Clone nat105

Hematoxylin and eosin slides of each archival specimen were evaluated, and a block representing the overall tumor was chosen for TMA preparation. Three cores of 1 mm diameter per case were selected. Sections were cut 4 µm thick and placed on positively charged slides for immunostaining. Staining was performed using the Ventana Discovery XT automated system (Ventana Medical Systems, Tucson, AZ, USA) using commercially available antibodies against PD-1 on tissue-infiltrating lymphocytes (clone NAT105, diluted 1:100; Abcam, Cambridge, UK) and PD-L1 on tumor cell membranes (clone E1L3N, diluted 1:200; Cell Signaling Technology, Beverly, MA, USA). These assays had been previously validated using Formalin-Fixed Paraffin-Embedded cell-line controls.^{10 (link)}All TMAs were reviewed twice, based on antibody expression and compared with a positive control. Scoring was performed by an experienced uropathologist who was blinded to clinical outcomes. PD-L1 expression was evaluated based on the intensity and proportion of tumoral cells showing membranous or cytoplasmic staining, and was scored as follows: 0, negative (no immunoreactivity); 1, weak (5% to less than 25% of cells); 2, moderate (between 25 and 60% of cells); and 3, strong (more than 60% of cells). The numbers of PD-1 cytoplasmic-positive lymphocytes were assessed semiquantitatively.^{11 (link)}