免疫荧光联合原位杂交技术检测骨肉瘤患者外周血中循环肿瘤细胞的表达及临床意义

Detection and clinical significance of circulating tumor cells in osteosarcoma using immunofluorescence combined with in situ hybridization

目的探讨骨肉瘤患者外周血中循环肿瘤细胞（CTCs）的表达及其与患者预后的关系。方法采用活体细胞荧光示踪技术验证了免疫磁珠富集法回收细胞的能力，应用流式细胞术检测HOS细胞、HepG2细胞和人白细胞中CD45和CK18的表达，采用荧光原位杂交检测HOS细胞染色体倍数异常情况。采用免疫荧光联合原位杂交技术（iFISH）检测23例骨肉瘤患者中外周血CTCs，分析CTCs计数与患者预后的关系。结果免疫磁珠CD45差减富集法富集HOS细胞的回收率〉90%。流式细胞术检测显示，HOS细胞呈CD45（－）和CK18（－）。CEP8杂交探针信号〉2的细胞占总细胞数的96.5%，其中探针信号数为3的细胞占异常细胞的65.0%。23例骨肉瘤患者中，19例患者外周血中检测到CTCs。复发和（或）转移患者CTCs计数为（2.846±1.281）个，无复发和转移患者的CTCs计数为（1.400±1.506）个，差异有统计学意义（P＝0.021）。结论利用iFISH可以检测到骨肉瘤患者外周血CTCs，且CTCs计数与患者的预后有关，CTCs可以作为骨肉瘤患者病情变化的预测指标。临床试验注册 中国临床试验注册中心，ChiCTR－OOC－15005925。

Objective To investigate the clinical significance of detection of circulating tumor ceils （CTCs） in peripheral blood from patients with osteosarcoma （OS） using the iFISH （immunofluoreseenee and fluorescence in situ hybridization） method. Methods The live cells recovery rate of immune-magnetic beads was evaluated by live-cell fluorescent tracer technology. The expression of CD45 and CK18 on the cell surface of HOS and HepG2 cells was measured by flow eytometry. And the chromosome aneuploidy was detected by centromeric FISH probe CEP8. Subsequently, 23 OS patients were enrolled and divided into two groups, relapse or metastasis group and primary group. And the prognostic significance of CTCs numbers was analyzed. Results The live cells recovery rate of immune-magnetic beads was higher than 90%. The flow cytometry results showed that HOS cells were double negative for the surface biomarkers of CD45 and CK18. In addition, the FISH-CEP8 signal abnormality rate were 96.5% in HOS cells. Thus, CTC was identified using the criteria as follows： the cells with CEP8-positive signal 〉2 accounted for more than 96.5% of the total cells, of which the cells with 〉3 positive signal were more than 65.0%. Among the enrolled patients, 19 patients had detectable CTCs in the peripheral blood. The CTCs numbers in the relapse or metastasis group and primary group were 2.846＋1.281 and 1.400＋ 1.506, respectively. The results showed that the CTCs in patients with recurrence or metastasis were significantly higher than those in primary patients （P= 0.021 ）. Conclusions To our knowledge, this is the first evidence of existence of CTCs in OS patients. The CTCs numbers were positively associated with disease progression and poor prognosis. These results may provide a potential prognostic tool for monitoring metastasis and recurrence in OS patients. Trial registration Chinese Clinical Trial Registry, ChiCTR-OOC-15005925