Plasma-Derived Exosomes Carrying Leukemia-Associated Antigens Associate with Leukemia Relapse in AML Patients after Chemotherapy

Introduction: Exosomes are the smallest (30-150nm) type of extracellular vesicles that are produced by normal and cancerous cells, including leukemia blasts. Exosomes accumulating in the plasma of patients with cancer carry molecular cargo resembling that of parental tumor cells. Patients newly diagnosed with AML prior to any therapy have significantly higher plasma levels of exosomes (in µg protein/mL plasma) compared to levels in the plasma of normal controls. We previously showed that exosomes in AML patients carry leukemia-associated antigens (LAAs) and hypothesized that exosomes could define disease progression and outcome. Here, we evaluated the molecular cargo of exosomes at AML diagnosis, monitored changes in the exosome cargo during and post therapy, and correlated these changes with the blast content of LAAs detected in leukemia blasts using multi-parameter flow cytometry.

Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML and following therapy. Exosomes were isolated from plasma by exclusion chromatography and characterized for protein levels, number and size by qNano, and for morphology by transmission electron microscopy. Exosome cargos, including the presence and level of LAA, were evaluated by Western blots using exosomes isolated from serial plasma specimens obtained during and post chemotherapy. The data were correlated for expression of LAAs.

Results: Exosomes isolated from the plasma of 15 patients newly diagnosed with AML carried CD34, CD117, C-type lectin-like molecule-1 (CLL-1), and interleukin-3 receptor subunit alpha (CD123). These antigens were carried by exosomes of all patients, but the antigen levels or profiles were distinct for each patient. The exosome antigen profile detected at AML diagnosis was comparable to that detected in each patient's leukemic blasts by multi-parameter flow cytometry. In AML patients with persistent leukemia following therapy, at the time when blasts were detectable in the bone marrow but not in the peripheral blood, circulating exosomes carried the LAAs. In all patients who achieved complete remission (n=7), at the time when no blasts were detected by morphologic and flow cytometric evaluation of the bone marrow, circulating exosomes carried LAAs with the same profile as that detected at AML diagnosis. All complete remission patients with LAA+ exosomes in the plasma eventually relapsed.

Conclusions: We demonstrated, for the first time, that exosomes in patients newly diagnosed with AML have the LAA profiles that are identical to the profiles detected on leukemic blasts by conventional multi-parameter flow cytometry. The persistence of LAA+ exosomes in patients' plasma post-chemotherapy at the time of complete remission suggests the presence of residual leukemia and may be predictive of leukemia relapse.