Androgen receptor and FOXA1 coexpression define a “luminal-AR” subtype of feline mammary carcinomas, spontaneous models of breast cancer

At least three factors justify that invasive mammary carcinomas that spontaneously arise in pet cats were proposed as relevant animal models for human breast cancer. The first is their high frequency: mammary tumors are reported to constitute 17% of neoplasms in female cats [1], are malignant in 80–90% of the cases [2, 3], and most of these malignant tumors are carcinomas. The second is their aggressive biological behavior: the median overall survival time of cats with invasive mammary carcinomas is 8–12 months post-diagnosis in most studies with follow-up [4‐9]. The third is their resemblance with the most aggressive subtypes of breast cancer, for which targeted therapies are still needed: feline mammary carcinomas (FMCs) often lack significant levels of Estrogen Receptor (ER) and Progesterone Receptor (PR) expression, are rarely positive to HER2 (Human Epidermal growth factor Receptor 2), and thus most of them are considered to be triple-negative mammary carcinomas [10‐19].

Transcriptome studies showed that triple-negative breast cancers (TNBCs) are a heterogeneous group. Among the major subtypes, the luminal-Androgen Receptor (luminal-AR) subtype appears to be a stable subgroup, characterized by high expression of downstream Androgen Receptor (AR) targets and coactivators [20]. Indeed, luminal-AR TNBC has been reported in multiple gene expression studies as reported by Lehmann et al. (2011 and 2016) [20, 21], Burstein et al. (2015) [22], and Jézéquel et al. (2015 and 2019) [23, 24]. Among others, luminal-AR breast cancers highly express the Forkhead box A1 (FOXA1) gene [20]. The transcription factor FOXA1 is able to bind to highly compacted chromatin, and acts as a pioneer factor with chromatin opening potential that allows other transcription factors and steroid hormone receptors to trigger transcriptional programs [25]. In the healthy mammary epithelial cell as well as in ER-positive breast cancers, FOXA1 is necessary for the expression of ERα target genes [25, 26]. In addition, preclinical studies suggest that FOXA1 allows AR to bind to DNA and thereby induce transcription of AR target genes and stimulate tumor proliferation [27].

Since transcriptome studies are expensive, some authors proposed an immunohistochemical definition of the luminal-AR subtype of triple-negative breast cancer. Guiu et al. (2015) hypothesized that these two markers, AR and FOXA1, are needed to identify the luminal-AR TNBC subtype by immunohistochemistry [28]. Other authors such as Astvatsaturyan et al. (2018) defined the luminal-AR subtype of TNBC by its positivity for AR and negativity for Epidermal Growth Factor Receptor (EGFR), a basal marker [29].

As most of feline invasive mammary carcinomas are triple-negative [11, 13, 19], the question arises of their heterogeneity. Here, we hypothesized that feline triple-negative mammary carcinomas may comprise, as in human TNBCs, a luminal-AR subtype, expected to be a non-basal-like TNBC with AR and FOXA1 coexpression, associated with a different biological behavior than other triple-negative FMCs.

The objectives of this study were thus 1) to investigate for the first time AR and FOXA1 expression in a large series of feline mammary carcinomas, 2) to assess their relationships with other clinical and pathological features of FMCs, 3) to investigate their prognostic significance in the feline patient, and 4) to put the first cornerstone for a better characterization of triple-negative FMCs, with identification of a luminal-AR subtype.

This study investigates for the first time AR and FOXA1 expressions in a large cohort of feline invasive mammary carcinomas. The first objective was thus to determine if AR and FOXA1 expressions are common in FMCs. Most of the carcinomas (97%) contained AR-positive neoplastic cells. At 10% threshold for AR positivity, 92% of the cases would be considered positive for AR. In women, the frequency of AR expression in invasive ductal mammary carcinomas varies considerably depending on the studies and the thresholds used [44‐51]. At the frequently used 10% threshold for AR positivity, from 58% [51] to 90% [46] of breast cancers are AR+, compared to 92% in cats with FMCs. Cats tend thus to have a higher AR expression in FMCs than women with breast cancer.

In our cohort, 43% of the carcinomas contained FOXA1-positive neoplastic cells. At FOXA1 index ≥1, 36% of the FMCs were considered positive for FOXA1. The prognostic cutoff for FOXA1 used in breast cancer varies considerably [52‐55], reaching 71% for hormone receptor-positive breast cancers [52]. The frequency of FOXA1 positivity in breast cancer ranges from 41.5 to 85.9% of cases [52, 56‐62]. Thus FOXA1 expression in breast cancer seems to be considerably higher than in FMCs. This difference may partly be due to the fact that most of the FMCs are triple-negative carcinomas, whereas FOXA1 is strongly associated with the luminal phenotype in women [57, 59].

The second objective of this study was to assess the relationships between AR, FOXA1, and the other clinical and pathological features of feline invasive mammary carcinomas. In the present study, there was a negative correlation between AR overexpression and pathologic tumor size, in agreement with results reported in human breast cancer [50, 51]. For instance, Ogawa et al. (2008) reported that 14% (19/134) of AR-positive breast cancers had a tumor size > 3 cm compared to 26% (21/81) of AR-negative carcinomas [50]. We also found a negative correlation between AR overexpression and lymphovascular invasion, including in the triple-negative subcohort. However, according to Tsang et al. (2014) in a large cohort of 1144 patients with primary invasive breast cancer, there is no significant correlation between AR positivity and lymphovascular invasion in human breast cancer [63]. We observed a negative correlation between AR expression and pathologic nodal stage, a finding also reported in breast cancer: Ogawa et al. (2008) reported that 73% (100/136) of AR-positive invasive mammary carcinomas were free of regional lymph node metastasis, compared to only 60% (49/81) of AR-negative breast cancers [50]. AR overexpression in FMCs was also negatively associated with the clinical stage at diagnosis, as in human breast cancer: according to Alshenawy et al. (2012), 91% (98/107) of AR-positive breast cancers were diagnosed at stages I–II, compared to 58% (25/43) of AR-negative carcinomas [44]. AR expression was negatively associated with the Elston and Ellis histological grades, including in triple-negative FMCs considered separately. This negative association between AR and histological grade has been reported in multiple publications on breast cancers, including in TNBCs alone [28, 64‐66]. For example Tang et al. (2012) reported that 70% (77/111) of AR-negative triple-negative carcinomas were grade III while about 70% (11/16) of AR-positive triple-negative carcinomas were grade I–II [66]. And finally AR expression was negatively correlated with tumor-associated inflammation visible on HES-stained sections. Interestingly, tumor-associated inflammation was associated with poor prognosis in cats of the present study and in the veterinary literature [67]. A similar finding was recently reported in human breast cancer: according to Gujam et al. (2018), AR expression is associated with reduced tumor-associated inflammation [68]. We can conclude that as in human breast cancer, AR positivity in FMCs is mainly associated with favorable prognostic features: smaller pathologic tumor size, negative nodal stage, lower clinical stage, lower histological grade, absence of lymphovascular invasion, and lower tumor-associated inflammation.

FOXA1 positivity in FMCs was negatively associated with pathologic nodal stage, including in triple-negative FMCs, as reported in human breast cancer [56, 69, 70]. According to Albergaria et al. (2009) 31% (30/97) of FOXA1-positive breast cancers were pN+ compared to 69% (67/97) in FOXA1-negative carcinomas. However in ER-negative breast cancers, no association between FOXA1 and nodal stage was found [56]. FOXA1 was negatively associated with the clinical stage of FMCs, including triple-negative FMCs considered separately. Whereas in human breast cancer, no significant correlations were found between clinical stage and FOXA1 [54]. We also observed a negative correlation between FOXA1 and Elston and Ellis histological grade that was also observed in triple-negative FMCs. In human breast cancer, many studies have reported a negative association between FOXA1 and grade in consecutive series of breast cancers [56, 58, 71], but not in ER-negative breast cancers considered separately [56]. FOXA1 index ≥1% was also negatively correlated with lymphovascular invasion, including in triple-negative FMCs, as sometimes reported in breast cancer: Albergaria et al. (2009) found that 37% (30/81) of FOXA1-positive breast cancers were LVI+ compared to 55% (67/122) of FOXA1-negative carcinomas [56]. FOXA1 in our cohort was negatively associated with the Ki-67 proliferation index, in agreement with data reported in human breast cancer [52, 69]. And finally FOXA1 was positively associated with PR, AR and the luminal phenotype. Similarly, in the medical literature, most publications report a very strong correlation between FOXA1 positivity in breast cancers and the expression of the hormone receptors ER and PR [56, 57, 69, 72], confirmed by two meta-analyses [70, 73], AR [74], and the luminal-A phenotype [56, 57, 69, 72]. Although the positive correlation between FOXA1 and PR was also observed in our cohort, it is however interesting to note that FOXA1 and ER did not show significant association in cats of our cohort. This suggests a loss of the normal interplay between FOXA1 and ER [26] in feline mammary carcinomas. Loss of FOXA1 expression in ER+ breast cancers has been associated with resistance to endocrine therapy [69, 75, 76], thus ER+ FOXA1– FMCs in our cohort may not be hormone-responsive mammary carcinomas. Overall, we found that as in human breast cancer, FOXA1 positivity in FMCs was associated with favorable features such as negative nodal stage, lower clinical stage, lower histological grade and lower proliferation index. Most importantly FOXA1 was positively associated with AR, including in triple-negative FMCs.

The third objective of this study was to investigate the prognostic significance of AR and FOXA1 in FMCs, in order to evaluate their significance in the feline patient. AR overexpression was associated with longer disease-free interval, overall survival, and cancer-specific survival by univariate and multivariate survival analyses, independently of clinical stage at diagnosis (tumor size, lymph node metastasis, distant metastasis). AR overexpression was associated with favorable outcomes in both luminal and triple-negative FMCs. In human breast cancer also, AR overexpression is associated with better outcomes in patients with luminal [51, 77] and triple-negative carcinomas [66, 77‐79]. In this study, FOXA1 index ≥1% was also associated with a longer disease-free interval, overall survival, and cancer-specific survival, by multivariate survival analyses, again with components of clinical stage as covariates of the models (tumor size, nodal status, distant metastasis). This finding was also observed in luminal but not in triple-negative FMCs. The significance of FOXA1 expression appears to be similar in human oncology, as FOXA1 positivity of breast carcinomas is associated with better outcomes especially in luminal carcinomas [52, 59, 72, 80]. By multivariate survival analysis, Hisamatsu et al. (2012) reported that FOXA1 was associated with longer relapse-free survival independently of the pathological size, histological grade and ER expression in luminal breast cancers [52]. In TNBC, FOXA1 expression alone was not associated with prognosis. However, Albergaria et al. (2009) reported a near significant result (p = 0.06) with the risk of disease progression being higher for FOXA1-negative TNBCs than for FOXA1+ TNBCs [56].

The fourth and last objective of our study was to better characterize AR-positive triple-negative FMCs in order to define a “luminal-AR” subtype. Indeed, gene expression studies have shown that TNBCs in women are a heterogeneous group, comprising a stable luminal-AR subtype, which has been reported by Lehmann et al. [20, 21], Burstein et al. [22], and Jézéquel et al. (referred to as Cluster 1 or C1 in their study) [23, 24]. Other groups have confirmed that the luminal-AR subtype is a distinct subtype of TNBC, characterized by high expression of the androgen receptor and enrichment in the hormone-regulated pathways that play an important role in steroid synthesis, and androgen/estrogen metabolism despite the absence of ER [81, 82]. Since transcriptome studies are expensive, Guiu et al. (2015) proposed a definition of the luminal-AR subtype using immunohistochemistry. The simple expression of AR is not enough to define a luminal-AR TNBC because AR-positive triple-negative carcinomas contain multiple subgroups with different behaviors and prognoses. FOXA proteins bind to DNA and induce rearrangements that facilitate DNA binding with other regulators such as steroid hormone receptors. In the healthy mammary epithelial cell, FOXA1 is necessary for the expression of ERα target genes [26]. In addition, preclinical studies suggest that FOXA1 allows AR to bind to ER DNA binding sites and thereby induce transcription of ER-related genes and thereby stimulate tumor proliferation [27]. Guiu et al. (2015) investigated whether AR and FOXA1 coexpression (evaluated by immunohistochemistry) could define the luminal-AR subtype of TNBC. In their study, 25.9% (126/487) of the triple-negative carcinomas expressed AR and 15.2% (70/460) were both AR- and FOXA1-positive, called luminal-AR [28]. This frequency was consistent with gene expression studies, in which 11 to 15.4% of triple-negative carcinomas were luminal-AR [20, 83]. Luminal-AR TNBCs defined by double positivity to AR and FOXA1 according to Guiu et al. were associated with a worse outcome than other TNBCs in terms of recurrence-free and overall survivals [84]. Other authors have used other immunohistochemical definitions of the luminal-AR subtype. For instance, Astvatsaturyan et al. (2018) defined luminal-AR TNBCs by their positivity to AR and negative expression of Epidermal Growth Factor Receptor (EGFR), a basal marker. In their study the luminal-AR subgroup (AR+ EGFR–, 9% of TNBCs) was associated with a better disease-free survival compared to other TNBCs [29]. In our current study, we decided to combine AR, FOXA1 and the basal marker CK14 in order to better characterize the luminal-AR subgroup of feline mammary carcinomas. And by doing so, we were able to find a non-basal like subgroup characterized by double positivity to AR and FOXA1 (AR+ FOXA1+ CK14–) that might represent the luminal-AR subtype of triple-negative FMCs, and a basal-like subgroup with AR positivity but loss of FOXA1 expression (AR+ FOXA1–CK14+). The non basal-like AR+ FOXA1+ CK14– subgroup (7/53 cases, i.e., 13% of AR+ triple-negative cases) was associated with a better outcome compared to the basal-like AR+ FOXA1–CK14+ subgroup by univariate and multivariate survival analyses. Inspired from the immunohistochemical definitions of luminal-AR TNBCs (AR+ FOXA1+ according to Guiu et al., AR+ EGFR– according to Astvatsaturyan et al.), we propose that luminal-AR triple-negative mammary carcinomas of cats can be defined by an AR+ FOXA1+ CK14– phenotype.

In conclusion, we have shown that AR and FOXA1 were mainly associated with favorable features such as lower stage and lower histological grade, but also correlated together, as reported in human breast cancer, despite the fact that AR was more commonly expressed in FMCs than in breast cancers, and conversely FOXA1 was less frequently expressed in cats than in women with invasive mammary carcinomas. Both AR and FOXA1 were strong and favorable prognostic factors, independently of tumor stage at diagnosis. Within AR-positive triple-negative FMCs, we identified a non-basal AR+ FOXA1+ CK14– subgroup with AR and FOXA1 coexpression, which appears similar to the luminal-AR TNBC subgroup described in humans, and is associated with a favorable outcome. Female cats with invasive mammary carcinomas may thus be interesting spontaneous animal models to investigate new cancer therapy strategies such as anti-androgen or anti-androgen receptor molecules, especially in the aggressive and potentially non-hormone-responsive triple-negative basal-like AR+ FOXA1–CK14+ subgroup.