Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer.Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule-based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors.Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels.Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs.

Detection of circulating tumor cells (CTCs) in whole blood from metastatic cancer patients by the CellSearch TM CTC Test (Veridex LLC, Warren, NJ, USA) has been shown to have clinical relevance. In addition to enumeration, there is great interest in molecular characterization of these CTCs. We aimed to establish a robust method to perform mRNA expression analysis of multiple genes by a real-time reverse transcriptase (RT)-PCR on small numbers of CTCs enriched from whole blood by the CellSearch TM system. Despite the 4 log depletion of leukocytes after CellSearch enrichment, the CTC-enriched fractions still contained leukocytes, in particular B-lymphocytes, which severely interfered with our CTC-specific gene expression profiling. After extensive washing and leukocyte-specific depletion by anti-CD45 coated magnetic beads prior to CellSearch TM enrichment, the number of leukocytes present in the enriched fraction was still high (range 60-929). However, by using a set of genes with no or minor expression by leukocytes, we succeeded to perform quantitative gene expression profiling specific for as little as one breast cancer CTC present in a CTC-enriched environment typically containing over 800 contaminating leukocytes. Our method allows molecular characterization specific for as little as one CTC, and can be used to expand the understanding of the biology of metastasis and, potentially, to improve patient management.

Cathepsin-D is a lysosomal aspartyl protease which is expressed in all tissues. In breast cancer, it was first identified as a 52-kDa oestrogen-regulated secretory glycoprotein with autocrine mitogenic activity (Westley and Rochefort, 1979, 1980;Vignon et al, 1986;Rochefort, 1994;Westley and May, 1996). In oestrogen receptor (ER)-positive breast cancer cells, its gene transcription is increased by oestrogen and growth factors, whereas in ER-negative breast cancer cells it is constitutively expressed by an unknown mechanism. Many biological roles have been attributed to cathepsin-D (reviewed by Westley and May, 1996), including, among others: degradation of the extracellular matrix (Briozzo et al, 1988), increasing cells' malignant phenotype and metastatic potential (Garcia et al, 1990), stimulation of (metastatic) cell proliferation by increasing the local bioavailability of growthstimulatory growth factors (Briozzo et al, 1991;Conover and De Leon, 1994), inactivation of a growth inhibitor (Liaudet et al, 1995), and prevention of apoptosis (Saftig et al, 1995).In patients with primary breast cancer, overexpression of cathepsin-D was found to be related to a poor prognosis (Thorpe et al, 1989;Spyratos et al, 1989), in analogy with observations made with the serine protease urokinase-type plasminogen activator (uPA), of which increased activity (Duffy et al, 1988) and antigen level (Jänicke et al, 1990) have been shown to be associated with a poor prognosis. The initial studies of Thorpe et al (1989) andSpyratos et al (1989), using quantitative immunoassays (enzyme-linked immunosorbent assay, immunoradiometric assay) to assess cytosolic cathepsin-D levels, have been confirmed by many others employing the same technique (reviewed by Rochefort, 1994;Westley and May, 1996). However, utilization of other methods to assess cathepsin-D status, i.e. Western blotting and immunohistochemistry, resulted in discrepant results (Henry et al, 1990;Tandon et al, 1990;Domagala et al, 1992, Isola et al, 1993Ravdin, 1993, Ravdin et al, 1994Westley and May, 1996). These conflicting results have been attributed to the use of different antibodies without standardized quantification (Cardiff, 1994; Rochefort, 1996, Westley andMay, 1996), a problem which is not encountered when quantitative immunoassays on cytosolic extracts are used (Benraad et al, 1992).Notwithstanding the drawbacks of immunohistochemistry and contrasting data (Cardiff, 1996;Emmert-Buck, 1996; Rochefort, 1996, Westley andMay, 1996), there is evidence that the expression of cathepsin-D by host stromal cells (Têtu et al, 1993;Joensuu et al, 1995;O'Donoghue et al, 1995;Nadji et al, 1996), or the cancer cells (Isola et al, 1993), is associated with prognosis. It has been suggested that measuring total cathepsin-D levels in tumour extracts (comprising tumour cells and host cells) has no practical value (Nadji et al, 1996). This firm conclusion drawn from data obtained from only 154 patients is surprising. In contrast to conflicting data obtained with immunohistochemistry,...