Immunohistochemical (IHC) analysis and assessment

IHC analyses of resected, paraffin-embedded lung cancer tissues were performed. After microtome sectioning (4 μm), the slides were processed for staining using an automated immunostainer (Nexes; Ventana, Tucson, AZ, USA). The streptavidin–biotin–peroxidase detection technique using diaminobenzidine as the chromogen was applied. The primary antibodies were used according to the manufacturer’s instructions. The slides were examined by two investigators who had no knowledge of the corresponding clinicopathological data. IHC was performed using the following antibodies: a mouse monoclonal anti-PD-L1 antibody (1:100, clone SP142; Spring Bioscience, Pleasanton, CA, USA), CD8 (1:50, clone C8/144B, Dako; Agilent Technologies, Inc., Santa Clara, CA, USA), and Cox-2 (1:50, clone CX-294, Dako; Agilent Technologies, Inc.) according to a previously described protocol.^8,18,27

The expression levels of each marker protein were examined and evaluated according to a previously reported original protocol. PD-L1 expression was categorized as positive when staining of the tumor cell membrane (at any intensity) was present. PD-L1 positive expression was observed at prespecified expression levels of 5% of all cells in a section that included at least 100 evaluable tumor cells.⁸ In order to evaluate the immunostaining of the CD8-positive TILs, 10 digital high-power field (HPF) images of the tumor area were selected, and the absolute number of CD8-positive TILs in these images was determined.¹⁸ For Cox-2, the slides were scored according to the intensity of staining (0–3) and the percentage of positively stained cells (0, 0%; 1, 1%–9%; 2, 10%–49%; and 3, 50%–100%). The IHC score (0–9) was calculated by multiplying the intensity and percentage scores. Cox-2 expression was considered high when the IHC score was ≥4.²⁷ Figure 1 depicts the IHC Cox-2 expression levels in the tissues.

Representative sections of lung adenocarcinoma tissues with cytoplasmic staining for Cox-2. (A) High expression; (B) low expression.

Abbreviation: Cox-2, cyclooxygenase-2.