Comparison of PD-L1 expression in CTCs and matched primary tumor prior to anti-PD1 immunotherapy. (A) Tumor specimens were analyzed by IHC staining for PD-L1, using DAB Peroxidase (HRP) staining technique. PD-L1 tumor score is the percentage of cells in each category: negative, low, medium, high. CTCs were analyzed by IF and semi-automated quantification of PD-L1 staining intensity (Supp. Figure 1), with CTC intensity normalized by the average staining intensity of H1703 as a control cell line. (B) PD-L1 CTC threshold level and IHC PDL1 threshold levels are quantified based on the percentage of cells in four different categories. (C) Bar plots showing percentage and PD-L1 level of matched tumor biopsy and CTC specimens (n = 4 patients).