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.

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.

Major pathologic response assessment and clinical significance of metastatic lymph nodes after neoadjuvant therapy for non-small cell lung cancer.

For neoadjuvant therapy in patients with non-small cell lung cancer, the major pathologic response of primary tumors may be an assessable and reliable surrogate measure of survival. Few studies have examined the pathologic evaluation of metastatic lymph node responses and their prognostic significance. This retrospective study enrolled 336 patients with non-small cell lung cancer (squamous cell carcinoma, n = 216; adenocarcinoma, n = 120) treated with neoadjuvant therapy including chemotherapy (n = 316) and targeted therapy (adenocarcinoma, n = 20). The treatment response of the primary tumor and lymph node metastases (LNM) were pathologically assessed according to the multidisciplinary recommendations of the International Association for the Study of Lung Cancer. The relationship of overall survival (OS) and disease-free survival (DFS) with the responses of the primary tumor or LNM was analyzed. The optimal cutoff value of the residual viable tumor (%RVT) of the primary tumor was 12% for both OS (P < 0.001) and DFS (P < 0.001). The pathologic assessment identified LNM in 208 patients. The optimal %RVT cutoff value in LNM was 8% for both OS (P = 0.003) and DFS (P < 0.001). The Spearman's rank correlation coefficient between primary tumors and corresponding LNM was 0.487 for %RVT (P < 0.001), which indicated a positive correlation. On multivariable analysis, an RVT of the primary tumor ≤12% was an independent prognostic factor for improved OS (P = 0.024), whereas an RVT of LNM ≤ 8% was an independent prognostic factor for increased DFS (P = 0.018). Furthermore, in the neoadjuvant chemotherapy group, the optimal %RVT cutoff values for OS in patients with squamous cell carcinoma and adenocarcinoma in the primary tumor were 12% and 58%, respectively. Considering its convenience and operability in clinical application, a 10% threshold RVT value can be used for prognostic evaluation of LNM and primary tumors of squamous cell carcinoma histology; further studies are needed to confirm the optimal cutoff value for primary tumors of adenocarcinoma.