RESUMEN
BACKGROUND: A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. METHODS: Blood samples from healthy volunteers were spiked with high (â¼500) and low (â¼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. RESULTS: Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (â¼500) had a â¼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. CONCLUSIONS: A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Criopreservación/métodos , Células Neoplásicas Circulantes/ultraestructura , Neoplasias de la Mama/metabolismo , Recuento de Células , Línea Celular , Proliferación Celular , Femenino , Humanos , Células MCF-7 , Células Neoplásicas Circulantes/metabolismo , Células Tumorales CultivadasRESUMEN
Comparison of two methods to detect circulating tumor cells (CTC) CytoTrack and CellSearch through recovery of MCF-7 breast cancer cells, spiked into blood collected from healthy donors. Spiking of a fixed number of EpCAM and pan-cytokeratin positive MCF-7 cells into 7.5 mL donor blood was performed by FACSAria flow sorting. The samples were shipped to either CytoTrack or CellSearch research facilities within 48 h, where evaluation of MCF-7 recovery was performed. CytoTrack and CellSearch analyses were performed simultaneously. Recoveries of MCF-7 single cells, cells in clusters, and clusters were determined. The average numbers of MCF-7 cells/cells in clusters/clusters recovered from blood by the CytoTrack and CellSearch methods were 103 ± 5.9/27 ± 7.9/11 ± 3.5 (95 % CI) and 107 ± 4.4/20 ± 7.1/10 ± 3.5, respectively, with no difference between the two methods (p = 0.37/p = 0.23/p = 0.09). Overall, the recovery of CytoTrack and CellSearch was 68.8 ± 3.9 %/71.1 ± 2.9 %, respectively (p = 0.58). In spite of different methodologies, CytoTrack and CellSearch found similar number of CTCs, when spiking was performed with the EpCAM and pan cytokeratin-positive cell line MCF-7. The results suggest that CytoTrack and CellSearch have similar abilities to identify CTC in vitro.
Asunto(s)
Antígenos de Neoplasias/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Moléculas de Adhesión Celular/genética , Células Neoplásicas Circulantes , Antígenos de Neoplasias/química , Biomarcadores de Tumor/química , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/química , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Técnicas In Vitro/métodos , Queratinas/química , Queratinas/genética , Células MCF-7 , PronósticoRESUMEN
BACKGROUND: Retracing and biomarker characterization of individual circulating tumour cells (CTCs) may potentially contribute to personalized metastatic cancer therapy. This is relevant when a biopsy of the metastasis is complicated or impossible to acquire. METHODS: A novel disc format was used to map and retrace individual CTCs from breast-cancer patients and nucleated cells from healthy blood donors using the CytoTrack platform. For proof of the retracing concept, CTC HER2 characterization by immunofluorescence was tested. RESULTS: CTCs were detected and enumerated in three of four blood samples from breast-cancer patients and the locations of each individual CTCs were mapped on the discs. Nucleated cells were retraced on seven discs with 96.6%±8.5% recovery on five fields of view on each disc. Shifting of field of view for retracing was measured to 4-29 µm. In a blood sample from a HER2-positive breast-cancer patient, CTC enumeration and mapping was followed by HER2 characterization and retracing to demonstrate downstream immunofluorescence analysis of the CTC. CONCLUSION: Mapping and retracing of CTCs enables downstream analysis of individual CTCs for existing and future cancer genotypic and phenotypic biomarkers. Future studies will uncover this potential of the novel retracing technology.