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

Abstract

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.

Circulating Cell-Free DNA and RNA Analysis as Liquid Biopsy: Optimal Centrifugation Protocol

“…In a multicenter study, we observed a negative influence on plasma volume upon longer storage of blood samples in Streck tubes. Increased centrifugation speed was found to negate this effect [20,21], which is recommended by Streck. As plasma volume is directly associated with cfDNA concentration [22], it is doubtful whether protocols based on low centrifugation speed, such as the platelet-generating and (adapted) basic protocol, will generate sufficient material for the detection of low abundant aberrations.…”

“…cfDNA isolation was performed using the Maxwell RSC ccfDNA large volume plasma kit (Promega, Leiden, The Netherlands) according to the manufacturer’s protocol. Detailed procedures are described elsewhere [20]. Plasma samples were processed immediately after thawing, with the exception of the adapted basic protocol.…”

Utilization of Archived Plasma to Detect Epidermal Growth Factor Receptor Mutation in Non-Small Cell Lung Cancer Patients

“…Liquid biopsy, consisting of biomarker testing, for example, mutations in cell free DNA (cfDNA), has emerged as an important approach to guide therapeutic decisions. 9–13 A liquid biopsy is generally accompanied by simple blood collection and can enable real-time follow-up. Because of challenges associated with the availability of archived tissue, liquid biopsies are often performed to detect cancer-specific mutations.…”

Pre-analytical issues in liquid biopsy – where do we stand?

“…Unfortunately, there are almost no data on how to handle these fluids in a way to optimize the isolation and characterization of extracellular nucleic acids. In a "seven at one blow" approach, Sorber and colleagues tried to analyze the effect of a variety of different pre-analytical factors on the sensitivity of a test for the detection of EGFR mutations in the plasma of lung cancer patients [88]. One of their conclusions reads that "A longer transit time was associated with a lower amount of plasma" in combination with a one-step low-speed centrifugation protocol for plasma processing.…”