Automated staining system

Tissues from UK patients, both core biopsies and surgical resections were collected according to ASCO/CAP guidelines, while patients’ tissues from Japan were collected following ISO (International Organization for Standardization) 15189 approved by the Japan Accreditation Board. Preparation of the Ki67 slides of the first cohort has been previously described⁹ . Briefly, the corresponding core-cut biopsy and surgical resection blocks were centrally cut and stained with Ki67, resulting in 60 Ki67 slides from 30 cases. The IHC was performed using monoclonal antibody MIB-1 at dilution 1:50 (DAKO UK, Cambridgeshire, UK) using an automated staining system (Ventana Medical Systems, Tucson, AZ, USA) according to the consensus criteria established by the International Ki67 Working Group⁶ . Sections from the same block were stained in a single immunohistochemistry run, except for four cases where the staining was performed in two different runs. This approach effectively controls for any technical variation in staining.

For immunohistochemical evaluation a representative tissue block of n = 11 prostate adenocarcinoma cases, including periurethral tumor manifestations if available, was selected and stained for specific antibodies (Table S2). Staining and detection was performed using an automated staining system (Ventana). Semi-quantitative evaluation for each antibody was performed by two experienced pathologists. For each tissue block a corresponding hematoxylin–eosin (HE)-stained slide was available for morphological identification of prostate cancer. For each immunohistochemical marker the expression in the tumor and normal prostatic tissue were evaluated separately by assigning a four-tiered score (0 = negative, 1 = weak, 2 = moderate, 3 = strong). The extent of stained benign and malignant glands was estimated in 10% increments. In addition, the cellular compartment of the staining for both tumor area and normal prostatic glands was specified, whereas in the normal prostatic glands further evaluation of the distinct stained cell type (luminal and basal cells) was recorded. The predominant staining pattern was assessed when considerable heterogeneity of the staining intensity was detected.