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Keywords: Cancer; Cell-free DNA; Genetic testing; Genomics; Genome sequencing

J. Lee 3; W. Park 1; S. Jang 2; S. Chun 3

1) Asan Institute for Life Sciences, Asan Medical Center, Seoul, Select a Country; 2) Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.; 3) Department of Medical Science, Asan Medical Institute of Convergence Science and Technology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.

Circulating cell-free DNA (cfDNA) has great potential in clinical filed including care of cancer patients. Previously, prognostic and predictive value of cfDNA in non-small cell lung cancer (NSCLC) has been reported by several studies. Genetic mutations of EGFR, KRAS and BRAF genes in tumor derived cfDNA in patient with lung cancer could be applied as biomarkers for monitoring the early steps of tumor progression or a recurrence. However, low amount and extremely low tumor-derived DNA rate are recognized as hurdles to be overcome of cfDNA application.
In this study, we developed NSCLC-UHS (Ultra High-Sensitive) panel, which targets hot-spot mutations of BRAF, KRAS and EGFR in multiplex fashion using low amount cfDNA. Using the serially diluted positive samples, detection sensitivity of this panel was evaluated to be 0.05%. We performed targeted NGS analysis for detection of genetic mutations using FFPE tumor tissue DNA and matched cfDNA extracted from 0.4 ml plasma of 30 NSCLC patients, then, NSCLC-UHS panel test was done using cfDNA to compare the mutation patterns of EGFR, KRAS and BRAF genes. Focusing EGFR mutations, 20 of 30 cfDNA from NSCLC patients (66%) showed same mutation pattern with those of tumor tissue DNA by targeted NGS analysis. When considering the amount of applied cfDNA was very low from 0.1 ng to 1 ng, 66% detection sensitivity of NSCLC-UHS assay is very impressive. We expect that the sensitivity will be dramatically improved by increasing the input amount of cfDNA with higher starting volume of plasma.
In conclusion, Onco-UHS method is fast and cost-effective methods for a targeted set of mutations and can be used for selection of chemotherapy and for validation of NGS.