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Licensed Unlicensed Requires Authentication Published by De Gruyter August 30, 2019

Next-generation sequencing for tumor mutation quantification using liquid biopsies

  • Mariano Provencio , Clara Pérez-Barrios , Miguel Barquin , Virginia Calvo , Fabio Franco , Estela Sánchez , Ricardo Sánchez , Daniel Marsden , Juan Cristóbal Sánchez , Paloma Martin Acosta , Raquel Laza-Briviesca , Alberto Cruz-Bermúdez and Atocha Romero ORCID logo EMAIL logo

Abstract

Background

Non-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations.

Methods

A total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR.

Results

Lin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p < 0.0001, respectively), indicating an excellent concordance between both measurements. Similarly, the agreement between NGS and dPCR for the detection of the resistance mutation p.T790M was almost perfect (K = 0.81; 95% CI = 0.62–0.99), with an excellent correlation in terms of MAFs (ρc = 0.991; 95% CI = 0.981–0.992 and Pearson’s r = 0.998; p < 0.0001). Importantly, cfDNA sequencing was successful using as low as 10 ng cfDNA input.

Conclusions

MAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.


Corresponding author: Atocha Romero, PharmD, PhD, Medical Oncology Department, Puerta de Hierro Hospital, C/ Manuel de Falla 1, Majadahonda, Madrid 28222, Spain; and Molecular Oncology Laboratory, Biomedical Sciences Research Institute, Hospital Universitario Puerta de Hierro-Majadahonda, Madrid, Spain
aMariano Provencio, Clara Pérez-Barrios and Miguel Barquin contributed equally to this work.

Acknowledgments

We would like to thank the patients for their participation in the study.

  1. Author contributions: AR conceived and coordinated the study. CBP, AR, MB and MM contributed to experimental design and data analysis. CBP, MB, ES, RS, RLB, ACB, PM and PM carried out the experiments. AR, MB and CPB performed the statistical analyses. MM, VC, JCS and FF selected the study population. AR and CPB contributed to manuscript preparation. All authors read and approved the final manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: This study was supported by the Carlos III Institute of Health, the Spanish Ministry of Science and Innovation and the European Regional Development Fund (grant numbers: PI16/01818 and PIE17/01977). MB was financed by i-PFIS predoctoral fellowship (grant number IFI18/00051) from the ISCIII-MINECO-AES-FEDER (Plan Estatal I+D+I 2013-2016). ES was financed by the Consejería de Educación, Juventud y Deporte of Comunidad de Madrid and by the Fondo Social Europeo (Programa Operativo de Empleo Juvenil, and Iniciativa de Empleo Juvenil, PEJ-2017-AI/SAL-6478).

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0745).


Received: 2019-07-21
Accepted: 2019-08-05
Published Online: 2019-08-30
Published in Print: 2020-01-28

©2019 Walter de Gruyter GmbH, Berlin/Boston

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