Gene Expression Profiling of Circulating Tumor Cells in Breast Cancer

Abstract

BACKGROUND Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5–200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples. RESULTS GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples. CONCLUSIONS Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.

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

“…In this regard, Mostert et al developed a 16 gene profile assay based on qPCR mRNA expression quantification, distinguishing patients showing no response to therapy or dying in less than nine months after start of first-line systemic therapy from those with a better prognosis [58]. Moreover, Fina et al compared CTC counts with gene expression patterns based on the PAM50 gene panel in CTC from seven MBC patients resulting in no correlation between CTC number and gene expression levels as well as molecular subtype specific differences [41]. …”

“…PIK3CA, AKT2, TWIST1, ALDH1, EPCAM, ERBB2, MUC1 and EGFR, respectively [14, 36–40]. Studies using a multimarker qPCR panel to follow up patients during the course of disease have rarely been published up to now [41]. …”

Signatures of breast cancer metastasis at a glance

“…When coupled to high-throughput microarray or RNAsequencing technologies and unbiased bioinformatics analyses, the aforementioned obstacles are significantly surmounted (Curtis, 2015;Magbanua and Park, 2014). Overall, these new-generation studies have allowed single-cell and bulk-cell analyses of CTCs, which efficiently capture the transcriptional heterogeneity and identify the molecular portraits of metastasizing cells (Ascolani et al, 2015;Cassatella et al, 2012;De Mattos-Arruda et al, 2013;Fina et al, 2015;Lang et al, 2015;Polzer et al, 2014;Powell et al, 2012). For example, one study has demonstrated that CTCs express various biomarkers and mediators of EMT (Yu et al, 2013), a phenotypic change crucial for the completion of the metastatic cascade (Bill and Christofori, 2015;Hanahan and Weinberg, 2011;Kalluri, 2009;Kalluri and Weinberg, 2009;Radaelli et al, 2009).…”

Sulfatase 2 facilitates lymphangiogenesis in breast cancer by regulating VEGF-D

“…In recent years more research oncologists are becoming interested in the mechanisms of tumor-induced lymphangiogenesis in various tumors (19,20). Breast cancer is one of the most common types of cancer among women worldwide (21,22). Over 50% of early-stage breast cancer patients have local lymph node metastasis (18).…”