p16 IHC

Samples with a historic p16 slide had the existing slide used in this study. Samples were recoded and read blinded. All other samples had p16 IHC performed at a diagnostic medical laboratory (MedLab Central Ltd, Palmerston North, New Zealand). Formalin fixed paraffin embedded (FFPE) tissue was used for IHC. Sections were cut at four microns and baked onto Superfrost Plus slides at 60°C for 60 minutes. Staining was performed on the Ventana Benchmark Ultra (Ventana Medical Systems, Tucson, USA), with antigen retrieval performed on board, using Ventana CC1 buffer for 32 minutes at 100°C. A 1:150 dilution of the p16 (INK4a) antibody (G175-405, product code 551153) (BD Biosciences, North Ryde, Australia), was incubated on the slide for eight minutes at 36°C. The detection of p16 was then visualised using the Ventana Optiview DAB kit (Ventana Medical Systems). A multi-tumour block positive control containing a serous ovarian carcinoma was included with each run of samples. All slides were read independently by two pathologists (BL and UV), and discordant cases reviewed to reach a consensus. Samples showing strong diffuse staining in the nucleus and cytoplasm of greater than 75% of tumour cells were considered positive. Samples showing staining in 10% to 75% of tumour cells were considered focally positive, and samples with staining in less than 10% of tumour cells were considered negative.