We performed immunohistochemical (IHC) staining for HER3 as previously described [34 (link), 35 (link)]. Briefly, sections of each sample were deparaffinized and antigen retrieval was performed at high pH (PT Link machine, Dako). The sections were then stained with a rabbit monoclonal antibody against HER3/ErbB3 (1:59 dilution; clone D22C5, Cell Signaling Technology Inc., Danvers, MA, USA) using the Dako autostainer Link48 (Dako, CA, USA) and EnVision Flex Mini Kit (Dako), according to the manufacturer’s instructions. Hematoxylin was used as a nuclear counterstain. HER3-high was defined as an IHC score of 3 + or 2 + , and HER3-low/zero was defined as an IHC score of 1 + or 0 in line with the HER2 testing guidelines for gastroesophageal cancer [36 (link)]. The H-score (range, 0–300) was calculated using the following formula: 3X + 2Y + Z, where X, Y, and Z are the percentages of tumor cells showing strong, moderate, and weak staining intensities, respectively [37 (link)] (Additional file 1: Table S1A–D). The specificity of this HER3 IHC staining method was validated by confirming no signal detected with an isotype control antibody. Both positive and negative cell line control slides were also incorporated into HER3 staining to verify the specificity at every batch of staining.
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