Workflow of FFPE sample preparation and selection. Eighty-two FFPE blocks  were stained for p16 of which eight samples were excluded from further analysis, showing mixed p16 staining. Eight samples were excluded after the LCM step, yielding insufficient amounts or quality of DNA and two further samples were excluded due to inconsistent or borderline results in repeat E6 qPCR measurements. In total, 22 confirmed HPV+ (p16+ and E6 qPCR+) and 34 HPV- (p16- and E6 qPCR-) samples were suitable for further analysis. Following age and gender matching, 20 HPV+ HNSCC samples (red) and 20 HPV- HNSCC samples (grey) were then selected for the final analysis (next-generation (NG) sequencing).