Prevalence of human papillomavirus infection in premalignant and malignant lesions of the oral cavity in U.K. subjects: a novel method of detection

Abstract

To evaluate the possible role of human papillomavirus (HPV) in oral neoplasms, 28 oral squamous cell carcinomas (SCC) and 12 potentially malignant lesions were analysed for the presence of HPV DNA. A nested polymerase chain reaction (PCR) approach, using two sets of HPV consensus primers to the L1 region, was used, which was able to detect a broad spectrum of HPV types. HPV DNA was detected in 14/28 (50%) carcinomas and 4/12 (33%) precancerous lesions. A novel approach based on labelling the PCR products with ³²P and the separation of radioactively labelled products on an 8% polyacrylamide gel increased the sensitivity of the detection and enabled the identification of the HPV types. The typing of HPV was subsequently confirmed by direct DNA sequencing. HPV 6 and HPV 16 were the only HPV types detected and seven tumours harboured both types. Our results suggest that HPVs may be an important aetiological factor in the development of oral cancer. The detection procedure ensured sensitivity and consistency of the detection of low copy numbers of the virus DNA. The presence of HPV in 33% of premalignant tissues suggests that HPV infection may be an early event in the malignant transformation of oral SCC. There was no statistically significant association between viral infection and tumour grade or stage.

Introduction

Oral and pharyngeal cancer together constitute the sixth most common malignancy in the world, accounting for almost 4% of all cancers and 2% of all cancer deaths1, 2. In the U.K., there are nearly 2000 new cases and 900 deaths from oral cancer itself each year[3]. In some developing countries, such as India and Sri Lanka, oral cancers are often the commonest form of malignancy, accounting for over 40% of all cancers. High incidence rates are also found in north-western France and Central Europe[3]. The vast majority of malignancies in the oral cavity are squamous cell carcinomas (SCCs). Despite major advances in the management of oral cancer, improvements in the prognosis of this disease have not so far been observed and the 5-year survival rate remains poor. The cause of oral cancer has been attributed to several factors, including smoking, heavy alcohol consumption and betel quid chewing4, 5. However, it is clear that mutagenesis by these environmental agents alone is not the sole cause of oral cancer and that additional agents, possibly viral, may be required for the acquisition of the malignant phenotype.

Aberrations in the p53 gene, associated with the loss of function of the p53 protein, have been reported in up to 80% of oral SCCs6, 7, 8. Loss of function of the p53 tumour suppressor gene can occur by mutations in the conserved region of the gene, as well as through its association with other cellular, MDM2 and viral oncoproteins, such as E6 of human papillomavirus (HPV)9, 10. HPV is a small DNA virus with over 70 different types identified so far based on the DNA sequence[11]. HPV plays an important role in the pathogenesis of cervical and anogenital SCC and with less certainty in SCCs of the skin and oral cavity12, 13. The high risk HPVs 16 and 18 are associated with the malignant transformation of the cervical epithelium of the uterine cervix. The low risk HPVs, including 6 and 11, are associated with benign skin and mucosal warts14, 15. More than 12 types of HPV, including types 1, 2, 4, 6, 7, 11, 13, 16, 18, 30, 32 and 57, have been identified in oral lesions and HPV 16 is the most common type to be associated with both cervical and oral cancers16, 17, 18. In vitro studies have also shown that primary human oral epithelial cells can be immortalised by high risk HPV types19, 20. However, exposure to tobacco related chemicals was required for these cells to progress to a fully malignant phenotype21, 22. Studies of HPV in patients with oral cancer report prevalences varying from 0[23]to 100%[24]. Possible reasons for such a large variation are the use of different detection methods and differences in the populations studied, not least with respect to prevailing environmental risk factors. One of the major difficulties in the detection of HPV infection in oral cancer is the presence of the virus in only a subpopulation of cells and at low copy number in the infected cells[25]: highly sensitive and controlled methods are therefore required.

In this study we used DNA extracted from primary oral lesions for polymerase chain reaction (PCR) amplification of the HPV genome. Using a nested PCR procedure and labelling the PCR products with ³²P dCTP, we increased the sensitivity and accuracy of viral detection.