Prevalence, distribution, and viral load of human papillomavirus 16 DNA in tonsillar carcinomas

Oncogenic human papillomaviruses (HPV) DNA have repeatedly been observed in many head and neck carcinomas (HNSCCs), and HPV infections are currently considered a possible factor in the etiology of these tumors. However, the reported prevalences of HPV-DNA in HNSCC are variable. In the current study the authors used highly sensitive polymerase chain reactions (PCRs) to analyze the occurrence of viral sequences in 98 carefully stratified HNSCCs. The authors determined the load and localization of HPV DNA in a subset of tonsillar carcinomas and their metastases.

Nested PCR and an HPV16 specific single step PCR were used to screen 98 HNSCCs for HPV DNA for genital- and Epidermodysplasia verruciformis (EV)-associated HPVs. Typing was performed by direct sequencing and/or sequencing of cloned amplimers. In two patients HPV16 subtypes in tonsillar carcinomas and their metastases were compared by amplification and sequencing of the long control region of the virus. In a subset of HPV16 positive tonsillar carcinomas and their metastases, localization and viral load were determined using laser assisted microdissection and real time fluorescent PCR, respectively.

Altogether 25 HNSCCs (26%) were found to be HPV positive. Stratified according to the tumor localization, the frequency of HPV positive lesions was 18% in the oral cavity, 45% for oropharynx, 25% for hypopharynx, 8% for nasopharynx, and 7% for larynx. The highest HPV DNA prevalence (58%) was found in tonsillar carcinomas. The high risk HPV type 16 was found in 84% of positive HNSCCs, in 14% of which EV-associated HPVs were detected. Human papillomavirus sequences were detected in 64% of biopsies with normal mucosa from 11 patients with positive carcinomas. As a control group, 14 tumor free tonsils were analyzed. In none of these specimens were HPV sequences detected. Viral long transcriptional control region sequences in homologous metastases were identical with those in primary tumors and the load values in both locations were roughly comparable. Viral loads differed substantially in different areas of one tumor. Statistical evaluation of data related to clinicopathologic parameters showed a significant linkage of HPV with tonsillar carcinomas compared to other locations. Furthermore, a significant correlation of HPV status of tonsillar carcinomas with tumor grading and alcohol consumption was found.

Our study shows a preferential association of HPV-DNA with tonsillar carcinomas. The data support the view of HPV negative and positive tonsillar carcinomas being different tumor entities and conventional cancer risk factors being of less importance in HPV-infected individuals. The HPV genome is located in the cancer cells, whereas the infection of normal mucosa is a rare event. Data on quantification of HPV16 in tonsillar tumors and their metastases showed mean viral loads comparable to other HPV associated malignancies.