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

Author(s): Wu W, Huang Y, Guo J, Xie X, Li H, Cao Z, Wei H, Wu C

Publication: Cancer Cytopathol, 2020, Vol. , Page

PubMed ID: 32286726 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare DNA yield and mutation status in the supernatant and cellular material from FNA specimens of NSCLC. The effects of extraction method used to obtain cfDNA from the supernatants and tumor cell content on DNA yields were also investigated. Computed tomography (CT)– or endoscopic transbronchial ultrasound–guided FNAs specimens were collected from 214 patients with NSCLC using 20- to 25-gauge needles. After onsite evaluation, the residual tissue was rinsed in CytoLyt. The cellular material was centrifuged at 3000 rpm for 5 min and the supernatants were frozen at -20°C until DNA extraction. The supernatants were recentrifuged at 10,000 rpm for 10 min. The resulting supernatants were aliquoted for cfDNA extraction using the QIAamp Circulating Nucleic Acid Kit (144 specimens) and the AmoyDx Circulating DNA Kit (70 specimens) while the DNA was extracted from the cellular pellet and stained smears using the AmoyDx Tissue DNA Kit. DNA integrity was evaluated using the Qubit DNA High Sensitivity Assay. EGFR mutational status was evaluated using the real-time PCR based Super-ARMS assay for cfDNA and the ARMS assay for DNA. Next-generation sequencing was conducted using an Amoy Diagnostics capture-based sequencing panel.

   Summary of Findings:

   The DNA yield was higher from cell pellets than from the supernatants. Although the median DNA concentration was higher when cfDNA extraction from the supernatant was with the QIAamp Kit rather than the AmoyDx method (0.77 ng/µL versus 0.48 ng/µL), the difference was not significant, and the range of the concentrations was the same. Further, the EGFR mutation rate was unaffected by extraction method. In the cellular pellets, the tumor cell content did not affect the DNA yield. The EGFR mutation positivity rate was 57.5% for both the cells and supernatants with consistent status between specimen types in 97.2% of the specimens. The discordant cases consisted of three with mutations identified only in the supernatant and three with mutations only identified in the pellet. Sequencing of four of these cases confirmed the status in pellets and supernatants and in one case this was thought to reflect a partial response to drug treatment.

   Biospecimens

   • Cell - Lung

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Next generation sequencing
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen components	Cells Supernatant ≤ 200 tumor cells 200-500 tumor cells ≥ 500 tumor cells
   Real-time qPCR Specific	Technology platform	NGS
   Analyte Extraction and Purification	Analyte isolation method	QIAamp circulating nucleic acid kit AmoyDx circulating DNA kit

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