Immunohistochemical (IHC) staining was performed on 5-μm-thick unstained sections from tissue microarray blocks using antibodies to cytokeratin 7 (CK7) (clone OV-TL12/30; Dako, Carpinteria, CA, USA; 1:300 dilution), to cytokeratin 20 (CK20) (clone Ks20.8; Dako; 1:200 dilution), to homeobox protein CDX2 (clone CDX2-88; Abcam, Cambridge, MA, USA; 1:100 dilution), and to epidermal growth factor receptor(EGFR) (clone 3C6; Roche Diagnostics, Mannheim, Germany; 1:100 dilution). IHC staining of human epidermal growth factor receptor 2 (HER2) was performed using the Ventana Ultra View DAB detection kit (Ventana Medical Systems, Tucson, AZ, USA) and the Ventana PATHWAY HER2/neu rabbit monoclonal antibody (4B5) on a Ventana BenchMark XT immunostainer (Ventana Medical Systems). All slides from each tumor were evaluated by a single pathologist independently based on the following criteria. Expression of CK7, CK20, and CDX2 was considered positive if 10% of the tumor cells showed immunoreactivity. For EGFR, both the percentage of positive tumor cells and the intensity of positive staining were graded according to a previous report [23 (link)]. Total grades were generated on a scale of 0–6 and considered positive if the score was 2–6. The staining for HER2 was graded according to the guidelines for such testing in gastric cancers [24 (link)].
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