Plasma Exosomes As Markers of Residual Disease in Patients with Acute Myeloid Leukemia

Background: Exosomes are virus-size (30–100 nm in diameter) membrane-bound microvesicles that are formed within the endocytic compartments and via fusion of multivesicles bodies are released into extracellular space. The exosomal cargo includes proteins/glycoproteins expressed on the cell membrane as well as molecules and soluble factors present in the cytosol of parental cells. While exosome secretion occurs under physiologic conditions, and all cells are capable of their release, tumor cells are avid exosome producers. Patients newly diagnosed with acute myeloid leukemia (AML) prior to any therapy have higher levels of exosomes compared to normal controls (NC). We hypothesize that the molecular content of isolated exosomes, which are thought to mimic that of leukemic blasts, could be informative about the presence in the bone marrow of leukemic blasts that might avoid detection by conventional hematopathological assays.

Methods: Samples of venous blood (20-50 mL) were obtained from patients newly diagnosed with AML prior to any treatment (n=13), after completion of initial induction chemotherapy in patients who achieved complete remission (n=8), during consolidation therapy and age-matched healthy volunteers. Exosome fractions were isolated from plasma by exclusion chromatography on Sephadex G50 columns followed by ultracentrifugation. Exosome protein content was determined and expressed in µg protein/mL plasma. Isolated exosomes were characterized by western blots for expression of classical exosomal markers and for expression of novel myeloid cell surface markers associated with AML, interleukin-3 receptor a chain (CD123) and C-type lectin-like molecule-1 (CLL-1).

Results: The exosome fractions isolated from AML patients’ plasma at diagnosis had a considerably greater mean protein content (81.5 ± 10.8 μg protein/mL plasma) than did exosome fractions isolated from the plasma of NC (13.1 ± 2.4 μg protein/mL plasma) with p < 0.005. The molecular profile of exosomes isolated from plasma of AML patients at diagnosis was distinct from that of exosomes isolated from plasma of NC. In addition to classical exosomal markers (MHC class I molecules, LAMP-1, CD81) exosomes isolated from AML patients at diagnosis contained CD34, CD117, CD123 and CLL-1. The exosome fractions isolated from the patients’ plasma who achieved complete remission (n=8) remained elevated, similar to the levels at the time of AML diagnosis (78.5 vs 77.5 μg protein/mL plasma). Exosomes in AML patients who achieved complete remission and in patients receiving consolidation therapy when leukemic blasts are undetectable in the bone marrow by conventional hematopathological methods contained CD123 and CLL-1.

Conclusions: Exosomes in AML patients have a unique and distinctive molecular profile. The exosomal profile suggest the presence of residual disease in patients considered to have achieved complete remission by conventional hematopathologic assays.