Introduction

Breast cancer (BC) is the most common cancer in women worldwide and the leading cause of cancer related mortality in women. (1) In India, it is second most common malignancy after cancer cervix among females. (2) BC is considered as a heterogeneous disease sharing similar histopathologic features but with distinctive clinical presentation, biologic features, behaviour, outcome and response to treatment, (3) which is attributed to molecular diversity among histologically similar tumours. (4) Hence, despite the progress in BC therapy, there is no incisive therapy for BC treatment and this highlights the importance to develop new therapeutic strategies, alternative to the currently used drugs, such as Tamoxifen or Trastuzumab or Lapatinib, which are only useful when the target proteins (Estrogen receptor or Her2) are expressed. It is in this scenario, the androgen receptor (AR) is emerging as a new marker and a potential new therapeutic target in the treatment of BC. (5,6)

Androgen receptor belongs to the steroid hormone nuclear receptor family similar to estrogen receptor (ER) and progesterone receptor (PR). The role of AR in development of prostate and progression of prostate cancer is well documented. However, its role in breast cancer is unclear (1,2) and the precise understanding of its action remains a challenging puzzle. (5) The role of AR also depends on the tumor microenvironment as well as the relative levels of circulating estrogens and androgens. (5) It has been hypothesized that androgen may influence breast cancer risk indirecty through their conversion to estradiol or by competing for steroid binding proteins, or directly by binding to the AR. (1,2) Thus, AR is thought to play a central role in its initiation, progression of breast cancer, and its response to therapy. (1)

Gene expression profiling characterized four major groups of BC, which classified patients into Luminal A, Luminal B, HER-2/neu enriched, and Triple Negative BC (TNBC); based on immunohistochemical staining for Estrogen Receptor (ER), Progesterone Receptor (PR), HER-2/neu and Ki-67 staining. (3,7) This helped stratification of BC patients for prognostic and therapeutic purposes. AR expression in relation to different molecular sub-types of BC is not clearly understood. (3) Currently, investigators suggest that AR positive tumours have favourable characteristics and that tumours expressing both AR and ER are associated with better outcome. (4) In studies by Hu et al. and Agoff et al., AR expression and patient's survival depend on the status of ER. (8,9) Hence the coexpression of these receptors may be needed to assess better prediction of patient’s survival. (1)

Whatever be the mechanism, AR stimulates or inhibits cellular proliferation and promotes metastatization or resistance to therapies in ER-positive BC cells. In ER-negative BC cells, it clearly promotes cell proliferation and spreading by acting at different levels. This scenario makes possible the use of AR modulators or blockers in BCs. (5) In ER-positive BCs and in a subset of ER-negative BCs in which AR activation inhibits tumor growth, natural and synthetic steroidal androgens have been used for therapeutic purpose. But these are known to induce many side effects. Thus the use of selective AR modulators (SARMs, i.e., enobosarm GTx-024) which have less side effects with favorable results has been considered for the therapy of ER-positive advanced BCs patients. This is still investigated in a phase II clinical trials in patients with ER positive BC. (5)

The prognostic and predictive value of AR in Triple negative breast cancer (TNBC) remains a challenging topic of research. There is a subtype of TNBC which positively express AR termed as luminal androgen receptor (LAR) subtype. The AR antagonist (enzalutamide) implicated in prostate cancer treatment, has shown promising results in some patients with advanced TNBC whose tumours were AR-positive. (4)

There is limited literature from India, on role of AR in breast cancer. (1) Hence this study was undertaken to evaluate the AR expression in primary carcinoma breast and to find its association with clinic-pathological features, ER,PR, HER2 and Ki67 status and its molecular subtypes.

Materials and Methods

Patient variables

A retrospective study was conducted in the department of Pathology, during the year 2017-2022, after approval from Institutional Ethics Committee. The study included 64 cases of mastectomy specimens of primary breast carcinoma in female patients with known hormone receptor status (ER, PR, Her2) and Ki 67 proliferation index. Patients with inadequate clinical data or cases with unavailable slides and blocks were excluded from the study. Patient demographic details such as age, laterality and histopathological parameters such as histological type, tumor size, lymphovascular emboli (LVI), grade of the tumor (Modified Bloom‑Richardson grade), lymph node involvement, metastasis, and hormone receptor status (ER, PR, and Her‑2 receptor), Ki 67 index were retrieved from the hospital laboratory information system and medical records department. Molecular classification of the tumor was done based on hormone receptor status into Luminal A, Luminal B, Her 2 enriched and basal type.

Immunohistochemistry

Immunohistochemistry (IHC) staining for AR was performed on 4–5 μm thickness tissue sections. The slides were incubated overnight at 60°C. Antigen retrieval was performed in citrate buffer using pressure cooker method. The slides were incubated with primary rabbit monoclonal antibody (clone EP120, Pathnsitu) at room temperature for 30 min. The slides were then incubated with secondary antibody and using diaminobenzidine as chromogen, immunoreactivity was detected. The slides were counterstained with Harris’s hematoxylin. Prostate tissue was used as AR‑positive controls. A negative control with exclusion of primary antibody was done done. Tumors with ≥10% nuclear staining of neoplastic cells were considered as positive. (1) Intensity was scored as 0 for no staining, 1 for weak, 2 for moderate, and 3 for strong intensity staining.

For ER and PR, tumor cells with at least 1% stained cells were considered as positive. Her‑2 status was interpreted according to the American Society of Clinical Oncology/College of American Pathologists guideline recommendations. (9) Both the score of 1+ and 2+ were considered as negative.

Ki67 proliferative index assessment was expressed as the percentage of ki 67- positive cells within the total number of malignant cells among five high-power fields (×400). (10) A percentage of 14% or more was considered to be a cutoff point. (3)

Molecular subtyping of breast carcinoma was done using a combination of four IHC markers (ER, PR, HER-2/neu, and Ki-67) according to St. Gallen international expert Consensus, 2013. (7)

Statistical analysis

The collected data were coded, tabulated, and statistically analyzed using statistical Package for Social Sciences version 26.0 software. Descriptive statistics were done for quantitative data; The Chi-square test was used to assess the association between clinicopathological variables and AR positivity. A value of P < 0.05 was considered as statistically significant.

Results

AR expression was noted in 67.2% (43/64) of tumors in this study population. The relationship between various clinicopathological parameters and biomarkers with AR expression is depicted in Table 1.

The patients ranged in age from 31 to 88 years, (mean=56.4years) and 21 cases (32.8%) out of 64 patients were younger than 50 years. 43 (67.2%) patients were above the age of 50 years. AR expression (71.4%) was noted in patients aged below 50 years and 65.1% in age above 50 years. No statistically significant association was noted between age and AR expression.

Thirty four (53.1%) cases has lump in the right breast and 30 (46.9%) in left breast. Laterality did not have any association with AR expression.

Majority 59/64 (92.2%) tumors were infiltrating duct carcinoma (IDC), four cases (6.2%) being mucinous carcinoma and one case (1.6%%) of invasive lobular carcinoma (ILC). 64.4% of IDC showed AR expression. All the mucinous carcinoma in this study showed AR expression. But no significant statistical association was noted with histological type of tumor. AR expression of various intensities in carcinoma breast is depicted in (Fig 1).

Fig 1: AR expression of various staining intensities in carcinoma breast. A-Strong intensity (Score 3), B- Moderate intensity (Score 2), C- Weak intensity (Score 1).

Out of 64 studied cases, 14 (21.9%) were grade I, 35 (54.7%) grade II, and 15 (23.4%) cases were grade III tumors. AR was positive in 12 (85.7%) of grade I, 23 (65.7%) of grade II, and 8 (53.3%) of grade III tumors. A higher AR positivity was noted in grade 1 and 2 tumors but was not statistically significant. No association between tumor grade and AR expression was identified

Among the histopathologic parameters evaluated, tumor size, LVI, lymph nodal status and metastasis did not show any association with AR expression.

Expression of ER, PR and Her‑2 receptors was noted in 65.6% (n = 42), 51.6% (n = 33), and 32.8% (n = 21), respectively. Ki 67 expression was <14% in 20.3% (n=13) and >14% in 79.7% (n= 51) cases. AR was expressed in 81% of ER‑positive tumors and 40.9% in ER negative tumor. AR expression was noted in 81.8% of PR positive tumors 51.6% of PR negative tumor. AR expression showed a significant association with ER (P = 0.001) and PR status of the tumors (P = 0.010). In Her‑2 positive tumors, AR expression was seen in 76.2% of the tumors. In spite of higher percentage of AR positivity in Her‑2 positive tumors, the P value was not statistically significant (p=0.329).

Among luminal A, luminal B, Her‑2 overexpression, and triple‑negative cancers, the rates of AR expression were as follows: 66.7%, 83.3%, 30.8%, and 55.6%, respectively. There was significant association between AR positive expression with Luminal B tumors (P = 0.002). AR expression was noted in 30.8% of triple negative tumors and was considered statistically significant (P = 0.003).

Discussion

Over the past two decades, hormone receptors (ER/PR) and Her‑2 growth factor receptors biomarkers have gained importance due to implications in prognosis and clinical management. (1) Several studies have examined the correlation of ER and PR with other prognostic indicators, but little is known about the role of AR and its prognostic value in breast carcinoma. (2)

Current concept is that different types of luminal cells in the normal breast epithelium might serve as a precursor for different subtypes of breast cancer. AR expression is found in luminal and metaplastic apocrine cells. Even through AR is expressed in the luminal cells, they are not biologically active. (1) Wang reported that there is an absence of downstream regulatory proteins (prostate‑specific antigen, gross cystic disease fluid protein) in the normal breast epithelium by IHC. Thus, further oncogenic events are required for the initiation and progression of breast cancer in these AR‑positive luminal cells. (11)

AR is highly expressed in breast cancer. The positive rates of expression of AR vary mostly from 60% to 80% in the literature. (1) Our study showed AR expression rate of 67.2% which was almost similar as compared to the Western literature and also study done by Ismael et al. (4) This varied AR expression rates may be due to the different methodology used and the geographical distribution of the population studied. (1)

The relationship between the AR expression status and the various clinicopathological parameters showed inconsistent results in literature published. The grade of the tumor is the most consistent parameter which correlated with AR status. Park et al. reported that, patients with smaller tumor size and lower histological grade had a higher AR expression, but not with age, menopausal status, body mass index, nodal involvement, preoperative CEA levels, and stage. (12) Agrawal et al. found a relation with only tumor grade. (13) On the other hand, Gonzalez et al. (14) and Samaka et al., (3) found no significant correlation between AR expression and the size of the tumour. Similar to study done by Ismael et al,(4) this study also found no between AR status and patients age, laterality, tumour size, histologic type, grade, lymphovascular invasion, T stage, N stage, Her-2/neu status and ki 67 status.

The frequency of AR expression is generally comparable with or higher than that of ER/PR expression. (1) In this study, the expression rates AR showed a significant association with ER (P = 0.001) and PR status of the tumors (P = 0.010). A significant correlation between AR expression and hormonal status was also found by Park et al.,(12) Qi et al., (15) Safarpour et al., (16) Vera-Badillo et al., (17) and Chottanapund et al.. (18) AR was expressed in 81% of ER positive tumors and 81.8% of PR positive tumors. In ER negative patients, AR is present in 40.9% of cases. Hence ER negative cases not responding to treatment are treated with medroxyprogesterone acetate which mediates its action by binding to AR. (2)

In our study, in spite of higher percentage of AR positivity in Her‑2 positive tumors, the P value was not statistically significant (p=0.329). Park et al., (12) and Qi et al., (15) found no significant relation between AR expression and Her-2/neu expression status, although Agrawal et al., (13), Chottanapund et al., (18) and Samaka et al., (3) found that AR expression is more in tumours expressing Her-2/neu. This could be due to different primary antibodies used.

Our study showed no significant relation between AR expression and Ki 67 expression similar to study done by Vera- Badillo et al., (17) in contrast to Qi et al., (15) and Samaka et al. (3) this might be due different cut-offs used.

ER, PR, and Her‑2 have been considered as immunohistochemical surrogate markers for molecular subtypes of cancer.(1) Regarding the correlation between AR expression and the molecular subtypes, AR expression was seen significantly higher in luminal breast carcinoma cases. This is by Collins et al., (19) Qi et al., (15) and Samaka et al. (3)

In triple‑negative tumors, the rates of AR expression varied from 6.6% to 75%. (20) In this study, AR expression in triple negative patients was 30.8% and it showed significant association with AR expression (p< .003) which was similar to study done by Ismael et al. (4) These subsets of patients are possible candidates for the promising anti-androgen target therapy. (4) Studies state that androgen can inhibit the growth of hormone negative breast cancer if there is strong expression of AR, because of conversion of androgen to estrogen by the aromatase enzyme. These androgens can also induce apoptosis regardless of ER and PR status. Thus, AR antagonist may be used in AR positive tumours regardless of their ER status. (2)

The strength of this study is that it has been done in a population of patients in whom the clinicopathological data regarding the role of AR expression is sparse. The limitations of this study are small sample size and that AR expression could not be correlated with overall survival and disease‑free survival as patients could not be followed up.

Conclusion

We conclude that breast cancer has significant AR expression which is significantly associated with ER and PR but not with HER2/neu status. AR is also significantly expressed in Luminal B subtype and triple negative cases. Also, a subset of TNBC cases showed significant positive AR expression. Further researche on AR expression in breast cancer are recommended on a larger scale with follow up and survival to validate the current results and to determine whether AR could be a therapeutic target in triple negative and other molecular subtype of breast cancer patients.

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