Abstract 3152: Breast cancer-derived extracellular vesicles: clinical and prognostic impact
Authors: König Lisa , Rebmann Vera , Hoffmann Oliver , Bittner Ann-Kathrin , Wagner Bettina , Santos Manvailer Luis Felipe , Schramm Sabine , Bankfalvi Agnes , Giebel Bernd , Kimmig Rainer , Horn Peter A. , Kasimir-Bauer Sabine 
Published: July 2016

Abstract
Background: Extracellular vesicles (EVs) are released from cancer cells into the tumor microenvironment, there participating in intercellular communication by altering recipient cell function. Breast cancer (BC) derived EVs are hypothesized to have an impact on tumor growth, immunosuppression or metastatic development, indicating analytical significance of EV concentrations in BC patients. For this reason, we investigated plasmatic EV numbers from locally advanced, neoadjuvant treated (NACT) BC patients in association with clinical parameters and prognostic impact.
Material and methods: Plasmatic extracellular vesicles were isolated using ExoQuick™ precipitation reagent (SBI Inc., Mountain View, CA, USA), according to manufacturer's instructions, from locally advanced BC patients before (n = 142) and after (n = 156) NACT as well as from healthy female controls (n = 16). Subsequently, number and EV particle size were analyzed using ZetaView Laser Scattering Video Microscope (Particle Metrix GmbH, Microtrac, Meerbusch, Germany). Samples were 1:50.000 in PBS pre-diluted to obtain particle concentrations of approx. 1 × 10⁁6 particles per ml. Circulating tumor cells (CTCs) and stem cell-like circulating tumor cells (slCTCs) were evaluated using the AdnaTests BreastCancer, StemCell and EMT, respectively (QIAGEN Hannover GmbH, Germany).
Results: EV particle concentrations (mean ± SEM in 10⁁9/ml) were significantly (p &lt; 0.0001) elevated in BC patients (n = 104, respectively) before (2370 ± 170) and after (3524 ± 523) NACT compared to healthy females (90 ± 19). Paired analysis before and after NACT revealed higher EV levels after NACT (p = 0.008). In association studies, high EV numbers before NACT were related to less differentiated carcinomas and to lymph node spread. ROC analysis showed optimal cut-off values of EV levels (i) before NACT: 3540 × 10⁁9/ml (sensitivity: 66.7%; specificity: 78.1%; AUC = 0.706) and (ii) after NACT: 2480 × 10⁁9/ml (sensitivity: 100%; specificity: 44,3%; AUC = 0.654). In Kaplan-Meier analysis, a decreased 3-year PFS was significantly associated with EV particle concentrations after NACT &gt; 2480 × 10⁁9/ml (p = 0.005). A reduced OS of BC patients was significantly associated with (i) EV levels before NACT &gt; 3540 × 10⁁9/ml) (p = 0.001) and (ii) EV levels after NACT &gt; 2480 × 109/ml) (p = 0.003). Decreased EV levels were found in BC patients after NACT with CTCs expressing ERCC1 (p = 0.025) or the stem cell marker ALDH1 (p = 0.004).
Conclusion: In conclusion, BC patients showed elevated plasmatic EV levels compared to healthy females, while increased EV levels after NACT might be due to therapeutic effects. The association between EV particle concentrations and clinical parameters as well as their prognostic impact on clinical outcome in BC indicate the importance of EVs as a mediator in the BC tumor microenvironment. Therefore, determination of plasmatic EV particle amount might serve as a biomarker for BC monitoring.
Citation Format: Lisa König, Vera Rebmann, Oliver Hoffmann, Ann-Kathrin Bittner, Bettina Wagner, Luis Felipe Santos Manvailer, Sabine Schramm, Agnes Bankfalvi, Bernd Giebel, Rainer Kimmig, Peter A. Horn, Sabine Kasimir-Bauer. Breast cancer-derived extracellular vesicles: clinical and prognostic impact. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3152.