1075 Background: Rapidly self-renewing stroma cells of the bone marrow (RS cells) are MUC-1 and cytokeratin positive at a rate of 56% (475/850). Elimination of RS cells by size exclusion results in a positivity rate of 2.6% (36/1407) in primary breast cancer patients. We have used preanalytical immunomagnetic tumor cell enrichment followed by molecular detection of CK19 and Mammaglobin1 (MA) transcripts for an independent verification of the bright-field immunocytology results. Material and Methods: Mononuclear cells (2x106) from bone marrow of 1,407 patients with primary breast cancer were analysed by standardized cytospin immunocytology techniques. Disseminated tumor cells (DTC) were stained with CK8/18/19 antibodies 5D3 and A45-B/B3 using either the APAAP technique (Dako Techmate 500) or the enhanced SA-FastRed method (Ventana). Parallel DTC consisted of immunomagnetic tumor cell selection of 1x107 BM cells using the cell membrane glycoproteins EpCAM and MUC-1 as targets. CK 19 and MA transcripts were determined by conventional and quantitative real time RT-PCR. Results: Using acetic acid and periodate blocking steps before immunocytology 36 from 1407 (2.6%) breast cancer patients operated between 1999 and 2005 at the University Hspital Heidelberg were scored DCT positive using computer assisted automated (Chromavision ACIS II) picture analysis. From this group 186 bone marrow samples were used for molecular tumor cell detection. By RT-PCR analysis of 1x107 immunomagnetically purified BM cells only 6 from 186 (3.2%) samples were CK19 and MG positive. Discussion: Detection rates of tumor cells in bone marrow from non-metastatic breast cancer patients has been reported to be in the range of 0% to 100%. We show here that proliferating bone marrow stroma cells of the RS type are positive for various antigens including CK polypeptides 7, 8, 18 and 19. Elimination of these cells by size exclusion results in a tumor cell detection rate of 2,6% in 1,407 BM probes. This low detection rate could be verified in 186 BM probes by CK19 and mammaglobin1 RT-PCR. We conclude that standard immunocytology in bone marrow is severely impaired by cytokeratin expression of RS-type stroma cells. No significant financial relationships to disclose.
Circulating tumor cell detection and transcriptomic profiles in early breast cancer patients