Circulating Tumor Cells (CTCs) and Epithelial Mesenchymal Transition (EMT) in Breast Cancer: Describing the Heterogeneity of Microscopic Disease.

Abstract

Abstract Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer (BC) patients. CTC are readily detected by CellSearch System based on their expression of EpCAM. Epithelial-mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased chemotherapy resistance. Human mammary epithelial cells (HMEC) transformed by the EMT transcription factor TWIST1 and spiked into normal peripheral blood (PB) are not detected by EpCAM enrichment based conventional detection methods compared to non-transformed HMECs. We hypothesize that CTCs undergoing EMT and resultant loss of epithelial markers may escape detection by conventional detection methods. The aim of this study was to detect CTCs based on expression of EMT genes in breast cancer patient's peripheral blood.Methods: This prospective ongoing study of breast cancer patients consisted of 16 (57.1%) patients with metastatic disease, 19 (67.9%) patients with inflammatory breast cancer (IBC) and 12 (42.9%) patients with primary, non-IBC breast cancer, respectively. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using anti-CD45 coated magnetic beads. RNA extracted from CD45-depleted (CD45-) PBMC were interrogated for expression of TWIST1, SNAIL1, SLUG, ZEB1, FOXC2 and EpCAM gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of healthy donors (HD). Expression of one or more gene transcripts was considered a positive result. Concurrently, a 7.5 mL PB sample was collected for determination of CTC by CellSearch.Results: Median age was 54 year (range: 34-72 years). Overall, the median CTC count by CellSearch was 2 (range; 0-750) per 7.5 mL of PB. TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2 were overexpressed in CD45- PBMC in 7%, 4 %, 4%, 0% and 14 % of patients, respectively. At least one of the EMT genes was overexpressed in 6 (21%) of patients. TWIST1 and SLUG were overexpressed only in IBC patients (10.5% and 5.3% of patients, respectively). Patients with triple negative breast cancer more commonly overexpressed EMT genes compared to non-triple negative patients (30.8% vs. 13.3%). There was no correlation between expression of EMT genes, EpCAM expression or CTC count measured by CellSearch, respectively.Conclusions: These data suggest that EMT genes may be involved in the dissemination of CTCs. Loss of epithelial antigen on CTC due to EMT, triggered by high expression of these genes, may be responsible for their undetection by conventional methods in a fraction of patients with early or advanced breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3011.