Purpose: To investigate the prognostic value of the molecular detection of circulating tumor cells (CTCs) using three markers [cytokeratin 19 (CK19), mammaglobin A (MGB1), and HER2] in early breast cancer. Experimental Design: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected using real-time (CK19) and nested (MGB1 and HER2) reverse transcription-PCR in the peripheral blood of 175 women with stage I to III breast cancer before the initiation of adjuvant chemotherapy. The detection of CTCs was correlated with clinical outcome. In 10 patients, immunofluorescence staining experiments were done to investigate the coexpression of cytokeratin, MGB1, and HER2 in CTCs. Results: CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells were detected in 41.1%, 8%, and 28.6% of the 175 patients, respectively. Patients had one of the following molecular profiles: CK19mRNA+/MGB1mRNA+/HER2 mRNA+ (n = 8), CK19mRNA+/MGB1mRNA+/ HER2mRNA-(n = 1), CK19mRNA+/MGB1mRNA-/HER2mRNA+ (n = 42), CK19mRNA+/ MGB1mRNA-/HER2mRNA-(n = 21), CK19mRNA-/MGB1mRNA+/HER2mRNA-(n = 5), and CK19mRNA-/MGB1mRNA-/HER2mRNA-(n = 98). Double-immunofluorescence experiments confirmed the following CTC phenotypes: CK+/MGB1+, CK+/MGB1-, CK-/MGB1+, CK+/ HER2+, CK+/HER2-, MGB1+/HER2-, and MGB1+/HER2+. In univariate analysis, the detection of CK19mRNA+, MGB1mRNA+, and HER2mRNA+ cells was associated with shorter diseasefree survival (DFS; P < 0.001, P = 0.001, and P < 0.001, respectively), whereas the detection of CK19mRNA+ and MGB1mRNA+ cells was associated with worse overall survival (P = 0.044 and 0.034, respectively). In multivariate analysis, estrogen receptor^negative tumors and the detection of CK19mRNA+ and MGB1mRNA+ cells were independently associated with worse DFS. Conclusion: The detection of peripheral blood CK19mRNA+ and MGB1mRNA+ cells before adjuvant chemotherapy predicts poor DFS in women with early breast cancer.

The aim of the present study was to decrease the incidence of false positives and to better characterize marginally cytokeratin-19 (CK-19) mRNA positive peripheral blood samples from patients with early stage breast cancer. A new set of highly specific primers for CK-19, which avoids amplification of contaminating genomic DNA, was designed and evaluated to improve the specificity and sensitivity of the previously described methodology. The primers were specifically designed to avoid amplification of contaminating genomic DNA and CK-19 pseudogenes. The breast cancer cell line MCF-7 was used as positive control for the development and analytical evaluation of the assay, while peripheral blood samples from 62 healthy female individuals and 160 patients with early breast cancer were used for the evaluation of the sensitivity and specificity of the new primer pair. The novel designed primer pair was highly sensitive, as it detects up to 1 MCF-7 cell, and specific as none of the healthy individuals had detectable CK-

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Supplementary Figures S1-S2 from Prognostic Value of the Molecular Detection of Circulating Tumor Cells Using a Multimarker Reverse Transcription-PCR Assay for Cytokeratin 19, Mammaglobin A, and HER2 in Early Breast Cancer