In patients with metastatic breast cancer the identification of circulating tumor cells in epithelial-to-mesenchymal transition is associated with a poor prognosis

Abstract

BackgroundAlthough recent models suggest that the detection of Circulating Tumor Cells (CTC) in epithelial-to-mesenchymal transition (EM CTC) might be related to disease progression in metastatic breast cancer (MBC) patients, current detection methods are not efficient in identifying this subpopulation of cells. Furthermore, the possible association of EM CTC with both clinicopathological features and prognosis of MBC patients has still to be demonstrated. Aims of this study were: first, to optimize a DEPArray-based protocol meant to identify, quantify and sort single, viable EM CTC and, subsequently, to test the association of EM CTC frequency with clinical data.MethodsThis prospective observational study enrolled 56 MBC patients regardless of the line of treatment. Blood samples, depleted of CD45pos leukocytes, were stained with an antibody cocktail recognizing both epithelial and mesenchymal markers. Four CD45neg cell subpopulations were identified: cells expressing only epithelial markers (E CTC), cells co-expressing epithelial and mesenchymal markers (EM CTC), cells expressing only mesenchymal markers (MES) and cells negative for every tested marker (NEG). CTC subpopulations were quantified as both absolute cell count and relative frequency. The association of CTC subpopulations with clinicopathological features, progression free survival (PFS), and overall survival (OS) was explored by Wilcoxon-Mann-Whitney test and Univariate Cox Regression Analysis, respectively.ResultsBy employing the DEPArray-based strategy, we were able to assess the presence of cells pertaining to the above-described classes in every MBC patient. We observed a significant association between specific CD45neg subpopulations and tumor subtypes (e.g. NEG and triple negative), proliferation (NEG and Ki67 expression) and sites of metastatic spread (e.g. E CTC and bone; NEG and brain). Importantly, the fraction of CD45neg cells co-expressing epithelial and mesenchymal markers (EM CTC) was significantly associated with poorer PFS and OS, computed, this latter, both from the diagnosis of a stage IV disease and from the initial CTC assessment.ConclusionThis study suggests the importance of dissecting the heterogeneity of CTC in MBC. Precise characterization of CTC could help in estimating both metastatization pattern and outcome, driving clinical decision-making and surveillance strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0687-3) contains supplementary material, which is available to authorized users.

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

“…In patients with advanced non-small cell lung cancer, the majority of isolated or clustered CTCs harbor a dual epithelial/mesenchymal phenotype, and only some CTCs express exclusively vimentin or fibronectin [140] . Importantly, a fraction of cells coexpressing epithelial and mesenchymal markers was associated with poorer PFS and OS in MBC [142] . In lung cancer patients it was shown that radiotherapy can induce release of single CTCs or CTC clusters [143] .…”

Critical issues in the clinical application of liquid biopsy in non-small cell lung cancer

“…On the other side, the EpCAMlow/− fraction of CTCs alone has not been shown to correlate with patient outcome (53). Consistently, in other types of metastatic tumors, mesenchymal CTCs alone have been only weakly associated with OS and PFS (78). All together, these data suggest that a complementary dual technology, detecting both epithelial and mesenchymal CTCs might allow to obtain a much clear picture of the potential of these cells in predicting patient outcome (18).…”

Liquid biopsy - emergence of a new era in personalized cancer care

“…In order to circumvent the issue of heterogeneous expression of EpCAM on tumor cells, Bulfoni and group adopted a CTC enrichment strategy based on red blood cell lysis followed by the immunomagnetic depletion of leukocytes from blood samples (i.e., a negative selection) and subsequently stained the recovered cells with a cocktail of antibodies recognizing epithelial and mesenchymal markers and sorted them by multiparametric fluorescence. They found presence of CTCs co-expressing epithelial and mesenchymal markers (EM CTC) were significantly associated with poorer progression free survival and overall survival [132]. Zhang et al [133], used anti-CD45 antibodies to deplete CD45 positive cells and enrich CTCs in blood collected from pancreatic cancer patients followed by immune-staining of CK and CD45, DAPI and fluorescence in situ hybridization with the centromere of chromosome 8 (CEP8) probe to identify CTCs in 31 cases of pancreatic cancers, and 30 healthy individuals.…”