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

doi:https://doi.org/10.1093/clinchem/hvaa248

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 journal › Article › Academic › peer-review

Abstract

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 language	English
Pages (from-to)	554-563
Number of pages	10
Journal	Clinical Chemistry
Volume	67
Issue number	3
DOIs	https://doi.org/10.1093/clinchem/hvaa248
Publication status	Published - 1 Mar 2021
Externally published	Yes

Keywords

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

Access to Document

https://doi.org/10.1093/clinchem/hvaa248

Cite this

Giménez-Capitán, A., Bracht, J., García, J. J., Jordana-Ariza, N., García, B., Garzón, M., Mayo-de-Las-Casas, C., Viteri-Ramirez, S., Martinez-Bueno, A., Aguilar, A., Sullivan, I.-G., Johnson, E., Huang, C.-Y., Gerlach, J. L., Warren, S., Beechem, J. M., Teixidó, C., Rosell, R., Reguart, N., & Molina-Vila, M. A. (2021). Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform. Clinical Chemistry, 67(3), 554-563. https://doi.org/10.1093/clinchem/hvaa248

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

abstract = "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.",

keywords = "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",

author = "Ana Gim{\'e}nez-Capit{\'a}n and Jillian Bracht and Garc{\'i}a, {Juan Jos{\'e}} and N{\'u}ria Jordana-Ariza and Beatriz Garc{\'i}a and M{\'o}nica Garz{\'o}n and Clara Mayo-de-Las-Casas and Santiago Viteri-Ramirez and Alejandro Martinez-Bueno and Andr{\'e}s Aguilar and Ivana-Gabriela Sullivan and Eric Johnson and Chung-Ying Huang and Gerlach, {Jay L} and Sarah Warren and Beechem, {Joseph M} and Cristina Teixid{\'o} and Rafael Rosell and Noem{\'i} Reguart and Molina-Vila, {Miguel A}",

note = "{\textcopyright} American Association for Clinical Chemistry 2021.",

year = "2021",

month = mar,

day = "1",

doi = "https://doi.org/10.1093/clinchem/hvaa248",

language = "English",

volume = "67",

pages = "554--563",

journal = "Clinical Chemistry",

issn = "0009-9147",

publisher = "American Association for Clinical Chemistry Inc.",

number = "3",

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Giménez-Capitán, A, Bracht, J, García, JJ, Jordana-Ariza, N, García, B, Garzón, M, Mayo-de-Las-Casas, C, Viteri-Ramirez, S, Martinez-Bueno, A, Aguilar, A, Sullivan, I-G, Johnson, E, Huang, C-Y, Gerlach, JL, Warren, S, Beechem, JM, Teixidó, C, Rosell, R, Reguart, N & Molina-Vila, MA 2021, 'Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform', Clinical Chemistry, vol. 67, no. 3, pp. 554-563. https://doi.org/10.1093/clinchem/hvaa248

TY - JOUR

T1 - Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform

AU - Giménez-Capitán, Ana

AU - Bracht, Jillian

AU - García, Juan José

AU - Jordana-Ariza, Núria

AU - García, Beatriz

AU - Garzón, Mónica

AU - Mayo-de-Las-Casas, Clara

AU - Viteri-Ramirez, Santiago

AU - Martinez-Bueno, Alejandro

AU - Aguilar, Andrés

AU - Sullivan, Ivana-Gabriela

AU - Johnson, Eric

AU - Huang, Chung-Ying

AU - Gerlach, Jay L

AU - Warren, Sarah

AU - Beechem, Joseph M

AU - Teixidó, Cristina

AU - Rosell, Rafael

AU - Reguart, Noemí

AU - Molina-Vila, Miguel A

N1 - © American Association for Clinical Chemistry 2021.

PY - 2021/3/1

Y1 - 2021/3/1

N2 - 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.

AB - 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.

KW - Adult

KW - Circulating Tumor DNA/genetics

KW - DNA Mutational Analysis/methods

KW - Female

KW - Humans

KW - Liquid Biopsy

KW - Male

KW - Middle Aged

KW - Mutation

KW - Neoplasms/genetics

KW - Nucleic Acid Hybridization

KW - Reproducibility of Results

KW - Retrospective Studies

U2 - https://doi.org/10.1093/clinchem/hvaa248

DO - https://doi.org/10.1093/clinchem/hvaa248

M3 - Article

C2 - 33439966

SN - 0009-9147

VL - 67

SP - 554

EP - 563

JO - Clinical Chemistry

JF - Clinical Chemistry

IS - 3

ER -