2.9. Immunohistochemistry (IHC)

Samples from the HIPO-HNC cohort ({"type":"entrez-geo","attrs":{"text":"GSE117973","term_id":"117973"}}GSE117973) were provided by the tissue bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany) in accordance with the regulations of the tissue bank and the approval of the Ethics Committee of the Heidelberg University Hospital (under protocols S-206/2011, S-220/2016 and S-786/2021). IHC staining was performed using anti-SOX2 (Cell Signaling, D6D9) or anti-SOX9 (Cell Signaling, D8G8H or ABCAM, 76997) antibodies as described previously [18]. The whole stained slices were scanned using a slide scanner (Axioscan, Zeiss, Oberkochen, Germany) with a 20× magnification objective. The immune-reactivity score (IRS) was computed as a product of the staining intensity (1: mild, 2: moderate and 3: strong) and the percentage of positively stained tumor cells ranging from 0–100%. The final immune-reactivity score (IRS) was calculated as described [18].