Egfr e746 a750

The enriched samples were cyto-centrifuged onto glass slides and CTCs identified by immunocytochemistry (ICC) using the CellSearch^® antibody cocktail (pan-Cytokeratin/CD45/DAPI; Menari-Silicon Biosystems, PA, USA). Briefly, the slides were air dried overnight and incubated with the combination of CellSearch^® (Menarini Silicon Biosystems Inc, PA, USA) reagents (20 µL staining reagent, 20 µL permeabilization buffer, 20 µL fixation buffer, 10 µL DAPI and 130 µL 1× PBS). The slide was incubated for 1.5 h at room temperature, washed three times in 1× PBS. Putative CTCs were further characterized for EGFR exon 19 deletion (1:100 dilution) (EGFR E746-A750, Cell Signaling, Beverly, MA, USA) and PD-L1 (1:200 dilution) (anti-PD-L1 (28-8)). The slides were mounted with Prolong Gold mounting medium (Molecular Probes, Eugene, OR, USA) to prevent photo-bleaching and preserve the fluorescent labelled molecules for long term storage, coverslipped and imaged using a Nikon Epifluorescence microscope (Nikon, Tokyo, Japan). CTCs were identified as intact, pan-cytokeratin⁺, DAPI⁺, CD45⁻ cells larger than 4 µm. The mean fluorescence intensity (MFI) was measured per CTC by determining the fluorescence intensity per CTC and subtracting this from the local background intensity. This was compared to the MFI of known NSCLC cancer cell lines and controls.