IntroductionThe presence of circulating tumor cells (CTC) in breast cancer might be associated with stem cell-like tumor cells which have been suggested to be the active source of metastatic spread in primary tumors. Furthermore, to be able to disseminate and metastasize, CTC must be able to perform epithelial-mesenchymal transition (EMT). We studied the expression of three EMT markers and the stem cell marker ALDH1 in CTC from 502 primary breast cancer patients. Data were correlated with the presence of disseminated tumor cells (DTC) in the bone marrow (BM) and with clinicopathological data of the patients.MethodsA total of 2 × 5 ml of blood was analyzed for CTC with the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1, HER2 and beta-Actin transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers [TWIST1, Akt2, phosphoinositide kinase-3 (PI3Kα)] and separately for the tumor stem cell marker ALDH1. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Two BM aspirates from all patients were analyzed for DTC by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3.ResultsNinety-seven percent of 30 healthy donor samples investigated were negative for EMT and 95% for ALDH1 transcripts, respectively. CTC were detected in 97/502 (19%) patients. At least one of the EMT markers was expressed in 29% and ALDH1 was present in 14% of the samples, respectively. Interestingly, 5% of the ALDH1-positive and 18% of the EMT-positive patients were CTC-negative based on the cut-off level determined for CTC-positivity applying the AdnaTest BreastCancer. DTC in the BM were detected in 107/502 (21%) patients and no correlation was found between BM status and CTC positivity (P = 0.41). The presence of CTC, EMT and ALDH1 expression was not correlated to any of the prognostic clinical markers.ConclusionsOur data indicate that (1) a subset of primary breast cancer patients shows EMT and stem cell characteristics and (2) the currently used detection methods for CTC are not efficient to identify a subtype of CTC which underwent EMT. (3) The clinical relevance on prognosis and therapy response has to be further evaluated in a prospective trial.

Introduction The role of circulating tumor cells (CTCs) in blood of primary breast cancer patients is still under investigation. We evaluated the incidence of CTCs in blood, we evaluated the correlation between CTCs and disseminated tumor cells (DTCs) in the bone marrow (BM), and we characterized CTCs for the expression of HER2, the estrogen receptor (ER) and the progesterone receptor (PR).

Extracellular vesicles (EVs) have been discussed as a diagnostic tool for minimal residual disease (MRD) evaluation in breast cancer (BC) in addition to the analysis of circulating tumor cells (CTCs). Therefore, we investigated circulating EV levels as surrogate markers for disease monitoring and prediction of prognosis in primary, non-metastatic, locally advanced BC patients. EVs were enriched from blood samples of BC patients before and after neoadjuvant chemotherapy (NACT) and from healthy females. EV marker expression analysis was performed and EV sizes and concentrations were determined by nanoparticle tracking analysis. The results were associated with disease status, outcome and CTC presence, evaluated by gene expression analysis after enrichment. We demonstrated that i) the EV concentration was 40-fold higher in BC patients compared to healthy females, ii) the EV concentration increased during therapy, iii) an increased EV concentration pre-NACT was associated with therapy failure and iv) an elevated EV concentration post-NACT was associated with a reduced three-year progression-free and overall survival. Of note, residual stem cell-like and/or resistant CTCs after therapy were associated with a lower EV concentration post-NACT. Our study highlights that the concentration of EVs within BC blood samples may serve as a complementary parameter reflecting the status of MRD as well as therapy and disease outcome in parallel with CTC investigation.

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Integrated PET/MRI does not provide diagnostic advantages for local tumor staging of breast cancer patients in comparison to MRI alone. Positron emission tomography/MRI and MRI enable an improved determination of the local tumor extent in comparison to PET/CT, whereas all 3 imaging modalities offer a comparable diagnostic performance for the identification of axillary disease.