Aims This 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).
Methods Fifty 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.
Results A 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.
Conclusions A 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.
- lung neoplasms
Data availability statement
Data are available on reasonable request. The data that support the findings of this study are available from the corresponding author on reasonable request.
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Handling editor Runjan Chetty.
JW and LM contributed equally.
Contributors JW and LM contributed equally to image analysis, data statistics, manuscript writing and participated in the experimental design. WS and XY scanned the slides for multiplex staining and verified PD-L1 scoring. HW, XL, KC and XH prepared the slides and performed multiplex immunofluorescence staining. DL conceived the study, participated in its design and coordination and helped draft and edit the manuscript.
Funding This work was supported by the National Natural Science Foundation of China (82003155 and 81871860), the Capital’s Funds for Health Improvement and Research (2020-2-1025) and Innovation Fund for Outstanding Doctoral Candidates of Peking University Health Science Centre (JW).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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