Analytical and clinical validation of a custom 15-gene next-generation sequencing panel for the evaluation of circulating tumor DNA mutations in patients with advanced non-small-cell lung cancer

PLoS One. 2022 Oct 18;17(10):e0276161. doi: 10.1371/journal.pone.0276161. eCollection 2022.

Abstract

Background: This is a pilot proof-of-concept study to evaluate the utility of a custom 15-gene circulating tumor DNA (ctDNA) panel as a potential companion molecular next-generation sequencing (NGS) assay for identifying somatic single nucleotide variants and indels in non-small-cell lung cancer (NSCLC) patients. The custom panel covers the hotspot mutations in EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET, KIT, PDGFRA, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 genes which serve as biomarkers for guiding treatment decisions in NSCLC patients.

Method: The custom 15-gene ctDNA NGS panel was designed using ArcherDX Assay Designer. A total of 20 ng or 50 ng input ctDNA was used to construct the libraries. The analytical performance was evaluated using reference standards at different allellic frequencies (0.1%, 1%, 5% and parental). The clinical performance was evaluated using plasma samples collected from 10 treatment naïve advanced stage III or IV NSCLC patients who were tested for tissue EGFR mutations. The bioinformatics analysis was performed using the proprietary Archer Analysis Software.

Results: For the analytical validation, we achieved 100% sensitivity and specificity for the detection of known mutations in the reference standards. The limit of detection was 1% allelic frequency. Clinical validation showed that the clinical sensitivity and specificity of the assay for detecting EGFR mutation were 83.3% and 100% respectively. In addition, the NGS panel also detected other mutations of uncertain significance in 6 out of 10 patients.

Conclusion: This preliminary analysis showed that the custom 15-gene ctDNA NGS panel demonstrated good analytical and clinical performances for the EGFR mutation. Further studies incorporating the validation of other candidate gene mutations are warranted.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers, Tumor / genetics
  • Carcinoma, Non-Small-Cell Lung* / pathology
  • Circulating Tumor DNA* / genetics
  • Class I Phosphatidylinositol 3-Kinases / genetics
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lung Neoplasms* / pathology
  • Mutation
  • Nucleotides
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins p21(ras) / genetics

Substances

  • Circulating Tumor DNA
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins p21(ras)
  • Proto-Oncogene Proteins
  • Class I Phosphatidylinositol 3-Kinases
  • Nucleotides
  • Biomarkers, Tumor

Grant support

This work was supported by the research fund granted by Sunway Medical Centre (SUNMED Research Fund, grant SRC/F/18/007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.