Her2 inform

As previously described [17 (link)], we evaluated ER, PR, HER2, and Ki67 expression using the following antibodies, ER (1:100 clone 6F11; Novocastra, Newcastle upon Tyne, UK), PR (clone 16; Novocastra), HER2 (4B5 rabbit monoclonal antibody; Ventana Medical Systems, Tucson, AZ, USA), and Ki-67 (MIB-1; Dako, Glostrup, Denmark). ER- and PR-positive IHC expression was defined according to the modified Allred system: positive, Allred score 3–8 and negative, Allred score 0–2. HER2 status was re-evaluated according to American Society of Clinical Oncology/College of American Pathologists guideline [18 (link)]. HER2 status was considered positive if the score was 3+, and was considered negative with a score of 0 or 1+. Tumors with a score of 2+ underwent FISH or SISH analysis, according to the manufacturer’s instructions (PathVysion kit; Vysis, Downers Grove, IL, USA or HER2 inform; Ventana). Ki-67 expression is presented as the percentage (range 0–100%) of positive tumor cells.
For the molecular subtyping, the following definitions were used: i) Luminal/HER2 negative: ER positive and/or PR positive and HER2 negative; ii) HER2 positive: HER2 positive regardless of ER and PR status; and iii) TNBC: ER negative, PR negative, and HER2 negative.

In our immunohistochemistry (IHC) study, core needle biopsy samples were stained using appropriate antibodies specific for four markers: ER (1:100 dilution, clone 6F11; Novocastra, Newcastle upon Tyne, UK), PR (clone 16; Novocastra, UK), HER2 (4B5 rabbit monoclonal antibody; Ventana Medical Systems, Tucson, AZ, USA), and Ki-67 (MIB-1; Dako, Glostrup, Denmark). ER- and PR-positive were defined as a cutoff value of ≥ 1% positively stained nuclei^{41 (link)}, or according to the modified Allred system: positive, Allred scores 2–8,and negative, Allred scores 0^{42 (link)}. The HER2 status was defined as positive with a score of 3+, and negative with a score of 0 or 1+. Tumors with scores of 2+ were sent for fluorescent in situ hybridization analysis according to the protocol given by the supplier (PathVysion kit; Vysis, Downers Grove, IL, USA, or HER2 inform; Ventana)^{43 (link)}. Staining positive for the nuclear antigen Ki-67 was evaluated in a quantitatively and visually way using light microscopes, and the positive tumor cell percentage was reported as the Ki-67 labeling index (LI)^{44 (link)}. We considered Ki-67 levels ≥ 14% as high^{45 (link)}.