Pr clone 16

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) were stained using formalin fixed paraffin-embedded tissue sections as previously described [23 ,24 ]. ER positivity and PR expression on immunohistochemistry were defined according to the modified Allred system. In our study, ER and PR positivity were defined as an Allred score ≥3. HER2 staining was analyzed according to the American Society of Clinical Oncology/College of American Pathologists guidelines [25 ]. HER2 immunostaining was considered positive when strong membranous staining (3+) was observed, whereas 0 and 1 + were regarded as negative. The cases showing 2 + HER2 expression were further evaluated for HER2 amplification via silver in situ hybridization. Nuclear positivity of Ki-67 staining was evaluated, and the percentage of positive tumor cell (range 0%–100%) was reported as the Ki-67 labeling index. For the breast cancer subtyping, the following definitions were used: i) luminal/HER2(−): ER positive and/or PR positive, and HER2 negative; ii) HER2(+): HER2 positive regardless of ER and PR status; iii) TNBC: ER negative, PR negative, and HER2 negative.

In our immunohistochemical study, formalin-fixed, paraffin-embedded tissue sections obtained from surgical specimens were stained using appropriate antibodies specific for the following four markers: ER (1:100 dilution, 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 positivity were defined using the modified Allred system as follows: positive, Allred scores 3–8; negative, Allred scores 0 and 2. HER2 status was defined as positive with a score of 3+ and negative with a score of 0 or 1+. Tumors with a score of 2+ were subjected to a silver-enhanced in situ hybridization analysis according to the manufacturer’s protocol (PathVysion kit; Vysis, Downers Grove, IL, USA or HER2 inform; Ventana Medical Systems). The Ki-67 level was considered high when the Ki-67 proliferation index was ≥ 14%. pCR was defined as no evidence of invasive cancer residues in the breast parenchyma and all axillary lymph nodes (ypT0/is, ypN0) based on the pathological evaluation of surgical specimens after neoadjuvant chemotherapy.