Detection of HPV in Japanese and Chinese oral carcinomas by in situ PCR
Introduction
The etiology of oral cancer is complex and many factors including tobacco and alcohol have been reported. It is well established that the presence of certain human papillomavirus (HPV) types causes cervical carcinoma [1]. More than 100 distinct HPV genotypes [2] have been identified in humans and the possible role of HPV infection in the etiology of oral cancer has been supported by direct studies of tumor materials. However, the association between HPV and oral squamous cell carcinomas (SCCs) is unknown and the results have been contradictory. The reported prevalence of HPV DNA in oral cancer tissue has varied from 0 to 100%. The variations in reported prevalence are likely to be due to differences in the populations studied and the methods used for HPV detection. Of all the HPV DNA detection techniques, the polymerase chain reaction (PCR) is the most sensitive under optimal experimental conditions. In situ PCR combines the sensitivity of solution PCR with the subcellular localization provided by traditional in situ hybridization (ISH). The purpose of this study was to examine the prevalence of HPV in oral SCCs of Japanese and Chinese using in situ PCR.
Several consensus or general primers such as MY09 and MY11 [3], GP5 and GP6 [4], CP1 and CP2 [5], L1C1 and L2C2 [6] are particularly useful for analyzing large numbers of clinical samples for epidemiological research. They can amplify a broad spectrum of HPV types that are conserved among most of the HPV genomes. MY09 and MY11 primers are the most popular and widely used of these [7], [8], [9], [10], [11], [12]. To our knowledge, no application of MY09 and MY11 primers to in situ PCR detection has been reported.
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Tissue samples
Oral specimens were obtained from our university hospital, from the Department of Oral and Maxillofacial Surgery, College of Stomatology, West China University of Medical Sciences, China, and from the Department of Oral Pathology, Francisco Marroquin University, Guatemala. All tissues were fixed in 10% buffered formalin for 5–36 h at room temperature and routinely embedded in paraffin. Formalin-fixed, paraffin-embedded tissue specimens were stored at room temperature for a period of 3 months–2
Results
The distribution of various lesions and results of ISH and in situ PCR experiments are shown in Table 1. All positive control samples of 10 anogenital lesions showed positive reactions by ISH (Fig. 1a) or in situ PCR (Fig. 1b, c). However, the positive reactions detected by in situ PCR showed a dramatically increased signal intensity compared with those processed by ISH. The images shown in Fig. 1b showed not only much more intense signals but also many more positive cells than those in Fig. 1a
Discussion
The in situ PCR method enabled strong visualization of a direct correlation of the presence of HPV infection with the histologic and cytologic features of individual SCC lesions, while detection of HPV DNA in anogenital condyloma lesions by ISH gave only a weak signal (Fig. 1a). In all our cases of oral SCCs from Japanese and Chinese (n=20), HPV-DNA-positive reactions were detected by in situ PCR (Fig. 2, Fig. 3, Fig. 4, Fig. 5), whereas ISH could not detect HPV DNA in oral SCCs at all.
Acknowledgements
This work between Osaka Dental University and West China University of Medical Sciences was supported by the International Academic Exchange Grant from Osaka Dental University.
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