Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18  E6  and  E7  oncogenes

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Abstract

Human papillomaviruses (HPV) play a key role in promoting human anogenital cancers. Current high-risk HPV screening or diagnosis tests involve cytological or molecular techniques mostly based on qualitative HPV DNA detection. Here, we describe the development of a rapid quantitative polymerase chain reaction (qPCR) detection test of HPV16 and HPV18 oncogenes ( E6 and E7) normalized on human gene encoding GAPDH. Optimized qPCR parameters were defined, and analytical specificities were validated. The limit of detection was 10 ¹ for all genes tested. Assay performances were evaluated on clinical samples (n = 96). Concordance between the Xpert HPV assay and the triplex assay developed here was 93.44% for HPV16 and 73.58% for HPV18. HPV co-infections were detected in 15 samples. The systems developed in the present study can be used in complement to traditional HPV tests for specifically validating the presence of HPV16 and/or HPV18. It can also be used for the follow-up of patients with confirmed infection and at risk of developing lesions, through the quantification of E6 and E7 oncogene expression (mRNA) normalized on the GAPDH expression levels.

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Worldwide burden of cancer attributable to HPV by site, country and HPV type

Catherine de Martel, Martyn Plummer, Jerome Vignat … (2017)

HPV is the cause of almost all cervical cancer and is responsible for a substantial fraction of other anogenital cancers and oropharyngeal cancers. Understanding the HPV‐attributable cancer burden can boost programs of HPV vaccination and HPV‐based cervical screening. Attributable fractions (AFs) and the relative contributions of different HPV types were derived from published studies reporting on the prevalence of transforming HPV infection in cancer tissue. Maps of age‐standardized incidence rates of HPV‐attributable cancers by country from GLOBOCAN 2012 data are shown separately for the cervix, other anogenital tract and head and neck cancers. The relative contribution of HPV16/18 and HPV6/11/16/18/31/33/45/52/58 was also estimated. 4.5% of all cancers worldwide (630,000 new cancer cases per year) are attributable to HPV: 8.6% in women and 0.8% in men. AF in women ranges from 20% in India and sub‐Saharan Africa. Cervix accounts for 83% of HPV‐attributable cancer, two‐thirds of which occur in less developed countries. Other HPV‐attributable anogenital cancer includes 8,500 vulva; 12,000 vagina; 35,000 anus (half occurring in men) and 13,000 penis. In the head and neck, HPV‐attributable cancers represent 38,000 cases of which 21,000 are oropharyngeal cancers occurring in more developed countries. The relative contributions of HPV16/18 and HPV6/11/16/18/31/33/45/52/58 are 73% and 90%, respectively. Universal access to vaccination is the key to avoiding most cases of HPV‐attributable cancer. The preponderant burden of HPV16/18 and the possibility of cross‐protection emphasize the importance of the introduction of more affordable vaccines in less developed countries.

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Human papillomavirus molecular biology and disease association

John Doorbar, Nagayasu Egawa, Heather Griffin … (2015)

Summary Human papillomaviruses (HPVs) have evolved over millions of years to propagate themselves in a range of different animal species including humans. Viruses that have co‐evolved slowly in this way typically cause chronic inapparent infections, with virion production in the absence of apparent disease. This is the case for many Beta and Gamma HPV types. The Alpha papillomavirus types have however evolved immunoevasion strategies that allow them to cause persistent visible papillomas. These viruses activate the cell cycle as the infected epithelial cell differentiates in order to create a replication competent environment that allows viral genome amplification and packaging into infectious particles. This is mediated by the viral E6, E7, and E5 proteins. High‐risk E6 and E7 proteins differ from their low‐risk counterparts however in being able to drive cell cycle entry in the upper epithelial layers and also to stimulate cell proliferation in the basal and parabasal layers. Deregulated expression of these cell cycle regulators underlies neoplasia and the eventual progression to cancer in individuals who cannot resolve high‐risk HPV infection. Most work to date has focused on the study of high‐risk HPV types such as HPV 16 and 18, which has led to an understanding of the molecular pathways subverted by these viruses. Such approaches will lead to the development of better strategies for disease treatment, including targeted antivirals and immunotherapeutics. Priorities are now focused toward understanding HPV neoplasias at sites other than the cervix (e.g. tonsils, other transformation zones) and toward understanding the mechanisms by which low‐risk HPV types can sometimes give rise to papillomatosis and under certain situations even cancers. Copyright © 2015 John Wiley & Sons, Ltd.

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The natural history of cervical HPV infection: unresolved issues.

Ciaran Woodman, Stuart Collins, Lawrence S Young (2007)

The identification of high-risk human papillomavirus (HPV) types as a necessary cause of cervical cancer offers the prospect of effective primary prevention and the possibility of improving the efficiency of cervical screening programmes. However, for these opportunities to be realized, a more complete understanding of the natural history of HPV infection, and its relationship to the development of epithelial abnormalities of the cervix, is required. We discuss areas of uncertainty, and their possible effect on disease prevention strategies.

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Author and article information

Contributors

Christelle Desnues: christelle.desnues@univ-amu.fr

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 17 February 2021

Publication date PMC-release: 17 February 2021

Publication date Collection: 2021

Volume: 11

Electronic Location Identifier: 4039

Affiliations

[1 ]GRID grid.5399.6, ISNI 0000 0001 2176 4817, Aix-Marseille Université, IRD 198, Assistance-Publique des Hôpitaux de Marseille, , UMR Microbes, Evolution, Phylogeny and Infections (MEPHI), ; Marseille, France

[2 ]GRID grid.500499.1, ISNI 0000 0004 1758 6271, Aix-Marseille Université, Université de Toulon, CNRS, IRD, , Mediterranean Institute of Oceanography (MIO), ; MEB, UM 110, 163 avenue de Luminy, Case 901, Bâtiment OCEANOMED-Méditerranée, 13288 Marseille Cedex 09, France

[3 ]GRID grid.483853.1, ISNI 0000 0004 0519 5986, IHU Méditerranée Infection, ; Marseille, France

Article

Publisher ID: 83489

DOI: 10.1038/s41598-021-83489-2

PMC ID: 7889863

PubMed ID: 33597592

SO-VID: abfbad79-d34c-409d-8933-69a93ce55d43

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 5 October 2020

Date accepted : 15 January 2021

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001665, Agence Nationale de la Recherche;

Award ID: n° 10-IAHU-03

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ScienceOpen disciplines: Uncategorized

Keywords: human papilloma virus,pcr-based techniques

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ScienceOpen disciplines: Uncategorized

Keywords: human papilloma virus, pcr-based techniques

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Development and validation of a multiplex qPCR assay for detection and relative quantification of HPV16 and HPV18 E6 and E7 oncogenes

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Most cited references 42

Worldwide burden of cancer attributable to HPV by site, country and HPV type

Human papillomavirus molecular biology and disease association

The natural history of cervical HPV infection: unresolved issues.

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