Sections (5 μm) were cut and fixed on Superfrost plus glass slides before dewaxing and rehydration. Expression of CDKN2A was examined using a CDKN2A monoclonal antibody (1:1500 for 1 hr; Ab-7; Labvision, Freemont, CA, USA) and the catalyzed signal amplification system (CSA system; DakoCytomation, Cambridgeshire, UK) according to the manufacturer's instructions. Endogenous biotin or biotin-binding proteins were blocked using the Avidin Biotin blocking kit (Vector Laboratories, Peterborough, UK) according to manufacturer's instructions and endogenous peroxidase activity was blocked using 3% hydrogen peroxide in water for 5 min (CSA system). Sections were counterstained with hematoxylin, dehydrated and mounted in Depex mounting medium (VWR International, Leicestershire, UK). Expression of CDKN2A was examined by light microscopy and scored in tumors as absent (0), expressed (1) or highly expressed (2).