New molecular method for the detection of human papillomavirus type 16 integration

Abstract

Human papillomavirus (HPV) infection is the cause of cervical cancer. Integration of HPV-16 DNA in cervical cells is considered to be a key event in the progression towards invasive cancer, but little is known about this event in anal carcinogenesis. The integration could be a useful biomarker for cancer progression. Optimized assays are needed to determine the value of real-time detection of HPV integration in longitudinal studies, and this approach is only possible with a high-throughput assay. The aim of this study was to develop a new multiplex real-time PCR assay based on simultaneous amplification of the E2 and E6 HPV open reading frames (ORFs) in order to assess the physical status (episomal and/or integrated) of HPV-16 in anal cells of HIV-positive men. The comparative threshold (Ct) cycle values for E2 and E6 obtained for SiHA cells and artificial mixtures of episomal and integrated DNA were as expected: similar Ct for episomal forms and absence of E2 amplification for integrated forms. The multiplex real-time PCR was tested in 77 consecutive samples from individual HIV-infected patients with HPV-16 anal infection. The integration of HPV-16 was detected in 25 (32%) patients: 23 as mixed (episomal and integrated) and two as completed integrated forms. The integration occurs in the early stage of anal lesions and was associated with the severity of the lesions (p 0.004). The multiplex real-time PCR assay developed in the course of this study was shown to be a simple, sensitive, specific and inexpensive technique which may be applied routinely to detect HPV-16 integration.

Human papillomavirus HPV-16, 18, 52 and 58 integration in cervical cells of HIV-1-infected women

“…These limitations could underestimate the detection of integrated forms. [11][12][13] However, our results are in agreement with previous studies, [14][15][16] suggesting that the viral integration of HPV could be a good biomarker for the evolution from HPV infection to cervical cancer.…”

“…11 The assay is based on the principle that, when integration occurs, the E2 gene is partially or totally disrupted while the E6 gene remains intact. It was developed to analyze the physical status of HPV-16 and has been adapted to HPV-18, HPV-52 and HPV-58 types.…”

“…PCR protocols and primers/probes have been previously described. 11,12 Real-time PCR was performed using an ABI 7300 PCR systems (Applied Biosystems, USA). Particular care was taken to prevent carry-over contamination by separating pre-and post-PCR areas.…”

MicroRNA-21 expression and susceptibility to HPV-induced carcinogenesis — role of microenvironment in K14-HPV16 mice model

“…The integration of HPV was assessed by amplification of HPV16-E6 and HPV16-E2 genes with specific primers (Table 1), which amplify a region of 130 bp and 184 bp, respectively, (adapted from a protocol described by Cañadas et al and Ribeiro et al (Canadas et al, 2010;). The PCR amplification reaction with HPV16-E6 and HPV16-E2 primers was carried in a 50 µl reaction mixture with 1x PCR Buffer, 2.5 mM MgCl2, 0.2 mM DNTP'S, 0.30 µM of each primer, 1 U of Taq DNA polymerase and 0.2 µg of genomic DNA.…”

Relationship between E6-E7 lineage sequences, viral loads, and integration of HPV16 in women with atypical squamous cells of undetermined significance (ASCUS) pap smears

“…The physical state of HPV16 was determined using qPCR that targeted the HPV16 genes E6 and E2, because the E2 gene is more frequently lost during viral integration. Thus, E2 and E6 genes are present in equivalent amounts in cells with only episomal HPV genomes, whereas in cells with 100% integrated HPV genomes, the E6 gene is present but the E2 gene is absent [38]. The percentage of integrated HPV DNA was expressed as the ratio of: the integrated viral load ([number of E6 DNA copies] -[number of E2 DNA copies] per cell) / the to number of E6 DNA copies x100.…”