A liquid biopsy-based method for the detection and quantification of circulating tumor cells in surgical osteosarcoma patients

Abstract

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.

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs

“…Limited studies of OSA CTC in humans also report a higher number than typically noted for carcinomas, but not nearly as high as those in our study (30,31,44). OSA is a highly aggressive neoplasm in dogs, and circulating OSA cells may at times be observed even on routine blood smear review (data not shown); therefore, frequent CTCs may be a biological feature of the tumor.…”

“…OSA is a highly aggressive neoplasm in dogs, and circulating OSA cells may at times be observed even on routine blood smear review (data not shown); therefore, frequent CTCs may be a biological feature of the tumor. Limited studies of OSA CTC in humans also report a higher number than typically noted for carcinomas, but not nearly as high as those in our study (30,31,44). Strengths of our study are that blood samples from dogs were analyzed multiple times, including immediately before death, which provided a dynamic picture of CTC frequency.…”

“…Over the past years, multiple commercial platforms involving negative and positive immune and nonimmune-based identification of leukocyte and tumor antigens, respectively, were used in oncology, and recently efforts are underway to assess agreement across platforms and methods (28). Enumeration of CTCs in humans with OSA using a combination of leukocyte depletion, immunochemistry, and fluorescent in situ hybridization identified significant differences in progression-free survival among patients with ≤2 versus >2 CTC/7.5 ml of blood (30). Enumeration of CTCs in humans with OSA using a combination of leukocyte depletion, immunochemistry, and fluorescent in situ hybridization identified significant differences in progression-free survival among patients with ≤2 versus >2 CTC/7.5 ml of blood (30).…”

“…The number of CTCs detected in humans with carcinoma is extremely low, and significant prognostic differences have been attributed to 3 versus 5 CTC/7.5 ml of blood (25,29). Enumeration of CTCs in humans with OSA using a combination of leukocyte depletion, immunochemistry, and fluorescent in situ hybridization identified significant differences in progression-free survival among patients with ≤2 versus >2 CTC/7.5 ml of blood (30). Furthermore, using quantitative PCR techniques, detection of osteospecific mRNA in blood of humans with OSA was significantly associated with the development of metastases during the subsequent 12-months period (31).…”

Circulating tumor cells detection in neuroblastoma patients by EpCAM-independent enrichment and immunostaining-fluorescence in situ hybridization

“…Therefore, we used an epithelial marker-independent enrichment method combined with immunostaining-fluorescence in situ hybridization (i-FISH) to detect CTCs from NB patients. This EpCAM-independent method has been described by Zhang et al [ 6 ] in a study that quantified CTCs from patients with osteosarcoma, a mesenchymal tumor type that does not express epithelial markers. This method has also been used in the detection of CTCs in gastric [ 7 ], pancreatic [ 8 ] and lung cancer [ 9 ].…”