Evaluation and classification of immunostaining

Evaluation of immunostainings was performed by a senior pathologist without knowledge of the disease outcome. Immunoreactivity was scored based on both staining intensity and percentage of immunoreactive epithelial cells within the tumor. Intensity was evaluated on a four-grade semi-quantitative scale; no staining, weak intensity, moderate intensity or strong intensity. For statistical purposes, intensity scores were grouped into no/weak and moderate/strong. The percentage of positive cells was grouped into ‘at or above median’ or ‘below median’ to obtain relatively even groups. For simplicity, at or above median is henceforth referred to as ‘high’ and below median as ‘low’ staining percentage. For p16^INK4a immunostaining, only cases with a strong nuclear and cytoplasmic expression in a continuous segment of at least 10–20 cells were considered positive.