Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens

J Biomol Tech. 2016 Jul;27(2):46-52. doi: 10.7171/jbt.16-2702-004. Epub 2016 Mar 7.

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.

Keywords: head and neck cancer; human papillomavirus; viral.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA, Viral / genetics
  • DNA, Viral / isolation & purification
  • Female
  • Formaldehyde
  • Genotyping Techniques
  • High-Throughput Nucleotide Sequencing
  • Human papillomavirus 16 / genetics*
  • Humans
  • Oropharyngeal Neoplasms / diagnosis*
  • Oropharyngeal Neoplasms / virology
  • Paraffin Embedding
  • Sensitivity and Specificity
  • Sequence Analysis, DNA
  • Tissue Fixation
  • Uterine Cervical Neoplasms / diagnosis*
  • Uterine Cervical Neoplasms / virology

Substances

  • DNA, Viral
  • Formaldehyde