Comparative study between the Hybrid Capture II test and PCR based assay for the detection of human papillomavirus DNA in oral submucous fibrosis and oral squamous cell carcinoma

OSCC was most prevalent in this region [4]. The habits of chewing tobacco and areca nut with or without betal quid are rampant in this area. Besides the main risk factors of tobacco, smoking and alcohol, infection by human papillomavirus (HPV) and genetic alterations are likely to play an important role in these lesions [5]. Oncogenic HPVs are a main causative agent for cervical cancer, but the role of HPV infection in OSMF and OSCC is less established. Human papillomavirus is about 55 nm in diameter. It has a single circular double stranded DNA molecule and belongs to the family papillomaviridae. Its genome is made up of 7,200-8,000 base pairs with a molecular weight of 5.2 × 106 D. Molecular evidences also provide support to the role of high risk HPV, particularly HPV16, in the pathogenesis of OSCC of the head and neck [6]. Kreimer et al reported that genomic DNA of oncogenic HPV has been detected approximately 26% of all OSCC of the head and neck worldwide [7] but the most accurate and consistent study for OSMF and OSCC, in which viral integration and the expression of viral oncogenes (E6 and E7) have been shown [8]. HPV detection in potentially malignant and malignant oral squamous cell carcinoma showed many discrepancies. Several studies reported the presence of HPV-DNA within these lesions with variable frequency. HPV16 and 18 genotypes were the most frequently found viruses in these lesions. Bouda et al suggested that high risk HPV E6/E7 transcripts and viral integration have also been detected in head and neck squamous cell carcinoma (HNSCC). Transcriptionally active HR-HPVs, particularly HPV-16 are found in a subset of HNSCC. HPV16- associated carcinogenesis is mediated by expression of the viral E6 and E7 oncoproteins, which cause deregulation of the cell cycle by inactivating p53 and pRb respectively [9]. Integration often disrupts the integrity and expression of the E1 and E2 open reading frames, which may affect the transcription of E6 and E7 genes [10-13]. In HPV-16 and HPV-18, the E2 proteins are active in virus proliferation, it control E6-E7 gene expression and are necessary for episomal virus production [10]. Specific viral genes (E6 and E7) from HPV types 16, 18, and 33 act as oncogenes [14,15]. Recentaly, a second-generation assay with improved diagnostic sensitivity has been developed known as hybrid capture II test (HC-II) and approved by the US food and drug administration (FDA). The performance characteristics of HC-II assay and the PCR for detection of HPV DNA have been compared in cervical lesions and results shown variation. [16-19]. Many testing techniques used to identify the prevalence rate of cervical as well as oral associated HPV infection have been developed but specific HPV testing may not be proper as a primary screening tool due to lack of specificity and sensitivity of the test. Newly developed molecular techniques have significantly assisted to indentifying the association of HR-HPV types with these oral lesions. The purpose of this study was to compare the efficacy of HC-II assay and PCR for the detection of specific HPV type (HPV 16 E6) in OSMF and OSCC cases as well as find out the prevalence rate of the HR-HPV in these lesions. Materials and methods Clinical data collection and sample collection Four hundred and thirty patients of the OSMF and OSCC cases were taken from the Department of Pathology & Department of Otorhinolaryngology, Moti Lal Nehru Medical College, Allahabad, India from Sept 2007-March 2010, in a random manner after obtaining consent from the institutional ethical committee. Of which 208 cases were OSMF and 222 cases were OSCC. Detailed demographic information of each patient, including the patient’s age, sex, addiction habits was obtained. Emphasis was given on their addiction habits. Usually those patients were considered who had no previous history of treatment for HPV infection. Figure 1 illustrating the oral sample collection and HPV testing strategy [Figure 1]. Detailed clinical examination of each patient was done to assess the site, size and type of lesions. For confirmation of the clinical diagnosis, histopathological examination was carried out. Patients with OSMF were included in this study and lesions with an abnormal epithelial surface like erythroplakia, leukoplakia and submucosal lesions including hemangiomas, mucoceles, papilloma, aphthous ulcers, melanoplakia and fibromas were excluded due to less number. A punch biopsy was performed as per standard protocol and the tissue was processed by paraffin embedding 2-3 micrometer thick sections were cut and stained by haematoxylin and eosin (H and E). Biopsies