Genie analysis tool

Representative hematoxylin and eosin-stained sections on glass slides containing prominent intratumoral regions from the 152 CCRCC specimens were chosen. Two cores were obtained from each representative paraffin block and transferred to recipient TMA blocks. Immunohistochemical staining was performed on the TMA blocks. Anti-MYOF monoclonal antibody (1:100 dilution, #ab76746, Abcam, UK), anti-VEGFR2 polyclonal antibody (1:50 dilution, #RB-1526-P1, Thermo Fisher Scientific, USA), and anti-CD31 monoclonal antibody (1:100 dilution, JC70, Cell Marque, USA) were used as the primary antibodies. The absolute number of positive CD31 endothelial cells was automatically counted using the Genie analysis tool (Leica Biosystems, Wetzlar, Germany).

All the IHC stained slides were digitally scanned at 200x magnification into a high-resolution digital image of the whole tissue (e-slide manager) using a pathology scanner (Aperio AT Turbo, Leica Biosystems, Buffalo Grove, IL). The images were visualized using the ImageScope software program (Leica Biosystems) and analyzed using the Aperio Image Toolbox and GENIE analysis tool (Leica Biosystems). The densities of immune cells markers including PD-1, ICOS, OX-40 CD3, CD4, CD8, CD57, granzyme B, CD45RO, and FOXP3 were evaluated using the Aperio nuclear algorithm, CD68 using Aperio cytoplasmic algorithm, and counting the cells positive for them in five square areas (1 mm² each) in the inside of the tumor compartment. Each area examined was overlapped with the sequential IHC slides to quantify each marker at the same location of the tumor specimen [36 (link)]. The average of total number of cells positive for each marker in the five square areas was expressed in density per mm².