Next-generation sequencing for simultaneous determination of human papillomavirus load, subtype, and associated genomic copy number changes in tumors

J Mol Diagn. 2012 Mar-Apr;14(2):104-11. doi: 10.1016/j.jmoldx.2011.10.003. Epub 2012 Jan 10.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Squamous Cell / diagnosis
  • Carcinoma, Squamous Cell / genetics
  • Carcinoma, Squamous Cell / virology
  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA, Viral / genetics
  • Female
  • Gene Dosage*
  • Head and Neck Neoplasms / diagnosis*
  • Head and Neck Neoplasms / genetics
  • Head and Neck Neoplasms / virology
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoenzyme Techniques
  • Middle Aged
  • Neoplasm Proteins / metabolism
  • Papillomaviridae / classification*
  • Papillomaviridae / genetics*
  • Papillomavirus Infections / diagnosis*
  • Papillomavirus Infections / genetics
  • Papillomavirus Infections / virology
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • Tumor Virus Infections / diagnosis*
  • Tumor Virus Infections / genetics
  • Tumor Virus Infections / virology
  • Viral Load / genetics*


  • CDKN2A protein, human
  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA, Viral
  • Neoplasm Proteins