Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens.
Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.
['Ambulos|Nicholas P|NP|', 'Schumaker|Lisa M|LM|', 'Mathias|Trevor J|TJ|', 'White|Ruth|R|', 'Troyer|Jennifer|J|', 'Wells|David|D|', 'Cullen|Kevin J|KJ|']
D004279:DNA, Viral D005260:Female D005557:Formaldehyde D060005:Genotyping Techniques D059014:High-Throughput Nucleotide Sequencing D052162:Human papillomavirus 16 D006801:Humans D009959:Oropharyngeal Neoplasms D016612:Paraffin Embedding D012680:Sensitivity and Specificity D017422:Sequence Analysis, DNA D016707:Tissue Fixation D002583:Uterine Cervical Neoplasms
head and neck cancer human papillomavirus viral