Circulating tumor cells (CTCs) are cancer cells that are released from a tumor into the bloodstream. The presence of CTCs in peripheral blood has been associated with metastasis formation in patients with breast cancer. Therefore, the molecular characterization of CTCs may improve diagnostics and support treatment decisions.We performed gene expression profiling to evaluate the enriched CTCs and peripheral blood mononuclear cells (PBMCs) of breast cancer patients using an expression panel of 55 breast cancer-associated genes. The study revealed several significantly differentially expressed genes in the CTC-positive samples, including a few that were exclusively expressed in these cells. However, the expression of these genes was barely detectable in the PBMC samples. Some genes were differentially expressed in PBMCs, and the expression of these genes was correlated with tumor grade and the formation of metastasis.In this study, we have shown that the enriched CTCs of breast cancer patients overexpress genes involved in proteolytic degradation of the extracellular matrix (ECM) as well as genes that play important roles in the epithelial-mesenchymal transition (EMT) process that may occur in these cells.

Receptor for advanced glycation end products (RAGE) plays a central role in the regulation of tissue homeostasis, regeneration and resolution of inflammation, but under pathological conditions RAGE-mediated pathways may induce diminished apoptosis, but enhanced autophagy and cell necrosis. These mechanisms may contribute to malignant transformation, cancer progression and metastases. Soluble RAGE may bind natural RAGE ligands and counteract some of the RAGE-mediated effects. Activation of RAGE was demonstrated in different types of cancer (including colon, pancreatic and breast cancer). Expression of RAGE and serum levels of soluble RAGE may serve as cancer biomarkers and strategies aimed at interfering with RAGE signaling might be promising anticancer drugs.

In conclusion, CTC detection may be a promising early marker of disease progression potentially enhancing the difficult therapeutic decisions. Further studies should, however, clearly demonstrate its utility for both the prediction of outcome and monitoring the effect of treatment.

BACKGROUND: Breast cancer (BC) patients still harbor a considerable risk of metastatic relapse caused by minimal residual disease (MRD) despite of complete removal of primary tumor. The aim of this study was to identify single disseminated tumor cells (DTC) in the bone marrow and circulating tumor cells (CTC) in the peripheral blood as potential biomarkers for therapy response monitoring and metastatic relapse risk prediction. Finally, the gene expression profiles of CTCs have been analyzed. METHODS: A total of 87 patients with diagnosed BC at stage I to III and 115 metastatic patients were enrolled into a prospective study between years 2008 - 2010. Peripheral blood (5ml) for CTC-detection was collected from primary BC patients before chemotherapy (CHT), after 2 cycles of CHT and after the CHT. Metastatic BC patients have been examined for CTCs before starting a new line of the treatment. Bone marrow aspirates from 16 premenopausal BC patients (mean age 31) with primary tumor were analyzed for the presence of DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3 (Epimet™, AS Diagnostik, Germany) before surgery. CTC detection in blood was performed by AdnaTest BreastCancerTM(AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC-lysates. cDNA from isolated CTCs has been further pre-amplified and used for multimarker qPCR gene expression profiling using Biomark® microfluidic 48x48 GE Dynamic arrays (Fluidigm, USA). qPCR results have been analyzed by GENEX vs. 5.2 software (MultiD Analysis). RESULTS: 286 CTC samples have been analyzed in total. The analysis has shown that the expression profiles of CTCs from primary BC patients have been significantly different comparing them to the CTC-profiles from metastatic BC patients for several of tested genes (e.g., CK19, GA7332, MLFIP1, SATB1, PTEN). Interestingly, before surgery of primary tumor DTCs were found in 5/16 patients (31 %) and CTCs in 7/16 (43 %). Both DTCs and CTCs together were found in 4/16 (25%) patients. In 18% of the primary BC patients no dissemination markers were found CONCLUSIONS: Information based on the CTCs-gene expression profiles could provide an additional support for therapy management. Metastatic potential of enriched CTCs will be further evaluated on the single cell level. This work has been supported by Grant Agency of Ministry of Health, Czech Republic (IGA NS9976) and Grant Agency of Charles University in Prague, Czech Republic no. 7709 and 59410. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-03-01.

breast cancer, disseminated tumor cells, circulating tumor cells, bone marrow aspiration, prognostic/predictive markers, therapy monitoring.

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