Ki67 staining, scanning, central reading and interpretation

Whole-tissue sections (thickness: 4 μm) were prepared from formalin-fixed paraffin-embedded tissue blocks from all patient samples and placed on positively charged Superfrost Plus microscope slides (Thermo Scientific, Waltham, MA). For the immunohistochemistry (IHC), slides were deparaffinized using xylene for 15 min and hydrated in a series of graded ethanol to water. Antigen retrieval was then carried out using Diva (BioCare Medical, Pacheco, CA) pretreatment reagent in a decloaking chamber (BioCare Medical) under pressure at 95°C for 45 min and cooled down in wash buffer. Peroxidase block, protein block, immunostaining with the rabbit primary monoclonal antibody targeting Ki67 (clone SP6, 1 : 100, BioCare Medical), primary antibody binding detection with an HRP-coupled polymer combined with 3,3′-diaminobenzidine chromogen (MACH 1 Universal HRP-Polymer Detection Kit, BioCare Medical) and counterstaining with hematoxylin were carried out in the IntelliPath FLX automated slide stainer (BioCare Medical) per manufacturer's protocol. Slides were then dehydrated in water to increasing ethanol concentration and mounted using Pertex (Histolab, Gothenburg, Sweden). Colon and skin tissues were used as positive controls and liver and kidney tissues as negative controls.

Ki67 tumor expression was evaluated centrally by an experienced certified pathologist (EL) who was blinded to the patient characteristics, Ki67 values from the diagnostic samples and outcome. Evaluation was carried out according to the recommendations provided by the International Ki67 Breast Cancer Working Group.32 Specifically, Ki67 score was calculated by dividing the total number of positively stained invasive tumor cell nuclei with the total number of tumor cell nuclei counted across each whole core-cut ×100. As adequate for scoring were considered core-cuts that comprised a minimum of 500 invasive tumor cells. Only nuclear staining (plus mitotic figures which are stained by Ki67) was incorporated into the Ki67 score. A nucleus was considered positively stained if presenting any definite (brown) staining above the surrounding background in the cytoplasm and extracellular matrix, regardless of the intensity of staining, and as negative when presenting only the blue counterstaining. Mitotic figures, normal ducts and lymphocytes served as the internal positive control. Foci of carcinoma in situ, areas of necrosis as well as non-tumoral tissue (stroma, inflammatory background) were excluded from counting.