Human papillomavirus (HPV) 16 genome integration into the host chromosome is a crucial event during the life cycle of the virus and a major step towards carcinogenesis. The integration of HPV16 DNA promotes a constitutive high expression level of E6 and E7 oncoproteins, resulting in the extensive proliferation of the infected epithelial cells. In the present report the physical status of the HPV16 genome was studied, through determination of E1/E6 and E2/E6 DNA copy number ratios in 61 cervical samples of low-and high-grade malignancy and 8 cervical cancer samples, all of them associated with HPV16 infection. The selection of E1, E2 and E6 amplification target regions was performed according to the most prevalent deleted/disrupted sites of E1 and E2 genes. For this target selection we also considered the most conserved regions of E1, E2 and E6 genes among the same HPV16 isolates that were recently reported by our group. The analysis of HPV16 DNA form revealed a significant association among the mixed DNA forms in low-grade and high-grade malignancies, (x 2 , P,0.01). The comparative analysis of E1/E6 and E2/E6 in the same cervical samples provides an accurate picture of HPV16 DNA form and may reveal whether different HPV16 DNA integrants coexist in the same cervical sample or not. This study proposes that E1/E6 and E2/E6 ratios determine with accuracy the HPV16 DNA integration pattern and may predict multiple integration events in the examined sample, thus providing significant information about the progression of cervical dysplasia. INTRODUCTIONCervical cancer is the third most common type of cancer among women worldwide, with a high mortality rate. The worldwide incidence of cervical carcinoma is more than 530 000 cases per year, whereas mortality reaches 275 000 deaths annually, of which approximately 85 % occur in developing countries (Jemal et al., 2011;Forman et al., 2012). The aetiological agents for the development of highgrade precancerous cervical lesions and invasive cervical cancer are the oncogenic human papillomavirus (HPV) types present as persistent infections (Muñoz et al., 2003).To date, more than 150 different HPV types have been characterized and about 40 of them are related with anogenital tract malignancy, grouped as high-risk (HR) or low-risk (LR) genotypes (zur Hausen, 1996;Bernard et al., 2006 Bernard et al., , 2010. Epidemiological studies revealed that HPV16 is the most commonly observed HR HPV type, followed by HPV18, 31, 33 and 45 (de Sanjose et al., 2010;Li et al., 2011).Persistent infection with HR HPV types is associated with an increasing risk of integration of the viral circular genome (episome) into the host chromosomes, leading to cancer development. The circular HPV genome is then linearized, but the long control region, and the E6 and E7 oncogenes, are always retained intact (Wentzensen et al., 2004; Xu et al., 2013;Akagi et al., 2014). Viral integration appears to coincide with the development of high-grade cervical intraepithelial neoplasia (CIN II, III) as a consequence...

The causal association between persistent human papillomavirus (HPV) infection and cervical cancer has lead to the development of a variety of molecular assays for HPV detection. The present study focused on the development of a simple, sensitive and cost-effective HPV genotyping method based on multiplex PCR methodology that could be easily performed in small laboratories. Three multiplex PCR assays were developed to identify the HPV genotypes 16, 18, 45, 35, 66, 33, 51, 58, and 31 together with an internal control. The method was established by designing nine type-specific primer sets that target conserved regions of the L1 gene. The assay was applied using HPV-positive cervical specimens, and cloning and sequencing of all of the amplicons that were generated were performed to examine the specificity of the newly designed primers. Moreover, an experimental cutoff value was determined through reconstitution experiments using HPV DNA plasmids. Amplicons of expected size were obtained, while cloning and sequencing of PCR products confirmed the genomic specificity of the amplicons. The sensitivity of this method was determined to be 10 copies of each individual HPV genotype per test. Multiple and single HPV infections were documented in 42.2 % and 57.8 % of cervical specimens, respectively. The most prevalent HPV genotype was HPV16, followed by HPV18, HPV66 and HPV51. The present multiplex PCR assay is a simple method with high specificity and sensitivity that can be applied in clinical or epidemiological analyses for rapid identification of the most clinically important HPV genotypes present in cervical intraepithelial neoplasias.