Anti pd l1 clone sp263

EUS-FNB specimens obtained from treatment-naïve patients were archived respecting formalin fixation and paraffin embedding protocols and were subjected to IHC staining. Briefly, Ventana BenchMark Ultra automated slide-staining system was used in order to stain 4 μm thick tissue sections utilizing the following antibodies: anti-PD-L1 (clone SP263 Ventana), MLH1 (clone ES05 Leica), MSH2 (clone G219-1129, Ventana), MSH6 (clone SP93 Novus Biological), and PMS2 (clone A16-4, Ventana). Diaminobenzidine was used as a chromogen for UltraView detection in order to visualize antigen–antibodies reactions. A specimen was considered adequate for evaluation only if a minimum of 100 tumor viable cells were found on the slides. Membranous staining was the hallmark for positive PD-L1 expression (Figure 1C). The tumor proportion score (TPS) has been calculated as the percentage of viable tumor cells with complete or partial membrane staining at any intensity. PD-L1 expression was considered in specimens with TPS > 1% and high PD-L1 expression was considered in patients with TPS > 50%. In the case of absent nuclear staining of DNA mismatch repair protein (PMS2, MSH2, MSH6, or MLH1), the tumor was targeted as dMMR (Figure 2).

Expression of the immune checkpoint molecules in CLL and RS lymph node samples was analyzed by IHC staining using antibodies specific for PD-L1 and PD1. Immune cell infiltration in CLL and RS lymph node samples was assessed by IHC staining using antibodies specific for CD3, CD8, FOXP3, and CD163. IHC staining was done on 4-μm FFPE sections on DAKO Autostainer Plus (Agilent, Santa Clara, CA) using standard protocol^{17 (link),20 (link)}. The antibodies used include anti-PD-L1 (clone SP263, Ventana Medical Systems, Inc., Tucson, AZ), anti-PD1 (clone NAT105, Abcam, Inc., Cambridge, MA), anti-CD3 (clone LN10, Leica Biosystems, Newcastle Upon Tyne, UK), anti-CD8 (clone C8/144B, Dako, Carpenteria, CA), anti-FOXP3 (clone 236A/E7, Abcam, Inc., Cambridge, MA), and anti-CD163 (clone10D6, Leica Biosystems, Newcastle Upon Tyne, UK). IHC images were taken using whole slide imaging technology with MoticEasyScan Pro (Motic digital pathology, San Francisco, CA), and saved in tagged image file format. Percentage of expression for each individual antigen was calculated by dividing number of cells with positive staining by number of total cells in the image using the Image-Pro premier 3D 9.1.4 software (Media Cybernetics, Silver Spring, MD).