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Int J Clin Exp Pathol 2013;6(1):24-30

Original Article
High-sensitivity epidermal growth factor receptor immunostaining for colorectal
carcinomas, compared with EGFR PharmDxTM: A study of diagnostic accuracy

Kazuya Shiogama, Trai Wongsiri, Yasuyoshi Mizutani, Ken-ichi Inada, Yutaka Tsutsumi

Department of Pathology, Fujita Health University School of Medicine, Toyoake, Japan; Department of Pathology, Faculty of Medicine, Khon
Kaen University, Khon Kaen, Thailand

Received October 18, 2012; Accepted November 6, 2012; Epub November 20, 2012; Published January 1, 2013

Abstract: 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 PharmDxTM (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, EnVisionTM FLEX+, and
MACH3, were evaluated to compare the results with those with EGFR PharmDxTM, employing a combination of 2-18-C9 as the primary
monoclonal antibody and EnVisionTM as the secondary reagent. Furthermore, we replaced EnVisionTM in the EGFR PharmDxTM 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 PharmDxTM. When EnVisionTM in EGFR PharmDxTM 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 PharmDxTM, but consistently revealed strong membrane staining in the two high-sensitivity
sequences. EGFR PharmDxTM frequently gave false negativity. Importantly, EGFR was consistently and sensitively detected when the
secondary polymer in the EGFR PharmDxTM kit was simply replaced by CSA II. (IJCEP1210007).

Keywords: Colorectal cancer, epidermal growth factor receptor, immunohistochemistry, sensitivity and specificity, monoclonal antibody

Address all correspondence to:
Dr. Yutaka Tsutsumi
Department of Pathology
Fujita Health University School of Medicine
Toyoake, Aichi 470-1192, Japan.
Tel: +81-562-93-2439; Fax: +81-562-93-3063
E-mail: tsutsumi@fujita-hu.ac.jp