Marianna Martinelli

Clinical Performance of OncoPredict HPV Screening Assay on Self‐Collected Vaginal and Urine Specimens Within the VALHUDES Framework

ABSTRACT The introduction of self‐sampling in cervical cancer screening has raised the importance of HPV test validation on self‐collected samples. This study aimed to evaluate the clinical accuracy of the OncoPredict HPV Screening (SCR) assay on self‐collected vaginal and first‐void urine (FVU) samples, relative to cervical specimens, using the VALHUDES Framework. FVU and vaginal self‐samples followed by a clinician‐collected cervical brushing were collected from 500 women referred to colposcopy and tested using OncoPredict HPV SCR assay. The assay demonstrated clinical sensitivity to detect cervical intraepithelial neoplasia grade 2 or worse (≥ CIN2) similar to cervical samples in FVU (ratio: 0.95, [95% CI: 0.88–1.02]) and vaginal self‐samples (ratio: 0.96 [95% CI: 0.90–1.02]). The clinical specificity for < CIN2 was lower in vaginal (ratio: 0.90 [95% CI: 0.84–0.96]) but not in FVU samples (ratio: 1.03 [95% CI: 0.96–1.12) when compared to cervical samples. However, the relative specificity improved following cut‐off optimization (ratio: 0.94, 95% CI: [0.88–1.01]). Moderate to excellent agreement in HPV detection between self‐collected and cervical samples was demonstrated (Kappa values: 0.53–1.00). To conclude, OncoPredict HPV SCR assay demonstrated similar accuracy on FVU and cervical samples. On vaginal compared to cervical samples sensitivity was similar with a lower specificity, which improved with cut‐off optimization.

Performance of BD Onclarity HPV assay on FLOQSwabs vaginal self-samples

ABSTRACT This study assessed the accuracy of high-risk human papillomavirus testing of BD Onclarity HPV (Onclarity) assay on vaginal self-collected FLOQSwab versus cervical samples to ensure similar accuracy to detect cervical intraepithelial neoplasia. Testing was performed on two automated platforms, BD Viper LT and BD COR, to evaluate the effect of machine and using two vaginal self-samples to analyze the influence of collection, transport, and freezing-unfreezing on the results. A cervical sample and two self-samples were collected from 300 women. The first collected vaginal and the cervical sample were tested on BD Viper LT, and the second swab was frozen and subsequently tested on both automated systems. Test results on vaginal and cervical specimens were considered the index and comparator, respectively; colposcopy and histology were reference standards. Relative sensitivity for ≥CIN2 on vaginal samples analyzed versus the cervical sample was 1.01 (0.97–1.06), 1.01 (0.97–1.06), and 1.00 (0.95–1.05), for the first, second self-collected sample tested on BD VIPER LT, and second self-collected sample tested on BD COR, respectively. Relative specificity was 0.83 (0.73–0.94), 0.76 (0.67–0.87), and 0.82 (0.73–0.92) using the three different workflows. Cut-off optimization for human papillomavirus (HPV) positivity defined at Ct ≤38.3 for HPV16, ≤ 34.2 for HPV18, and ≤31.5 for all other types showed an increased relative specificity with similar sensitivity. No significant difference was observed between self-samples tested with the two platforms and between first- and second-collected swabs. Onclarity assay on FLOQSwab using both platforms showed similar sensitivity but lower specificity to detect ≥CIN2 compared to cervical samples. By cut-off optimization, non-inferior specificity could be reached. IMPORTANCE Human papillomavirus (HPV) testing on self-collected vaginal samples has been shown to improve women’s participation to cervical cancer screening programs, particularly in regions with limited access to health care. Nevertheless, the introduction of self-sampling in cervical cancer screening programs requires prior clinical validation of the HPV assay in combination with a self-sample collection device, including also the laboratory workflow and automation required for high-throughput testing in screening. In this study, the performance of BD Onclarity HPV on FLOQSwab-collected vaginal self-samples has been compared to clinician-taken liquid-based cytology samples, to detect high-grade cervical intraepithelial neoplasia using two high-throughput platforms, BD Viper LT and BD COR. The study findings have shown a similar performance of BD Onclarity on testing self-collected samples, confirming the validation of the proposed pre-analytical and analytical protocols for their use in cervical cancer screening programs based on self-collected vaginal samples.

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.

Accuracy of Human Papillomavirus (HPV) Testing on Urine and Vaginal Self-Samples Compared to Clinician-Collected Cervical Sample in Women Referred to Colposcopy

In the context of cervical cancer prevention, where human papillomavirus (HPV) infection is pivotal, HPV testing is replacing Pap Smear in primary screening. This transition offers an opportunity for integrating self-sampling to enhance coverage. We evaluated the accuracy of HPV testing using self-collected urine and vaginal samples, comparing them to physician-collected cervical swabs. From a cohort of 245 women with abnormal cytology, we collected self-sampled vaginal, urine, and clinician-administered cervical specimens. Employing Anyplex™II HPV28 assay, outcomes revealed HPV positivity rates of 75.1% (cervical), 78.4% (vaginal), and 77.1% (urine). Significant, hr-HPV detection concordance was observed between self-taken cervical samples and clinical counterparts (k = 0.898 for vaginal; k = 0.715 for urine). This study extends beyond accuracy, highlighting self-collected sample efficacy in detecting high-grade cervical lesions. The insight underscores self-sampling’s role in bolstering participation and aligns with WHO’s goal to eliminate cervical cancer by 2030.