Detection and comparison of EGFR mutations from supernatants that contain cell‐free DNA and cell pellets from FNA non–small cell lung cancer specimens

Abstract

BackgroundEpidermal growth factor receptor (EGFR) is an important marker for targeted therapy in patients with advanced non–small cell lung cancer (NSCLC). The samples obtained with minimally invasive biopsy techniques are usually small, and this limits their application in tissue subtyping or molecular profiling. The supernatants obtained after centrifugation of fine‐needle aspiration (FNA) samples are typically discarded. However, these fractions might contain cell‐free DNA that could be tested for EGFR mutations by genotyping methods that are normally used for plasma analysis.MethodsIn this study, 214 patients with known or suspected NSCLC who underwent FNA were enrolled. The workflow of the supernatants before molecular detection was as follows. The discarded FNA samples (15 mL) were stored in CytoLyt, a cleaning, fixation solution, and 10 mL of each sample was placed in a preservation solution for separation by low‐speed centrifugation. The primary supernatants (8 mL) were then separated by high‐speed centrifugation to obtain secondary supernatants. DNA was extracted from the supernatants with QIAamp circulating nucleic acid kits (Qiagen) and circulating DNA kits (AmoyDx), and EGFR mutations were assessed with Super‐ARMS EGFR detection kits (AmoyDx). The DNA was then extracted from corresponding cell pellets with tissue DNA kits (AmoyDx), and the EGFR status was analyzed with the amplification refractory mutation system and next‐generation sequencing methods.ResultsAll 214 samples yielded an adequate amount of cell‐free DNA for EGFR detection. The use of different DNA commercial extraction kits and the DNA contents of tumor cells did not affect the yield of DNA from the supernatants. The external controlled cycle threshold value of the EGFR test was affected by the concentration of the DNA in the supernatants (P < .05). However, the difference in the concentrations of the DNA in the supernatants did not affect the EGFR mutation status. The EGFR‐positive rate was 57.5% (123 of 214) in both the supernatants and the pellets from the 214 FNA samples. The concordance between EGFR variants in the supernatants and the corresponding pellets was 97.2%. EGFR mutations were also detected in 3 pellets but not in their corresponding supernatants and in 3 supernatants but not in their corresponding pellets. The supernatants of FNA biopsy samples might represent a new source for gaining information regarding the molecular characteristics of patients for targeted therapy.ConclusionsDiscarded supernatants provided an adequate amount of cell‐free DNA for EGFR detection, and this means that the pellets can be reserved for additional morphological and molecular analyses or to avoid repeat biopsies. Analyzing the EGFR status in cell supernatants and pellets might improve detection sensitivity and confer benefits to patients.

Validating cell‐free DNA from supernatant for molecular diagnostics on cytology specimens

“…The supernatant from this process is typically discarded, but recent studies have suggested that supernatant fluid from cell block formation is a potential alternative source of DNA for biomarker testing. [11][12][13][14][15][16][17][18][19] Multiple studies have shown very good concordance rates between supernatant (either from body fluids or FNA specimens) and FFPE cell blocks for detecting mutations, and some studies have shown an improved mutation detection rate with DNA from supernatant as compared with cell blocks. 11,15,19 In addition, in cases where neither cell blocks nor aspirate smears were prepared (eg, bile duct brushings with only ThinPrep slides), molecular studies performed on the supernatant has been shown to have a higher sensitivity than cytology alone.…”

Sputum cell‐free DNA for detection of alterations of multiple driver genes in lung adenocarcinoma

“…Mutation status of 10 driver genes in matched tumor samples was detected by a NGS panel (Amoy Diagnostics), 17,18 which can detect hotspot mutations/fusions in genes of EGFR, KRAS, BRAF, NRAS, HER2, PIK3CA, ALK, ROS1, RET, and MET in one single test. The detection process described by the manufacturer was followed.…”

Diagnostic and prognostic biomarkers in oligometastatic non-small cell lung cancer: a literature review

“…Traditional or liquid-based preparations are suitable for the application of exome sequencing or customized panels and, especially, May Grunwald Giemsa (MMG)-stained slides can be easily microdissected to obtain enriched samples. Supernatants may provide adequate material for the detection of driver mutations in oncogene-addicted cancers, helping to avoid the sacrifice of diagnostic stained smears (14). Especially during the natural history of longlasting cancers, the possible contribution of core-needles as repeat biopsies is debated.…”