Immunohistochemical analysis and scoring

The expression of the estrogen receptor (ER), progesterone receptor (PR), HER2, p53 and Ki-67 was evaluated in the representative tumor sections of the surgical specimens at the time of diagnosis. With regards to the cases with missing data, immunohistochemical staining on representative tissue sections was carried out in a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) using an UltraView detection kit (Ventana Medical Systems). The following antibodies were used: anti-ER (1:100; clone SP1; LabVision, Fremont, CA), anti-PR (1:70; PgR 636; Dako, Carpinteria, CA), anti-HER2 (ready to use; 4B5; Ventana Medical Systems), anti-p53 (1:600; D07; Dako), and anti-Ki-67 (1:250; MIB-1; Dako).

A tumor was regarded as positive for ER or PR if it showed at least 1% positive nuclear staining with the relevant antibody. A tumor was considered as HER2 positive, if it was 3 + on immunohistochemistry or if gene amplification was seen on FISH. Nuclear staining in 10% or more of the tumor cells was considered positive for p53. Nuclear staining in 20% or more of the tumor cells was considered an indication of high Ki-67 proliferation index.

Immunohistochemical expression of the standard biomarkers were used to categorize the tumor samples into breast cancer subtypes. Breast cancer subtypes were categorized according to the criteria used in our previous study¹⁰: luminal/HER2-negative subtype (ER+ and/or PR+, HER2-), luminal/HER2-positive subtype (ER+ and/or PR+, HER2+), HER2-positive subtype (ER-, PR-, HER2+), and triple-negative subtype (ER-, PR-, HER2-).