FFPE blocks from ONB tissue were generated at the time of primary surgery from radiotherapy (RT)-naïve patients. All cases were reviewed by two expert head and neck pathologists (S.B. and L.A.) and the diagnosis confirmed using hematoxylin and eosin staining. In case of disagreement about grading between the two pathologists, a final grade was reached by consensus.
All immunohistochemical analyses were performed on serial 5 µm sections of a selected FFPE representative tumor block. The markers used for diagnostic purposes were chromogranin A (clone LH2H10, DB-Biosystem, Franklin Lakes, NJ, USA), synaptophysin (clone DAK-SYNAP, Dako, Glostrup, Denmark), S100 protein (polyclonal, Leica, Wetzlar, Germany), pan-cytokeratin (clone MNF116, Dako, Glostrup, Denmark) and Ki-67 proliferation index (clone 30-9, Ventana, AZ, USA). Immunohistochemical analysis was performed on the entire slide of the selected block and the percentage of positive neoplastic cells was determined. Quantification of the Ki-67 index was done by counting the positive neoplastic cells among all tumor cells present in the area of interest (hotspot area) and by determining the percentage of positive neoplastic cells in the entire tumor area.
All immunohistochemical analyses were performed on serial 5 µm sections of a selected FFPE representative tumor block. The markers used for diagnostic purposes were chromogranin A (clone LH2H10, DB-Biosystem, Franklin Lakes, NJ, USA), synaptophysin (clone DAK-SYNAP, Dako, Glostrup, Denmark), S100 protein (polyclonal, Leica, Wetzlar, Germany), pan-cytokeratin (clone MNF116, Dako, Glostrup, Denmark) and Ki-67 proliferation index (clone 30-9, Ventana, AZ, USA). Immunohistochemical analysis was performed on the entire slide of the selected block and the percentage of positive neoplastic cells was determined. Quantification of the Ki-67 index was done by counting the positive neoplastic cells among all tumor cells present in the area of interest (hotspot area) and by determining the percentage of positive neoplastic cells in the entire tumor area.