Ruth Chinyere Njoku

Clinical performance of the novel full‐genotyping OncoPredict HPV Quantitative Typing assay using the VALGENT framework

AbstractClinical 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.

Quality Assurance in Cervical Cancer Screening: Evaluation of Sample Adequacy in HPV DNA Testing

ABSTRACTIn HPV‐primary screening, sample quality significantly influences test accuracy. Unlike cytology‐based screening, no consensus guidelines presently exist for sample quality assessment in HPV testing. This study aims to evaluate the impact of sample cellularity on HPV testing. A total of 37 592 liquid‐based cytology (LBC) samples from women undergoing HPV‐primary screening (aged 30–64, median 48; IQR: 40–56 years) were analyzed using Cobas 4800 HPV Test (Roche). Sample adequacy was assessed by the assay's β‐globin internal control and by an independent quantitative cellularity assessment (OncoPredict HPV, Hiantis). HPV positivity rates (PR) were stratified according to β‐globin Ct values. Among the analyzed samples, 50.0%, 47.1%, 2.3%, and 0.6% had β‐globin Ct values of ≤ 28, > 28 to ≤ 32, > 32 to ≤ 34, and > 34, respectively. Overall HPV‐PR was 7.7% (2891/37 592). PR reached 9.7% in samples with β‐globin ≤ 28 Ct (1820/18 801), decreasing markedly to 1.4% for β‐globin > 34 Ct (3/214), (p < 0.001). Quantitative analysis showed that Cobas 4800 β‐globin Ct = 34 corresponds to approximately 1.5 × 10^3 nucleated cells/reaction. A subset of 195 HPV‐negative samples with β‐globin Ct ≥ 34 was evaluated by liquid based cytology (LBC): 19% had inadequate cellularity according to LBC guidelines, 8% were ≥ ASC‐US and 73% NILMs. 65% of adequate LBC showed cellular atrophy. These findings emphasize the importance of assessing cellularity in HPV‐screening to avoid potentially false‐negative results due to inadequate samples. Future research should focus on establishing standardized cellularity thresholds to improve screening accuracy.