Evaluation of dsDNA from extracellular vesicles (EVs) in pediatric AML diagnostics.

Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by a collection of genetic and epigenetic changes. As a consequence, AML can evolve towards more aggressive subtypes during treatment, which require additional therapies to prevent future relapse. As we have previously detected double-stranded DNA (dsDNA) in tumor-derived extracellular vesicles (EVs), in this current study we attempted to evaluate the potential diagnostic applications of AML EV-dsDNA derived from primary bone marrow and peripheral blood plasma samples. EVs from plasma of 29 pediatric AML patients (at initial diagnosis or during treatment) were isolated by ultracentrifugation, after which dsDNA was extracted from obtained EVs and analyzed for leukemia-specific mutations using next generation sequencing (NGS) and GeneScan-based fragment-length analysis. In 18 out of 20 patients, dsDNA harvested from EVs mirrored the (leukemia-specific) mutations found in the genomic DNA obtained from primary leukemia cells. In the nanoparticle tracking analysis (NTA), a decrease in EV numbers was observed in patients after treatment compared with initial diagnosis. Following treatment, in 75 samples out of the 79, these mutations were no longer detectable in EV-dsDNA. In light of our results, we propose the use of leukemia-derived EV-dsDNA as an additional measure for mutational status and, potentially, treatment response in pediatric AML.

Detection of AML-specific mutations in pediatric patient plasma using extracellular vesicle-derived RNA.

Despite high remission rates, almost 25% of patients with AML will suffer relapse 3-5 years after diagnosis. Therefore, in addition to existing diagnostic and MRD detection tools, there is still a need for the development of novel approaches that can provide information on the state of the disease. Extracellular vesicles (EVs), containing genetic material reflecting the status of the parental cell, have gained interest in recent years as potential diagnostic biomarkers in cancer. Therefore, isolation and characterization of blood and bone marrow plasma-derived EVs from pediatric AML patients could be an additional approach in AML diagnostics and disease monitoring. In this study, we attempt to establish a plasma EV-RNA-based method to detect leukemia-specific FLT3-ITD and NPM1 mutations using established leukemia cell lines and primary pediatric AML plasma samples. We were successfully able to detect FLT3-ITD and NPM1 mutations in the EV-RNA using GeneScan-based fragment-length analysis and real-time PCR assays, respectively, in samples before therapy. This was corresponding to the gDNA mutational analysis from leukemic blasts, and supports the potential of using EV-RNA as a diagnostic biomarker in pediatric AML.