University of Groningen Imaging hormone receptors in metastatic breast cancer patients Venema, Clasina Marieke

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Imaging hormone receptors in metastatic breast cancer patients

Venema, Clasina Marieke

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Venema, C. M. (2018). Imaging hormone receptors in metastatic breast cancer patients. Rijksuniversiteit Groningen.

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Chapter 6

Targeting the androgen receptor: a potentially more valuable

therapeutic strategy in breast cancer when additional tumour

characteristics are taken into account

Clasina M Venema, MD1, Rico D Bense, BSc1, Tessa G Steenbruggen, MD1, Hilde H Nienhuis, MD1, Si-Qi Qiu, MD1,2, Michel van Kruchten, MD1, Myles Brown, MD3, Rulla M Tamimi, ScD 4,5, Prof Geke AP Hospers, MD1, Carolina P Schröder, MD1, Rudolf SN Fehrmann, MD1, Prof Elisabeth GE de Vries, MD1

1

Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

2

The Breast Center, Cancer Hospital of Shantou University Medical College, Shantou, China

3

Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, United States of America

4

Channing Division of Network Medicine, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

5

Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA

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Summary

The androgen receptor (AR) is a drug target in breast cancer, and AR-targeted therapies have induced tumour responses in breast cancer patients. In this review, we summarized the role of AR in breast cancer based on preclinical and clinical data. Moreover, as the prognostic value of AR for patients remains uncertain due to the use of various antibodies and cut-off values for immunohistochemical assessment, we performed an in

silico analysis to determine the prognostic value of the AR using mRNA

profiles of 7,270 primary breast tumours. A higher AR mRNA level was associated with improved disease outcome in patients with oestrogen receptor-positive/human epidermal growth factor receptor 2 (HER2)-negative tumours, but with worse disease outcome in HER2-positive subgroups. These findings indicate that next to AR expression, incorporation of additional tumour characteristics will potentially make AR-targeting a more valuable therapeutic strategy in breast cancer.

Keywords: breast cancer, androgen receptor, therapeutic target, hormonal therapy, prognostic value

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Introduction

Breast cancer is the most common cancer in women.1 Among invasive breast cancers, 75% express the oestrogen receptor (ER) and 20-30% overexpress the human epidermal growth factor receptor 2 (HER2). These patients can benefit from therapy that targets ER or HER2 receptors, resulting in superior overall survival (OS) in both the curative and non-curative setting.2-4 However, there is still a need to improve disease outcome, leading to a constant search for new drug targets. In recent studies, androgen receptor (AR) has shown interesting potential as a drug target in breast cancer.

In prostate cancer, AR is a key driver of proliferation, and AR-targeted drugs are currently part of standard care.5 Interestingly, AR is considered to be overexpressed in 70%-90% of breast cancers, including up to 30% of the triple negative breast cancers (TNBC), and tumour response has been observed following AR-directed therapy.6 This makes AR a potentially interesting drug target for many breast cancer patients.

However, AR status is not routinely assessed in breast tumours. Currently, for immunohistochemical (IHC) analysis, a broad range of cut-off values is used, and AR status is determined by various antibodies. This variance makes it difficult to interpret the role of AR based on expression data obtained with IHC in breast cancer. Therefore, in silico analyses using gene expression to determine the association between AR status and disease-free survival (DFS) and OS in breast cancer patients is of interest. To address this, we first performed a literature review to summarize preclinical and clinical data concerning the role of AR in breast cancer, including its role in physiology, and its use in targeted therapy in prostate cancer. We also predicted AR positivity based on RNA data obtained from the public domain in primary breast tumours using normal breast tissue samples as a reference.

Literature search

PubMed was searched for articles published until May 2017 with the following terms: ‘androgen receptor’, ‘expression’, ‘cancer’, ‘molecular imaging’, and ‘tumour’ in various combinations. Only articles in English

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were reviewed. The abstracts were screened for relevance. We included in

vitro studies with breast cancer cell lines and in vivo and clinical studies

using androgens or AR targeted drugs studies. Outside of PubMed, we searched abstracts of the American Society of Clinical Oncology (ASCO) in 2015 and 2016 and San Antonio Breast Cancer Symposium in 2014, 2015 and 2016 with the same terms. Finally, ClinicalTrials.gov was searched for AR-targeted therapy trials in breast cancer patients.

Physiological function of AR

AR is expressed in hair follicles, bone, brain, liver, cardiovascular and breast tissue in both sexes and in males also in testes and prostate tissue.7 AR belongs to the type I nuclear receptors. These receptors are intracellular transcription factors that directly regulate gene expression in response to their ligand (figure 1). Androgens are ligands that bind to AR, and are produced in ovaries of women, the prostate and testes of men, and by hair follicles and the zona reticularis of the adrenal glands of both sexes.8-10 After the lipophylic androgens diffuse through the cell membrane into the cytoplasm they bind to intracellular AR. This leads to dissociation of heat shock proteins and activation and dimerization of AR. The AR dimer then translocates to the nucleus. Binding of the AR dimer to the androgen response element in the promoter and enhancer regions of target genes leads to upregulation or downregulation of DNA transcription (figure 1). Depending on tissue type this leads to cell division, differentiation, apoptosis, proliferation or angiogenesis.

Female AR knockout mice experience impaired follicular growth and

dysfunctional ovulation, illustrating that AR is essential for normal

female fertility.

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In women, low serum androgen levels lead to

reduced libido, reduced muscular strength and vaginal dryness,

whereas high levels result in hirsutism, a lower voice and acne.

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Germline AR mutations result in androgen insensitivity syndromes,

which cause disorders in secondary sex characteristics such as

clitoromegaly, absence of internal genital structures or presence of

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In men, low serum androgen levels are associated with depression

and can lead to low libido and erectile dysfunction. High levels are

associated with aggressive behaviour and increased bone density.

Cardiovascular disease and coagulation abnormalities have been

related to high doses of androgens used in men, but these effects

have not been reported in women.

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Figure 1. Effect of androgens on androgen receptor (AR) in a physiological

setting in an androgen-responsive cell. Testosterone is converted to

dihydrotestosterone (DHT) by 5α-reductase. In the cell DHT binds to the AR intracellular, which leads to dissociation of heat shock proteins (HSPs), activation and dimerization of the AR. Binding of AR dimer to the androgen response element in the promoter regions of target genes, depending on the tissue, leads to up- or downregulation of DNA transcription.

Mechanism of AR targeted therapy in prostate cancer

The AR signalling cascade can be inhibited for therapeutic use in several ways. Firstly, it can be inhibited indirectly by androgen deprivation therapy by lowering circulating androgen levels. This can be done with drugs such as luteinizing hormone releasing hormone (LHRH)-agonists or 17α-hydroxylase/C17,20 (CYP17α1) lyase inhibitors such as abiraterone acetate

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or by orchidectomy (see table 1). In up to 90% of prostate cancer patients, lowering plasma testosterone levels below 1.7 nmol/l, stabilizes or decreases the prostate specific antigen, which is considered a surrogate marker for response.16 In metastatic prostate cancer patients, the addition of abiraterone acetate to prednisone resulted in a median OS of 15·8 months for the combination versus 11·2 months for prednisone alone.17 Secondly, the AR can be directly blocked by administering AR antagonists. The First-generation AR antagonists approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) are bicalutamide, flutamide and nilutamide which inhibit the effects of autocrine testosterone production by the tumour.18 Unlike these AR antagonists, the second-generation AR antagonist enzalutamide not only competitively binds to the AR ligand-binding domain, but also inhibits nuclear translocation of AR, DNA binding and coactivator recruitment.19 Thirdly, degradation of AR serves as a novel strategy for interfering the AR signalling. The AR degraders such as ARV-330 are currently in preclinical development.20

Mechanisms of actions of AR in breast cancer, preclinical evidence

In vitro the androgens testosterone and dihydrotestosterone (DHT) reduced

proliferation, while AR antagonists stimulated proliferation of ER-positive/AR-positive breast cancer cell lines.21-31 However, in the most extensively studied ER-positive/AR-positive cell line MCF-7, the androgen influence was dose-dependent. 21,26-36 Increased proliferation has been observed at very high androgen doses (100 nM-1,000 nM), while reduction in proliferation occurred at lower (1-10 nM) doses.27,30,37-40 These proliferative effects of androgen treatment observed at very high doses in ER-positive cell lines might be due to conversion of DHT to oestrogen.41 In addition, AR agonists and AR antagonists both reduced tumour growth in in

vivo ER-positive/AR-positive breast cancer models.30,42-46 This phenomenon was also seen with ER-targeted therapy in breast cancer patients. Although anti-oestrogen therapy is the cornerstone of endocrine therapy, high dose oestrogens have also induced tumour regression.44

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In comparison to ER-positive/AR-positive breast cancer cell lines, an opposite effect of androgens and AR antagonists is seen in in vitro ER-negative/AR-positive cell lines. In these cell lines, androgens stimulated proliferation, while AR antagonists lowered proliferation.35,47-50 Also, in in

vivo ER-negative/AR-positive human breast cancer xenografts AR agonists

stimulated tumour growth while AR-antagonists inhibited androgen-mediated growth of ER-negative/AR-positive breast tumours.30,51,52 Bicalutamide, an AR-antagonist with partial agonistic effects, did not affect growth of human ER-negative breast cancer xenografts.53

Increased proliferation and cell survival has been associated with the AR-mediated activation of the mitogen-activated protein kinase (MAPK) signalling pathway.54 Simultaneous stimulation of the epidermal growth factor receptor (EGFR) and AR hyperactivated the MAPK pathway. In ER-negative/AR-positive ARIBE and MDA-MB-231 cells this led to reduced proliferation, while stimulation of the EGFR or AR separately increased proliferation.55

Crosstalk between AR and ER, where signal transduction of the ER can affect the AR and vice versa, appears to increase proliferation. These receptors can co-localize in breast cancer cells, as shown with immunofluorescence and immunoprecipitation.56 Interestingly, blocking the AR in tamoxifen-resistant, ER-positive/AR-positive MCF-7 cells did restore sensitivity to tamoxifen. This suggests that the AR/ER ratio may influence tumour response to ER-targeted therapy.30

Crosstalk between AR and HER2 has also been indicated. Testosterone exposure of MDA-MB-453 cells increased HER2 mRNA levels, and exposure to the HER2 ligand heregulin increased AR mRNA levels. Moreover, inhibition of HER2 signalling reduced androgen-stimulated cell growth in ER-negative/HER2-positive/AR-positive cell lines.47,48

Crosstalk between AR and the Wingless proteins (Wnt) signalling pathway has also been observed in ER-negative/AR-positive MDA-MB-453 cells.48 Stimulation of AR with DHT directly upregulated WNT7B mRNA levels, resulting in catenin activation. Nuclear translocation of activated β-catenin stimulates HER3 transcription, which then forms heterodimers with

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HER2 and activates the mTOR/PI3K/AKT pathway, resulting in cell proliferation.48

In quadruple-negative breast cancer cell lines, comprising TNBC cell lines without AR expression, androgens did not affect proliferation, independent of the dose.23,26,31,33,34,49,55

In conclusion, the effect of AR-targeted therapies differs according to the ER status of the breast cancer cells. Whereas high doses of androgens mainly inhibit tumour growth in ER-positive breast cancer cell lines, androgens stimulate tumour growth in ER-negative cell lines, and anti-androgens were most effective in ER-negative/AR-positive cells. The effects of AR-targeted drugs per breast cancer cell line are described in supplemental table S1.

Targeting AR in breast cancer: clinical evidence

The ovaries are a main source of androgens. Theoretically, this means that LHRH analogues as well as oophorectomy, which are both used in breast cancer patients with ER-positive tumours, likely result in a reduction of androgen (figure 2). In 13 premenopausal patients with ER-positive breast cancer, androgen serum levels were lower following treatment with the LHRH-analogue goserelin and aromatase inhibitor.57 Aromatase inhibitors, also part of standard care for breast cancer patients with ER-positive tumours, inhibit the conversion of androgens into estrogens.4 However, this does not result in a significant increase in serum testosterone levels. To date few data is available with regards to the use of aromatase inhibitors combined with androgen deprivation therapy in AR-positive breast cancer patients. In a phase II study in 53 women with AR-positive/triple negative metastatic breast cancer androgen deprivation by abiraterone acetate 1000 mg once daily combined with prednisolone 5 mg twice daily resulted in one complete response and five patients with stable disease.58

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Figure 2. Influencing the androgen receptor (AR) in women. 1. Luteinizing

hormone releasing hormone (LHRH) agonists suppress luteinizing hormone and follicle stimulating hormone (FSH), which stimulate the ovaries to produce oestrogens and androgens. 2. Selective AR blockers bind the AR and inhibit its action in the ovaries, breasts and adrenal glands. 3. Oophorectomy removes the source of oestrogens and androgens. 4. Abiraterone acetate inhibits the conversion from progesterone to androgen by cytochrome P17 (CYP17) in the adrenal glands, which are stimulated by the adrenocorticotropic hormone (ACTH). 5. Aromatase inhibitors prevent conversion from androgen to oestrogen.

Until recently, studies exploring the effect of AR-targeted therapy included breast cancer patients regardless of tumour AR expression levels. Non-tissue-selective androgens, such as testosterone propionate and fluoxymesterone, have been used for treatment of metastatic breast cancer since late 1940s.59 High doses of androgens such as fluoxymesterone and

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testosterone administered to metastatic breast cancer patients showed 19% and 36% tumour response rates, respectively, without selection for AR expression. The treatment coincided with masculinizing side effects such as acne, hirsutism and lowering of the voice in 15-25% of the patients.60-64 Testosterone propionate administered to patients with ER-positive breast cancer, refractory to ER-targeted therapy, resulted in a complete or partial tumour response in nine out of 53 patients and a median OS of 12 months.65 A retrospective analysis of 103 metastatic breast cancer patients with ER-positive tumours treated with fluoxymesterone provided a clinical benefit, defined as objective tumour response or stable disease ≥6 months, in 43% of the patients. However, 33 patients discontinued treatment due to

side effects.

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Until recently, studies exploring the effect of AR-targeted therapy included breast cancer patients regardless of tumour AR expression levels. Non-tissue-selective androgens, such as testosterone propionate and fluoxymesterone, have been used for treatment of metastatic breast cancer since late 1940s.59 High doses of androgens such as fluoxymesterone and testosterone administered to metastatic breast cancer patients showed 19% and 36% tumour response rates, respectively, without selection for AR expression. The treatment coincided with masculinizing side effects such as acne, hirsutism and lowering of the voice in 15-25% of the patients.60-64 Testosterone propionate administered to patients with ER-positive breast cancer, refractory to ER-targeted therapy, resulted in a complete or partial tumour response in nine out of 53 patients and a median OS of 12 months.65 A retrospective analysis of 103 metastatic breast cancer patients with ER-positive tumours treated with fluoxymesterone provided a clinical benefit, defined as objective tumour response or stable disease ≥6 months, in 43% of the patients. However, 33 patients discontinued treatment due to side effects.66

Direct blocking of AR in breast cancer patients was first described in 1988. Flutamide, 750 mg orally daily administered, resulted in one partial tumour response and five stable diseases out of 29 patients.45 Due to gastrointestinal side effects and limited anti-tumour activity, the study was discontinued prematurely. In postmenopausal women, two out of 14

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patients experienced disease stabilization for 20-26 weeks when treated with the AR antagonist nilutamide 100 mg orally per day.51 The side effects and disappointing results of AR-targeted therapies, combined with evidence of the conversion of androgens into oestrogens, resulted in the discontinuation of this approach. However, with the emerging AR-targeted therapies in the prostate cancer setting and the awareness of the high frequency of AR expression in breast cancer, more studies have focussed on AR-targeted therapies in breast cancer.

The first study to select patients based on AR expression evaluated efficacy of the AR blocker bicalutamide 150 mg per day orally in 28 postmenopausal women with ER-negative (IHC positivity ≤10% tumour cells), progesterone receptor-negative and AR-positive (IHC ≥10%) metastatic breast cancer. A clinical benefit rate was seen in 19% of the patients, while the drug was well tolerated.67 However, most patients in this study were heavily pre-treated, which may explain the low overall response rate. One case study reported a complete response to bicalutamide in a woman with AR-positive metastatic breast cancer.

Preliminary results of new AR-targeted therapies, such as the second-generation AR antagonist enzalutamide and the selective AR modulator enobosarm, are of interest: stable disease for ≥6 months has been reported in up to 35% of heavily pre-treated patients (table 2.68,69 Efficacy results are not yet available for combined AR- and ER-targeted therapies or AR- and HER2-targeted therapies. Other ongoing combination trials are listed in the supplemental table S2.

AR expression measured immunohistochemically in breast cancers

Breast cancer patients with various tumour characteristics have experienced clinical benefit from AR-targeted therapies. However, selecting patients for such therapies has been challenging. Clear guidelines on IHC interpretation of the AR have not been established thus far. Most studies use IHC to determine AR expression and base their cut-off value on 10% tumour cells staining positive. Data concerning the response of AR-targeted therapies in patients with low-level AR expression, in the range of 1% to 10% positive cells by IHC, are less frequently described (supplemental table

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3). In the current ER setting, even patients with low ER expression (1%-10%) are eligible for therapy, and guidelines now use the 1% cut-off value.70 For AR measurements, different antibodies with varying sensitivity and specificity have been used. Most experience in clinical breast cancer trials has been obtained with the AR441 mouse monoclonal IgG antibody from DAKO.

Studies on the role of AR in breast cancer have shown that AR expression in the primary tumour is associated with better OS and reduced recurrence rate (figure 3).71-97 This effect is most profound in patients with ER-positive tumours.71-75,82,83,87,88,90,91 In patients with ER-negative breast cancer, data have been contradictory (figure 3 and supplemental table 3).74-79,87,89,98,99 AR expression in HER2-positive primary tumours has not resulted in a significant effect on DFS or OS, probably due to limited patient numbers.71,74,75,81,82,95

In silico analysis of mRNA expression of AR in breast cancer

Given the limited available data on IHC, in silico analyses using mRNA is very interesting. Recently a meta-analysis on gene expression data demonstrated that higher AR mRNA levels is associated with favourable clinical outcome in women with early stage breast cancer.100 This analysis was based on intrinsic molecular subtypes, but in current practice IHC determined receptor status is used. Therefore, we used publicly available mRNA profiles to assess associations between predicted AR status as well as AR mRNA levels and DFS and OS per breast cancer subgroup based on ER and HER2 status, as well as per intrinsic molecular subtypes based on the PAM50 classifier.101

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Figure 3. Overview of literature results with regards to disease free survival (A) and overall survival (B) per breast cancer subgroup, depicted

as a bubble plot. The y-axis shows the study represented by the first author

reference and the x-axis shows the whole cancer population and the subgroups. A blue bubble indicates a positive association (HR < 1.0) between the androgen receptor measured immunohistochemically and disease-free survival (A) or overall survival (B). The red bubble indicates an inverse association (HR > 1.0). The size of the bubble indicates the number of tumour samples used. Black delineation indicates a correlation with a p-value ≤ 0.05

We analysed 7,270 expression profiles of primary tumour samples of non-metastatic breast cancer patients, and we assembled a reference group of 172 normal breast tissue samples obtained during reduction mammoplasty. Whenever information on receptor status was missing, we determined

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these by inference using gene expression data. Detailed analysis methods information has previously been published.102 Overall, ESR1 mRNA and ERBB2 functional genomic mRNA expression clearly discriminated between immunohistochemically determined positive and negative receptor statuses (supplemental figure S1).103 AR status in the tumour samples was considered positive when the AR mRNA level was above a certain threshold. We explored multiple thresholds by calculating the 2.5th, 25th, 50th, 75th and 97.5th percentiles of AR mRNA level in normal breast tissue. Differences in survival between predicted AR-positive and negative tumours was determined with Kaplan-Meier curves and log-rank test. In addition, the association between AR mRNA levels and DFS and OS in the tumour samples was determined with Cox regression.

For the group as a whole, DFS and OS were prolonged in patients with AR-positive tumours in comparison to those with AR-negative tumours (figures 4 and 5). The difference in survival is more pronounced when lowering the thresholds defining AR-positivity (supplemental figure S2 and S3). Cox regression, also showed that a higher AR mRNA level was associated with prolonged DFS and OS in the whole group. However, this association did not remain significant when corrected for relevant clinicopathological parameters (table 3).

In patients with ER-positive/HER2-negative tumours, AR-positivity was also significant associated with a prolonged DFS, depending on the threshold used (figures 4 and 5, supplemental figure S2 and S3). We observed a similar, but less pronounced, trend for prolonged OS with AR-positivity. Higher AR mRNA level was significant associated with prolonged DFS and OS in univariate analyses. This association also did not remain significant when corrected for relevant clinicopathological parameters (table 3). For patients with ER-negative/HER2-positive and ER-positive/HER2-positive tumours, AR positivity was associated with a shorter DFS (figures 4 and 5). The difference in survival is more pronounced when a higher threshold is used for defining AR-positivity (supplemental figure S2 and S3). In line with this observation, Cox regression showed that a higher AR mRNA level was associated with shorter DFS and OS in patients with

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positive breast cancer and remained significant when corrected for relevant clinicopathological parameters (table 3).

Figure 4. Disease-free survival curves for different thresholds for androgen

receptor (AR)-positivity in breast cancer subgroups

.

Non-transparent lines

show the threshold discriminating best between AR-positive and AR-negative cases in terms of disease-free survival. Hazard ratios (HR) and corresponding 95% confidence intervals are shown for non=transparent curves. ER = oestrogen receptor, HER2 = human epidermal growth factor receptor 2.

For the intrinsic molecular subtypes, a higher AR mRNA level was independently associated with prolonged DFS and OS in the luminal B subtype, independent of other relevant clinicopathological parameters (table 4). In the HER2-enriched molecular subtype, a higher AR mRNA level was associated with shorter OS independent of clinicopathological parameters.

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The results above show that the effect of AR status and AR mRNA levels on DFS and OS varies between receptor status based subgroups as well as between intrinsic molecular subtypes.

Figure 5. Overall survival curves for different thresholds for androgen

receptor (AR)-positivity in breast cancer subgroups. Non-transparent lines

show the threshold discriminating best between AR-positive and AR-negative cases in terms of overall survival. Hazard ratios (HR) and corresponding 95% confidence intervals are shown for non-transparent curves. ER = oestrogen receptor, HER2 = human epidermal growth factor receptor 2.

We also explored mRNA expression of AR in breast cancer subgroups. Whereas AR expression was comparable in ER-positive/HER2-negative and negative/HER2-positive tumours, it was evidently lower in

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negative/HER2-negative tumours (figure 6). However, in the luminal AR (LAR) TNBC subtype, AR mRNA levels were similar to those found in ER-positive or HER2-ER-positive tumours. ESR1 and ERBB2 expression levels in the LAR subtype were similar to other TNBC subtypes (supplemental figure S4). Furthermore, in the ER-negative/HER2-positive subgroups AR mRNA levels positively correlated with HER2 (R 0.47, 95% conficende interval [CI] 0.41 – 0.52) and HER3 mRNA levels (R 0.43. 95% CI 0.37 – 0.48). AR mRNA expression levels did not correlate with WNT or the more downstream c-Myc and β-catenin.

Figure 6. Scatter dot plot of standardized androgen receptor mRNA

expression in breast cancer subgroups.The left panel shows mRNA expression

in subgroups based on receptor statuses as determined with immunohistochemistry. The right panel shows subgroups based on complementation of missing receptor statuses with inference. Error bars indicate median mRNA expression and interquartile range. BL1 = like 1. BL2 = basal-like 2. ER = estrogen receptor. HER2 = human epidermal growth factor receptor 2. IM = immunomodulatory. LAR = luminal androgen receptor. M = mesenchymal. MSL = mesenchymal stem-like. US = unstable.

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Discussion and future perspectives

This review summarises information on preclinical and clinical data concerning the role of AR in breast cancer as well as on immunohistochemical and mRNA measurement of AR.

For further implementation of AR directed therapy in breast cancer insight in patient selection criteria seems to be critical. The in silico associations of AR mRNA levels with DFS and OS in the different ER and HER2 status based subgroups reported by us, are in agreement with the associations reported in current literature based on IHC data. However, the association between predicted AR-positivity as well as a higher AR mRNA level and shorter survival we observed in the ER-negative/HER2-positive subgroup and the HER2-enriched intrinsic molecular subtype is in contrast with another recent mRNA based analysis.100 That analysis showed that a higher AR mRNA level is associated with prolonged survival. The discrepancy indicates that the in silico analyses should be interpreted with some caution as they are based on retrospective, publicly available data that can contain potential confounders. However, based on our in silico analysis, targeting both HER2 and AR might be of interest for patients with ER-negative/HER2-positive/AR-positive tumours. This is supported by a currently ongoing trial in breast cancer patients with HER2-positive/AR-positive tumours assessing the effect of trastuzumab plus enzalutamide (NCT02091960). Preliminary results have shown a 24-week clinical benefit rate of 27.3% in patients receiving a median of four prior anti-HER2 therapies.104

The limited amount of data on the role of AR in breast cancer suggests that a discrepancy in AR status between primary and metastatic breast cancer lesions can exist in up to 30% of the patients.105 Obtaining a biopsy during the course of disease is currently considered the gold standard, but is not always feasible. Furthermore, a single biopsy from a metastatic lesion is not necessarily representative for the patient’s complete AR status.

A different approach to obtain potentially whole body information about tumour hormonal receptor status is via circulating tumour cells or circulating tumour DNA.106,107 Also, whole body in vivo expression of AR with intact ligand binding domain is possible by using molecular imaging of the AR with 18F-fluorodihydrotestosterone (18F-FDHT) positron emission tomography (PET). This tracer showed selective uptake in prostate cancer

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metastases and could be blocked by flutamide and enzalutamide.108,109 In metastatic breast cancer patients, 18F-FDHT tumour uptake showed good correlation with IHC staining for AR in representative tumour biopsies (p=0.01) of 13 patients.105

Although the results of AR-targeted therapies in metastatic breast cancer patients are interesting, all patients eventually showed progression while on treatment. Mechanisms that may be related to resistance to AR-targeted therapies in metastatic prostate cancer are amplification or overexpression of AR, ligand-independent activation, overexpression of coactivators and the expression of active AR splice variants.110-112 The most frequently studied AR splice variant in tumours and circulating tumour DNAs in the context of prostate cancer is AR-V7, in which AR is activated without ligand binding; this variant is also predictive of resistance to both enzalutamide and abiraterone.113 Analysis of different splice variants showed AR-V7 mutations in 53.7% of the primary breast cancer samples (n=54).114,115 The clinical relevance of these limited data and the possible relationships to subtypes require further study.

Conclusions

Increased understanding of the role of AR in breast cancer, and optimal selection for AR-targeting therapies, can potentially improve treatment options for breast cancer patients. With novel (selective) AR antagonists becoming available along with new patient selection methods, AR-targeted therapies deserve further evaluation in clinical breast cancer studies. The response rates of AR-targeted therapies in unselected patient populations are relatively low. Preclinical and clinical data show that AR antagonists could be potential therapy for patients with ER-negative/AR-positive tumours. In addition, based on our in silico analysis, patients with HER2-positive/AR-positive tumours may be a preferred subgroup to treat with combined HER2-targeted and AR-targeted treatment. These data indicate that patient selection, using additional tumour characteristics, might increase the role of AR-targeting therapy in patients with breast cancer.

Contributors

All authors contributed to the literature search, data interpretation and writing of the report. CMV, RDB and RSNF performed in silico analyses.

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Declaration of interests

EGEdV reports consulting fees from Synthon, Medivation and Merck; and grants from Novartis, Amgen, Roche/Genentech, Servier, Chugai, Synthon, AstraZeneca and Radius all to the institution and outside the submitted work.

CMV, RDB, TGS, HHN, SQ, MvK, RSNF, CPS, GAPH declare no competing interests.

Acknowledgments

This research was supported by NWO-VENI grant (916-16025), the Bas Mulder award of Alpe d’HuZes/Dutch Cancer Society (RUG 2013-5960), Ubbo Emmius Fund grant (510215), v/d Meer Boerema Foundation, Anna Dorothea den Hingst Foundation, a Mandema Stipendium and European Research Council advanced grant OnQview (293445).

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List of abbreviations 18

F-FDHT 16β-fluoro-5-α-dihydrotestosterone AR androgen receptor

ASCO American Society of Clinical Oncology BRCA breast cancer gene

CBR clinical benefit rate CI confidence interval CYP17α1 17α-hydroxylase/C17,20 DHEA Dehydroepiandrosterone DFS disease free survival DHT 5α-dihydrotestosterone

EGFR epidermal growth factor receptor EMA European Medicines Agency ER oestrogen receptor

FDA Food and Drug Administration GnRH gonadotropin releasing hormone

HER2 human epidermal growth factor receptor 2 HR hazard ratio

HRE hormone response element IHC Immunohistochemistry LAR luminal androgen receptor

LHRH luteinising hormone releasing hormone MAPK mitogenic activated protein kinase OR odds ratio

OS overall survival

PET positron emission tomography

RPPLM reverse phase protein lysate microarray SABCS San Antonio Breast Cancer Symposium TNBC triple negative breast cancer

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Tables

Table 1. AR-targeted therapies in use as standard care

Class Subclass

Drugs Current indication Mechanism of action

Androgen deprivation Luteinising hormone releasing hormone (LHRH) analogues Leuprorelin, goserelin Prostate cancer, endometriosis Suppresses luteinising hormone and follicle stimulating hormone, which stimulate the testicles androgens. CYP17α1 inhibitor Abiraterone acetate Metastatic castration-resistant prostate cancer Blocks conversion of precursors pregnenolone and 17α-hydroxypregnenolone into dehydroepiandrosteron e and androstenediol AR blocking AR antagonists Bicalutamide, flutamide, nilutamide Metastatic prostate cancer

Competes directly with (dihydro)-testosterone for AR binding site AR degrading Selective AR antagonist Enzalutamide Metastatic prostate cancer Blocks androgen binding to AR, inhibits nuclear translocation, inhibits AR association with DNA

High dose androgens

Androgens Testosterone Testosterone

deficiency, breast cancer in post-menopausal women

Binds directly to AR

Lixisenatide Diabetes mellitus type 2

Glucagon peptide agonist,

little AR stimulation CYP17α1 inhibitor = 17α-hydroxylase/C17,20-lyase inhibitor. EMA = European Medicines Agency. FDA = Food and Drug Administration. AR = androgen receptor.