2.3. Digital Image Analysis Using QuPath

Stained slides were scanned on a slide scanner (P250 Flash, 3DHistech, Budapest, Hungary) and annotated by two pathologists (M.E., V.W.) in QuPath (https://qupath.github.io; RRID:SCR_018257, last access: 5 January 2021) [28]. TMA cores were quality controlled for the presence of tumor tissue and proper staining results. Cores with either absence of representative tumor tissue (complete absence, or less than 5% of the TMA core area) or staining artifacts (e.g., DAB shades) were excluded from the analysis. Total number of positive cells expressing CD3, CD8, FOXP3, CD56, CD68, CTLA-4, LAG3, PD-1, and GZMB were quantified per mm2 in all viable TMA cores via QuPath. For further analysis, median counts were log2 transformed. Log2-transformed values were Z-score-transformed to build a combined immune cell infiltration score (ICIS).