Abstract P1-04-02: Correlation between blood markers of hemostasis and circulating tumor cells (CTCs) in early breast cancer patients
Authors: Mego M , Karaba M , Minarik G , Benca J , Sedlackova T , Tothova L , Vlkova B , Cierna Z , Janega P , Manasova D , Luha J , Gronesova P , Pechan J , Fridrichova I , Celec P , Reuben JM , Cristofanilli M , Mardiak J 
Published: August 2014

Abstract
Background: Cancer is a risk factor for venous thromboembolism (VTE) and plasma D-dimer (DD) and tissue factor (TF) are established blood markers associated with VTE. CTCs are an independent predictor of survival in early and metastatic breast cancer (BC) patients (pts) and CTCs are associated with the risk of VTE in metastatic BC pts. In this study, we hypothesized correlation between plasma DD and TF with presence of CTCs. Moreover, we hypothesized, that activation of urokinase plasminogen activator system (uPA) could be involved in CTCs released into peripheral blood (PB) as well as coagulation activation.
Methods: This prospective study included 119 early BC patients treated by primary surgery followed by systemic therapies when indicated, at the NCI in Slovakia from March to December 2012. Isolated peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells (CD45+) using RossetteSepTM negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by qRT-PCR. Expressions of gene transcripts in CD45- PBMC of pts and healthy donors (HD) were compared. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of HD (n = 60) were considered as CTC positive. Plasma DD, TF, uPA (urokinase-type plasminogen activator) and PAI-1 (plasminogen activator inhibitor) levels were detected by ELISA, while expressions of TF and uPA system in surgical specimens were evaluated by immunohistochemistry.
Results: CTCs were detected in 27.7% pts. The majority of CTCs exhibited either epithelial differentiation (13.5% pts) or mesenchymal phenotype (EMT markers in 15.1% of pts). A small fraction of CTCs showed co-expression of both markers (0.8%). Pts with any CTCs in PB had significantly higher mean ± SEM plasma DD levels (ng/mL) than those of pts without CTCs (632.4 ± 75.9 vs. 365.4 ± 47.0, p = 0.00002). This association was observed for both, epithelial CTCs (688.4 ± 110.4 vs. 400.7 ± 43.5 (p = 0.026) and mesenchymal CTCs (573.1 ± 105.8 vs. 415.6 ± 44.7, p = 0.0007). There was no association between plasma TF levels or breast tumor TF expression and CTCs. Patients with any CTCs had higher mean ± SEM plasma uPA (ng/mL) (374.6 ± 32.6 vs. 307.1 ± 20.3, p = 0.02) and PAI-1 (pg/mL) levels (5.3 ± 0.7 vs. 4.4 ± 0.4, p = 0.28), than those of patients without CTCs, however, there was no correlation between plasma uPA system activation and DD or TF levels. In multivariate analysis CTCs, patients age and tumor grade were independent factors associated with plasma DD levels.
Conclusion: This prospective study showed for the first time a positive association between plasma D-dimer levels and CTCs indicating a potential role for activation of the coagulation cascade in the early metastatic process. We hypothesize that CTCs could be directly involved in coagulation activation in breast cancer patients, or alternatively an increased CTCs count could be a marker of more aggressive disease and increased risk of VTE. Future studies will need to address the prognostic implications of these observations with the potential for therapeutic interventions for the metastatic process.
Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-02.