Ultra-deep massively parallel sequencing with unique molecular identifier tagging achieves comparable performance to droplet digital PCR for detection and quantification of circulating tumor DNA from lung cancer patients

PLoS One. 2019 Dec 16;14(12):e0226193. doi: 10.1371/journal.pone.0226193. eCollection 2019.

Abstract

The identification and quantification of actionable mutations are of critical importance for effective genotype-directed therapies, prognosis and drug response monitoring in patients with non-small-cell lung cancer (NSCLC). Although tumor tissue biopsy remains the gold standard for diagnosis of NSCLC, the analysis of circulating tumor DNA (ctDNA) in plasma, known as liquid biopsy, has recently emerged as an alternative and noninvasive approach for exploring tumor genetic constitution. In this study, we developed a protocol for liquid biopsy using ultra-deep massively parallel sequencing (MPS) with unique molecular identifier tagging and evaluated its performance for the identification and quantification of tumor-derived mutations from plasma of patients with advanced NSCLC. Paired plasma and tumor tissue samples were used to evaluate mutation profiles detected by ultra-deep MPS, which showed 87.5% concordance. Cross-platform comparison with droplet digital PCR demonstrated comparable detection performance (91.4% concordance, Cohen's kappa coefficient of 0.85 with 95% CI = 0.72-0.97) and great reliability in quantification of mutation allele frequency (Intraclass correlation coefficient of 0.96 with 95% CI = 0.90-0.98). Our results highlight the potential application of liquid biopsy using ultra-deep MPS as a routine assay in clinical practice for both detection and quantification of actionable mutation landscape in NSCLC patients.

Publication types

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

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Carcinoma, Non-Small-Cell Lung / diagnosis
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Circulating Tumor DNA / analysis*
  • Circulating Tumor DNA / genetics
  • DNA Barcoding, Taxonomic / methods
  • DNA Mutational Analysis / methods
  • DNA, Neoplasm / analysis
  • DNA, Neoplasm / genetics
  • DNA, Neoplasm / isolation & purification
  • ErbB Receptors / genetics
  • Female
  • GTP Phosphohydrolases / genetics
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Limit of Detection
  • Liquid Biopsy
  • Lung Neoplasms / diagnosis
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / pathology
  • Male
  • Membrane Proteins / genetics
  • Middle Aged
  • Polymerase Chain Reaction / methods*
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Reproducibility of Results
  • Sequence Tagged Sites*

Substances

  • Circulating Tumor DNA
  • DNA, Neoplasm
  • KRAS protein, human
  • Membrane Proteins
  • EGFR protein, human
  • ErbB Receptors
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • GTP Phosphohydrolases
  • NRAS protein, human
  • Proto-Oncogene Proteins p21(ras)

Grants and funding

This research is funded by The Vietnam National Foundation for Science and Technology Development (NAFOSTED, https://nafosted.gov.vn/) under grant number 108.01-2017.306 (to HNN) and Ho Chi Minh city Department of Science and Technology under grant number 257/2017/HD-SKHCN (to HNN) and Gene Solutions (GS-003). We confirm that the funder Gene Solutions provided support in the form of salaries for authors LS Tran, HAT Pham, VU Tran, TT Tran, BT Vo, HTT Dao, NH Nguyen, HN Do, M-D Phan and H Giang but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.