Envision dab detection system

EGFR gene mutations were analyzed by a peptide nucleic acid (PNA) clamping-based sensitive method (PNA Clamp™ or PANAMutyper™ EGFR mutation detection kit) in formalin-fixed paraffin-embedded tissues (16 (link)-18 (link)). EGFR-activating mutations were defined as mutations associated with EGFR-TKI sensitivity, including exon 19 deletion, exon 21 L858R, and L861Q.
Diagnostic assays based on PD-L1 immunohistochemistry were performed according to the manufacturer’s instructions. The following antibodies and detection systems were utilized: mouse monoclonal primary anti–PD-L1 antibody, 22C3 pharmDx (prediluted, clone 22C3, Dako, Carpinteria, CA, USA) with an Autostainer Link 48 and an EnVision DAB Detection System (Agilent Technologies, Santa Clara, CA, USA); rabbit monoclonal primary anti-PD-L1 antibody, Ventana SP263 (prediluted, Ventana Medical Systems, Tucson, AZ, USA) with a Benchmark XT staining system and Ultra with OptiView Universal DAB Detection Kit (Ventana Medical Systems). The staining results were interpreted by expert lung pathologists. The percentage of tumor cells with membranous PD-L1 staining of any intensity (tumor proportion score, TPS) was categorized into three ranges and the corresponding groups (<1%, negative expression; 1–49%, weak expression; ≥50%, strong expression).

IHC analysis was conducted with the PD‐L1 IHC 22C3 pharmDx and the Ventana PD‐L1 (SP263) assays on the DAKO Autostainer Link 48 and Ventana BenchMark platforms, respectively. Consecutive 4 μm thick sections cut from the same core specimen were pretreated and stained with the PD‐L1 antibody 22C3 mouse monoclonal primary antibody from pharmDx on a Dako Autostainer Link 48 with EnVision DAB Detection System (Agilent/Dako, Santa Clara, CA, USA) with negative control reagents and cell line run controls, as described in the PD‐L1 IHC 22C3 pharmDx, and PD‐L1 antibody SP263 rabbit monoclonal primary antibody from Ventana on a Ventana Benchmark Ultra with OptiView Universal DAB Detection Kit (Ventana Medical Systems, Tucson, AZ, USA) with a matched rabbit immunoglobulin G–negative control, in accordance with the manufacturers’ instructions, respectively. The detection and quantification of the percentage of immunoreactive tumor cells was performed according to the manufacturers’ recommendations. Briefly, neoplastic cells were considered positive when any cell membrane staining (partial or complete) was present, ignoring pure cytoplasmic immunoreaction. Staining on immune cells was also disregarded. Quantification of immunoreactive neoplastic cells was obtained by evaluating the ratio between stained carcinoma cells and all viable carcinoma cells.