Immunohistochemical staining

The fixed tumor tissues were embedded in paraffin and cut into 3–5 µm sections (20). The experiment was performed on with a streptavidin-peroxidase system (OriGene Technologies, Inc., Beijing, China). EGFR and phosphorylated EGFR (p-EGFR) were detected with a rabbit anti-human EGFR antibody (cat. no. ab52894; Abcam) and a rabbit anti-human p-EGFR antibody (cat. no. 36-9700; Thermo Fisher Scientific, Inc.), respectively. Imaging was performed by fluorescence microscopy (DS-Ri1; Nikon Corporation). To determine the IOD indices of EGFR and p-EGFR, 5 representative visual areas that were positive for EGFR or p-EGFR were examined from each tumor. The EGFR and p-EGFR indices for each selected area were analyzed using IPP 6.0 software.