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

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 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 from 17 of the 30 patients. Overall design: Bone Marrow aspirates where taken from EBC patients. Patients were considered positive (DTC≥1 cell/ml) via immunomagnetic enrichment followed by flow cytometry (IE/FC). When a sample was determined positive, the remainder of the bone marrow was processed and sorted via IE/FACS. 36 aspirates where sorted for RNA analysis. Quality control (QC) measures were implemented as follow: Sorted cells ≥10, expression of house keeping genes RPS18 and ACTB (Ct value ≤26 and ≤36 respectively), and detection rate ≥ 20% of 64 gene panel. 31 samples from 30 patients passed all QC.