title: Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens.
abstract: 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.
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Ambulos
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Schumaker
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Trevor J
authors.initials: TJ
authors.lastname: Mathias
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Ruth
authors.initials: R
authors.lastname: White
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Troyer
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: David
authors.initials: D
authors.lastname: Wells
authors.affiliation: 1 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, Maryland 21201, USA; and 2 Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Cullen
mesh_terms: D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005260:Female; D005557:Formaldehyde; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D009959:Oropharyngeal Neoplasms / Q000175:diagnosis* / Q000821:virology; D016612:Paraffin Embedding; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D016707:Tissue Fixation; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000821:virology
chemical_list: D004279:DNA, Viral; D005557:Formaldehyde
keywords: head and neck cancer; human papillomavirus; viral