Cm405 a

The detailed methods and results of the IHC analysis of expression of: KRT5/6 (PRB-160P, Covance, 1/2000; 0.5 µg/mL), KRT14 (PRB-155P, Covance, 1/2000; 0.5 µg/mL), GATA3 (CM405 A, Biocare Medical, 1/300) and FOXA1 (ab170933, Abcam, 1/100; 10 µg/mL) in this patient series have been reported elsewhere (13 (link)). Tumors were classified in three subtypes according to the IHC results: BASQ-like (FOXA1/GATA3 low; KRT5/6/14 high), luminal-like (FOXA1/GATA3 high; KRT5/6/14 low), and mixed (FOXA1/GATA3 high; KRT5/6 high; KRT14 low).

TURBT formalin-fixed paraffin-embedded (FFPE) blocks were reviewed. Representative tumor areas were selected to extract 3 cores and construct tissue microarrays (TMAs). Cores were positioned non-consecutively to avoid artefacts; TMAs were constructed following the established guidelines and sectioned; and the slides were embedded in paraffin and stored at 4 °C. The following antibodies were used: KRT5/6 (PRB-160P, Covance; 1/2000), KRT14 (PRB-155P, Covance, 1/2000), GATA3 (CM405 A, Biocare Medical, 1/300), FOXA1 (ab170933, Abcam, 1/100), FGFR3 (B9, Santa Cruz), KRT20 (Ks20.8, Dako), and STAG2 (J-12, Santa Cruz). After deparaffinization, antigen retrieval, and endogenous peroxidase blockade, the sections were incubated with antibodies overnight, washed, and Envision secondary reagents (Agilent) were added for 1 h. After washing, the reactions were developed with DAB, the sections were counterstained with hematoxylin, and mounted. Scoring was performed blind to clinical/pathological information by an experienced investigator (FXR). The proportion of reactive cells (0–100%) and staining intensity (0–3) were recorded; the histoscore was calculated as the product: Intensity x % reactive cells (range 0–300). The average of the scores was used for analysis.