Analytical and clinical validation of a custom 15-gene next-generation sequencing panel for the evaluation of circulating tumor DNA mutations in patients with advanced non-small-cell lung cancer.

BACKGROUND: This is a pilot proof-of-concept study to evaluate the utility of a custom 15-gene circulating tumor DNA (ctDNA) panel as a potential companion molecular next-generation sequencing (NGS) assay for identifying somatic single nucleotide variants and indels in non-small-cell lung cancer (NSCLC) patients. The custom panel covers the hotspot mutations in EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET, KIT, PDGFRA, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 genes which serve as biomarkers for guiding treatment decisions in NSCLC patients. METHOD: The custom 15-gene ctDNA NGS panel was designed using ArcherDX Assay Designer. A total of 20 ng or 50 ng input ctDNA was used to construct the libraries. The analytical performance was evaluated using reference standards at different allellic frequencies (0.1%, 1%, 5% and parental). The clinical performance was evaluated using plasma samples collected from 10 treatment naïve advanced stage III or IV NSCLC patients who were tested for tissue EGFR mutations. The bioinformatics analysis was performed using the proprietary Archer Analysis Software. RESULTS: For the analytical validation, we achieved 100% sensitivity and specificity for the detection of known mutations in the reference standards. The limit of detection was 1% allelic frequency. Clinical validation showed that the clinical sensitivity and specificity of the assay for detecting EGFR mutation were 83.3% and 100% respectively. In addition, the NGS panel also detected other mutations of uncertain significance in 6 out of 10 patients. CONCLUSION: This preliminary analysis showed that the custom 15-gene ctDNA NGS panel demonstrated good analytical and clinical performances for the EGFR mutation. Further studies incorporating the validation of other candidate gene mutations are warranted.

Analysis of genetic variants in myeloproliferative neoplasms using a 22-gene next-generation sequencing panel.

BACKGROUND: The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythaemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF), are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of MPNs by allowing for the simultaneous screening of many disease-associated genes. METHODS: The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. RESULTS: The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5% (4 of which were putatively novel variants with potential biological significance) and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. CONCLUSIONS: The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in MPL.