Clone sp2

Tumors obtained from the surgical specimens were detail sampled at the Breast Pathology Division. The pathological features of the tumors were evaluated briefly in all cases and included tumor type, tumor size, presence of lymphovascular invasion (LVI), the type and extent of in situ carcinoma, evaluation of TILs, surgical margins, and axillary lymph node status. All cases were routinely assessed for immunohistochemical findings regarding estrogen receptor (ER) (clone SP1, 1:100 dilution; Biocare Concord, CA, USA), progesterone receptor (PR) (clone SP2, 1:400 dilution; Spring Pleasanton, California, USA), human epidermal growth factor receptor-2 (HER-2; clone SP3, 1:200 dilution; Thermo Waltham, Massachusetts, USA), and Ki67 (clone SP6, 1:100 dilution; Biocare Concord, CA, USA). The cutoff value for ER and PR positivity was at least 1% of tumor cells. Immunohistochemical analysis for HER-2 was scored according to the current guidelines of American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP).

RNAscope assay (Advanced Cell Diagnostics, Cat. No. 323110) was performed according to manufacturer’s protocol on 4 μm deparaffinized sections and hybridised with probes: Mm-Esr1 (ACD, Cat. No. 432861), Mm-Areg (ACD, Cat. No. 430501), Mm-Ppib (ACD, Cat. No. 313911, positive control) and DapB (ACD, Cat. No. 310043, negative control) at 40 °C for 2h and revealed with TSA Plus-Cy3 (Perkin Elmer, Cat. No. NEL744001KT). Rabbit anti-PgR (1:400, clone SP2, Thermo Fisher, Cat. No.: RM-9102-P) and rabbit anti-ERα (1:100; MC20, sc-542 SantaCruz) was incubated overnight at 4 °C and detected with Alexa 488 or Alexa 568 conjugated goat anti-rabbit (1:1000, Life Technology), respectively. Images were captured on confocal Zeiss LSM700 and spots quantified using QuPath and an in-house script, code available from O. Burri at the Bioimaging and Optics platform, EPFL, based on the guide for RNAscope Data Analysis.