CD14 and Tissue Factor Positive Extracellular Vesicles Predict Response to Dovitinib in Patients with GBM: A Pilot Study
Authors: Papadantonakis Nikolaos , Ahluwalia Manmeet S , Khoury Micheal , Chaturvedi Shruti , McCrae Keith R. 
Published: October 2019

Abstract
BACKGROUND: Glioblastoma (GBM) is most common primary malignant brain tumor, and has a median survival of 15-18 months. Dovitinib, an oral multi-tyrosine kinase inhibitor of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet derived growth factor (PDGF) is currently under study in a phase II trial for GBM at the Cleveland Clinic. Dovitinib is administrated 5 days on and 2 days off every 4 weeks until progressive disease (PD) or intolerable toxicity are observed. Extracellular vesicles (EV) are submicron particles that express or contain cellular proteins and nucleic acids and are released from a variety of non-malignant cells (e.g. endothelial cells, platelets, leucocytes) and malignant cells. In some settings, EV may serve as biomarkers of inflammation, thrombosis and tumor spread/burden.
OBJECTIVE: The aim of our study was to characterize levels of circulating EV and their relation to disease course in patients with GBM enrolled in the Dovitinib study (with or without prior treatments with anti-angiogenic agents). We also examined the association between EV levels and the development of venous thromboembolism (VTE).
METHODS: Patients previously treated with anti-angiogenic therapy (Group 1, n=14) or without prior anti-angiogenic treatment (Group 2, n=14) were examined separately. EV were measured at study enrollment (pre-treatment), at the end of cycle 1 (day 28), and at PD. EV were isolated from citrated whole blood by differential centrifugation and incubated with fluorochrome-conjugated monoclonal antibodies to CD144-PE (endothelial cells), CD41-PECy4 (platelets), CD14-PE (monocytes) and CD142 (tissue factor, Alexa Fuor 647), then analyzed by flow cytometry. Depending on sample size, the Student t-test or Wilcoxon test was used to compare EV levels (due to the small sample size and skewed distribution of EV levels). P<0.05 was considered significant for all analyses.
RESULTS: Three patients from group 1 and 6 patients from group 2 were not included in the analysis secondary to lack of an EV sample, withdrawal of consent or complications leading to early drug discontinuation. Of theremaining 11 patients in Group 1, 3 had PD and 8 had stable disease (SD) at the end of cycle 1. Of the 8 patients in group 2 available for analyses after cycle 1, 2 had PD and 6 had SD (one of these developed VTE but continued on the study).
In the pretreatment sample of patients from group 1, patients who developed PD had significantly higher levels of CD14+ EV (89977±12121 vs. 42237±27651, p =0.048) and CD142+ EV (68701±9010 vs. 9695±12462, p=0.048) compared to those with SD. However, there was no statistically significant difference in EV levels (all sub-populations) from pre-treatment to the end of cycle 1 in patents with either PD or SD. EV levels did not correlate with peripheral blood counts. Due to the small number of patients in group 2 with progressive disease, we were unable to assess the correlation with EV.
Six (2 in group 1, 4 in group 2) of the 27 patients for which pre-treatment EV were available developed VTE during the study. The EV levels were not significantly different between patients who developed VTE compared to those who did not both at pretreatment and at the day 28 evaluation. However, most patients who developed VTE demonstrated profound increases in EV before or in association with their thrombotic event.
CONCLUSIONS: In patients with GBM receiving Dovitinib without prior exposure to anti-angiogenic therapy, elevated pre-treatment levels of CD14+ and CD142+ EV were associated with progressive disease, suggesting their potential role as a predictor of poor response to Dovitinib. Due to the relatively small sample size, no significant differences were observed between patients that developed VTE and those that did not, either pretreatment or at the Day 28 evaluation; however, these studies are ongoing. In the majority of patients with VTE, EV levels increased substantially before or in association with VTE development.
Acknowledgment: This work was supported by a grant from the Scott Hamilton Cares Initiative

Disclosures
No relevant conflicts of interest to declare.