The Prognostic Role of Androgen Receptor in Patients with Early

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Published OnlineFirst November 9, 2016; DOI: 10.1158/1078-0432.CCR-16-0979

Cancer Therapy: Clinical Clinical Cancer Research The Prognostic Role of Androgen Receptor in Patients with Early-Stage Breast Cancer: A Meta- analysis of Clinical and Gene Expression Data Ivana Bozovic-Spasojevic1,2, Dimitrios Zardavas3, Sylvain Brohee 4, Lieveke Ameye5, Debora Fumagalli3, Felipe Ades1,6, Evandro de Azambuja1, Yacine Bareche4, Martine Piccart7, Marianne Paesmans5, and Christos Sotiriou4

Abstract

Purpose: Androgen receptor (AR) expression has been as evaluable for the current study after the qualitative assess- observed in about 70% of patients with breast cancer, but its ment. AR positivity defined by IHC was associated with prognostic role remains uncertain. improved DFS in all patients with breast cancer [multivariate Experimental Design: To assess the prognostic role of AR (M) analysis, HR 0.46; 95% confidence interval (CI) 0.37– expression in early-stage breast cancer, we performed a meta- 0.58, P < 0.001] and better OS [M-HR 0.53; 95% CI, 0.38– analysis of studies that evaluated the impact of AR at the protein 0.73, P < 0.001]. Thirty-five datasets including 7,220 patients and gene expression level on disease-free survival (DFS) and/or were eligible for the pooled gene expression analysis. High AR overall survival (OS). Eligible studies were identified by system- mRNA levels were found to confer positive prognosis overall atic review of electronic databases using the MeSH-terms "breast in terms of DFS (HR 0.82; 95% CI 0.72–0.92;P ¼ 0.0007) and neoplasm" and "androgen receptor" and were selected after a OS (HR 0.84; 95% CI, 0.75–0.94; P ¼ 0.02) only in univariate qualitative assessment based on the REMARK criteria. A pooled analysis. gene expression analysis of 35 publicly available microarray data Conclusion: Our analysis, conducted among more than sets was also performed from patients with early-stage breast 17,000 women with early-stage breast cancer included in clinical cancer with available gene expression and clinical outcome data. and gene expression analysis, demonstrates that AR positivity is Results: Twenty-two of 33 eligible studies for the clinical associated with favorable clinical outcome. Clin Cancer Res; 1–11. meta-analysis, including 10,004 patients, were considered 2016 AACR.

Introduction with metastatic breast cancer, ranging from 14% to 53% (1). However, due to their adverse effects associated with virilization, The androgen receptor (AR) is a steroid hormone nuclear lack of solid understanding of their biological mode of action receptor, which is frequently expressed in breast cancer. Still, the and the development of aromatase inhibitors, androgens were contribution of AR signaling in breast cancer carcinogenesis and not further pursued as therapeutic modality for patients with its clinical relevance as potential prognostic factor and therapeutic breast cancer. target remain largely unknown. The AR expression in primary In vitro evidence indicated that AR has both inhibitory and breast cancer was discovered long ago, and androgens such as stimulatory effects on different breast cancer cell lines' growth, fuoxymesterone showed promising response rates in patients which is considered to be modulated by the presence or absence of estrogen receptor (ER) expression (2). fl 1Breast Data Centre, Medical Oncology Department, Institut Jules Bordet, Epidemiologic studies also provided con icting results, report- Universite Libre de Bruxelles, Brusells, Belgium. 2Institute for Oncology and ing significant association between androgen serum level and risk Radiology of Serbia, National Cancer Research Centre, Belgrade, Republic of for developing breast cancer or no association at all. Those results Serbia. 3Breast International Group, Brussels, Belgium. 4Breast Cancer Transla- were dependent on age and more importantly on menopausal tional Research Laboratory, Institut Jules Bordet, Universite Libre de Bruxelles, status and estrogen milieu suggesting that androgen could act as 5 Brusells, Belgium. Data Centre, Institut Jules Bordet, UniversiteLibrede anti-estrogen in premenopausal women, whereas it acts as an Bruxelles, Brussels, Belgium. 6Hospital Albert Einstein, Sao~ Paulo, Brazil. 7Med- estrogen agonist in postmenopausal women (3). Indeed, some ical Oncology Department, Institut Jules Bordet, Universite Libre de Bruxelles, Brussels, Belgium. epidemiologic studies demonstrate that high circulating androgen level in postmenopausal women was linked with higher risk Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). for developing breast cancer, but it is not known if these effects are mediated through AR signaling (4). I. Bozovic-Spasojevic and D. Zardavas contributed equally to this article. More recent results originated by gene expression profiling Corresponding Author: Christos Sotiriou, Jules Bordet Institute, 121 Blvd. de studies renewed the interest in AR receptor signaling and its Waterloo, 1000, Brussels 1000, Belgium. Phone: 322-541-3428; Fax: 322-538- potential clinical relevance for breast cancer. One of these studies 0858; E-mail: [email protected] identified ER-negative, AR-positive breast cancer as a subtype doi: 10.1158/1078-0432.CCR-16-0979 showing a distinct transcriptome profile, called the molecular 2016 American Association for Cancer Research. apocrine subtype (2, 5). Of note, these studies indicated possible

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together and, whenever possible, within different breast cancer Translational Relevance subtypes, defined as follows: (i) ER-positive (ERþ); (ii) ER-neg- Androgen receptor (AR) recently regained interest as a ative (ER); (iii) HER2þ and (iv) TN (ERHER2). Patients were possible therapeutic option in breast cancer treatment; considered to have AR-positive tumors according to the cut-off however, little is known about the clinical significance of points defined by the authors in each of the eligible studies. AR in breast carcinogenesis. To our knowledge, we report the largest combined clinical and gene expression meta-analy- Identification and selection of studies fi sis, assessing the prognostic signi cance of AR expression in The eligible studies were identified by an electronic search on early-stage breast cancer indicating that AR expression at PubMed/Medline, Cochrane Review, and Google Scholar using both protein and mRNA level serves as a positive prognos- the MeSH terms "breast neoplasm" and "androgen receptor." The ticator for women with early-stage breast cancer. In addi- literature search was conducted independently by two investigation, we explore connection among AR and pathologic tors (I. Bozovic-Spasojevic and D. Zardavas) up to June 2015. complete response after neoadjuvant chemotherapy in the Identified studies were eligible for this meta-analysis if they met gene expression publicly available datasets, and also corre- the following inclusion criteria: studies conducted in patients with lation of AR and genes and gene signatures of interest. early-stage breast cancer that assessed AR expression in primary Additional information about distinct prognostic relevance breast cancer, studies that compared clinical outcomes (DFS of AR expression in different breast cancer subtypes was and/or OS) in association with AR status, studies published in fi provided; however, further studies are warranted to con rm extenso and reported in the English language. fi these ndings. It is apparent that AR has many effects on the Of note, no minimum threshold for either number of patients biology of breast cancer and deserves more clinical and or duration of follow-up period was mandated. Cross-referencing translational research attention. from relevant studies was performed to confirm retrieval of all potentially eligible studies. To avoid duplication of data, when the same patient population was reported in several publications, only study with the highest number of events was included in the fi antagonism between AR and ER mediated by competition for the current analysis. In terms of study eligibility, nal decisions were DNA-binding sites in the cells and a possible adoption of AR in taken upon consensus between two investigators (I. Bozovic- ER-like oncogenic role in the absence of ER (2, 5). Spasojevic and D. Zardavas). AR expression by IHC has been observed in up to 90% of primary breast cancer, and in up to 75% of breast cancer metas- Data extraction tases, depending on the method, patient's population and cutoffs Data, such as authors, publication date, number of patients, used. These studies indicate that the frequency of AR positivity subgroup analysis, method of AR assessment, and AR threshold differs between different breast cancer subtypes; the highest were extracted by two investigators (I. Bozovic-Spasojevic and positivity observed in ER-positive tumors in up to 80%–90% D. Zardavas) using a predesigned abstraction form. Survival data (6–10) and the lowest in triple-negative (TN) tumours, in up to within all patients and whenever possible within different breast 30% (6, 7, 11). Of note, a phase II trial assessing enzalutamide in cancer subtypes [including HR and P value] were extracted inde- metastatic TNBC, reported AR positivity (defined as 1%) in 79% pendently by two statisticians (L. Ameye and M. Paesmans). of 404 cases analyzed (12). Retrospective clinical studies showed that AR by IHC adds prognostic information beyond the estab- Quality of the eligible studies lished clinicopathologic parameters in all patient groups and in The study methodology of each eligible study was indepen- patients with early-stage ER-positive breast cancer (9, 13, 14). dently scored by three reviewers [two medical doctors (I. Bozovic- Tumors coexpressing AR and ER are smaller, have lower Notting- Spasojevic and D. Zardavas) and one statistician (M. Paesmans)], ham grade and low proliferative index (14), lymph node involve- according to REMARK (Reporting Recommendations for Tumor ment is less frequent, and they are more likely to be found in Marker Prognostic Studies; ref. 15). Each of the 20 criteria listed in fi postmenopausal women (4). However, the clinical signi cance of REMARK was scored for each eligible study, using an ordinal scale AR in patients with ER-negative breast cancer is less clear. with possible values 0, 1, and 2, making a maximal score of 40. To address the prognostic role of AR status in early-stage breast The overall score evaluated several scopes of the methodology cancer, we conducted a meta-analysis of published clinical studies including the scientific rational and design, the description of the fi to evaluate the impact of AR protein expression de ned by IHC on methods used to identify AR expression, data generation, analysis disease-free survival (DFS) and overall survival (OS). Further- of the study data, and discussion of their relevance. The attributed more, we conducted a pooled gene expression analysis of publicly value per item was 2 points if clearly defined in the article, 1 point fi available microarray datasets to assess the prognostic signi cance if description incomplete or unclear, and 0 point if not defined, AR of mRNA status in early-stage breast cancer and explore inadequate, or not applicable. The scores for each individual study AR potential associations of mRNA expression with the expres- were compared and consensus for each item was reached among sion of other individual genes and gene signatures (GS) of interest. the three investigators. We did not predefine a threshold for the REMARK score of each study as inclusion criterion in the meta- Methods analysis. However, scoring was performed as quality assessment Systematic review of published clinical studies of the clinical studies data included in this meta-analysis and used Meta-analysis of clinical studies that evaluated the impact of AR for sensitivity analyses (Supplementary Table S1). protein expression on DFS and OS in patients with early-stage The studies being eligible for the systematic review are called breast cancer was performed within all breast cancer subtypes "eligible," and those providing data for the clinical meta-analysis

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with at least retrievable HR in one of the defined endpoints are manuscript of Haibe-Kains and colleagues, with three additional called "evaluable." We excluded studies with insufficient or with sets: TCGA (TCG Data Portal; ref. 16), PNC (GSE20713; ref. 17), unreliable data to estimate clinical outcome. In terms of defining and METABRIC (European Genome-Phenome Archive under the evaluable studies, final decisions were taken upon consensus accession number EGAS00000000083; ref. 18). between the two physicians (I. Bozovic-Spasojevic and D. Zarda- To ensure comparability of expression values across multiple vas) and the two statisticians (L. Ameye and M. Paesmans). datasets, we performed a 0.95-quantile normalization of all genes and GS of interest. Pooled gene expression analysis AR expression levels were calculated as continuous and cate- Thirty-five datasets of gene expression profiling analysis of gorical variable where classified as low (corresponding to the more than 7,220 primary breast cancer were retrieved from public lower tertile across all available expression values), medium databases or authors' websites, 32 previously described in the (intermediate tertile), and high (upper tertile).

Table 1. Eligible clinical studies for clinical meta-analysis and their characteristics Type of NARþ Method of AR Antibody AR threshold for positivity RS ER threshold for First author study (total) assessment used (chosen by) positivity Collett K. (46), Retrospective analysis 137/269 Dextran-coated 10nM3 H-labeled 43 fmol/mg Median value 12 >15 fmol/mg 1996 charcoal methyltrienolone technique R-1881 Agoff A. (6), Retrospective analysis 51/88 IHC F39.4.1 >5% arbitrary 13 >5% of cells 2003 of random cases Rahka EA. Retrospective analysis 36/282 IHC on TMA F39.4.1. 1% arbitrary/literature 16 1% of cells (41), 2006 of consecutive cases based Soiland H. Retrospective analysis WS-ARn: 103 IHCARc and AR441; R1881 charcAR30 pmol/g; 25 10% of cells (42), 2008 TMA-Arn: 94 ArnonWS IHC-Arn15%; IHC-Arc>0 IHC-Arc: 58 and TMA plus Calculated ROC curve charc-AR: 91 charc-AR Gonzalez-Angulo Retrospective analysis of 174/347 RPPL Microarray Primary AR antibody - >0.0852 dichotomized 15 Not reported AM. (8), 2009 selected cases/frozen Epitomics by mean Peters A. Retrospective analysis 116/215 IHC on TMA AR U407 antiserum Median % of positive 6 10% of cells (40), 2009 cells 75% (range 0–96%) Castellano I. Retrospective analysis of 609/859 IHC on TMA AR441 1% literature based 26 1% of cells (9), 2010 consecutive cases Luo X. (39), Retrospective analysis 148/269 IHC ZYMED PV-6000-G Kit 2 (intensity %) arbitrary 13 Not reported 2010 AR antibody not specified Micello D. Retrospective analysis of 128/226 IHC AR27 >10% literature based 16 10% of cells (7), 2010 consecutive cases Loibl S. (44), Prospective study 358/673 IHC on TMA F39.4.1 >3 (% x intensity) 25 10% of cells 2010 Remmele score Park S. (11), Prospectively collected 541/931 IHC on TMA AR 441 10% arbitrary 26 10% of cells 2011 tissues Yu Q. (43), Retrospective analysis of 237/327 IHC AR441 >1 Allred score (intensity x %) 18 >1% of cells 2011 consecutive cases Peters KM. Retrospective analysis 41/73 IHC on TMA Anti-AR (Biocare 1% not reported 11 Not reported (54), 2012 Medical) Hu R. (53), 2012 Prospective study 1154/1467 IHC on TMA AR441 >1% (1%—10% þ > 10%) arbitrary 24 Not reported He J. (47), 2012 Retrospective analysis 73/287 IHC on TMA AR 441 Dako 5% nuclear staining arbitrarily 11 Not reported Honma N. Retrospective analysis 212/403 IHC on FFPE AntiAR Ab clone 10% nuclear staining literature 13 10% of cells (48), 2013 AR27; Novocastra based Laboratories Witzel I. (52), Retrospective analysis of Cohort 1 126/165 AR mRNA Affymetrix HG-U 133A Expression value of AR mRNA 18 Not reported 2013 consecutive cases plus Cohort 2 133/200 GeneChip System higher than 75% The most population-based cohort Cohort 3 167/223 significant difference among expression values Tokunaga E. Retrospective analysis of 155/250 IHC AR441; Dako 75% nuclear staining 9 1% of cells (50), 2013 consecutive pts literature based Takeshita T. Retrospective analysis 295/379 IHC AR318 Leica Histoscore >10 literature 11 1% of cells (65), 2013 Biosystems based Thike AA. Retrospective 267/699 IHC on TMA AR 27 NCL-AR-318 1% nuclear staining 11 1% of cells (49), 2013 cohort analysis literature based Tsang J. Retrospective analysis of 549/1144 IHC on TMA AR 441 Dako 1% nuclear staining 12 >1% of cells (51), 2014 consecutive histologic literature based files of 3 Institutions Pistelli M. Retrospective analysis 15/81 IHC F39.4.1 Biogenex 10% nuclear staining 18 10% of cells (45), 2014 arbitrarily Abbreviations: AR, androgen receptor; ARc, cytoplasmic androgen receptor; ARn, nuclear androgen receptor; charc-AR, charcoal androgen receptor; RPPL, reverse-phase protein lysate microarray; RS, REMARK score (15)—reporting recommendations for tumor marker prognostic studies; please refer to Supplementary Table S1 for details; TMA, tissue microarray; WS, whole section.

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Breast cancer molecular subtypes were defined on the basis of continuous and categorical variable following the UICC-TNM the PAM50 classifier as luminal A, luminal B, HER2-enriched, and classification); grade (grade 1, 2, and 3); nodal status (positive basal-like. As no clear consensus has ever been established on the vs negative); ER status (positive vs negative); and HER2 status existence of this PAM50 group, the normal like subgroup of tumor (positive vs negative). Furthermore, we assessed potential asso- was not specifically considered in the subgroup analyses. More- ciations of AR mRNA expression with the expression levels of the over, as not every study had complete information about ER and previously mentioned individual genes and GS using a Wilcoxon HER2 status, ESR1 and ERBB2 status were derived from the test. bimodal distribution of these two genes expression. For the survival analysis, the two endpoints were disease-free We chose to evaluate potential associations between AR gene survival (DFS) and overall survival (OS). Survival plots according expression levels and individual genes and GS that can be clas- to the groups were drawn using the Kaplan–Meier method, and sified as follows: estrogen GS (ESR1), proliferation-based prog- the differences were evaluated with a log-rank test. The median nostic GS [AURKA (19), CIN70 (20), GENE70 (21), GGI (22)], follow-up was calculated with the reversed Kaplan–Meier method immune-related individual genes and GS (CTLA4 gene, IDO1 and data were censored after 10 years of follow-up. gene, IFNG gene, IGKC gene, CD3D gene, CD80 gene, CD8A gene, To compute HR and 95% CI in a univariate analysis we used a FOXP3 gene, Immune1 GS (23), Immune2 GS (24), PD1 gene, Cox linear regression model (adjusting only for the dataset). PDL1 gene), stroma-related genes, and GS [CXCL13 gene, CXCL9 Multivariate analysis was computed by the linear Cox regression gene, RANKL gene, Stroma1 GS (25), Stroma2 GS (24)], as well as model, adjusted for the ESR1 and ERBB2 gene expression, patient other individual genes and GS related to further oncogenic age, tumor grade, tumor size, and the lymph node involvement. signaling pathways [RAS GS (26), SRC GS (26), MYC GS (26), In the 8 neoadjuvant datasets, we looked for the association of E2F3 GS (26), BetaCatenin GS (26), BRCA1 and BRCA2 genes, AR mRNA expression level among breast cancer subtypes and AKT/mTOR GS (27), HER3 gene, IGF1 GS (28), MAPK GS (29), percentage of pCR by using a logistic regression model (uni- and PIK3CA GS (30), PTEN GS (31), VEGFA gene, WNT7B gene]. multivariate adjusted for dataset, tumor grade, and treatment). Furthermore, we assessed the possible impact of AR expression pCR was defined as loss of the invasive component of the primary on pathologic complete response (pCR), using neoadjuvant tumor in one study (33) and no residual invasive cancer in the datasets previously published in Ignatiadis and colleagues (32), breast and axillary lymph nodes in other seven studies (34–38). with availability of gene expression profiling data, and pCR status. Results Statistical analysis Clinical meta-analysis Clinical meta-analysis. We used as a measure of the prognostic We retrieved a total of 913 references. After applying secondary effect of AR protein expression the HR for the comparison of the exclusion criteria (Supplementary Fig. S1A), a total of 33 studies DFS or OS distributions using AR-negative patients as a reference. were eligible for the analysis and were scored on the basis of For each study and each considered subtype (when applicable and REMARK criteria (15). We then excluded studies where none of possible), we extracted the individual HR estimates and their the HR of interest was available or retrievable with its variance or variances if reported. When only the HR estimates were reported, where the number of observed events was < 15. (Supplementary fi we calculated their variances using the con dence interval for the Fig. S1A; Supplementary Table S1). HR estimate or the log-rank statistic value and the number of Out of the 33 eligible studies, 22 studies were found to be events. If none of these methods was applicable, we read the evaluable, including 10,004 patients, with 5,860 cases of AR survival curves and calculated back the HRs with their variances. positivity (Table 1). Twenty studies (6–9, 11, 39–52) were evalu- Whenever available, we used both the individual HRs from the able for the DFS and sixteen (7–9, 11, 39–42, 44–49, 53, 54) for univariate and the multivariate analyses in separate analyses. We the OS (Supplementary Tables S2A and S2B); among studies only fi report combined HRs with 95% con dence intervals (CIs) using two (44, 53) were prospective, the rest was retrospective. fi xed-effect models or random-effects models depending on the Of note, differences were noted among the studies concerning detected heterogeneity between the individual HRs estimates; if the methods of AR status assessment, antibodies used, cut-off heterogeneity was detected, random effect was reported. Hetero- values implemented, and thresholds for AR positivity selection. geneity of HRs between the different studies was assessed using a There was heterogeneity between evaluable studies in regards to c2 test for heterogeneity. the study population with variation of breast cancer subtypes definition, as well with treatment given and follow-up time. Pooled gene expression mRNA analysis. We calculated potential associations between AR mRNA levels with clinicopathologic Prognostic significance of AR expression variables among all patients with breast cancer and within breast For the overall population, univariate analysis showed longer cancer subtypes using t tests. The clinicopathologic variables that DFS [HR ¼ 0.61; 95% CI, 0.52–0.72; P < 0.001] and OS [HR 0.62; were assessed are the following: age (both as continuous and 95% CI, 0.51–0.75; P < 0.001] among AR-positive patients categorical variable and > 50 years); tumor size (both as (Table 2A; Fig. 1A). Multivariate analysis was performed on the

Table 2A. Univariate and multivariate analyses of AR prognostic role in all patients with breast cancer HR (95% CI) P Univariate analysis DFS (21 studies, N ¼ 8036) Random-effects Heterogeneity, P ¼ 0.01 0.61 (0.52–0.72) <0.001 OS (18 studies, N ¼ 8301) Random-effects Heterogeneity, P ¼ 0.005 0.62 (0.51–0.75) Multivariate analysis DFS (13 studies, N ¼ 5648) Random-effects Heterogeneity, P ¼ 0.01 0.46 (0.37–0.58) <0.001 OS (8 studies, N ¼ 5773) Random-effect heterogeneity, P < 0.001 0.53 (0.38–0.73)

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basis of available information, confirming good prognostication Subgroup analysis was performed on the basis of available data, conferred by AR positivity in regard to DFS (HR 0.46; 95% CI, whenever feasible for the following breast cancer subtypes: þ þ 0.37–0.58; P < 0.001) and OS (HR 0.53; 95% CI, 0.38–0.73; P < ER ;ER; HER2 ER (representative of the HER2-enriched) 0.001; Table 2A; Fig. 1B). and TN (representative the basal-like subtype) (Table 2B). This analysis revealed that AR positivity resulted in significantly þ improved DFS and OS in ER patients, both in univariate (HR 0.53; 95% CI, 0.44–0.63, P < 0.001 and HR ¼ 0.59; 95% CI, 0.49– 0.72, P < 0.001, respectively) and multivariate analysis (HR 0.40; 95% CI, 0.31–0.52, P < 0.001 and HR ¼ 0.37; 95% CI 0.16–0.85, P ¼ 0.02, respectively). Similar positive association was seen in TNBC patients (HR 0.64; 95% CI 0.51–0.81, P < 0.001 and HR 0.64 95% CI, 0.49–0.88, P < 0.001, respectively), in univariate þ analysis. In HER2 ER breast cancer, AR positivity was not significantly associated with DFS (HR 1.20; 95% CI, 0.86–1.69, P ¼ 0.28) while overall survival was worse (HR 1.50; 95% CI, 1.01–2.22 P ¼ 0.04). No significant association was found either for DFS and OS in ER breast cancer subgroup (HR 0.33; 95% CI, 0.04–2.44, P ¼ 0.28 and HR 1.32; 95% CI, 0.98–1.80, P ¼ 0.08 respectively).

Pooled gene expression mRNA analysis Datasets. A total of 35 datasets with data from 7,737 patients, and median follow-up time of 10.17 years were available (Supple- mentary Fig. S1B; Supplementary Table S3), including eight studies with 1,005 patients in the neoadjuvant setting (Supple- mentary Table S4; ref. 32). Clinicopathologic characteristics of these patients according to the datasets can be found in Supple- mentary Table S3.

Associations of AR mRNA levels with clinicopathologic variables among all patients with breast cancer. Associations between AR mRNA expression and clinicopathologic variables were evaluated among all patients: a significant inverse correlation was observed between AR mRNA expression and tumor size (P ¼ 0.03). Women older than 50 years had higher AR levels (P < 0.001). In addition, AR expression was positively correlated with ERBB2 expression (P < 0.001; Table 3; Supplementary Fig. S2A). We also evaluated AR expression among different breast cancer subtypes defined by PAM50 classifier (Table 3, Supplementary Fig. S2B). High AR expression was seen in luminal A tumors with tumors less than 2 cm. High AR expression was found in Luminal B tumors in women older than 50 years and with lymph node–negative disease (P ¼ 0.05); AR mRNA levels were positively correlated with ESR1 (P < 0.001).

Survival analysis according to AR mRNA expression levels. To address the impact of AR mRNA expression levels on prognosis, we compared the risk of relapse among three subgroups of patients corresponding to low, intermediate, and high AR mRNA levels (level 1, 2, and 3, respectively) among all patients, and within different breast cancer subtypes (see Methods). Overall, high AR mRNA levels were found to confer better prognosis in terms of DFS (HR 0.82; 95% CI, 0.72–0.92;P ¼ 0.0007) and OS (HR 0.84; 95% CI, 0.75–0.94; P ¼ 0.02) in univariate analysis, but did not remain signiﬁcant in the multivariate analysis (DFS: HR 0.97; 95% CI 0.84–1.13; P ¼ 0.72 and OS HR 0.98; 95% CI 0.85–1.12, P ¼ 0.72; Table 4). In contrast, higher AR mRNA expression levels were found to be Figure 1. associated with improved OS in both, uni- and multivariate Clinical meta-analysis. A, Forest plots for DFS by AR protein expression; B, analyses for all women with HER2-enriched breast cancer Forest plots for OS by AR protein expression. (HR 0.75; 95% CI, 0.56–1.00, P ¼ 0.05 and HR 0.72; 95% CI,

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Table 2B. Univariate analysis of AR prognostic role in breast cancer (BC) subtypes and multivariate analysis in ERþ subtype Univariate analysis HR (95% CI) P Multivariate analysis HR (95% CI) P ERþ ERþ DFS (9 studies, N ¼ 2,843) fixed effects, 0.53 (0.44–0.63) <0.001 DFS (5 studies, N ¼ 1,571) fixed effects, 0.40 (0.31–0.52) <0.001 Heterogeneity P ¼ 0.53 Heterogeneity P ¼ 0.65 OS (6 studies, N ¼ 3,383) fixed effects, 0.59 (0.49–0.72) <0.001 OS (3 studies, N ¼ 2,180) random effects, 0.37 (0.16–0.85) 0.02 Heterogeneity P ¼ 0.26 Heterogeneity P ¼ 0.003 ER DFS (3 studies, N ¼ 383) random effects, 0.33 (0.04–2.44) 0.28 Heterogeneity P ¼ 0.05 OS (4 studies, N ¼ 687) fixed effects, 1.32 (0.98–1.80) 0.08 Heterogeneity P ¼ 0.11 HER2þ/ER DFS (4 studies, N ¼ 410) fixed effects, 1.20 (0.86–1.69) 0.28 Heterogeneity P ¼ 0.99 OS (3 studies, N ¼ 358) fixed effects, 1.50 (1.01–2.22) 0.04 Heterogeneity P ¼ 0.50 TN DFS (9 studies, N ¼ 1,373) fixed effects, 0.64 (0.51–0.81) <0.001 Heterogeneity P ¼ 0.16 OS (5 studies, N ¼ 1,285) fixed effects, 0.64 (0.49–0.88) <0.001 Heterogeneity P ¼ 0.07

0.53–0.97, P ¼ 0.03 respectively). Similar results were found in Discussion HER2-enriched subgroup treated with hormone therapy only To our knowledge, we report the largest combined clinical and – P ¼ (multivariate HR 0.50; 95% CI, 0.26 0.97, 0.04). We did gene expression meta-analysis, assessing the prognostic signifi- fi fi not nd any other signi cant association with clinical outcome in cance of AR expression in early-stage breast cancer indicating that the luminal A, luminal B, and basal-like breast cancer subtypes AR expression at both protein and mRNA level serves as a positive (Supplementary Table S5). prognosticator for women with early-stage breast cancer. Our results show that AR protein expression confers a DFS and Associations of AR mRNA expression levels with individual genes and OS advantage among all patients with breast cancer. A possible AR gene signatures. We next looked for associations between explanation for this is the documented association of AR expres- mRNA expression and expression of individual genes and GS of sion with favorable prognostic factors such as small tumor size potential clinical relevance. Among all breast cancer subtypes, the (39), low grade (8, 39, 44, 52), negative lymph node status, ER fi following signi cant associations were found: negative correla- and/or PR positivity (44) and older age (8). These associations tion with proliferation-based GS such AURKA GS (19), CIN70 were confirmed by our AR mRNA pooled analysis, which showed P < (20), GGI (22), and GENE70 (21; all 0.001); negative that higher AR mRNA expression levels serve as positive prog- correlation with immune-related genes and GS such as Immune nosticator among all women with early-stage breast cancer. P ¼ P < 1 (23) and Immune 2 (24) GS ( 0.004 and 0.001, In addition, we assessed the prognostic relevance of AR posi- CTLA4, IDO1, IFNG, IGKC, PD1, CXCL9, respectively), as well as tivity among different breast cancer subtypes, defined differently CXCL13 PDL1 P < , and genes (all, 0.001); positive correlation with in the clinical and transcriptomic meta-analysis (IHC and PAM50 P < stroma1 GS (ref. 25; 0.001) but not with stroma2 GS (ref. 24; based respectively). In the former, a strong better prognostication P ¼ fi þ 0.13); and nally, positive correlations with several oncogenic was conferred by AR positivity among patients with ER breast WNT7B, ESR1, HER3, PIK3CA, BRCA1 pathways including genes cancer both in univariate and multivariate analysis, which is in P < (all 0.001), and negative correlations with IGF1 (28), line with previously published studies (8, 9, 11, 41, 42, 53). BRCA2, BetaCatenin, PTEN, MAPK, MYC, RAS, SRC GS (26) and Preclinical evidence suggesting an AR-ER cross-talk also support VEGFA, CD80, CD8A, CD3D SQLE P < , and genes (all 0.001; these findings, indicating that AR can antagonize ER signaling AR Supplementary Table S6A). Associations between mRNA and depending on the relative levels of these two steroid receptors (2). expression levels of individual genes and GS among different Interesting results have been reported by Cochrane and collea- breast cancer subtypes are presented in Supplementary Table S6B gues, assessing the potential prognostic/predictive relevance of fi in supplementary le. AR:ER ratio at the protein level assessed through IHC in the setting of ER-positive breast cancer (55). The study was fueled by previ- Response to neoadjuvant CT according to AR mRNA levels. We ous observations that in ER-positive breast cancer responding to further evaluated whether AR mRNA expression were associated endocrine treatment, AR downregulation at the protein and with response to neoadjuvant CT (anthracyclines taxanes). mRNA level is observed, whereas no such effect is seen in non- Out of 1,005 patients from eight datasets (33–38), 235 (23%) responsive tumors (56, 57). In a cohort of 192 patients with early- patients achieved pCR, 765 (76%) did not, while five samples stage ER-positive breast cancer receiving adjuvant tamoxifen were inadequate for the analysis. High AR expression levels treatment and another one of a randomized phase II trial assessing showed significant association with lower pCR rate (OR, 0.57; exemestane with or without tamoxifen, high AR:ER ratio was 95% CI, 0.44–0.74, P < 0.001) in the univariate analysis while not found to predict resistance to endocrine therapy in a statistically significant in a multivariate model (OR, 0.74; 95% CI, 0.54–1.01, significant manner. These findings need further confirmatory P ¼ 0.063; Fig. 2).

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Androgen Receptor in Early Breast Cancer: Meta-analysis P studies; however, the data available in our study do not allow Kw further evaluation.

P According to the recently proposed "androgen excess" theory, increased androgen activity is one of the most important hor- þ monal abnormities seen in the patients with both ER and ER breast cancer (58). This theory suggests that androgens could þ stimulate ER tumors by increased conversion into estrogens cor Wilcox 0.038 NA NA within the tumor tissue; concentration of estradiol within tumor tissues about 20 times greater than in the circulation has been Wallis test; TU size cat, tumor size –

P reported, an observation that could be explained by the local hormone production as androgen precursors are highly present within the tumor. According to this theory, ER tumors could also value

P be stimulated via androgen activity, and depending on testoster- one serum levels and AR positivity, either through EGFR or direct Kw stimulation via androgen pathway (58). P Another notable ﬁnding of our clinical meta-analysis is the worse prognostication conferred by AR positivity for women with þ 0,0010,001 NA NA NA NA NA NA 0.001 0.001HER2 NA NA ER (Her2-enriched) disease, albeit the analysis included < < three studies with 358 patients (7, 11, 44). Similar ﬁndings have been reported, with AR positivity associated with worse clinical cor Wilcox 0.002 NA NA 0.26 þ þ

outcome among HER2 (7) as well as patients with HER2 ER (44) breast cancer. The biologic basis for the interaction between AR and HER2 in breast cancer cell lines was recently studied through an analysis of the androgen regulated gene expression in PP þ HER2 ER breast cancer, indicating that AR signaling results in Kw ligand-dependent Wnt- and HER2-signaling pathways activation

P through direct transcriptional induction of WNT7B and HER3, the latter being a dimerization partner to HER2. These ﬁndings

0,001 NA NA NA suggest that anti-androgens merit clinical evaluation as anticancer < þ agents in the setting of HER2 ER breast cancer (59). There is an ongoing phase II, single-arm clinical trial assessing the combina- cor Wilcox tion of trastuzumab plus enzalutamide in the setting of HER2/AR- positive metastatic pretreated breast cancer (NCT02091960,

0.001 0.09 NA NA 0.86 0.005 NA NA 0.79https://clinicaltrials.gov/ct2/show/NCT02091960, 0.008 NA NA accessed on < July 15, 2016). However, contrasting to our clinical meta-analysis PP our gene expression analysis ﬁnd signiﬁcantly better OS for AR Kw mRNA expression within all HER2-enriched breast cancer s and

P within HER2-enriched subgroup treated only with hormone therapy. Caution is warranted when we compare data from IHC subtyping with data from gene expression proﬁling as these two 0,001 NA NA NA < approaches may not produce identical calls for the same tumors and published data suggested modest overlap between 0.01 NA NA cor Wilcox 0.09 NA NA 0.193 0.032 NA NA 0.96 AR IHC and transcriptional proﬁles(2).Itmustbenoted, however, that there is no subset of patients included in the present analysis, for whom information concerning the AR 0.001 < status at both the mRNA and protein level, as assessed through

PP IHC, is available. 50 years; cor, correlation; ESR1, estrogen gene level; ERBB2, erb-b2 avian erythroblastic leukemia viral oncogene homolog 2; KW, Kruskal

Kw We also investigated the prognostic value of AR in the more aggressive TN phenotype, with the clinical meta-analysis indicat- P

50 vs. ing that it serves as positive prognosticator in the univariate < analysis, conﬁrming previously published results (6, 8, 39, 0,001 NA NA0,001 NA 0.30 NA NA NA NA NA 0.46 NA 0.015 NA NA NA NA NA 0.85 NA 0.86 NA NA NA NA NA NA 0.44 NA ALL LUMINAL A LUMINAL B HER2 enriched BASAL like < < 41, 44). This might be partly explained by the negative correlation 5 cm; Wilcox, Wilcoxon test.

> of Ki67 as marker of decreased tumor cell proliferation and AR cor Wilcox 0,03 NA NA expression, which was seen in TNBC (60). However, other studies did not show any effect of AR on the TNBC outcome (11, 40, 53). 5 cm vs.

– ﬁ

P In the gene-expression pro ling analysis, AR mRNA levels were not prognostic in basal-like breast cancer. Recent ﬁndings iden-

2 vs. 2 ﬁ

< ti ed a luminal androgen subtype within basal-like breast cancer Correlation of AR mRNA with tumors' and patients' characteristics in all and in breast cancer subtypes conﬁrming the extensive heterogeneity of basal-like breast cancer (60), and the role of AR within this breast cancer subtype requires TU SIZE CATGRADE NANODE CATESR1 bimod NA NA NA NA NA NA NA NA NA 0,65 0,458 0 NA NA 0,425 NA NA NA NA NA NA NA NA 0.41 NA 0.142 0.512 NA NA NA NA NA NA NA NA NA NA 0.05 0.495 NA NA NA 0.474 NA NA NA NA NA NA 0.492 0.63 0.492 NA NA NA NA NA NA NA NA NA 0.45 0.65 0.43 NA AGE CAT NA NA ERBB2 bimodAbbreviations: NA Age cat, age category: NA TU SIZE 0,03 AGE 0 0,13 NAcategory: NA 0.60 Table 3. further assessment.

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Table 4. Disease free survival (DFS) and overall survival (OS) analysis according to AR mRNA expression levels in all patients with breast cancer HR Uni (95% CI) HR Multi (95% CI) ALL patients (no. of patients) Treatment DFS P DFS P 3,190 treated_and_not_treated 0.82 (0.72–0.92) 0.0007 0.97 (0.84–1.13) 0.72 1,219 untreated 0.89 (0.72–1.09) 0.26 0.90 (0.69–1.19) 0.49 1,268 HT_only 0.91 (0.71–1.18) 0.12 0.99 (0.76–1.32) 0.99 463 CT_only 0.98 (0.76–1.28) 0.91 0.87 (0.61–1.23) 0.43 240 HT_and_CT 0.82 (0.52–1.29) 0.40 1.05 (0.65–1.71) 0.87 OS OS 2,671 treated_and_not_treated 0.84 (0.75–0.94) 0.002 0.98 (0.85–1.12) 0.72 914 untreated 0.87 (0.70–1.08) 0.22 0.86 (0.66–1.21) 0.26 1,143 HT_only 1.05 (0.86–1.29) 0.62 0.99 (0.79–1.23) 0.93 374 CT_only 1.02 (0.79–1.32) 0.85 0.92 (0.67–1.27) 0.63 240 HT_and_CT 0.89 (0.60–1.30) 0.62 1.01 (0.66–1.57) 0.93 Abbreviations: CT, chemotherapy; HT, hormonotherapy.

The different prognostic relevance of AR positivity found of 673 core biopsies, evaluated the predictive value of AR for pCR among the different breast cancer subtypes indicates that the after neoadjuvant chemotherapy in patients with primary breast þ broader molecular profile constituting the tumor influences the cancer (44). This study found that AR tumors had lower rates of functional output of AR signaling and its relevance for the clinical pCR in comparison with AR tumors, 12.8% versus 25.4%, þ outcome of patients. Thus, studies conducted among homoge- respectively P < 0.0001, but AR tumors had significantly better neous breast cancer populations in terms of subtypes are needed DFS and OS including those not reaching pCR (44). Moreover, to better delineate the prognostic relevance of AR positivity in AR, ER, and HER2 were independent predictors for pCR in a early-stage breast cancer. Ideally, analyses should be performed multivariate model (44). When pCR was analyzed across different within the context of prospective randomized clinical trials, where IHC-assessed breast cancer subtypes, it was found for all sub- þ the abovementioned conditions are fulfilled, along with homo- groups that AR tumors had the lower rate of pCR, although the geneous treatment approaches, along with high-level clinical differences were not significant. Hormone receptor positivity is annotation in terms of clinical outcomes. It is important to have known to be one of the strongest negative predictive biomarkers a clear definition of what is considered as AR positivity in such for pCR after neoadjuvant chemotherapy (61). Indeed, we did studies, as mentioned before, different cutoffs were used in the confirm that AR positivity was associated with lower pCR rate, but studies we included in this meta-analysis. only in univariate analysis. We also investigated the potential impact of AR expression in Finally, we looked for correlations among AR mRNA and other pCR rates among all and different breast cancer subtypes. Only genes and GS of interest; it must be noted that none of these genes one study, using prospectively collected samples from the Gepar- or GS is validated and they are therefore not routinely assessed Trio trial, in which AR expression was evaluated by IHC on a TMA in clinical practice. As expected and in accordance with clinical

Figure 2. II Gene expression analysis. Forest plots of univariate and multivariate analysis of AR mRNA expression and pCR in all and in breast cancer subtypes.

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Androgen Receptor in Early Breast Cancer: Meta-analysis

data, AR expression was highly correlated with expression of subgroups is not possible, due to lack of published data charac- ESR1. Positive correlations were also found with expression terizing them. In addition, this is a literature-based meta-analytic of PIK3CA, recently published as possible target in combination study without individual patient data, thus limiting our ability to þ with antiandrogens in AR TNBCs (62). Of note, there is address certain issues, such as interaction of AR expression status an ongoing phase I/II trial assessing the combination of taselisib, with other prognostic factors. Regarding gene expression analysis, an alpha-selective PI3K inhibitor, with enzalutamide, in the we used data from publicly available data sets, which are not setting of pretreated metastatic AR-positive TNBC, with AR pos- without limitations either. Such databases could be biased toward itivity defined as AR 10% (NCT02457910 https://clinicaltrials. highly expressed genes. gov/ct2/show/NCT02457910, accessed on July 15, 2016). Other In conclusion, our study shows that AR expression at both positive correlations of interest were with HER3 and WNT7B, protein and mRNA levels serves as positive prognosticator among for whom molecular basis for correlation was published by Li more than 17,000 women with early-stage breast cancer. Addi- and colleagues (59) and with BRCA1 which requires further tional information about distinct prognostic relevance of AR investigation. expression in different breast cancer subtypes was provided; Recently, a systematic review and meta-analysis of AR prog- however, further studies are warranted to confirm these findings. nostic role in early breast cancer has been published (63). Vera- It is apparent that androgens and AR have many effects on the Badillo and colleagues have analyzed 19 studies with 7,693 biology of breast cancer and deserve more clinical and transla- patients with early breast cancer, providing data for the pooled tional research attention. Currently, AR is being actively investi- analysis evaluating two endpoints, DFS and OS, at 3 and 5 years gated as therapeutic target in breast cancer. and AR expression defined by IHC. Of note, out of the 19 and 22 studies included in Vera-Badillo and colleagues and our meta- Disclosure of Potential Conflicts of Interest analysis, 13 studies show overlap between both studies, encom- No potential conflicts of interest were disclosed. passing 6,015 patients; this corresponds approximately to 60% of the total number of patients included in our meta-analysis. Their Authors' Contributions results are in line with ours, as they also showed that AR positivity Conception and design: I. Bozovic-Spasojevic, D. Zardavas, E. De Azambuja, confers better DFS and OS in all patients with early breast cancer. M. Piccart, M. Paesmans, C. Sotiriou Development of methodology: I. Bozovic-Spasojevic, D. Zardavas, F. Ades, AR was a good prognostic factor within ER patients, contrasting E. De Azambuja, M. Paesmans our results, in which ER patients tended to have worse OS, Acquisition of data (provided animals, acquired and managed patients, without reaching statistical significance. Differently, for the ER provided facilities, etc.): I. Bozovic-Spasojevic, D. Zardavas, L. Ameye, F. Ades, subgroup analysis they include six studies (8, 39, 40, 44, 53) in M. Paesmans, C. Sotiriou comparison with four (40, 48, 53) in our analysis. One of the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, explanations could be more restricted criteria for inclusion in computational analysis): I. Bozovic-Spasojevic, D. Zardavas, S. Brohee, L. Ameye, D. Fumagalli, F. Ades, E. De Azambuja, M. Paesmans, C. Sotiriou our analysis as we excluded from the analysis studies with sig- fi Writing, review, and/or revision of the manuscript: I. Bozovic-Spasojevic, ni cant heterogeneity. Of note, currently there is an ongoing D. Zardavas, L. Ameye, D. Fumagalli, F. Ades, E. De Azambuja, Y. Bareche, program under the umbrella of an international consortium M. Piccart, M. Paesmans, C. Sotiriou focusing on male breast cancer, the International Male Breast Administrative, technical, or material support (i.e., reporting or organizing Cancer Program. Both, data and biosamples from 1473 patients data, constructing databases): I. Bozovic-Spasojevic, D. Fumagalli, E. De have been collected, with the intention being to delineate the Azambuja, M. Paesmans, C. Sotiriou Study supervision: I. Bozovic-Spasojevic, E. De Azambuja, M. Paesmans, clinical relevance of several biomarkers including AR expression, C. Sotiriou assessed through IHC (64). Our analysis has some limitations. The studies included in the The costs of publication of this article were defrayed in part by the payment of clinical meta-analysis suffer from methodologic issues, such as page charges. This article must therefore be hereby marked advertisement in different populations of patients, different methods and cut-offs accordance with 18 U.S.C. Section 1734 solely to indicate this fact. used for determine AR positivity, small numbers of patients, retrospective nature of the studies, and intergroup heterogeneity. Received April 28, 2016; revised October 3, 2016; accepted October 27, 2016; The identification of sources of heterogeneity among different published OnlineFirst October 27, 2016.

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Androgen Receptor in Early Breast Cancer: Meta-analysis

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www.aacrjournals.org Clin Cancer Res; 2017 OF11

The Prognostic Role of Androgen Receptor in Patients with Early-Stage Breast Cancer: A Meta-analysis of Clinical and Gene Expression Data

Ivana Bozovic-Spasojevic, Dimitrios Zardavas, Sylvain Brohée, et al.

Clin Cancer Res Published OnlineFirst November 9, 2016.

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