P4-07-17: Isolation of Highly Pure Circulating Tumor Cells (CTCs) from Metastatic Breast Cancer (MBC) Patients for Gene Expression Analysis.

Abstract

Abstract Background: The paucity of information regarding the molecular nature of CTCs can be largely attributed to the difficulty of isolating these rare cells. As such, efforts on RNA profiling of CTCs have been limited to studies using immuno-enriched samples containing a high background of leukocytes. Methods: We developed a two-step process: immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) to isolate CTCs away from leukocytes and performed expression analysis on a limited panel of genes. Magnetic beads coated with EpCAM mAb were used to enrich for tumor cells. Enriched samples were then subjected to FACS using fluorescently labeled mAbs to distinguish tumor cells (EpCAM+) from leukocytes (CD45+) during sorting. CTCs and matched leukocytes were isolated from 67 sequential patients with MBC. Total RNA from isolated 20–50 cells was reverse-transcribed into cDNA. Sixty four (64) CTC-related genes from previous published reports were chosen for expression analysis. cDNA of these genes were pre-amplified and analyzed in triplicate via Taqman®-based RT-PCR in a low density array format. Statistical analysis of gene expression data was performed using Realtime Statminer®. Results: Unsupervised hierarchical clustering analysis showed that CTCs clustered away from the leukocytes. Differential gene expression analysis revealed the up-regulation of several genes including EPCAM, MUC1, and KRT19 in CTCs (adjusted p <0.05). In addition, CTCs showed a significant down-regulation of the leukocyte-specific marker PTPRC (CD45) as well as CD44 and VIM, markers associated with stem cellness and epithelial to mesenchymal transition, respectively. Discussion: We demonstrate the feasibility of isolation and gene expression analysis of CTCs. Molecular profiling confirmed that isolated CTCs were highly pure with little or no evidence of leukocyte contamination. This approach can serve as a non-invasive source of tumor tissue for further molecular characterization of CTCs and for monitoring of therapeutic efficacy of targeted therapies in clinical trials. This study was supported by CALGB and BCRF. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-17.