Abstract 3691: Molecular profiling using next generation sequencing with high purity enrichment of circulating tumor cells
Authors: Park Sehwan , Choi Jaesung , Jeong Aejin , Kim Hanbyeol , Oh Jiyoun , Jang Ingeon , Kang Hoin , Lee Jihyun , Pak Sehyung , Song Min-Ji , Kim Jung Won , Lee Hai-Chon 
Published: March 2024

Abstract
Purpose: In the area of liquid biopsy, coupled with next generation sequencing (NGS), circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) can serve as an alternative substrate to tumor tissue for mutation detection and companion diagnostic purpose. Emerging evidence shows that complementary sequential CTCs and ctDNA profiling provide promising benefit for prognosis and treatment. In contrast to well-established ctDNA extraction methods, the process of isolating intact CTCs for NGS analysis is yet to be fully elucidated. Here, we show that the isolation and harvest of viable CTCs by CytoGen’s Smart Biopsy™ System enable the attainment of sufficiently high purity for NGS analysis in metastatic breast cancer.
Methods: Targeted panel sequencing for CTC was developed to detect representative mutated genes, using the Oncomine™ Comprehensive Assay Plus (501 genes) and the Oncomine™ Pan-Cancer Cell-Free Assay (52 genes). Analytical validation was performed with breast cancer cells MDA-MB-231 (50 to 5 cells) which were spiked into 5 mL healthy blood, to mimic CTC. The CytoGen’s Smart Biopsy™ system was applied to isolate the CTC. Then, CD45 depletion and whole genome amplification were performed. Sequencing was carried out on Ion S5™ XL systems using an Ion 550™ Chip Kit. Subsequently, sequencing data was analyzed using Ion Torrent software with default configurations.
Results: As a result of targeted panel sequencing using Oncomine™ Comprehensive Assay Plus, representative mutated genes (BRAF, KRAS, TP53, NF2 and NOTCH3) in breast cancer were detected in at least 20 MDA-MB-231 cells. In the results using Oncomine™ Pan-Cancer Cell-Free Assay, mutated genes (BRAF, KRAS and TP53) were successfully detected even with only 5 MDA-MB-231 cells. These results suggest that clinical sample of breast cancer could be applied with promising sensitivity and specificity for detection of representative mutated genes.
Conclusion: Molecular analysis of CTCs is a challenge since CTCs are very rare in the blood and there are technical limitations in isolating CTCs with sufficient purity. In this study, we developed the process for NGS analysis on very low number of CTCs with high purity, isolated using CytoGen's Smart Biopsy™ system. Overall, these process could provide information for diagnosis and appropriate treatment of metastatic breast cancer along with the results of our ongoing clinical trials.
Citation Format: Sehwan Park, Jaesung Choi, Aejin Jeong, Hanbyeol Kim, Jiyoun Oh, Ingeon Jang, Hoin Kang, Jihyun Lee, Sehyung Pak, Min-Ji Song, Jung Won Kim, Hai-Chon Lee. Molecular profiling using next generation sequencing with high purity enrichment of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3691.