Androgen receptor positive triple negative breast cancer: Clinicopathologic, prognostic, and predictive features

Introduction Overexpression of the androgen receptor (AR) characterizes a distinct molecular subset of triple negative breast carcinomas (TNBC). The role of AR as a prognostic/predictive biomarker in TNBC is controversial, but increasing evidence suggests that this subset may respond to therapeutic agents targeting AR. Evaluation of AR has not been standardized, and criteria for selection of patients for antiandrogen therapy remain controversial. In this study we determine the appropriate threshold of AR immunoreactivity to define AR positive (AR+) TNBC, describe the clinicopathologic features of AR+ TNBC, and discuss the utility of AR positivity as a prognostic and predictive marker in TNBC. Materials and methods 135 invasive TNBC processed in accordance with ASCO/CAP guidelines, were immunostained for AR. Clinicopathologic features of AR+ TNBC were analyzed and compared to AR negative (AR-) TNBC. Patients’ age, tumor size, tumor grade, lymph node status, proliferation rate, immunopositivity for EGFR, CK5/6, Ki-67, and disease free survival (DFS) were evaluated statistically. Results A 1% cutpoint was confirmed as the appropriate threshold for AR positivity. Using this cutpoint 41% of 135 TNBC were AR+. AR+ TNBC occurred in older women, were larger, had lower mean proliferation rate and increased incidence of axillary metastasis than AR- TNBC. 76% of TNBC with apocrine morphology were AR+. A subset of AR+TNBC expressed basal markers (EGFR and CK5/6). A prognostic model was created. Summary AR identifies a heterogeneous group of TNBC. Additional evaluation of EGFR expression allowed us to stratify TNBCs into 3 risk groups with significant differences in DFS and therapeutic implications: low-risk (AR+ EGFR-) which represents the LAR molecular subtype with the best prognosis and may benefit the most from anti-androgen therapies; high-risk (AR- EGFR+) which represents the basal molecular subtype with the worst prognosis and may benefit the most from chemotherapy regimens; intermediate-risk (AR+EGFR+ and AR-EGFR-) TNBC with an intermediate prognosis. Prospective trials are required to further validate this prognostic and predictive grouping.


Introduction
Triple negative breast carcinomas (TNBC) are defined by absence of expression for estrogen receptor (ER) and progesterone receptor (PR) by immunohistochemistry (IHC), absence of overexpression for human epidermal growth factor receptor HER2/neu (HER2) by IHC, and absence of amplification of HER2 by fluorescent in situ hybridization (FISH). TNBC accounts for 10-20% of newly diagnosed breast cancers [1]. They tend to be larger and higher grade, have a higher incidence of axillary lymph node metastasis and are associated with worse overall survival than other types of breast carcinoma [2][3][4][5][6][7][8][9].
Molecular analyses have shown that TNBC is a heterogeneous disease. As reported by Lehmann et al., TNBC can be further classified into four molecular subtypes [basal-like1, basal-like2, mesenchymal, and luminal androgen receptor (LAR)], each characterized by different clinicopathologic features and different driver signaling pharmacologically targetable pathways [10]. In another study, Jezequel et al. demonstrated three molecular subtypes (basal with low immune response, basal with high immune response, and LAR) [11]. Subsequently, other groups have confirmed LAR as a distinct subtype of TNBC characterized by high AR expression and enrichment of hormonally regulated pathways that are important in steroid synthesis, porphyrin metabolism, and androgen/estrogen metabolism despite absence of ER [12,13].
This study was designed to determine the threshold of AR for immuohistochemical evaluation (minimum staining required for a tumor to be considered AR+), to analyze the clinicopathologic features of TNBCs expressing AR, and to determine the utility of AR immunoexpression as a prognostic and predictive marker in TNBC. AR expression was also integrated into a prognostic model that stratified TNBC into risk groups that may be used to help guide personalized therapy.
TNBCs diagnosed from 2008 to 2012 were retrieved from our database. TNBC were defined by lack (<1% positivity) of ER and PR immunoreactivity and a HER2/neu score of 0 or 1+ by immunohistochemistry (IHC) and absence of amplification by FISH. Clinical parameters (age, sex, tumor location, type of surgery, adjuvant chemotherapy) and pathologic features (tumor size, grade, proliferation rate, lympho-vascular invasion, axillary lymph node status, results of Ki-67, p53, EGFR and CK5/6 immunostains) were recorded. All tissue samples were fixed in 10% buffered formalin and embedded in paraffin wax for routine histological examination in accordance with the 2007 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines [33]. Hematoxylin and eosin (H&E) stained sections of all cases were reviewed, the diagnosis of invasive carcinoma was confirmed, and a representative block was selected for AR immunostain. 135 cases contained sufficient tumor; these formed the study cohort. No additional selection criteria were applied.

Immunohistochemistry
IHC stains were performed on whole sections using a Polymer and/or SA-HRP Detection System with appropriate positive and negative controls. ER, PR, Ki-67, p53, and HER2/neu labeling indices were determined using SP1, 1E2, K-2, DO7, and 4B5 antibodies (Ventana Medical Systems, Tucson, AZ, USA), respectively. ER, PR, Ki-67 and p53 immunoexpression was evaluated as the percentage of cells exhibiting nuclear staining. Cell proliferation (Ki-67) was assessed by counting at least 500 tumor cell nuclei (depending upon the availability of tumor) and graded as low (<11%), intermediate (11-20%), and high (>21%) [34]. Immunostaining for EGFR and CK 5/6 was performed using the monoclonal antibody 2-18C9 for EGFR (Dako, Glostrup, Denmark) and D5/16 B4 for CK5/6 (Dako, Glostrup, Denmark). Results were recorded as the percentage of invasive carcinoma cells showing cytoplasmic and/or membrane staining. For EGFR, staining in >15% of tumor cells was interpreted as positive and staining in 15% of tumor cells was interpreted as negative. For CK5/6, staining in >50% of tumor cells was interpreted as positive and staining in 50% of tumor cells was interpreted as negative, as previously reported [16].
Immunohistochemical detection of Androgen Receptor was performed on 4-μm whole tissue sections with antibody clone F39.4.1 (Biogenex, Fremont, CA) applied at 1:100 dilution and incubated for 45 minutes at room temperature. Pretreatment was performed by the Dako PT Link module with low pH buffer (Carpenteria, CA). Staining was done on the Dako Autostainer and visualized by the Dako Envision mouse detection system at 30-minute incubation using Dako DAB and subsequent counterstaining with Mayer's hematoxylin. The percentage of tumor nuclei that stained was recorded.
All cases were evaluated independently by two pathologists. Consensus results were recorded and discordances were resolved by review and discussion.

Statistical analyses
Statistical analysis was performed using R software 3.1.1 (The R foundation for statistical computing, Vienna, Austria) and SPSS 17.0 statistical software (SPSS Inc, Chicago, IL, USA). The Wilcoxon test was used to compare the clinicopathologic characteristics of AR+ and AR-TNBCs. Survival differences between the two groups were compared using log-rank tests and Kaplan-Meier curves. Disease-free survival (DFS), defined as the interval between period after curative treatment and the date of first recurrence or progression of disease, or the date of death from any cause, was used as the endpoint for survival analysis. Patients who were not reported to be dead at the time of the analysis were censored at the date they were last known to be alive.

Determination of the threshold for AR positivity
In order to determine the appropriate threshold for AR positivity the 135 cases were divided into 2 groups: Group A (the study set; n = 35) and Group B (the validation set; n = 100). Tumor characteristics and DFS survival were similar in the two groups. Additionally, DFS survival of EGFR+ and EGFR-cases in the two groups was also similar (S1 Fig). DFS was analyzed with AR defined as positive when 1%, 10%, 20%, 25%, and 30% of tumor nuclei stained.

Creation of prognostic model
Prognostic factors were determined using Cox proportional hazard regression analysis. The resultant significant biomarkers/variables were selected as candidate prognostic indicators, and the prognostic contributions of a combination of biomarkers were identified by multivariate Cox analysis [31]. Patients were further stratified into different risk categories based on the expression values of the various biomarkers. The difference in DFS between the various risk categories was evaluated by log-rank tests and Kaplan-Meier curves. A p-value less than 0.05 was considered significant.

Establishing a threshold to define AR positivity
No significant difference was observed in DFS within Group A or within Group B when 1%, 10%, 20%, 25% or 30% staining was used as the threshold for AR positivity (Fig 2, S1A and S1B Table). Clinicopathologic features of all 135 cases were also evaluated using 1% and 25% staining as the threshold for AR positivity. At the 1% cutpoint AR positive (AR+) tumors were significantly larger and showed a higher incidence of lymph node metastasis than AR negative (AR-) tumors. At the 25% cutpoint, no significant differences in tumor characteristics were observed between AR+ and AR-tumors. Thus, AR immunoreactivity in at least 1% of tumor cell nuclei was considered the most appropriate threshold to define AR positivity.

Clinical and pathologic features of AR+TNBC
Prevalence. When 1% was utilized as the threshold to define AR positivity, 41% (55/135) of the TNBC were AR+ (Table 1). Among the AR+ tumors, staining was strong (!50% positive, Fig 3)  Age of patients. Women with AR+TNBC ranged from 29 to 89 years. The mean age of women in the AR+ group was significantly older than that of women in the AR-group (61.4 vs. 54.8 yrs; p = 0.015).
Summary of clinical and pathological features of AR positive TNBC. Using !1% AR immunoreactivity to define AR+TNBC, 41% of our study cohort were AR+. When compared to AR-TNBC, AR+TNBC were larger and more frequent in older women, showed a higher incidence of apocrine differentiation, a higher incidence of axillary lymph node metastasis, and lower proliferation rates. No siginificant difference was observed in mean DFS between the two groups. 24% exhibited prominent apocrine differentiation. AR+ApoCA+TNBC were also more frequent in older women, were larger tumors with lower proliferation rates and increased lymph node metastasis when compared to AR+ApoCA-TNBC. A subset of AR +TNBC demonstrated EGFR and CK5/6 positivity suggesting a basal phenotype.

Prognostic model
We previously identified EGFR immunoreactivity in !15% of tumor cell nuclei as a negative correlate of DFS in TNBC [16]. In the current study, EGFR+ (!15%) TNBC also experienced a lower DFS than the EGFR-(<15%) TNBC (mean DFS 31.7 vs 43.8 months; p = 0.005, Fig 5). Although AR+TNBC did not demonstrate any survival benefit, we were able to develop a statistically significant (p = 0.0374) prognostic model using a combination of EGFR and AR which was successful in stratifying TNBC into the following three prognostic groups (Table 2 Group 3 (high-risk TNBC): AR-EGFR+ tumors. This group had a mean DFS of 29.8 months and the highest event rate with !1 events having occurred in 34% of patients. We also noted that the mean proliferation rate (Ki-67%) increased from 30% in the low-risk group to 57% in the high-risk group.

Discussion
AR is a nuclear steroid hormone receptor that is normally expressed in benign breast tissue where it is coexpressed with ER and PR in 5-30% of luminal epithelial cells [32]. AR is also expressed in metaplastic apocrine cells, a frequent component of fibrocystic change which occurs commonly in the breast. Apocrine cells show uniform and diffuse positivity for AR but do not show expression for ER or PR. AR is also reported in 60-90% of all breast cancers [35]. The biologic role of AR in breast is poorly understood. Androgen has been described as a potential tumor suppressor in ER-positive breast cancers with its anti-proliferative effect presumed to result from cross talk between steroid receptor signaling pathways [36]. However, studies investigating AR in TNBC have reported conflicting results. For example, Birell et al. noted that AR had a proliferative effect in ER and PR negative cell lines [37] which was confirmed by Garay et al. and by Doanne et al., who raised the possibility of targeting the androgen pathway [6,38].
AR is reported in 7-75% of TNBC [13][14][15][16][17][18][19]. This wide range in reported incidence of AR expression in TNBC can be attributed, at least in part, to differences in immunoreactivity threshold used to define immunopositivity, in tissue fixation, in AR analysis, and/or in tumor heterogeneity. The use of tissue microarrays is another confounding factor. Currently, there are no standard or consensus guidelines for scoring AR immunoreactivity in tissue sections. We used 1% as the cutpoint to define AR+ and evaluated AR immunoreactivity in whole sections of TNBC. 1% is also the cutpoint that should be used to evaluate ER and PR positivity in breast cancers according to the ASCO/CAP guidelines [39]. A few published studies also used 1% as the cutpoint to evaluate AR immunoexpression [19,26] while others, including a recent clinical trial showing benefit of anti-AR therapy in the metastatic setting, used a 10% as their cutpoint [27,30,31].  In an effort to determine the most appropriate threshold, we analyzed a cohort of 135 TNBC using several different cutpoints and found no difference in DFS of patients with cutpoints >1%. Although similar clinicopathologic correlates (occurrence in older women, apocrine morphology, and the inverse correlation with proliferation rate (Ki-67%) were noted when 25% and 1% were used as cutpoints, additional correlations with larger tumor size and increased incidence of node metastasis were observed only with 1% as the cutpoint. Therefore, 1% was considered the most appropriate cutpoint for evaluation of AR by immunohistochemistry. Adoption of 1% immunoreactivity to define AR+ TNBC would also allow the largest number of patients to benefit from targeted therapies.
Several studies have reported AR immunoexpression as a favorable prognostic factor associated with lower clinical stage, lower histologic grade, lower mitotic score, and better outcome (5-year DFS and overall survival (OS) [18][19][20][21][22][23][24]40]. Although we observed a lower mitotic rate in our AR+ TNBC, the AR+TNBC in our study were larger (mean size) and had a higher Androgen receptor positive triple negative breast cancer incidence of axillary lymph node metastasis than the AR-tumors. Moreover, in our study there was no significant difference in mean DFS between AR+ and AR-TNBC. Our findings are similar to those reported by Pistelli et al., who observed an inverse relationship between AR expression and Ki-67% as well as a higher incidence of lymphovascular invasion, but no association with DFS or OS [30].
AR expression in the LAR molecular subtype has been shown to be 10X that in non-LAR TNBC. LAR TNBC usually express low levels of basal biomarkers and are predominantly subclassified in the non-basal subgroup [38]. Conversely, the basal-like TNBCs have low levels of AR expression. Variable levels of EGFR and CK5/6 (basal markers) positivity were noted in our AR+ cases, consistent with the expression of AR in both the LAR and basal molecular subtypes of TNBC. In our prognostic stratification, the AR+EGFR-tumors showed the best prognosis and probably represent the LAR molecular subtype, whereas, the AR-EGFR+ tumors had the worst prognosis and likely represent the basal TNBC. AR+EGFR+ tumors and AR-EGFR-tumors had an intermediate prognosis. Our prognostic groups also showed differences in proliferative rates (Ki-67%). The low-risk group had the lowest Ki-67 index, a feature that is consistent with the LAR molecular subtype. Given that chemotherapeutic agents used in the treatment of TNBC are most effective in tumors with a high proliferative rate, this group would be expected to show a poor response to chemotherapy. This may, at least in part, explain the poor response of AR+ tumors to chemotherapy, resulting in an outcome similar to that in the AR-TNBC in our study. Thus, the low-risk (AR+EGFR-) TNBC may benefit the most from antiandrogen targeted therapies. The high-risk (AR-EGFR+) TNBC had the highest proliferation rate and, therefore, might be expected to benefit the most from chemotherapy. Our findings warrant further studies for validation in larger cohorts of AR+TNBC and in controlled prospective clinical trials. Androgen receptor positive triple negative breast cancer