Molecular Biomarker Analyses Using Circulating Tumor Cells

BackgroundEvaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.
Methodology/Principal FindingsUsing spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.
Conclusions/SignificanceOur data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.

背景：
从血液样本中开展癌症生物标志物评估，可通过获取相对无创的代表性肿瘤来源材料，显著推动生物标志物的检测与评价工作。从转移性癌症患者血液中分离得到的循环肿瘤细胞（Circulating Tumor Cells, CTCs）在该研究领域展现出显著的应用潜力。

研究方法与主要结果：
本研究采用人工掺入肿瘤细胞的方式，对包括CellSearch®平台与两种生物芯片平台在内的多款循环肿瘤细胞捕获技术平台进行性能评估；同时利用分离得到的循环肿瘤细胞，开发并优化用于其分子特征分析的检测方案。研究结果表明，各受试平台在循环肿瘤细胞捕获方面的整体性能相近，且捕获效率取决于上皮细胞黏附分子（Epithelial Cell Adhesion Molecule, EpCAM）的表达水平。
本研究证实，捕获获得的循环肿瘤细胞可适用于多种生物标志物分析，包括人表皮生长因子受体2（Human Epidermal Growth Factor Receptor 2, HER2）状态检测、乳腺癌亚型标志物的实时荧光定量聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）分析、KRAS突变检测，以及采用免疫荧光（Immunofluorescence, IF）技术进行的EGFR染色检测。我们对转移性肺癌患者样本中的EGFR细胞表面表达水平进行了定量分析。此外，通过免疫荧光（IF）与荧光原位杂交（Fluorescence In Situ Hybridization, FISH）技术，我们检测了转移性乳腺癌患者循环肿瘤细胞的HER2状态。在大部分患者（89%）中，循环肿瘤细胞的HER2状态与患者原发肿瘤组织的HER2状态一致，但在11%的患者亚组中，循环肿瘤细胞的HER2状态与原发肿瘤存在差异。
值得注意的是，我们发现雌激素受体阳性（Estrogen Receptor-positive, ER+）患者的循环肿瘤细胞计数高于HER2阳性（HER2-positive, HER2+）与三阴性乳腺癌患者，这一现象可通过乳腺癌基底样分子亚型肿瘤的EpCAM表达水平较低且具有更显著的间质表型加以解释。

结论与意义：
本研究数据显示，从捕获得到的循环肿瘤细胞中开展分子特征分析具备可行性，且有望提供生物标志物状态的实时检测信息。就此而言，循环肿瘤细胞作为肿瘤来源材料，在推动临床生物标志物评估方面具有重要的应用前景。然而，仅基于EpCAM的捕获系统存在一定局限性，若添加针对间质标志物的特异性抗体，可进一步提升循环肿瘤细胞的捕获效率，从而实现基于循环肿瘤细胞的常规化生物标志物分析。