Abstract Background: We present a highly specific immunomagnetic separation technique in combination with an improved multimarker gene panel for molecular identification and characterization of circulating breast tumor cells in blood. Here we describe the creation of a new composite score in order to manage results and avoid false positives. Methods: Blood from breast cancer patients with primary or metastatic disease was drawn into two 10mL EDTA-tubes. The high affinity antibodies BM7 (MUC1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment. Separated cells were lysed and used for mRNA isolation and c-DNA synthesis (Qiagen®). A real-time quantitative RT-PCR approach using MESA FAST SYBR Assay (Eurogentec®) and primers selected from the UniversalProbeLibrary system (Roche AG®) for the epithelial markers cytokeratin 19 (CK19), mammaglobin 1 (MG1), epithelial cell adhesion molecule (EpCAM), baculoviral IAP repeat-containing 5 (Sur), immunosuppressive CD276 (CD276), carcinoembryonic antigen-related cell adhesion molecule 5 (CEA), HER-2, aldehyde dehydrogenase 1 family, member A1 (ALDH1), hypoxia inducible factor (HIF-1alpha) and CD44 molecule (CD44) was used for tumor cell identification and characterization. The ß-actin transcript was used for internal control and matched calibrator probes containing 2 or 10 tumor cells were used for quantitative expression analysis of tumor associated genes present in blood.Results: Positivity rate was based on a score that consists of 2 different characteristics: Marker detection (ranking according to the number of positive markers, varies from 0 – negative - to 1 – positive - for each marker) in combination with the marker expression level (ranking according to the C(t) values and varies from 0 – less then 2 tumor cells – to 1 – more then 2 tumor cells). This composite score is based on the expression of CK19, MG1, EpCAM, Sur and CD276 and has a ranking from 0 to 10. Negative cases are classified with score 0 and 1, cases ranked with score 2 need retesting and finally scores above 2 indicate clearly positive patients. The additional surrogate markers CEA, HER-2, ALDH, HIF and CD44 are analysed in positive patients in order to obtain further information. In our group of metastatic breast cancer patients, 53% were classified as positive, 42% of the patients were negative and in 5% a retesting is needed. Conclusion: The results of the composite score clearly show an increase of sensitivity and specificity for this assay. The implementation of this test in the routine monitoring of patients should help us to evaluate treatment response and create individualized treatment schedules. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3015.