Gene panels covering hundreds of mutation hotspots for detection of cell-free DNA (cfDNA) have been developed for the establishment of clinically practical circulating tumor (ctDNA) detection system. Although amplicon-based or capture-based genomic profiling of genes commonly mutated in cancer using next-generation sequencing (NGS) system have been developed, they are not sufficiently sensitive nor accurate to be used for clinical diagnosis in hospital. As a result of preliminary study using 87 advanced pancreatic cancer (stage lll/lV), we observed that one or more KRAS mutation(s) could be detected in 60.9% of patients with advanced pancreatic cancer by using the amplicon-based deep sequencing system. To develop more sensitive and accurate ctDNA detection system, we optimized conditions of our DNA chip system. We firstly used 100 synthetic DNA samples, whose KRAS mutant allele frequencies were from 0.1% to 10.0%, and investigated the quantitativity of our DNA chip system. We amplified target regions by PCR to obtain 2,000 copies of synthesized DNA. The PCR product was hybridized with the probe on the DNA chip. The fluorescence ratio (mutant allele fluorescence intensity / total fluorescence intensity) of 100 synthetic DNA samples were clearly distinguished to that of negative control by using the DNA chip system. We defined an appropriate cut-off values of small quantities (0.1%) of mutations. Furthermore, to confirm the accuracy and reliability of this method, we verified our DNA chip system using 15 plasma cfDNA samples from patients with mutant KRAS (0.1~10.0%) and 15 samples from patients without mutant KRAS as negative control. 10 ng of ctDNA which extracted from blood sample is used in PCR. We observed that detected mutant status by DNA chip was completely concordant (100%) with those by amplicon-based deep sequencing system. Our data confirmed that mutant allele in cfDNA can be sensitively and accurately detected by DNA chip system. These results suggest that the ctDNA detection system developed in this study could be a novel diagnostic tool for tumor burden, and could realize the ultra-early detection of refractory tumors including pancreatic cancer.

Citation Format: Noriaki Nakamura, Hiroshi Okamura, Hirofumi Yamano, Hiromichi Ito, Hitoshi Zembutsu. The development of novel detection system for circulating tumor DNA using DNA chip system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2420.