Immunohistochemical Profiling of ccRCC

Immunohistochemical staining was performed as described previously (26 (link)). Briefly, IHC was performed on the paraffin-embedded ccRCC tissues using Musashi-2 (Abcam, #ab76148), PD-L1 (GeneTech, #GT228002, Colone: ZR3), HIF1A (CST, (E1V6A), #48085), CA9 (MXB Biotechnologies, RAB-0615), KIT (MXB Biotechnologies, KIT-0029, Colone: YR145) and MKI67 (MXB Biotechnologies, MAB-0672, Colone: MX006) antibodies. IHC images were captured with a Leica Aperio Versa 200 microscope. Musashi-2 expression levels were scored by multiplying the percentage of positive cells by the staining intensity. The percentage was scored as follows: no positive cells, 0 point; 1%‐25%, 1 point; 26%‐50%, 2 points; 51%‐75%, 3 points; and 76%‐100%, 4 points. The staining intensity was scored as follows: no positive staining, 0 point; weak staining, 1 point; moderate staining, 2 points; and strong staining, 3 points. The final Musashi-2 IHC scores were classified as follows: 0‐4, low expression; and 6-12, high expression. For PD-L1 IHC evaluation, positive expression was defined as a tumor proportion score exceeding 1% (27 (link)).