Image analysis software

PD-L1 expression on tumor cells and immune cells was assessed at a central laboratory using the Ventana PD-L1 (SP263) assay (Ventana Medical Systems; 740–4907). The threshold of ≥1% of tumor-infiltrating immune cells staining positive within the tumor area of the tested tissue sample defined the official PD-L1 status of a given sample, but tumor cell expression was also evaluated as an exploratory variable. CD8 expression was assessed by immunohistochemistry using clone C8/144B (M710301–2) and scored via a quantitative method using image analysis software (Definiens).^{20 (link)} A central tumor region was delineated by a pathologist. At the interface between malignant and adjacent normal tissue, a 1000-μm wide immune margin (IM), an immunologically active region and site of PD-L1 expression,^{21 (link)} centered around the perimeter was generated. For both the central tumor region and the IM, the relative area of marker-positive cells (ie, the CD8⁺ area relative to the total tumor area) was calculated. CD8 expression was reported in terms of the percentage of CD8⁺ cells in relation to the total number of CD8⁺ cells in the total tumor area, tumor center, or IM, with the median as the cut-point value.^{20 (link)}

Tumor sections were harvested and fixed in 3% PFA overnight, then placed in 50% ethanol (EtOH) until paraffin embedding. Tumor sections (4 μm) were stained with F4/80 (1:500; Serotec), MMP-9 (1:1000; Abcam), CSF1R (1:200; Santa Cruz) CSF1R-Y723 (1:50; Santa Cruz), Ki67 (1:500, Vector Labs). Histological images were taken by the Nikon Eclipse 90i microscope. Five to six fields per slide of the Ki67 samples were analyzed at 10× magnification and quantified using ImageJ Version 1.34s (National Institute of Health). IHC and IHF slides were scanned by the Aperio and Arial whole slide scanner, respectively, as service provided by the UCLA Translational Pathology Core Laboratory (TPCL). Quantification of staining was analyzed by the Definiens image analysis software.