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Immunohistochemical studies were performed on 3-μm sections cut from paraffin blocks using a fully automated system (“Benchmark XT System”, Ventana Medical Systems Inc, 1910 Innovation Park Drive, Tucson, Arizona, USA) and the following antibodies: pancytokeratin (clone AE1/AE3, 1:40, Zytomed, Berlin, Germany), CK7 (OV-TL, 1:1000, Biogenex), p63 (4A4, 1:100, Zytomed), CK5 (clone XM26, 1: 50, Zytomed), chromogranin A (clone LK2H10, 1:500, Beckman-Coulter GmbH), synaptophysin (clone SY38, 1:50, Dako), CD56 (clone MRQ-42, 1:100, CELL MARQUE), CD117 (polyclonal rabbit antibody, 1:100; Dako), p16 (clone JC8, 1:100, Santa Cruz Biotechnology), anti-NUT (clone C52B1, 1:45, Cell Signaling), SMARCB1 (INI1) (MRQ-27, 1:50, Zytomed), SMARCA2 (polyclonal antibody, 1:100, Atlas Antibodies AB, Stockholm, Sweden), SMARCA4 (anti-BRG1 antibody, clone EPNCIR111A, 1:100, Abcam; Cambridge, UK) and ARID1A (rabbit polyclonal antibody, ab97995, 1:100; Abcam). Epstein Barr virus (EBV) in-situ hybridization (EBER 1/2 probes, ZytoVision, Bremerhaven, Germany) was performed according to the manufacturer instructions. Human papillomavirus (HPV) testing was performed using either PCR-based method or RNA in-situ hybridization (ISH) by the RNAscope method as detailed previously.12,13 Assessment of the staining results of the SWI/SNF components was done as recently described21, i.e. only the nuclei of viable tumor tissue (away from necrotic areas) were assessed. As a control, the presence of a homogeneous strong nuclear staining of stromal fibroblasts, inflammatory cells, vascular endothelial cells or normal epithelial cells in the background was a prerequisite for assessable staining in the tumor. Three staining grades were defined: intact (strong staining in the neoplastic cells that is similar to normal background cells), lost (indicating clean neoplastic cell nuclei as opposed to strong staining in normal cells) and reduced if very weak but still discernible as opposed to strong staining in normal cells). Tumors with an admixture of these three patterns were specifically reported. Cases with absent or very weak staining in the normal background cells were considered equivocal or not assessable (no results=NR).
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