Abstract P2-01-05: Parallel DNA and RNA profiling of EpCAM-positive cells in blood of metastatic breast cancer (MBC) patients confirm their malignant nature

• Published in: Cancer research (Chicago, Ill.) Vol. 72; no. 24_Supplement; pp. P2 - P2-01-05
• Main Authors: Magbanua, MJM, Hauranieh, L, Sosa, EV, Pendyala, P, Scott, JH, Rugo, HS, Park, JW
• Format: Journal Article
• Language: English
• Published: 15.12.2012
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.SABCS12-P2-01-05

Abstract Background: Molecular studies of EpCAM-positive cells found in the blood of cancer patients have been limited to immuno-enriched samples containing a high background of leukocytes. We developed a process to isolate rare EpCAM-positive cells and subjected them to DNA and RNA profiling. Methods: Blood 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+). Duplicate samples of 20 cells were isolated from the same enriched blood from 53 MBC patients and then subjected to DNA and RNA profiling in parallel. 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 blood samples from 44 of the 53 patients. Results: Differential gene expression analysis between EpCAM-positive cells and matched leukocytes confirmed the up-regulation of EPCAM and other genes including MUC1 and KRT19 (adjusted p <0.05). In addition, EpCAM-positive cells showed a significant down-regulation of the leukocyte-specific marker PTPRC (encodes CD45) as well as CD44 and VIM, markers associated with stem cellness and epithelial to mesenchymal transition, respectively. Unsupervised hierarchical clustering analysis of RNA profiles showed that EpCAM-positive cells clustered away from the leukocytes. Genome-wide copy number analysis of EpCAM-expressing cells revealed gains (e.g. 1q and 8q), losses (e.g. 8p and 16q), and focal amplifications (e.g. on 8q and 11q including CCND1) frequently seen in primary breast cancers. Discussion: We demonstrate the feasibility of isolation and molecular analysis of highly pure EpCAM-positive cells in the blood. Our results strongly suggest that epithelial cells captured from blood of MBC patients are malignant in nature and are therefore circulating tumor cells. This approach can serve as a non-invasive source of highly pure metastatic tumor tissue for further molecular characterization. This study was supported by CALGB and BCRF. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-01-05.