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Myers-Squibb, Agenus and Torque Therapeutics. All other authors declared no conflicts of interest.

Background Programmed death–ligand 1 (PD‐L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non–small cell lung cancer (NSCLC) for anti‐programmed cell death protein 1 (PD‐1)/PD‐L1 therapy. The current study evaluated the feasibility and efficacy of PD‐L1 immunostaining and quantitation on direct Papanicolaou‐stained cytological smears compared with formalin‐fixed paraffin‐embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD‐L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona). Methods PD‐L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC). Results In 57 sets of samples, both PD‐L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin‐fixed paraffin‐embedded samples was observed in 97.3% of the cases. Conclusions The quantification of PD‐L1 expression on direct Papanicolaou‐stained cytology smears is feasible and reliable for both PD‐L1 assays.

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Objective Understanding the immune environment of non‐small cell lung cancer (NSCLC) is important for designing effective anticancer immunotherapies. We describe the use of multiplex immunofluorescence (mIF) assays to enable characterisation of the tumour‐infiltrating immune cells and their interactions, both across and within immune subtypes. Methods Six cytological samples of NSCLC taken by transoesophageal ultrasound‐guided fine needle aspiration were tested with an mIF assay designed to detect the expression of key immune cell markers such as CD3, CD8, CD20, CD11b, and CD68. Pan‐cytokeratin was used to detect the NSCLC cells. Fluorescence images were acquired on a Vectra‐Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). Results MIF assay was able to reliably detect and quantify the myeloid cell markers CD11b, CD68, CD3+ and CD8+ T cells, and CD20+ B lymphocytes on cytological samples of NSCLC. Whole‐tissue analysis and its correlation with the corresponding H&E stains allowed a better understanding of the tissue morphology and the relationship between tumour and stroma compartments. Additionally, a uniform, specific, and correct staining pattern was seen for every immune marker. Conclusion The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC.