Quentin Lepiller

Reproducibility of HPV Testing With Two Versions of the Papilloplex High‐Risk HPV Assay

ABSTRACT Validation of HPV tests usable in cervical cancer screening require demonstration of noninferior clinical accuracy and sufficient reproducibility. The Papilloplex HR‐HPV assay [version1] (Genefirst, UK) has been validated only for clinical accuracy. Here, we assessed its reproducibility, aiming to complete the validation process. A panel of 550 cryopreserved cervical cell samples collected from women attending the cervical cancer screening program in Belgium was used to assess the intra‐ and inter‐laboratory reproducibility of Papilloplex HR‐HPV [version1], a full genotyping assay that identifies separately 14 high‐risk HPV (hrHPV) types using multiple probe amplification technology. We assessed whether the reproducibility fulfils validation criteria (lower 95% confidence interval [CI] bound ≥ 87% and κ ≥ 0.50). Subsequently, we compared the concordance between version1 and a new version6 of the assay and between three analysis methods for PCR curve interpretation. Papilloplex HR‐HPV version1 assay showed an excellent reproducibility for hrHPV (97.5% [CI: 95.8%–98.7%], κ = 0.94 for intra‐ and 93.5% [CI: 91.0%–95.4%], κ = 0.85 for inter‐laboratory reproducibility). Concordance analyses exhibited an excellent agreement between two assay versions and between three PCR curve analysis methods. Papilloplex HR‐HPV version1 assay exhibited excellent reproducibility, completing the international validation criteria. Papilloplex HR‐HPV version6 showed excellent concordance with version1 but still lacks clinical validation.

The Sansure® Human Papillomavirus DNA Diagnostic Kit offers excellent reproducibility performance for the detection of high‐risk HPV

AbstractCervical cancer screening is a cornerstone of cervical cancer elimination. Detection of high‐risk human papillomavirus (hrHPV) is recommended as the first step in screening provided that the assay used has been adequately validated. The Sansure® Human Papillomavirus DNA Diagnostic Kit is a new assay designed to detect HPV16, HPV18 and 13 other HPV in aggregate. The study aimed to evaluate the intra‐ and interlaboratory reproducibility of the assay according to international guidelines. Five hundred and fifty cervical residual cell samples from women attending cervical cancer screening were selected from the biobank of the HPV National Reference Centre in Belgium and used in this study. After DNA extraction, HPV was tested using the Sansure® Human Papillomavirus DNA Diagnostic Kit. The lower 95% confidence limit around the general reproducibility of this assay should be greater than or equal to 87%, with κ ≥ 0.50. Five hundred and thirty‐three samples had valid results. The Sansure® Human Papillomavirus DNA Diagnostic Kit demonstrated an excellent intra‐laboratory reproducibility of 93.8% (95% confidence interval [CI]: 91.4–95.7, κ = 0.85). The interlaboratory reproducibility was 93.4 (95% CI: 91.0–95.4, κ = 0.84). Intra and interlaboratory reproducibility were also excellent at the genotype level. Excluding HPV53 single infection samples from the analyses also resulted in excellent agreement. These data show that the Sansure® Human Papillomavirus DNA Diagnostic Kit is highly reproducible.

The level of expression of HPV16 early transcripts is not associated with the natural history of cervical lesions

Abstract The natural history of cervical cancer is closely linked to that of high‐risk human papillomaviruses (HPV) infection. It is recognized that upon HPV DNA integration, partial or complete loss of the E2 open reading frame precludes expression of the corresponding protein, resulting in upregulation of the E6 and E7 viral oncoproteins. To better characterize HPV16 infection at the cervical level, viral load, viral DNA integration, and viral early transcript expression (E2, E5, and E6) were analyzed in a series of 158 cervical specimens representative of the full spectrum of cervical disease. Overall, the frequency of early transcript detection varied from 45% to 90% and tended to increase with lesion severity. In addition, the levels of E2, E5, and E6 transcript expression were slightly higher in high‐grade lesions than in cervical specimens without abnormalities. Notably, early transcript expression was clearly associated with viral load, and no inverse correlation was found between the expression of E2 and E6 transcripts. No clear association was found between early transcript expression and HPV16 DNA integration, with the exception that samples with a fully integrated HPV16 genome did not harbor E2 or E5 transcripts. In conclusion, early HPV16 transcript expression appears to be associated with viral load rather than lesion grade. From a practical point of view, quantification of HPV16 early transcripts is difficult to translate into a relevant biomarker for cervical cancer screening.

Prevalence and significance of HPV DNA detection below the clinical threshold of the commercial kit Alinity m HR‐HPV assay (Abbott)

Abstract The positive clinical threshold of human papillomavirus (HPV) tests validated for primary cervical cancer screening (CCS) is designed to offer an optimal balance between clinical sensitivity and specificity. However, there may be a gap between the analytical sensitivity of the test and the positive clinical threshold, referred to here as the “gray‐zone.” This study aims to determine the prevalence and significance of HPV results obtained in the gray‐zone in routine practice. Cervical samples obtained in our institution for CCS over a 22‐month‐period were tested with the Alinity m HR‐HPV Assay (Abbott). Clinical and biological data, including cytological results and patients' HPV history were collected. Of the 6101 samples collected, 1.7% had an HPV result in the gray‐zone (102 patients). The proportion of gray‐zone results varied according to HPV genotype, reaching 11.8% of samples with detectable HPV DNA in the case of HPV31/33/52/58 genotypes. Reflex cytologies showed no abnormalities or Atypical Squamous Cells of Undetermined Significance results in 74.6% and 17.9% of cases, respectively. A previous or subsequent HPV‐positive result with a (possibly) identical genotype was observed in 58% and 38% of cases, respectively. Two women with a history of persistent HPV detection had a CIN2+ lesion 1 year after the gray‐zone result. In conclusion, the proportion of HPV results in the gray‐zone varies according to genotype. No cytological abnormality is observed in the majority of cases, but a few rare patients with a history of persistent HPV infection should be closely monitored even if the HPV result is transiently located in the gray‐zone.

Repurposing the Hybrid Capture 2 (HC2) screening test for whole-genome sequencing of human papillomaviruses

Simple and standardized approaches for genome analysis of human papillomavirus (HPV) by next-generation sequencing are needed. The aim of the study was to develop a protocol for direct deep sequencing of high-risk (hr) HPV strains, based on the widely used commercial Hybrid Capture 2 (QIAGEN) test, without any additional probe design. This protocol was applied to 15 HPV-positive and two HPV-negative cervical samples or cell lines and validated at the genotype level by comparing the sequencing results to those obtained using a commercial genotyping kit. The performance of our protocol, presented in this proof-of-principle study, supports its use for accurate characterization of genetic variants of hrHPV.

Longitudinal follow‐up of HPV16 sequence after cervical infection: Low intrahost variation and no correlation with clinical evolution

AbstractHuman papillomavirus (HPV) 16 exhibits different variants that may differ in their carcinogenic risk. To identify some high‐risk variants, we sequenced and compared HPV16 whole genomes obtained from a longitudinal cohort of 34 HPV16‐infected women who had either spontaneously cleared their infection (clearance group or “C”), or developed cervical high‐grade lesions following a viral persistence (group persistence or “P”). Phylogenetic analysis of paired samples obtained at the beginning (C0 or P0) and at the end (C2 or P2) of the follow‐up (median intervals between C0–C2 and between P0–P2 were 16 and 36.5 months, respectively) revealed a low genetic variability within the host compared to the genetic interhost diversity. By comparing our HPV16 sequences to a reference sequence, we observed 301 different substitutions, more often transitions (60.9%) than transversions (39.1%), that occurred throughout the viral genome, but with a low frequency in E6 and E7 oncogenes (10 and 9 substitutions), suggesting a high conservation of these genes. Deletions and insertions were mostly observed in intergenic regions of the virus. The only significant substitution found between the subgroups C2 and P2 was observed in the L2 gene (L330F), with an unclear biological relevance. Our results suggest a low longitudinal intrahost evolution of HPV16 sequences and no correlation between genetic variations and clinical evolution.