Interestingly, phospho-PI3K and phospho-Akt expression levels were similar at 88% (14 out of 16) and 81% (13 out of 16), respectively, in circulating tumor cells of patients with early and metastatic disease. early and metastatic disease. Phospho-EGFR was observed in circulating tumor cells of two (33%) early and six (86%) metastatic EGFR-positive patients. Immunomagnetic separation of peripheral blood mononuclear cells, using EpCAM antibody, and subsequent double-staining experiments of circulating tumor cells showed that EGFR was co-expressed with HER2, phospho-Akt and phospho-PI3K kinases, indicating activation of the corresponding survival signaling pathway. Conclusions Our findings demonstrate that circulating tumor cells express receptors and activated signaling kinases of the EGFR/HER2/PI3K/Akt pathway, which could be used as targets for their effective elimination. Introduction Circulating tumor cells (CTCs) have been identified in the blood of patients bearing a wide range of malignancies [1,2], but not in healthy individuals or in patients with nonmalignant diseases [1]. CTCs have also been identified in significant proportions of patients with both early and metastatic breast cancer, and their presence carries significant prognostic information [3,4]. Indeed, the detection of CTCs before adjuvant chemotherapy as well as the persistence of CTCs after the completion of systemic adjuvant treatment is associated with an unfavorable RO4987655 clinical outcome [4-6]. Similarly, in patients with metastatic disease, elevated CTC numbers before or soon after the initiation of chemotherapy is an indicator of poor prognosis [7,8]. The malignant nature of CTCs is supported by the presence of RO4987655 chromosomal alterations [9-12]. However, it appears that only a small proportion of CTCs are capable of forming overt tumor deposits [13]. The molecular characteristics of these cells may play an important role in their survival and could therefore be used to guide effective treatment strategies. Epidermal growth factor receptor (EGFR; human epidermal growth factor receptor [HER]1) is a member of the ErbB family of receptors that also includes HER2, HER3, and HER4. RO4987655 EGFR ligand binding induces the formation of homodimers and heterodimers [14,15] and triggers the activation of downstream signaling pathways, such as the phosphoinositide-3 kinase (PI3K)/Akt pathway (among others), which control cell proliferation, survival, and migration [16]. HER2 is the preferred partner for heterodimerization with the other members of ErbB family of receptors [17], and its over-expression has been reported to amplify EGFR signaling [18]. EGFR and ligands such as transforming growth factor- and amphiregulin are over-expressed in a large subset of primary breast carcinomas [19,20]. Co-expression of these factors in breast cancer confers poor prognosis and resistance to hormonal therapy [21]. Moreover, inappropriate activation [22] or over-expression [23] of EGFR was associated with poor patient outcome. Recent studies have reported expression of growth factor receptors on CTCs of patients with breast and prostate cancer [15,24-26]. However, little is known about the presence of activated receptors and downstream signaling kinases that regulate pro-survival pathways in the CTCs of breast cancer patients. The objective of this study was to investigate whether EGFR and phosphorylated EGFR are expressed on CTCs isolated from Rabbit Polyclonal to RAB31 the blood of patients with breast cancer. The expression of HER2 and the activation status of PI3K and Akt kinases operating downstream of EGFR were also evaluated in adjuvant as well as in metastatic settings. Materials and methods Patient samples and cytospin preparation A total of 38 patients with detectable cytokeratin (CK)-19 mRNA positive cells [27,28] in peripheral blood were screened using immunofluorescence, and 32 (84.2%) of them with early (n = 16) and metastatic (n = 16) breast cancer who were found to harbor occult tumor cells were enrolled in RO4987655 the study. In addition 20 female normal blood donors were included in the study as negative control individuals. Specifically, peripheral blood (10 ml in EDTA) was obtained before the initiation of RO4987655 adjuvant treatment (usually 3 to 4 4 weeks after primary surgery) or first-line chemotherapy for metastatic disease. All blood samples were obtained at the middle of vein puncture after the first 5 ml of blood was discarded. These precautions was undertaken in order to avoid contamination of the blood sample with epithelial cells from the skin during sample collection. All patients gave their informed consent to participate in the study, which has been approved by the Ethics and Scientific Committees of our institution. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque.