In comparison to cytology, HPV-based primary cervical cancer screening provides greater protection against invasive cervical cancer, has better sensitivity for detection of underlying cervical intraepithelial neoplasia grade 2 or worse (CIN2+), and allows prolonged intervals between screening rounds. Despite recommendations that only clinically validated HPV tests with optimal balance between clinical sensitivity and clinical specificity should be used, several clinically non-validated HPV tests are used worldwide in daily practice. At least 246 commercial HPV tests and 214 test variants were available on the global market in October 2017, however only a small subset of commercial HPV tests has a documented clinical performance and performance evaluations were frequently not performed in line with agreed standards in the HPV community. To assess suitability and facilitate the acceptance of novel high-risk HPV tests for primary cervical cancer screening, carefully designed cohorts must be used to obtain conclusive evidence.

Slovenian HPV Prevalence Study
In 2009, a cross-sectional study was conducted in Slovenia to provide baseline data for pre-vaccination HPV prevalence.^1-3 Within 16 outpatient gynecology services with a nationwide coverage, a representative cohort of women attending the routine national cervical cancer screening program was established. Between December 2009 and August 2010, we prospectively enrolled 4,432 women aged 20-64 years. During the gynecological examination, two samples were obtained from each woman – one sample for routine cervical cytology testing and one sample for HPV DNA testing. The sample for HPV DNA testing was collected in PreservCyt ThinPrep solution (Hologic, Marlborough, MA) and transported to the laboratory within a week of collection. Upon the arrival to the laboratory, the samples were divided into several aliquots. The aliquots that were not immediately used for HPV testing were stored at −70°C. All cervical smear specimens obtained for cytological examination were processed under routine screening conditions. Women were referred to colposcopy at cytology threshold of atypical squamous cells-cannot exclude high-grade lesion (ASC-H) or worse in accordance with the Slovenian Cervical Cancer Screening Guidelines. In addition, all HPV16 and/or HPV18 positive women were invited for colposcopy regardless of their cytology result. Colposcopically directed punch biopsies obtained from the suspicious areas were histopathologically assessed by certified pathologists, who were blinded to the HPV status.

To provide longitudinal data, second screening round was commenced in December 2012 using similar approach as in the first screening round.⁴ To collect data for possible follow-up visits that occurred between two screening rounds two questionnaires (patient-based and physician-based) were used, as well as data from national screening registry for non-responders.

In addition to cervical samples, 3,195 and 2,041 serum samples were collected from women enrolled in the first and second screening round, respectively, in order to assess the cumulative exposure to HPV in this cohort of women, to evaluate the correlation between persistence and/ or clearance of HPV infection and to estimate the protective effect of naturally acquired serum antibodies against incident HPV infections.^5-7

The Slovenian HPV Prevalence Study is the largest screening cohort in this part of Europe to date, with reliable longitudinal data and thus represents valuable collection of clinical samples that can be used for ongoing and future validation studies of HPV tests designed to be used in primary HPV screening setting.

VALGENT 3
The VALGENT (VALidation of HPV GENotyp- ing Tests) framework is an international collab- oration designed to promote clinical validation and to assess the comparative performance of HPV tests with limited, extended or full genotyping ability.⁸ The study protocol is comprised of continuous samples obtained from women participating in a screening program, enriched with samples obtained from women with cytopathological abnormalities.

From sample collection of Slovenian HPV Prevalence Study, a total of 1,300 sequential cervical samples (screening population) were included in the VALGENT 3 framework (Figure 1). In addition, enrichment population consisted of 100 women with atypical squamous cervical cells of undetermined significance (ASC-US), 100 women with low-grade squamous intraepithelial lesion (LSIL) and 100 women with high-grade squamous intraepithelial lesion (HSIL) (Figure 1). The average age of women in the total study population (screening and enrichment population) was 39 years (range, 20-77), with 18.4% of the population below 30 years old.

VALGENT is a powerful tool for providing comprehensive evidence of the performance of HPV tests used in primary HPV screening setting. Using internationally recognized uniform criteria, validation is based on comparison of the novel HPV tests to the standard comparator (e.g., Hybrid Capture 2 (Qiagen Gaithersburg, MD, USA) and/or GP5+/6+ PCR). AML laboratory in Antwerp, Belgium, provided samples for VALGENT 1 and Scottish HPV Reference Laboratory in Edinburgh, Scotland, provided samples for VALGENT 2. VALGENT 4 is still ongoing and the samples are being collated in Copenhagen, Denmark.

HPV tests included in the VALGENT 3 are summarized in Table 1. Majority of the tests validated within the VALGENT 3 framework, fulfill the Meijer’s criteria for use in clinical practice. Based on the samples enrolled in the Slovenian HPV Prevalence Study, VALGENT 3 framework will provide important head-tohead data on clinical accuracy of the majority of the most important HPV tests currently available on the market.

References

1. Poljak M, Oštrbenk A, Seme K, et al. Comparison of clinical and analytical performance of the Abbott RealTime High Risk HPV Test to the performance of Hybrid Capture 2 in population-based cervical cancer screening. J Clin Microbiol 2011;49(5):1721-9.
2. Učakar V, Poljak M, Klavs I. Pre-vaccination prevalence and distribution of high-risk human papillomavirus (HPV) types in Slovenian women: a cervical cancer screening based study. Vaccine 2012;30(2):116-20.
3. Učakar V, Poljak M, Oštrbenk A, et al. Pre-vaccination prevalence of infections with 25 non-high-risk human papillomavirus (HPV) types among 1000 Slovenian women in cervical cancer screening. J Med Virol 2014;86:1772-9.
4. Poljak M, Oštrbenk A, Seme K, et al. Three-year longitudinal data on the clinical performance of the Abbott RealTime High Risk HPV test in a cervical cancer screening setting. J Clin Virol 2016;76 Suppl 1:S29-S39.
5. Učakar V, Jelen MM, Faust H, et al. Pre-vaccination seroprevalence of 15 human papillomavirus (HPV) types among women in the population-based Slovenian cervical screening program. Vaccine 2013;31(43):4935-9.
6. Faust H, Jelen MM, Poljak M, et al. Serum antibodies to human papillomaviruses (HPV) pseudovirions correlate with natural infection for 13 genital HPV types. J Clin Virol 2013;56(4):336-41.
7. Triglav T, Artemchuk H, Oštrbenk A, et al. Effect of natural- ly acquired type-specific serum antibodies against human papillomavirus type 16 infection. J Clin Virol 2017;90:64-9.
8. Arbyn M, Depuydt C, Benoy I, et al. VALGENT: a protocol for clinical validation of human papillomavirus assays. J Clin Virol 2016;76 Suppl 1:S14-S21.