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Clinical validation of human papillomavirus (HPV) assays according to international criteria is prerequisite for their implementation in cervical cancer screening. OncoPredict HPV Quantitative Typing (QT) assay (Hiantis Srl, Milan, Italy) is a novel full‐genotyping multiplex real‐time PCR quantitative assay targeting E6/E7 genes, allowing individual viral load determination of 12 high‐risk (HR) HPV types. Quality controls for sample adequacy, efficiency of nucleic acid extraction and PCR inhibition are included in the assay. Clinical performance of OncoPredict HPV QT test was assessed as part of the "Validation of HPV Genotyping Tests" (VALGENT‐2) framework, consisting of 1300 cervical liquid‐based cytology (LBC) samples of women aged between 20 and 60 years who had originally attended for routine cervical screening in Scotland. The clinical accuracy of the OncoPredict HPV QT (index test) for the detection of CIN2+ was assessed relative to the GP5+/6+ Enzyme ImmunoAssay (GP5+/6+ EIA) (comparator test), using noninferiority criteria. Intra‐ and interlaboratory reproducibility of the assay was assessed on a subpopulation, comprising 526 samples. The relative sensitivity and specificity for OncoPredict HPV QT vs GP5+/6+‐PCR‐EIA were 1.01 (95% CI: 0.99‐1.03) and 1.03 (95% CI: 1.0‐1.06) respectively. The P‐values for noninferiority were ≤0.001. The intra‐ and inter‐laboratory reproducibility demonstrated a high concordance (>98.7%) with kappas for individual types ranging from 0.66 to 1.00. OncoPredict HPV QT fulfills the international validation criteria for the use of HPV tests in cervical cancer screening. [ABSTRACT FROM AUTHOR]

Background High-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia (CIN) grade 2/3 lesions in human papillomavirus (HPV)–positive women <30 years of age have high spontaneous regression rates. To reduce overtreatment, biomarkers are needed to delineate advanced CIN lesions that require treatment. We analyzed the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in HPV-positive women aged <30 years, aiming to identify CIN2/3 lesions in need of treatment. Methods A European multicenter retrospective study was designed evaluating the FAM19A4/miR124-2 methylation test and HPV16/18 genotyping in cervical scrapes of 1061 HPV-positive women aged 15–29 years (690 ≤CIN1, 166 CIN2, and 205 CIN3+). A subset of 62 CIN2 and 103 CIN3 were immunohistochemically characterized by HPV E4 expression, a marker for a productive HPV infection, and p16ink4a and Ki-67, markers indicative for a transforming infection. CIN2/3 lesions with low HPV E4 expression and high p16ink4a/Ki-67 expression were considered as nonproductive, transforming CIN, compatible with advanced CIN2/3 lesions in need of treatment. Results FAM19A4/miR124-2 methylation positivity increased significantly with CIN grade and age groups (<25, 25–29, and ≥30 years), while HPV16/18 positivity was comparable across age groups. FAM19A4/miR124-2 methylation positivity was HPV type independent. Methylation-positive CIN2/3 lesions had higher p16ink4a/Ki-67-immunoscores (P =.003) and expressed less HPV E4 (P =.033) compared with methylation-negative CIN2/3 lesions. These differences in HPV E4 and p16ink4a/Ki-67 expression were not found between HPV16/18–positive and non-16/18 HPV–positive lesions. Conclusions Compared with HPV16/18 genotyping, the FAM19A4/miR124-2 methylation test detects nonproductive, transforming CIN2/3 lesions with high specificity in women aged <30 years, providing clinicians supportive information about the need for treatment of CIN2/3 in young HPV-positive women. [ABSTRACT FROM AUTHOR]

Human papillomavirus (HPV) assays used in cervical cancer screening should be clinically validated according to international criteria. OncoPredict HPV® Screening (SCR) is a partial genotyping multiplex real‐time PCR assay targeting E6/E7 genes of 13 high‐risk (hr) HPVs. OncoPredict HPV® SCR (index assay) identifies HPV‐16 and HPV‐18 separately, 11 other hrHPV in aggregate and includes quality controls for sample adequacy, DNA extraction efficiency and PCR inhibition. 1300 VALGENT‐2 study samples (from women aged 20–60 attending the Scottish cervical cancer screening program) were tested with the index assay and the GP5+/6+ PCR enzyme immunoassay (standard comparator assay). Non‐inferior accuracy detecting cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) of the index versus comparator was verified. Intra‐ and interlaboratory reproducibility of the index was evaluated by overall concordance and Cohen's kappa, using a sub‐population (n = 526). Relative sensitivity and specificity for CIN2+ of the index versus comparator were 1.01 (95% confidence interval [CI]: 0.99–1.03) and 1.02 (95% CI: 1.0–1.04), respectively. Noninferiority p values were all ≤0.05, except for CIN3+ in patients ≥30 years. Excellent intra‐ and interlaboratory reproducibility was shown with concordance >98% and kappas >0.95. OncoPredict HPV® SCR fulfills the three international validation criteria for hrHPV DNA tests in cervical cancer screening. [ABSTRACT FROM AUTHOR]

Sodium bisulphite conversion of DNA to separate methylated from unmethylated cytosines is a standard for methylation analysis. This study evaluated a direct cell conversion protocol on cervical samples as alternative to isolated genomic DNA as input. Clinician-collected cervical samples (n = 120) were subjected to a direct conversion protocol, or genomic DNA was isolated with a fixed amount used for subsequent bisulphite conversion. Converted samples were compared for ACTB control gene and methylation of FAM19A4 and miR124-2 genes using quantitative methylation-specific PCR (QIAsure Methylation Test). Direct conversion resulted in a high success rate, i.e., 119/120 (99.2%) samples reported a valid test result. ΔΔCq values of FAM19A4 and miR124-2 were significantly correlated between both protocols (Spearman Rho 0.708 and 0.763, respectively, all p-values = 0.000). Agreement between both the bisulphite protocols was demonstrated by Bland–Altman plots. A direct cell conversion protocol shows good technical and analytical performance and offers a streamlined workflow for methylation analysis. [ABSTRACT FROM AUTHOR]

Aims: Human papillomavirus (HPV) molecular testing targets either the late gene L1 or early genes E6 and/or E7. Loss of L1 during integration is suggested to compromise sensitivity in samples associated with cancer, however, clear evidence for this is lacking. Our aim is to address this by performing a head-to-head comparison between assays targeting L1 vs E6/E7, using a series of high-grade and invasive disease samples within different biological matrices and anatomical sites., Methods: We obtained 298 samples comprising of liquid-based cytology and biopsies of cervical cancer and cervical intraepithelial neoplasia grade 3, in addition to biopsies of penile and oropharyngeal cancers. Two commercially available HPV primary screening assays and two assays with extended genotyping were applied to the sample set targeting L1 (Abbott RealTime HR HPV Assay and Optiplex HPV Genotyping Test) and E6/E7 genes (Xpert HPV Test and EuroArray HPV Test)., Results: Agreement for high-risk HPV (hrHPV) for all samples types between the screening assays is over 88% and over 96% for the two genotyping assays. For HPV 16 agreement is over 90% for both screening and genotyping assays. Kappa statistics show good to very good agreement between the screening and genotyping assays for hrHPV and HPV 16., Conclusions: Analysis of the valid results from our data indicates that L1 and E6/E7 targeting assays show similar performance for detection of hrHPV in high grade cervical lesions and cancers of cervix, penis and oropharynx., Competing Interests: Competing interests: KC, RB, SM and EAB’s institution has received research funding or gratis consumables to support research from the following commercial entities in the last 3 years: Cepheid, Euroimmun, GeneFirst, SelfScreen, Hiantis, Seegene, Roche, Abbott and Hologic., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The ability to distinguish which hrHPV infections predispose to significant disease is ever more pressing as a result of the increasing move to hrHPV testing for primary cervical screening. A risk-stratifier or "triage" of infection should ideally be objective and suitable for automation given the scale of screening., Results: CCL2, CCL3, CCL4, CXCL1, CXCL8 and CXCL12 emerged as the strongest, candidate biomarkers to detect underlying disease [cervical intraepithelial neoplasia grade 2 or worse (CIN2+)]. For CIN2+, CCL2 had the highest area under the curve (AUC) of 0.722 with a specificity of 82%. A combined biomarker panel of six chemokines CCL2, CCL3, CCL4, CXCL1, CXCL8, and CXCL12 provides a sensitivity of 71% and specificity of 67%., Conclusion: The present work demonstrates that the levels of five chemokine-proteins are indicative of underlying disease. We demonstrate technical feasibility and promising clinical performance of a chemokine-based biomarker panel, equivalent to that of other triage options. Further assessment in longitudinal series is now warranted., Methods: A panel of 31 chemokines were investigated for expression in routinely taken archived and prospective cervical liquid based cytology (LBC) samples using Human Chemokine Proteomic Array kit. Nine chemokines were further validated using Procartaplex assay on the Luminex platform., Competing Interests: CONFLICTS OF INTEREST None.