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

• Published in: Cancers 2018-08, Vol.10 (9), p.290
• Main Authors: Sorber, Laure, Zwaenepoel, Karen, De Winne, Koen, Van Casteren, Kaat, Augustus, Elien, Jacobs, Julie, Zhang, Xiang Hua, 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
• Format: Article
• Language: English
• ISSN: 2072-6694; 2072-6694
• DOI: 10.3390/cancers10090290

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 ( 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 ( < 0.001) and short transit time ( = 0.018). Multistep, high-speed centrifugation both increased plasma generation ( < 0.001) and reduced genomic DNA (gDNA) contamination. Longer transit time increased the risk of hemolysis ( < 0.001) and low temperatures were shown to have a negative effect. Metastatic sites were found to be strongly associated with ctDNA detection ( < 0.001), as well as allele frequency ( = 0.034). Activating mutations were detected in a higher concentration and allele frequency compared to the T790M mutation ( = 0.003, and = 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.