Abstract B56: Neuroblastoma patient-derived tumor-specific mRNA and DNA in platelets and extracellular vesicles
Authors: Lak Nathalie , van Zogchel Lieke , Zappeij-Kannegieter Lily , Stutterheim Janine , van der Schoot Ellen , Tytgat Lieve 
Published: June 2020

Abstract
Background: Neuroblastoma is the most common extracranial pediatric solid tumor. Current diagnostic strategies involve imaging and histopathology of tumor tissue and bone marrow. PHOX2B is a specific mRNA marker for neuroblastoma that can be used for detection of circulating tumor cells (CTCs) in peripheral blood and bone marrow. Methylated RASFF1A and ALK F1174L mutation can be used as a tumor-specific cell-free DNA marker. In this study, we aim to detect tumor-specific mRNA in neuroblastoma patient-derived platelets and plasma. Furthermore, we investigate if there is a diagnostic potential for extracellular vesicles (EV) as a source of neuroblastoma-specific mRNA and DNA.
Method: Blood was collected in EDTA tubes and was centrifuged to separate platelets, platelet-free plasma (PFP), and mononuclear cells (MNC, consisting of leukocytes and CTCs). Using size exclusion chromatography (SEC), extracellular vesicles were isolated from 500uL PFP. The presence of EV was confirmed with Western blot, NanoSight, and electron microscopy. RNA was isolated from PFP, platelets, MNC, and SEC fractions using TRIzolTM Reagent. DNA was isolated using the QIAamp DNA Blood Mini Kit®. To detect neuroblastoma-specific RNA, PHOX2B and GUSB as reference gene was measured by RT-qPCR. To detect cell-free tumor DNA, methylated RASSF1A was detected with RT-qPCR, and the ALK F1174L mutation by digital droplet PCR. As a reference gene for DNA, albumin was used.
Results: We collected blood from one healthy control and two patients with neuroblastoma (one at relapse and one in remission). GUSB could be detected in PFP, platelets, and MNC from the control and both patients. PHOX2B was detected in PFP, platelets, and MNC from the patient with a relapse and not in the control or other patient sample. EV were isolated from plasma from one healthy control and one patient at diagnosis. GUSB was present in vesicle-enriched fractions from both samples. PHOX2B was only detected in the neuroblastoma patient vesicle-enriched fractions. In vesicle-enriched fractions of the patient, more total DNA (albumin) was present than in the control. Tumor-specific DNA (ALK F1174L mutation and methylated RASSF1A) was predominantly present in the vesicle-poor, protein-rich fractions.
Conclusion: This study demonstrates that neuroblastoma-specific mRNA and DNA can be detected in platelets, PFP and EV. In particular, mRNA is enriched in EV. This finding suggests that cell-free mRNA from plasma is conserved in EV and implies a role for cell-free RNA from liquid biopsies for diagnostic approaches. In the future, we will investigate the presence and prognostic importance of cell-free RNA and DNA in neuroblastoma and other solid tumors, especially those with few circulating tumor cells.
Citation Format: Nathalie Lak, Lieke van Zogchel, Lily Zappeij-Kannegieter, Janine Stutterheim, Ellen van der Schoot, Lieve Tytgat. Neuroblastoma patient-derived tumor-specific mRNA and DNA in platelets and extracellular vesicles [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B56.