Signicance of Immunohistochemical Biomarker in Breast Carcinoma from a Single Tertiary Care Hospital of Southern Assam

Breast carcinoma is the most common malignancy in female worldwide, leading cause of death in women.immunohistochemistry plays a very important role in the prognostication and treatment determination of breast carcinoma patients. To analyze the immunohistochemical markers in invasive carcinoma of breast and to correlate the expression of hormonal receptors with age of the patient, tumor size, histological grade and lymph node metastasis. Materials and method: The study was conducted on 88 inltrating ductal breast carcinoma sample in a tertiary care hospital of Southern Assam for a period of two year (January2018- December 2019). Data including age, tumor size, and histologic grade and lymph node status retrieved from pathology department. Chi- square was used to determine the statistical signicance between ER/PR status HER2/neu status along with their correlation with various clinicopathological parametres with respect to inltrating ductal breast carcinoma. The mean age of the patients was 56.6 years. We observed correlation between ER and PR expression with age, tumor size, tumor grade. There was correlation between HER2/neu expression and age only. None of the markers showed correlation with lymph node involvement (P>0.05). Our ndings showed the importance of biomarkers (ER, PR, HER2/neu) expression as prognostic factors for therapeutic


Introduction
Breast cancer is a major concern and one of the leading causes of cancer related death throughout the world. Breast cancer like many other types of cancer is a complex heterogeneous disease controlled by a multitude of genetic and epigenetic alterations [1]. During the past two decades the mortality rate has declined signi cantly, primarily due to the early use of adjuvant chemotherapy as well as detection of earlier stage tumours due to increased screening. [2] Prognosis and management of breast cancer is in uenced by the classical variables such as histological type and grade, tumour size, lymph node status, and status of hormonal receptors, Estrogen receptors (ER) and progesterone receptors (PR) of the tumour and more recently Her2/neu oncoprotein status. [3] ER expression is undoubtedly the most important biomarker in breast cancer, because it provides the index for sensitivity to endocrine treatment. ER positive tumours (80% of breast cancer) use the steroid hormone estradiol as their main growth stimulus; ER is therefore direct target of endocrine therapies. PR expression is strongly dependent on the presence of ER.
Tumours expressing PR but not ER are uncommon and represent < 1% of all breast cancer.
The tumors that are estrogen receptor (ER) positive and/or progesterone receptor (PR) positive have lower risks of mortality after their diagnosis compared to women with ER and/or PR negative disease. Clinical trials have also shown that the survival advantage for women with hormone receptor-positive tumors is enhanced by treatment with adjuvant hormonal and/or chemotherapeutic regimens [4]. In breast cancer the average incidence of estrogen receptor and progesterone receptor positivity is 57% and 43% respectively as shown in the studies. However, lower rates of positive estrogen and progesterone receptor breast cancers are found in Indian population from the western literature. The frequency of negative estrogen receptor and progesterone receptor is much more common in India (46.5%) than in the West (10%). Breast cancer patients of Indian origin tend to be younger, tumors are often large when rst diagnosed, and of a high grade as compared to western series [5].
The purpose of this study was to analyze the immunohistochemical markers in invasive carcinoma of breast and to correlate the expression of hormonal receptors with age of the patient, tumor size, histological grade and lymph node metastasis.

Material And Methods Study Design
Eighty eight patients with a diagnoisis of in ltrating ductal carcinoma of breast in a tertiary care hospital of Southern Assam over a period of two year (January2018-December2019) were included in this study. Data including age, tumor size, and histologic grade and lymph node status retrieved from pathology department. We analysed the expression of ER, PR, and HER2/neu by immunhistoochemistry (IHC), with each other and to various clinicopathological parametres. Institutional ethical committee approval was taken Inclusion criteria All patients with histologically con rmed in ltrating ductal carcinoma of the breast were included in the study.

Exclusion criteria
Patients with in ammatory breast lesions, posttraumatic breast lesions, benign breast diseases and patients with breast cancer who received neoadjuvant chemotherapy were excluded from the study.
Para n blocks containing cancer tissue were selected from histopathologically con rmed cases of in ltrating ductal carcinoma. After preparing slides from blocks, immunohistochemical staining was done for ER, PR, and HER2/neu by standard procedure [6].

Preparation of slides
Para n sections were cut and mounted on salinized slides. Slides were melted at 65°C and then dipped into xylene to remove the para n. After rehydrating tissues, slides were washed with distilled water. Then, slides were dipped into a fresh aqueous solution of 3% peroxide for 3 min and rinsed with Tris buffer.

Antigen retrieval and detection of antigens
Heat retrieval was done with citrate buffer in the Decloaking chamber for 40 min at 95°C and then brought to room temperature after removing from the Decloaking chamber and by placing the slides in Tris-Saline buffer. 1% mouse serum was added to the tissue section to block nonspeci c immunostaining. The sections were exposed to the primary antibody for about 1 h, and then primary antibody was washed with Tris buffer.
Secondary detection of the primary antibody Sections were incubated with biotinylated mouse anti-species antibody for 10 min, and then rinsed in Tris buffer. A solution of chromogen, 3, 3'-diaminobenzidine (DAB) at 1 mg/ml in Tris buffer with 0.016% fresh H 2 O 2 was prepared and added to the slides. DAB from the slides was washed with tap water.
Counterstaining A solution of hematoxylin diluted 1:1 with distilled water was made slides were dipped into hematoxylin solution for staining. Then, slides were washed in distilled water and dehydrated by dipping in ethanol.

Washed in xylene and coverslip was applied for viewing and reporting
Reporting Reporting done as per ER/PR scoring system and criteria as per Allred scoring system [7]. 1 + = incomplete membrane staining which is faint and barely perceptible and within > 10% of tumor cells.
2 + = circumferential membrane staining that is incomplete and/or weak/moderate and within > 10% of the invasive tumor cells; or complete and circumferential membrane staining that is intense and within ≤ 10% of the invasive tumor cells.
FISH will be done for equivocal HER2/neu positivity. Hence, HER2/neu 2 + was taken as negative along with HER2/neu 0 and 1+. Only 3 + on IHC was taken as positive.

Statistical analysis
Chi-square was used to determine the statistical signi cance between ER/PR status HER2/neu status along with their correlation with various clinicopathological parametres such as patient's age, tumor size, tumor grade and axillary lymph node status with respect to in ltrating ductal carcinoma breast. A value of P < 0.05 was considered as statistically signi cant.

Receptor status
Fifty tumors were ER-positive and thirty eight were ER-negative. ER-positive tumors showed weak, moderate to strong nuclear positivity in > 1% of tumor cells (Fig. 1).
Forty six tumors were PR positive and forty two were PR negative. PR poisitive cases showed weak, moderate to strong nuclear positivity in > 1% of tumor cells (Fig. 2). Seven tumors that were positive for HER2/neu showed complete and intense staining and within > 10% of tumors cells (Fig. 3).
Out of 88 cases, 46 cases were ER and PR positive, 38 cases were negative for both ER and PR. 4 cases showed different expressions of ER and PR.

Age
Patients were in the age group between 24 and 80 years, with mean age 56.6yrs years. The majority 35.22%, 29.54% were in the age group 51-60and > 60 years respectively. About 82% ER positive and 80.43% PR positive cases were of age group > 50 years whereas 57.14% HER2/neu positive were in age < 40 years. (Table 1). It was statistically concluded that ER, PR, and HER2/ neu expression shows signi cant correlation with age.

Discussion
Female breast cancer is the most common malignancy worldwide, with over two million cases diagnosed in 2018 [9].
Breast cancer is the most common cancer in female, representing approximately 25% of all cancers. It is also ranked number one cancer among Indian females with age adjusted incidence rate of 25.8 per 1, 00,000 women and mortality 12.7 per 1,00,000 women [10]. Treatment of breast cancer includes combined therapy; surgery, radiotherapy, chemotherapy, endocrine therapy, and targeted therapy and so forth.
Hormone therapy can be started before surgery (as neoadjuvant therapy) or used after surgery (as adjuvant therapy) or as a prophylactic treatment of high risk populations as in BRCA mutation carriers. Evaluation of hormone receptor on surgically resected specimen or core biopsy material is essential to assess the utility of hormone therapy and thus the College of American Pathologists and American Society of Clinical Oncology recommend ER and PR testing for all newly diagnosed cases of invasive breast cancer and breast cancer recurrences [11].
Various biomarkers such as hormone receptors, vascular endothelial growth factors, epithermal growth factor, tumor suppressor genes, multidrug resistant genes and adhesion molecules have been identi ed [12]. Currently, determination of estr ogen receptor (ER), progesterone receptor (PR) and human epidermal receptor growth factor 2 (HER2/neu) receptor is routine in the diagnosis of breast cancer [13] with atleast 1% positivity is necessary for commencement for hormone therapy.
ER is a biomarker found in over 56.82% of in ltrating breast cancer in this study and contributes signi cantly to its pathobiology. ER positivity makes it responsive to hormonal therapy, resulting in a more favourable outcome. PR, like ER, is also a transcription factor, which is largely controlled by ER and to a lesser extent by growth factors. About 43.8% of in ltrating breast cancers show PR positivity. PR commonly coexists with ER. Studies from other region have documented lower positivity for ER and PR receptors. Desai et al [14] from India have reported 32.6% and 46.1% positivity for ER and PR respectively.
Another study by Suvarchala et al [15]  HER2/neu has the potential of enhancing proliferation and survival of tumor cells. In this study its overexpression occurs in about 7.95% of in ltrating breast cancer, results in a more aggressive growth and poor response to treatment. In this study the sample showing equivocal HER2/neu expression will be evaluated by uorescence in situ hybridization. Unlike our study, Ranvijay et al [19] and Rashmi et al [20] reported 34.2% and 69.2% HER2/neu expression.
The mean age of breast cancer is 56.6yrs in our study which is much lower than the mean age of 62 year reported in UK [21] whereas in the US [22], peak is observed at the age 75 years. Our study corroborates with the study done by Elsayed et al [23] at Egypt where the mean age is 50.4 years. In India the incidence rates begin to rise in the early thirties and peak at ages 50-64 years. Though the reason entirely is still not clear but a major factor could be ignorance, lack of awareness and under reporting amongst the elderly population in India. Majority of ER and PR positive cases were of age > 60 years and HER2/neu positive were of age < 40 years, as seen in the study conducted by Alzaman et al [18]. A signi cant correlation was observed between age of the patient and ER (0.000), PR (0.000) and Her2/neu (0.012) expression as shown in studies by Dodiya et al [24].  [29 ].
Metastasis in axillary lymph nodes was seen in 37.5% of patients. About 54.55% ER 57.58% PR and %HER2/neu positive cases had positive axillary lymph nodes positive for metastasis. In our study, we found that ER/PR expression had no signi cant correlation with lymph node metastasis. Also HER2/neu overexpression showed no signi cant association with lymph node metastasis and this result is in agreement with Almasri et al [30]. Unlike our study, Siadati et al 2015, showed signi cant association between HER2/neu overexpression and lymph node status [29].

Conclusion
Invasive ductal carcinoma of the breast cancer was seen in the age of 24 and 80 yrs, with a mean age of 56.6 years. The maximum number of cases were seen in the age above 50 years (64.77%). Majority of the tumors that were ER and PR positive were of Grade II, whereas majority of HER2/neu positive tumors were of Grade III. This study showed that ER and PR are correlated with age, tumor size and tumor grade but not with lymph node status. HER2/neu expression are correlated with age only but not with tumor size, tumor grade and lymph node status. Therefore it is strongly recommended to assess the hormone receptors for clinical management of a breast cancer patient to provide prognostic information and therapeutic measurement.

Declarations
Compliance with Ethical Standards: This work is approved by institutional ethical committee.
Con ict of interest: Immunohistochemical staining showing positive for HER2/neu