DNA profiling of EpCAM-positive cells in bone marrow of early breast cancer (EBC) patients. DNA profiling of EpCAM-positive cells in bone marrow of early breast cancer (EBC) patients

Parallel DNA and RNA profiling of EpCAM-positive cells in bone marrow and primary tumor tissue with positive disseminated tumor cell (DTC) count via immunomagnetic Enrichment/Flow Cytometry (IE/FC) of metastatic breast cancer (MBC) patients confirm their malignant nature We developed a novel approach to isolate tumor cells with high purity from bone marrow which was subjected to immunomagnetic enrichment using EpCAM beads followed by fluorescence activated cell sorting (IE/FACS) to isolate EpCAM-positive cells away from leukocytes (CD45+). For DNA profiling, sorted cells were subjected to BAC array comparative genomic hybridization analysis following whole genome amplification. For RNA profiling, QPCR analysis was performed on sixty four (64) cancer-related genes using Taqman® low density arrays. For non-tumor controls, RNA profiling was performed on matched leukocytes (CD45+) isolated from the same enriched bone marrow samples. Overall design: Bone marrow aspirates were collected from early breast cancer patients. Disseminated tumor cells (DTCs) were enumerated via immunomagnetic enrichment followed by flow cytometry (IE/FC). For patients considered positive for DTCs, the remaining volume of bone marrow sample was subjected to IE/FACS to isolate DTCs for gene expression (QPCR) and copy number analysis. Differential gene expression analysis between EPCAM-positive cells and matched leukocytes confirmed the up-regulation of EPCAM and other genes including MUC1 (adjusted p <0.05). In addition, EPCAM-positive cells showed a significant down-regulation of the leukocyte-specific marker PTPRC (encodes CD45). Matched primary tumor samples from a subset of patients were subjected to copy number analysis. Genomic profiling of DTCs revealed fewer aberrations compared to matched primary tumors.

本数据集针对转移性乳腺癌（metastatic breast cancer, MBC）患者，通过免疫磁珠富集/流式细胞术（immunomagnetic Enrichment/Flow Cytometry, IE/FC）计数骨髓及原发肿瘤组织中播散肿瘤细胞（disseminated tumor cell, DTC）呈阳性的上皮细胞黏附分子阳性细胞（EpCAM-positive cells），并对其开展平行DNA与RNA谱分析，以证实其恶性特性。我们开发了一种从骨髓中高纯度分离肿瘤细胞的新方法：先使用EpCAM磁珠完成免疫磁珠富集，随后通过荧光激活细胞分选（fluorescence activated cell sorting, IE/FACS）将EpCAM阳性细胞与白细胞（CD45+）分离。DNA谱分析环节中，分选后的细胞先进行全基因组扩增，再采用细菌人工染色体阵列比较基因组杂交（BAC array comparative genomic hybridization）分析；RNA谱分析则使用Taqman®低密度阵列芯片，对64个癌症相关基因开展定量聚合酶链反应（quantitative polymerase chain reaction, QPCR）分析。非肿瘤对照样本采用从同一富集骨髓样本中分离的配对白细胞（CD45+）进行RNA谱分析。总体实验设计如下：从早期乳腺癌患者中采集骨髓穿刺液，通过免疫磁珠富集-流式细胞术（IE/FC）计数其中的播散肿瘤细胞（DTCs）；对于DTC阳性患者，取剩余骨髓样本通过IE/FACS分离DTCs，用于基因表达（QPCR）及拷贝数分析。对EpCAM阳性细胞与配对白细胞进行差异基因表达分析，结果证实EpCAM及包括黏蛋白1（MUC1）在内的其他基因均呈上调表达（校正后P<0.05）；此外，EpCAM阳性细胞中白细胞特异性标志物PTPRC（编码CD45）的表达显著下调。对部分患者的配对原发肿瘤样本开展拷贝数分析后发现，DTCs的基因组谱异常数量少于配对原发肿瘤。