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Open AccessArticle

A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting

1
Center for Oncological Research Antwerp (CORE), University of Antwerp, 2610 Wilrijk, Belgium
2
Laboratory of Pathological Anatomy, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
3
Biomedical Quality Assurance Research Unit, KU Leuven, 3000 Leuven, Belgium
4
Department of Pulmonology & Thoracic Oncology, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
5
Pneumology, ZNA Middelheim, 2020 Antwerp, Belgium
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Thoracic Oncology Group Antwerp (TOGA), University of Antwerp, 2610 Wilrijk, Belgium
7
Pneumology, ZNA STER, 2140 Antwerp, Belgium
8
Pneumology, AZ Sint-Jozef, 2880 Bornem, Belgium
9
Lung diseases/Allergology, AZ Heilige Familie, 2840 Reet, Belgium
10
Pneumology/Allergology, CHR de la Citadelle, 4000 Liège, Belgium
11
Department of Pulmonary diseases, AZ Delta, 8800 Roeselare, Belgium
12
Pneumology, Cliniques Universitaires Saint-Luc, 1200 Bruxelles, Belgium
13
Pneumology, AZ Maria Middelares, 9000 Gent, Belgium
14
Pneumology-Thoracic Oncology, AZ Groeninge, 8500 Kortrijk, Belgium
15
Pneumology, AZ Nikolaas, 9100 Sint-Niklaas, Belgium
16
Oncology & Phase I Unit-Early Clinical Trials, Antwerp University Hospital (UZA), 2650 Edegem, Belgium
17
Marlene and Steward Greenebaum Comprehensive Cancer Center, Thoracic Medical Oncology & Early Clinical Trials, Baltimore, MD 12116, USA
18
Antwerp University Hospital (UZA), Tumor Bank, 2650 Edegem, Belgium
*
Author to whom correspondence should be addressed.
Cancers 2018, 10(9), 290; https://doi.org/10.3390/cancers10090290
Received: 16 July 2018 / Revised: 21 August 2018 / Accepted: 23 August 2018 / Published: 27 August 2018
(This article belongs to the Special Issue Cancer Biomarkers)
A multicenter study was performed to determine an optimal workflow for liquid biopsy in a clinical setting. In total, 549 plasma samples from 234 non-small cell lung cancer (NSCLC) patients were collected. Epidermal Growth Factor Receptor (EGFR) circulating cell-free tumor DNA (ctDNA) mutational analysis was performed using digital droplet PCR (ddPCR). The influence of (pre-) analytical variables on ctDNA analysis was investigated. Sensitivity of ctDNA analysis was influenced by an interplay between increased plasma volume (p < 0.001) and short transit time (p = 0.018). Multistep, high-speed centrifugation both increased plasma generation (p < 0.001) and reduced genomic DNA (gDNA) contamination. Longer transit time increased the risk of hemolysis (p < 0.001) and low temperatures were shown to have a negative effect. Metastatic sites were found to be strongly associated with ctDNA detection (p < 0.001), as well as allele frequency (p = 0.034). Activating mutations were detected in a higher concentration and allele frequency compared to the T790M mutation (p = 0.003, and p = 0.002, respectively). Optimization of (pre-) analytical variables is key to successful ctDNA analysis. Sufficient plasma volumes without hemolysis or gDNA contamination can be achieved by using multistep, high-speed centrifugation, coupled with short transit time and temperature regulation. Metastatic site location influenced ctDNA detection. Finally, ctDNA levels might have further value in detecting resistance mechanisms. View Full-Text
Keywords: liquid biopsy; non-small cell lung cancer (NSCLC); EGFR; ctDNA; ddPCR liquid biopsy; non-small cell lung cancer (NSCLC); EGFR; ctDNA; ddPCR
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Figure 1

MDPI and ACS Style

Sorber, L.; Zwaenepoel, K.; De Winne, K.; Van Casteren, K.; Augustus, E.; Jacobs, J.; Zhang, X.H.; Galdermans, D.; De Droogh, E.; Lefebure, A.; Morel, A.-M.; Saenen, E.; Bustin, F.; Demedts, I.; Himpe, U.; Pieters, T.; Germonpré, P.; Derijcke, S.; Deschepper, K.; Van Meerbeeck, J.P.; Rolfo, C.; Pauwels, P. A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting. Cancers 2018, 10, 290. https://doi.org/10.3390/cancers10090290

AMA Style

Sorber L, Zwaenepoel K, De Winne K, Van Casteren K, Augustus E, Jacobs J, Zhang XH, Galdermans D, De Droogh E, Lefebure A, Morel A-M, Saenen E, Bustin F, Demedts I, Himpe U, Pieters T, Germonpré P, Derijcke S, Deschepper K, Van Meerbeeck JP, Rolfo C, Pauwels P. A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting. Cancers. 2018; 10(9):290. https://doi.org/10.3390/cancers10090290

Chicago/Turabian Style

Sorber, Laure; Zwaenepoel, Karen; De Winne, Koen; Van Casteren, Kaat; Augustus, Elien; Jacobs, Julie; Zhang, Xiang H.; Galdermans, Daniëlla; De Droogh, Els; Lefebure, Anneke; Morel, Ann-Marie; Saenen, Erika; Bustin, Frédérique; Demedts, Ingel; Himpe, Ulrike; Pieters, Thierry; Germonpré, Paul; Derijcke, Sofie; Deschepper, Koen; Van Meerbeeck, Jan P.; Rolfo, Christian; Pauwels, Patrick. 2018. "A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting" Cancers 10, no. 9: 290. https://doi.org/10.3390/cancers10090290

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