Abstract 3099: Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients

Abstract

Abstract Purpose Circulating tumor DNA (ctDNA) can potentially be used to monitor cancer treatment response. In this study, we developed and used a sensitive and selective ultra-deep next-generation sequencing (NGS) approach for detection of ctDNA in lung cancer patients. Method Most studies use fixed gene panels for detection of ctDNA by NGS. Deep sequencing of large target regions is expensive and generates a myriad of variants, which are difficult to interpret. For the purpose of using ctDNA for monitoring, we try to overcome these challenges by sequencing only selected regions where mutations were previously found in the tumor. This method is more sensitive and cost-efficient. In this study, we included about 80 lung cancer patients. Tumor DNA was sequenced with a large gene panel and somatic mutations were identified in each tumor. We then defined the target region according to the tumor mutations and selected primers for constructing NGS libraries. Since the coverage region is small it enables ultra-deep sequencing of several samples simultaneously. We evaluated the lower limit of detection for this method by sequencing a series of artificially constructed samples with known mutant allele frequencies (MAFs). DNA from peripheral blood leucocytes was sequenced in parallel with plasma DNA to exclude variants from clonal hematopoiesis. Preliminary results Using 40 ng as input DNA, which contains approximately 12,000 haploid genomes, we detected mutations down to 0.02% MAF. This corresponds to about 6 mutated copies per mL plasma. Currently, we are applying this method on patient plasma DNA samples. Conclusion We have developed a sensitive method for ctDNA detection, and we will present the results of the lung cancer patient samples. Citation Format: Anine Larsen Ottestad, Elisabeth F. Emdal, Sissel G. Wahl, Bjørn Henning Grønberg, Hong Yan Dai. Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3099.