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

Objective: To investigate the clinical significance of detection of circulating tumor cells (CTCs) in peripheral blood from patients with osteosarcoma (OS) using the iFISH (immunofluorescence 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 cytometry. 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.