Multiplex IHC for Tumor Immunophenotyping

Multi-color multiplex immunohistochemistry (IHC) with CD8 for cytotoxic T lymphocytes (clone MRQ26, mouse, Ventana), CD163 for macrophages (clone SP57, rabbit, Ventana), and PD-L1 (clone SP263, rabbit, Ventana) was performed on freshly cut whole sections from pretreatment biopsies as described before^{60 (link),61 (link)}. A membranous PD-L1 staining in tumor cells or immune cells was considered as specific staining. The immunohistochemistry was evaluated with consensus viewing by two pathologists (Y. Hou and Z. Li). The percentage of PD-L1 positively-stained cells were recorded and used for machine learning models (features that generated by pathologists). The parameters assessed were as follows: PD-L1 expression in tumor cells (PD-L1 TC), PD-L1 expression in immune cells (PD-L1 IC), PD1 expression in immune cells, intratumoral CD8+ immune cells (IT-CD8+), peritumoral CD8+ immune cells (PT-CD8+), intratumoral CD163+ macrophages (IT-CD163+), and peritumoral CD163+ macrophages (PT-CD163+).