Abstract 4594: Detection of colorectal cancer related gene mutations from CTC and ctDNA
Authors: Takeda Kohki , Yamada Takeshi , Koizumi Michihiro , Shinji Seiichi , Yokoyama Yasuyuki , Takahashi Goro , Hotta Masahiro , Iwai Takuma , Hara Keisuke , Furuki Hiroyasu , Uchida Eiji 
Published: August 2018

Abstract
[Background] It is well known that emerging mutations which is not found in the primary tumor exist in metastatic tumor and molecular therapy induce emerging mutation. Liquid biopsy, which includes circulating tumor cell (CTC) and circulating tumor DNA (ctDNA), may help detecting this spatial and temporal heterogeneity. We have reported that emerging KRAS mutation can be detected by using ctDNA (Yamada et al, Cancer Science 2016). However, mutation detection by using CTC has been difficult because enough amount of DNA cannot be extracted from CTC. Currently we have been able to collect more CTC than before, by using a new device which uses 3 antibodies (EpCAM, Her2, Trop2). In this study, we evaluated the potential to detect colorectal cancer (CRC) related gene mutations from CTC, and compared it with ctDNA.
[Methods] Cohort 1: This cohort included untreated CRC patients. Tumor tissue was collected from each patient, either by primary surgery or by colonoscopic biopsy. DNA was extracted from tumor tissue and was analyzed using Next Generation Sequencing (NGS). Ten mL of whole blood was also collected from the same patient. CTC, serum and white blood cell (WBC) was collected by using the CTC recovery machine (Ion Torrent Liquid Biopsy Instrument®). Cytokeratin positive, DAPI positive, CD45 negative cells were defined as CTC. DNA was extracted from each sample (CTC-DNA, ctDNA, WBC-DNA) and was analyzed using NGS. Cohort 2: This cohort included unresectable CRC patients with KRAS mutation in their primary tumor. All patients in this cohort were under treatment or after completion of chemotherapy. CTC and ctDNA was collected in the same method as cohort 1. KRAS mutations of CTC and ctDNA were detected by using digital PCR (dPCR).
[Results] Cohort 1: We enrolled 16 CRC patients (stage II: n=2, stage III: n=2, stage IV: n=12). A total of 30 somatic, hotspot mutations were detected from tumor tissue DNA. The median number of the detected mutation for each patient was 2 (0-4). The most frequent gene mutation was APC, followed by KRAS and TP53. In all patients, CTC was successfully collected. The median number of the CTC was 34 cells (5-94). However, only 6 somatic, mutations were detected from CTC-DNA. Conversely, 16 somatic mutations were detected from ctDNA. Cohort 2: We enrolled 14 stage IV CRC patients with KRAS mutation in their primary tumor. CTC was collected from 9 patients but not from 5 patients. In the 9 patients CTC was collected, the median number of the collected CTC was 26 cells (5-121). By dPCR, KRAS mutation was detected in 2 patients (2/14) from CTC-DNA, and 4 patients (4/14) from ctDNA.
[Conclusions] The new CTC capturing technology using 3 antibodies can improve detection rate and yield of CTC. However, in patients undergoing chemotherapy, the amount of CTC and ctDNA drastically reduces. At present, ctDNA is superior to CTC in potential to detect mutations, and dPCR is more sensitive than NGS to detect mutations.
Citation Format: Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Hiroyasu Furuki, Eiji Uchida. Detection of colorectal cancer related gene mutations from CTC and ctDNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4594.