Abstract 1389: Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients

Abstract

Abstract Circulating tumor cells (CTCs) are shed from primary or metastatic tumors into blood circulation, which may carry specific genetic characteristics related to hematogenous metastasis. Drawbacks of conventional EpCAM or cell size-dependent CTC detection and relevant single-cell sequencing technologies have limited discovery of specific genetic variants in CTCs. In the current study, a novel integrated SE-iFISH strategy, despite cell size or epithelial marker status, was applied to detect CTCs in advanced gastric cancer (GC) patients. Obtained CTCs were classified into diverse subtypes upon different chromosome ploidy. Fifty-three aneuploid CTCs identified by iFISH in 6 of naïve GC patients were isolated by a novel non-laser microscopic single cell manipulator (NMSCM), and subjected to modified Multiple Displacement Amplification (MDA) and subsequent single-cell sequencing. Compared with the conventional PCR, MDA generates 2 to 100-kb products with a lower error rate and achieves high fidelity whole genome amplification. Sequencing targeting 50 tumor genes of single CTC as well as paired primary tumor specimens was performed. Moreover, leukocytes genomic DNA as germline controls were included. Obtained results showed that high frequent SNV in genes such as TP53 were detected in both CTCs and the paired primary tumor mass. Furthermore, CTCs had considerable genetic heterogeneity compared to the paired primary tumor lesions. In particular, the CDKN2AA68V variant is frequently detected in CTCs but the paired primary GC tumor. We propose that the specific CDKN2AA68V variant carried by GC CTCs with high frequency, but low in the examined whole population of cancer cells in paired primary tumor lesions, may serve as the driver to promote CTCs shed into peripheral blood and to further facilitate hematogenous metastasis. Systematic in vitro and ex vivo studies are in progress to investigate roles of CDKN2A mutation in hematogenous dissemination in GC. Citation Format: Yilin Li, Xiaotian Zhang, Jifang Gong, Daisy Dandan Wang, Mingxia Qi, Yanyan Li, Dan Liu, Chunxiang Wang, Peter Ping Lin, Lin Shen. Single-cell sequencing to identify genetic variant accelerating shedding of CTCs into peripheral blood in gastric cancer patients [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 1389.