NuclearandCytoplasmicExpressionofERB1, ERB2,andERB5 IdentifiesDistinctPrognosticOutcomefor BreastCancerPatients AbeerM.Shaaban, 1 Andrew R. Green, 2 SuchitaKarthik, 1 Yalda Alizadeh, 1 Thomas A.Hughes, 1 LynnHarkins, 1 Ian O. Ellis, 2 JohnF.Robertson, 3 Emma C. Paish, 2 PhilippaT.K.Saunders, 4 Nigel P. Groome, 5 and Valerie Speirs 1 Abstract Purpose: Previous conflicting results about the prognostic significance of estrogen receptor (ER)-h in breast cancer may be explained by contribution of isoforms, of which five exist. Our aim was to elucidate the prognostic significance of ERh1, ERh2, and ERh5 by immuno- histochemistryinalarge cohortofbreastcarcinomaswithlong-termfollow-up. ExperimentalDesign: Tissue microarrays were stained with ERh1, ERh2,andERh5antibodies and scored as percentage of positive tumor cells and using the Allred system. Nuclear and cytoplasmic staining was evaluated and correlated with histopathologic characteristics, overall survival (OS), and disease-free survival (DFS). Results: Nuclear ERh2 and ERh5, but not ERh1, significantly correlated with OS (P = 0.006, P =0.039,and P =0.099,respectively),andERh2 additionally with DFS (P = 0.013). ERh2also predicted response to endocrine therapy (P = 0.036); correlated positively with ERa,progester- onereceptor,androgenreceptor,andBRCA1;andcorrelatedinverselywithmetastasisandvascu- lar invasion.Tumors coexpressing ERh2 and ERa had better OS and DFS. Cytoplasmic ERh2 expression, alone or combined with nuclear staining, predicted significantly worse OS. Notably, patients with only cytoplasmic ERh2 expressionhad significantly worse outcome (P = 0.0014). Conclusions: This is the first study elucidating the prognostic role of ERh1, ERh2,andERh5ina large breast cancer series. ERh2 is a powerful prognostic indicator in breast cancer, but nuclear and cytoplasmic expression differentially affect outcome. Measuring these in clinicalbreast cancer could provide a more comprehensive picture of patient outcome, complementing ERa. Estrogen receptor (ER)-a remains the most important marker of response to hormonal therapy in breast cancer. The role of ERh is much less clear with many conflicting studies published to date (reviewed in ref. 1). Although structurally related and homologous at the DNA and ligand binding domains, both receptors are genetically distinct (2, 3). Furthermore, ERh is abundant in normal mammary tissue (4) with loss or diminished expression implicated in mammary carcinogenesis (5–7). ERh exists as five full-length versions, ERh1toERh5 (8, 9), each formed by alternative splicing of the last coding exon. ERh1istheonlyfullyfunctionalisoform(10).ERh2isidentical toERhcx(11).ERh3seemstobetestisspecific(8)andERh4is notfoundinbreasttissue(9).AlthoughERh2andERh5cannot bindligand,transienttransfectionstudiessuggestthattheymay antagonizeERa functionthroughheterodimerization(11–13). FunctionalstudieshaveshownthatoverexpressionofERh1has antiproliferative and proapoptotic effects (14, 15). ERh2 and ERh5 have no intrinsic activities with conflicting data on whether they can (8) or cannot homodimerize (10). However, they can heterodimerize with ERh1 and enhance its transcrip- tional activity in a ligand-dependent fashion (10). If expressed in breast tumors, these could have important clinical implica- tions in influencing response to hormone therapy. DatafromallpreviousimmunohistochemicalstudiesonERh in breast cancers have to be interpreted with caution. First, the number of cases included in many studies was insufficient to infer generalization of results to the general breast can- cer population. Second, in many studies, antibodies used did not discriminate between different ERh isoforms. Third, cutoff Imaging, Diagnosis, Prognosis Authors’Affiliations: 1 YCR and Liz Dawn Pathology andTranslational Sciences Centre, LeedsTeaching Hospitals and Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom; 2 Department of Histopathology and 3 Professorial Unit of Surgery, Nottingham University HospitalsTrust and University of Nottingham, Nottingham, United Kingdom; 4 Medical Research Council Human Reproductive Sciences Unit, Queen’s Medical Research Institute, Edinburgh, United Kingdom; and 5 OxfordBrookesUniversity,Oxford,UnitedKingdom Received10/3/07;revised5/19/08;accepted5/19/08. Grantsupport: LeedsTeachingHospitalsNHSTrustNewInvestigatorAward(A.M. Shaaban);BreastCancerCampaign(A.R.GreenandT.A.Hughes);YorkshireCancer Research, Liz Dawn Breast CancerAppeal, and Breast Cancer Research Action Group (V. Speirs); Breast Cancer ResearchTrust (V. Speirs and A.M. Shaaban); and MedicalResearchCouncil(P.T.K.Saunders). Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely toindicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online(http://clincancerres.aacrjournals.org/). Requests for reprints: Valerie Speirs, Leeds Institute of Molecular Medicine, WellcomeTrust Brenner Building, St. James’s University Hospital, Leeds LS9 7TF, United Kingdom. Phone: 44-113-3438633; Fax: 44-113-3438431; E-mail: v.speirs@ leeds.ac.uk. F 2008AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-07-4528 www.aacrjournals.org ClinCancerRes2008;14(16)August15,2008 5228 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/14/16/5228/1976034/5228.pdf by guest on 17 May 2023