Abstract P1-13-07: Standardized Immunocytological and Molecular Analysis of Tumor Cells and Mesenchymal Stromal Cells in Bone Marrow of Patients with Primary Breast Cancer

Abstract

Abstract Background: Using criteria for classification of disseminated tumor cells in compliance with the publication of Fehm et al. (Cancer, Vol.107, 2006) we have reported a cytological DTC-positivity rate of 1.9% (39/2050) in patients with primary breast cancer (T1-4, N0/N+, M0/M1). However automated picture analysis revealed cytokeratin positive stroma cell types in DTC negative samples. Here we describe the cytological and molecular characterization of these mesenchymal stromal cell (MSC) types. Material and Methods: Mononuclear cells were enriched by Ficoll density-gradient centrifugation. Cytospin slides (mean 6) with 1x106 BM-cells were stained by CK antibodies 5D3 and A45-B/B3 using automated immunostainers with the APAAP-and the enhanced SA-FastRed-method. Slides were analysed by automated picture analysis with the ACIS I and ACIS II systems (ChromaVision). Plastic adherent cells (1x106) from culture passage one from 71 breast cancer patients were analysed for expression of CK19, MUC1 and EpCAM by FACS analysis. Trypsinized cells (n=100) were lysed and used for mRNA isolation and c-DNA synthesis (Qiagen®). A real-time quantitative RT-PCR approach (MESA FAST SYBR Assay, Eurogentec®) and primers selected from the UniversalProbeLibrary system (Roche AG®) was used for the determination of the markers EpCAM, survivin, CD144, CD146, CD 105, and CD271. Results: Small cells characterized by an excentrically located small and clear nucleus were detected in 56% (486/866) of the bone marrow samples in a mean frequency of 1-2 cells per slide. However a subgroup of patients is characterized by elevated MSC levels. More than 5 MSC cells per 106 BM cells were detected in 19,7% (96/486) samples of the DTC negative group with elevated MSC levels in 5,2% NO, 6% N+ and 9% T3/T4 tumors. By molecular expression analysis of 71 bone marrow samples 8 probes (11%) were CK19 positive. Corresponding MSC cultures had significantly elevated expression levels of the markers vimentin, survivin, CD144, CD105, CD146 and CD271. Discussion: We have identified CK positive bone marrow stroma cells in 56% of patients with primary breast cancer. Immunobead RT-PCR based on 107 BM cells can be used to eliminate this background and to increase DTC-sensitivity from 2 to 11%. Frequency and expression profile of MSC in these probes may give valuable additional information to understand in the complex interrelation of tumor cells with tumor-associated mesenchymal stromal cells, erythroblasts and endothelial cells in the process of metastasis. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-13-07.