Lee Cham Han, Lee Soo Jeong, Choi Sung Ho, Ahn Sei Hyun, Son Byung Ho, Lee Jong Won, Yu Jong Han, Kwon Nak-Jung, Lee Woo Chung, Yang Kap-Seok, Lee Dong Hyoung, Han Du Yeol, Choi Mi So, Park Pyeong-Soo, Lee Hyun Kyung, Kim Myoung Shin, Lee Jinseon, Jeon Byung Hee
Cytogen, Inc., Seoul 05838, Republic of Korea.
Department of Surgery, College of Medicine, University of Ulsan and Asan Medical Center, Seoul 05505, Republic of Korea.
Oncol Lett. 2018 Jul;16(1):612-618. doi: 10.3892/ol.2018.8646. Epub 2018 May 7.
Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM cells were detected in seven out of eight patients, and the average number of EpCAM cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.
使用循环肿瘤细胞（CTC）的液体活检是一种非侵入性且可重复的程序，因此对分子检测很有用。然而，CTC的稀有性仍然是一个挑战。为了克服这个问题，我们团队开发了一种基于细胞大小差异分离CTC的新技术。本研究使用该方法从乳腺癌患者中分离出CTC，然后将这些细胞用于癌症基因panel分析。收集了8例乳腺癌患者的血样，并使用基于大小的过滤方法富集CTC。使用上皮细胞粘附分子（EpCAM）和CD45抗体的免疫荧光染色对富集的CTC进行计数。提取、扩增CTC基因组DNA，并使用Ion AmpliSeq Comprehensive Cancer Panel筛选400个基因中的突变。同一患者的白细胞（WBC）用作阴性对照，并比较CTC和WBC中的突变。8例患者中有7例检测到EpCAM细胞，EpCAM细胞的平均数量为8.6。扩增DNA的平均量为32.7μg，相对于所有映射到参考序列的reads，映射到任何目标区域的reads百分比为98.6%。CTC特异性突变的检测率为62.5%。CTC特异性突变包括zeste多梳抑制复合体2亚基增强子、Notch 1、富含AT的相互作用结构域1A、丝氨酸/苏氨酸激酶11、fms相关酪氨酸激酶3、MYCN原癌基因、bHLH转录因子、APC、WNT信号通路调节因子以及磷酸酶和张力蛋白同源物。本研究使用的技术被证明能够有效地以足够高的纯度分离CTC用于基因组分析，并支持将综合癌症panel分析作为精准医学的一种潜在应用。
