Immunohistochemistry

Immunohistochemical staining for FAK was performed according to standard procedures. Briefly, 4-µm-thick serial section of formalin-fixed and paraffin-embedded (FFPE) tumor tissue slides were incubated with the primary antibody in a dilution of 1:100 (rabbit anti-human-FAK antibody, Cell Signaling Technology), followed by polymeric biotin-free visualization system (Envision™; DAKO Diagnostic Company, Hamburg, Germany). Finally, the sections were counterstained with Mayer's hematoxylin solution (Sigma-Aldrich®). We used a BC sample strongly positive for FAK as a positive control. Negative controls were prepared by substituting normal mouse serum for primary antibody, and no detectable staining was evident. FAK immunostaining was evaluated by 2 of the authors (S.H. and M.S.) experienced in histological and immunohistochemical diagnostics unaware of the clinical outcome. Since evaluation of FAK expression is not yet standardized, we used the scoring system for estrogen receptor (ER) and progesterone receptor (PR) in accordance to Remmele and Stegner [10]. It was defined by the product of a proportion (0, none; 1, <10%; 2, 10–50%; 3, 51–80%; 4, 81–100%) and an intensity score (0, no staining; 1, weak; 2, moderate; 3, strong) [10]. The median was used to dichotomize the samples into FAK positive (high expression) and negative (low expression) tumors.