A standard immunohistochemical (IHC) protocol was followed and 2 µm unstained tumor sections from formalin-fixed paraffin-embedded tissue were deparaffinized in xylene and rehydrated through graded alcohols. After antigen retrieval, the sections were stained with primary antibodies, PD-L1 (28-9; Abcam; dilution: 1:200), CD45 (EP322Y; Abcam; dilution: 1:250), and β-catenin (E247; Abcam; dilution: 1:250), followed by antibody localization using the Dako Envision + HRP-labeled polymer (DAKO). Staining was visualized by 5-minute incubation with diaminobenzidine.
Positivity for PD-L1 and PD-1 was defined as positive staining in both tumor cells and tumor-infiltrating lymphocytes (TILs). Expression of PD-L1 on tumor cell was scored as 0 (absence of staining), 1 (1%–10% of staining), 2 (10%–50% of staining), and 3 (>50% of staining). The extent of staining in TILs was recorded as absent (0), focal (1), mild (2), moderate (3), and strong (4) with score 0 or 1 considered negative. IHC expression of β-catenin in tumor cell was evaluated and scored as percentage of cytoplasmic and/or nuclear positivity multiplied by intensity (weak, 1; moderate, 2; and strong, 3), as described previously. All sections were reviewed by two independent pathologists until consensus was made.
Positivity for PD-L1 and PD-1 was defined as positive staining in both tumor cells and tumor-infiltrating lymphocytes (TILs). Expression of PD-L1 on tumor cell was scored as 0 (absence of staining), 1 (1%–10% of staining), 2 (10%–50% of staining), and 3 (>50% of staining). The extent of staining in TILs was recorded as absent (0), focal (1), mild (2), moderate (3), and strong (4) with score 0 or 1 considered negative. IHC expression of β-catenin in tumor cell was evaluated and scored as percentage of cytoplasmic and/or nuclear positivity multiplied by intensity (weak, 1; moderate, 2; and strong, 3), as described previously. All sections were reviewed by two independent pathologists until consensus was made.