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 freshfrozen 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 ␤-actin. Key words: HPV; tonsillar cancer; physical state; viral load; prognosisAccumulating molecular and epidemiological data indicate that the high-risk types of human papillomavirus (HPV) are not only associated with cervical cancer, but may also be associated with certain subtypes of cancer in the head and neck. [1][2][3][4][5] The strongest association has been found for oropharyngeal squamous cell carcinoma, especially tonsillar cancer, where HPV DNA is present in 45-70% of the cases. [1][2][3][5][6][7][8][9] In a nested case-control study it was found that patients who were sero-positive for HPV-16 had a 14.4 excess risk of developing oropharyngeal cancer later in life, and in another study patients with a history of HPV-related anogenital cancer had a 4.3 higher risk of developing tonsillar cancer. 4,10 Furthermore, patients with HPV positive tonsillar cancer seem less likely to be heavy smokers and drinkers, and to have a better prognosis than patients with HPV negative tonsillar cancer. 3,7,8 Similar to cervical cancer, the oncogenes of HPV-16, E6 and E7, are generally expressed in HPV positive tonsillar cancer. 1,9,11,12 The viral protein E6 promotes degradation of p53, whereas E7 inactivates pRb. 13,14 P53 mutations have been reported to be less frequent in HPV positive tonsillar cancer as compared to HPV negative tonsillar cancer, although when assayed by immunohistochemistry the proportion of elevated p53 levels are similar in both HPV positive and negative tumours. 3,12,15 In addition, HPV positive tonsillar cancers seem to have a decrease of pRb, possibly due to E7 activity. 2,9 In cervical cancer the HPV genome is mainly integrated in the host genome. 16,17 Integration leads to disruption and deletion of the viral genes E1 or E2 open reading frame (ORF), which are of importance for viral replication and viral transc...

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