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Metastasis describes the process of migration of a frequently clinically occult circulating tumor cell (CTC) from the primary lesion to a new location and the subsequent formation of an overt growth. We and others have shown that the detection and quantitation of these cells has significant prognostic value, however there still remains no consensus as to the optimal methods to achieve this. The work described herein therefore considered various techniques, from storage and sample processing to data acquisition and analysis, to find an optimal combination of methods for an effective and practical platform for the detection of CTCs in peripheral blood. A dual-antigen epithelial cell enrichment procedure followed by a multi-marker QPCR analysis demonstrated the highest sensitivity and specificity, with the ability to detect as few as 10 tumor cells from a background of 10(6) peripheral blood mononuclear cells. Using these techniques in conjunction with a quadratic linear discriminant analysis (QDA) resulted in a platform able to generate this data and then combine it a single score for each patient, in which positivity reflected tumor cell presence, and negativity represented tumor cell absence. This assay was able to correctly determine tumor cell presence or absence in 100% of healthy controls and 84% of metastatic patients in a validation cohort of 39 individuals. This platform represents a highly sensitive and specific assay which could augment current routine assays for CTCs in the clinic.

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#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.

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 platform for the detection of CTCs in peripheral blood, which proved to be prognostic in a cohort of metastatic breast cancer patients.In the current study, we aimed to build on this work by determining the prognostic value of our CTC assay in an independent, prospectively collected, retrospective patient series of 776 non-metastatic, stage I-III breast cancer patients (median follow-up 7.3 years). Assay positivity was observed in 83 patients (10.7%), while maintaining 96% specificity in a group of 25 healthy female controls (however a second blood sample from the healthy control that gave a positive result was found to be negative). CTC positivity as determined by our assay corresponded to a significantly poorer relapse-free survival (RFS) (HR = 1.9, 95% CI = 1.3-2.9, p = 0.02) and overall survival (HR = 1.8, 95% CI = 1.1-2.8, p = 0.02) in this group. This platform demonstrated similar prognostic power to the pan-anticytokeratin (AE1/AE3) immunohistochemistry-based detection of disseminated tumor cells (DTCs) in the bone marrow of these patients (RFS HR = 1.9, 95% CI = 1.3-2.7, p = 0.02). Importantly, multivariate analyses including tumor size, histological grade, lymph node status, and hormone receptor status, both CTC and DTC status remained significant, independent predictors of RFS (CTC status HR = 1.6, 95% CI = 1.0-2.6, p = 0.037; DTC status HR = 1.6; 95% CI = 1.1-2.5, p = 0.023). However, CTC status was prognostic in both lymph node negative patients (HR = 2.0, p = 0.04) and lymph node positive patients (HR = 1.7, p = 0.043), whereas DTC status was only prognostic in lymph node positive patients (HR = 2.0, p = 0.002).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(24 Suppl):Abstract nr 3002.