ABSTRACT
Circulating tumor cells (CTCs) play a pivotal role in tumor dissemination and progression, and are considered to be a critical part of the metastatic cascade. The aim of the present research article was to examine breast cancer-specific mutations in primary breast cancer (PBC) using targeted resequencing. A total of 78 patients with PBC were enrolled into this translational study. Reverse transcription-quantitative PCR assay for the expression of epithelial markers (CK19) or epithelial-to-mesenchymal transition (EMT)-related genes (TWIST1, SNAIL1, SLUG and ZEB1) was applied for identification of CTCs prior to surgery. Total DNA was isolated from fresh frozen primary tumors. Sequencing was performed by Agilent SureSelect target enrichment and Illumina paired-end sequencing on the MiSeq platform. The most commonly affected genes were TP53 (mutated in 21 tumors; 26.9%), followed by PIK3CA (mutated in 16 tumors; 20.5%) and BRCA1/2 (mutated in 7 tumors, BRCA1 n=2 and BRCA2 n=5; 9.0%). In our cohort, a significantly higher proportion of patients with epithelial CTCs harbored mutations in the BRCA1/2 genes in the tumor tissue. There were no mutations in specific genes associated with CTCs with the EMT phenotype. To the best of our knowledge, this study is the first to report a correlation between the presence of epithelial CTCs in the peripheral blood and mutations of the BRCA1/2 genes in primary tumor tissue.
ABSTRACT
BACKGROUND/AIM: Annexin A2 (ANXA2) is a phospholipid-binding protein involved in fibrinolysis, cell proliferation, migration and metastatic dissemination. Circulating tumor cells (CTCs) are cells responsible for tumor dissemination and have a prognostic value in several types of cancers including breast cancer. Previously, we found correlation between CTCs and activation of coagulation. This study aimed to correlate CTCs with ANXA2 expression on CTCs, tumor cells and tumor associated stroma in primary breast cancer (PBC) patients. PATIENTS AND METHODS: This prospective study included 101 PBC patients treated by primary surgery. CTCs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) assay for the expression of epithelial (CK19) or epithelial-mesenchymal transition (EMT) genes [TWIST1, SNAI1, SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1)]. ANXA2 expression on CTCs was detected by qRT-PCR, while expression of ANXA2 in tumor specimen was evaluated by immunohistochemistry and expressed by a weighted histoscore, evaluating both the percentage of positive cells and the intensity of membrane and cytoplasmic staining. Results of hormone receptors, HER2 status, B-cell lymphoma 2 (bcl-2) protein expression and protein p53 were reported as either positive or negative on histopathology report without further quantification. RESULTS: CTCs were detected in 24.8% patients. Patients with epithelial CTCs had a significantly higher ANXA2 expression on CTCs than those of patients without CTCs (p=0.01). There was no association between CTCs and ANXA2 protein expression in tumor cells. However, patients, whom CTCs with EMT phenotype were detected in, had higher ANXA2 expression in tumor stroma when compared to those with absent EMT CTCs (p=0.04). Hormone-negative tumors had significantly higher ANXA2 expression in tumor cells compared to hormone-positive tumors (p=0.03). Similarly, tumors without bcl-2 protein expression had higher tumor levels of ANXA2 compared to tumor cells that were bcl-2 positive (p=0.05). CONCLUSION: ANXA2 stromal expression might play a key role in aggressive tumor phenotype associated with increased EMT CTCs release, however, other factors beyond ANXA2 are responsible for coagulation activation mediated by CTCs in breast cancer patients.
