Sequencing-based counting and size profiling of plasma Epstein-Barr virus DNA enhance population screening of nasopharyngeal carcinoma

Proc Natl Acad Sci U S A. 2018 May 29;115(22):E5115-E5124. doi: 10.1073/pnas.1804184115. Epub 2018 May 14.


Circulating tumor-derived DNA testing for cancer screening has recently been demonstrated in a prospective study on identification of nasopharyngeal carcinoma (NPC) among 20,174 asymptomatic individuals. Plasma EBV DNA, a marker for NPC, was detected using real-time PCR. While plasma EBV DNA was persistently detectable in 97.1% of the NPCs identified, ∼5% of the general population had transiently detectable plasma EBV DNA. We hypothesized that EBV DNA in plasma of subjects with or without NPC may have different molecular characteristics. We performed target-capture sequencing of plasma EBV DNA and identified differences in the abundance and size profiles of EBV DNA molecules within plasma of NPC and non-NPC subjects. NPC patients had significantly higher amounts of plasma EBV DNA, which showed longer fragment lengths. Cutoff values were established from an exploratory dataset and tested in a validation sample set. Adopting an algorithm that required a sample to concurrently pass cutoffs for EBV DNA counting and size measurements, NPCs were detected at a positive predictive value (PPV) of 19.6%. This represented superior performance compared with the PPV of 11.0% in the prospective screening study, which required participants with an initially detectable plasma EBV DNA result to be retested within 4 weeks. The observed differences in the molecular nature of EBV DNA molecules in plasma of subjects with or without NPC were successfully translated into a sequencing-based test that had a high PPV for NPC screening and achievable through single time-point testing.

Keywords: circulating tumor DNA; ctDNA; liquid biopsy; massively parallel sequencing; size-based diagnostics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Carcinoma* / blood
  • Carcinoma* / diagnosis
  • Circulating Tumor DNA / blood*
  • Cohort Studies
  • DNA, Viral / blood*
  • DNA, Viral / chemistry
  • DNA, Viral / genetics
  • Female
  • Herpesvirus 4, Human / genetics*
  • Humans
  • Liquid Biopsy / methods
  • Male
  • Middle Aged
  • Molecular Diagnostic Techniques / methods
  • Nasopharyngeal Carcinoma
  • Nasopharyngeal Neoplasms* / blood
  • Nasopharyngeal Neoplasms* / diagnosis
  • Reproducibility of Results
  • Viral Load / methods*


  • Circulating Tumor DNA
  • DNA, Viral