Exosomes from Plasma of Neuroblastoma Patients Contain Doublestranded DNA Reflecting the Mutational Status of Parental Tumor Cells.

Chiara Degli Esposti,Cristina Beltrami,Patrizia De Marco,Barbara Iadarola,Federico Zara,Annalisa Pezzolo,Martina Morini,Massimo Delledonne,Alberto Garaventa,Marzia Ognibene,Simone Maestri,Denise Lavezzari,Giovanni Erminio

doi:10.3390/ijms22073667

Abstract

Neuroblastoma (NB) is an aggressive infancy tumor, leading cause of death among preschool age diseases. Here we focused on characterization of exosomal DNA (exo-DNA) isolated from plasma cell-derived exosomes of neuroblastoma patients, and its potential use for detection of somatic mutations present in the parental tumor cells. Exosomes are small extracellular membrane vesicles secreted by most cells, playing an important role in intercellular communications. Using an enzymatic method, we provided evidence for the presence of double-stranded DNA in the NB exosomes. Moreover, by whole exome sequencing, we demonstrated that NB exo-DNA represents the entire exome and that it carries tumor-specific genetic mutations, including those occurring on known oncogenes and tumor suppressor genes in neuroblastoma (ALK, CHD5, SHANK2, PHOX2B, TERT, FGFR1, and BRAF). NB exo-DNA can be useful to identify variants responsible for acquired resistance, such as mutations of ALK, TP53, and RAS/MAPK genes that appear in relapsed patients. The possibility to isolate and to enrich NB derived exosomes from plasma using surface markers, and the quick and easy extraction of exo-DNA, gives this methodology a translational potential in the clinic. Exo-DNA can be an attractive non-invasive biomarker for NB molecular diagnostic, especially when tissue biopsy cannot be easily available.

Highlights

Neuroblastoma (NB), the most common extracranial solid tumor occurring in childhood, arises from neural crest cells of the sympathetic nervous system [1,2]
Given the recent findings on the biological content of exosomes, including chromosomal DNA [21,25], we focused on characterization of the NB exosomal DNA (exo-DNA) and its potential use as a non-invasive biomarker for screening of somatic mutations present in the parental cells
Exosomes were purified from peripheral blood samples of the nineteen NB patients at onset of the disease and, in four cases, at the time of relapse too

Summary

Introduction

Neuroblastoma (NB), the most common extracranial solid tumor occurring in childhood, arises from neural crest cells of the sympathetic nervous system [1,2]. NB patients diagnosed with metastatic disease, or older than 18 months, or when the tumor carries genomic amplification of MYCN oncogene and/or segmental chromosome abnormalities, are considered to be at high risk of death [5]. Besides to MYCN amplification, the rearrangements of TERT gene, encoding the catalytic subunit of telomerase, or inactivating mutations in ATRX and ARID1A loci, encoding chromatin-remodeling proteins, have been detected mainly in high-risk NB [6,7,8,9,10]. Mutations in genes of the RAS and p53 pathways have been described in relapsed NB, and they occur in both high- and nonhigh-risk tumors at lower frequencies in the latter group [13,14]. Other somatic mutations have been detected in primary NB albeit rarely (

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Conclusion