Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16‐positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods—the mapping of E2 integration breakpoint at the mRNA level, the 3’ RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV‐positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV‐positive integrated vs. extrachromosomal/mixed forms of the virus.

The association of high‐risk human papillomaviruses (HR HPVs) with tonsillar cancer (TC) has been documented. Because patients with HPV‐associated tumors show better survival rates, modification of their treatment regimen is being considered. It is therefore crucial to find markers for the identification of patients whose tumors are linked to viral infection. A cohort of 109 patients with primary TC was screened for HPV DNA presence in the tumor tissues and HPV‐specific antibodies in sera. Data regarding risk factors and clinical parameters were collected. Forty‐five specimens were analyzed for the expression of viral E6 and E2‐region mRNA, and the p16 and p53 protein expression status was assessed by immunohistochemistry. The overall prevalence of HPV DNA in TC tissues was 65.1%. Ninety‐three percent of HR HPV DNA‐positive samples expressed E6*I mRNA. E2‐region mRNA expression was detected in 36% of positive samples, which implies that the virus is integrated in 64% of HPV DNA/RNA‐positive tumors. p16 overexpression and the presence of antibodies specific to HPV16 E6/E7 oncoproteins correlated well with HPV DNA and RNA presence. The disease‐specific survival rate of patients with HPV DNA‐positive tumors was significantly higher than that of HPV DNA‐negative patients. In addition to providing further evidence of the involvement of HPV infection in the etiopathogenesis of a proportion of TC cases, our study demonstrates that p16 immunostaining and anti‐E6/E7 antibodies as surrogate markers of HPV involvement represent specific, sensitive and clinically accessible assays for the identification of TC patients who have a considerably better prognosis.