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M Arbyn et al. (July 2024). Validated HPV tests usable in cervical cancer screening on clinician-collected cervical specimens. www.HPVWorld.com, 270

HPV testing and clinical validation of HPV assays
Ample evidence indicates that cervical cancer screening using assays targeting carcinogenic human papillomavirus (HPV) types, followed by management of HPV-positive women offers stronger protection against future cancer of the cervix uteri than cytology-based screening^1,2. Therefore, an increasing number of countries and international cancer organisations have switched or are in the phase of switching to virological screening and recommend screening using clinically validated tests that identify nucleic acid sequences of carcinogenic HPV types³. Whereas hundreds of different HPV assays are commercially available, only a small minority of them have demonstrated fulfilment of the international validation criteria defined by Meijer et al in 2009^4-6. The clinical validation of a new index HPV assay for use in cervical cancer screening requires that the new assay demonstrates non-inferior sensitivity and non-inferior specificity to detect cervical intraepithelial neoplasia of grade 2 (CIN2) or worse compared to a first or second-generation comparator test. The benchmarks for relative sensitivity and specificity (index versus comparator assay) are ≥0.90 and ≥0.98, respectively⁶. Practically this means that the left 90% confidence interval bound (CIB) around the relative sensitivity or relative specificity is not lower than these benchmarks.

Initially, only Hybrid Capture 2^® (HC2, Qiagen, Gaithersburg, MD, USA) and GP5+/6+ PCR-EIA^® (Diassay, Rijkswijk, the Netherlands) were accepted as comparator tests since these assays were evaluated in population-based randomised trials that proved lower cumulative incidence of invasive cancer than cytology. Since these two assays are no longer widely used as most laboratories have moved to newer-generation HPV tests, new criteria for second-generation comparators have been defined. Four assays fulfil the second-generation standard comparator criteria: RealTime High-Risk HPV Test^® (Abbott, Wiesbaden, Germany), Cobas 4800 HPV Test^® (Roche Molecular System, Pleasanton, CF, USA), Onclarity HPV Assay^® (BD Diagnostics, Sparks, MD, USA); and Anyplex II HPV HR Detection^® (Seegene, Seoul, South Korea) (Arbyn 2024, submitted). Besides fulfilling the criteria of non-inferior clinical accuracy, sufficient intra- and inter-laboratory reproducibility of the index HPV assay should be documented where the lower 95% CIB ≥87% and kappa ≥0.5⁶.

The international validation criteria outlined above concern only HPV DNA assays applied to cervical specimens. Assays that target other molecules than HPV DNA sequences also have to provide evidence of the longitudinal safety, which means documentation of a similar or lower risk of CIN3+ after a negative test result compared to the risk after a negative result of a validated HPV DNA test, over a period of at least five years⁵.

In this article we update previous lists of clinically validated HPV tests^5,7. Systematic reviews and meta-analyses on the accuracy for CIN2+ of new HPV tests compared to a first or second-generation comparator assay were extended in time to cover reports published up to April 2024. The systematic review also incorporates recently published data concerning intra- and inter-reproducibility of new assays and longitudinal performance of HPV mRNA assays.

Compared to the latest list incorporated in the 2023 ESGO Book of Gynaecologic Oncology, containing 16 validated assays, four additional assays that fulfil international validation criteria can be added: OncoPredict HPV QT^® (Hiantis, Milano, Italy), RIATOL HPV genotyping qPCR assay^® (AML, Antwerp, Belgium), Allplex HPV HR Detection assay^® (Seegene, Seoul, South Korea) and Vitro HPV Screening assay^® (Vitro S, Sevilla, Spain). The results are summarized in Table 1. The updated list contains 19 HPV DNA tests that fulfilled the cross-sectional validation criteria and one HPV mRNA test, which also demonstrated non-inferior longitudinal performance compared to validated DNA assays⁸. Three assays target only an aggregate of 13 to 14 high-risk HPV types and do not provide any genotyping detail; eight assays provide limited genotyping capacity enabling separate identification of the most carcinogenic types HPV16 and HPV18 (with or without HPV45); three assays provide extended genotyping (more genotyping detail compared to tests with limited genotyping capacity) whereas six assays enable separate genotyping of all targeted types. Two test manufacturers produced assays with limited or no genotyping, but provide a second reflex assay allowing for more genotype granularity when the initial assay is positive.

CONFLICTS OF INTERESTS
MA, SKD and MP were supported by the Horizon 2020 Framework Programme for Research and Innovation of the European Commission, through the RISCC Network (Grant No. 847845).

Sciensano, the employer of MA and SD, received funding from the European Society of Gynaecological Oncology (ESGO), the World Health Organization (Geneva, Switzerland, via the Agreement for Performance of Work for guidelines on Screening and Treatment of Pre-Invasive Cervical Disease), the European Commission Initiative on Cervical Cancer (EC-CvC), and the VALGENT and VALHUDES projects.

University of Ljubljana, the employer of MP has received research funding, free-of-charge reagents, and consumables to support research from the following commercial entities in the last three years: Qiagen, Self-screen, Seegene, Abbott, and Roche. MP has been a co-investigator on the VALGENT projects.

This research work has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No.847845 (Project RISCC).

References

1. Arbyn M, Ronco G, Anttila A, et al. Evidence regarding human papillomavirus testing in secondary prevention of cervical cancer.
Vaccine. 2012;30Suppl5:F88-F99. Available from: https://www.sciencedirect.com/science/article/pii/S0264410X12010055?via%3Dihub

2. Ronco G, Dillner J, Elfstrom KM, et al. Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials. Lancet. 2014;383(9916):524-32. Available from: https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(13)62218-7/abstract

3. IARC. IARC Handbooks of Cancer Prevention, Vol 18: Cervical Cancer Screening. Lyon; 2022. Available from: https://publications.iarc.fr/Book-And-Report-Series/Iarc-Handbooks-Of-Cancer-Prevention/Cervical-Cancer-Screening-2022

4. Poljak M, Ostrbenk Valencak A, Cuschieri K, Bohinc KB, Arbyn M. 2023 global inventory of commercial molecular tests for human papillomaviruses (HPV). J Clin Virol. 2024;172:105671. Available from: https://www.sciencedirect.com/science/article/pii/S1386653224000337?via%3Dihub

5. Arbyn M, Simon M, Peeters E, et al. 2020 List of human papillomavirus assays suitable for primary cervical cancer screening. Clin Microbiol Infect. 2021;27(8):1083-95. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1198-743X(21)00219-6

6. Meijer CJLM, J Berkhof, Castle PE, Hesselink AT, et al. Guidelines for human papillomavirus DNA test requirements for primary cervical cancer screening in women 30 years and older. Int J Cancer. 2009;124(3):516-20. Available from: https://onlinelibrary.wiley.com/doi/epdf/10.1002/ijc.24010

7. Arbyn M, Rousta P, Dhillon SK, et al. Chapter 10: HPV testing on cervical cancer specimens: which assay can be used in cervical cancer screening. In: Ayhan A, ed. ESGO Textbook of Gynaecological Oncology. Istanbul: Günes Publishing;2023:115-20. Available from: https://flipflashpages.uniflip.com/3/34834/1129203/pub/html5.html

8. Arbyn M, Simon M, de Sanjose S, et al. Accuracy and effectiveness of HPV mRNA testing in cervical cancer screening: a systematic review and meta-analysis. Lancet Oncol. 2022;23(7):950-60. Available from: https://www.thelancet.com/journals/lanonc/article/PIIS1470-2045(22)00294-7/abstract

9. Bouvard V, Baan R, Straif K, et al; WHO International Agency for Research on Cancer Monograph Working Group. A review of human carcinogens--Part B: biological agents. Lancet Oncol. 2009;10(4):321-2. Available from: https://doi.org/10.1016/s1470-2045(09)70096-8



This article is included in the HPW monograph Cervical cancer prevention in Europe

Scientific coordinators:
Johannes Berkhof, Kate Cuschieri, Clàudia Robles, Xavier Bosch

HPW editors:
Patricia Guijarro, Paula Peremiquel, Valentina Rangel

On behalf of the editorial team, we would like to thank all the authors who contributed to this special monograph


OTHER ARTICLES INCLUDED IN THIS MONOGRAPH:

J Wang, MK Elfström, J Dillner. The impact of cervical cancer screening for different HPV genotypes.

E van den Borst, S Van Keer, A Vorsters. Urine as sample for primary cervical cancer screening.

M Poljak, A Oštrbenk Valenčak. Global overview of commercially available HPV molecular tests.

M Lehtinen, K Louvanto. Low progression potential of cervical HSIL among HPV-vaccinated women with persistent non-vaccine type HPV infections.

M Elfstrom, J Dillner. Evaluation of co-testing with cytology and human papillomavirus testing in cervical screening.

L Baandrup, SK Kjaer. Profile of HPV infections in vaccinated cohorts and implications for future screening.

IM de Kok, E E.L. Jansen, J A.C. Hontelez. Harms and benefits of cervical cancer screening and its future.

H JA Bogaards, V Pimenoff, M Lehtinen. HPV type replacement in vaccinated cohorts.

J Berkhof. Main results of RISCC Consortium.

K Cuschieri, L Connor, S Arroyo Muhr. Key elements of quality assurance for practical HPV testing to support cervical screening.

T McGlacken, M Fitzgerald, N Russell. Allowing safe exit from cervical cancer screening – which strategy to use.

J Bonde. A Silver bullet - HPV self-sampling in Denmark.

S Hawco, A Woolner. Obstetric effects of introducing the HPV vaccination.