Evaluation and validation of HPV real-time PCR assay for the detection of HPV DNA in oral cytobrush and FFPE samples

Abstract

Specific HPV genotypes have been recognized as risk factors inducing head and neck cancers (HNC). The aim of this study was to validate a real-time PCR assay to detect accurately High Risk HPV DNA in Formalin Fixed Paraffin Embedded (FFPE) and oral cytobrush samples and compare the results with conventional PCR. Repeatability, reproducibility and limit of detection of Cobas assay were estimated for oral cytobrush and FFPE samples of patients with HNC. 53 samples of patients with a HNC were then used for assay comparison with conventional PCR. Finally, 26 samples of patients with anogenital neoplasia cancer were analyzed as control and assays comparison. Among the 53 samples of patients with HNC, 12 (26.7%) were HPV positive, 33 (73.3%) were HPV negative and 8 (15.1%) were non contributive with the Cobas assay. Among the 26 samples of patients with anogenital neoplasia, 15 (57.7%) were HPV positive and 11 were HPV negative (42.3%). One sample was found with an HPV 16 and HPV 18 co-infection. Only 3 samples were found with discrepant results. Cobas assay was found suitable for routine HPV detection with a very good repeatability and reproducibility for all HPV genotypes (CV < 0.6% and <0.4% respectively). Sensitivity and specificity for Cobas assay were 91.7% [61.5%;99.8%] and 96.9% [83.8%;99.9%] respectively. Ten nanograms of DNA were sufficient for the detection of HPV 16, HPV 18 and HPV in FFPE and oral cytobrush samples. Cobas assay was found comparable to conventional PCR and can detect accurately and rapidly HPV DNA in FFPE and oral cytobrush samples for the management of HNC and other types of HPV-associated neoplasia.

High yield gold nanoparticle‐based DNA isolation method for human papillomaviruses genotypes from cervical cancer tissue samples

“…The current gold standard for the viral load is qPCR. However, various PCR‐based quantitative methods such as qPCR‐enzyme immunoassay [35], TaqMan probes [36], fluorescence resonance energy transfer (FRET) probes [37], AllGlo probes [38], and more recently digital droplet PCR have been used for the detection and quantification of HPV DNA extracted from tissue samples [39]. Since the accurate limit of detection and limits of quantification of a PCR method mainly depend on the DNA extraction step, it is difficult to compare the detection limit of our method with others.…”

Comparison of HPV detection rate in formalin‐fixed paraffin‐embedded tissues of head and neck carcinoma using two DNA extraction kits and three amplification methods

“…Several studies have shown that this method is very useful in HPV detection in FFPE tissue samples of HNSCC. The authors of these studies suggested that the higher efficiency of real‐time PCR in HPV detection in FFPE tissue samples is based on the amplification of short DNA fragments (31,43).…”

A high yield gold nanoparticle-based DNA isolation method for human papillomaviruses genotypes from cervical cancer tissue samples

“…The current gold standard for viral load is qPCR. However, various PCR-based quantitative methods such as quantitative PCR-enzyme immunoassay [34], TaqMan probes [35], FRET probes [36], AllGlo probes [37], and more recently digital droplet PCR have been used for the detection and quantification of HPV DNA extracted from tissue samples [38]. Since the accurate limit of detection and limits of quantification of a PCR method mainly depend on the DNA extraction step, it is difficult to compare the detection limit of our method with others.…”