High-sensitivity epidermal growth factor receptor immunostaining for colorectal carcinomas, compared with EGFR...

Immunostaining for epidermal growth factor receptor (EGFR) is important in the contemporary therapeutic strategy of colorectal carcinomas. We tried to increase detection sensitivity, and compared the high-sensitivity EGFR immunostaining with a worldwide standard, EGFR PharmDx™ (Dako). In order to pursue high-sensitivity EGFR detection, deparaffinized sections were pressure-cooked in 1 mM EDTA solution, pH 8.0. Two mouse monoclonal antibodies against EGFR, clone EGFR2.5 and DAK-H1-WT, and six kinds of secondary detection reagents, including biotin-free catalyzed signal amplification (CSA II), Simple Stain MAX-PO, PolyVue, Novolink, EnVision™ FLEX+, and MACH3, were evaluated to compare the results with those with EGFR PharmDx™, employing a combination of 2-18-C9 as the primary monoclonal antibody and EnVision™ as the secondary reagent. Furthermore, we replaced EnVision™ in the EGFR PharmDx™ kit with CSAII. EGFR detection sensitivity was higher with DAK-H1-WT than with EGFR2.5, and among the secondary reagents, the strongest signals were observed with Novolink. All 30 colorectal carcinomas showed distinct expression of EGFR with our high-sensitivity EGFR immunostaining, while only 16 (53%) gave focal positivity with EGFR PharmDx™. When EnVision™ in EGFR PharmDx™ was replaced by CSA II, strong signals were seen in all cases, and the expression pattern was comparable with our sequence. Non-neoplastic crypt epithelial cells often showed weakly signal with the standard EGFR PharmDx™, but consistently revealed strong membrane staining in the two high-sensitivity sequences. EGFR PharmDx™ frequently gave false negativity. Importantly, EGFR was consistently and sensitively detected when the secondary polymer in the EGFR PharmDx™ kit was simply replaced by CSA II.