4.7. Immunohistochemistry

As mentioned previously [17], IHC biomarkers, namely endogenous hypoxic (Glut1, CAIX, and HIF-1α), angiogenesis or metastasis (VEGF, c-Met), cell proliferation (EGFR, c-Myc, insulin-like growth factor 1 receptor (IGF-1R)), cell-to-cell adhesion (E-cadherin, Vimentin), evasion to apoptosis (B-cell lymphoma 2 (BCL2), Bax, MCL1), immunogenic or inflammatory biomarkers (programmed cell death protein ligand 1 (PD-L1), tumor necrosis factor-α (TNF-α), calretinin, galectin-9, and chemokine ligand 5 (CCL5)) were analyzed using tissue microarrays from incisional biopsy specimens before treatment. Each tumor was represented by one tissue core on a tissue microarray. Furthermore, 4 µm thick paraffin sections were deparaffinized and microwaved according to standard procedures before being processed for IHC staining.

The microscopic stains were scored by two pathologists blinded to the clinical outcome. Except for PD-L1, IHC results from the aforementioned biomarkers were scored by a semiquantitative approach used to assign an H-score to tumor samples [17,28].

The tumor PD-L1 biomarker was evaluated through IHC staining using the DAKO clone 22C3 pharmDx kit (DAKO, Carpinteria, CA, USA). PD-L1 expression was scored according to the combined positive score (CPS), which is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) at any intensity divided by the total number of viable tumor cells, multiplied by 100 [29].