A PCR approach to discriminate between integrated and episomal HPV DNA in small clinical specimens

doi:10.1006/mcpr.1993.1042

Molecular and Cellular Probes

Volume 7, Issue 4, August 1993, Pages 285-292

https://doi.org/10.1006/mcpr.1993.1042 Get rights and content

Abstract

HPV infection has long been implicated in the development of cervical carcinoma. There is strong evidence for association of high-risk HPV types 16 and 18 with cervical intraepithelial neoplasia (CIN) grades 2 and 3, and integration of viral DNA of these types into the host genome has been suggested to play an important role in progression to invasive cervical carcinoma. However, the existing techniques for detection of integrated DNA in clinical specimens are time-consuming and require large amounts of template DNA, often unavailable for small premalignant CIN lesions. In this study, a novel, two stage PCR assay was designed to discriminate between integrated and episomal HPV 16 DNA. The first stage was designed to determine whether intact HPV genomes were present. This initial PCR analysis of the entire viral genome in eight segments was successfully applied to authentic human cervical cancers. The second stage consisted of an ANCHOR-PCR-based analysis, developed specifically to discriminate between integrated and episomal HPV DNA, that was successfully tested with cloned HPV containing plasmids used to mimic both episomal and integrated viral DNA. Further optimization and validation will be required for application of the second stage to clinical specimens. The entire assay was developed to be applicable to small colposcopic biopsies or cervical scrape samples, representative of those acquired in routine clinical investigation of CIN 2 or CIN 3, in which determination of the physical state of HPV DNA may provide prognostically valuable information.

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Cited by (11)

Understanding the HPV integration and its progression to cervical cancer
2018, Infection, Genetics and Evolution
Citation Excerpt :
The first techniques used for the detection of HPV in cells were immunohistochemistry and Fluorescent in situ hybridization (FISH), using monoclonal antibodies targeting cell proteins such as p16, which is produced in the presence of HPV infection, primarily when E7 oncogene is expressed (Park et al., 1999a; Sano et al., 1998; Rufforny et al., 2005), and probes for HPV sequences allowing the fluorescent detection of small segments of the viral genome in the chromosomes of the host cell (Lizard et al., 1994; Siadat-Pajouh et al., 1994; Adler et al., 1997). Later, HPV detection was performed by polymerase chain reaction (PCR) (Carmody et al., 1996), and it was possible to detect the presence of the E2, E6, and E7 genes, allowing researchers to determine the viral state in the infected cell (episomal/integrated) by measuring the amplification of these sequences (Donaldson et al., 1993). These integration-detecting methods improved with real-time PCR, with the ability to observe with higher precision the E2/E6 or E2/E7 ratio in real time (Zheng et al., 2006; Fujii et al., 2005).
Cervical cancer is one of the main causes of female cancer death worldwide, and human papilloma virus (HPV) its causal agent. To investigate viral oncogenesis several studies have focused on the effects of HPV oncoproteins E6 and E7 and the mechanisms by which these proteins stimulate the cellular transformation process. However, phenomena such as the physical state of the viral genome (episomal or integrated) and the effects of this integration on cell proliferation contribute new clues to understand how HPV infection causes carcinogenesis. New molecular technologies are currently facilitating these discoveries. This paper reviews the tumor development process initiated by HPV, recent findings on the process of viral integration into the host genome, new methods to detect HPV integration, and derived associated effects.
Detection and identification of human papilloma viral DNA, types 16, 18, and 33, by a combination of polymerase chain reaction and a colorimetric solid phase capture hybridisation assay
1998, Journal of Virological Methods
A colorimetric microplate hybridization assay was developed previously to simplify detection procedures of DNA fragments resulting from polymerase chain reactions (PCR). This format has now been adapted for the simultaneous detection and identification of three human papillomavirus (HPV), types 16, 18 and 33, associated frequently with cervical cancer. This post-PCR detection system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated oligonucleotidic probes for detection. It therefore offers a double specificity; the first is conferred by pairs of primers, specific of each type of virus tested, and the second, by the sets of capture and detection probes which are complementary to internal regions of the amplified DNA fragments. The detection format outperformed agarose gel electrophoresis of amplified DNA products in sensitivity and specificity. The rapidity and simplicity of this hybridisation system would justify its use in routine diagnostic examination of cervical specimens (smears and biopsies).
Use of the polymerase chain reaction to specifically amplify integrated HPV-16 DNA by virtue of its linkage to interspersed repetitive DNA
1996, Molecular and Cellular Probes
A polymerase chain reaction (PCR) based technique that combines a virus specific primer and a human interspersed repetitive sequence (IRS) specific primer in order to detect integration of human papilloma virus type 16 (HPV-16) is described. Amplification of viral-host DNA junctions occurs when viral integration results in placement of the virus specific primer binding site near (less that 3–4 kb) the primer binding site within a repetitive sequence element. The method relies on enzyme labeled oligonucleotide probes to achieve rapid, specific, and nonradioisotopic detection of viral integration related PCR products since episomal forms of the viral DNA do not lead to exponential accumulation of hybridizable PCR products. The technique is demonstrated for human genomic DNA derived from clinical cervical swab specimens and archival paraffin embedded blocks. Viral integration was detected in 41% of the HPV-16 positive samples (n=34). In this positive subset, 64% were classified as invasive neoplasias, 29% CIN III and 7% CIN II. Analyzing the positive invasive neoplasias, 6 of 9 (66%) of the fingerprint results were obtained when an HPV primer was paired with anAluprimer. Interestingly, 100% ofAluprimed fingerprint results obtained were derived from samples presenting invasive neoplasia (P<0·025 by chi square).
Risk of Cervical Intraepithelial Neoplasia Grade 2 or Worse and HPV Integration Status Conversion in HPV Integration-Positive Women: A One-Year Follow-Up
2023, SSRN
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2003, Journal of Clinical Pathology
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Original ArticlesA PCR approach to discriminate between integrated and episomal HPV DNA in small clinical specimens

Abstract

Original Articles
A PCR approach to discriminate between integrated and episomal HPV DNA in small clinical specimens