Mario Poljak

Criteria for second generation comparator tests in validation of novel HPV DNA tests for use in cervical cancer screening

Abstract While HC2 and GP5+/6+ PCR‐EIA were pivotal in test validation of new HPV assays, they represent the first generation of comparator tests based upon technologies that are not in widespread use anymore. In the current guideline, criteria for second‐generation comparator tests are presented that include more detailed resolution of HPV genotypes. Second‐generation comparator tests should preferentially target only the 12 genotypes classified as carcinogenic (IARC‐group I), and show consistent non‐inferior sensitivity for CIN2+ and CIN3+ and specificity for ≤CIN1 compared to one of the first‐generations comparators, in at least three validation studies using benchmarks of 0.95 for relative sensitivity and 0.98 for relative specificity. Validation should take into account used storage media and other sample handling procedures. Meta‐analyses were conducted to identify the assays that fulfill these stringent criteria. Four tests fulfilled the new criteria: (1) RealTime High‐Risk HPV Test (Abbott), (2) Cobas‐4800 HPV test (Roche Molecular System), (3) Onclarity HPV Assay (BD Diagnostics), and (4) Anyplex II HPV HR Detection (Seegene), each evaluated in three to six studies. Whereas the four assays target 14 carcinogenic genotypes, the first two identify separately HPV16 and 18, the third assay identifies five types separately and the fourth identifies all the types separately.

2023 global inventory of commercial molecular tests for human papillomaviruses (HPV)

To suit the needs of the human papillomaviruses (HPV) community comprehensively, a range of commercial HPV tests with different performance characteristics are required. Four periodic inventories of commercial HPV molecular tests present in the global market were published previously in 2010, 2012, 2015 and 2020. For the fifth inventory, data were retrieved from internal files and a detailed search using the main bibliographic databases as well as general internet search without period or language restrictions was performed in December 2023. At least 264 distinct HPV tests (and 511 test variants) were available globally in December 2023. A small 2020-2023 net increase in total numbers was observed, but with a strong introduction/withdrawal dynamic: 86 new distinct HPV tests (and 141 variants) were introduced and 76 tests (and 55 variants) were withdrawn from the market in the last four years. Although quality improvement of some tests was recorded, half of all HPV tests are still without a single peer-reviewed publication, and 79 % of tests are without published evidence that demonstrate performance characteristics are in line with requirements agreed in the HPV community. Only a relatively small pool of tests fulfill the operational/performance characteristics required to meet the global cervical cancer screening challenge. Although clinical and analytical performance characteristics of many commercial HPV tests are largely unknown, such tests are used worldwide in daily clinical practice and research, with potentially deleterious consequences. Due to this long-lasting unfavorable situation, significant scope for improvement persists for both manufacturers of HPV tests and the HPV community.

Stratified Mucin-Producing Intraepithelial Lesion (SMILE) of the Uterine Cervix: High-Risk HPV Genotype Predominance and p40 Immunophenotype

Stratified mucin-producing intraepithelial lesion (SMILE) is a rare high-grade cervical precancerous lesion designated a variant of adenocarcinoma in situ (AIS) in the WHO classification. We aimed to determine HPV genotypes, immunohistochemical phenotype and mucin presence in SMILE. Between 2010 and 2018, SMILE was diagnosed in 34 out of 6958 (0.5%) cervical biopsies, in 23 patients. Twenty-six tissue samples from twenty-one patients were available for further analysis, including 13 with SMILE alone, 12 with SIL and/or AIS and one with HSIL, AIS and endocervical adenocarcinoma. HPV genotyping was performed using the Seegene Anyplex II HPV 28 assay. Of the 26 samples, a single HPV genotype was identified in the majority of cases (n = 22), including 12/13 SMILEs associated with SIL/AIS. All but one were high-risk HPV genotypes (23/24; 96.8%). We identified seven different HPV genotypes, the most common being HPV16 (n = 10; 43.5%), HPV18 (n = 8, 34.8%) and HPV 31 (n = 5, 21.7%). All SMILEs showed a strong positive reaction to p16, CK7, CK19 and high Ki67 expression comparable to adjacent HSIL and/or AIS if present. SMILE showed variable mucin presence and p40-positive squamous differentiation suggesting phenotypic diversity in cervical precancerous lesions infected by single HPV.

The European response to the WHO call to eliminate cervical cancer as a public health problem

AbstractThe age‐standardised incidence of cervical cancer in Europe varies widely by country (between 3 and 25/100000 women‐years) in 2018. Human papillomavirus (HPV) vaccine coverage is low in countries with the highest incidence and screening performance is heterogeneous among European countries. A broad group of delegates of scientific professional societies and cancer organisations endorse the principles of the WHO call to eliminate cervical cancer as a public health problem, also in Europe. All European nations should, by 2030, reach at least 90% HPV vaccine coverage among girls by the age of 15 years and also boys, if cost‐effective; they should introduce organised population‐based HPV‐based screening and achieve 70% of screening coverage in the target age group, providing also HPV testing on self‐samples for nonscreened or underscreened women; and to manage 90% of screen‐positive women. To guide member states, a group of scientific professional societies and cancer organisations engage to assist in the rollout of a series of concerted evidence‐based actions. European health authorities are requested to mandate a group of experts to develop the third edition of European Guidelines for Quality Assurance of Cervical Cancer prevention based on integrated HPV vaccination and screening and to monitor the progress towards the elimination goal. The occurrence of the COVID‐19 pandemic, having interrupted prevention activities temporarily, should not deviate stakeholders from this ambition. In the immediate postepidemic phase, health professionals should focus on high‐risk women and adhere to cost‐effective policies including self‐sampling.

Evaluation and Optimization of the Clinical Accuracy of Hybribio's 14 High-Risk HPV with 16/18 Genotyping Assay within the VALGENT-3 Framework

Hybribio’s 14 High-Risk HPV with 16/18 genotyping real-time PCR (HBRT-H14) is a human papillomavirus (HPV) assay with approval from the China Food and Drug Administration that is widely used in China. VALGENT (VALidation of HPV GENotyping Tests) is an established framework for evaluating HPV tests’ clinical performance relative to validated comparators. The aim of this study was to assess the clinical accuracy of HBRT-H14 following international validation criteria. Within VALGENT-3, clinical performance of HBRT-H14 was compared with Hybrid Capture 2 (HC2), Linear Array HPV genotyping test (Linear Array), and Cobas 4800 HPV test (Cobas).

Comparison of the clinical and analytical performance of Alinity m HR HPV and cobas 4800 HPV assays in a population-based screening setting

The recently launched Abbott Alinity m HR HPV (Alinity) assay separately identifies high-risk human papillomavirus (hrHPV) genotypes HPV16, HPV18, and HPV45, and reports 11 other genotypes as two aggregates. Clinical and analytical performance of Alinity was compared with the cobas 4800 HPV assay on 4,334 women aged 20-64 years attending routine, population-based organized cervical cancer screening during 2009/2010. After 36 months, they were invited to participate in the second screening round (2012-2014) and later followed-up through centralized national cervical cancer screening registry. In women 30 and older, the clinical sensitivity for cervical intraepithelial neoplasia grade 2+ (CIN2+) was 100.0% (95% CI, 88.2-100.0%) for Alinity and 100.0% (95% CI, 88.2-100.0%) for cobas, and for CIN3+ 100.0% (95% CI, 78.9-100.0%) for both assays. The clinical specificity for ≤ CIN1 in women 30 and older was 92.4% (95% CI, 91.4-93.3%) and 92.9% (95% CI, 91.9-93.7%), respectively. The assays demonstrated excellent overall agreement for hrHPV detection (97.9%) and genotype-specific agreement for HPV16 (99.6%), HPV18 (99.8%), and other hrHPV (98.1%). Overall positive agreement and positive agreements for HPV16, HPV18, and other hrHPV genotypes were 84.3%, 89.1%, 73.2%, and 82.3%. Based on a 5-year CIN3+ risk, slightly more HPV-positive women would require referral to immediate colposcopy after testing with Alinity vs. cobas (4.1% vs. 3.8%; p = 0.470), but significantly fewer Alinity-tested women would need a 6- to 12-month follow-up visit compared with those tested with cobas (5.0% vs. 8.6%; p < 0.0001). Alinity and cobas have comparable clinical performance and showed excellent overall and genotype-specific agreement. The Alinity's extended genotyping ability could help predict the 5-year CIN3+ risk and cost-saving management of HPV-screen-positive women.

Clinical and Analytical Evaluation of the Alinity m HR HPV Assay within the VALGENT-3 Framework

Only clinically validated human papillomavirus (HPV) tests should be used in cervical cancer screening. VALGENT provides a framework to validate new HPV tests.

Classification of high‐grade cervical intraepithelial neoplasia by p16ink4a, Ki‐67, HPV E4 and FAM19A4/miR124‐2 methylation status demonstrates considerable heterogeneity with potential consequences for management

AbstractHigh‐grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a, Ki‐67 and host‐cell DNA methylation) could provide guidance for clinical management in women with high‐grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0‐3) and Ki‐67 (Scores 0‐3), referred to as the “immunoscore” (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124‐2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0‐2 (6.0%), 151 lesions within IS group 3‐4 (30.4%) and 316 lesions within IS group 5‐6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P &lt; .001) and with increasing immunoscore group (Ptrend &lt; .001). Methylation positivity increased significantly from CIN2 to CIN3 (P &lt; .001) and with increasing immunoscore group (Ptrend &lt; .001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5‐6 (38/316). Notably, in a minority (43/497, 8.7%) of high‐grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high‐grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a “wait and see” policy to reduce overtreatment of high‐grade CIN lesions.

Clinical Validation of the Fully Automated NeuMoDx HPV Assay for Cervical Cancer Screening

The NeuMoDx HPV assay is a novel fully automated, real-time PCR-based assay for the qualitative detection of high-risk human papillomavirus (HPV) DNA in cervical specimens. The assay specifically identifies HPV16 and HPV18 and concurrently detects 13 other high-risk HPV types at clinically relevant infection levels. Following the international guidelines, the clinical performance of the NeuMoDx HPV assay for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) against the reference standard Hybrid Capture 2, as well as intra- and inter-laboratory reproducibility were assessed on PreservCyt samples. The clinical accuracy of the assay was additionally evaluated against the clinically validated Alinity m HR HPV and COBAS 4800 HPV Test on PreservCyt samples, and against the clinically validated HPV-Risk assay on SurePath samples. The NeuMoDx HPV assay performance for CIN2+ was non-inferior to the reference methods on both sample types (all p &lt; 0.05), and showed excellent intra- and inter-laboratory reproducibility (95.7%; 95% CI: 93.9–97.3; kappa value 0.90 (95% CI: 0.86–0.94); and 94.5%; 95% CI: 92.6–96.2; kappa value 0.87 (95% CI: 0.82–0.92), respectively). In conclusion, the NeuMoDx HPV assay meets international guideline criteria for cross-sectional accuracy and reproducibility, and performs equally well on cervical screening specimens collected in two widely used collection media. The NeuMoDx HPV assay fulfils the requirements to be used for primary cervical screening.

Testing for Human Papillomaviruses in Urine, Blood, and Oral Specimens: an Update for the Laboratory

Twelve high-risk alpha human papillomavirus (HPV) genotypes cause approximately 690,000 cancer cases annually, with cervical and oropharyngeal cancer being the two most prominent types. HPV testing is performed in laboratory settings for various applications of a clinical, epidemiological, and research nature using a range of clinical specimens collected by clinicians or by individuals (self-collected specimens).

Allplex HPV HR Detection assay fulfils all clinical performance and reproducibility validation requirements for primary cervical cancer screening

Human papillomavirus (HPV)-based screening offers better protection against cervical cancer compared to cytology, but HPV screening assays must adhere to validation requirements of the international guidelines to ensure optimal performance. Allplex HPV HR Detection (Allplex) assay, launched in the late 2022, is a fully automated real-time PCR-based assay utilizing innovative technology that enables quantification and concurrent distinction of 14 high-risk HPV genotypes (HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68). We assessed the validity of the Allplex for cervical cancer screening purposes, via comparison to a clinically validated comparator assay (Hybrid Capture 2; HC2), and through assessment of intra-laboratory reproducibility and inter-laboratory agreement. A clinical validation panel comprised of 973 residual ThinPrep samples was obtained from women aged 30-64 years participating in the organized Slovenian screening program, of these 863 were from women undergoing their regular screening visit after a previous negative screen test while 110 were from women with underlying cervical intraepithelial neoplasia grade 2 or worse (CIN2+) lesions. The Allplex's relative clinical sensitivity for detection of CIN2+ and CIN3+ were 1.01 (95%CI;0.98-1.04) and 0.98 (95%CI;0.95-1.02), compared to that of HC2. At recommended thresholds of ≥98% and ≥90%, the Allplex's clinical sensitivity and specificity (p=0.0004 and p=0.02, respectively) were non-inferior to HC2. High intra-laboratory reproducibility and inter-laboratory agreement, both overall (98.1% and 97.9%, respectively) and at genotype level (>98.7%) was observed. In addition, analytical genotype-specific performance of Allplex was compared to that of its predecessor Anyplex HPV HR; high overall agreement was observed (96.3%; kappa value 0.88), with some variations in performance. In conclusion, Allplex met all validation criteria described in the international guidelines on sensitivity, specificity and laboratory reproducibility and can be considered clinically validated for primary cervical cancer screening.