Validation of multiplex immunofluorescence and digital image analysis for programmed death-ligand 1 expression and immune cell assessment in non-small cell lung cancer: comparison with conventional immunohistochemistry
AimsThis study aimed to validate the application of combined multiplex immunofluorescence (mIF) and digital image analysis (DIA) in formalin-fixed and paraffin-embedded tissues for the quantitative assessment of programmed death-ligand 1(PD-L1) and immune cells (ICs) in non-small cell lung cancer (NSCLC).MethodsFifty resected samples of NSCLC were sequentially stained with a DNA-tagged mIF (panel including PD-L1, CKpan, CD8, CD68 and 4′,6-diamidino-2-phenylindole (DAPI)) and conventional immunohistochemistry (cIHC). The assessment of cell density and consistency of tumour proportion score (TPS) via DIA were compared with those by pathologists.ResultsA strong correlation in the cell population of immune markers was obtained between mIF and cIHC (for PD-L1: R=0.9304, CKpan: R=0.8231, CD8: R=0.9314 and CD68: R=0.8366) within 95% limits of agreement. The continuous TPS calculated using mIF was highly consistent with the IHC staining results which were evaluated by pathologists (R=0.9362). However, in the comparison of TPS using interval variables, a poor agreement was obtained at a cut-off of 1% (κ=0.197), whereas excellent agreement was achieved at cut-offs of 50% (κ=0.908) and 5% (κ=0.823). DIA on mIF showed that PD-L1 commonly colocalised with CD68+ macrophages and CD8+ cytotoxic cells were closer to PD-L1-/CK+ tumour cells (TCs) than to PD-L1+/CK+ TCs in spatial distribution.ConclusionsA combination of mIF and DIA is useful for the quantification of PD-L1 expression and IC populations in NSCLC. Further validation of TPS at a cut-off of 1% and assay harmonisation is essential for translating this method in a diagnostic setting.