Evolution of PD-1 and PD-L1 Gene and Protein Expression in Primary Tumors and Corresponding Liver Metastases of Metastatic Bladder Cancer  Markus Eckstein, Danijel Sikic, Pamela L. Strissel, Franziska Erlmeier  European Urology  Volume 74, Issue 4, Pages 527-529 (October 2018) DOI: 10.1016/j.eururo.2018.06.028 Copyright © 2018 European Association of Urology Terms and Conditions

Fig. 1 (A) Distribution of sTILs in PTs and LMs. Overall sTIL infiltration was significantly higher in PTs. The table shows statistical comparison of sTILs in PD-L1-positive and -negative LMs. PD-1 and PD-L1 positivity in TCs and ICs was defined as expression of ≥1%. PD-L1 mRNA positivity was defined as gene expression of ≥36.5 40−ΔCt. (B) mRNA and protein expression of PD-L1 in PTs and corresponding LMs. IHC stains (SP263 assay; Ventana, Tucson, AZ, USA) show a PT and LM with the absence and presence of PD-L1 expression. Graphs show the gene and protein expression in PD-L1-positive TCs and ICs. (C) IHC images show a PT and LM with the absence and presence of PD-1 expression. Graphs show mRNA and protein expression in a PD-1-positive PT and the corresponding LM. All TCs in PTs and LMs were negative for PD-1 (data not shown). (D) Dynamic evolution of sTIL infiltration and PD-1 and PD-L1 protein and gene expression in matched PT/LM pairs (tumor numbers 1–14). sTIL presence was defined as a sTIL score of ≥1% according to the standard methodology [4]. PD-1 and PD-L1 positivity in TCs and ICs (IHC; Abcam, Cambridge, UK) was defined as expression of ≥1%. If sTILs were absent, PD-1 and PD-L1 expression on ICs was classified as absent. PD-L1 mRNA positivity was defined as gene expression of ≥36.5 40−ΔCt; the cutoff was estimated using engineered cell lines from Horizon Discovery (Cambridge, UK) with different levels of PD-L1 gene and protein expression. Statistical analyses were performed using JMP13.2 (SAS Institute, Cary, NC, USA) and GraphPad Prism 7 (San Diego, CA, USA). A nonparametric Mann-Whitney test was used for statistical comparisons of continuous variables, with p<0.05 considered significant. All tests were two-sided. sTIL=stromal tumor-infiltrating lymphocyte; IHC=immunohistochemistry; PT=primary tumor; LM=liver metastasis; IC=immune cell; TC=tumor cell; SD=standard deviation; IQR=interquartile range; Ct=cycle threshold. European Urology 2018 74, 527-529DOI: (10.1016/j.eururo.2018.06.028) Copyright © 2018 European Association of Urology Terms and Conditions