Biomarker potential of mRNA in extracellular vesicles in pediatric AML

Background:

Relapse is the major cause of death in pediatric AML. Due to heterogeneity caused by clonal evolution, it is almost impossible to predict relapse. Therefore, novel methods for accurate prediction of relapse need to be established. Analysing mRNA from extracellular vesicles (EV) that are released into the blood by evolving leukemic cells may be useful in forecasting the future relapse in pediatric AML.

Cell lines and Methods:

1. EV isolation, using SEC and ultracentrifugation, from conditioned media of OCI-AML3 and MV4 – 11 cell lines;

2. Characterisation of EVs using BCA and Zeta view;

3. Isolation of mRNA from cell lines and EVs;

4. real-time PCR for detection of mutations.

Results:

EV isolation methods were successfully optimized. Analysis of EV protein concentration, number and average diameter were determined. Interestingly, using RT-PCR, the leukemia-specific NPM1 mutation was detectable in mRNA isolated from EVs.

Conclusions:

Our EV-based approach to analyze RNA-based leukemic mutations can be a powerful tool in pediatric clinical diagnostics for detecting mutational clonal shifts primarily responsible for AML relapse.