Abstract
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. 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. 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. 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.
Highlights
Our study shows that molecular profiling of low numbers of Circulating tumor cells (CTC) in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs
We show for the first time the feasibility of extensive molecular characterization of CTCs at both the mRNA and microRNA level in a high background of leukocytes and show its applicability in a cohort of 50 metastatic breast cancer patients
To select the gene transcripts, we used the approach described in detail in the Materials and Methods section, resulting in 43 putative breast CTC–specific miRNAs, 85 putative breast CTC–specific mRNAs, and 5 control mRNAs
Summary
Molecular characterization of primary tumors has already greatly contributed to the personalized treatment of cancer. Combined with classical tumor characteristics, these models are increasingly used to guide individualized treatment of patients, thereby aiming to avoid over- or undertreatment. Most of these prognostic and predictive models have been developed based on primary tumor tissue, whereas metastases, rather than the primary tumor, determine the clinical outcome of cancer patients. It is anticipated that molecular characterization of metastases will improve the currently available prognostic and predictive models. Taking biopsies from metastases in patients, is an invasive and often painful procedure, and frequently impossible due to the lack of accessible lesions
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