adibas journl clup presenation on pathology

gata binding protein expression evaluation as prognosis factor in breast cancer

1. JOURNAL CLUB Presenter: Dr. Adiba Khan Moderator: Prof. Kafil Akhtar

2. GATA Binding Protein 3 (GATA-3) expression evaluation as prognostic factor in breast cancer and its relationship with other immunemarkers . Author- Hedieh M. Tabriz, Elahe Farmani , Elham Nazar , Arezoo E. Javadi . Place of Study: Department of Pathology, Tehran University of Medical Sciences, Tehran, Iran. Journal: Indian J Pathol Microbiol , 2023 April – June;66:286-290 DOI: 10.4103/ ijpm .

3. OBJECTIVE To evaluate GATA binding protein 3 ( GATA-3) expression as prognostic factor in breast cancer and its relationship with other immune markers.

4. INTRODUCTION Breast cancer is the most common malignancy in women, with more than 1.7 million cases occurring worldwide annually . Majority of cases are detected in postmenopausal females, however it can develop at any age. Despite a decline in breast cancer related mortality due to increased rate of diagnosis and effective treatment , BC is still considered as a burden on society.

5. WHO Globocan 2020

6. WHO Globocan 2020 India

7. Hence, novel strategies are required to predict disease course as well as the response to therapies. Earlier grade and stage of tumor were main prognostic factors. Nowadays immunohistochemistry markers such as estrogen receptors(ER) , progesterone receptors (PR) and human epidermal growth factor 2(HER2) and their considerable correlation with treatment options are used. There are atleast 4 subtypes of breast cancer based on these biomarkers such as luminal A, Luminal B , her2 overexpressed and triple negative breast cancer.

8. Recognition of these subtypes is essential as they exhibit different clinical and molecular prognosis. Triple negative and ER/PR- HER2+ subtypes have generally worse prognosis whereas ER/PR+ have better prognosis and respond to hormonal therapy more efficiently. However in this study a new marker GATA-3 transcription factor was used to evaluate prognosis in breast cancer.

9. GATA 3 is a member of GATA family which are considered zinc finger transcription factors which plays important role in cell proliferation, development and differentiation in various tissues and cell types such as breast epithelial cells. Therefore, GATA -3 could significantly act in development of breast tumors. GATA-3 drives invasive breast cancer cells to undergo the reversal of epithelial- mesenchymal transition, leading to suppression of cancer metastasis. Overexpression of GATA has been observed in luminal type of breast cancer however expression of this protein reduces with increasing grade of tumor. Also overexpression GATA has been has shown to be associated improved survival outcome in ER + premenopausal women. GATA-3

10. GATA -3 expression in breast carcinoma

11. Various mutations in GATA -3 protein are associated in pathogenesis of luminal breast cancer. These include overexpression , silencing by methylation or interference by protein that bind GATA factors. Loss of ER/PR receptor loss represent maintained level of GATA expression. All these findings are representative of the growing attention regarding the usefulness of GATA-3 as prognostic marker in breast carcinoma.

12. MATERIAL AND METHODS 166 patients with breast cancer were assigned which were obtained via radical/partial mastectomy between february 2010 and october 2016 from the electronic registry of the department of Pathology, Sina hospital affiliated to Tehran University of Medical Sciences. Cross sectional study was done on this population. Following data was recorded from all patients: Demographic information Tumor size Tumor stage and grade Lymph node invasion Formlin -fixed paraffin-embedded primary tumor samples Tumor staging was performed according to the College of American pathologists protocols 2019. Tumor grading was based on Nottingham algorithm.

13. Immunohistochemical analysis IHC staining was performed on 3 micron formalin-fixed, paraffin-embedded sections including ER, PR and HER2 as per College of American protocols 2019. GATA -3 staining - Mouse monoclonal anti-GATA3 antibody (1:200 dilution) used. Sections from block deparaffinized with xylene , rehydrated in a series of ethanol solutions with decreasing concentration. Antigen retrieval was achieved in cell conditioning solution CC1 for 60min. Endogenous peroxidase was inhibited by 3% H2O2 in methanol solution for 10 min. Slides were incubated with monoclonal antibodies for 120 min at room temperature. The color reaction product was developed with 3,3’-diaminobenzidine, tetrahydroxychloride as a substrate and nuclear contrast was achieved with hematoxylin /ammoniac water counterstaining.

14. POSITIVE CONTROL - Section from normal breast gland. NEGATIVE CONTROL- Tissue sections stained without primary antibody. SCORING METHOD- Score Expression 0 % - 5 % 1+ 6 % - 25% 2+ 25 % - 50% 3+ 51 % - 75% 4+ 76 % or above

15. GATA -3 Stain expression : 4 plus = 76% or above

16. GATA-3 stain expression: 3 plus = 51-75% GATA-3 stain expression: 2 plus = 26-50%

17. GATA-3 stain expression: 1 plus = 6 - 25% GATA-3 stain expression: 0 = 0 - 5%

18. Statistical analysis Statistical Package of Social Science Software (SPSS version 24) used. P value level of 0.05 or below was considered as significant. Shapiro- Wilk test and probability graphs were used to test the normality of baseline data. Categorical variable were reported in percentage whereas continuous data as mean +- SD. Continuous and categorical variables were compared using an independent t-test and Levene’s test for equality of variance and Fisher’s exact test, respectively.

19. Positive Negative ER staining 123 (74.1%) 43 (25.9%) PR staining 110 (66.3%) 56 (33.7%) HER 2 staining 91 (54.8%) Equivocal – 48 (28.9%) Negative – 27 (16.3%) RESULT GATA-3 staining Negative 1 plus 2 plus 3 plus 4 plus Frequency 38 9 6 13 100 (Number /%) (22.9%) (5.4%) (3.6%) (7.8%) (60.2%)

20. In the same study it was found that, 31 patients had negative ER staining & GATA-3 staining, 96 patients had positive ER staining & GATA-3 staining. Dominant relationship between the ER and the GATA-3 staining ( P value : 0.001) 33 patients had negative PR staining and GATA-3 staining , 90 patients had positive PR and GATA -3 staining. Significant relationship between PR and GATA-3 staining (P value: 0.001) Only 19 patients had negative staining for HER 2 & GATA-3 11 patients had positive HER2 and GATA -3 staining. No specific relationship between HER-2 and GATA -3 staining ( P value : 0.372).

21. In 22 patients with triple negative carcinoma , 15 patients had negative 7 patients had positive GATA-3 staining In 144 patients with luminal carcinoma, 4 patients had negative & 100 patients had positive GATA- 3 staining. Direct relationship between luminal carcinoma and higher GATA - 3 expression( P value 0.001). Major relationship between triple negative carcinoma and lower GATA - 3 expression ( P value 0.001).

22. Relationship between GATA-3 staining and other immunomarkers and cancer types- ER + PR + HER-2 + Triple negative Luminal carcinoma GATAA -3 staining 123 110 27 22 99 Frequency/% (74.1%) (66.3%) (16.3%) (13.3%) (59.6%)

23. Furthermore, there was no significant relationship between the tumor size, local recurrence and lymph node involvement by tumor with GATA -3 staining ( P value: 0.233, 0.101, and 0.122, respectively). However, a dominant relationship between the metastasis rate and grade with GATA -3 staining ( P value : 0.000 and 0.001, respectively). Grade/ GATA-3 staining Negative 1 plus 2 plus 3 plus 4 plus Total Grade 1 4 1 2 3 32 42 Grade 2 16 4 3 6 57 86 Grade 3 18 4 1 4 11 38 Total 38 9 6 13 100 166

24. DISCUSSION The current challenge for cancer biologist is to establish the relationship between specific mutations and tumor biology, and informing on clinical parameters including aggressiveness, response to therapy, and potential for metastasis. ER, PR, HER-2 are well known breast cancer markers , but in this study a new marker, GATA-3 was used. GATA-3 is the most abundant transcription factor in normal luminal epithelial cells of mammary gland. Many studies have examined the prognostic significance of GATA -3 expression in breast carcinoma . GATA-3 mutations appear to occur mainly in patients with luminal subtype breast cancer and are associated with favorable prognosis.

25. In this study, a significant relationship between GATA-3 expression with tumor grade and metastasis rate is established (P value 0.001 and 0.000 , respectively ). In addition, GATA-3 immunohistochemistry marker was found to be useful in diagnosis of both primary and metastatic disease. Particularly in metastatic triple negative breast carcinoma, which lacks specific markers of mammary gland origin, GATA-3 had a role in diagnosis. It was reported that GATA -3 is connected to less aggressive, ER/PR+, HER2 – phenotype, therefore to favorable survival outcome in breast cancer patients.

26. Based on these findings, the expression assessment of GATA-3 in breast cancer patients can provide important clinical information not only regarding the favorable prognostic nature but it can also constitute an important tool to hormone response assessment in breast cancer. On the other hand, low GATA-3 expression is associated with high histologic grade, poor differentiation, positive lymph node, ER/PR negative status and HER2 overexpression .

27. SUMMARY Breast cancer is the most frequent cancer in which mortality can be decreased by proper management. Alternative to grade and stage of breast tumor, immunohistochemical (IHC) markers such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) have considerable correlations with treatment response and prognosis. Novel marker GATA-3 is estimated to be most abundant transcription factor in luminal epithelial cells of mammary gland, therefore is most frequently mutated in breast cancer. In the study, there was a direct relationship between luminal subtype carcinoma and higher GATA-3 expression ( P value: 0.001) and between triple negative carcinoma and lower GATA -3 expression. Also there was a direct relationship between metastasis rate and tumor’s grade with GATA-3 staining.

28. CONCLUSION GATA-3 expression is useful as a prognostic indicator in breast cancer, and is related to other hormonal receptors, and therefore recommended to be routinely included in panel of immunohistochemical markers on surgical specimen.

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