p=0.979) and grade (p=0.095) of the tumour. Although scoring pattern I and III had no remarkable findings. Notably, scoring pattern II showed higher tumour sizes, lymph node positivity, higher stage and grade and basal-like tumours demonstrated a higher Ki-67 index.

Conclusion: Evaluation of Ki-67 as a continuous variable yielded significant association with other prognostic markers of breast cancer. There was no single “best” scoring pattern identified. A direct association of Ki-67 was found with molecular subtypes of breast cancer.

Keywords

Breast Neoplasms, Immunohistochemistry, Molecular Subtypes, Prognosis

According to GLOBOCAN 2020 (Global Cancer Incidence, Mortality and Prevalence), female breast carcinoma has emerged as the most common cancer found worldwide. It accounted for 2.3 million new cases (11.7 %) of the total cancer cases across the globe in 2020 (1).

Breast cancer can be classified according to the histopathological type {World Health Organization (WHO) 2019} (2), tumour grade (Elston Ellis modification of Scarff-Bloom-Richardson grading system) (3), tumour stage {American Joint Committee on Cancer (AJCC) 8th edition} (4) and different molecular subtypes of breast cancer (Luminal A, Luminal B, HER2 rich and Basal-like/Triple Negative) (5).

The proliferative potential of any malignancy can be estimated using various methods like the historically used method of counting mitotic figures, incorporation of labelled nucleotides into DNA, flow cytometric assessment of S phase as well as by Immunohistochemical (IHC) identification of markers like Proliferating Cell Nuclear Antigen (PCNA), cyclin E and Ki-67 antigen (6),(7).

The Ki-67 antigen is also known as MKI67 or MIB-1 (6),(8). It is a non histone protein (8). It was first described by Gerdes J et al., in 1980’s while raising mouse monoclonal antibodies to the nuclei of Hodgkin’s disease cell line. Ki-67 is present within the nucleus in all active phases of cell cycle, except the Go phase that is the resting phase (9).

There are innumerable studies in literature on the correlation of Ki-67 Proliferation Index (PI) in breast cancer with other prognostic markers like tumour size, lymph node status, tumour stage and grade and different molecular subtypes. The results of each study were found to differ when compared with other well established prognostic markers of breast carcinoma. Multiple studies evaluating Ki-67 as continuous variable and categorising Ki-67 PI into different scoring patterns/cut-off points have been performed (7),(10),(11),(12),(13). However, standardisation of a definite method or an accepted cut-off point distinguishing Ki- 67 into a low or a high PI is still a matter of debate (7),(8),(10). The aim of this study was to analyse the effectiveness of Ki-67 as a prognostic indicator, to establish an association between Ki-67 with other prognostic markers of breast cancer by evaluating Ki-67 both as continuous (mean±SD) and categorisation into different scoring patterns. A comparative analysis was also performed of the different scoring patterns of Ki-67 used in this study.

Material and Methods

A cross-sectional study was carried out in the Department of Pathology, Dr. D.Y. Patil Medical College, Hospital and Research Centre, Pune, Maharashtra, India, between August 2019 to August 2021. Institutional Ethical Committee clearance was obtained before the start of the study-(I.E.S.C/280/2019).

Inclusion criteria: All female patients diagnosed with breast carcinoma were included in the study.

Exclusion criteria: Male breast, inadequate sample for histopathology and inconclusive IHC results due to technical errors of any of the molecular markers- ER, PR, HER2 and Ki-67 were excluded from the study.

Sample size: The study included 50 cases of breast cancer which were diagnosed as invasive breast carcinoma on the basis of histology. Sample size was calculated through convenient sampling method. There were total 55 cases collected over a period of 2 years. Five cases were lost due to lack of follow-up or were discarded from the study due to insufficient data.

Procedure

Relevant demographic and clinical presentation data was obtained. The breast tissue obtained in the form of Modified Radical Mastectomy (MRM), an excision and or a tru-cut biopsy was fixed in 10% of buffered formalin (formalin buffered to pH 7.0-7.4) for 12-24 hours. Grossing of the specimen was done according to the standard protocols followed by the department (14). The tissue was processed, and the sections were stained with Haematoxylin and Eosin (H&E). The H&E sections were studied under a light microscope for the histologic type (WHO 2019) (2), histologic grade (Elston Ellis modification of Scarff-Bloom-Richardson grading system) (3) and pathologic stage classification (AJCC 8th edition) (4) of the breast tumour.

Immunohistochemical Staining

A representative block with maximum tumour tissue and adjacent normal breast tissue was selected for IHC. Four serial sections were obtained on Poly-L-lysine coated slide. These sections were used for IHC staining of ER, PR, HER2 and Ki-67.

For IHC all the reagents and staining materials were brought down to room temperature. The sections were incubated in a peroxide block for 5-10 minutes at room temperature followed by washing with SuperSensitiveTM Wash Buffer. Antigen retrieval was performed at a pH of 6.0 by microwave technique. PowerBlock, a proteinaceous blocking reagent was used to prevent non specific binding of antibodies. The slides were further incubated with primary antibodies for 60 minutes. The sections were counter stained using haematoxylin, dehydrated and cleared using ethyl alcohol and xylene respectively. The primary antibodies used for ER was clone SP1 (Thermoscientific), for PR was clone SP2 (Thermoscientific), for HER2 was clone SP3 (Thermoscientific), and Ki-67 was a rabbit monoclonal antibody, Clone SP6 (Epredia) (15).

According to the guidelines recommended by American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), Allred system of scoring was used for the reporting of ER and PR receptors (16).

Allred Score= Proportion Score (PS) + Intensity Score (IS)

The PS is the number of cells that are stained and IS is the intensity of staining that is pale or dark. The two scores were added together for the final score that is the Allred score. A total score of 0-2 was considered negative and a score of 3-8 was considered positive (16).

HER2 staining (ASCO and CAP guidelines) was considered positive (Score 3+) when complete, intense, circumferential membrane staining was seen in >10% of invasive tumours cells and it was negative (0 or 1+) when no or incomplete, faint membrane staining was seen in >10% of invasive tumour cells (17). In this study, molecular subtyping of breast tumours was done according to (Table/Fig 1) (13).

Ki-67 score or PI was evaluated as percentage of positively stained cells against the total number of tumour cells scored (0-100%). For each case a minimum of 500 cells were counted under high power. Areas with highest concentration of positively stained Ki-67 cells (tumour edge or hot spots) were selected (18).

Ki-67 (PI) = No of positive cells/Total no of cells × 100 (18).

For each case, IHC reporting of ER, PR, HER2 and Ki-67 was done by two individual authors (MN & AB). It was primarily done by MN using the above said methods and guidelines. These results were re-checked and confirmed by AB.

Ki-67 was evaluated as a continuous variable (mean ± standard deviation) to establish a relationship with other prognostic markers of breast cancer using different statistical tests. Ki-67 was also categorised into different scoring patterns. The three scoring patterns used in this study were:

• Scoring Pattern I: ≤14%, >14% (Low, High) (11)
• Scoring Pattern II: ≤15%, 16-30%, >30% (Low, Intermediate, High) (10),(12)
• Scoring Pattern III: <20%, 20-50%, ≥50% (Low, Intermediate, High) (13)

Statistical Analysis

Ki-67 was analysed as a continuous variable by expressing it in terms of mean±SD. An association between the mean difference of Ki-67 index and other prognostic markers of breast cancer was established using different statistical tests like Kruskal-Wallis test (tumour size, tumour grade and molecular subtypes), Mann-Whitney Rank Sum Test (lymph node status) and ANOVA (staging) respectively. A p-value <0.05 was regarded as significant.

Results

From the overall of 50 patients with confirmed breast carcinoma, 40 (80%) cases were older than 50 years of age. Twenty six (52%) of the cases affected the left breast. A total of 49 (98%) cases were diagnosed as IDC. The frequency of grade III was the highest 26 (52%). The frequency of Luminal A, Luminal B, Her2 enriched and Basal-like tumours were 25 (50%),10 (20%), 7 (14%) and 8 (16%) respectively (Table/Fig 2).

To establish an association between Ki-67 and various other prognostic markers, Ki-67 was analysed both as a continuous variable (Mean±SD) and by using different scoring patterns for categorising Ki-67 into a low or a high PI. These findings are shown in (Table/Fig 3),(Table/Fig 4),(Table/Fig 5),(Table/Fig 6).

The mean values of Ki-67 varied with increasing tumour size. The mean Ki-67 was higher in presence of lymph node involvement. The mean difference in Ki-67 values showed a surge through the progressive stages of the tumour. It did not show any significant change with different grades of tumour. A statistically significant relationship between Ki-67 and these parameters was not established. The molecular subtyping of the tumours revealed that the mean value of Ki-67 was highest in basal-like tumours indicating the aggressive nature of these tumours. This was statistically significant with p-value=0.002 (p-value <0.05) (Table/Fig 3).

In scoring pattern I, with low (≤14%) and high (>14%) index categorisation of Ki-67, 5 (71.4%) of T1, 21 (65.62%) of T2 and 8 (72.72%) of T3) cases, 18 (64.28%) and 16 (72.72%) cases of lymph node negativity and positivity, 5 (71.4%), 18 (64.28%) and 11 (73.33%) cases of stage I, II and III respectively and 4 (66.66%), 9 (50%), and 21 (80.7%) cases of grade I, II and III, respectively belonged to higher Ki-67 index. Similarly, 14 (56%), 6 (60%) and 6 (85.71%) of Luminal A, B and HER2 had a higher Ki-67 PI. However, the percentages were significantly higher in basal-like tumours 8 (100%) (Table/Fig 4).

According to scoring pattern II, Ki-67 was divided into low (≤15%), intermediate (16-30%) and high (>30%) indices. Five (45.45%) of the T3, 10 (45.45%) of lymph node positive tumours and 14 (53.84%) of grade III showed a higher PI of Ki-67. However, for stage III, equal number of cases 5 (33%) belonged to low, intermediate and high Ki-67 index category. A higher Ki-67 index was found in 7 (88%) of basal-like subtype of breast (Table/Fig 5).

In scoring pattern III (<20%, 20-50%, ≥50%), 4 (57.14%) of T1 and 5 (45.45%) of T3 cases belonged to low Ki-67 PI category, whereas equal number of cases 13 (40.62%) of T2 had a low and intermediate Ki-67 index. Thirteen (46%) and 9 (40.90%) cases of lymph node negativity and positivity showed a low Ki-67 PI. Four (57.14%) and 7 (46.66%) cases of stage I and III had low Ki-67 index and equal number of cases 11 (39.28%) of stage III had a low and intermediate Ki-67 index. In regards to grade, equal number of cases 2 (33.33%) belonged to low, intermediate and high Ki-67 index. Thirteen (72.22%) of grade II had low Ki-67 PI and 12 (46.15%) of grade III had higher Ki-67 index. Similarly, 15 (60%) of Luminal A and 3 (42.85%) of HER2 had a low Ki-67 PI whereas 5 (50%) of Luminal B had an intermediate index. However, 6 (75%) of basal-like tumours had a higher Ki-67 PI (Table/Fig 6).

Photomicrographs demonstrating the Ki-67 index value in different molecular subtypes of breast cancer have been depicted in (Table/Fig 7),(Table/Fig 8),(Table/Fig 9),(Table/Fig 10).

Discussion

Prognostication of breast cancer through evaluation of Ki-67 proliferation marker has been utilised widely over more than a decade now. However, Ki-67 is yet to achieve a standardised cut-off value in order to upsurge its efficacy as a prognostic marker for oncologists and pathologists.

The present cross-sectional study indicated that the expression of Ki-67 index as a continuous variable (mean±SD) yielded better statistical association with respect to other prognostic parameters of breast cancer than the different scoring patterns used. It was also found that the three scoring patterns used in this study showed varied distribution of Ki-67 with respect to other prognostic markers. Therefore, none could be selected as a single “best” scoring pattern. However, scoring pattern II showed notable findings with respect to other factors affecting prognosis of breast cancer.

It was also noticed that all three scoring patterns showed a higher Ki-67 PI in basal-like breast tumours signifying its aggressive behaviour and poor outcome. The study also indicated that, irrespective of the method of Ki-67 evaluation, Ki-67 and molecular subtyping can be considered as important prognostic factors in breast cancer.

Soliman NA and Yussif SM reported the mean age of patients in his study was 54.6±12. 94.4%, 42% and 41% of the cases in his study were reported as IDC, grade II tumours and Luminal A tumours respectively (19). These findings were in accordance with the present analysis. Another study by Amer MH showed a predominance of left-sided breast cancer, with a left to right ratio of 1.1. A relative excess incidence of left to right breast cancer has been well-documented in the literature, with the left to right side ratio ranging from 1.05 to 1.26 (20). These results were in accordance with the current study.

In a review by Inwald EC et al., a strong association was observed between mean Ki-67 and grade of the tumour. Higher nodal status was associated with higher mean Ki-67. The mean Ki-67 values were lower in ER and PR positive tumours and higher in HER2 positive tumours (7). Similar findings were reported by Ahmed ST et al., and Haroon S et al., (11),(12) Additionally, Ahmed ST et al., revealed that the mean Ki-67 was higher in the triple negative groups with no significant association. No association was seen between Ki-67 and tumour size and lymph node status (11). Haroon S et al., also exhibited the mean Ki-67 values were significantly higher in T3 tumours (12). In another study by Awadelkarim, nothing significant could be demonstrated with tumour size, stage and hormone receptors- ER, PR and HER2 (12). In present analysis, the mean values of Ki-67 had a significant difference between the different tumour sizes (p-value=0.608), lymph node status (p-value=0.506) stages (p-value=0.979) and grades (p-value=0.095) of breast tumour but no statistically significant association was found. However, it was found that Ki-67 statistically correlated with different molecular subtypes of breast cancer (p-value=0.002).

Ahmed ST et al., used a threshold of 14% to categorise patients into low and high Ki-67 PI index. Accordingly, Ki-67 was reported to be significantly associated with tumour grade. Higher PI of Ki-67 was present in HER2 positive and the triple negative breast cancer. No significant association was observed with tumour size and lymph node status (11). However, in the current study according to scoring pattern I (≤14%, >14%), majority of different tumour sizes, absence or presence of lymph node positivity, different stages and grades of breast tumour belonged to higher Ki-67 index. Majority of the different molecular subtypes had a higher Ki-67 PI, which was significantly higher in basal-like tumours.

Haroon S et al., divided the tumour cases into low (<15%), intermediate (16-30%) and high (>30%) Ki-67 index levels. Significant correlation was found between Ki-67 and tumour grade, HER2 neu, PR positivity and lymph node status. High Ki-67 index was seen in T2 and T3 tumours. HER2 neu and lymph node status showed direct correlation with Ki-67 and PR status was inversely related. Ki-67 and tumour size and ER status showed no significant association (12). Similar findings were reported by Abebe E et al., (10). This was in agreement with the current study, where scoring pattern II (≤15%, 16-30%, >30%) depicted that higher tumour sizes, lymph node positivity, higher stage and grade of the tumour and basal-like breast tumours had a higher Ki-67 PI.

Nishimura R et al., found the median value of Ki-67 to be 20% and divided breast tumours into low (<20%), intermediate (20-50%) and high (≥50%) Ki-67 categories. Accordingly, Luminal A tumour had a low Ki-67 PI. A higher PI was observed in triple negative tumours. Large sized tumours, younger age, lymph nodes positivity, a higher grade, negative ER/PR status and positive HER2 receptor status was significantly associated with high Ki-67 index (13). However, scoring pattern III (<20%, 20-50%, ≥50%) used in present study, showed that majority of the cases of different tumour sizes, absence or presence of lymph node positivity, different stages and grades of breast tumour belonged either to low or intermediate Ki-67 PI. Maximum cases of basal-like tumours had a higher Ki-67 index.

According to literature, significant difference in Ki-67 PI between biopsies and surgical specimens has been observed due to several factors like poor tissue fixation, poor staining and intratumoural heterogenicity to name a few. Studies also suggest performing immunostaining for Ki-67 PI on biopsies as well as on surgical specimens for verification (21),(22).

Limitation(s)

The major limitation of the current study was a small sample size and the various types of breast specimens which were included in the study. However, immunostaining of Ki-67 on biopsy and corresponding surgical specimens for confirmation was not feasible in present study. Some pathological parameters like distant metastasis and overall survival could not be accessed due to lack of follow-up. Intratumoural heterogenicity in terms of Ki-67 expression could also be considered. A distribution error could also be implicated.

Conclusion

The present study highlights that the association of Ki-67 with other prognostic markers of breast cancer was established appropriately when evaluated as a continuous variable rather than using different two or three tier scoring patterns. However, amongst the different scoring patterns used here in this study, scoring pattern II (≤15%, 16-30%, >30%) showed remarkable findings with Ki-67. Moreover, Ki-67 showed a direct relationship with different molecular subtypes of breast tumours irrespective of the method of Ki-67 evaluation. Thus, suggesting that of all the various prognostic markers of breast cancer, Ki-67 and molecular subtyping could be considered the best.

References

1.
Sung, H, Ferlay, J, Siegel, RL, Laversanne, M, Soerjomataram, I, Jemal, A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209-49. [crossref] [PubMed]
2.
WHO Classification of Tumours Editorial Board. Breast tumours. 5th ed. Lyon (France): International Agency for Research on Cancer; 2019;10-250.
3.
Ellis IO, Elston CW. Histologic grade. In: O’Malley FP, Pinder SE, editors. Breast Pathology. Philadelphia, PA: Elsevier; 2006;225-233. [crossref] [PubMed]
4.
American Joint Committee on Cancer. Breast. In: AJCC Cancer Staging Manual. 8th ed.New York, NY: Springer; 2017;589.
5.
Kondov B, Milenkovikj Z, Kondov G, Petrushevska G, Basheska N, Bogdanovska-Todorovska M, et al. Presentation of the molecular subtypes of breast cancer detected by immunohistochemistry in surgically treated patients. Open Access Maced J Med Sci. 2018;6(6):961-67. [crossref] [PubMed]
6.
Dowsett M, Nielsen TO, A’Hern R, Bartlett J, Coombes RC, Cuzick J, et al. A. Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group. Journal of the National Cancer Institute. 2011;103(22):1656-64. [crossref] [PubMed]
7.
Inwald EC, Klinkhammer-Schalke M, Hofstädter F, Zeman F, Koller M, Gerstenhauer M, et al. Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry. Breast Cancer Research and Treatment. 2013;139(2):539-52. [crossref] [PubMed]
8.
Mannell A. The role of Ki-67 in breast cancer. South African Journal of Surgery. 2016;54(2):10-13.
9.
Gerdes J, Schwab U, Lemke H, Stein H. Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer. 1983;31(1):13-20. [crossref] [PubMed]
10.
Abebe E, Mekonen W, Seifu D, Bekurtsion Y, Bekele A, Kantelhardt EJ. Assessment of proliferation index and pathological features as prognostic potential of breast cancer in Ethiopia. J Cancer Sci Ther. 2019;11:106-14.
11.
Ahmed ST, Ahmed AM, Musa DH, Sulayvani FK, Al-Khyatt M, Pity IS. Proliferative Index (Ki67) for prediction in breast duct carcinomas. Asian Pac J Cancer Prev. 2018;19(4):955-59.
12.
Haroon S, Hashmi AA, Khurshid A, Kanpurwala MA, Mujtuba S, Malik B, et al. Ki67 index in breast cancer: correlation with other prognostic markers and potential in pakistani patients. Asian Pac J Cancer Prev. 2013;14(7):4353-58. [crossref] [PubMed]
13.
Nishimura R, Osako T, Okumura Y, Hayashi M, Toyozumi Y, Arima N. Ki-67 as a prognostic marker according to breast cancer subtype and a predictor of recurrence time in primary breast cancer. Exp Ther Med. 2010;1(5):747-54. [crossref] [PubMed]
14.
Desai Saral S, Bal Munita, Rekhi Bharat, Jambhekar Nirmala A, editors. Grossing of Surgical Oncology Specimens. A Practical Guide Towards Complete Pathology Reporting. Mumbai: Department of Pathology, TATA Memorial Hospital; 2011;56–59.
15.
Shi S-R, Guo J, Cote RJ, Young L, Hawes D, Shi Y, Thu S, Taylor CR. Applied Immunohistochemistry & Molecular Morphology. 1999;7:201-08. [crossref]
16.
Harvey JM, Clark GM, Osborne CK, Allred DC. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J Clin Oncol. 1999;17(5):1474-81. [crossref] [PubMed]
17.
Wolff AC, Hammond MEH, Allison KH, Harvey BE, Mangu PB, Bartlett JMS, et al. Human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update. Arch Pathol Lab Med. 2018;142(11):1364-82. [crossref] [PubMed]
18.
Kinra P, Malik A. Ki 67: Are we counting it right? Indian J Pathol Microbiol. 2020;63:98-99. [crossref] [PubMed]
19.
Soliman NA, Yussif SM. Ki-67 as a prognostic marker according to breast cancer molecular subtype. Cancer Biol Med. 2016;13(4):496-504. [crossref] [PubMed]
20.
Amer MH. Genetic factors and breast cancer laterality. Cancer Manag Res. 2014;6:191-203. [crossref] [PubMed]
21.
Ahn S, Lee J, Cho M-S, Park S, Sung SH. Evaluation of Ki-67 Index in Core Needle Biopsies and Matched Breast Cancer Surgical Specimens. Arch Pathol Lab Med. 2018;142(3):364-68. [crossref] [PubMed]
22.
Chen J, Wang Z, Lv Q, Du Z, Tan Q, Zhang D, et al. Comparison of core needle biopsy and excision specimens for the accurate evaluation of breast cancer molecular markers: a report of 1003 cases. Pathol Oncol Res. 2017;23(4):769-75. [crossref] [PubMed]

DOI and Others

DOI: 10.7860/JCDR/2022/52704.16231

Date of Submission: Oct 03, 2021
Date of Peer Review: Nov 11, 2021
Date of Acceptance: Jan 08, 2022
Date of Publishing: May 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. Yes

PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Oct 05, 2021
• Manual Googling: Dec 27, 2021
• iThenticate Software: Jan 24, 2022 (9%)

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)
  • www.omnimedicalsearch.com