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Background: Human papilloma virus (HPV) infection, p16 expression and hypoxia may play important roles in the carcinogenesis, treatment response and toxicities of head and neck squamous cell carcinoma (HNSCC). The aim of this analysis was to assess whether there is any correlation between pre-radiotherapy (RT) anemia, p16 expression and toxicities and local control for patients undergoing definitive therapy. Methods: 79 HNSCC patients who had undergone radiochemotherapy (RCT) or RT-antibody therapy were retrospectively analyzed. p16 (INK4A) expression was detected by immunohistochemical analysis. Factors predisposing for acute side effects were examined by uni- and multivariate analysis. Results: p16 overexpression was detected in 32 cases. Pretreatment anemia was present in one third of patients. Only 5% of patients were characterized by both pre-RT anemia and p16 overexpression. p16 expression was significantly associated with acute grade 3 toxicity. 47% of p16-positive patients developed grade ≥ 3 radiodermatitis compared to 26% of p16-negative patients (p = 0.04). A reduced risk of severe skin toxicities was noted for patients with hypoxic blood values before RT. p16 expression was significantly correlated with local control (p = 0.002). Conclusions: p16 expression is associated with better response to definitive combined treatment (RCT, RT + cetuximab), but also significantly related to acute high-grade toxicity.