A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

PLoS One. 2021 Jun 24;16(6):e0253687. doi: 10.1371/journal.pone.0253687. eCollection 2021.

Abstract

Background: A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen® EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies.

Results: Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven-log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen® EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R2≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen® EGFR Liquid assay platform showed better sensitivity than the Therascreen® EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT® Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level.

Conclusion: For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen® EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice.

Publication types

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

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Carcinoma, Non-Small-Cell Lung / blood
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Cell Line, Tumor
  • Circulating Tumor DNA / blood
  • Circulating Tumor DNA / genetics*
  • DNA Mutational Analysis
  • ErbB Receptors / blood
  • ErbB Receptors / genetics
  • Female
  • Humans
  • Liquid Biopsy
  • Lung Neoplasms / blood
  • Lung Neoplasms / genetics*
  • Male
  • Middle Aged
  • Mutation*
  • Neoplasm Proteins / blood
  • Neoplasm Proteins / genetics*

Substances

  • Circulating Tumor DNA
  • Neoplasm Proteins
  • EGFR protein, human
  • ErbB Receptors

Grants and funding

The project was supported by EuroStars (project number E!8421). EuroStars did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. PentaBase and ICP played the major role in study design, data collection, and preparation of the manuscript, as mentioned in the authors contribution. ICP is the scientific leader of the consortium. The role played by the different authors is reported in the “Authors contribution” section.