European Journal of Cardiovascular Medicine

Breast cancer affects more women than any other type of cancer, except for lung cancer, and is a leading cause of cancer-related deaths worldwide [1]. In India, breast cancer has the highest incidence rate among females, with an age-adjusted rate as high as 25.8 per 100,000 women and a mortality rate of 12.7 per 100,000 women [2,3]. The rise of urbanization and changes in lifestyle have contributed to the increasing prevalence of breast carcinoma, making it a significant cause of morbidity and mortality among women in developing countries like India [4]. Therefore, there is a crucial need to strengthen and expand the current diagnostic and therapeutic facilities to address this growing problem.

Over the past few years, there has been increasing use of molecular markers such as ER, PR, and HER2 in breast carcinoma to determine the aggressiveness of tumors and develop personalized treatment plans [5]. This approach helps to reduce drug resistance, non-response, and toxicity. Furthermore, a better understanding of the molecular mechanisms underlying breast cancer development and progression has resulted in the identification of several new and novel molecular targets. One of these emerging molecular targets is GATA3, which has been found to have a diverse range of functions in cancer cells.

GATA binding protein 3 (GATA3) belongs to a family of six zinc finger transcription factors and was initially recognized for its role in regulating immune cell function. It plays a significant regulatory role in the development of various tissues, including hematopoietic cells, skin, breast, kidney, and the central nervous system [6,7]. GATA3 is responsible for maintaining the quiescent state of differentiated luminal cells in the adult mammary gland, suggesting its involvement in the pathogenesis of breast carcinoma. Supporting this hypothesis, a study conducted on a mouse model found that the loss of GATA3 was associated with a loss of tumor differentiation, marked progression from adenoma to early carcinoma, and the onset of tumor dissemination, whereas the restoration of GATA3 in late carcinomas resulted in tumor differentiation and the suppression of tumor dissemination [8]. Although GATA3 is expressed in a wide range of tissues, it has been demonstrated to be a highly specific marker for breast carcinomas [9] and urothelial carcinomas only through GATA3 labeling.

Hence the study was planned with the objectives to study GATA3 expression in invasive breast carcinoma and to determine its correlation with modified Scarf-Bloom-Richardson grading of breast cancer.

It was a cross-sectional study conducted in KR Hospital, government tertiary care hospital in Karnataka for a year (January, 2021 to January, 2022). Data was obtained from mastectomy specimen, received in the Department of Pathology, K.R. Hospital attached to Mysore Medical College and Research Institute, Mysuru, Karnataka.

All ductal and lobular invasive breast carcinoma specimens which were received by the study setting were included in the study. Specimen of Sarcomas / Metastatic lesions / benign lesions / in-situ carcinomas of the breast and other malignant subtypes of breast cancers were not included in the study. Also, the specimens were excluded if they were autolyzed / were from the patients who were receiving Chemotherapy and Radiotherapy. Timeframe sampling technique was adopted, according to which all cases encountered during the study period which satisfied the eligibility criteria were taken up for the study.

Upon receipt of the specimens in 10% formalin, patient details were reviewed, and the mastectomy specimens were fixed in fresh formalin for 24 hours. The fragments were thoroughly examined, and the processing was carried out accordingly. The sections were stained using hematoxylin and eosin; and confirmed through immunohistochemistry. Each stained section was evaluated for the following:

1. a) The histological type of the tumor.
2. b) The histological grade of the tumor, classified according to the modified Bloom-Richardson-Elston grading system as Grade 1 (well-differentiated), Grade 2 (moderately differentiated), or Grade 3 (poorly differentiated) [10].
3. c) Pathological Staging pTNM, wherever feasible [11].

Immunohistochemistry

Formalin-fixed, paraffin-embedded breast tissue specimens were subjected to immunohistochemistry (IHC) for Estrogen Receptor (ER), Progesterone Receptor (PR), Human epidermal growth factor receptor 2 (HER2/neu), Ki 67, and GATA3 using their corresponding antibodies. GATA3 IHC was conducted using a pre-diluted mouse monoclonal antibody from Biocare Medical [12]. Surrogate Molecular Classification was determined through immunohistochemistry for ER, PR, and HER2/neu, which is categorized as follows [10]:

1. GATA3 score: GATA3 immunostaining was scored in semiquantitative manner. The benign lobules' ductal epithelial cells serve as the positive internal controls for GATA3 staining.
2. GATA3 labeling was scored on a scale of 0 to 4+ based on the extent of nuclear staining, graded as follows (8): 0 (0–5%), 1+ (6%–25%), 2+ (26%–50%), 3+ (51%–75%) and 4+ (>75%).

Informed consent was obtained from all individual participants included in the study.

Statistical Analysis

For the purpose of statistical analysis and better understanding, GATA3 staining was dichotomized i.e. grouped into 2 categories only i.e. Negative (0) and Positive (1+ or more). The staining intensity was noted independently as weak, moderate, or strong, and any staining intensity with more than 5% distribution was deemed positive.

The data was entered in Microsoft Excel Spreadsheet v2016 and was analyzed using trial version of SPSS version 20.0. Quantitative data has been summarized as mean and standard deviation while qualitative data has been summarized as proportions. Association between GATA3 expression and modified Scarf-Bloom-Richardson grading of breast cancer was assessed using Pearson’s chi-square test and its modifications wherever applicable. A p value of < 0.05 was considered as statistically significant.

In our study most of the study participants belong to the age group of 51–60 years (37.5%) and  41–50 years (30.0%)

(Table 1).

Table 1: Age distribution of study participants

More than 90% cancer patients were females (Table 2).

Table 2: Gender distribution of study participants

 With respect to laterality of breast cancer, almost half of the patients had right sided cancer, and the other half had left sided cancer (Table 3).

Table 3: Laterality of invasive breast Ca in patients

Around one-third of the cases were of IDC type, and the other two-thirds were of IBC-NST type (Table 4A).

Table 4A: Histological type of breast Ca in patients

Age distribution of the IDC cases was equivocal while most of the IBC-NST cases (77%) occurred in age group of 41-60 years. Only in individuals aged ≤ 40 years, IDC was very common while in all the other age groups, common type of Ca was IBC-NST (Table 4B).

Table 4B: Association between age distribution categories and histological type of breast Ca in patients

However, age was not significantly associated with Histological type of breast cancer. Around half of the cancers were of grade 2 category. Another third was of grade 1 and the remaining 17.5% were of grade 3 (Table 5A).

Table 5A: Grading of breast Ca among patients

Age was significantly associated with grade of invasive breast cancer in the sense that, as the age increased, probability of breast cancers being grade 3 also increased (Table 5B).

Table 5B: Association between age distribution categories and grading of breast Ca in patients

GATA 3 was not expressed in 10% cases. GATA-3 expression was weakly positive in 7.5% of the breast cancer cases (Figure 5).

Figure 1A and 1B: GATA3 staining in well differentiated Invasive breast carcinoma (Nuclear score:3) (IHC;10X and 40X).

GATA-3 expression was moderately positive in 32.5% of the breast cancer cases (Figure 3 &4).

Figure 2A and 2B: GATA3 staining in Moderately differentiated Invasive breast carcinoma (Nuclear score;2) (IHC;10X and 40X).

GATA-3 expression was strongly positive in 50% of the breast cancer cases (Figure 1 & 2).

Figure 3: GATA3 staining in poorly differentiated Invasive breast carcinoma (Nuclear score;0/1) (IHC;40X).

The GATA3 expression was not significantly associated with age (Table 6 A,B).

Table 6A: Expression of GATA3 among invasive breast Ca patients

Table 6B: Association between age distribution categories and expression of GATA3 among invasive breast Ca patients

In the present study, GATA3 expression was not associated with histological study (Table 7).

Table 7: Association between histological type and GATA3 expression among invasive breast Ca patients

However, Grade of breast cancer was significantly associated with GATA3 expression. Higher the grade of breast cancer, weaker was the GATA3 expression. All cancers with  grade 1 cancer had expressed GATA3 expression strongly (Table 8).

Table 8: Association between Ca grade and GATA3 expression among invasive breast Ca patients

GATA3 is a transcription factor that regulates the expression of genes involved in cell differentiation, proliferation, and survival. It is expressed in luminal epithelial cells of the breast, and reduced GATA3 expression has been observed in a subset of breast cancer patients. Studies have shown that reduced GATA3 expression is associated with poor prognosis, resistance to endocrine therapy, and decreased survival in breast cancer patients. Therefore, GATA3 expression is a potential biomarker for breast cancer diagnosis and treatment, as well as a therapeutic target for the development of novel therapies for breast cancer patients with reduced GATA3 expression.

The study aimed to investigate the expression of GATA3 in invasive breast carcinoma and establish its correlation with the modified Scarf-Bloom-Richardson grading of breast cancer.

The Scarf-Bloom-Richardson grading system is a histological grading system used to evaluate the aggressiveness of breast cancer cells [13]. It assesses the features of tumor cells, such as their size, shape, and differentiation, and assigns a score based on these criteria. The modified version of this grading system includes the addition of an intermediate category between grades 2 and 3 to improve the accuracy and reliability of the grading system. This grading system is widely used in clinical practice to guide treatment decisions and predict the prognosis of breast cancer patients. Hence this grading system was used to determine correlation with GATA3 expression.

The present study included only invasive mammary carcinomas just like many other contemporary studies. Most belonged to age group of 51 to 60 years in present study, which was similar to age distribution of invasive carcinoma as per the studies conducted by Suri T et al. in Delhi in 2020 [14], Tabriz HM et al. in Iran in 2022 [15] etc. More than 97% were females similar to gender distribution of pattern of study conducted by Banik L et al. in 2022 in Kolkota [16].

In present study, one-third of the carcinomas are of ductal type. This proportion was very less as compared to 97% ductal carcinomas in the study conducted by Suri T et al. in Delhi in 2020 [14] and to 87% ductal carcinomas in the study conducted by Banik L et al. in 2022 in Kolkota [16].

With respect to GATA-3 expression, the present study showed following distribution pattern: 10% showed no GATA-3, 7.5% were weakly positive, 32.5% were moderately positive and 50.0% were strongly positive. Proportion of cancers with negative GATA-3 expression was higher in other studies i.e. it was 28% as per the study conducted by Ismail AM et al. in Egypt in 2017 [17]; GATA-3 expression was negative in 22% as per the study conducted by Tabriz HM et al. in Iran in 2022[15]; and GATA-3 expression was negative in 18% as per the study conducted by  Shaoxian T et al. in China in 2017 [18].

To understand the importance of GATA-3 expression in cancer prognostication / management plan, it is crucial to assess the association of GATA-3 expression with known prognostic factors. There are many known and proven prognosticating factors with respect to breast cancer. Firstly, tumors of a higher grade exhibit a greater tendency to metastasize to other parts of the body, including lymph nodes, as compared to tumors of a lower grade. As a result, tumors of a higher grade are generally associated with a worse prognosis than those of a lower grade. Secondly, hormone receptor-positive tumors, which possess a significant number of hormone receptors, can be effectively treated with hormone therapies such as tamoxifen, aromatase inhibitors, and ovarian suppression. In contrast, hormone receptor-negative tumors, which lack sufficient hormone receptors, cannot be managed with hormone therapies and typically have a higher likelihood of recurrence. Other prognostic factors are lymph node involvement, Ki-67 expression, gene expression, type of cancer etc. Comparisons are made with all these parameters in various studies while Scarf-Bloom-Richardson grading system, another prognosticating factor, is used to correlate with GATA-3 expression in the present study.

Almost half the patients with Scarf-Bloom-Richardson grade 3 of breast cancer do not express GATA-3 while the other half expressed GATA-3 in a weak manner. Around two-thirds of patients with Scarf-Bloom-Richardson grade 2 exhibited moderate expression of GATA-3 while the remaining one-third exhibited GATA-3 strongly. All the patients with Scarf-Bloom-Richardson grade 1 exhibited strong GATA-3 expression. These 2 parameters were significantly associated.

Similar observation was reported by the study conducted by Tabriz HM et al. in Iran in 2022, where most of the patients with grade 3 carcinoma did not express GATA-3; and the difference in proportions was statistically significant. Also, the study conducted by Banik L et al. in 2022 in Kolkota [16] showed similar distribution of GATA-3 expression among breast carcinomas of different histological grades. As the histological grade increased, it was observed that GATA-3 expression got significantly fewer.

With the above pooling of information, it could be stated that GATA-3 expression has the potential to be considered as a prognosticating biomarker or may even be included in planning the management protocol. But larger multicentric studies with various subtypes of breast cancers and with follow-up of patients must be conducted before generalizing the conclusion.

The predominant limitation would be that chi-square test had to be used to determine statistical significance; and due to the limited numbers in some of the cross-tabulation categories, the trends must be considered preliminary. Additionally, as there was no available data on follow-up, it was not possible to assess the prognostic significance of GATA-3 in breast carcinoma.

GATA-3 is expressed by most invasive breast cancers and are strongly associated with histological grade 1 followed by grade 2.

Acknowledgement:

None.

 Conflicts of interest:

The authors declare no conflict of interest.

1. Basic Information about Breast Cancer. Centers for Disease Control and Prevention. 2022. Retrieved from https://www.cdc.gov/cancer/breast/basic_info/index.htm and accessed on 21^st April 2023
2. Ferlay J., Soerjomataram I., Dikshit R., Eser S., Mathers C., Rebelo M., Parkin DM., Forman D, Bray F., 2015. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015 Mar;136(5):E359-86. https://doi.org/10.1002/ijc.29210
3. Gupta A., Shridhar K., Dhillon PK., 2015. A review of breast cancer awareness among women in India: Cancer literate or aware-ness deficit? Eur J Cancer. 2015 Sep;51(14):2058-66. doi: 10.1016/j.ejca.2015.07.008
4. Kaarthigeyan K., 2012. Cervical cancer in India and HPV vaccination. Indian J Med Paediatr Oncol. 2012;33(1):7-12. doi: 10.4103/0971-5851.96961
5.  
6. O’Connell PR., McCaskie AW., Sayers RD., 2023. Bailey and Love’s Short Practice of Surgery. 27^th ed. Boca Raton: CRC Press; 2023. DOI https://doi.org/10.1201/9781315111087
7. Kumar V., Abbas AK., Aster JC., 2018. Robbins basic pathology. 10^th Philadelphia: Elsevier; 2018. https://shop.elsevier.com.
8. Kouros-Mehr H., Slorach E., Sternlicht M., Werb Z., 2006. GATA-3 maintains the differentiation of the luminal cell fate in the mammary gland. Cell. 2006 Dec;127(5):1041-55.  DOI: 1016/j.cell.2006.09.048
9. Liu H., Shi J., Wilkerson ML., Lin F., 2012. Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: A useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol. 2012 Jul;138(1):57-64. DOI: 1309/AJCP5UAFMSA9ZQBZ
10. Mehra R., Varambally S., Ding L., Shen R., Sabel MS., Ghosh D., Chinnaiyan AM., Kleer., 2005. Identification of GATA3 as a breast cancer prognostic marker by global gene expression metaanalysis. Cancer Res. 2005 Dec;65(24):11259-64. DOI: 1158/0008-5472.CAN-05-2495
11. Collins LC., 2017. Breast. In: Rosai and Ackerman (eds.), Surgical Pathology. 11^th Philadelphia: Elsevier; 2017. pp1434-527. Hardback ISBN: 9780323263399
12. Singletary SE., Allred C., Ashley P., Berry D., Bland KI., Borgen PI., Gary clark., Stephen B Edge., Daniel F Hayes., Lorie L Hughes., Robert V P Hutter., Monica Marrow., David L Page., Abram Recht., Richard L Theriault, Ann Thor., Donald L Weaver., H Samuel Wieand., Frederick L Greene., 2002. Revision of the American Joint Committee on Cancer staging system for breast cancer. J Clin Oncol. 2002 Sep;20(17):3628-36. DOI: 1200/JCO.2002.02.026
13. GATA-3 antibody. Biocare Medical; 2023. Retrieved from https://biocare.net/product/ gata-3-antibody/ and accessed on 1^st May 2023
14. Bansal C., Singh US., Misra S., Sharma KL., Tiwari V., Srivastava AN., 2012. Comparative evaluation of the modified Scarff-Bloom-Richardson grading system on breast carcinoma aspirates and histopathology. Cytojournal. 2012;9:4. DOI: 4103/1742-6413.92550
15. Suri T., Ahluwalia C., 2021. Immunohistochemical analysis of expression of GATA3 in carcinoma breast and its correlation with prognostic parameters. In: Annals of Pathology and Laboratory Medicine. 2021;8(2):A26-32. DOI: https://doi.org/10.21276/apalm.3012
16. Tabriz HM., Farmani E., Nazar E., Javadi AE., 2022. GATA Binding Protein 3 (GATA-3) expression evaluation as prognostic factor in breast cancer and its relationship with other immunomarkers. Indian J Pathol Microbiol. 2022 Apr-Jun;66(2):286-90. DOI: 4103/ijpm.ijpm_453_21
17. Banik L., Pal M., Mayur N., 2022. Study of GATA-3 expression in breast carcinoma in a tertiary care hospital in Eastern India. Arch Breast Cancer. 2022;9(4):450-5. DOI https://doi.org/10.32768/abc.202294450-455
18. Ismail AM., Khalifa SE., Saied EM., Tamamy MME., 2018. Immunohistochemical study of GATA-3 expression versus estrogen and progesterone receptor in invasive mammary carcinomas. Kasr Al Ainy Medical Journal. 2018:24(1):40. URL: http://www.kamj.eg.net/text.asp?2018/24/1/40/233107
19. Shaoxian T., Baohua Y., Xiaoli X., Yufan C, Xiaoyu T, Hongfen L, Rui B., Xiangjie S., Ruohong S., Wentao., 2017. Characterisation of GATA3 expression in invasive breast cancer: differences in histological subtypes and immunohistochemically defined molecular subtypes. J Clin Pathol.  2017 Nov;70(11):926-34.  DOI: 1136/jclinpath-2016-204137 .