Analysis of Circulating Tumor Cells Lab, Department of Chemistry, University of Athens, Athens, Greece.
Institute for Cell Biology, Histology and Embryology, Center of Molecular Medicine, Medical University of Graz, Graz, Austria.
Clin Chem. 2018 Feb;64(2):297-306. doi: 10.1373/clinchem.2017.275503. Epub 2017 Nov 9.
Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch, PSA-EPISPOT, and immunofluorescence of CellCollector in vivo-captured CTCs-using the same blood draw.
EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (, , , , , , , , , , , , , and ), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers.
We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining.
The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.
循环肿瘤细胞（CTC）的分子特征对于选择接受靶向治疗的患者非常重要。我们首次展示了对高风险前列腺癌（PCa）患者体内分离的 CTC 与通过 3 种基于蛋白质的检测方法（CellSearch、PSA-EPISPOT 和 CellCollector 体内捕获的 CTC 的免疫荧光）检测到的 CTC 进行基因表达谱分析的结果，这些检测均使用相同的血液样本。
使用 CellCollector 从 108 名高危 PCa 患者和 36 名健康志愿者中体内分离 EpCAM 阳性 CTC。对于 27 名患者，在治疗前后都有样本。我们开发了用于 14 个基因（、、、、、、、、、、和）的高灵敏度多重 RT-qPCR 检测，包括上皮标志物、干细胞标志物和上皮间质转化（EMT）标志物。
我们观察到每个患者捕获的 CTC 中基因表达存在高度异质性。在 105 名患者中的 74 名（70.5%）至少检测到 1 个标志物，在 105 名患者中的 45 名（40.9%）检测到 2 个标志物，在 105 名患者中的 16 名（15.2%）检测到 3 个标志物。在 31 名（29.5%）患者中检测到上皮标志物，在 46 名（43.8%）患者中检测到 EMT 标志物，在 15 名（14.3%）患者中检测到干细胞标志物。在治疗前，EMT 标志物阳性率非常低（27 名患者中的 2 名，7.4%），但在治疗后增加（27 名患者中的 17 名，63.0%），而上皮标志物在治疗后（27 名患者中的 2 名，7.4%）与治疗前（27 名患者中的 13 名，48.1%）相比趋于下降。至少 2 个标志物在 40.9%的患者中表达，而 CellSearch 的阳性率为 19.6%，EPISPOT 的阳性率为 38.1%，CellCollector 基于免疫荧光的染色的阳性率为 43.8%。
体内 CTC 分离与下游 RNA 分析相结合，作为一种高通量、特异性和超灵敏的基于液体活检的分子诊断方法具有很大的前景。
