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.

Results of this nationwide study give a general overview of the outcome of unselected patients treated with unified guidelines. Patients with T2 tumors, usually treated with radiotherapy (RT), had a worse prognosis than expected. This patient group warrants further investigation and possibly treatment intensification.

Fiberoptic videolaryngoscopy during bicycle ergometry was a well-tolerated and relatively easily established diagnostic tool that could induce dyspnea in more than one half the patients examined. If the symptom of dyspnea appeared, the most frequent diagnosis was exercise- induced vocal cord dysfunction.

According to PCR, the prevalences of human papillomavirus (HPV) DNA were 6.3% (13 of 206) in tonsillitis or hypertrophic tonsillar tissues and 0.6% (1 of 174) in exfoliated cells from normal tonsils. HPV-16 was the only type detected in tonsillar tissues, but it did not appear to lead to L1 antibody production.Of the squamous cell carcinomas of the head and neck, human papillomavirus (HPV) DNA has been repeatedly reported in tonsillar carcinoma, suggesting a role for HPV in tonsillar carcinogenesis (5,9,10,13,22). The reason for the strong association of HPV with tonsils remains unclear. Previous data on the presence of HPV DNA in tumor-free tonsils are based on small series of experiments (24). A significantly increased risk for squamous cell carcinomas of the head and neck is associated with HPV-16 seropositivity (11), but serum antibodies against HPV-16 proteins have been detected in healthy individuals as well (6). Our aim was to study HPV in tumor-free tonsils and to correlate these findings with the seroprevalence of HPV antibodies.We enrolled 212 patients operated on in 2001 and 2002 because of tonsillitis (n ϭ 135) or tonsillar hypertrophy (n ϭ 77) at Helsinki University Central Hospital (Table 1). After tonsillectomy, a piece of tissue was immediately frozen at Ϫ70°C. A 100-m cryo-section per tonsil per patient was subjected to DNA extraction. Five-micrometer sections from both ends of the tissue were toluidine blue stained to verify that the tissue section consisted of at least 30% epithelial cells. After every tenth sample, only optimal cutting temperature compound without tonsillar tissue was cryo-sectioned as a negative control for the DNA extraction; none of these sections showed positive ␤-globin or HPV by PCR. Serum samples were drawn on the day of surgery.Tonsillar exfoliated cells from 189 control subjects (age and sex matched to patients) with normal tonsils were collected bilaterally from tonsillar surfaces with a Cytobrush Plus Cell Collector (Medscand Medical). The brushed samples were stored in phosphate-buffered saline at Ϫ70°C. Serum samples were collected on the same day.DNA was extracted from tonsillar tissue with the DNA mini kit (QIAGEN) and from exfoliated cells with the MagNA Pure LC DNA isolation kit I (Roche). In order to assess the quality of the DNA submitted to HPV PCR, we applied ␤-globin PCR (15) and found that 97% (206 of 212) of DNA samples from tonsillar tissue and 92% (174 of 189) of DNA samples from tonsillar exfoliated cells were positive for ␤-globin.HPV DNA was identified by nested PCR with consensus primers MY09/11 (14) and GP5ϩ/6ϩ (3). The genotype was determined by direct sequencing of the approximately 150-bp amplicons with an ABI PRISM Dye Terminator and analyzed on an ABI 377 DNA sequencer (Applied Biosystems) and on the basis of Ͼ90% homology with HPV sequences deposited in GenBank with BLAST software.Serum antibodies against HPV-16 L1, E6, and E7 were analyzed by a newly developed method. The HPV proteins were expressed as fusion proteins with N-terminal glut...

In our series HPV was not important in LSCC. Heavy drinking led to major mortality in LSCC and promoted early carcinogenesis.