Breast cancer and the related deaths, as seen by the global and national figures have increased rapidly over the past decade. The GLOBOCAN 2020 statistics have shown that, worldwide, breast cancer accounted for 24.6% of all cancers and around 16% of all deaths among females. In India, from the year 1965 to 1985 there was an almost 50% rise in breast cancer cases. Recent statistics show that breast cancer accounted for 13.5% of all cancers and 10.6% of all deaths with a cumulative risk of 2.81 in India 1 . Studies have also shown that the incidence of breast cancer is rising among younger women. Although the breast cancer figures among Indian women is lower as compared to the West, epidemiological studies have estimated that breast cancer cases will reach almost 2 million by 2030. 23 As breast cancer cases continue to rise, there has also been significant progress in the diagnosis of breast cancer malignancies, with significant advances in molecular genomics and onco-pathological studies for earlier detection of disease and subsequent development of more effective targeted therapy for patient management. This may contribute to the reduction in the mortality and morbidity rates of breast cancer in the developed countries.

Breast cancer exhibits significant heterogeneity in terms of histopathological features, metastatic patterns, molecular features, outcome and response to therapy. This has an impact on the clinical outcome of the breast cancer patients as well. Prognostic variations occur with respect to tumor size, histologic grade, histologic type, and biological markers such as estrogen receptor (ER), progesterone receptor (PR), and Her-2-2/neu expression profile 4 .

Presently IHC is accepted as an adequate surrogate marker for molecular subtypes. This surrogate IHC method aids the determination of molecular subtypes 5 . Immunohistochemistry markers like estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2(Her-2) proteins give valuable information and help in patient management. 6 Knowing the hormone receptor status of these tumors helps in predicting the patient response to the treatment. If the tumor is hormone receptor positive, i.e. ER PR positive, it is more likely that the patient will respond to endocrine therapy. The hormone therapy is unlikely to be effective if the tumor is hormone receptor negative. Hormone receptor expression is thus a weak prognostic but a strong predictive biomarker in breast cancer cases. All invasive breast cancers should be tested for the hormone receptor status as well as Her-2/neu. 7 Combined expression of these three hormone receptors is important in the further molecular classification of breast cancer cases for the clinical assessment and deciding on further treatment. Molecular classification of breast tumors is the grouping of cases that share characteristics and hormone receptor status, which can guide the response of tumors to various lines of hormonal therapies. 8 Triple-negative cases of breast cancer are known to grow and spread faster and are less responsive to hormonal or targeted therapy. Apart from this, with latest advances in the field of molecular diagnostics and therapeutics, newer noninvasive prognostic biomarker tests aid the detection of breast cancer cases in the early stages itself. Molecular subtyping using immunohistochemistry can provide additional prognostic and predictive information.

In India, the last decade has seen an exponential rise in the incidence of breast cancer cases, with the age of onset also markedly reducing. Utilizing genomics to understand India-specific differences with respect to breast cancer cases may enable the identification of women at high risk of developing cancer, where targeted screening may be costeffective. There is an urgent need to identify Indianspecific genetic/epigenetic biomarkers. These may have the potential to be used as biomarkers for early detection at the screening stage. 9 The current study aims to analyze the hormone receptor positivity status and the subsequent morphological classification in the Indian population.

-To study the immunohistochemical markers across breast cancer patients in the Indian population over the period of seven years from January 2015 to June 2022. -To understand the association of molecular subtypes of breast cancers with respect to demographics and tumor grade.

A retrospective study was conducted at Global Reference Laboratory, Mumbai, Metropolis Healthcare Ltd. In this study, 12808 malignant breast tumor samples received in seven years [2015 to 2022] were studied. The type and grade of the tumor were assessed as per WHO 5 th edition classification of breast tumors. 10

IHC was considered positive if >1% of tumor cell nuclei were immunoreactive to respective hormone receptor. College of American Pathologists (CAP) guidelines were used for ER, PR, Her-2/ Neu and Ki-67 assessment. 11 As per CAP guidelines, the wet tissue was fixed in 10% neutral buffered formalin for 6 to 72 hours and processed overnight in an automated tissue processor. In many cases, paraffin blocks were received. Four-micron thick sections were cut and stained with hematoxylin and eosin. ER/PR scoring was done as per the Allred scoring system. 11

The results were scored from 0 to 3+ according to the criteria of the HercepTest™. HercepTest™ mAb pharmDx (Dako Omnis) is a semi-quantitative immunohistochemical assay based on a primary monoclonal rabbit antibody (clone DG44) and an assay-specific visualization reagent. The assay determines HER2 protein overexpression in formalinfixed, paraffin-embedded (FFPE) breast cancer tissues processed for histological evaluation. HercepTest™ mAb pharmDx (Dako Omnis) is indicated as an aid in the assessment of breast cancer patients for whom Herceptin® (trastuzumab) treatment is being considered. 13 Her-2 by FISH For 509 cases where IHC was done, Fluorescence in situ hybridization (FISH) was also performed using HER-29 PathVysion® HER-2 DNA Probe Kit II, a dual probe assay. The PathVysion HER-2 DNA Probe Kit II (PathVysion Kit II) has been designed to detect amplification of the HER-2/neu gene via fluorescence in situ hybridization (FISH) in formalinfixed, paraffin-embedded human breast and gastric cancer tissue specimens. 14 FDA approved automated result scanning is available for PathVysion. 12,16 HER-2 scoring was done as per ASCO/CAP guidelines.

Details of tumor type, grade, ER, PR, Her-2 and Ki-67 were used for further molecular classification. 17 Further comparisons between age, gender, histological type, and grade were made.

The data were analyzed using "R Studio version 1.4.1103". Descriptive analyses were done to obtain the frequency and percentage of Morphological classification, Hormone Receptor status, Her-2 expression, Molecular Subtype in this given population, in addition to the characteristics of the sample Age, Gender and Scarff Bloom Richardson (SBR) grading. Comparison of Molecular Subtype with Age, Gender, Scarff Bloom Richardson (SBR) grading and Morphological classification was done by the Chi-square test. Concordance between the results of Her-2 by IHC & Her-2 by FISH was calculated in cases in which both tests had been performed. A P-value of less than 0.05 was considered to be statistically significant.

The formula below was used to calculate the concordance rate:

CONR=C/SA*100, where CONR was the concordance rate, "C" was the number of subjects with concordant results, and "SA" was the total number of subjects assessed for concordance.

In this study, 12808 malignant breast cancer cases were analyzed over a period of seven years; the mean age of patients seen was 53.63+13.08 years, with the minimum age of 18 years and the maximum age of 92 years (Table 1).

Morphologically, invasive duct carcinoma of NST, (12018, 93.83%) was the most prevalent subtype followed by Invasive lobular carcinoma (380, 2.97%). There were also some extremely rare subtypes observed, such as apocrine carcinoma (5, 0.04%), adenoid cystic carcinoma (2, 0.02%) and neuroendocrine carcinoma (1, 0.01%) ( Table 2). The relationship of ER PR positivity with respect to grade showed that ER PR positivity was higher in grade 1 and 2 as compared to grade 3 cases (Table 4).

Her-2 by FISH was performed in 4617 cases. Out of this, Her-2 by FISH was positive in 1631 cases (35.33%) while 39 cases (0.84%) showed equivocal results.

In addition, 509 cases were tested for both Her-2 by IHC and Her-2 by FISH and the concordance was established. Her-2 by IHC and Her-2 by FISH showed a concordance rate of 97.53% (Table 5). On comparison of Ki-67proliferation index with respect to grade, Ki-67 proliferation index was seen to be lower in grade 1 breast cancer cases as compared to the grade 2 and 3 cases (Table 7). The further classification of molecular subtypes is as follows: There was a significant association seen between molecular subtype with respect to age, gender, SBR grade, and histological type (P<0.0001) ( Table 8).

Breast cancer burden is rapidly increasing worldwide as well as in India. 13 As observed in a study by Upadhyay et al. and in some previous studies as well, the mean age of breast cancer patients in the Indian population is seen to be almost a decade younger as compared to the western counterparts. 14 However, carcinomas related to the BRCA1 and BRCA2 gene mutation may occur at younger ages as well. 15 Majority of the patients in this study had grade 3 carcinomas (45.43%), followed by grade 2 and grade 1 cases (Figure 1). Maximum cases seen in our study were invasive duct carcinoma, comprising around 93.83% cases ( Table 2). As breast carcinoma is a heterogeneous tumor with multifaceted features, its classification into subtypes is important. As stated in the study by Britta et al., clinicopathological classification of breast carcinoma is crucial from the diagnostic, theranostic and prognostic perspective. 16 The statistical significance seen in the present study further reinforces this. Tumor differentiation is highly important in the management of breast carcinomas. Hormonal estrogen receptors are major markers of tumor differentiations. 17 The treatment of breast cancer includes a multi-disciplinary approach with surgery as the mainstay. The other important adjuvant types of treatment are hormone receptor status based endocrine therapy and anti-Her-2 drugs based on the Her-2 expression. 18 In the present study, overall we saw an ER and PR receptor positivity of 58.44% and 47.06%, respectively, with maximum hormone receptor positivity seen in the grade 1 and 2 cases as compared to grade 3 cases. The overall Her-2 positivity seen was 18.67% (Table 3 and 4). In a study done in the African population by Dimitri et al. in the Republic of Congo among 150 patients, maximum cases belonged to grade 2 tumors, with an overall hormone positivity seen of 68%. 19 However, an Indian study done in Kerala showed an ER positivity of 52%. 7 Ambroise et al. reported that the hormone expression seen in the Indian studies is much lower than that in the western counterparts, which again could be due to the late stage diagnosis or the grade 3 cases seen among Indians. 13 Among those patients tested for Ki67 proliferation index, 44.44% showed a Ki67 percentage <20% (Table 3). This is of value prognostically as breast cancers with high Ki67 index are known to have poor prognosis and survival rates as well. Even in the current study, most of the grade 3 tumor cases showed a high Ki67 proliferation index >20% (Figure 4 Figure 5). These results were somewhat similar to those seen in the study by Dimitri et al. 19 In our study, maximum cases of luminal A were seen. In 2021, Jonnada et al. performed a meta-analysis on breast cancer studies among the Indian population which also showed similar results. 20 A 2015 study by Kumar et al. showed Luminal A subtype as the most prevalent (34%), followed by Basal like/Triple negative subtype (25%). Luminal B and Her-2/neu subtypes had a lower prevalence, i.e. 18% each. This study was, however, done on a small data set of 50 patients with maximum (54%) belonging to grade 2. 21 Luminal A is known to have a good response to hormonal therapy, whereas non-Luminal A cancers are at greater risk of recurrence. Luminal B cases have a variable response to hormonal therapy but a good response to chemotherapy. 2223 Majority of the invasive duct carcinoma cases in our study were triple negative cases (37.88%) ( Table 8). Triple negative tumors show aggressive clinical behavior, indicate a high histological grade, often present with advanced disease, and may show early metastases. These have been found to be less responsive to treatment and are known to have the worst prognosis. 20 The probable factors for high rate of triple negative cases in the Indian population could be the mean age of cancer origin, family history, lifestyle factors like obesity, reproductive status, multiparity, socio-economic status and cancer screening. 24 The correlation of molecular subtypes with age, histological type and SBR grade performed in our study showed that there was a significant association between molecular subtype and each of these parameters. The percentage of Luminal A cases was seen to increase with age, whereas a contradictory finding was seen in the triple negative cases (Table 8). Thus, based on the results of our study, the triple negative cases were more predominant in the younger age group <40 years, while the luminal A cases were more frequent in the older age group. This finding was in contradiction to the finding by Ambroise et al. where maximum triple negative cases were grade 3 and were seen in the older age group (>50 years). However, some other worldwide studies have also shown that triple negative cases are more common in younger women< 40 years. 13252627 Some factors that may contribute to this observation could be racial predisposition, family history, drug history like oral contraceptive pills and the presence of BRCA genes. Further research may be required to understand the increased prevalence of triple negative cases in the Indian scenario as well. In our study, maximum percentage of triple negative cases were grade 3. This further reinstates that triple negative tumors are more aggressive clinically and poor prognostically. There is continuous ongoing research in the field of targeted therapy and precision medicine to overcome this hurdle. Newer technologies and platforms like next generation sequencing and microarrays have shown that the tumor response to treatment is hugely dependent on the intrinsic molecular makeup of these tumors and less on the anatomic structure. Hence, newer assays and comprehensive panels may help identify these otherwise overlooked and biologically aggressive tumors with the long term aim of guiding therapeutic strategies to deliver optimum patient outcome. 28

It was observed that grade 3 tumor cases were most common and most of the cases of triple negative tumors were seen among women, mostly concentrated in those <40 years. Her-2 gene status and Ki-67 proliferation index are major biomarkers that predict the tumor behavior and, thus, are important clinically for tumor responsiveness to various lines of therapies. We can conclude that molecular sub-classification of tumors is primarily guided by immunohistochemistry, and it can be recommended that the use of molecular analysis and gene expression studies can further augment the histopathological diagnosis to assist strategies of targeted therapy and precision medicine resulting in better patient outcomes.

All patients signed an informed consent for the unanimous presentation of their data in a medical journal. The local ethical committee has approved the protocol of this retrospective study.