IHC investigation for various surrogate markers was performed to substantiate the results of gene expression analysis and to identify basal-like breast carcinomas. Markers investigated included the RTK EGFR, HER2 and IGF-1R, the hormone receptors ER and PR, and the basal-like marker CK5/6 32 . Whole FFPET sections cut at 3 µm were stained with a Ventana Benchmark XT autostainer (Ventana Medical Systems). Details of the antibodies and methods employed are given in Table 3.
Slides were counterstained with haematoxylin (Ventana). System and isotype controls were included. IHC investigation for ER and PR and for HER2 was performed according to up-to-date histopathological guidelines and recommendations 28 , 29 (link).
Immunostaining for EGFR and IGF-1R was evaluated semiquantitatively with a scoring system similar to that established for HER2 (0: no membrane-specific staining, 1+: weak, incomplete cell membrane staining in <10% of cells, 2+: weak or moderate staining of the complete cell membrane in > 10% of cells, 3+: strong, complete membrane staining in > 10% of cells. Immunostaining for CK 5/6 was evaluated according to the criteria of Dabbs et al. 33 (link) (0: no staining, R: single cells stained, 1+: 5-30% of cells stained, 2+: >30-60% of cells stained, 3+: > 60% of cells stained). The scores 0 and R were considered negative, 1+ to 3+ positive.