Originl Article Oncol Res Tret 2017;40:167 172 DOI: 10.1159/000458156 Received: September 13, 2016 Accepted: Jnury 26, 2017 Published online: Mrch 15, 2017 Overexpression of FOXM1 Is Potentil Prognostic Mrker in Mle Brest Cncer Syrine Abdeljoued Ilhem Bettieb Meher Nsri b Olf Adouni Aid Gouch Olf El Amine Hmmoud Boussen c Khled Rhl d Amor Gmoudi Deprtment of Immunohistocytology, Slh Azïz Cncer Institute, Tunis, Tunisi; b Deprtment of Medicl Oncology, Slh Azïz Cncer Institute, Tunis, Tunisi; c Deprtment of Oncology, Abderrhmen Mmi Hospitl, Arin,Tunisi; d Deprtment of Surgicl Oncology, Slh Azïz Cncer Institute, Tunis, Tunisi Keywords FOXM1 Mle brest cncer Prognosis Outcome Therpeutic trget Summry Bckground: Severl studies hve outlined biologicl differences between femle nd mle brest cncer (MBC) nd concluded tht MBC should be considered s n entirely seprte disese. Whether FOXM1 hs ny indiction for prognosis in MBC ptients remins unknown. We sought to exmine the expression levels of FOXM1 in MBC nd to identify the reltionship between FOXM1 expression nd ptient survivl. Ptients nd Methods: FOXM1 expression ws evluted in totl of 130 MBC specimens. Results: FOXM1 ws overexpressed in 37% of the MBC smples. FOXM1 overexpression ws significntly ssocited with tumor size (p = 0.045), histologicl grde (p = 0.048), lymph node metstsis (p = 0.012), Ki-67 prolifertion index (p = 0.016), nd moleculr subtypes (p < 0.001). Multivrite nlyses indicted tht FOXM1 ws n independent prognostic fctor for overll survivl in MBC ptients (p < 0.001, hzrd rtio = 0.69 (0.43 0.96)). Conclusions: Overexpression of FOXM1 ws ssocited with well-estblished mrkers of poor prognosis; thus FOXM1 my represent potentil novel prognostic mrker for MBC. 2017 S. Krger GmbH, Freiburg Introduction Mle brest cncer Is n uncommon disese ccounting for less thn 1% of ll cncers in men nd only 1% of ll brest cncers [1, 2]. In 2016, the Americn Cncer Society estimted tht bout 2,600 new cses of mle brest cncer will be dignosed in the United Sttes nd tht bout 440 men will die of the disese [3]. Despite its incresing incidence, mle brest cncer remins n understudied disese, thus prognostic fctors nd tretment decisions for ptients with mle brest cncer re extrpolted from studies crried out on femle brest cncer ptients [4, 5]. Ltely, number of studies conducted on mle brest cncer outlined some notble differences compred to femle brest cncer in prognosis nd survivl [6 9], genomic profiling, driver genes, nd microrna [10 15]. These findings highlight the biologicl differences between mle brest cncer nd its femle counterprt nd suggest tht it should be considered s n entirely seprte disese tht needs further investigtion in order to define n optiml clinicl mngement for these ptients. The Forkhed box protein M1, FOXM1, is member of the Forkhed superfmily of trnscription fctors [16, 17]. FOXM1 plys key role in cell prolifertion nd cell cycle progression by promoting both G1/S trnsition nd G2/M progression [18]. Furthermore, mny studies demonstrted tht FOXM1 contributes to ll hllmrks of cncer [19, 20], which explins the numerous emerging studies indicting FOXM1 s n importnt trget for cncer therpy in the ner future tht offers exciting prospects [21 24]. Overexpression of FOXM1 hs been reported in lrge vriety of humn cncers including femle brest cncer [25 27], nd it correlted with tumor progression nd poor prognosis [28]. However, the prognostic vlue of FOXM1 overexpression in mle brest cncer remins unknown. In this study, we sought to investigte the FOXM1 expression level in mle brest cncer nd to evlute its prognostic impct in mle ptients with brest cncer. Fx +49 761 4 52 07 14 Informtion@Krger.com www.krger.com 2017 S. Krger GmbH, Freiburg Accessible online t: www.krger.com/ort Dr. Syrine Abdeljoued Deprtment of Immunohistocytology Slh Azïz Cncer Institute Bb Sdoun, 1006, Tunis, Tunisi bdeljoued.syrine@hotmil.fr
Ptients nd Methods Ptients nd Tissue Smples We reviewed 130 cses of mle brest cncer dignosed t the Tunisin Slh Azïz Cncer Institute between 2004 nd 2013. Only ptients with vilble formlin-fixed prffin-embedded mteril nd complete clinicopthologicl dt were included. Clinicl dt included ge, tumor size, histologicl type, histologicl grde, lymph node sttus, TNM stge, distnt metstsis, nd follow-up informtion. 2 experienced pthologists independently grded ll tumors following the Nottinghm grding system [29]. The study ws pproved by the institutionl ethics committee. Immunohistochemistry Hemtoxylin & eosin-stined slides of ech mle brest cncer ptient were reviewed; then representtive res of invsive crcinom were mrked, nd their corresponding tissue blocks were selected. Immunohistochemicl ssys were performed on 4-μm-thick prffin-embedded tumor tissue sections. Briefly, the formlin-fixed prffin-embedded tissue slides were dewxed in xylene nd rehydrted grdully through 3 lcohol chnges. All sections were incubted in boiling citrte buffer (0.01 M, ph 6.0) for ntigen retrievl, nd endogenous peroxidse ctivity ws blocked in 3% hydrogen peroxide. A Snt Cruz blocking serum kit (ImmunoCruz TM rbbit ABC Stining System: sc-2018, Snt Cruz Biotechnology Inc., Snt Cruz, CA, USA) ws used to decrese bckground stining. The slides were then incubted with primry ntibody (nti-foxm1; 1: 300; H-300, Snt Cruz Biotechnology Inc.) t 4 C overnight. Sections were incubted with Snt Cruz biotinylted secondry ntibody nd vidin-biotinylted horserdish peroxidse complex s detection regent, counterstined with hemtoxylin, dehydrted, nd mounted. A brest cncer smple known to express FOXM1 ws used s positive control, while omission of the primry ntibody ws used s negtive control. In ddition, immunohistochemicl nlysis of estrogen receptor (ER), progesterone receptor (PR), humn epiderml growth fctor receptor 2 (HER2), nd Ki-67 prolifertion index were used s immunohistochemicl surrogte mrkers for brest cncer intrinsic subtype definition ccording to the St. Gllen criteri [30]. Evlution of Immunohistochemicl Rection Intensity All slides were evluted by 2 experienced pthologists who were blinded to the ptients dt. FOXM1 expression ws determined bsed on stining intensity nd percentge of cells showing nucler stining with or without cytoplsmic stining using semiquntittive scoring system. Stining intensity ws ssessed s: 0 (negtive), 1 (wek), 2 (moderte), nd 3 (strong). The percentge of FOXM1-positive cells ws scored s follows: 0 (negtive), 1 (1 25%), 2 (26 50%), 3 (51 75%), nd 4 (76 100%). The finl FOXM1 stining score ws determined by multiplying the intensity score nd the percentge score. Score 6 ws used s cutoff to define low FOXM1 expression group nd high FOXM1 expression group (tble 1) [31]. Sttisticl Anlysis Correltion of the expression of FOXM1 with clinicopthologicl fctors ws tested using χ 2 test. The Kpln-Meier method ws used to estblish survivl curves, nd the log-rnk test ws used to perform survivl nlysis. Fctors with significnt prognostic vlue in the univrite Cox regression model were evluted with the multivrite Cox regression model to explore the independent effects on survivl. A p vlue of < 0.05 ws considered sttisticlly significnt. All nlyses were performed using the SPSS 20.0 softwre pckge (IBM Corp., Armonk, NY, USA). Results Clinicopthologicl Fetures of Mle Brest Cncer Ptients The medin ge t dignosis ws 66 yers (rnge 30 96 yers). The medin tumor size ws 2 cm (rnge 0.5 10 cm). 48.5% hd T4 Fig. 1. Immunohistochemicl (IHC) stining of FOXM1 in mle brest cncer tissue: negtive stining for FOXM1 (IHC 250); b wekly positive nucler stining for FOXM1 (IHC 250), inset (IHC 400); c modertely positive nucler stining for FOXM1 (IHC 250), inset (IHC 400); d Strongly positive nucler stining for FOXM1 (IHC 400). Tble 1. Interprettion of FOXM1 immunohistochemicl stining tumors t dignosis. A centrl review of the histopthology reports reveled tht most mle brest cncers were invsive ductl crcinoms (97%; n = 126), 63.8% (n = 83) of cses were grde 2, nd 59.2% (n = 77) hd positive xillry lymph node sttus. Of the 130 mle brest cncer ptients, 90.8% (n = 118) were ER+, 83% (n = 108) were PR+, 7.7% (n = 10) were HER2+, nd 54.6% (n = 71) hd high Ki67 prolifertion index. Using immunohistochemicl surrogtes, 45.4% (n = 59) of our ptients were clssified s luminl A-like, 44.6% (n = 58) s luminl B-like, 5% (n = 6) s HER2-enriched, nd 5% (n = 7) s triple-negtive. 46% (n = 60) of ptients hd distnt metstsis. FOXM1 Expression in Mle Brest Cncer Tissue To investigte the protein expression of FOXM1 in mle brest cncer, we immunostined 130 tissue sections with n nti- FOXM1 ntibody (fig. 1). A predominntly nucler stining pttern ws observed in the mle brest cncer smples. 93% (n = 121) of the mle brest crcinoms expressed FOXM1, only 7% (n = 9) of cses were FOXM1-negtive. However, overll, only 37% (n = 48) were clssified s belonging to the high FOXM1 expression group. SI PCS 0 1 2 3 1 0 1 2 3 2 0 2 4 6 3 0 3 6 9 4 0 4 8 12 High FOXM1 expression group. PCS = Positive cell score; SI = stining intensity. 168 Oncol Res Tret 2017;40:167 172 Abdeljoued/Bettieb/Nsri/Adouni/Gouch/ El Amine/Boussen/Rhl/Gmoudi
Tble 2. FOXM1 expression nd clinicopthologicl chrcteristics in mle brest cncer Chrcteristics Ptients, n (%) FOXM1 expression, n (%) p high low Totl 130 48 (37.0) 82 (63.0) Age, yers 0.102 < 60 34 (26.1) 12 (25.0) 22 (26.8) 60 96 (73.9) 36 (75.0) 60 (73.2) Pthologicl type 0.737 IDC 126 (97.0) 47 (98.0) 79 (96.0) IPC 4 (3.0) 1 (2.0) 3 (4.0) Tumor size, cm 0.045 < 2 41 (31.5) 9 (18.75) 32 (39.0) 2 89 (68.5) 39 (81.25) 50 (61.0) SBR grde 0.048 1 21 (16.2) 3 (6.0) 18 (22.0) 2 83 (63.8) 35 (73.0) 48 (58.5) 3 26 (20.0) 10 (21.0) 16 (19.5) Axillry lymph node sttus 0.012 Negtive 53 (40.8) 13 (27.1) 40 (48.8) Positive 77 (59.2) 35 (72.9) 42 (51.2) Stge 0.487 I II 58 (44.6) 22 (45.8) 36 (43.9) III IV 72 (55.4) 26 (54.2) 46 (56.1) ER sttus 0.474 Negtive 12 (9.2) 5 (10.4) 7 (8.5) Positive 118 (90.8) 43 (89.6) 75 (91.5) PR sttus 0.250 Negtive 22 (17.0) 10 (20.8) 12 (14.6) Positive 108 (83.0) 38 (79.2) 70 (85.4) HER2 sttus 0.880 Negtive 120 (92.0) 42 (87.5) 78 (95.1) Positive 10 (8.0) 6 (12.5) 4 (4.9) Ki-67 index, % 0.016 < 20 59 (45.4) 11 (22.9) 48 (58.5) 20 71 (54.6) 37 (77.1) 34 (41.5) Intrinsic moleculr subtypes < 0.001 Luminl A-like 59 (45.4) 11 (23.0) 48 (58.5) Luminl B-like 58 (44.6) 30 (62.5) 28 (34.2) HER2-enriched 6 (5.0) 1 (2.0) 5 (6.1) Triple-negtive 7 (5.0) 6 (12.5) 1 (1.2) Distnt metstsis 0.111 No 70 (54.0) 22 (45.8) 48 (58.5) Yes 60 (46.0) 26 (54.2) 34 (41.5) IDC = Invsive ductl crcinom; IPC = invsive ppillry crcinom; SBR = Scrff-Bloom-Richrdson; ER = estrogen receptor; PR = progesterone receptor. Assocition of FOXM1 Expression with Clinicopthologicl Fetures We nlyzed the correltion between FOXM1 expression nd clinicopthologicl fetures in order to determine the clinicopthologicl significnce of this mrker in mle brest cncer. FOXM1 expression ws significntly relted to lrger tumor size (p = 0.045), higher histologicl grde (p = 0.048), lymph node metstsis (p = 0.012), high Ki-67 prolifertion index (p = 0.016), nd intrinsic moleculr subtypes (p < 0.001). Regrding the intrinsic moleculr subtypes, FOXM1 ws more expressed in luminl B-like nd triplenegtive subtypes. 51.7% of the luminl B-like cses nd 85.7% of the triple-negtive cses belonged to the high FOXM1 expression group, wheres only 19% of the luminl A-like cses were observed in the high FOXM1 expression group. Ptients with FOXM1 expression hd significntly poorer overll survivl (OS) (p < 0.001). No sttisticlly significnt correltion between FOXM1 nd ge, histologicl type, ER sttus, PR sttus, HER2 sttus, stge, nd distnt metstsis ws observed (tble 2). Prognostic Significnce of FOXM1 Expression Due to high rte of dvnced-stge disese t presenttion nd distnt metstsis t the time of dignosis, 50% of the ptients died Prognostic Impct of FOXM1 in Mle Brest Cncer Oncol Res Tret 2017;40:167 172 169
Tble 3. Univrite nd multivrite Cox regression nlyses of different prognostic fctors for overll survivl in mle brest cncer ptients Vribles Univrite nlysis Multivrite nlysis HR p 95% CI HR p 95% CI FOXM1, low vs. high 0.329 < 0.001 0.190 0.572 0.695 < 0.001 0.430 0.960 Age, 60 vs. > 60 yers 0.558 < 0.001 0.497 0.626 4.268 0.590 0.100 19.918 Pthologicl type, IDC vs. IPC 1.13 0.696 0.599 2.154 Size, < 2 vs. 2 cm 0.877 0.646 0.500 1.537 Grde, 1 vs. 2 3 0.852 0.454 0.559 1.297 Axillry lymph node sttus, negtive vs. positive 0.848 0.552 0.493 1.459 Stge, I II vs. III IV 0.849 0.193 0.664 1.086 ER sttus, negtive vs. positive 0.840 0.803 0.214 3.299 PR sttus, negtive vs. positive 0.585 0.179 0.268 1.279 HER2 sttus, negtive vs. positive 1.07 0.196 0.964 1.196 Ki67 index, < 20 vs. 20% 1.67 0.388 0.518 5.432 Intrinsic moleculr subtypes, LA vs. LB vs. HER2-0.809 0.645 0.329 1.990 nriched vs. TN Distnt metstsis, no vs. yes 0.838 0.556 0.464 1.512 HR = Hzrd rtio; CI = confidence intervl; IDC = invsive ductl crcinom; IPC = invsive ppillry crcinom; ER =estrogen receptor; PR = progesterone receptor; LA = luminl A-like; LB = luminl B-like; TN = triple-negtive. in less thn 12 months fter being dignosed. The medin followup ws 12.5 months (rnge 1 132 months); 118 deths were reported. In the overll popultion, the 5-yer OS rte ws 43% (95 confidence intervl (CI) 0.04 0.52). The Cox proportionl hzrds regression model ws used to exmine the ssocition between clinicopthologicl fetures nd OS. Univrite nlysis showed tht high FOXM1 expression (p < 0.001) nd ge (p < 0.001) were significntly ssocited with OS (tble 3). Moreover, multivrite nlysis identified only FOXM1 expression s n independent prognostic fctor for OS in mle brest cncer ptients (p < 0.001, hzrd rtio (HR) = 0.69 (0.43 0.96)) (tble 3). Furthermore, Kpln-Meier survivl curves reveled tht high FOXM1 expression ws ssocited with poorer prognosis in mle brest cncer (fig. 2). We lso ssessed the prognostic vlue of FOXM1 expression in terms of disese-free survivl (DFS). Univrite nlysis showed tht ge (p < 0.001) nd lymph node metstsis (p < 0.001) were significntly ssocited with DFS, but not FOXM1 expression (p = 0.238). Multivrite nlysis identified ge s the sole independent prognostic fctor for DFS in mle brest cncer ptients (p < 0.001, HR = 1.32 (1.02 16.588)) (tble 4). Discussion Mle brest cncer is n understudied disese tht for long time ws thought to be identicl to its femle counterprt. Ltely, the mle brest cncer reserch field hs witnessed the emergence of new studies tht hve provided evidence tht mle brest cncer is biologiclly different from femle brest cncer [10 15]. Thus, extrpolting from studies crried out on femle ptients with brest cncer is something to be reconsidered. Given the fct tht there re currently few well-defined prognostic mrkers for mle brest cncer, we ssessed FOXM1 s potentil prognostic mrker. Fig. 2. Kpln-Meier nlysis of overll survivl curves in mle brest cncer ptients ccording to FOXM1 expression. FOXM1 is trnscriptionl ctivtor with Forkhed domin tht plys mjor role in the regultion of cell cycle genes nd is thus bckbone of the highly prolifertive nture of cncer cells [32]. Additionlly, long with its min role of stimulting cell cycle progression nd cell growth, FOXM1 promotes ngiogenesis [33, 34] nd contributes to metstsis by stimulting migrtion nd invsion s well s epithelil-mesenchyml trnsition (EMT) [35 37]. Furthermore, FOXM1 countercts senescence nd increses tumor cell resistnce to poptosis [38 40]. FOXM1 lso plys role in promoting the expnsion of undifferentited cncer cells [41]. In short, 170 Oncol Res Tret 2017;40:167 172 Abdeljoued/Bettieb/Nsri/Adouni/Gouch/ El Amine/Boussen/Rhl/Gmoudi
Tble 4. Univrite nd multivrite Cox regression nlyses of different prognostic fctors for disese-free survivl in mle brest cncer ptients Vribles Univrite nlysis Multivrite nlysis HR p 95% CI HR p 95% CI FOXM1, low vs. high 1.450 0.238 0.782 2.686 Age, 60 vs. > 60 yers 0.965 0.000 0.948 0.982 1.32 0.000 1.02 16.588 Pthologicl type, IDC vs. IPC 9.808 0.927 9.226 10.717 Size, < 2 vs. 2 cm 1.119 0.776 0.516 2.427 Grde, 1 vs. 2 3 0.833 0.485 0.500 1.389 Axillry lymph node sttus, negtive vs. positive 4.760 0.000 1.989 11.390 0.400 0.472 0.672 1.472 Stge, I II vs. III IV 0.831 0.235 0.613 1.128 ER sttus, negtive vs. positive 0.541 0.415 0.124 2.367 PR sttus, negtive vs. positive 1.366 0.575 0.459 4.067 HER2 sttus, negtive vs. positive 1.095 0.139 0.971 1.236 Ki67 index, < 20 vs. 20% 0.990 0.493 0.961 1.020 Intrinsic moleculr subtypes, LA vs. LB vs. HER2- enriched vs. TN 0.551 0.323 0.169 1.796 HR = Hzrd rtio; CI = confidence intervl; IDC = invsive ductl crcinom; IPC = invsive ppillry crcinom; ER =estrogen receptor; PR = progesterone receptor; LA = luminl A-like; LB = luminl B-like; TN = triple-negtive. FOXM1 plys centrl role in the mechnistic concepts involved in the development of cncer. In comprtive microrry nlysis conducted by Pilrsky et l. [25], FOXM1 ws consistently nd independently identified s one of the most commonly upregulted genes in solid tumors. Moreover, overexpression of FOXM1 hs been reported in wide rnge of cncers, nmely brest cncer, lung cncer, ovrin cncer, prostte denocrcinom, testiculr cncer, bldder cncer, nd colorectl cncer [17, 42]. In generl, high FOXM1 expression is recognized s n indictor of poor prognosis [43]. Numerous studies reported tht FOXM1 overexpression correlted with tumor histologicl grde or stge nd incidence of metstsis, nd inversely correlted with OS [26, 28, 44]. All in ll, these results suggest tht FOXM1 overexpression correltes with tumor ggressiveness nd progression. Moreover, in severl trils, downregultion of FOXM1 resulted in the inhibition of cncer cell prolifertion nd tumor growth [23, 45]. Tken together, these findings showcse the significnt nd diverse role plyed by FOXM1 in cncer, nd highlight the potentil of FOXM1 trgeted therpy. Despite the fct tht the pnel of mle brest cncer prognostic fctors is limited nd tht there is emerging evidence of the importnce of FOXM1 s prognostic mrker nd therpeutic trget, the prognostic impct of FOXM1 in mle brest cncer hs not been nlyzed so fr. We therefore ssessed FOXM1 s potentil prognostic mrker in mle brest cncer. In this study, we used immunohistochemistry to evlute FOXM1 expression in mle brest cncer tumors. We found FOXM1 to be expressed in 94% of the tumors nd highly expressed in 37% of cses. Interestingly, these results show tht FOXM1 is more expressed in mle thn in femle brest cncer, since previous studies demonstrted tht 87% of femle brest tumors express FOXM1 nd 20% of these were clssified s high-expression [26, 27]. This confirms the ggressive phenotype of mle brest cncer. Additionlly, we found tht FOXM1 expression ws significntly ssocited with lrger tumor size, higher histologicl grde, lymph node metstsis, nd intrinsic moleculr subtypes, which is in line with the literture on femle brest cncer nd other cncer types [31, 43]. Furthermore, FOXM1 expression highly correlted with high Ki-67 prolifertion index (p < 0.001); therefore, we could show tht FOXM1 is prolifertion-ssocited protein in mle brest cncer. It hs been reported tht FOXM1 expression is tightly correlted with HER2 sttus in femle brest cncer, pointing to potentil interction between FOXM1 nd HER2 [26]; however, in our report, we filed to demonstrte significnt ssocition between FOXM1 expression nd HER2 sttus in mle brest cncer, which could be explined by the lower rte of HER2-positive mle brest tumors compred to femle brest cncer [46]. Finlly, we demonstrted tht FOXM1 is n independent prognostic fctor for OS in mle brest cncer, but not for DFS. All in ll, the vst mjority of mle brest tumors expressed FOXM1, referred to by some s the Achilles heel of cncer. While less thn hlf of the cses overexpressed FOXM1, this rte ws higher thn tht reported for femle brest cncer, supporting the emerging evidence tht cler differences between mle nd femle brest cncer re hidden behind biologicl similrities. High expression of FOXM1 ws strongly ssocited with well-estblished mrkers of poor prognosis nd ws identified s n independent prognostic fctor for OS in mle brest cncer. Our study hs some limittions such s the retrospective study design nd the smple size. However, despite these limittions, our findings demonstrte tht FOXM1 hs relible prognostic significnce in mle brest cncer nd my thus become n importnt trget for mle brest cncer therpy in the future. In recent yers, mny studies hve demonstrted the prognostic vlue of FOXM1 for wide rnge of cncers but not mle brest cncer. In our study, we defined FOXM1 s potentil novel prognostic mrker tht could be included in the limited mrker pnel for mle brest cncer. Prognostic Impct of FOXM1 in Mle Brest Cncer Oncol Res Tret 2017;40:167 172 171
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