Nejlund Sarah, Smith Julie, Kraan Jaco, Stender Henrik, Van Mai N, Langkjer Sven T, Nielsen Mikkel T, Sölétormos György, Hillig Thore
1 CTC Center of Excellence, Nordsjællands Hospital , Hillerød, Denmark .
2 CytoTrack ApS , Lyngby, Denmark .
Biopreserv Biobank. 2016 Aug;14(4):330-7. doi: 10.1089/bio.2015.0074. Epub 2016 Apr 19.
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.
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.
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.
A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.
含有循环肿瘤细胞（CTC）的血样可作为浸润性癌症转移的替代物。冷冻保存将为实验室临床样本管理提供新机会，并允许随时间收集样本，以便对现有和即将出现的癌症生物标志物进行未来分析。
从健康志愿者的血样中加入来自培养物的高数量（约500个）和低数量（约50个）肿瘤细胞。样本与冷冻保护剂二甲基亚砜混合于罗斯威尔帕克纪念研究所1640培养基中，保存在-80°C。流式细胞术检测冷冻保存是否影响常用于检测CTC的特定生物标志物，即细胞角蛋白（CK）、上皮细胞粘附分子（EpCAM）和白细胞特异性淋巴细胞共同抗原（CD45）。在不同时间间隔（长达6个月）后，将样本解冻并检查肿瘤细胞回收率（计数）。临床样本可能与细胞系研究不同，因此在17例浸润性乳腺癌患者中测试了冷冻保存方案并检查了肿瘤细胞回收率。从每位患者采集两份血样。
生物标志物CK、CD45和EpCAM不受冻融程序影响。加入大量肿瘤细胞（约500个）的冷冻保存样本（n = 2）与加入新鲜样本相比，回收率约为90%。在加入少量肿瘤细胞的样本（n = 5个样本中中位数为43个）中，冷冻保存后的回收率为63%（中位数27个肿瘤细胞），p = 0.03。加入的肿瘤细胞数量更低（n = 8个样本中中位数为3个）时，冷冻保存后肿瘤细胞的回收率似乎未受影响（中位数 = 8），p = 0.09。冷冻保存时间不影响回收率。在测试冷冻保存对临床样本计数的影响时，基于n = 17对样本，新鲜样本和冷冻保存样本之间的CTC数量未观察到差异，p = 0.83；然而，变异很大。通过临床配对新鲜样本（n = 六十四对）证实了这种大变异，其中95%的样本（<30个CTC）数量变化高达±15个CTC，p = 0.18。
冷冻保存后可能预期CTC会有少量损失；然而，冷冻保存CTC用于临床应用的生物标志物表征似乎很有前景。
