Abstract B43: Genetic analysis of circulating tumor cells of colorectal cancer patients captured by multiantibodies technique
Authors: Takeda Kohki , Yamada Takeshi , Koizumi Michihiro , Shinji Seiichi , Matsuda Akihisa , Ohta Ryo , Yokoyama Yasuyuki , Takahashi Goro , Hotta Masahiro , Iwai Takuma , Hara Keisuke , Ueda Koji , Kuriyama Sho , Yoshida Hiroshi 
Published: June 2020

Abstract
Background: Accurate genetic analysis is essential for molecular therapy. Conventionally, DNA derived from tumor tissue has been the source of the gold standard of DNA information in solid tumors including colorectal cancer (CRC). However, it is well known that molecular therapy induces emerging mutations that are not found in primary tumor; thus, real-time monitoring of gene information is ideal. Circulating tumor cells (CTCs) are circulating malignant cells of solid tumor origin that are found in the bloodstream and can be a powerful candidate to play an important role. Capturing a large amount of CTCs improves the accuracy of genetic analysis of DNA derived from CTC (ctcDNA). We used 3 (EpCAM, Her2, Trop2) or 4 (EpCAM, Her2, Trop2, EGFR) antibodies to capture CTCs, and analyzed ctcDNA using next-generation sequencing (NGS).
Methods: Cohort 1: Untreated CRC patients were enrolled. Ten mL of whole blood was collected from each patient. The blood was processed using 3 antibodies (EpCAM, Her2, Trop2) and CTCs were collected. Tumor tissue was also collected from each patient. Tumor tissue DNA and ctcDNA were extracted and analyzed using NGS. Cohort 2: CRC patients, both treated and untreated, were enrolled. Twenty mL of whole blood was collected from each patient. Ten mL of the blood was processed using 3 antibodies (EpCAM, Her2, Trop2), and the remaining 10 mL was processed using 4 antibodies (EpCAM, Her2, Trop2, EGFR). The numbers of collected CTCs were counted and compared.
Results: Cohort 1: We enrolled 34 patients (stage II: n=4, stage III: n=7, stage IV: n=23). Median number of extracted CTC was 34 cells. From tumor tissue DNA, 53 mutations were detected. The most frequent mutation was within TP53 (n=18), followed by mutations in APC (n=13) and KRAS (n=12). From ctcDNA, 16 mutations, including 5 mutations which were not found in tissue DNA, were detected. The most frequent mutation was within TP53 (n=5), followed by mutations in KRAS and APC (n=4 each). Cohort 2: We enrolled 10 patients (stage II: n=1, stage III: n=1, stage IV: n=8). Using 3 antibodies, the median number of collected CTCs was 27 cells (range, 2–112). Using 4 antibodies, the median number of collected CTCs was 33 cells (range, 7–260). There were no statistically significant differences between the 2 groups (p=0.40).
Conclusions: Mutations not detected in primary tumors can be identified in ctcDNA, indicating the potential of CTCs in complementing gene analysis. The technique to capture CTCs using 3 antibodies appears to increase the detection rate and yield of CTCs. However, the present study did not show advantages of the 4-antibodies method, and future studies should investigate the best combination of antibodies to extract more CTCs with higher specificity.
Citation Format: Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Akihisa Matsuda, Ryo Ohta, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Koji Ueda, Sho Kuriyama, Hiroshi Yoshida. Genetic analysis of circulating tumor cells of colorectal cancer patients captured by multiantibodies technique [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B43.