Immunohistochemical staining was performed on 10% formalin-fixed paraffin-embedded tissue blocks using a Discovery Auto-Stainer with automated protocols (Ventana Medical Systems, Inc.). The primary antibodies used were anti-E-cadherin (1:400; cat. no. 3195) and anti-vimentin (1:200; cat. no. 5741) from Cell Signaling Technology, Inc., and anti-ZEB1 (1:100; cat. no. ab203829) from Abcam, in addition to the Discovery Universal secondary antibody (cat. no. 760-4205). Two clinical pathologists (FS and YM) independently scored the staining intensity in ATC and non-cancerous tissues using a light microscope (magnification, ×40; Nikon Eclipse TS2; Nikon Corporation) for E-cadherin, vimentin and ZEB1 expression. Staining scores were recorded as follows: 0, negative staining in the cell membrane; 1, <10% of weak staining in the cell membrane; 2, >10% of weak or moderate staining in the cell membrane; and 3, >50% of strong staining in the cell membrane (15 (link)).