Department of Gynecology and Obstetrics, University Hospital of Essen, University of Duisburg-Essen, D-45122 Essen, Germany.
Breast Cancer Res. 2012 Jan 20;14(1):R15. doi: 10.1186/bcr3099.
The presence of circulating tumor cells (CTC) in breast cancer might be associated with stem cell-like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTC must be able to perform epithelial-mesenchymal transition (EMT). We studied the expression of three EMT markers and the stem cell marker ALDH1 in CTC from 502 primary breast cancer patients. Data were correlated with the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and with clinicopathological data of the patients.
A total of 2 × 5 ml of blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1, HER2 and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [TWIST1, Akt2, phosphoinositide kinase-3 (PI3Kα)] and separately for the tumor stem cell marker ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Two BM aspirates from all patients were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.
Ninety-seven percent of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. CTC were detected in 97/502 (19%) patients. At least one of the EMT markers was expressed in 29% and ALDH1 was present in 14% of the samples, respectively. Interestingly, 5% of the ALDH1-positive and 18% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. DTC in the BM were detected in 107/502 (21%) patients and no correlation was found between BM status and CTC positivity (P = 0.41). The presence of CTC, EMT and ALDH1 expression was not correlated to any of the prognostic clinical markers.
Our data indicate that (1) a subset of primary breast cancer patients shows EMT and stem cell characteristics and (2) the currently used detection methods for CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.
乳腺癌患者循环肿瘤细胞（CTC）的存在可能与具有干细胞样肿瘤细胞有关，这些细胞被认为是原发性肿瘤转移扩散的活跃来源。此外，为了能够进行扩散和转移，CTC 必须能够进行上皮-间充质转化（EMT）。我们研究了 502 例原发性乳腺癌患者的 CTC 中三种 EMT 标志物和干细胞标志物 ALDH1 的表达情况。将数据与骨髓（BM）中播散性肿瘤细胞（DTC）的存在以及患者的临床病理数据进行了相关性分析。
使用 AdnaTest BreastCancer（AdnaGen AG）对 2×5ml 血液进行 CTC 分析，以检测 EpCAM、MUC-1、HER2 和 β-肌动蛋白转录本。回收的 cDNA 还进行了三种 EMT 标志物 [TWIST1、Akt2、磷酸肌醇激酶-3（PI3Kα）]的多重检测，以及单独检测肿瘤干细胞标志物 ALDH1。如果在样本中检测到至少一种标志物，则认为 EMT 标志物的鉴定为阳性。对所有患者的两个 BM 抽吸物均通过使用泛细胞角蛋白抗体 A45-B/B3 的免疫细胞化学法分析 DTC。
在 30 名接受调查的健康供体样本中，97%的样本 EMT 和 95%的样本 ALDH1 转录本均为阴性。在 502 例患者中有 97/502（19%）例患者检测到 CTC。在 29%的样本中至少表达了一种 EMT 标志物，在 14%的样本中存在 ALDH1。有趣的是，根据 AdnaTest BreastCancer 确定的 CTC 阳性截断值，在 5%的 ALDH1 阳性患者和 18%的 EMT 阳性患者中为 CTC 阴性。在 502 例患者中有 107/502（21%）例患者的 BM 中检测到 DTC，并且在 BM 状态与 CTC 阳性之间未发现相关性（P=0.41）。CTC、EMT 和 ALDH1 表达与任何预后临床标志物均无相关性。
我们的数据表明：（1）一部分原发性乳腺癌患者具有 EMT 和干细胞特征；（2）目前用于 CTC 检测的方法不能有效地识别经历 EMT 的 CTC 亚群；（3）其对预后和治疗反应的临床相关性需要在前瞻性试验中进一步评估。
