Key words: HPV; viral physical status; squamous cell carcinoma of the head and neckThe etiology of squamous cell carcinoma of the head and neck (HNSCC) is considered to be multifactorial. In addition to wellknown risk factors such as tobacco and alcohol, 1 human papillomaviruses (HPVs) may play an etiologic role in a subset of the head and neck cancers. 2 Particularly HPV-16 infection may be a risk factor for oropharyngeal HNSCC. 3 Integration of high-risk HPV DNA into the human cellular genome is considered an important step in malignant transformation. 4,5 Viral DNA integration often leads to disruption of the HPV-16 E2 gene, 6 which in an intact genome controls the expression of E6 and E7 oncogenes, resulting in increased expression of these oncogenes. 4,7 Application of a real-time PCR technique in determination of HPV-16 viral physical status has been recently evaluated. 8,9 It is based on the assumptions that 1) in integrated viral DNA, the E2 gene is disrupted; 2) episomal viral DNA has equivalent copy numbers of the E2 and the E6 genes; however, deleted forms, including E2 deletion, of episomal HPV DNA, although not very common, have been observed in the tonsillar carcinoma; 10 and 3) mixed forms have smaller copy numbers of the E2 than the E6 genes. 8 The aims of our study were to determine the prevalence, genotypes, and physical status of HPV DNA in HNSCC samples at diagnosis and to correlate these findings with clinical outcome. Moreover, we wanted to find out whether cutaneous HPV types play a role in head and neck carcinogenesis. PATIENTS, MATERIAL AND METHODS PatientsWe assessed HNSCC patients treated at Helsinki University Central Hospital during the years 1993 to 2002 whose fresh-frozen samples were available and obtained at diagnosis. These patients (n ϭ 61; 11 females, mean age 63.7 years, range 57-91; 50 males, mean age 59.6, range 40 -85) whose tumor biopsies contained at least 40% of neoplastic cells were included in the study. Clinical data concerning TNM classification, treatment of the primary tumor and the neck and overall survival at the end of follow-up were collected on data sheets from case records. Of these 61 patients, 2 were excluded from survival analysis because their samples had been taken after the defined endpoint for survival analysis. The mean follow-up time of the patients was 24.5 months (range 1.4 -89.6 months). Specimens and DNA extractionBiopsies from primary tumors were taken at diagnosis and stored immediately at Ϫ70°C. 50 -100 m thick cryo-sections, containing a minimum of 40% (typically 60 -70%) neoplastic cells, as examined by toluidine blue staining at both ends of the section, served for DNA extraction (QIAGEN GmbH, Hilden, Germany). Distilled H 2 O and human embryonic skin (HES) fibroblasts served as negative controls. DNA was stored at Ϫ20°C until PCR analysis. HPV PCR and genotypingThe prevalence of HPV DNA was analyzed with a broadspectrum SPF10 PCR and a microtiterplate-based probe hybridization assay as described earlier. 11 Amplicons from HPV-positive sam...

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MCPyV DNA occurs in tonsils more frequently in adults than in children. By contrast, WUPyV DNA is found preferentially in children. MCPyV occurs also in nasal swabs and NPAs, in a frequency similar to that of KIPyV and WUPyV. The tonsil may be an initial site of WUPyV infection and a site of MCPyV persistence.

Although laryngeal papillomatosis is clinically well defined, the mechanisms and treatment modalities of laryngeal HPV infection need further investigations.