No clinical relevance of standardized immunocytological and molecular methods for detection of disseminated tumor cells in bone marrow of patients with primary breast cancer.

Abstract

Abstract Abstract #306 Background: Bone marrow samples from 2093 patients operated for primary breast cancer (stage T1-4, N0/N+, M0/M1) at the University Hospital Heidelberg between 1999 und 2007 were analysed for disseminated tumor cells (DTC) by standard cytology on cytospin slides. In parallel a subgroup of 250 samples was analysed by immunobead-RT-PCR methods for CK19 expression.
 Material and Methods: Mononuclear cells were enriched by Ficoll density-gradient centrifugation. Cytospin-slides (mean 6) with 1x106 BM-cells were fixed in 3.5% buffered formalin at RT and endogenous AP and Ig were blocked with 10% acetic acid and 2.28% NaJO4. DTC were identified by CK antibodies 5D3 and A45-B/B3 (automated immunostainers, AP-techniques). Slides were analysed by automated picture analysis with the ACIS I and II systems (ChromaVision). Criteria for classification of disseminated tumor cells were in compliance with the publication of Fehm et al. (Cancer, Vol.107, 2006). 1x107 BM cells were used for immunomagnetic enrichment of epithelial cell types using antibodies targeting the cell membrane glycoproteins MUC1 and EpCAM. The markers cytokeratin 19 (CK19) and MG1 were analysed by real-time qRT-PCR using primers and FAM-labeled TaqMan probes of the UniversalProbeLibrary system (Roche AG, Basel, CH). The newly developed embedded tumor cell calibrator (ETC) technique was introduced for cell quantification.
 Results: Small cells characterized by an excentrically located small and clear nucleus were detected in 56% of the bone marrow samples. These cytokeratin-positive cells were identified as bone marrow stroma stem cells. Identical cell types were present in 27 samples from normal BM-donors. In contrast, tumor cells were characterized by strong and irregular cytoplasmic cytokeratin staining, a granular nucleus and a clearly enlarged nuclear size. These DTC had a mean value for the parameter area of 309 (n=13) according to the ACIS picture analysis. Using published tumor classification critera 40 (1.9%) from 2093 patients with primary breast carcinoma were scored DTC positive. At the basis of 2x106 BM-cells the molecular detection methods of a) endpoint RT-PCR for CK19 and b) real-time RT-PCR for CK19 and MG1 had positivity rates of 3.5% and 3.8%, respectively.
 Discussion: We have shown that bone marrow stroma cells are positive for various epithelial antigens including CK. Elimination of these cells either by size exclusion in picture analysis or by immunomagnetic separation results in tumor cell detection rates of less than 4% in 2x106 bone marrow cells. We conclude that cytological DTC analysis and the technique of immunomagnetic enrichment followed by molecular CK19 detection have no clinical relevance in patients with primary breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 306.