Feasibility of Leukemia-Derived Exosome Enrichment and Co-isolated dsDNA Sequencing in Acute Myeloid Leukemia Patients: A Proof of Concept for New Leukemia Biomarkers Detection

SIMPLE SUMMARY: The present pilot study aimed at investigating the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA target re-sequencing for adult Acute Myeloid Leukemias (AML) marker detection. To our knowledge, this is the first time that a proof-of-concept combining a leukemia-derived exosome enrichment strategy based on a commercial CE-IVD kit and next-generation sequencing was applied in a cohort of adult AML patients. The reported approach is easy, quick and user friendly and gives the possibility of obtaining a good quantity of exosomal dsDNA (composed of exosomal cargo and surrounding DNA) suitable for further analysis. The time-effective procedure opens up future effective clinical applications. This pilot study presents the potential of a proof-of-concept based on exosome analysis to be applied in clinical practice, as well as the feasibility of this kind of investigations using a certified kit, avoiding many additional analyses. It may encourage further studies regarding extracellular vesicles in myeloid neoplasia. ABSTRACT: Exosomes are extracellular vesicles playing a pivotal role in the intercellular communication. They shuttle different cargoes, including nucleic acids from their cell of origin. For this reason, they have been studied as carriers of tumor markers in different liquid biopsy approaches, in particular for solid tumors. Few data are available concerning exosomes as markers of myeloid neoplasia. To better understand their real potential and the best approach to investigate leukemic exosomes, we present the results of a pilot feasibility study evaluating the application of next-generation sequencing analysis of dsDNA derived from exosomes isolated in 14 adult patients affected by acute myeloid leukemias. In particular, leukemia-derived exosome fractions have been analyzed. The concentration of dsDNA co-extracted with exosomes and the number and types of mutations detected were considered and compared with ones identified in the Bone Marrow (BM) and Peripheral Blood (PB) cells. Exosomal DNA concentration, both considering the cargo and the DNA surrounding the lipid membrane resulted in a linear correlation with leukemic burden. Moreover, exosomal DNA mutation status presented 86.5% of homology with BM and 75% with PB. The results of this pilot study confirmed the feasibility of a leukemia-derived exosome enrichment approach followed by exosomal dsDNA NGS analysis for AML biomarker detection. These data point to the use of liquid biopsy in myeloid neoplasia for the detection of active leukemic cells resident in the BM via a painless procedure.