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The aim of our study was to investigate the physical state and the viral load of HPV-16 in tonsillar cancer and to correlate these findings with clinical outcome. To distinguish between integrated and episomal forms of HPV, 22 fresh-frozen tonsillar cancer samples were analysed by a method based on restriction enzyme cleavage, ligation and PCR (rliPCR). HPV-16 was detected in 11/22 and HPV-33 in 1/22 of the cancers, hence 12/22 (55%) of the tumours were HPV positive. Only extrachromosomal forms of HPV-16 were observed. Full-length episomal HPV was detected exclusively in 7/11 of the cancers, whereas both full-length and deleted forms of episomal HPV-16 were found in parallel in 2 other tumours. In 1 tumour only a deleted episomal form of HPV-16 was present. In the remaining HPV-16 positive tumour both full-length episomal as well as an 11 kbp PCR product were detected and if the 11 kbp product contained integrated HPV, or was off-size linearised episomal could not be determined. In 2 cervical cancer controls, HPV-16 was integrated and could be chromosome located. HPV-16 was quantified by real-time PCR and most tonsillar cancers contained between 10 to a few hundred copies of HPV per beta-actin. The 6 patients with tumour sections with > or =190 HPV-16 copies/beta-actin remained tumour free (p = 0.026) and had a better survival rate (p = 0.039) when compared to the 5 patients with tumours sections with < or =60 HPV-16 copies/beta-actin. In conclusion, HPV-16 is mainly episomal in tonsillar cancer. The viral load showed a wide distribution and the clinical outcome in our study was better when the HPV load was higher.
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