Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients

580 Background: The treatment of advanced breast cancer demands systemic therapies that can address disease heterogeneity and the development of treatment resistance without a “real-time” molecular window into disease biology. New technologies have focused on increased capture and molecular analysis of circulating tumor cells (CTCs) including cells undergoing epithelial mesenchymal transition (EMT). We conducted a pilot experiment to test the efficiency of capture and cytokeratin (CK) detection and the presence of single point variants (SNV) to determine the best utility of scoring alternatives for CTC. Methods: EpCAM expressing CTC were recovered from breast cancer patients using CellSearch (Veridex) and LiquidBiopsy (Cynvenio Biosystems). EpCAM recovery and CK scoring were indexed in spiked samples and in 12 inflammatory breast cancer (IBC) patient samples using antibodies against CKs 7, 8 or CKs 1-8, 10, 13-16, 18, 19. Additionally, LiquidBiopsy template was analyzed using an Ampliseq 1.0 panel on the IonTorrent PGM. SNV present in the CTC but not white blood cell (WBC) negative controls were identified and where possible, compared to tissue biopsy SNV analyzed using Foundation One (Foundation Medicine). Results: CTCs were detected using CellSearch 10/12 (83%) (range 0-2502 CTC/7.5ml) and LiquidBiopsy 12/12 (100%) (range 6-2800 CTC/7.5mL). More CK positive events were scored using CKs 1-8, 10, 13-16, 18, 19 than CKs 7, 8 in patient samples. Upon sequencing, shared germline polymorphisms were observed in CTC and WBC. Conversely, 1 or 2 SNV were detected in the Epcam selected population but not WBC controls from 6/12 patients (frequency 1.1%-2.1% with 520-5160x coverage) with SNV observed in TP53, MPL, PIK3ca, MET and IDH1. All but one of the PIK3ca mutations were absent in evaluable tissue biopsy. Conclusions: CTC recovery and scoring are two separate events. Altered CK detection emphasized the need to tailor CTC classification to specific disease settings. Sequence analysis showed one correlated SNV among 6 evaluable comparisons to tissue reflecting variable analysis as well as the biologic disparity of metastatic disease. This pilot demonstrates the feasibility of using CTC for molecular analysis.

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Heterogeneity of Stemlike Circulating Tumor Cells in Invasive Breast Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21082780 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2780 ◽

Cited By ~ 3

Author(s):

Olga E. Savelieva ◽

Liubov A. Tashireva ◽

Evgeniya V. Kaigorodova ◽

Angelina V. Buzenkova ◽

Rustam Kh. Mukhamedzhanov ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Epithelial Mesenchymal Transition ◽

Receptor Expression ◽

Stem Cell Markers ◽

Breast Cancer Patients ◽

Mesenchymal Transition ◽

Tumor Cell Heterogeneity ◽

Stromal Cell Derived Factor

The presence of stem and epithelial–mesenchymal-transition (EMT) features in circulating tumor cells (CTCs) determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. It also allows them to fulfil the role of metastatic “seeds”. We evaluated the heterogeneity of stem CTCs by their CD44, ALDH1, and CD133 expression depending on N-cadherin expression in breast-cancer patients. A total of 38 female patients were selected for this study. CTC phenotypes were determined by flow cytometry before any type of treatment. Multiplex immunofluorescence was used for the evaluation of tumor-cell heterogeneity in primary lesions. In patients who had CD44-CD24- CTCs, a subset of cells with the expression of other stem-cell markers (CD133 and ALDH1) were detected. Expression of CD133 and/or ALDH1 may be associated with expression of N-cadherin: all populations of N-cadherin+ CTCs demonstrate stem features; in the absence of N-cadherin expression, true nonstem (CD44-CD24-CD133-ALDH1-) cells are found. The heterogeneity of stem marker expression in CTCs was observed regardless of N-cadherin expression. In our study, stromal cell-derived factor-1 (SDF-1) receptor expression in CTCs did not depend on stemlike traits, but was instead associated with N-cadherin expression. Subpopulations of tumor cells, detected both in tumors and blood, were identified. Breast cancer was characterized by pronounced interpersonal and intrapersonal heterogeneity of CTCs by the presence and combination of various stem features and N-cadherin expression. To complete the characterization of stemlike features of CTCs, we suggest the simultaneous use of the three stem markers.

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Expression of Stem Cell and Epithelial-Mesenchymal Transition Markers in Circulating Tumor Cells of Breast Cancer Patients

BioMed Research International ◽

10.1155/2014/415721 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 54

Author(s):

Natalia Krawczyk ◽

Franziska Meier-Stiegen ◽

Malgorzata Banys ◽

Hans Neubauer ◽

Eugen Ruckhaeberle ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cell ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Epithelial Mesenchymal Transition ◽

Metastatic Breast ◽

Current Data ◽

Stem Cell Markers ◽

Breast Cancer Patients ◽

Mesenchymal Transition

Evaluation and characterization of circulating tumor cells (CTCs) have become a major focus of translational cancer research. Presence of CTCs predicts worse clinical outcome in early and metastatic breast cancer. Whether all cells from the primary tumor have potential to disseminate and form subsequent metastasis remains unclear. As part of the metastatic cascade, tumor cells lose their cell-to-cell adhesion and undergo epithelial-mesenchymal transition (EMT) in order to enter blood circulation. During EMT epithelial antigens are downregulated; thus, such tumor cells might elude classical epithelial marker-based detection. Several researchers postulated that some CTCs express stem cell-like phenotype; this might lead to chemoresistance and enhanced metastatic potential of such cells. In the present review, we discuss current data on EMT and stem cell markers in CTCs of breast cancer and their clinical significance.

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THE PRESENCE OF VARIOUS POPULATIONS OF CIRCULATING TUMOR CELLS IN THE BLOOD OF BREAST CANCER PATIENTS BEFORE TREATMENT: ASSOCIATION WITH FIVE-YEAR METASTASIS-FREE SURVIVAL

Siberian Journal of Oncology ◽

10.21294/1814-4861-2020-19-6-57-65 ◽

2020 ◽

Vol 19 (6) ◽

pp. 57-65

Author(s):

E. V. Kaigorodova ◽

N. A. Tarabanovskaya ◽

P. V. Surkova ◽

R. V. Zelchan ◽

E. Yu. Garbukov

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Survival Rate ◽

Cancer Patients ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Breast Cancer Patients ◽

Free Survival ◽

Mesenchymal Transition ◽

Different Populations

Localized and metastatic tumors are known to lead to the formation of circulating tumor cell (CTC ) clusters in the blood. Currently, there is a heightened interest in the study of molecular and biological characteristics of CTC s. Recent studies have shown the presence of different populations of CTC s in the blood of cancer patients. Some cells are cancer stem cells, some tumor cells undergo epithelial-mesenchymal transition (EMT), and most CTC s do not have features of either stem cells or EMT.The aim of the study was to evaluate the five-year metastasis-free survival rate in patients with invasive breast carcinoma, depending on the presence of various populations of circulating tumor cells in the blood before treatment.Material and Methods. A prospective study included 47 patients with newly diagnosed invasive breast cancer (T1–4N0–3M0), who were treated at Cancer Research Institute, Tomsk National Research Medical Center. The patients aged 31 to 69 years. The presence of different populations of CTC s in the blood of patients before treatment was determined by multicolor flow cytometry on the BD FACS Canto system, using different fluorochrome-labeled monoclonal antibodies to EpCam, CD 45, CD 44, CD 24, and N-cadherin. Five-year metastasis-free survival was evaluated by the Kaplan–Meier method. The differences were considered significant at p<0.05.Results. The results obtained demonstrated that the presence of both stem-like and non-stem CTC s showing signs of EMT with Epcam+CD 45-CD 44-CD 24-Ncadherin+, Epcam+CD 45-CD 44+CD 24-Ncadherin+, and Epcam(m)- CD 45-CD 44+CD 24-Ncadherin+ phenotypes in the blood of breast cancer patients before treatment reduced the five-year metastasis-free survival rate (p=0.0016, p=0.017 and p=0.011, respectively).Conclusion. Thus, CTC s in the EMT state are informative for liquid biopsy to assess the risk of hematogenous metastasis and can be considered as targets for selection of personalized chemotherapy.

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