Androgen Receptor Expression Shows Distinctive Significance in ER Positive and Negative Breast Cancers

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1 Ann Surg Oncol (2014) 21: DOI /s ORIGINAL ARTICLE BREAST ONCOLOGY Androgen Receptor Expression Shows Distinctive Significnce in ER Positive nd Negtive Brest Cncers Juli Y. S. Tsng, PhD 1, Yun-Bi Ni, MMSc 1, Siu-Ki Chn, FHKCPth 2, Mu-Min Sho, MMSc 3, Bonit K. B. Lw, FRCS 4, Puy Hoon Tn, FRCPA 5, nd Gry M. Tse, FRCPC 1 1 Deprtment of Antomicl nd Cellulr Pthology, Prince of Wles Hospitl, The Chinese University of Hong Kong, Hong Kong, Chin; 2 Deprtment of Pthology, Kwong Wh Hospitl, Hong Kong, Chin; 3 Deprtment of Pthology, Shenzhen Affilited Hospitl, Gungzhou University of Trditionl, Chinese Medicine, Shenzhen, Chin; 4 Brest Center, Union Hospitl, Hong Kong, Chin; 5 Deprtment of Pthology, Singpore Generl Hospitl, Singpore, Singpore ABSTRACT Bckground. Androgen receptor (AR), nucler steroid hormone receptor, is differentilly expressed in rest cncer sugroups with distinct clinicl implictions. Methods. To investigte the clinicl significnce of AR in rest cncers more precisely, the expression of AR in lrge cohort of rest cncer ws correlted with clinicopthologicl fetures, iomrker expression, nd ptients survivl ccording to different moleculr groupings in this study. Results. Higher AR expression ws found in ER? (57.8 %) thn in ER- (24.7 %) cncers. In the ER? cncers, AR expression ws ssocited with fvorle clinicopthologicl fetures, including lower grde (p \.001), lower pt stge (p \.001), nd positivity for PR (p \.001). It ws n independent prognostic fctor for longer disese-free survivl, minly in the HER2? luminl B cncers (hzrd rtio [HR] = 51, 95 % CI , p =.045). In ERcncers, AR expression ws ssocited with fetures distinct from sl-like rest cncer, nd such fetures were found in moleculr pocrine (MA) cncers. AR correlted with presence of extensive in situ component (p =.006) nd pocrine phenotype (p \.001), HER2 (p =.026), nd EGFR (p =.048), ut negtively with c-kit (p =.041), Electronic supplementry mteril The online version of this rticle (doi: /s ) contins supplementry mteril, which is ville to uthorized users. Ó Society of Surgicl Oncology 2014 First Received: 5 Novemer 2013; Pulished Online: 18 Mrch 2014 G. M. Tse, FRCPC e-mil: grytse@cuhk.edu.hk CK5/6 (p \.001), CK14 (p =.002), nd B-crystllin (p =.038). However, AR expression ws found only in 37.8 % of immunohistochemiclly defined MA. Of note, AR-MA ppered to hve trend of worse overll survivl thn AR?MA. Conclusions. AR expression ws different in ER? nd ER- cncers nd hd different clinicl implictions. AR lone my not e good mrker for MA sutype. Its expression in MA my hve sustntil prognostic impliction nd s such wrrnts further vlidtion. In rest cncer, the oncogenic role of nucler steroid hormone receptor signling, notly estrogen receptor (ER) nd progesterone receptor (PR) hs een extensively studied nd exploited to develop nti-estrogen therpy. Another nucler steroid hormone receptor, ndrogen receptor (AR), is lso highly expressed in femle rest cncers ([60 70 %) regrdless of ER sttus. 1 5 In contrst to ER nd PR, the roles of AR in rest crcinogenesis re less clerly defined. Different moleculr rest cncer sutypes re chrcterized y ctivtion of different iologicl pthwys. AR is expressed in most of ER? cncers nd ER- cncers tht overexpressed humn epiderml growth fctor receptor 2 (HER2)(ER-HER2? cncers), ut t lower level in triple negtive rest cncers (TNBC). 4,6,7 In ER? cncers, propoptotic effect of AR signling hs een reported. 8,9 AR cn ind directly to ER or compete for estrogen responsive elements, to lock ctivtion of ER downstrem trgets in these cncers. 10,11 The role of AR signling in ER- rest cncer is less cler. Gene profiling hs identified suset of ERcncers, the moleculr pocrine (MA) cncers tht expressed genes of AR signling. 12 MA cncers hve worse outcome compred with other ER- susets, suggesting the oncogenic

2 Distinctive Significnce in ER? &ER- Brest Cncers 2219 potentil of AR signling. 13 In cell line models, AR signling hs een shown to fcilitte tumor cell growth, possily vi upregultion of Wnt/-ctenin signling with HER3, the ctivting inding lignd of HER AR-trgeted therpy hs een developed in prostte cncer. 17 Given its high prevlence nd potentil oncogenic effect in rest cncer, it ws proposed to e therpeutic trget, prticulrly for AR? TNBCs. However, there were other studies showing the opposite result of AR signling in ER- cncers These differences could e ttriuted to vritions in reltive exposure to steroid stimuli nd in cell line models nd suggested possile plsticity in its oncogenic role in ER- rest cncer. Severl recent studies lso demonstrted the prognostic vlue of AR expression in rest cncer. AR expression in ER? rest cncer predicted fvorle prognosis. 6,7,11,21 Aprt from smll proportion of PR- cncers, the mjority of the ER? cncers elonged to PR?HER2- group which hs good prognosis, nd PR?HER2? group tht hs comprtively worse outcome. The prognostic role of AR in these 2 groups of ER? cncers hs not een fully explored. In the ER- cncers, the dt re less cler, vrile results on its prognostic effects hve een reported. 6,7,21 24 Little ws known on the prognostic significnce of AR expression in MA cncers. The im of this study is to investigte the clinicl significnce of AR in rest cncer. We nlyzed the expression of AR in lrge cohort nd correlted with vrious clinicopthologicl fetures, iomrker expression nd its ssocited moleculr groupings. Furthermore, AR expression ws correlted with outcome dt to evlute its prognostic impliction. MATERIALS AND METHODS Ptients Dt The histologic files of the 3 involved institutions were serched for rest crcinom over period of 4 ( ), 7 ( ), nd 2 ( ) yers. All consecutive cses with excision specimens were included. The study ws pproved y Joint Chinese University of Hong Kong New Territories Est Cluster clinicl reserch ethics committee. All the specimens were routinely processed nd stined with hemtoxylin nd eosin (H&E). Dignosis ws confirmed (WHO criteri) nd grded (Bloom nd Richrdson grding). 25,26 Lymphovsculr invsion (LVI), mrgin sttus, necrosis, phenotypic pocrine fetures, extensive in situ components (EIC), nd firotic focus were evluted s previously. 27,28 Ptients ge, tumor size, lymph node involvement, pn stge, pt stge, nd outcome dt were retrieved from the medicl records. Overll survivl (OS) ws defined s the time intervl from the dte of initil dignosis to the dte of rest cncer relted deth. Disesefree survivl (DFS) ws defined s the durtion from the dte of initil dignosis to the first detection of rest cncer specific relpse or deth. Tissue Microrry Construction nd Immunohistochemistry TMAs contining representtive tumor res were constructed with duplicted 1.5-mm cores s previously descried. 28 One section from ech TMA ws stined with H&E nd reviewed to confirm the presence of representtive tumors. Immunohistochemicl stining ws performed on the TMA with the selected ntiodies using Ultrview Universl DAB Detection Kit (Ventn, Tucson, AZ) fter deprffiniztion, rehydrtion, nd ntigen retrievl of the slides. All slides were counterstined with hemtoxylin. The TMA slides were ssessed for the stining intensity, nd the ctul percentge of stined cells in the nucleus, cytoplsm, or memrne ccording to different ntiodies y 2 of the uthors linded to the clinicl informtion nd the stining results of other mrkers. The stining ws considered positive when there ws moderte or strong immunorectivity t the pproprite loction over the cutoff point. Further detils of stining nd their ssessment re shown in Supplementry Tle S IHC surrogtes for moleculr sutype clssifiction re: luminl A (ER/nd PR?, HER2-,Ki67-), luminl B (ER/ nd PR?, HER2?/nd Ki67?), HER2-overexpressed (HER2-OE) (ER-, PR-, HER2?), sl-like rest cncer (BLBC) (ER-, PR-, HER2-, CK5/6?/, nd EGFR?), nd unclssified (5NP) (ER-, PR, HER2-, CK5/6-, EGFR-). 32,33 Sttisticl Anlysis The findings were nlyzed using the sttisticl softwre SPSS for Windows, Version 18. Chi-squre nlysis or Fisher exct test ws used to test for the ssocition of AR expression with tumor grde, lymph node sttus, pn, pt, lymphocyte infiltrtion, LVI, EIC, pocrine, necrosis, moleculr sutypes, nd iomrkers. Mnn-Whitney U test ws used for nlyzing the differences in ptients ge nd tumor size with AR expression. Survivl dt were evluted with Kpln Meier nlysis. Multivrite nlysis with ckwrd Wld model ws used. Sttisticl significnce ws estlished t p \.05. RESULTS A totl of 1144 primry invsive rest cncers were included in this cohort. Detils of the clinicopthologicl

3 2220 J. Y. S. Tsng et l. TABLE 1 Correltion with clinicopthologicl fctors Grde AR, N (%) Totl p vlue Negtive Positive \.001, 1 56 (32.4) 117 (67.6) (47.7) 250 (54.3) (64.4) 181 (35.6) 509 Totl 594 (52.0) 548 (48.0) 1142 LVI.124 Asent 389 (50.3) 385 (49.7) 774 Present 167 (55.5) 134 (44.5) 301 Totl 556 (51.7) 519 (48.3) 1075 EIC.003 Asent 491 (54.3) 413 (45.7) 904 Present 97 (43.3) 127 (56.7) 224 Totl 588 (52.1) 540 (47.9) 1128 Apocrine.256 Asent 543 (52.4) 493 (47.6) 1036 Present 45 (46.4) 52 (53.6) 97 Totl 588 (51.9) 545 (48.1) 1133 FF.647 Asent 436 (51.5) 410 (48.5) 846 Present 144 (53.1) 127 (46.9) 271 Totl 580 (51.9) 537 (48.1) 1117 Necrosis \.001, Asent 408 (46.3) 474 (53.7) 882 Present 165 (76.7) 50 (23.3) 215 Totl 573 (52.2) 524 (47.8) 1097 Mrgin.001, Round 122 (61.9) 75 (38.1) 197 Infiltrtive 337 (48.1) 363 (51.9) 700 Totl 459 (51.2) 438 (48.8) 897 LN involvement.058 Asent 276 (5) 276 (5) 552 Present 308 (55.7) 245 (44.3) 553 Totl 584 (52.9) 521 (47.1) 1105 pt \.001, (44.6) 257 (55.4) (52.3) 251 (43.7) (65.6) 21 (34.4) (73.7) 5 (26.3) 19 Totl 585 (52.3) 534 (47.7) 1119 pn (50.7) 264 (49.3) (54.8) 157 (45.2) (54.0) 57 (46.0) (62.0) 30 (38.0) 79 Totl 577 (53.2) 508 (46.8) 1085 Sutype \.001, Lum A 245 (45.3) 296 (54.7) 541 TABLE 1 continued AR, N (%) Totl p vlue Negtive Positive Lum B 134 (42.2) 182 (57.8) 316 HER2-OE 77 (72.0) 30 (28.0) 107 BLBC 61 (84.7) 11 (15.3) 72 5NP 66 (73.3) 24 (26.7) 90 Totl 583 (51.8) 543 (48.2) 1126 Age.891 Men SD Rnge Size \.001 Men SD Rnge Sttisticlly significnt Clinicopthologicl fctors tht re significntly ssocited with AR sttus in multivrite nlysis fetures of this cohort re summrized in Tle 1. Overll, 549 cses (48.0 %) were positive for AR nd 595 cses (52.0 %) were negtive. AR nd Tumor Clinicopthologicl Chrcteristics AR expression ws ssocited with lower tumor grde (p \.001), presence of EIC (p =.003), infiltrtive mrgin (p =.001), sence of necrosis (p \.001), nd lower pt stge (p \.001), ut not relted to LVI, phenotypic pocrine fetures, nd nodl sttus (Tle 1). Except EIC, ll fctors remined ssocited with AR expression in multivrite nlysis (Supplementry Tle S2). Among the 1126 invsive cncers with complete dt for moleculr clssifiction, 541 (48.0 %) were luminl A, 316 (28.1 %) were luminl B, 107 (9.5 %) were HER2-OE nd 162 (14.4 %) were TNBC (including 72 cses [6.4 %] of BLBC nd 90 cses [8.0 %] unclssified).the expression rte of AR ws 54.7 % in luminl A, 57.6 % in luminl B, 28.0 % in HER2-OE, nd 21.6 % in TNBC (15.3 % in BLBC nd 26.7 % in unclssified). Significnt difference in AR positivity ws found mong different moleculr sutypes (p =.001). AR correlted positively with ER (p \.001), PR (p [.001), nd GCDFP-15 (p \.001), ut negtively with c-kit (p =.035), CK5/6 (p \.001), CK14 (p \.001), nd B-crystllin (p =.002) (Tle 2). However, there ws no significnt correltion with other iomrkers, including HER2, EGFR, nd p63.

4 Distinctive Significnce in ER? &ER- Brest Cncers 2221 TABLE 2 Correltion with other iomrkers ER AR, N (%) Totl p vlue Negtive Positive Negtive 256 (75.3) 84 (24.7) 340 Positive 337 (42.2) 461 (57.8) 798 Totl 593 (52.1) 545 (47.9) 1138 PR \.001 \.001 Negtive 270 (70.5) 113 (29.5) 383 Positive 311 (41.9) 432 (58.1) 743 Totl 581 (51.6) 545 (48.4) 1126 GCDFP-15 \.001 Negtive 468 (57.3) 349 (42.7) 817 Positive 116 (37.5) 194 (62.5) 310 Totl 584 (51.2) 543 (48.2) 1127 Ki Low 346 (5) 343 (49.8) 689 High 237 (54.4) 199 (45.6) 436 Totl 583 (51.8) 542 (48.2) 1125 HER2.055 Negtive 468 (5) 460 (49.6) 928 Positive 118 (57.8) 86 (42.2) 204 Totl 586 (51.8) 546 (48.2) 1132 HER2 IHC score (55.3) 248 (44.7) (40.7) 150 (59.3) (42.3) 62 (51.7) (58.1) 86 (42.2) 204 Totl 586 (51.8) 546 (48.2) 1132 EGFR.163 Negtive 543 (51.3) 516 (48.7) 1059 Positive 38 (61.3) 25 (39.7) 63 Totl 581 (51.8) 541 (48.2) 1122 C-kit.035 Negtive 470 (5) 462 (49.6) 932 Positive 109 (58.9) 76 (41.1) 185 Totl 579 (51.8) 538 (48.2) 1117 P Negtive 556 (51.6) 522 (48.4) 1078 Positive 25 (56.8) 19 (43.2) 44 Totl 581 (51.8) 541 (48.2) 1122 CK5/6 \.001 Negtive 489 (48.9) 512 (51.1) 1001 Positive 95 (77.9) 27 (22.1) 122 Totl 584 (52.0) 539 (48.0) 1123 CK14 Negtive 530 (50.3) 524 (49.7) 1054 Positive 52 (73.2) 19 (26.8) 71 Totl 582 (51.7) 543 (48.3) 1125 \.001 TABLE 2 continued B-crystllin AR, N (%) Totl p vlue Negtive Positive Negtive 199 (42.7) 267 (57.3) 466 Positive 39 (63.9) 22 (36.1) 61 Totl 238 (45.1) 289 (54.9) 527 Sttisticlly significnt Spermn correltion AR in ER Positive nd ER Negtive Tumors AR ws expressed in 57.8 % (461 of 798) of ER? nd 24.7 % (84 of 340) of ER- cncers. In the ER? cncers, similr to the entire cohort, AR ws ssocited with lower grde (p \.001), the sence of necrosis (p \.001), the presence of infiltrtive mrgin (p =.044), nd lower pt stge (p \.001).The former 2 were ssocited with AR in multivrite nlysis. AR ws lso positively ssocited with PR (p \.001) nd GCDFP-15 (p =.001) (Tle 3). In the ER- cncers, AR correlted with sence of necrosis (p =.014), presence of EIC (p =.006), nd pocrine phenotype (p \.001), ut not tumor grde, pt stge, or mrgin sttus. Necrosis nd pocrine were shown to e independently ssocited with AR expression. With the iomrkers, AR ws ssocited positively with GCDFP-15 (p \.001), HER2 (p =.026), nd EGFR (p =.048), ut negtively with c-kit (p =.041), CK5/6 (p \.001), CK14 (p =.002), nd Bcrystllin (p =.038) (Tle 3). The negtive ssocitions with sl mrkers were oserved within TNBC, suggesting tht AR ws genuinely nd preferentilly expressed in the nonsl TNBC (Supplementry Tle S3). Recently, IHC definition using ER- nd HER2? nd/or GCDFP-15? ws proposed to identify MA cncers with 94 % sensitivity nd 100 % specificity. 34 Using this pnel, MA cncers were found in 52.1 % (176 of 338) of ERcses, nd these MA cncers showed little overlp with BLBC (8 of 176 cses of IHC defined were lso clssified s BLBC). So defined, MA cncers in this cohort were ssocited negtively with sl mrker expression ut positively with LVI, EIC, LN sttus, nd phenotypic pocrine fetures. Among these MA cncers, 66 cses (37.8 %) were AR positive. A significnt ssocition of AR expression with MA cncers ws oserved (p \.001) (Supplementry Tle S4). Reltionship with Ptient Outcome in Different Sugroup of Brest Cncer.002 Follow-up dt were ville in 994 cses with men follow-up durtion of 62.8 months (rnge, months).

5 2222 J. Y. S. Tsng et l. TABLE 3 Assocition of AR expression of clinicopthologicl fetures nd iomrker expression ccording to ER sttus ER-ve ER?ve AR, N (%) Totl p vlue AR N (%) Totl p vlue Negtive Positive Negtive Positive Clinicopthologicl fetures Grde.756 \.001, 1 9 (81.8) 2 (18.2) (29.0) 115 (7) (71.6) 21 (28.4) (4) 228 (59.4) (76.1) 61 (23.9) (53.2) 118 (46.8) 252 Totl 256 (75.3) 84 (24.7) (42.2) 461 (57.8) 798 LVI Asent 169 (74.1) 59 (25.9) (4) 323 (59.6) 542 Present 73 (81.1) 17 (18.9) (44.5) 117 (55.5) 211 Totl 242 (76.1) 76 (23.9) (41.6) 440 (58.4) 753 EIC Asent 221 (78.6) 60 (21.4) (43.4) 351 (56.6) 620 Present 33 (61.9) 21 (38.9) (37.9) 105 (62.1) 169 Totl 254 (75.9) 81 (24.1) (42.2) 456 (57.8) 789 Apocrine \.001,.461 Asent 224 (79.4) 58 (2) (42.4) 433 (57.6) 752 Present 30 (53.6) 26 (46.4) (36.6) 26 (63.4) 41 Totl 254 (75.2) 84 (24.8) (42.1) 459 (57.9) 793 FF Asent 195 (75.0) 65 (25.0) (41.2) 343 (58.8) 583 Present 57 (78.1) 16 (21.9) (44.2) 110 (55.8) 197 Totl 252 (75.7) 81 (24.3) (41.9) 453 (58.1) 780 Necrosis.014, \.001, Asent 145 (71.4) 58 (28.6) (38.9) 413 (61.1) 676 Present 105 (83.3) 21 (16.7) (67.4) 29 (32.6) 89 Totl 250 (76.0) 79 (24.0) (42.2) 442 (57.8) 765 Mrgin Round 60 (82.2) 13 (17.8) (5) 62 (5) 124 Infiltrtive 130 (71.8) 51 (28.2) (4) 310 (6) 517 Totl 190 (74.8) 64 (25.2) (42.0) 372 (58.0) 641 LN involvement Asent 118 (75.7) 38 (24.3) (39.8) 237 (6) 394 Present 134 (76.1) 42 (23.9) (46.4) 201 (53.6) 375 Totl 252 (75.9) 80 (24.1) (43.0) 438 (57.0) 769 pt.328 \ (72.6) 31 (27.4) (35.6) 224 (64.4) (76.9) 42 (23.1) (46.9) 208 (53.1) (79.3) 6 (20.7) (53.1) 15 (46.9) (8) 2 (2) 10 6 (67.7) 3 (33.3) 9 Totl 253 (75.7) 81 (24.3) (42.4) 450 (57.6) 781 pn (76.3) 36 (23.7) (4) 227 (59.6) (77.2) 23 (22.8) (45.9) 132 (54.1) (75.0) 10 (25.0) (44.0) 47 (56.0) (75.8) 8 (24.2) (52.2) 22 (47.8) 46 Totl 249 (76.4) 77 (23.6) (43.3) 428 (56.7) 755

6 Distinctive Significnce in ER? &ER- Brest Cncers 2223 TABLE 3 continued ER-ve ER?ve AR, N (%) Totl p vlue AR N (%) Totl p vlue Negtive Positive Negtive Positive Sutype Lum A 18 (78.3) 5 (21.7) (43.8) 291 (56.2) 518 Lum B 25 (64.1) 14 (35.9) (39.4) 168 (6) 277 HER2-OE 77 (72.0) 30 (28.0) 107 BLBC 61 (84.7) 11 (15.3) 72 5NP 66 (73.3) 24 (26.7) 90 Totl 247 (74.6) 84 (25.4) (42.3) 459 (57.7) 795 Age Men SD Rnge Size.301 \.001 Men SD Rnge Biomrker PR.277 \.001 Negtive 206 (76.0) 65 (24.0) (57.7) 47 (42.3) 111 Positive 43 (69.4) 19 (3) (39.4) 413 (6) 681 Totl 249 (74.8) 84 (25.2) (41.9) 460 (58.1) 792 KI Low 109 (74.7) 37 (25.3) (42.7) 306 (56.3) 543 High 140 (74.9) 47 (25.1) (39.1) 151 (60.9) 248 Totl 249 (74.8) 84 (25.2) (42.2) 457 (57.8) 791 GCDFP15 \.001 \.001 Negtive 210 (83.3) 42 (16.7) (45.8) 305 (54.2) 563 Positive 44 (51.2) 42 (48.8) (32.1) 152 (67.9) 224 Totl 254 (75.1) 84 (24.9) (41.9) 457 (58.1) 787 HER Negtive 165 (78.9) 44 (21.1) (42.3) 414 (57.7) 717 Positive 85 (68.0) 40 (32.0) (41.8) 46 (58.2) 79 Totl 250 (74.9) 84 (25.1) (42.2) 460 (57.8) 796 HER2 IHC score.001,c.093 c (85.3) 20 (14.7) (54.2) 226 (54.2) (68.4) 12 (31.6) (35.8) 138 (64.2) (65.7) 12 (34.3) (41.2) 50 (58.8) (68.0) 40 (32.0) (41.8) 46 (58.2) 79 Totl 250 (74.9) 84 (25.1) (42.2) 460 (57.8) 796 HER2/GCDFP15 \ Negtive 145 (89.0) 18 (1) (42.1) 271 (53.9) 503 Positive 102 (62.3) 66 (37.7) (34.6) 185 (65.4) 283 Totl 254 (75.1) 84 (24.9) (42.0) 456 (58.0) 786 EGFR Negtive 222 (76.8) 67 (23.2) (41.8) 447 (58.2) 768 Positive 27 (62.8) 16 (37.2) (55.0) 9 (45.0) 20 Totl 249 (75.0) 83 (25.0) (41.2) 456 (57.8) 788

7 2224 J. Y. S. Tsng et l. TABLE 3 continued ER-ve ER?ve AR, N (%) Totl p vlue AR N (%) Totl p vlue Negtive Positive Negtive Positive C-kit Negtive 174 (71.6) 69 (28.4) (43.0) 393 (57.0) 689 Positive 72 (82.8) 15 (17.2) (37.8) 61 (62.2) 98 Totl 246 (74.5) 84 (25.5) (42.3) 454 (57.7) 787 P Negtive 230 (74.7) 78 (25.3) (42.3) 444 (57.7) 770 Positive 17 (73.9) 6 (26.1) 23 8 (38.1) 13 (61.9) 21 Totl 247 (74.6) 84 (25.4) (42.2) 457 (57.8) 791 CK5/6 \ Negtive 167 (70.5) 70 (29.5) (42.3) 439 (57.7) 761 Positive 83 (89.2) 10 (1) (41.4) 17(58.6) 29 Totl 250 (75.8) 80 (24.2) (42.3) 456 (57.7) 790 CK Negtive 206 (72.8) 81 (28.2) (42.4) 441 (57.6) 765 Positive 43 (93.5) 3 (6.5) 46 9 (36.0) 16 (64.0) 25 Totl 249 (74.8) 84 (25.2) (42.2) 457 (57.8) 790 B-crystllin Negtive 54 (61.4) 34 (38.6) (38.4) 233 (61.6) 378 Positive 32 (8) 8 (2) 40 7 (33.3) 14 (66.7) 21 Totl 86 (67.2) 42 (32.8) (38.1) 247 (61.9) 399 -ve nonoverexpressing,?ve overexpressing Sttisticlly significnt Clinicopthologicl fctors tht re significntly ssocited with AR sttus in multivrite nlysis c Spermn correltion Of these, 152 cses (15.3 %) hd rest cncer specific mortlity or relpse. AR expression ws ssocited with etter OS nd DFS (log rnk = 9.266, p =.002; log rnk = , p \.001, respectively) (Supplementry Fig. S1A nd S1B). Strtifying y ER sttus, in ER? cncers, AR expression ws ssocited with longer DFS (log rnk = 5.009, p =.025) (Fig. 1), prticulrly for PR?HER2? cses. In the ER?HER2- cncers cses, no significnt difference in OS nd DFS ws detected (Fig. 1). In the ER?HER2? cses, AR?PR? expression showed significntly etter DFS thn AR-PR? cncers (log rnk = 4.778, p =.029) while there ws no significnt differences etween PR- AR? nd PR-AR- cncers (Fig. 1c). Although AR ws not found to e n independent prognostic fctor for DFS in the overll ER? cohort, it ws found to hve significnt prognostic vlue in the ER?HER2?PR? suset (HR = 51, 95 % CI , p =.045) in multivrite nlysis (Supplementry Tle S5). In ER- cses, AR expression did not show ny significnt reltionship with ptients survivl. Compring MA with BLBC, ptients with MA cncers ppered to hve n intermedite survivl compred with BLBC nd non-(ma nd BLBC). While significnt differences in DFS nd OS were found etween BLBC nd non-(ma nd BLBCs) (log rnk = 5.252, p =.022; log rnk = 7.259, p =.007, respectively) (Fig. 2 nd 2), MA cncers showed mrginlly worse OS (log rnk = 3.132, p =.077) thn non-(ma nd BLBCs) (Fig. 2). Interestingly, though not with DFS (Fig. 2d), mrginlly worse OS compred to non-(ma nd BLBCs) ws found with AR-MA (log rnk = 3.207, p =.077) ut not AR?MA (log rnk = 0.927, p =.336) (Fig. 2c). It ppered tht worse outcome in MA ws more relted to the AR-MA rther thn AR?MA. DISCUSSION Brest cncer is highly hormone dependent. Recent moleculr studies indicted tht AR signling could ply n importnt role in rest cncer, reviving interest of AR in rest crcinogenesis. 12,35 This study nlyzed AR expression

8 Distinctive Significnce in ER? &ER- Brest Cncers 2225 FIG. 1 Kpln Meier nlysis on DFS ccording to AR expression in ER? cncers () ER?HER2- (), nd ER?HER2? cncers (c) AR negtive AR positive A 0 Log rnk=5.009, p= DFS B Log rnk=2.530, p=0.112 Log rnk=05, p= DFS C Log rnk=4.778, p=29 Log rnk=0.109, p= DFS AR-PR+ AR+PR+ AR-PR- AR+PR- 200 in lrge cohort of rest cncers in reltionship with comprehensive list of clinicopthologicl fetures nd iomrker pnel. Anlysis on different susets of rest cncer ws lso performed. Although the expression rte of AR ws slightly lower thn in other studies (with[60 %), its rte in ER- cncers ws comprle to tht previously reported (Tle 1).7 The lower rte could e relted to the reltive younger ge in our cohort s AR expression hs een shown to e correlted with postmenopusl sttus. 1 AR expression ws found predominntly in ER? cncers, nd ssocited with PR expression, low grde, nd lymph node involvement (Tles 1 nd 2). 4,7,36 Interestingly, the ssocitions of AR with these vrious fctors differed in ER? nd ER- cncers (Tle 3), suggesting potentil different roles of AR pthwy. In ER? cncers, AR expression ws found to e ssocited with lower pt, lower grde, nd PR positivity (Tle 3) nd concordntly etter outcome (Fig. 1). The fvorle effect of AR expression could due to the inhiition y AR in ER signling. 10,11 Nonetheless, in the current study, the previously suggested independent prognostic role AR hs not een demonstrted (Supplementry Tle S5). 7,11 Here, ptients survivl ws further nlyzed sing on the 2 different profiles of luminl B cncers. 32 In HER2? luminl B, AR expression showed n independent fvorle effect, ut no difference ws found in HER2- luminl B (Fig. 1). AR expression ws suggested to identify ptients with etter prognosis in luminl B cncers. 37 Our results hve further refined the notion showing tht its fvorle effect my e limited to HER2? luminl B cncers. Notly, loss of AR cn ccelerte HER2-induced rest cncer in mice. 38 AR ws shown to regulte gene expression of HER2 pthwys in ER- cncers. 39 Similr mechnisms could possily operte in ER? cncers. In ER- cncers, AR expression ws ssocited with phenotypic pocrine feture, lck of necrosis, positive EGFR, nd HER2 expression, s well s the sence of sl mrkers expression (including C-kit, CK5/6, CK14, nd B-crystllin) (Tle 3). Gene expression profiling studies hve lso descried the MA sutype s hving phenotypic pocrine fetures, ctive AR pthwy, nd frequent HER2 mplifiction. 12 Ptients outcome of MA ws proposed to e similr to BLBCs. ER-PR-AR? ws used to define the MA sugroup. 4,12 Our findings lso indicted tht AR expression could identify distinct sugroup of ER- cncers. A new IHC signture for MA (ER- HER2? nd/or GCDFP15?) ginst gene expression profiling ws

9 2226 J. Y. S. Tsng et l. FIG. 2 Comprison of DFS etween BLBC nd MA ( nd, respectively) nd etween BLBC nd MA ccording to AR expression (c nd d, respectively) Non-BLBC/MA BLBC MA A 0 Log rnk=7.259, p=07 Log-rnk=3.132, p= OS B Log rnk=5.252, p=22 Log-rnk=1.462, p= DFS Non-BLBC/MA BLBC AR- MA AR+ MA C Log rnk=3.207, p=77 Log-rnk=0.927, p= OS D Log rnk=2.329, p=0.127 Log-rnk=26, p= DFS very recently proposed. 34 This definition ws used to identify MA cncers in this cohort. In the correltion studies, the results with MA cncer were very similr to those of AR? cncers (Tle 3 nd Supplementry Tle S4). However, [50 % of IHC-defined MA cncers did not express AR, prticulrly in the ER-HER2? cses. Likewise, [50 % of cncers with phenotypic pocrine fetures were AR- (Tle 3). The result indicted tht AR ssessed y IHC my not e sufficient for MA identifiction. In fct, the MA sutype is chrcterized y the ctivtion of AR pthwy. AR-trgeted gene ctivtion could possily occur through lso other signling pthwys in n AR independent mnner. 39 In ddition, the IHC-defined MA cncers did not show worse outcome compred with BLBC, contrry to previous suggestions (Fig. 2). AR?MA cncers pper to e less ggressive thn AR-MA cncers (Fig. 2c). Further vlidtion is required. A mjor clinicl interest is the use of AR-trgeted therpy in ER- rest cncer. Our results rised the need to further chrcterize the role of AR signling in ER- cncers. One limittion of the current study ws tht FISH nlysis ws not performed on HER2 equivocl cses; thus some of them could e misclssified s negtive. However, equivocl cses constituted low percentge in HER2 negtive cses (Tle 2), nd mong them, sed on our previous nlysis, only minor proportion could hve gene mplifiction. It is unlikely to ffect the overll conclusion. Even if ll equivocl cses were considered s HER2 positive in our nlysis, the finl conclusions remins the sme. In summry, AR expression ws different in ER? nd ER- cncers. In ER? cncers, AR expression ws ssocited with fvorle clinicopthologicl fetures nd etter outcome, prticulrly in the ER?HER2? cncers, suset of luminl B cncers. In ER- cncers, AR expression ws ssocited with fetures distinct from BLBC; nd such fetures were found in MA cncers. However, AR lone did not pper to e good mrker for MA cncers when correlted with the newly proposed IHC definition. AR in MA cncers my hve sustntil prognostic impliction nd s such wrrnts further vlidtion. REFERENCES 1. Agoff SN, Swnson PE, Linden H, Hwes SE, Lwton TJ. Androgen receptor expression in estrogen receptor-negtive rest cncer. Immunohistochemicl, clinicl, nd prognostic ssocitions. Am J Clin Pthol. 2003;120:

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Prognostic significance of pretreatment serum levels of albumin, LDH and total bilirubin in patients with nonmetastatic

Prognostic significance of pretreatment serum levels of albumin, LDH and total bilirubin in patients with nonmetastatic Crcinogenesis, 2015, Vol. 36, No. 2, 243 248 doi:10.1093/crcin/bgu247 Advnce Access publiction December 18, 2014 Originl Mnuscript originl mnuscript Prognostic significnce of pretretment serum levels of