HPV involvement in head and neck (HN) cancer is still under active investigation. Fresh frozen and archival clinical samples from 115 patients affected by HN carcinomas were analysed by PCR-based methods and direct sequencing. HPV types, intra-type variants, physical status, viral load and viral transcript presence were determined. HPV positivity was correlated with the main clinical-pathological features, including smoker and drinker status, and the clinical outcome. Twenty-one tumours were HPV positive (18.3%) with HPV16 being the most frequent type (n=14) followed by HPV6 (n=4), HPV33, HPV35, and HPV58 (n=1, each type). Tonsil carcinomas contained more high-risk HPV types (6/8; 75%) than all other sites (p=0.0004). HPV16 genome was integrated in all analysed tumours, as pure integrated form or mixed with concomitant episomal forms (4 cases). The viral load showed a wide variability (range, 0.7-485 copies per cell) with the highest value detected in a larynx tumour and the lowest one in a case of cancer of the oral cavity. In 9 HPV-positive samples where mRNA was available, transcripts of viral early oncogenes originating by integrated, episomal or mixed forms of the viral genome were found. A statistically significant correlation was evidenced between HPV and tumour differentiation, being the virus more associated with tumour grade G3/G4. Multivariate Cox regression analysis revealed that lymph-node and grade status were significant independent factors for a worse disease-free survival and overall survival, whereas the HPV status was associated with a better overall survival (OR, 0.33; 95% CI, 0.13-0.81; p=0.01). Taken together these results indicate that distinct pathological mechanisms for the malignant transformation in each single HN subsite should be taken in account; HPV molecular analyses should be considered a valid tool to distinguish subsets of oropharyngeal tumours and HPV presence could be useful for the prognostic assessment of HNSCC.

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