Retrospective Trial to Assess HPV Epidemiology in Squamous Cell Carcinoma of Head and Neck

To determine the prevalence and classify the high risk human papillomavirus types associated with head and neck cancer in Israel. We will determine the HPV genotype distribution in Israel in order to study the ethnic uniqueness of the region. Many studies have shown HPV 16 to be the most prevalent type in HNSCC (NEJM). In Israeli Jewish women the HPV types 16, 39, 52, and 18 were the most prevalent genotypes found (15). In our aim, we will determine the prevalence of various HPV genotypes in HNSCC specimens.Study population The group will include patients treated with primary surgery or biopsy, with or without adjuvant radiotherapy between 1998 and 2010 for SCC of the oral cavity, oropharynx, hypopharynx, larynx and paranasal sinuses.

The anatomic site and extent of the primary tumor will be documented. The TNM classification of will be based on the staging system revised by the American Joint Committee on Cancer (AJCC). Patients will be recruited from seven national cancer referral hospitals, including Soroka Medical Center in the Negev (Beer Sheva), Sourasky Medical Center (Tel Aviv), Rabin Medical Center (Petach Tikva), Hadassa Medical (Jerusalem), Rambam Medical Center (Haifa), Sheba medical center (Ramat- Gan) and French Hospital Nazareth (Nazareth). These centers cover almost the entire population in Israel, including Israeli Jews, Bedouins, Druze and Israeli Arab population.

Data Entry Data will be entered into a commercially available spreadsheet (Microsoft Excel 2000, Microsoft Corporation, Seattle, WA) and statistical analysis will perform using a computerized software package (SPSS). We have previously used a similar system to report the results of 146 patients with skull base sarcomas in an international multicenter study including 17 cancer centers (17).

Tumor specimens Specimens will be collected retrospectively, and comprise of paraffin sections of primary tumors or biopsies retrived from the pathology department archives at each center. Transportation of paraffin blocks to Tel Aviv for deparaphinization and HPV testing will take place personally by the investigators. Histopathological analysis Primary tumor specimens will be first evaluated by pathologists at each participating cancer. All specimens will be than re-analyzed and evaluated by head and neck pathologist, Dr. Kaplan Ilana. Specimen dissection and tissue sampling of the primary tumor will be in accordance with the current guidelines for the histopathological assessment of head and neck cancer carcinoma.

HPV detection at the laboratory Tissue sections will be subjected to deparaffinization, heatinduced target retrieval, and digestion with proteinase K (Roche Diagnostics, Indianapolis, IN), as described previously (18). DNA is then purified from paraffin by deparaffinization, proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation. We will use the HPV genotyping system using a PCR based HPV GenoArray test kit for genotyping of 37 HPV taypes (Hybribio Limited, Hong Kong). Specimens are placed into PreservCytR LBC medium (ThinPrep liquid Pap vial; Cytyc Corporation). The Amplicor HPV test kit contains a pool of HPV-specific primers designed to amplify HPV DNA from 13 HR genotypes (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). According to the manufacturer's specification, the Amplicor HPV test detects HPV genotypes 31, 52, 58, and 59 at 240 copies/ml and HPV genotypes 16, 18, 33, 35, 39, 45, 51, 56, and 68 at 100 copies/ml with a positivity rate greater than 95%. All genotypes are detected with a 100% positivity rate at 480 copies/ml.