Quantitative multimarker analysis in circulating tumor cells of patients with breast cancer

Abstract

1021 Background: The purpose of this study was clinical application of new methods for quantitative gene expression analysis of circulating tumor cells (CTC) in breast cancer patients. Methods: The high affinity antibodies BM7 (MUC-1) and VU1D9 (EpCAM) were used for immunomagnetic tumor cell enrichment in 10 mL of peripheral EDTA-blood of patients with primary breast cancer (n=167) and metastatic disease (n=131). Separated cells were lysed and used for mRNA isolation and c-DNA synthesis. The breast carcinoma- associated markers MUC1 and EpCAM were determined by conventional RT-PCR. The markers cytokeratin 19 (CK19), mammaglobin 1, survivin, HER-2, CXCR4 and prostate-specific ets factor (PSE) were amplified by real-time quantitative RT-PCR using primers and FAM-labeled TaqMan probes selected with the UniversalProbeLibrary system (Roche AG, Basel, CH). The β-actin transcript served as internal process quality control. Specificity of the RT-PCR was confirmed by examination of blood of healthy donors. Results: Sensitivity for every single transcript was adjusted to 2 tumor cells per 5 ml blood. Tumor-associated transcripts were detected in 31 of 167 (18.5%) patients with primary breast cancer. The marker with the highest incidence of 81% was MUC1. Patients with metastatic disease (n=131) had a positivity rate of 56% and were characterized by multiple marker expression. During a 12 (9–15) months follow-up of 81 patients with primary breast cancer, multimarker positivity was determined in 10 patients, and in three of these patients early metastasis was clinically confirmed. In patients with metastatic disease the CK19 positive CTCs co-expressed either survivin, PSE, CXCR4 or HER2 in more than 70% of the cases. Conclusion: Using a combination of preanalytical immunomagnetic tumor cell enrichment, followed by a multigene quantitative RT- PCR approach, we describe a sensitive detection system for rare circulating breast carcinoma cells. By quantitative expression analysis, survivin, PSE, CXCR4 and HER2 together with CK19 were identified as reliable markers for early detection of metastasis and for prediction of risk of recurrence. The marker panel can be extended further to match patients to best drugs and to predict treatment benefits. No significant financial relationships to disclose.