Abstract
Enumeration of circulating tumor cells (CTCs) may reflect the metastatic potential of breast cancer (BC). By using the DEPArray, we investigated CTCs with respect to their epithelial-to-mesenchymal transition phenotype and compared their genomic heterogeneity with tissue biopsies. Seventeen stage IV BC patients were enrolled. Pre-enriched CTC suspensions were stained with fluorescent-labeled antibodies to epithelial (E) and mesenchymal (M) markers. CTC samples were processed by DEPArray system and clustered in relation to their markers. DNA from CTCs, as well as from primary tumor samples, was sequenced by next generation sequencing to assess the mutational state of 50 major cancer-related genes. We identified four different CTC subsets that harbored different gene variants. The most heterogenous CTC subsets included the M+/E− phenotype, which, however, expressed only 7 repeatedly mutated genes, while in the M−/E+ subset multiple mutations affected only 2 out of 50 genes. When matching all gene variants among CTC subsets, a small number of mutations was shared by only 4 genes, namely ATM, FGFR3, PIK3CA, and TP53 that, however, were absent in primary tumors. Our results postulate that the detected mutations in all CTC subsets may be considered as genomic markers of metastatic dissemination to be investigated during early stages of BC.
Highlights
Enumeration of circulating tumor cells (CTCs) may reflect the metastatic potential of breast cancer (BC)
Concerning the histologic patterns, in both groups we found a variable occurrence of all tumor grading (G1-G3) as well as of Ki67 expression, whereas in addition to the variability of hormonal receptors, Her-2neu was detected in a single patient from the treatment-naïve group (A: pt. #407) and in two pre-treated patients (B: pt. #253; pt. #399)
Recent evidence suggests that a variable proportion of epithelial cell adhesion molecule (EpCAM)-negative BC cells can be detected in the peripheral blood of patients, in relation to the expression of epithelial-to-mesenchymal transition (EMT) markers, providing more information for metastatic evolution and management of the disease[12,13,14,15,21,41]
Summary
Enumeration of circulating tumor cells (CTCs) may reflect the metastatic potential of breast cancer (BC). Since BC cells usually metastasize and undergo the epithelial-to-mesenchymal transition (EMT), which implies the loss of epithelial (E) morphologic and molecular pattern and the acquisition of mesenchymal (M) markers[11], the correlation between the EMT functional status of CTCs and BC clinical outcome has been extensively investigated. In this regard, the expression of M markers emerged as an adverse prognostic factor, whereas the variations of M CTC counting during treatments, as increase or decrease, correlated with worsening of the metastatic disease www.nature.com/scientificreports or response to treatment, respectively[12,13,14]. The genomic assessment of CTCs needs standardization of methods as well as the comparison of relative molecular variations with other genomic sources as DNA from the primary tumor
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