Zhang Haoqiang, Gao Peng, Xiao Xin, Heger Michal, Geng Lei, Fan Bo, Yuan Yulin, Huang Chen, Chen Guojing, Liu Yao, Hu Yongchen, Yu Xiuchun, Wu Sujia, Wang Ling, Wang Zhen
Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China.
Department of Experimental Surgery, Academic Medical Center, University of Amsterdam, 1105AZ Amsterdam, The Netherlands.
Int J Oncol. 2017 Apr;50(4):1075-1086. doi: 10.3892/ijo.2017.3905. Epub 2017 Mar 8.
A method for the enumeration and quantification of osteosarcoma (OS) circulating tumor cells (CTCs) is currently not available. A correlation between the number of CTCs and progression-free survival (PFS) has been established for other cancers, but not for OS CTCs. A method was therefore developed for CTC quantification in OS and validated in a prospective cohort of surgical patients with primary and recurrent/metastatic OS (N=23). Human OS cells, acting as CTCs, were enumerated from spiked human peripheral blood (PB) following erythrocyte and leukocyte depletion. The OS cells were quantified microscopically based on aneuploidy and a CK18-/CD45- phenotype. Aneuploidy was assayed by fluorescence in situ hybridization (FISH) using fluorescence-labeled alpha-satellite probes for the centromeres of chromosome (CEP 8). CK18 and CD45 phenotyping was performed with immunocytochemistry. HOS cells in spiked PB could be effectively retrieved with the FISH-based enumeration method, which was subsequently employed in an OS patient cohort. PB of recurrent/metastatic OS patients contained more CTCs than the PB of primary OS patients. OS patients with ≥2 CTCs per 7.5 ml of PB had worse PFS than patients whose PB contained <2 CTCs. In 2 cases, CTCs were present in PB of OS patients with negative X-ray and chest CT scans. In conclusion, our method was able to quantitate CTCs in liquid biopsies of OS patients. The number of CTCs has diagnostic and prognostic value.
目前尚无用于骨肉瘤（OS）循环肿瘤细胞（CTC）计数和定量的方法。其他癌症已证实CTC数量与无进展生存期（PFS）之间存在相关性，但骨肉瘤CTC尚未证实。因此，开发了一种用于骨肉瘤CTC定量的方法，并在一组前瞻性手术患者队列中进行了验证，这些患者患有原发性和复发性/转移性骨肉瘤（N = 23）。在去除红细胞和白细胞后，从加标的人外周血（PB）中计数作为CTC的人骨肉瘤细胞。基于非整倍体和CK18-/CD45-表型，通过显微镜对骨肉瘤细胞进行定量。使用荧光标记的α-卫星探针进行染色体着丝粒（CEP 8）的荧光原位杂交（FISH）检测非整倍体。通过免疫细胞化学进行CK18和CD45表型分析。加标PB中的HOS细胞可以通过基于FISH的计数方法有效回收，随后该方法应用于骨肉瘤患者队列。复发性/转移性骨肉瘤患者的PB中所含CTC比原发性骨肉瘤患者的PB更多。每7.5 ml PB中≥2个CTC的骨肉瘤患者的PFS比PB中<2个CTC的患者更差。在2例病例中，X射线和胸部CT扫描阴性的骨肉瘤患者的PB中存在CTC。总之，我们的方法能够对骨肉瘤患者液体活检中的CTC进行定量。CTC数量具有诊断和预后价值。
