To study the utility of COX-2 as immunohistochemical prognostic marker in comparison to various histopathological parameters and TNM staging in breast carcinoma: an observational, cross-sectional study protocol

Jayashree Bhawani; Samarth Shukla; Sourya Acharya; Sunita Vagha; Miheer Jagtap

doi:10.12688/f1000research.138776.1

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Study Protocol

To study the utility of COX-2 as immunohistochemical prognostic marker in comparison to various histopathological parameters and TNM staging in breast carcinoma: an observational, cross-sectional study protocol

[version 1; peer review: 1 approved, 1 approved with reservations]

Jayashree Bhawani ¹, Samarth Shukla¹, Sourya Acharya², Sunita Vagha¹, Miheer Jagtap¹

Jayashree Bhawani ¹, Samarth Shukla¹, [...] Sourya Acharya², Sunita Vagha¹, Miheer Jagtap¹

PUBLISHED 30 Aug 2023

Author details Author details

¹ Department of Pathology, Jawaharlal Nehru Medical college, Sawangi(Meghe), Wardha, Maharashtra, 442001, India
² Department of General Medicine, Jawaharlal Nehru Medical College, Sawangi(Meghe), Wardha, Maharashtra, 442001, India

Jayashree Bhawani
Roles: Conceptualization, Investigation, Methodology, Project Administration, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Samarth Shukla
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Review & Editing

Sourya Acharya
Roles: Formal Analysis, Resources, Supervision, Validation, Writing – Review & Editing

Sunita Vagha
Roles: Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Review & Editing

Miheer Jagtap
Roles: Formal Analysis, Investigation, Methodology, Resources, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Datta Meghe Institute of Higher Education and Research collection.

Abstract

Background: Breast cancer is the most prevalent cancer among women worldwide and is a well-known cause for cancer mortality in females. COX-2 (cyclooxygenase) plays a vital role in development of some human cancers such as lung, colon and breast. It is a potent enzyme that is important for the conversion of arachidonic acid into prostaglandins. These prostaglandins mediate cellular proliferation, apoptosis and angiogenesis which contributes to carcinogenesis. Overexpression of COX-2 has been detected in several malignancies including breast cancer. COX-2 overexpression is regarded as a poor prognostic marker of breast cancer.
The present study will aim to study the immunohistochemical expression of COX-2 in breast cancer and compare it with known histopathological parameters thus assessing its prognostic value.
Methods: This will be an observational study conducted in the Department of Pathology, JNMC, Wardha (Sawangi). Radical mastectomy specimens will be studied for COX-2 expression by immunohistochemistry in patients diagnosed with breast carcinoma. COX-2 expression will be quantified as immunohistochemical score and results will be correlated with various histopathological parameters.
Results: The expected result of our study will suggest an association of COX-2 expression to the factors associated with poor prognosis in breast carcinoma. A positive correlation is expected between larger tumor size, positive lymph node status, higher T stage and N stage and lymphovascular invasion.
Conclusions: Conclusions will be drawn from the obtained results of the immunohistochemical study by using COX-2- for detection of overexpression of COX-2 when evaluated with TNM staging, histological grading and molecular types of breast cancer.

Keywords

Breast Cancer, COX-2, histopathological parameters, prognostic marker, immunohistochemistry.

Corresponding authors: Jayashree Bhawani, Sunita Vagha

Competing interests: No competing interests were disclosed.

Grant information: This research work is funded by the Indian Council of Medical Research, Delhi (grant ref: MD22JUN-0241). Grant is assigned to Dr. Jayashree Bhawani, Department of Pathology, JNMC, Sawangi (Meghe), Wardha, Maharashtra.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Bhawani J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Bhawani J, Shukla S, Acharya S et al. To study the utility of COX-2 as immunohistochemical prognostic marker in comparison to various histopathological parameters and TNM staging in breast carcinoma: an observational, cross-sectional study protocol [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 12:1057 (https://doi.org/10.12688/f1000research.138776.1) First published: 30 Aug 2023, 12:1057 (https://doi.org/10.12688/f1000research.138776.1) Latest published: 18 Jun 2024, 12:1057 (https://doi.org/10.12688/f1000research.138776.2)

Introduction

Breast carcinoma is the most frequent malignancy and the second leading cause of death in women worldwide.¹ It has surpassed lung cancer with an estimated 2.3 million new cases worldwide. In recent years, incidence rates of breast cancer have elevated by 0.5% every year, as breast cancer incidence varies with age and race/ethnicity.

In the year 2021, 2.6 million women were newly diagnosed with breast cancer.² In India, breast cancer accounts for 13.5% of cancer cases. It is assumed that the chance of a woman’s death due to breast carcinoma is about 1 in 39 or about 2.6%.³

The etiology is multifactorial, implicating hormonal imbalance, genetic predisposition, diet, metabolic factors, and reproductive factors.

The diagnosis of breast cancer is achieved by adopting a triple assessment approach which includes clinical evaluation, radiographical investigation and tissue diagnosis by biopsy, out of which tissue biopsy is currently the gold standard for cancer diagnosis and predicting prognosis.

There are certain indicators available which establish the prognosis of breast cancer,⁴ these are tumor size, age, site of tumor, lymph node status, stage and grade of tumor and lymphovascular invasion.

Various evaluation methods or systems are available for the determination prognosis of breast carcinoma, including the: - Nottingham Histologic Grading system which is also referred as The Elston-Ellis modification of Scarff-Bloom-Richardson grading system. The other method is the categorization of breast cancer according to TNM (tumor, nodal status, metastasis) staging, specified by the American Joint Committee on Cancer (AJCC). This is a more advanced diagnostic modality, which determines the molecular phenotypes of carcinoma breast by implementing immunohistochemistry.

Over the years additional prognostic and predictive factors are evolving and one of them is COX-2. This is the rate limiting enzyme in the biosynthesis of prostaglandins from arachidonic acid.⁵ It is an empirical catalyst formed under few definitive circumstances of inflammation and tumor -microenvironment. COX-2 is an important tumor marker which regulates tumor growth, invasion and metastasis.⁶ In carcinogenesis, inflammatory cytokines, growth factors and oncogenes trigger induction of COX-2, which results in tumor progression. COX-2 is strongly associated with tumor proliferation, angiogenesis and apoptosis resistance mechanism.⁷ Therefore, COX-2 overexpression is associated with poor prognosis in post-menopausal women, those above 50 years of age, those in the advanced stage of the disease, those with a larger tumor size, higher grade, and metastasis of lymph nodes.

Various studies⁸ showed that, among different types of breast tumors, triple negative breast cancer [ER (estrogren) negative, PR (progesterone) negative, HER2 neu negative)] are associated with aggressive progression of tumor and COX-2 expression seemed to be increased in triple negative tumors. Also, there is a positive correlation of COX-2 with these histopathological prognostic parameters.

To further evaluate the role of COX-2 as marker of tumor behavior and to correlate its expression with other essential parameters which play a vital role in the assessment of tumor behavior this study is being conducted.

The aim of this study is to analyze the relationship between COX-2 expression and histopathological parameters, pathological TNM staging and molecular immunophenotypes of carcinoma breast.

Objectives

• To confirm breast carcinoma by histopathological examination and to determine the histopathological grade of breast carcinoma by Elston-Ellis modification of Scarff- Bloom-Richardson grading system).
• To establish pathological TNM stage of breast carcinoma.
• To determine the immunophenotypes of carcinoma breast by evaluating ER/PR and Her 2 neu expression by immunohistochemistry.
• To evaluate immunohistochemical expression of COX-2 in breast carcinoma.
• To understand the inter relation between expression of COX-2, histopathological prognostic markers, molecular immunophenotypes, and pathological TNM staging of carcinoma breast.

Methods

Study design

An observational, cross-sectional study will be conducted on breast cancer patients for a span of two years (June 2022 to June 2024) in the Histopathology and Immunohistochemistry section of the Pathology Department, Jawaharlal Nehru Medical College, Sawangi (Meghe) in co-ordination with General Surgery Department, Acharya Vinoba Bhave Rural Hospital, Sawangi (Meghe).

Sample size

Desired sample size will be calculated according to Krejcie and Morgan’s formula⁹:

n = (Z^{α} {/2)}^{2} x P x (1 - P) / d^{2}

here,

Z^α/2= significance level of 5% i.e. 95% (CI) confidence interval = 1.96

P = incidence of breast cancer in female = 13.5¹⁰ %= 0.135

d = error of margin = 0.08

\begin{array}{c} n = (1.96 \times 1.96) \times 0.135 \times (1 - 0.135) / (0.08 \times 0.08) \\ = 70.96 \\ = 70 cases of patients diagnosed withbreast cancerby histopathological examination . \end{array}

The study will include approximately 60-70 resected specimens from confirmed and planned modified radical mastectomy of breast carcinoma in the General Pathology Department, J.N.M.C.

Ethics and consent

The Institutional Ethics Committee has given approval for the study ((DMIMS (DU)/IEC/2022/1068) on 27^th June 2022), and informed consent will be taken from the targeted patients who will be participating in this study.

Inclusion criteria

• Female patients diagnosed with breast carcinoma on histopathological examination.
• Women with breast carcinoma having no history of treatment.
• Female patients presenting with breast carcinoma arising de novo.
• All cases that underwent mastectomy/modified radical mastectomy (MRM) procedure.

Exclusion criteria

• Patients with lesions other than breast carcinoma i.e., benign breast tumors, myoepithelial tumors, mesenchymal tumors.
• Patients with breast carcinoma who have received neoadjuvant chemotherapy.
• Male patients with a diagnosis of breast carcinoma.
• Patients who underwent incisional biopsy, excisional biopsy, or tru-cut biopsy.

Approach to the study

The study will collect patients’ personal details including name, age, gender, registration number, IPD/OPD numbers, department and also clinical details such as symptoms, test interpretations and diagnosis. Radiological investigations such as mammography report, ultrasonography of breast, and CT scan reports will be noted from patient’s case file. The present study will take into consideration, the biopsy report of the lesion.

The mastectomy specimen will be received in the Histopathology Department, in 10% formalin and will be kept for proper fixation. After a period of 24 hours of fixation the specimen will be grossed and sections from the representative areas will be taken.

Method of tissue processing

The sections will be processed by automated histokinette. The paraffin blocks will be prepared with the help of Leuckhart’s mould and sections of 3-4 microns thick will be taken using microtome. These sections will be then transferred to glass slides, precoated with egg albumin. Hematoxylin and eosin staining of paraffin sections will be carried out using standard methods. Slides will be scanned and a diagnosis along with histological grading will be made, using the College of American Pathologists (CAP) protocol. The 8^th American Joint Committee on Cancer (AJCC) guidelines will be followed to carry out staging on the specimen.

Methodology of interpretation

Using the Nottingham modification of Bloom-Richardson System, histological grades of tumor will be determined. This will include factors such as tubule formation, nuclear size and mitotic count¹¹ and score will be assigned accordingly:

Tubule formation

Score 1: > 75% of tumor shows tubules

Score 2: 10-75% of tumor has tubules

Score 3: < 10% of tumor has tubules

Nuclear size

Score 1: Small regular nuclei: similar to normal ductal nuclei (nuclei with minimal variation in size and shape)

Score 2: Intermediate size: 1.5-2 times the size of normal ductal nuclei (nuclei with moderate variation in size and shape)

Score 3: High grade nuclei: > twice the size of normal ductal nuclei (nuclei with marked variation in size and shape)

Number of mitotic figures counting 10 high power fields (Table 1):

Table 1. Score Categories According to Field Diameter and Mitotic Count.¹²

Scoring categories of mitotic counts
Final diameter (mm)	Area (mm²⁾	Number of mitosis per 10 fields corresponding to:
Final diameter (mm)	Area (mm²⁾	Score 1	Score 2	Score 3
0.44	0.152	0-5	6-10	>11
0.59	0.274	0-9	10-19	>20
0.63	0.312	0-11	12-22	>23

Table 2. Evaluation of COX-2 immunostaining status based on proportion score i.e percentage of positive cells.¹

Percentage of positive cancer cells	COX-2 quantity score
No staining	0
1%-10%	1
11%-50%	2
51%- 80%	3
>81%	4

Table 3. Evaluation of COX-2 immunostaining status by measuring intensity of staining.¹

Staining intensity	Staining intensity score
No staining	0
Weak staining	1
Moderate staining	2
Strong staining	3

Score 1: 0-9 mitoses/10 HPF

Score 2: 10-19 mitoses/10 HPF

Score 3: > 20 mitoses/10 HPF

Add up to the score to reach the total points:

1-5 points: grade 1: well differentiated

6-7 points: grade 2: moderately differentiated

8-9 points: grade 3: poorly differentiated

After assessing tumor grade, tumor stage will be determined by TNM staging (8^th AJCC edition)

Hence, interpretation of overall stage will be as follows:

Stage 0: Tis

Stage I: T1, N0

Stage II: T2, N0; T3, N0; T0, N1; T1, N1 or T2, N1

Stage III: T3N1, T0N2, T1N2, T2N2, T3N2, any T-N3, locally advanced carcinoma breast, invasion in ribs, skin, matted lymph nodes

Stage IV: M1; advanced breast cancer.

Immunohistochemical assessment¹³

Immunohistochemistry (IHC) will be performed on a paraffin embedded segment of a surgically removed specimen that has been diagnosed as a malignant lesion of the breast. Only representative blocks from the lesion will be chosen for immunohistochemistry. Immunohistochemistry will be performed utilizing monoclonal antibodies and the biotin avidin peroxidase complex technique. For performing IHC, slides will be coated with Poly L- Lysine and 2 sections of 3-4 microns thick will be placed on the slide. Sections will be deparaffinized and then rehydrated with descending grades of alcohol (100% to 90% to 70% to at last 50% concentration). Antigen retrieval will be performed and followed by transfer of slide for wash in tris buffer solution (TBS). The next step will be the peroxidase blocking for 30 minutes using 3,5 hydrogen peroxide along with methanol. The wash will be repeated 3 times with TBS. Now the section will be exposed to DAKO monoclonal mouse anti human antibodies, i.e., mouse anti human ER/PR/Her2 antibodies and COX-2 antibody. It will be again washed in the buffer thrice for 5 minutes. Now chromogen 3,3’- diaminobenzydene (DAB) will be applied for 15-20 minutes. The DAB-stained slide will be washed in buffer for 10 minutes and then will be dipped in distilled water. Harris hematoxylin will be used for counterstaining. Lastly, after final wash with tap water, mounting of slides will be done to scan under microscope.

Evaluation of COX-2 expression by immuno-histochemistry

On immunohistochemical staining COX-2 will be observed in tumor cells. Immunohistochemical staining will be evaluated with a light microscope. A minimum of 500 cells will be counted per x 40 fields for immunohistochemical evaluation per antibody. Cyclooxygenase-2 positivity will be indicated by the presence of brown cytoplasmic- staining. For the evaluation of cytoplasmic staining for COX-2, a predefined scoring system which is based on the product of percentage of immunoreactive tumor cells with staining intensity will be studied.¹⁴ Immuno-reactivity for C0X-2 in tumor cells will be assessed using a scoring system based on the staining intensity.

COX-2 immunohistochemistry score is obtained by multiplying the staining quantity score and staining intensity score.

0-3 =Negative/faint staining

4-8= Moderate/intermediate staining

Statistical-analysis

The software used for analysis will be SPSS version 27.0. Statistical analysis will be carried out by using suitable statistical tests eg: ‘Chi-Square Test’, ‘Fisher’s Exact Test’ with significance set at p value < 0.05.

Results

Observations and results will be collected and combined together over the period of two years and will be analyzed statistically.

Discussion

In this study a statistically significant expression of COX-2 with histopathological parameters which are associated with poor prognosis such as tumor size, grade, lymph node metastasis is expected. As COX-2 is overexpressed in triple negative breast cancer, it indicates a poor outcome in terms of prognosis. COX-2 expression is increased when tested in post- menopausal status, those of age above 50 years, in an advanced stage of disease, with a larger tumor size, a high grade, and metastasis of lymph nodes. Few studies have shown that COX-2 expression is increased when correlated with HER-2/neu expression in breast cancer patients; COX-2 was expressed in ER negative, PR negative and HER-2/neu positive tumors and with high histological grade in breast carcinoma. Our study will help in assessing the role of COX-2 in carcinogenesis and its association with poor prognostic markers.

A key strength of this study is that it will use a useful biomarker for estimating tumor aggressiveness. This study will focus on COX-2 expression in breast carcinoma and its comparison with other histopathological parameters. This would also give clinicians a framework for evaluating the function of selective COX-2 inhibitors in human breast cancer chemoprevention as well as treatment.

Studies such as those conducted by Ibrahim Metin Ciris et al., Debashri Jana et al., Ari Ristimaki et al., Pereira et al. and Costa et al. have stated that COX- 2 overexpression has the potential to be a useful biomarker for estimating tumor aggressiveness, and that COX-2 expression is associated with aggressive tumor biology, and can behave as a predictor of tumors with bad prognosis. Hence, the above studies concluded that there is a correlation of COX-2 expression with histopathological prognostic parameters in breast cancer. The limitation of the study is that it will be a single center study.

Conclusion

The purpose of the current investigation is to determine COX-2's precise prognostic function in breast cancer. This would give clinicians a framework for evaluating the function of selective COX-2 inhibitors in human breast cancer chemoprevention as well as treatment. This study will correlate prognostic relation between COX-2 expression in breast carcinoma patients with routinely studies histopathological prognostic parameters.

Dissemination

The results of this study will be presented in peer reviewed publications.

Study status

Recruitment has begun, and currently 25 patients have been enrolled for the study.

Ethics and consent

The research work is approved by Institutional Ethical Committee of the Datta Meghe Institute of Medical Sciences (DMIMS (DU)/IEC/2022/1068) on 27^th June 2022.

Author contributions

Contributors: Dr. Samarth Shukla conceptualized the study and provided the idea of how to conduct it. Dr. Jayashree Bhawani will do the research work, collect the data, analyze it and under guidance of Dr. Samarth Shukla and Dr. Sunita Vagha will come to the conclusion.

Data availability

No data are associated with this article.

Acknowledgements

My sincere gratitude to all the patients whose sample will help us to conduct this study. I would also like to express my gratitude to all the faculties of department of pathology and surgery, and technical staffs for their support. I would also like to use this opportunity to express my gratitude towards Indian Council of Medical Research (ICMR) for providing their valuable grant for my research study. The completion of my study would not have been possible without their help and insights.

References

1. Solanki R, Agrawal N, Ansari M, et al.: COX-2 Expression in Breast Carcinoma with Correlation to Clinicopathological Parameters. Asian Pac. J. Cancer Prev. 2018; 19(7): 1971–1975. PubMed Abstract
2. Harbeck N, Penault-Llorca F, Cortes J, et al.: Breast cancer. Nat. Rev. Dis. Primers. 2019; 5(1): 66. Publisher Full Text
3. American Cancer Society: Breast Cancer Facts and Figures 2019-2020. Atlanta, Ga: American Cancer Society; 2020.
4. Dai X, Xiang L, Li T, et al.: Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes. J. Cancer. 2016; 7(10): 1281–1294. PubMed Abstract | Publisher Full Text | Free Full Text
5. Department of Pathology, Mugla Sitki Kocman University, School of Medicine, Mugla, TurkeyTekin L, Celik SY, et al.: Immunohistochemical Expression of Cyclooxygenase-2 and Its Relationship With Prognostic Parameters in Breast Cancer. Cyprus J. Med. Sci. 2021; 6(1): 39–43.
6. Liu B, Qu L, Yan S: Cyclooxygenase-2 promotes tumor growth and suppresses tumor immunity. Cancer Cell Int. 2015; 15: 106. PubMed Abstract | Publisher Full Text | Free Full Text
7. Desai SJ, Prickril B, Rasooly A: Mechanisms of phytonutrient modulation of cyclooxygenase-2 (COX-2) and inflammation related to cancer. Nutr. Cancer. 2018 Apr 3; 70(3): 350–375. PubMed Abstract | Publisher Full Text | Free Full Text
8. Pastor N, Ezquerra LJ, Santella M, et al.: Prognostic significance of immunohistochemical markers and histological classification in malignant canine mammary tumours. Vet. Comp. Oncol. 2020 Dec; 18(4): 753–762. PubMed Abstract | Publisher Full Text | Free Full Text
9. Chaokromthong K, Sintao N: Sample size estimation using Yamane and Cochran and Krejcie and Morgan and green formulas and Cohen statistical power analysis by G* Power and comparisions. Apheit International Journal. 2021 Dec 24; 10(2): 76–86.
10. Mehrotra R, Yadav K: Breast cancer in India: Present scenario and the challenges ahead. World J Clin Oncol. 2022 Mar 24; 13(3): 209–218. PubMed Abstract | Publisher Full Text | Free Full Text
11. Bansal C, Singh US, Misra S, et al.: Comparative evaluation of the modified Scarff-Bloom-Richardson grading system on breast carcinoma aspirates and histopathology. Cytojournal. 2012; 9: 4. PubMed Abstract | Publisher Full Text | Free Full Text
12. van Dooijeweert C , van Diest PJ , Ellis IO: Grading of invasive breast carcinoma: the way forward. Virchows Arch. 2022; 480: 33–43. PubMed Abstract | Publisher Full Text | Free Full Text
13. Sanderson T, Wild G, Cull AM, et al.: 19—Immunohistochemical and immunofluorescent techniques. Bancroft’s Theory and Practice of Histological Techniques. 8th ed. Suvarna SK, Layton C, Bancroft JD, editors. 2018 Feb 27; 337–94.
14. Half E, Tang XM, Gwyn K, et al.: Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res. 2002; 62(6): 1676–1681. PubMed Abstract

Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 30 Aug 2023

Author details Author details

Jayashree Bhawani
Roles: Conceptualization, Investigation, Methodology, Project Administration, Resources, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Samarth Shukla
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Visualization, Writing – Review & Editing

Sourya Acharya
Roles: Formal Analysis, Resources, Supervision, Validation, Writing – Review & Editing

Sunita Vagha
Roles: Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Review & Editing

Miheer Jagtap
Roles: Formal Analysis, Investigation, Methodology, Resources, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This research work is funded by the Indian Council of Medical Research, Delhi (grant ref: MD22JUN-0241). Grant is assigned to Dr. Jayashree Bhawani, Department of Pathology, JNMC, Sawangi (Meghe), Wardha, Maharashtra.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

version 2

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Published: 18 Jun 2024, 12:1057

https://doi.org/10.12688/f1000research.138776.2

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Published: 30 Aug 2023, 12:1057

https://doi.org/10.12688/f1000research.138776.1

© 2023 Bhawani J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Bhawani J, Shukla S, Acharya S et al. To study the utility of COX-2 as immunohistochemical prognostic marker in comparison to various histopathological parameters and TNM staging in breast carcinoma: an observational, cross-sectional study protocol [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 12:1057 (https://doi.org/10.12688/f1000research.138776.1)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

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Reviewer Report 05 Jun 2024

Shaan Khetrapal, Department of Pathology, Hamdard Institute of Medical Sciences and Research, New Delhi, Delhi, India

Approved

https://doi.org/10.5256/f1000research.152003.r202391

In exclusion criteria the point of male patients can be omitted as the inclusion criteria specifically states female patients.

Comparative elaboration of discussion with respect to other studies can be done, which can be beneficial for further ... Continue reading

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Histopathology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Reviewer Report 02 Oct 2023

Amit Kumar Chowhan, Department of Pathology, AIIMS, RAIPUR, RAIPUR, TirupatiCHHATTISGARH, India

Approved with Reservations

https://doi.org/10.5256/f1000research.152003.r202390

Comment

In Introduction it is mentioned ‘This is a more advanced diagnostic modality, which determines the molecular phenotypes of carcinoma breast by implementing immunohisto[1]chemistry’. – Kindly check whether this line has been kept at appropriate place.

Comment

In Introduction it is mentioned ‘This is a more advanced diagnostic modality, which determines the molecular phenotypes of carcinoma breast by implementing immunohisto[1]chemistry’. – Kindly check whether this line has been kept at appropriate place.
Sample size should be calculated in reference to prevalence of positivity for COX2 in breast cancer (data to be taken from other similar study). For example, in reference no 5, the positivity of COX2 is mentioned as 42.7% in tumour cells, so while calculating sample size ‘P’ should be 42.7%.
First point of Inclusion criteria is • ‘Female patients diagnosed with breast carcinoma on histopathological examination.’

Therefore, there is no need of keeping an exclusion criteria as ‘Male patients with a diagnosis of breast carcinoma.’
Second point of Inclusion criteria is ‘Women with breast carcinoma having no history of treatment.’Therefore, there is no need of keeping an exclusion criteria as ‘Patients with breast carcinoma who have received neoadjuvant chemotherapy.’
Third point of Inclusion criteria is ‘Female patients presenting with breast carcinoma arising de novo.’’ Kindly explain what is the exact meaning of ‘’de novo.’’
Fourth point of Inclusion criteria is ‘’All cases that underwent mastectomy/modified radical mastectomy (MRM) procedure’’, therefore there is no need of keeping an exclusion criteria as ‘’Patients who underwent incisional biopsy, excisional biopsy, or tru-cut biopsy’’.
Under the heading ‘’Evaluation of COX-2 expression by immuno-histochemistry’’, the reference article mentioned is reference no 14, whereas in table 2 and table 3, the reference mentioned is reference no 1, kindly explain the disparity.
Under the heading ‘’Evaluation of COX-2 expression by immuno-histochemistry’’, it has been further mentioned as ‘’COX-2 immunohistochemistry score is obtained by multiplying the staining quantity score and staining intensity score. 0-3 =Negative/faint staining 4-8= Moderate/intermediate staining’’, which is not matching with table 2 and table 3, where the scores are 0-4 and 0-3. Kindly explain the disparity.
Under the section ‘’Immunohistochemical assessment’’, it has been mentioned that ER/PR/Her2 antibodies and COX-2 antibody will be applied, kindly mention the scoring system for ER/PR/Her2 antibodies also, which will be followed in the study, along with reference.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Oncopathology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 18 Jun 2024

Jayashree Bhawani, Department of Pathology, Jawaharlal Nehru Medical college, Sawangi(Meghe), Wardha, 442001, India

18 Jun 2024

Author Response
1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.
... Continue reading
1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.

2. Sample Size
Using the formula:

N= https://f1000research.s3.amazonaws.com/linked/653957.image.png

Where:

N = Sample size

Z(1-α/2) = 1.96 (value of the standard normal variate corresponding to the level of significance α of 0.05)

d = Specified precision on either side of the mean (10% or 0.1)

P = Expected prevalence of COX2 expression in the study population (76% or 0.76)

From the previous study by Fatma Z Abd Elrahman et al., the prevalence of COX2 expression in carcinoma breast patients was found to be 76%. Taking d=10%, the total sample size calculated is 70.07.

N=        (1.96)²X 0.76X(1−0.76)
                                                         (0.1)²

N= 70.07
Rounding up to the nearest whole number, the required sample size is 70.
Reference- Abdel-rahman FZ, Zaky AH, Zedan A, Elsaba TM, Gabr A. COX-2 “Cyclooxygenase 2“as a Prognostic marker in Breast Cancer. SVU-International Journal of Medical Sciences. 2021 Feb 2;0(0):0–0.

3. Male patients are kept in exclusion criteria because exclusion criteria comes as an opposite if inclusion criteria and also because in males immunophenotyping cannot be performed.

4. Again here exclusion criteria is considered as exact opposite of inclusion criteria therefore patients with neoadjuvant therapy are excluded.

5. Here recurrence cases and secondaries from other malignancy are not counted therefore primary breast malignancy are referred as de novo.

6. Here I wanted to be specific for considering only mastectomy patient in inclusion criteria therefore opposites are mentioned in exclusion

7. I agree with you Sir, reference no. 1 should have been there at the place of 14th

8. Here COX-2 immunohistochemical score will be calculated by multiplying the values of Table 2 and Table 3 and values obtained will be put under 3 categories i.e

0-3 = Negative / Faint
4-8= Moderate
9-12 = High

9. ER/PR/Her2 antibodies assessment according to Allred scoring system as depicted in CAP( college of American Pathologists) guidelines.
1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.

2. Sample Size
Using the formula:

N= https://f1000research.s3.amazonaws.com/linked/653957.image.png

Where:

N = Sample size

Z(1-α/2) = 1.96 (value of the standard normal variate corresponding to the level of significance α of 0.05)

d = Specified precision on either side of the mean (10% or 0.1)

P = Expected prevalence of COX2 expression in the study population (76% or 0.76)

From the previous study by Fatma Z Abd Elrahman et al., the prevalence of COX2 expression in carcinoma breast patients was found to be 76%. Taking d=10%, the total sample size calculated is 70.07.

N=        (1.96)²X 0.76X(1−0.76)
                                                         (0.1)²

N= 70.07
Rounding up to the nearest whole number, the required sample size is 70.
Reference- Abdel-rahman FZ, Zaky AH, Zedan A, Elsaba TM, Gabr A. COX-2 “Cyclooxygenase 2“as a Prognostic marker in Breast Cancer. SVU-International Journal of Medical Sciences. 2021 Feb 2;0(0):0–0.

3. Male patients are kept in exclusion criteria because exclusion criteria comes as an opposite if inclusion criteria and also because in males immunophenotyping cannot be performed.

4. Again here exclusion criteria is considered as exact opposite of inclusion criteria therefore patients with neoadjuvant therapy are excluded.

5. Here recurrence cases and secondaries from other malignancy are not counted therefore primary breast malignancy are referred as de novo.

6. Here I wanted to be specific for considering only mastectomy patient in inclusion criteria therefore opposites are mentioned in exclusion

7. I agree with you Sir, reference no. 1 should have been there at the place of 14th

8. Here COX-2 immunohistochemical score will be calculated by multiplying the values of Table 2 and Table 3 and values obtained will be put under 3 categories i.e

0-3 = Negative / Faint
4-8= Moderate
9-12 = High

9. ER/PR/Her2 antibodies assessment according to Allred scoring system as depicted in CAP( college of American Pathologists) guidelines.
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Author Response 18 Jun 2024

Jayashree Bhawani, Department of Pathology, Jawaharlal Nehru Medical college, Sawangi(Meghe), Wardha, 442001, India

18 Jun 2024

Author Response
1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.
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1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.

2. Sample Size
Using the formula:

N= https://f1000research.s3.amazonaws.com/linked/653957.image.png

Where:

N = Sample size

Z(1-α/2) = 1.96 (value of the standard normal variate corresponding to the level of significance α of 0.05)

d = Specified precision on either side of the mean (10% or 0.1)

P = Expected prevalence of COX2 expression in the study population (76% or 0.76)

From the previous study by Fatma Z Abd Elrahman et al., the prevalence of COX2 expression in carcinoma breast patients was found to be 76%. Taking d=10%, the total sample size calculated is 70.07.

N=        (1.96)²X 0.76X(1−0.76)
                                                         (0.1)²

N= 70.07
Rounding up to the nearest whole number, the required sample size is 70.
Reference- Abdel-rahman FZ, Zaky AH, Zedan A, Elsaba TM, Gabr A. COX-2 “Cyclooxygenase 2“as a Prognostic marker in Breast Cancer. SVU-International Journal of Medical Sciences. 2021 Feb 2;0(0):0–0.

3. Male patients are kept in exclusion criteria because exclusion criteria comes as an opposite if inclusion criteria and also because in males immunophenotyping cannot be performed.

4. Again here exclusion criteria is considered as exact opposite of inclusion criteria therefore patients with neoadjuvant therapy are excluded.

5. Here recurrence cases and secondaries from other malignancy are not counted therefore primary breast malignancy are referred as de novo.

6. Here I wanted to be specific for considering only mastectomy patient in inclusion criteria therefore opposites are mentioned in exclusion

7. I agree with you Sir, reference no. 1 should have been there at the place of 14th

8. Here COX-2 immunohistochemical score will be calculated by multiplying the values of Table 2 and Table 3 and values obtained will be put under 3 categories i.e

0-3 = Negative / Faint
4-8= Moderate
9-12 = High

9. ER/PR/Her2 antibodies assessment according to Allred scoring system as depicted in CAP( college of American Pathologists) guidelines.
1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.

2. Sample Size
Using the formula:

N= https://f1000research.s3.amazonaws.com/linked/653957.image.png

Where:

N = Sample size

Z(1-α/2) = 1.96 (value of the standard normal variate corresponding to the level of significance α of 0.05)

d = Specified precision on either side of the mean (10% or 0.1)

P = Expected prevalence of COX2 expression in the study population (76% or 0.76)

From the previous study by Fatma Z Abd Elrahman et al., the prevalence of COX2 expression in carcinoma breast patients was found to be 76%. Taking d=10%, the total sample size calculated is 70.07.

N=        (1.96)²X 0.76X(1−0.76)
                                                         (0.1)²

N= 70.07
Rounding up to the nearest whole number, the required sample size is 70.
Reference- Abdel-rahman FZ, Zaky AH, Zedan A, Elsaba TM, Gabr A. COX-2 “Cyclooxygenase 2“as a Prognostic marker in Breast Cancer. SVU-International Journal of Medical Sciences. 2021 Feb 2;0(0):0–0.

3. Male patients are kept in exclusion criteria because exclusion criteria comes as an opposite if inclusion criteria and also because in males immunophenotyping cannot be performed.

4. Again here exclusion criteria is considered as exact opposite of inclusion criteria therefore patients with neoadjuvant therapy are excluded.

5. Here recurrence cases and secondaries from other malignancy are not counted therefore primary breast malignancy are referred as de novo.

6. Here I wanted to be specific for considering only mastectomy patient in inclusion criteria therefore opposites are mentioned in exclusion

7. I agree with you Sir, reference no. 1 should have been there at the place of 14th

8. Here COX-2 immunohistochemical score will be calculated by multiplying the values of Table 2 and Table 3 and values obtained will be put under 3 categories i.e

0-3 = Negative / Faint
4-8= Moderate
9-12 = High

9. ER/PR/Her2 antibodies assessment according to Allred scoring system as depicted in CAP( college of American Pathologists) guidelines.
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Amit Kumar Chowhan, AIIMS, RAIPUR, RAIPUR, India
Shaan Khetrapal, Hamdard Institute of Medical Sciences and Research, New Delhi, India

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22 Jul 2024 | for Version 2

Amit Kumar Chowhan, Department of Pathology, AIIMS, RAIPUR, RAIPUR, TirupatiCHHATTISGARH, India

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No further comments.

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05 Jun 2024 | for Version 1

Shaan Khetrapal, Department of Pathology, Hamdard Institute of Medical Sciences and Research, New Delhi, Delhi, India

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Approved

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests

No competing interests were disclosed.

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02 Oct 2023 | for Version 1

Amit Kumar Chowhan, Department of Pathology, AIIMS, RAIPUR, RAIPUR, TirupatiCHHATTISGARH, India

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Approved With Reservations

Comment

In Introduction it is mentioned ‘This is a more advanced diagnostic modality, which determines the molecular phenotypes of carcinoma breast by implementing immunohisto[1]chemistry’. – Kindly check whether this line has been kept at appropriate place.
Sample size should be calculated in reference to prevalence of positivity for COX2 in breast cancer (data to be taken from other similar study). For example, in reference no 5, the positivity of COX2 is mentioned as 42.7% in tumour cells, so while calculating sample size ‘P’ should be 42.7%.
First point of Inclusion criteria is • ‘Female patients diagnosed with breast carcinoma on histopathological examination.’

Therefore, there is no need of keeping an exclusion criteria as ‘Male patients with a diagnosis of breast carcinoma.’
Second point of Inclusion criteria is ‘Women with breast carcinoma having no history of treatment.’Therefore, there is no need of keeping an exclusion criteria as ‘Patients with breast carcinoma who have received neoadjuvant chemotherapy.’
Third point of Inclusion criteria is ‘Female patients presenting with breast carcinoma arising de novo.’’ Kindly explain what is the exact meaning of ‘’de novo.’’
Fourth point of Inclusion criteria is ‘’All cases that underwent mastectomy/modified radical mastectomy (MRM) procedure’’, therefore there is no need of keeping an exclusion criteria as ‘’Patients who underwent incisional biopsy, excisional biopsy, or tru-cut biopsy’’.
Under the heading ‘’Evaluation of COX-2 expression by immuno-histochemistry’’, the reference article mentioned is reference no 14, whereas in table 2 and table 3, the reference mentioned is reference no 1, kindly explain the disparity.
Under the heading ‘’Evaluation of COX-2 expression by immuno-histochemistry’’, it has been further mentioned as ‘’COX-2 immunohistochemistry score is obtained by multiplying the staining quantity score and staining intensity score. 0-3 =Negative/faint staining 4-8= Moderate/intermediate staining’’, which is not matching with table 2 and table 3, where the scores are 0-4 and 0-3. Kindly explain the disparity.
Under the section ‘’Immunohistochemical assessment’’, it has been mentioned that ER/PR/Her2 antibodies and COX-2 antibody will be applied, kindly mention the scoring system for ER/PR/Her2 antibodies also, which will be followed in the study, along with reference.

Is the rationale for, and objectives of, the study clearly described?

Yes
Is the study design appropriate for the research question?

Yes
Are sufficient details of the methods provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Oncopathology

Respond to this report

Responses (1)

Author Response

18 Jun 2024

Jayashree Bhawani, Department of Pathology, Jawaharlal Nehru Medical college, Sawangi(Meghe), Wardha, 442001, India

1. Instead of this is a more advanced diagnostic modality, It should be there is a more advanced diagnostic modality which is immunohistochemistry that determines molecular phenotype of breast carcinoma.

2. Sample Size
Using the formula:

N= https://f1000research.s3.amazonaws.com/linked/653957.image.png

Where:

N = Sample size
Z(1-α/2) = 1.96 (value of the standard normal variate corresponding to the level of significance α of 0.05)
d = Specified precision on either side of the mean (10% or 0.1)
P = Expected prevalence of COX2 expression in the study population (76% or 0.76)
From the previous study by Fatma Z Abd Elrahman et al., the prevalence of COX2 expression in carcinoma breast patients was found to be 76%. Taking d=10%, the total sample size calculated is 70.07.

N=        (1.96)²X 0.76X(1−0.76)
                                                         (0.1)²

N= 70.07
Rounding up to the nearest whole number, the required sample size is 70.
Reference- Abdel-rahman FZ, Zaky AH, Zedan A, Elsaba TM, Gabr A. COX-2 “Cyclooxygenase 2“as a Prognostic marker in Breast Cancer. SVU-International Journal of Medical Sciences. 2021 Feb 2;0(0):0–0.

3. Male patients are kept in exclusion criteria because exclusion criteria comes as an opposite if inclusion criteria and also because in males immunophenotyping cannot be performed.

4. Again here exclusion criteria is considered as exact opposite of inclusion criteria therefore patients with neoadjuvant therapy are excluded.

5. Here recurrence cases and secondaries from other malignancy are not counted therefore primary breast malignancy are referred as de novo.

6. Here I wanted to be specific for considering only mastectomy patient in inclusion criteria therefore opposites are mentioned in exclusion

7. I agree with you Sir, reference no. 1 should have been there at the place of 14th

8. Here COX-2 immunohistochemical score will be calculated by multiplying the values of Table 2 and Table 3 and values obtained will be put under 3 categories i.e

0-3 = Negative / Faint
4-8= Moderate
9-12 = High

9. ER/PR/Her2 antibodies assessment according to Allred scoring system as depicted in CAP( college of American Pathologists) guidelines.

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Competing Interests

No competing interest

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Solanki R, Agrawal N, Ansari M, et al.: COX-2 Expression in Breast Carcinoma with Correlation to Clinicopathological Parameters. Asian Pac. J. Cancer Prev. 2018; 19(7): 1971–1975. PubMed Abstract

[2] 2. Harbeck N, Penault-Llorca F, Cortes J, et al.: Breast cancer. Nat. Rev. Dis. Primers. 2019; 5(1): 66. Publisher Full Text

[3] 3. American Cancer Society: Breast Cancer Facts and Figures 2019-2020. Atlanta, Ga: American Cancer Society; 2020.

[4] 4. Dai X, Xiang L, Li T, et al.: Cancer Hallmarks, Biomarkers and Breast Cancer Molecular Subtypes. J. Cancer. 2016; 7(10): 1281–1294. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Department of Pathology, Mugla Sitki Kocman University, School of Medicine, Mugla, TurkeyTekin L, Celik SY, et al.: Immunohistochemical Expression of Cyclooxygenase-2 and Its Relationship With Prognostic Parameters in Breast Cancer. Cyprus J. Med. Sci. 2021; 6(1): 39–43.

[6] 6. Liu B, Qu L, Yan S: Cyclooxygenase-2 promotes tumor growth and suppresses tumor immunity. Cancer Cell Int. 2015; 15: 106. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Desai SJ, Prickril B, Rasooly A: Mechanisms of phytonutrient modulation of cyclooxygenase-2 (COX-2) and inflammation related to cancer. Nutr. Cancer. 2018 Apr 3; 70(3): 350–375. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Pastor N, Ezquerra LJ, Santella M, et al.: Prognostic significance of immunohistochemical markers and histological classification in malignant canine mammary tumours. Vet. Comp. Oncol. 2020 Dec; 18(4): 753–762. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Chaokromthong K, Sintao N: Sample size estimation using Yamane and Cochran and Krejcie and Morgan and green formulas and Cohen statistical power analysis by G* Power and comparisions. Apheit International Journal. 2021 Dec 24; 10(2): 76–86.

[10] 10. Mehrotra R, Yadav K: Breast cancer in India: Present scenario and the challenges ahead. World J Clin Oncol. 2022 Mar 24; 13(3): 209–218. PubMed Abstract | Publisher Full Text | Free Full Text

[11] 11. Bansal C, Singh US, Misra S, et al.: Comparative evaluation of the modified Scarff-Bloom-Richardson grading system on breast carcinoma aspirates and histopathology. Cytojournal. 2012; 9: 4. PubMed Abstract | Publisher Full Text | Free Full Text

[12] 12. van Dooijeweert C , van Diest PJ , Ellis IO: Grading of invasive breast carcinoma: the way forward. Virchows Arch. 2022; 480: 33–43. PubMed Abstract | Publisher Full Text | Free Full Text

[13] 13. Sanderson T, Wild G, Cull AM, et al.: 19—Immunohistochemical and immunofluorescent techniques. Bancroft’s Theory and Practice of Histological Techniques. 8th ed. Suvarna SK, Layton C, Bancroft JD, editors. 2018 Feb 27; 337–94.

[14] 14. Half E, Tang XM, Gwyn K, et al.: Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res. 2002; 62(6): 1676–1681. PubMed Abstract

To study the utility of COX-2 as immunohistochemical prognostic marker in comparison to various histopathological parameters and TNM staging in breast carcinoma: an observational, cross-sectional study protocol

Abstract

Keywords

Introduction

Objectives

Methods

Study design

Sample size

Ethics and consent

Inclusion criteria

Exclusion criteria

Approach to the study

Method of tissue processing

Methodology of interpretation

Table 1. Score Categories According to Field Diameter and Mitotic Count.12

Table 2. Evaluation of COX-2 immunostaining status based on proportion score i.e percentage of positive cells.1

Table 3. Evaluation of COX-2 immunostaining status by measuring intensity of staining.1

Immunohistochemical assessment13

Evaluation of COX-2 expression by immuno-histochemistry

Statistical-analysis

Results

Discussion

Conclusion

Dissemination

Study status

Ethics and consent

Author contributions

Data availability

Acknowledgements

References

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Table 1. Score Categories According to Field Diameter and Mitotic Count.¹²

Table 2. Evaluation of COX-2 immunostaining status based on proportion score i.e percentage of positive cells.¹

Table 3. Evaluation of COX-2 immunostaining status by measuring intensity of staining.¹

Immunohistochemical assessment¹³