The 4046 tumors assembled into tissue microarrays, in BCCA series, were examined with immunohistochemistry and fluorescent in situ hybridization. Immunohistochemistry for ER, PR, HER2, and Ki67 was performed concurrently on serial sections with the standard streptavidin–biotin complex method with 3,3′-diaminobenzidine as the chromogen. Staining for ER, PR, and HER2 interpretation was as described previously (20 (link)). Briefly, the Ki67 antibody (clone SP6; ThermoScientific, Fremont, CA) was applied at a 1:200 dilution for 32 minutes, by following the Ventana Benchmark automated immunostainer (Ventana, Tucson AZ) standard Cell Conditioner 1 (CC1, a proprietary buffer) protocol at 98°C for 30 minutes. ER antibody (clone SP1; ThermoFisher Scientific, Fremont CA) was used at 1:250 dilution with 10-minute incubation, after an 8-minute microwave antigen retrieval in 10 mM sodium citrate (pH 6.0). Ready-to-use PR antibody (clone 1E2; Ventana) was used by following the CC1 protocol as above. HER2 staining was done with the SP3 antibody (ThermoFisher Scientific) at a 1:100 dilution after antigen retrieval in 0.05 M Tris buffer (pH 10.0) with heating to 95°C in a steamer for 30 minutes. For HER2 fluorescent in situ hybridization assay, slides were hybridized with probes to LSI (locus-specific identifier) HER2/neu and to centromere 17 by use of the PathVysion HER-2 DNA Probe kit (Abbott Molecular, Abbott Park, IL) according to manufacturer's instructions, with modifications to pretreatment and hybridization as previously described (29 (link)). Slides were counterstained with 4′,6-diamidino-2-phenylindole, stained material was visualized on a Zeiss Axioplan epifluorescent microscope, and signals were analyzed with a Metafer image acquisition system (Metasystems, Altlussheim, Germany). Biomarker expression from immunohistochemistry assays was scored by two surgical pathologists (T. O. Nielsen and D. Gao), who were blinded to the clinicopathological characteristics and outcome and who used previously established and published criteria for biomarker expression levels that had been developed on other breast cancer cohorts (12 (link),30 (link)). Tumors were considered positive for ER (27 (link)) or PR (31 ) if immunostaining was observed in more than 1% of tumor nuclei, as described previously. Tumors were considered positive for HER2 if immunostaining was scored as 3+ according to HercepTest criteria, with an amplification ratio for fluorescent in situ hybridization of 2.0 or more being the cut point that was used to segregate immunohistochemistry equivocal tumors (scored as 2+) (32 (link)). Ki67 was visually scored for percentage of tumor cell nuclei with positive immunostaining above the background level by two pathologists (T. O. Nielsen and D. Gao). Tissue microarray core samples with fewer than 50 tumor cells were considered uninterpretable (27 (link),28 (link)). All the stained tissue microarrays were digitally scanned, and primary image data are available for public access (http://www.gpecimage.ubc.ca; username, luminalB; password, luminalb).