Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform

Ana Giménez-Capitán, Jillian Bracht, Juan José García, Núria Jordana-Ariza, Beatriz García, Mónica Garzón, Clara Mayo-de-Las-Casas, Santiago Viteri-Ramirez, Alejandro Martinez-Bueno, Andrés Aguilar, Ivana-Gabriela Sullivan, Eric Johnson, Chung-Ying Huang, Jay L Gerlach, Sarah Warren, Joseph M Beechem, Cristina Teixidó, Rafael Rosell, Noemí Reguart, Miguel A Molina-Vila

Research output: Contribution to journalArticleAcademicpeer-review


BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids.

METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes.

RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease.

CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

Original languageEnglish
Pages (from-to)554-563
Number of pages10
JournalClinical Chemistry
Issue number3
Publication statusPublished - 1 Mar 2021
Externally publishedYes


  • Adult
  • Circulating Tumor DNA/genetics
  • DNA Mutational Analysis/methods
  • Female
  • Humans
  • Liquid Biopsy
  • Male
  • Middle Aged
  • Mutation
  • Neoplasms/genetics
  • Nucleic Acid Hybridization
  • Reproducibility of Results
  • Retrospective Studies

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