Development and validation of a new HPV genotyping assay based on next-generation sequencing

Am J Clin Pathol. 2014 Jun;141(6):796-804. doi: 10.1309/AJCP9P2KJSXEKCJB.

Abstract

Objectives: We developed a new human papillomavirus (HPV) genotyping assay based on multiplex polymerase chain reaction and next-generation sequencing (NGS) methods for large-scale cervical cancer screening.

Methods: We first trained the assay on 1,170 self-collected samples, balancing the cutoff points for high-risk types. Then using 4,262 separate self-collected specimens, we compared concordance, sensitivity, and specificity for cervical intraepithelial neoplasia type 2 (CIN2) or higher and CIN type 3 (CIN3) or higher of the HPV sequencing assay with that of Hybrid Capture 2 (HC2) direct samples and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay self-samples.

Results: All assays had a good agreement. The sensitivity for CIN2 or higher and CIN3 or higher of the self-sampling specimens tested with the sequencing assay run on both MiSeq and Ion Torrent Personal Genome Machine sequencer was similar to that of direct-sampling specimens tested with HC2 (P > .05), but the specificity of the sequencing assay for CIN2 or higher and CIN3 or higher was significantly higher than that of HC2 (P < .01).

Conclusions: This population-based study has demonstrated the applicability of a new NGS high-risk HPV assay for primary cervical cancer screening based on self-collection.

Keywords: Cervical cancer screening; HPV genotyping test; Ion Torrent PGM; MiSeq; Next-generation sequencing (NGS); Self-sampling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Alphapapillomavirus / classification
  • Alphapapillomavirus / genetics
  • Alphapapillomavirus / isolation & purification*
  • Carcinoma, Squamous Cell / diagnosis
  • Carcinoma, Squamous Cell / virology*
  • DNA, Viral / chemistry
  • DNA, Viral / genetics
  • Early Detection of Cancer
  • Female
  • Genotyping Techniques / standards*
  • High-Throughput Nucleotide Sequencing
  • Human Papillomavirus DNA Tests
  • Humans
  • Multiplex Polymerase Chain Reaction
  • Papillomavirus Infections / complications
  • Papillomavirus Infections / diagnosis
  • Papillomavirus Infections / virology*
  • ROC Curve
  • Sensitivity and Specificity
  • Specimen Handling / methods
  • Specimen Handling / standards
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Uterine Cervical Dysplasia / diagnosis
  • Uterine Cervical Dysplasia / virology*
  • Uterine Cervical Neoplasms / diagnosis
  • Uterine Cervical Neoplasms / virology*
  • Vaginal Smears

Substances

  • DNA, Viral