Immunohistochemical Analysis of LKB1, NRF2, and NQO1

Formalin-fixed, paraffin-embedded (FFPE) tissue sections were prepared as per standard protocol for IHC. Epitope retrieval was performed on the Dako PT link (Dako, Denmark). Immunostaining was completed using the automated staining systems, Dako Autostainer Link48 (Dako, Denmark). The LKB1 antibody (1:100, clone D60C5F10, Cell Signaling Technology), NRF2 antibody (1:50, PA5-27,882, ThermoFisher), and NQO1 antibody (1:1000; ab28947; Abcam) were added for 30 min at room temperature. The slides were counterstained with hematoxylin. LKB1/NRF2/NQO1 expression was evaluated in the background non-neoplastic tissue providing an internal negative control. LKB1/NRF2/NQO1 staining was scored as previously described^{26 (link),27 (link)}. The staining intensity was graded as 0 (no staining), 1 + (weak), 2 + (moderate), and 3 + (intense). The percentage of stained tumor cells was recorded and the H-score was calculated using the following formula: 1 × (%cells 1 +) + 2 × (%cells 2 +) + 3 × (%cells 3 +). A final H-score of 0 was assigned as negative, 1–100 as weak, 101–200 as medium, and 201–300 + as strong. All slides were evaluated by a pathologist (P.C.) who was blinded from the patients' outcomes.