Immunohistochemistry

Immunostaining for p53 protein was performed as previously described⁹. Briefly, formalin-fixed, paraffin-embedded tissue specimens were sectioned (4 μm thick), deparaffinized and rehydrated in a series of graded ethanol. Immunohistochemical staining was performed using a mAb against p53 (DO-7, Dako, Carpinteria, CA, USA), which recognizes an epitope in the N-terminus between amino acid 35 and 45 and reacts with the wt and most mutant forms of p53 protein, followed by the avidin-biotin-peroxidase method to visualize the p53 according to the manufacturer’s instructions (Dako). Positive and negative controls were included in each run for quality control of the immunoreactivity. IgG isotype mAb was used as a negative control. Normal-appearing salivary gland tissue or skeletal muscle from patients with HNSCC served as an internal non-tumor control. A tumor was considered p53 positive when >25% of the tumor cells showed staining intensity of 2+ and higher on a scale of 0–4+. For p16 staining, mouse monoclonal antibody specific for p16 (1:100 dilution, clone DCs-50; neomarkers, Fremont, CA, USA) was used as introduced before¹⁴. p16 expression was scored as positive if there was strong and diffuse nuclear and cytoplasmic staining in >60% of the tumor. Three independent experienced observers, who were blinded to the patient clinical information, performed semiquantitative evaluation of the slides.