Coordinated activation of Yes-associated protein (YAP), Notch1, and mammalian target of rapamycin (mTOR) in human intrahepatic cholangiocarcinoma (iCCA) samples. Representative immunohistochemical pattern of YAP, phosphorylated/inactivated (p-) YAP at Ser127 (S127), Notch1, RAPTOR, and p-4EBP1 staining in sections from nontumorous surrounding livers (ST) and two human iCCAs (iCCA1 and iCCA2). In ST, faint to moderate nuclear positive staining for Notch1, YAP, and p-4EBP1 was limited to biliary cells, whereas hepatocytes exhibited absent or weak nuclear and cytoplasmic immunolabeling for the same proteins. Immunoreactivity for RAPTOR was moderate in nonneoplastic biliary cells and hepatocytes. In striking contrast, both tumors displayed strong immunoreactivity for nuclear and cytoplasmic YAP and Notch1, RAPTOR, and p-4EBP1. Intense immunoreactivity for p-YAP (S127) was detected in the nonneoplastic liver, especially in biliary cells; levels of p-YAP were low in tumors. Cytokeratin 19 (CK19) staining was used as a biliary marker. Arrows indicate representative biliary cells or tracts. Scale bars = 100 μm. Original magnification, ×200. HE, hematoxylin and eosin.