Materials and methods

Tissue slices from a series of 43 PTC, 8 LN, 15 MTC were collected from Policlinico Umberto I at University of Roma ‘Sapienza’, after having obtained the informed consent by each patient and the approval of the local ethical committee. Clinical–pathological characteristics of the samples, including, when available, the genetic analysis for detection of V600E BRAF mutation in PTCs and the mutational status of RET and RAS for MTCs, are shown in Fig. 1a. Tissue specimen of MTCs were from an archival series previously described [8]. Immunohistochemistry was performed on dewaxed and rehydrated 4 μm tissue sections, treated with citrate buffer (0.01 M, pH 6) to retrieve epitope and then incubated with H 2 O 2 to block endogenous peroxidase activity. Sections were incubated overnight with the polyclonal anti-YAP1 antibody (1: 200 dilution)(Cell signaling, Danvers, MA, USA) and, after washing, treated with biotinylated goat anti-polyvalent antibody (Detection IHC kit, Abcam, Cambridge, United Kingdom). Finally, staining was visualized using 3-3 diaminobenzidinetetrahydrochloride (Detection IHC kit, Abcam). The sections were slightly counterstained with Mayer hematoxylin (Carlo Erba Reagents srl, Milan, Italy) and examined by two pathologists, who expressed concordant opinions for all the cases examined. Positivity was indicated when a rate of stained cells> 5% was detected. Percentage of stained cells and estimated intensity of staining were used to assign a score (low, moderate, high)[9]. Analysis was performed using a binocular microscope with a 20× objective, a digital image-capture computer system and the software supplied with the microscope.