Prognostic Role of Androgen Receptor Expression in HER2+ Breast Carcinoma Subtypes

HER2+ breast cancer (BC) is an aggressive subtype representing a genetically and biologically heterogeneous group of tumors resulting in variable prognosis and treatment response to HER2-targeted therapies according to estrogen (ER) and progesterone receptor (PR) expression. The relationship with androgen receptors (AR), a member of the steroid hormone’s family, is unwell known in BC. The present study aims to evaluate the prognostic impact of AR expression in HER2+ BC subtypes. A total of 695 BCs were selected and reviewed, AR, ER, PR and HER2 expression in tumor cells were examined by immunohistochemical method, and the SISH method was used in case of HER2 with equivocal immunohistochemical score (2+). A high prevalence of AR expression (91.5%) in BC HER+ was observed, with minimal differences between luminal and non-luminal tumor. According to steroid receptor expression, tumors were classified in four subgroups, including BC luminal and non-luminal HER2+ expressing or not AR. The luminal BC HER2 + AR+ was associated with lower histological grade, lower tumor size, higher PR expression and lower HER2 intensity of expression (2+). Also, the non-luminal tumors AR+ showed lower tumor size and lower prognostic stage but frequently higher grade and higher HER2 intensity of expression (3+). These findings should suggest a different progression of luminal and non-luminal tumors, both expressing AR, and allow us to speculate that the molecular mechanisms of AR, involved in the biology of BC HER2 + AR+, differ in relation to ER and PR expression. Moreover, AR expression may be a useful predictor of prognosis for overall survival (OS) in HER2+ BC subtypes. Our findings suggest that AR expression evaluation in clinical practice could be utilized in clinical oncology to establish different aggressiveness in BC HER2+ subtypes.


Introduction
Breast cancer (BC) is a heterogeneous disease enclosing several entities with different morphologic, prognostic and therapeutic features [1]. Invasive breast cancer is classified tissue sections of FFPE specimens were cut for hematoxylin and eosin staining (H&E), immunohistochemistry, and SISH analysis.
The study protocol was approved by the local research ethics committee (File number PG/2021/14264); and followed the Italian law on guidelines for the implementation of retrospective observational studies (G.U. n. 76, 31 March 2008). Only coded data were collected in order to protect patient confidentiality.

Immunohistochemistry
The immunohistochemistry analysis was performed using specific antibodies against ER, Clone SP1 (Ventana Medical Systems Inc., Tucson, AZ, USA); PR Clone 1E2 (Ventana Medical Systems); Ki67, Clone 30-9 (Roche Diagnostics K.K., Tokyo, Japan); AR Clone SP107 (Cell-MarqueTM, Rocklin, CA, USA); HER2 PATHWAY Clone 4B5 (Ventana Medical Systems). Immunostaining was performed using the Ventana Benchmark XT staining system with Optiview DAB detection kit. In cases of HER2 with equivocal immunohistochemical score (2+), we performed HER2 gene amplification by ultra-View SISH Detection Kit (Ventana Medical Systems). Evaluation of immunostaining and SISH for HER2 was based on American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations [6]. ER, PR and AR expression was considered positive if at least 10% immunostained tumor nuclei were detected in the sample [17]. Ki67 was scored low if <14% of tumor nuclei were positive and high if ≥14% of tumor nuclei were positive [35].

Baseline Data
Demographic and clinico-pathological information were extracted from medical records, as well as age at diagnosis, histologic type, histologic grade, tumor site, and TNM classification. Histologic tumor type was established according to the UICC-WHO criteria [5]. Histologic tumor grade was determined in accordance with the Nottingham guideline [36]. TNM staging was described in accordance with the 8th edition of the American Joint Committee on Cancer criteria (AJCC) [37]. Lymph node ratio was described as the ratio between the positive lymph nodes number and the evaluated lymph nodes number. The cut-off points of lymph node ratio were: <0.21, 0.21-0.65, and >0.65 [38]. The time between the date of BC diagnosis and the date of death define the OS.

Statistical Analysis
Quantitative variables were described by median and interquartile range (IQR) according to non-normal distribution of variables, whereas absolute and relative (percentages) frequencies were used for qualitative variables. Statistical differences for qualitative variables were evaluated using Chi2 or Fisher's exact tests, when appropriate. Logistic regression analysis was performed to evaluate the association between mortality, IHC BC subtypes, clinico-pathological features, and molecular variables. A Kaplan-Meier curve and Log-Rank test were performed to describe OS according to IHC BC subtypes, clinicopathological and molecular variables. The statistical significance was set up at p < 0.05. Statistical analysis was carried out using STATA ® 16 (StataCorp, College Station, TX, USA).
AR positivity was detected in 91.5% of all tumors, specifically in 95.2% of luminal and in 84.2% of non-luminal subtypes.

Discussion
BC HER2+ is an aggressive subtype including heterogeneous tumors with variable prognosis and treatment response to HER2-targeted therapies. On this matter, Staaf et al. identified three genetic BC HER2+ subtypes showing distinct clinical outcomes according to molecular profiling analysis [39]. The same group of researchers strengthened the molecular and biological complexity of BC HER2+ by showing the presence of high-level amplifications at multiple sites that also involved the HER2-amplicon at 17q12-q21 [39].
AR is a steroid hormone receptor frequently expressed in BC, including the ER-negative subtypes for which it could represent a complementary target for therapy, though the clinical significance and functional role of AR has not been outlined in BC yet [40,41]. The AR signaling pathway differs according to molecular breast cancer subtypes. It is known that in non-luminal HER2+ AR+ BCs, AR starts the WNT/β-catenin activation,

Discussion
BC HER2+ is an aggressive subtype including heterogeneous tumors with variable prognosis and treatment response to HER2-targeted therapies. On this matter, Staaf et al. identified three genetic BC HER2+ subtypes showing distinct clinical outcomes according to molecular profiling analysis [39]. The same group of researchers strengthened the molecular and biological complexity of BC HER2+ by showing the presence of high-level amplifications at multiple sites that also involved the HER2-amplicon at 17q12-q21 [39].
AR is a steroid hormone receptor frequently expressed in BC, including the ERnegative subtypes for which it could represent a complementary target for therapy, though the clinical significance and functional role of AR has not been outlined in BC yet [40,41]. The AR signaling pathway differs according to molecular breast cancer subtypes. It is known that in non-luminal HER2+ AR+ BCs, AR starts the WNT/β-catenin activation, stimulating HER3 gene transcription, subsequently the heterodimers between HER3 and HER2 support cell proliferation [42]. Nevertheless, the AR signaling pathway in luminal HER2+ AR+ BCs is unclear, and the differences between AR signaling pathways in HER2+ BCs subtypes are not fully understood.
AR expression is higher than ER and PR in BC [16,43]. We found a high prevalence (91.5%) of AR expression in BC HER2+, with minimal differences between luminal and non-luminal tumors; the absence or low level of AR expression being detected in a minority of tumors. These results are consistent with previous reports, which did not show significant differences between HER2-positive/ER-positive and HER2-positive/ER-negative tumors [29,34,[44][45][46]. The higher percentage of AR expression in our cohort could depend on the definition of the AR expression, or on methods such as the use of the complete core section, population, and cut-off value.
Furthermore, our results showed that AR+ tumors are associated with older age at diagnosis and favorable clinical and pathological features, as well as lower histologic grade (G2), pT1 and prognostic stage (I), suggesting a prognostic value of AR in BC HER2+ subtypes. These findings are supported by previous studies showing the association of AR expression with lower grade, smaller size, more frequently tubule formation, and less pleomorphism and mitotic counts, although they evaluated ER-negative and ER-positive subgroups [17,47]. Besides, AR positivity was associated with high ER and PR expression in our cohort. Recently, Cruz-Tapias et al. evaluated the association of AR gene expression in accordance with intrinsic BC subtypes by meta-analysis of extensive microarray transcriptomic datasets. AR overexpression was prevalently observed in patients affected by less aggressive intrinsic molecular subtypes expressing either ER or PR and having a lower histological grade, such as Luminal A and B compared to Basal-like subtype. High AR mRNA levels can be defined as a prognostic biomarker for the detection of the less aggressive BC subtypes [48]. In the current study, the distinction of BC HER2+ in luminal and non-luminal showed that the lobular histotype is prevalent in luminal AR+ and AR− tumors compared to non-luminal subtypes. The apocrine phenotype can be found in non-luminal HER2 + AR+ tumors, whereas AR negativity increases in the mucinous phenotype in the non-luminal subtype. These data consent to distinguish a different phenotype between luminal and non-luminal BC HER2 + AR+ subtypes, adding more detail to Park's results [17].
Moreover, the luminal BC HER2+ AR+ was associated with lower histological grade (G2) and lower tumor size, higher PR expression, and a lower HER2 intensity of expression (2+). Also, non-luminal tumors AR+ showed smaller size and prognostic stage (I) but higher grade (G3) and higher HER2 intensity of expression (3+). These results agree with the observations of other studies that have detected HER2 overexpression in G3-AR+ carcinomas [16,29,49]. These findings could suggest a different progression of luminal and non-luminal tumors both expressing AR, and how AR−related molecular mechanisms of BC HER2+ AR+ could differ depending on ER and PR expression.
Traditionally, the BC HER2+ subtype is distinguished in luminal HER2+ and nonluminal HER2+. In our study, AR positivity was detected in 91.5% of all tumors analyzed. Moreover, AR is expressed in 84.2% of BC HER2+, in the absence of other hormonal receptors, namely ER and PR. From a clinical point of view, tumors with AR expression show a slightly better clinical outcome. These findings suggest that the immunophenotype classification is not completely exhaustive; like TNBC [50], the presence of AR could confer a luminal phenotype. The concurrent presence with the estrogen receptor could make the androgen receptor a favorable prognostic marker also in the BC HER2+ subtype.
The role of androgens and AR might vary depending on cancer cell types and/or on the level of expression of other steroid hormone receptors. In ER-positive tumors, AR has an anti-proliferative effect by antagonizing ER, by binding to a subset of estrogen response elements (EREs); it can prevent the activation of target genes which mediate the stimulatory effects of 17-beta-estradiol on breast cancer cells [51,52]. Recently, Hickey et al. demonstrated that AR performs a tumor suppressor role in the ER-positive BC subtype, and the AR activation induces potent antitumor activity in multiple disease contexts, including resistance to endocrine therapy and CDK4/6 inhibitors. These data reinforce AR agonism as the optimal AR−directed treatment strategy, showing a rational therapeutic opportunity for this tumor subtype.
However, androgens may have a proliferative effect through AR in ER-AR+ tumor cells. In HER2+ tumors, AR triggers the WNT/β-catenin pathway causing HER3 upregulation; through HER2/HER3 heterodimers, it can cause the activation of the PI3K/AKT pathway and may lead to cell proliferation through MYC [53,54]. Furthermore, in nonluminal BC HER2+, AR induces HER2 expression, which in turn leads to ERK activation, which requires HER2 and AR activity. These findings suggest that HER2 is an upstream connector between the AR and ERK signaling pathways. Another feature of this feedback loop is an ERK-mediated regulation of AR [55]. Previous findings should have potential clinical relevance; considering the He's study, the inhibition of AR with enzalutamide or shRNAs decreases the growth of HER2+ BC cells in vitro and in vivo, having a sensitivity similar to the trastuzumab. Interestingly, the inhibition of AR diminished the phosphorylation of HER2 and the activation of AKT and ERK without involving HER2 and HER3 protein expression levels. These findings indicate a new role of AR in HER2 signaling, and anti-AR target therapy may be beneficial in HER2+ BC patients that are unresponsive or that develop resistance to anti-HER2 therapies [56].
The prognostic role of AR in BC ER-negative and PR-negative has been poorly evaluated, in comparison with BC ER-positive PR-positive variants [57]. Our study, which was focused on luminal and non-luminal BC HER2+ showed that histological type and AR expression are good independent prognostic factors for OS in the HER2+ BC patients. Furthermore, it was found that a poorer OS in BC HER2-positive patients with higher histological grade (G3), higher level of lymph node ratio and PR expression <10%. Wang et al. demonstrated the association between BC HER2+ expressing AR and longer progressionfree survival, increased five years OS rate, and the efficacy of trastuzumab therapy [34]. Akashi et al. found that AR expression was associated with the significant effectiveness of neoadjuvant chemotherapy and prognosis in HER2+ tumors [46]. Our study does have some limitations, that are primarily focused on its retrospective strategy. Hence, some information on clinical follow-up data were not completely included in the medical records. Additionally, the analysis should be expanded to more patients in the coming years to consolidate and reproduce our results.

Conclusions
AR is frequently expressed in BC HER2+ subtypes. Our results showed that AR expression has a prognostic value in BC HER2+ subtypes, with better clinical outcomes. A better prognosis was highlighted in luminal HER2+ AR+ tumors subtype compared to the non-luminal HER2+ AR+ subtype, based on clinico-pathological data. AR expression should be assessed to evaluate the prognosis of BC HER2+ subtypes. Moreover, the understanding of the complex interactions between AR and the HER2 signaling pathway could pave the way to the use of AR as a therapeutic target in BC HER2+ subtypes.