IHC analysis of EGFR expression

Formalin-fixed and paraffin-embedded tissue blocks were cut into 3-μm sections and deparaffinized, rehydrated, and autoclaved at 121°C for 5 min in Target Retrieval solution (pH 6.0; Dako, Glostrup, Denmark) to retrieve antigens. Endogenous peroxidase was blocked using 3% hydrogen peroxide for 5 min at room temperature. After washing with a Tris buffer solution, the sections were incubated with EGFR for 1 h at room temperature. Then, DAKO REAL EnVision Detection System-HRP (Dako) was applied for 30 minutes at room temperature. Finally, the sections were incubated in 3′,3-diaminobenzidine for 5 minutes, before being counterstained with Mayer’s hematoxylin. The sections were dehydrated through two changes of 95% ethanol and two changes of 100% ethanol, and the samples were cleared in three changes of xylene and then mounted. Negative controls were obtained by replacing the primary antibody with nonimmune serum. The immunoreactivity of EGFR was evaluated by two independent researchers who were blinded to patient s’ outcomes.

The expression patterns of EGFR were determined in a semiquantitative manner through light microscopy. Immunoreactivity for EGFR (membrane staining) was categorized according to the presence of tumor cell staining and staining intensity. The intensity of EGFR immunoreactivity was scored using a three-tier system as follows [7, 33]: 1+ (weak intensity), 2+ (moderate intensity), and 3+ (strong intensity) (Figure ​(Figure4).4). A negative EGFR expression is defined as the absence of membrane staining above the background in all tumor cells, whereas a positive EGFR expression is defined the complete or incomplete IHC membrane staining of tumor cells, including intensities of 1+, 2+, or 3+.

(A) Negative expression (magnification, 100×). (B) 1+ expression (weak intensity of membrane staining) (magnification, 100×). (C) 2+ expression (moderate intensity of membrane staining) (magnification, 100X). (D) 3+ expression (strong intensity of membrane staining) (magnification, 100×).