During intravasation, circulating tumor cells (CTCs) detach from the epithelium of origin and begin the epithelial‐to‐mesenchymal transition (EMT) process, where they lose epithelial features and pass through the endothelium to enter circulation. Although detachment from the extracellular matrix is a strong source of metabolic stress, which induces anoikis, CTCs can survive. Recently, the tumor suppressor liver kinase B1 (LKB1) has gained attention for its role as a proto‐oncogene in restoring the correct ATP/AMP ratio during metabolic stress. The aim of this study was to assess LKB1 expression in epithelial‐negative CTCs isolated from patients with metastatic breast cancer and to characterize its possible association with EMT and stemness features. Transcriptome analysis of EpCAM‐negative CTCs indicated that over 25% of patients showed enhanced LKB1 levels, while almost 20% of patients showed enhanced levels of an EMT transcription factor known as ZEB1. Transcriptome and immunofluorescence analyses showed that patients with enhanced LKB1 were correspondingly ZEB1 negative, suggesting complementary activity for the two proteins. Only ZEB1 was significantly associated with cancer stem cell (CSC) markers. Neither LKB1 nor ZEB1 upregulation showed a correlation with clinical outcome, while enhanced levels of stemness‐associated CD44 correlated with a lower progression‐free and overall survival. Ex vivo models showed that MDA‐MB‐231, a mesenchymal tumor cell line, grew in suspension only if LKB1 was upregulated, but the MCF‐7 epithelial cell line lost its ability to generate spheroids and colonies when LKB1 was inhibited, supporting the idea that LKB1 might be necessary for CTCs to overcome the absence of the extracellular matrix during the early phases of intravasation. If these preliminary results are confirmed, LKB1 will become a novel therapeutic target for eradicating metastasis‐initiating CTCs from patients with primary breast cancer.

BackgroundmicroRNAs (miRNAs) are considered promising cancer biomarkers, showing high reliability, sensitivity and stability. Our study aimed to identify associations between whole blood miRNA profiles, presence of circulating tumor cells (CTCs) and clinical outcome in post-operative early breast cancer patients (EBC) to assess the utility of miRNAs as prognostic markers in this setting.MethodA total of 48 post-operative patients, recruited in frame of the SUCCESS A trial, were included in this retrospective study and tested with a panel of 8 miRNAs (miR-10b, −19a, − 21, − 22, −20a, − 127, − 155, −200b). Additional 17 female healthy donors with no previous history of cancer were included in the study as negative controls. Blood samples were collected at different time points (pre-adjuvant therapy, post-adjuvant therapy, 2 years follow up), total RNA was extracted and the relative concentration of each miRNA was measured by quantitative PCR and compared in patients stratified on blood collection time or CTC detection. Furthermore, we compared miRNA profiles of patients, for each time point separately, and healthy donors. CTCs were visualized and quantified with immunocytochemistry analysis. Data were analyzed using non-parametric statistical tests.ResultsIn our experimental system, miR-19a, miR-22 and miR-127 showed the most promising results, differentiating patients at different time points and from healthy controls, while miR-20a, miR-21 and miR-200b did not show any difference among the different groups. miR-10b and miR-155 were never detectable in our experimental system. With respect to patients’ clinical characteristics, we found a significant correlation between miR-200b and lymph node status and between miR-20a and tumor type. Furthermore, miR-127 correlated with the presence of CTCs. Finally, we found a borderline significance between Progression Free Survival and miR-19a levels.ConclusionsThis pilot study suggests that profiling whole blood miRNAs could help to better stratify post-operative EBC patients without any sign of metastasis to prevent later relapse or metastatic events.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4020-7) contains supplementary material, which is available to authorized users.

BACKGROUND: We evaluated both estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status on disseminated tumor cells (DTCs) in the bone marrow of 54 patients with early breast cancer and compared these with the corresponding primary tumor (PT). MATERIALS AND METHODS: Bone marrow aspirates were obtained at the time of first surgery, and ER and HER2 status on DTCs was assessed simultaneously by immunocytochemistry using a triple fluorescence staining method. RESULTS: The median number of DTCs was 13 (range 1-95). The concordance rate between ER status on DTC and PT was 74%. Patients with an ER-positive PT were significantly more likely to have at least one ER-positive DTC (34 out of 42) than patients with an ER-negative PT (6 out of 12; P = .031). Thirty-nine (93%) of the 42 patients with ER-positive PT had at least one ER-negative DTC. The concordance rate between HER2 status on DTC and PT was 52%. The probability of having at least one HER2-positive DTC was not related to the HER2 status of the PT (P = 0.56). Twenty-two (46%) of the 48 patients with a HER2-negative PT had at least one HER2-positive DTC. All the six patients with a HER2-positive PT had at least one HER2-negative DTC. CONCLUSION: Taken together, our study confirms that ER and/or HER2 status may differ between DTC and PT. This discordance could be important for patients lacking ER or HER2 expression on the PT but showing ER-positive or HER2-positive DTC because they might benefit from an endocrine and/or HER2-targeted therapy.

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Background: Metastasis are thought to be induced by occult spreading of tumor cells already during the early phases of the disease. Circulating Tumor Cells (CTCs) are regarded as precursors of distant metastasis, while detaching from the primary tumor and originating micrometastases in distant organs. Recent evidences pointed to CTC heterogeneity, showing that CTCs can present different phenotypes. Goal of this study was to identify in metastatic breast cancer (mBC) patients CTCs with EMT features and to further characterize them with respect to cellular heterogeneity. Methods: This prospective ongoing study included mBC patients (n=12) with a median age of 58,5 years (range: 35-78 years), enrolled in a time frame of 7 months while undergoing therapy. The majority of patients were estrogen and/or progesterone receptor positive (11/12) and HER2 non-amplified (10/12). Patients had metastatic lesions in viscera as well as bone (6/12), only bone (2/12), only viscera (2/12), only viscera in combination with locoregional recurrence (1/12), or visceral and bone metastases in combination with locoregional recurrence (1/12). Current therapy was endocrine therapy (4/12), chemotherapy (3/12), chemotherapy and HER2-targeted therapy (2/12), HER2-targeted monotherapy (1/12), antiangiogenic therapy (1/12), or surgical therapy only (1/12). Blood samples, withdrawn at any time point during treatment, were depleted of EpCAM+ cells and CD45+ white blood cells (EpCAM/CD45 depleted fraction) (Mego et al., Int J Cancer 2012;130(4):808-816) and expression of epithelial markers (EpCAM, E-Cadherin, Cytokeratin 8,18,19), mesenchymal markers (n-Cadherin, Vimentin), EMT-inducing factors (Twist1, Snail1, SLUG, Zeb1 and FoxC2), anoikis markers (TrkB1, Bcl2) and stem cell markers (CD24, CD44, CD133) were analyzed by qRT-PCR. CTC counting with the CellSearchTM system (Veridex, Raritan NJ) was run in parallel. Healthy donors (n=10) were included in the study as negative controls. Results: The data collected so far showed that 50% of the patients were positive for CTCs in the EpCAM+ fraction as detected with the CellSearchTM system, while 33% were still CTC positive in the EpCAM/CD45 depleted fraction. 50% of the patients, found CTC negative with CellSearchTM, were nevertheless positive for the epithelial and EMT markers in the EpCAM/CD45 depleted fraction (EpCAM 25%, E-Cadherin 25%, CK8 16,6%, CK18 16,6%, CK19 16,6%, SLUG 8,3%, Zeb1 25%, Twist1 8,3%, Vimentin 58,3%). N-cadherin, FoxC2, Snail1, TrkB1, CD24, CD44 and CD133 were never detectable. Conclusions: These preliminary results suggest that mBC patients undergoing different lines of therapy present heterogeneous CTCs. 50% of the patients with undetectable EpCAM+ CTCs, were found CTC positive in the EpCAM/CD45 depleted fraction. mBC patients might be insensitive to treatment due to a selection of resistant CTCs subpopulations with different phenotypes. Additional patients with the same clinical characteristics will be analyzed in the next 6 coming months and updated results will be included. Citation Format: Elisabeth K Trapp, Brigitte Rack, Leonie Majunke, Julian Koch, Simone Hofmann, Thomas WP Friedl, Julia Neugebauer, Julia Jückstock, Bernadette Jäger, Jens Huober, Wolfgang Janni, Marianna Alunni-Fabbroni. Detection of EMT, anoikis and stem cell markers in metastatic breast cancer patients under different lines of treatment [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-16.