Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.
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 (∼500) and low (∼50) 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 (∼500) had a ∼90% 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.
1 CTC Center of Excellence, Nordsjællands Hospital , Hillerød, Denmark .|2 CytoTrack ApS , Lyngby, Denmark .
Sarah
S
Nejlund
3 Department of Technology, Faculty of Health and Technology, Metropolitan University College , Copenhagen, Denmark .
Julie
J
Smith
4 Department of Medical Oncology, Erasmus MC Cancer Institute , Rotterdam, the Netherlands .
Jaco
J
Kraan
1 CTC Center of Excellence, Nordsjællands Hospital , Hillerød, Denmark .|2 CytoTrack ApS , Lyngby, Denmark .
Henrik
H
Stender
4 Department of Medical Oncology, Erasmus MC Cancer Institute , Rotterdam, the Netherlands .
Mai N
MN
Van
5 Department of Oncology, Aarhus University Hospital , Aarhus, Denmark .
Sven T
ST
Langkjer
1 CTC Center of Excellence, Nordsjællands Hospital , Hillerød, Denmark .|2 CytoTrack ApS , Lyngby, Denmark .
Mikkel T
MT
Nielsen
1 CTC Center of Excellence, Nordsjællands Hospital , Hillerød, Denmark .|6 Department of Clinical Biochemistry, Nordsjællands Hospital , Hillerød, Denmark .
György
G
Sölétormos
1 CTC Center of Excellence, Nordsjællands Hospital , Hillerød, Denmark .|6 Department of Clinical Biochemistry, Nordsjællands Hospital , Hillerød, Denmark .
Thore
T
Hillig
D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002452:Cell Count; D002460:Cell Line; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000648:ultrastructure*; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor