Establishment of a multimarker qPCR panel for the molecular characterization of circulating tumor cells in blood samples of metastatic breast cancer patients during the course of palliative treatment

Abstract

BackgroundCirculating tumor cells (CTC) are discussed to be an ideal surrogate marker for individualized treatment in metastatic breast cancer (MBC) since metastatic tissue is often difficult to obtain for repeated analysis. We established a nine gene qPCR panel to characterize the heterogeneous CTC population in MBC patients including epithelial CTC, their receptors (EPCAM, ERBB2, ERBB3, EGFR) CTC in Epithelial-Mesenchymal-Transition [(EMT); PIK3CA, AKT2), stem cell-like CTC (ALDH1) as well as resistant CTC (ERCC1, AURKA] to identify individual therapeutic targets.ResultsAt TP0, at least one marker was detected in 84%, at TP1 in 74% and at TP2 in 79% of the patients, respectively. The expression of ERBB2, ERBB3 and ERCC1 alone or in combination with AURKA was significantly associated with therapy failure. ERBB2 + CTC were only detected in patients not receiving ERBB2 targeted therapies which correlated with no response. Furthermore, patients responding at TP2 had a significantly prolonged overall-survival than patients never responding (p = 0.0090).Patients and Methods2 × 5 ml blood of 62 MBC patients was collected at the time of disease progression (TP0) and at two clinical staging time points (TP1 and TP2) after 8–12 weeks of chemo-, hormone or antibody therapy for the detection of CTC (AdnaTest EMT-2/StemCell Select™, QIAGEN Hannover GmbH, Germany). After pre-amplification, multiplex qPCR was performed. Establishment was performed using various cancer cell lines. PTPRC (Protein tyrosine phosphatase receptor type C) and GAPDH served as controls.ConclusionsMonitoring MBC patients using a multimarker qPCR panel for the characterization of CTC might help to treat patients accordingly in the future.

CTCs Expression Profiling for Advanced Breast Cancer Monitoring

“…Compared with previous articles [23,25,32,33], we reported a higher detection rate of epithelial, EMT, and stem markers expression on CTCs, probably due to the isolation method of choice. In addition, compared to the study of Aaltonen and colleagues, we observed discordances in the ER expression between the patient's solid tumor and CTCs [34], emphasizing the dynamic nature of tumors and the need for real-time monitoring in cancer patients.…”

The Landscape of Circulating Tumor Cell Research in the Context of Epithelial-Mesenchymal Transition

“…Cluster formation in cell culture was affected by the presence and duration of systemic therapy, and its persistence may reflect therapeutic resistance as it correlated with shorter OS [154] . Also gene expression profiles in CTCs were reported to predict response to therapy with aromatase inhibitors [155] and during the course of palliative treatment in MBC [156] .…”

Prognostic significance of a combined gene expression analysis in EpCAM(+) CTCs in metastatic breast cancer based on a long follow-up

“…Our group has shown the prognostic signi cance of CK-19 positive CTCs in peripheral blood of breast cancer patients (13,14). A variety of markers have been studied in CTCs at the RNA level (15)(16)(17) and there is some evidence that the expression of these markers can change during systemic treatment of MBC patients (18). Moreover, the gene signature of CTCs in breast cancer is correlated with the risk for brain metastasis (3).…”

“…In primary tumors, it has been shown that subpopulations of tumor cells that display the CD44 high /CD24 −/low pro le and are positive for aldehyde dehydrogenase 1 expression (ALDH1 + ) are of high tumorigenic potential (22,23). It is now known that the putative stem cell phenotype (CD44 + /CD24 − and/or ALDH1 + /CD24 − ) has been detected both in Disseminated Tumor Cells (DTCs) and CTCs (18,24), and that the tumor microenvironment regulates the plasticity of cancer stem cells through transition between epithelial to mesenchymal-like (EMT) and mesenchymal to epithelial-like (MET) states (25).…”