Abstract 1444: High-throughput four color detection and analysis of circulating epithelial cells in early and late breast cancer.
Authors: Habets Leo , Körber Wolfgang , Frenken Bettina , El Ghali Ilham , Danaei Mahmoud , Kusche Manfred , Peisker Uwe , Kroll Torsten , Pachmann Katharina 
Published: August 2014

Abstract
The Maintrac technique (RBClysis,fluorometric detection and analysis on Olympus ScanR) detects more circulating epithelial cells (CEC) then other enrichment- based methods. In our ongoing early breast cancer study (n=177), we found &gt;125 CEC/ml in 35% of patients. In advanced breast cancer 60% of patients had cell counts&gt;125 cells/ml . In controls (n=98) we found 125-250 cells/ml in 2%. Extended evaluation of a non cancer control group showed &gt;125 cells/ml in 90% of patients with liver affections such as nonalcoholic fatty liver disease(NAFLD) . Using a three colour- based technique living and dead (DAPI) EPCAM + cells coexpressing vimentin and in parallell EPCAM+ cells coexpressing Vimentin and CD44 were measured. We saw a very inhomogeneous EPCAM+ population with different expression patterns,which made analysis very tedious. Moreover, there was no detectable difference between the CEC in early and late breast cancer and in liver affections, in both situations CEC had epithelial-mesenchymal transition (EMT)- and stem cell characteristics. We aimed to perform a more comprehensive analysis of these CEC by introducing a fourth color.We modified the basic procedure of the Maintrac technique in order too allow analysis and detection of CEC on the AMNIS FlowSight. Cells are aspirated from 96 well microplates allowing four color detection and automated analysis with AMNIS IDEAS software. Total plate run is completed within 9 hours. Beside the fastness new items like detection of tumor microemboli (TME) and EPCAM- independent analysis of cells with a stem cell phenotype (CD44high,CD24 low) or metabolic markers (HIF-1,carboanhydrase 9) is possible. We currently use DAPI, EPCAMAF660, vimentin -PE and CD45-FITC as basic combination . A second run uses CD24-PE, CD45-FITC, CD44-PacBlue and EPCAM- AF660 . Double measurement of the EPCAM+CD45neg population as basic population for comparison is achieved . Comprehensive analysis of more defined circulating stem cells (CSC) is thus possible.This approach will clarify the differences between liver- and tumor-derived CEC thus avoiding false positive CTC counts in early breast cancer . Our preliminary results show detectable differences between patients with advanced cancer and NAFLD concerning highly defined CEC with with "stemness" expression (CD44high, CD24low, EPCAM+, CD45neg). Our focus is on a cell type with very high "dotted " expression of CD44, EPCAM and vimentin, seen already with three colour analysis. We are currently introducing other markers like ALDH1 and beta-catenin for better definition of CSC. Furthermore, detection of Her2 or other targets like PD-1 or phosphatidylserine are possible on this defined CEC. We will present extended data with the four color approach for CEC analysis in early and late breast cancer at the conference.
Citation Format: Leo Habets, Wolfgang Körber, Bettina Frenken, Ilham El Ghali, Mahmoud Danaei, Manfred Kusche, Uwe Peisker, Torsten Kroll, Katharina Pachmann. High-throughput four color detection and analysis of circulating epithelial cells in early and late breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1444. doi:10.1158/1538-7445.AM2013-1444