title: Next-generation sequencing for simultaneous determination of human papillomavirus load, subtype, and associated genomic copy number changes in tumors.
abstract: Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.
authors.affiliation: Section of Experimental Therapeutics, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, United Kingdom. c.a.conway@leeds.ac.uk
authors.forename: Caroline
authors.initials: C
authors.lastname: Conway
authors.forename: Rebecca
authors.initials: R
authors.lastname: Chalkley
authors.forename: Alec
authors.initials: A
authors.lastname: High
authors.forename: Kenneth
authors.initials: K
authors.lastname: Maclennan
authors.forename: Stefano
authors.initials: S
authors.lastname: Berri
authors.forename: Preetha
authors.initials: P
authors.lastname: Chengot
authors.forename: Melissa
authors.initials: M
authors.lastname: Alsop
authors.forename: Philip
authors.initials: P
authors.lastname: Egan
authors.forename: Joanne
authors.initials: J
authors.lastname: Morgan
authors.forename: Graham R
authors.initials: GR
authors.lastname: Taylor
authors.forename: John
authors.initials: J
authors.lastname: Chester
authors.forename: Mehmet
authors.initials: M
authors.lastname: Sen
authors.forename: Pamela
authors.initials: P
authors.lastname: Rabbitts
authors.forename: Henry M
authors.initials: HM
authors.lastname: Wood
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000175:diagnosis / Q000235:genetics / Q000821:virology; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D018628:Gene Dosage; D006258:Head and Neck Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007124:Immunoenzyme Techniques; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014412:Tumor Virus Infections / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D019562:Viral Load / Q000235:genetics*
chemical_list: C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004279:DNA, Viral; D009363:Neoplasm Proteins