#305 The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a promising tool for risk stratification, treatment tailoring, and monitoring of the disease state. We previously developed a QPCR-based detection platform for the semi-quantitation of circulating tumor cells in the peripheral blood of breast cancer patients. This CTC assay combines gene expression data from 4 tumor marker genes into a single score using a quadratic discriminant analysis, and is optimized such that positivity indicates tumor cell presence, and negativity indicates tumor cell absence. An initial cohort of metastatic patients demonstrated that score positivity was correlated to a significantly shorter time to disease progression.
 The current study aimed to validate these results by using the CTC assay in an independent, retrospective patient series consisting of 111 non-metastatic stage I-III breast cancer patients (median followup 7.3 years), 16 metastatic breast cancer patients, and 28 healthy controls. Assay score positivity was observed in 14 out of 16 (88%) metastatic patients, 16 out of 111 (14%) non-metastatic stage I-III patients, and 0 out of 28 (0%) healthy female controls.
 In the early stage patient group, a positive CTC score was correlated to significantly poorer relapse-free survival (HR = 3.6, 95% CI = 1.7-7.9, p < 0.01) and overall survival (HR = 3.2, 95% CI = 1.6-6.1, p < 0.01). The assay was particularly powerful in patients with no lymph node metastases at time of diagnosis (relapse-free survival HR = 13.0, 95%CI = 3.0-55.7, p < 0.01, overall survival HR = 6.3, 95% CI = 1.9-21.1, p < 0.01). This platform compared very favorably to the traditional immunohistochemistry-based assay for disseminated tumor cell detection in bone marrow utilizing pan-anticytokeratin staining (AE1/AE3), which was not a significant predictor of survival in this patient group (HR = 1.3, 95% CI = 0.6-2.4, p = 0.49). In multivariate analyses, our CTC assay was a significant, independent predictor of overall survival.
 Work is currently ongoing to expand the study to include an additional 500-600 patients which should serve as a robust validation of the assay. This CTC detection platform represents an objective assay with both high specificity and prognostic power, and could be an effective clinical tool for outcome prediction in breast cancer patients.
 This work was supported by the Sixth Framework Program of the European Commission as part of the international DISMAL collaboration for research into disseminated epithelial malignancies. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 305.