A Multicenter Study to Assess EGFR Mutational Status in Plasma: Focus on an Optimized Workflow for Liquid Biopsy in a Clinical Setting.
Laure SorberKaren ZwaenepoelKoen De WinneKaat Van CasterenElien AugustusJulie JacobsXiang Hua ZhangDaniëlla GaldermansEls De DrooghAnneke LefebureAnn-Marie MorelErika SaenenFrédérique BustinIngel DemedtsUlrike HimpeThierry PietersPaul GermonpréSofie DerijckeKoen DeschepperJan P Van MeerbeeckChristian C RolfoPatrick PauwelsPublished in: Cancers (2018)
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.
Keyphrases
- circulating tumor
- cell free
- epidermal growth factor receptor
- high speed
- small cell lung cancer
- tyrosine kinase
- squamous cell carcinoma
- atomic force microscopy
- risk assessment
- newly diagnosed
- signaling pathway
- high resolution
- dna methylation
- copy number
- human health
- ionic liquid
- prognostic factors
- quantum dots
- mass spectrometry
- nucleic acid
- heavy metals