Construction of a reference material panel for detectingKRAS/NRAS/EGFR/BRAF/METmutations in plasma ctDNA

Abstract

BackgroundThe absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China.ObjectiveThis study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal–epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA).MethodsMutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel.Results20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation.ConclusionRM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.

Prognostic Value of Circulating Tumor DNA (ctDNA) in Oncogene-Driven NSCLC: Current Knowledge and Future Perspectives

“…Attention has been called to the detection of MET mutations in plasma ctDNA [123]. For example, in a comprehensive genomic profiling study of ctDNA from 1552 NSCLC patients, METex14 skipping mutations were detected in 1.9% of cases.…”

The Overview of Perspectives of Clinical Application of Liquid Biopsy in Non-Small-Cell Lung Cancer

“…There are also some reports stating that, in NSCLC patients with ALK gene rearrangement, the L1196M and S1206Y substitutions in the ALK gene may be screened in liquid biopsy as possible factors of resistance to crizotinib—the first line of ALK TKI [ 127 ]. Moreover, there are many studies describing the potential clinical utility of mutation testing in the liquid biopsy of NSCLC patients in ROS1 [ 128 , 129 ], MET [ 130 , 131 ], and BRAF [ 119 , 131 ] genes.…”

Development of a Duck Genomic Reference Material by Digital PCR Platforms for the Detection of Meat Adulteration

“…Certified reference materials (CRMs) are reference materials (RMs) characterized using a metrologically valid procedure for one or more specified properties, accompanied by an RM certificate [14], which can be used for calibration of a measurement system, assessment of a measurement procedure, for assigning values to other materials, and quality control. Several DNA-based CRMs have been developed, such as for genetically modified organisms [15][16][17], cancer diagnosis [18][19][20], foodborne pathogens [21], and forensic science [22,23].…”