All stained slides were scanned with whole-slide scanner 250 Flash II (3DHISTECH) to obtain a whole-slide image. A 20 × Plan Apo objective (0.80 numerical aperture) and a CIS (VCC-FC60FR19CL) charge-coupled device progressive scan color camera with a resulting image resolution of 0.24 μm/pixel was used. JPEG image encoding with quality factor 80 and an interpolated focus distance of 15 with stitching in the scan options were chosen. For every slide, a specific scan profile was configured. Scanned images were examined in Pannoramic Viewer (3DHISTECH) to check for image quality and whole-tissue coverage.
Object-based image analysis for Pan-CK, CD45, CD3, CD8, CD4, CD163, CD20, PD-L1, and PD-1 was done with the commercial software VIS version 6.2 (Visiopharm) at a commercial laboratory. Manual delineation of the tumor region, guided by a histopathology report, was done in the Image Analysis module of VIS. Artifacts due to tissue processing and whole-slide imaging, such as ruptured tissue, dirt, or out-of-focus regions, were excluded. For every marker, an APP was written that calculated the area of the manually delineated tumor region and the area of the marker-positive regions.
Object-based image analysis for Pan-CK, CD45, CD3, CD8, CD4, CD163, CD20, PD-L1, and PD-1 was done with the commercial software VIS version 6.2 (Visiopharm) at a commercial laboratory. Manual delineation of the tumor region, guided by a histopathology report, was done in the Image Analysis module of VIS. Artifacts due to tissue processing and whole-slide imaging, such as ruptured tissue, dirt, or out-of-focus regions, were excluded. For every marker, an APP was written that calculated the area of the manually delineated tumor region and the area of the marker-positive regions.