Detection and Characterization of Estrogen Receptor α Expression of Circulating Tumor Cells as a Prognostic Marker.

CTCs have increasingly been used as a liquid biopsy analyte to obtain real-time information on the tumor through minimally invasive blood analyses. CTCs allow for the identification of proteins relevant for targeted therapies. Here, we evaluated the expression of estrogen receptors (ER) in CTCs of patients with metastatic breast cancer. From sixty metastatic breast cancer patients who had ER-positive primary tumors (range of 1−70% immunostaining) at initial cancer diagnosis, 109 longitudinal blood samples were prospectively collected and analyzed using the CellSearch System in combination with the ERα monoclonal murine ER-119.3 antibody. Prolonged cell permeabilization was found to be required for proper staining of nuclear ER in vitro. Thirty-one cases were found to be CTC-positive; an increased number of CTCs during endocrine and chemotherapy was correlated with disease progression, whereas a decrease or stable amount of CTC number (<5) during treatment was correlated with a better clinical outcome. Survival analyses further indicate a positive association of CTC-status with progression-free survival (HR, 66.17; 95%CI, 3.66−195.96; p = 0.0045) and overall survival (HR, 6.21; 95%CI, 2.66−14.47; p < 0.0001). Only one-third of CTC-positive breast cancer patients, who were initially diagnosed with ER-positive primary tumors, harbored ER-positive CTCs at the time of metastasis, and even in those patients, both ER-positive and ER-negative CTCs were found. CTC-positivity was correlated with a shorter relapse-free survival. Remarkably, ER-negative CTCs were frequent despite initial ER-positive status of the primary tumor, suggesting a switch of ER phenotype or selection of minor ER-negative clones as a potential mechanism of escape from ER-targeting therapy.

Emerging Insights into Keratin 16 Expression during Metastatic Progression of Breast Cancer.

Keratins are the main identification markers of circulating tumor cells (CTCs); however, whether their deregulation is associated with the metastatic process is largely unknown. Previously we have shown by in silico analysis that keratin 16 (KRT16) mRNA upregulation might be associated with more aggressive cancer. Therefore, in this study, we investigated the biological role and the clinical relevance of K16 in metastatic breast cancer. By performing RT-qPCR, western blot, and immunocytochemistry, we investigated the expression patterns of K16 in metastatic breast cancer cell lines and evaluated the clinical relevance of K16 expression in CTCs of 20 metastatic breast cancer patients. High K16 protein expression was associated with an intermediate mesenchymal phenotype. Functional studies showed that K16 has a regulatory effect on EMT and overexpression of K16 significantly enhanced cell motility (p < 0.001). In metastatic breast cancer patients, 64.7% of the detected CTCs expressed K16, which was associated with shorter relapse-free survival (p = 0.0042). Our findings imply that K16 is a metastasis-associated protein that promotes EMT and acts as a positive regulator of cellular motility. Furthermore, determining K16 status in CTCs provides prognostic information that helps to identify patients whose tumors are more prone to metastasize.