Circulating tumor cells (CTCs) bearing phenotypes related to cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) have been identified in breast cancer; however, their clinical significance is not clear. In the current study, we investigated the prognostic relevance of single CSC þ /partial-EMT þ CTCs in patients with metastatic breast cancer and the effect of first-line chemotherapy on their incidence. For this purpose, triple immunofluorescence against cytokeratin, ALDH1, and TWIST1 was performed in peripheral blood mononuclear cell (PBMC) cytospins from 130 patients before and after first-line chemotherapy. CSC þ / partial-EMT þ CTCs were characterized as cells co-expressing cytokeratin, high levels of ALDH1, and nuclear TWIST1. CSC þ /partial-EMT þ CTCs were evident in 27.7% of patients at baseline and were correlated to lung metastases (P ¼ 0.010) and decreased progression-free survival [PFS; median 10.2 (8.9-11.6) vs. 13.5 (11.3-15.7) months; P ¼ 0.024].Their detection was an independent factor predicting for increased risk of relapse [multivariate analysis; HR (95% confidence interval (CI)): 1.785 (1.171-2.720); P ¼ 0.007]. In HER-2-negative patients, CSC þ /partial-EMT þ CTCs were additionally associated with reduced overall survival (OS) [median 39 (26.2-51.9) vs. 51 (15.7-86.4) months; P ¼ 0.020] and increased risk of death [multivariate analysis; HR (95% CI): 2.228 (1.066-4.655); P ¼ 0.033]. Chemotherapy resulted in a significant increase in the incidence of CSC þ /partial-EMT þ CTCs (mean CTC% per patient: 59.4% post vs. 39.5% pre; P ¼ 0.018), which was subsequently confirmed only in HER2-negative patients (P ¼ 0.040) and in non-responders at the end of treatment (P ¼ 0.020). In conclusion, CSC þ /partial-EMT þ CTCs represent a chemoresistant subpopulation, which independently predicts for unfavorable outcome in metastatic breast cancer. Efficient targeting of these CTCs could potentially increase patient survival.

Background: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). Methods: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearch^TM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. Results: Using the CellSearch^TM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearch^TM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearch^TM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearch^TM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. Conclusion: A significant concordance was observed between the CellSearch^TM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

Background Circulating miRNAs (miRs) are increasingly recognized as potential biomarkers in cancer. We aimed to evaluate the differential expression of miR-23b and miR-190 which are involved in tumor dormancy, miR-21 involved in metastasis and miR-200b and miR-200c involved in epithelial-mesenchymal transition (EMT) and metastasis, in the plasma of patients with early and metastatic breast cancer (MBC). We also aimed to identify associations of the expression levels with patient and disease characteristics and outcomes in metastatic patients treated with first-line chemotherapy. Results miR-21 ( p < 0.001), miR-23b ( p = 0.033), miR-200b ( p < 0.001) and miR-200c ( p < 0.001) expression was higher in metastatic compared to early breast cancer. ROC curve analysis showed that miR-21 (AUC = 0.722; p < 0.001) and miR-200b (AUC = 0.720; p < 0.001) distinguished with high accuracy among the two disease states, whereas the combination of miR-21, miR-190, miR-200b and miR-200c, further improved accuracy (AUC = 0.797; p < 0.001). High miR-200b expression independently predicted for shorter OS ( p = 0.026) in MBC. High expression of both miR23b and miR-190 emerged as a strong independent factor associated with shorter PFS ( p = 0.001) in de novo metastatic patients and high miR-200b independently predicted for decreased OS in the HER2-negative subgroup ( p = 0.007). Materials and Methods Blood samples were obtained from patients with early ( n = 133) and MBC ( n = 110) before adjuvant or first-line chemotherapy, respectively. Plasma miRNA expression levels were assessed by RT-qPCR and were classified as high or low according to the median values. Conclusions Our results are in support of the concept that circulating miRNAs represent a tool with significant diagnostic and prognostic implications in breast cancer.

INTRODUCTION: The presence of KRAS mutations in patients with metastatic colorectal cancer (mCRC) predicts poor response to agents targeting the EGFR. Even in patients with RAS wild type (WT) tumors, resistance eventually develops due to multiple mechanisms, including the expansion of previously undetected KRAS mutated clones. In this feasibility study, we aimed to detect KRAS exon 2 mutations in serial samples of circulating tumor cells (CTCs) of RAS WT patients with mCRC captured by the Isolation by Size of Epithelial Tumor cells (ISET) system. METHODS: CTC isolation using the ISET system was performed from prospectively collected blood samples obtained from patients with RAS and BRAF WT mCRC prior to first-line therapy initiation, at first imaging assessment and on disease progression. CTCs were enumerated using hematoxylin & eosin and CD45 double stain on a single membrane spot. DNA was extracted from 5 spots and KRAS exon 2 mutations were detected using a custom quantitative Polymerase Chain Reaction (qPCR) assay. RESULTS: Fifteen patients were enrolled and 28 blood samples were analyzed. In 9 (60%) patients, at least one sample was positive for the presence of a KRAS exon 2 mutation. In 11 out of 28 samples (39.2%) with detectable CTCs a KRAS mutation was detected; the corresponding percentages for baseline and on progression samples were 27% and 37.5%, respectively. The most commonly detected mutations were G13D and G12C (n = 3). The presence of KRAS mutated CTCs at baseline was not prognostic for either PFS (P = .950) or OS (P = .383). CTC kinetics did not follow tumor response patterns. CONCLUSION: The results demonstrate that using a qPCR-based assay, KRAS exon 2 mutations could be detected in CTCs captured by the ISET system from patients with RAS WT primary tumors. However, the clinical relevance of these CTCs remains to be determined in future studies.

<p>Table S2 shows the associations between the type of chemotherapy and the presence and phenotype of CTCs before and after treatment. Among HER2-positive patients, there was no correlation between the type of therapy and the detection of CTC phenotypes before or after treatment. In the HER2-negative cohort, no associations were observed regarding the baseline detection of distinct CTC subpopulations. Post-treatment, monotherapy with Taxanes or Anthracyclines was strongly associated with negative CTC status (p=0.000) and with absence of CSC+/partial-EMT+ CTCs (p=0.002). In contrast, combination treatment without Taxanes or Anthracyclines resulted in a 100% positivity for CTCs and a higher incidence of CSC+/partial-EMT+ CTCs. All the associations were investigated Exact two-sided Chi-square tests.</p>