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The small extracellular vesicles (sEV) accumulating in acute myeloid leukemia (AML) patients’ plasma are mixtures of vesicles produced by leukemic and non-malignant cells. sEV originating from leukemia blasts could serve as potential non-invasive biomarkers of AML response to therapy. To isolate blast-derived sEV from patients’ plasma, we developed a bioprinted microarray-based immunoassay using monoclonal antibodies (mAbs) specific for leukemia-associated antigens (LAAs) and mAbs specific for a mix of tetraspanins (CD9, CD63, and CD81). We determined the proportion of LAA+ sEV relative to total plasma sEV (the LAA+/total sEV ratio) in serially collected samples of newly diagnosed AML patients prior to, during, and after chemotherapy. At AML diagnosis, the LAA+/total sEV ratio was significantly higher in patients than in healthy donors (HDs). In patients who achieved complete remission (CR) after induction chemotherapy, the LAA+/total sEV ratios significantly decreased after each chemotherapy cycle to levels seen in HDs. In contrast, the LAA+/total sEV ratios in AML patients with persistent leukemia after therapy remained elevated during and after therapy, as did the percentage of leukemic blasts in these patients’ bone marrows. The LAA+/total sEV ratio emerges as a promising non-invasive biomarker of leukemia response to therapy.

Immune checkpoint inhibitors can block inhibitory molecules on the surface of T cells, switching them from an exhausted to an active state. One of these inhibitory immune checkpoints, programmed cell death protein 1 (PD-1) is expressed on T cell subpopulations in acute myeloid leukemia (AML). PD-1 expression has been shown to increase with AML progression following allo-haematopoeitic stem cell transplantation, and therapy with hypomethylating agents. We have previously shown that anti-PD-1 can enhance the response of leukemia-associated antigen (LAA)-specific T cells against AML cells as well as leukemic stem and leukemic progenitor cells (LSC/LPCs) ex vivo. In concurrence, blocking of PD-1 with antibodies such as nivolumab has been shown to enhance response rates post-chemotherapy and stem cell transplant. The immune modulating drug lenalidomide has been shown to promote anti-tumour immunity including anti-inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenicity. The effects of lenalidomide are distinct from chemotherapy, hypomethylating agents or kinase inhibitors, making lenalidomide an attractive agent for use in AML and in combination with existing active agents. To determine whether anti-PD-1 (nivolumab) and lenalidomide alone or in combination could enhance LAA-specific T cell immune responses, we used colony-forming immune and ELISpot assays. Combinations of immunotherapeutic approaches are believed to increase antigen-specific immune responses against leukemic cells including LPC/LSCs. In this study we used a combination of LAA-peptides with the immune checkpoint inhibitor anti-PD-1 and lenalidomide to enhance the killing of LSC/LPCs ex vivo. Our data offer a novel insight into how we could improve AML patient responses to treatment in future clinical studies.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The standard of care in medically and physically fit patients is intensive induction therapy. The majority of these intensively treated patients achieve a complete remission. However, a high number of these patients will experience relapse. In patients older than 60 years, the results are even worse. Therefore, new therapeutic approaches are desperately needed. One promising approach in high-risk leukemia to prevent relapse is the induction of the immune system simultaneously or after reduction of the initial tumor burden. Different immunotherapeutic approaches such as allogenic stem cell transplantation or donor lymphocyte infusions are already standard therapies, but other options for AML treatment are in the pipeline. Moreover, the therapeutic landscape in AML is rapidly changing, and in the last years, a number of immunogenic targets structures eligible for specific therapy, risk assessment or evaluation of disease course were determined. For example, leukemia-associated antigens (LAA) showed to be critical as biomarkers of disease state and survival, as well as markers of minimal residual disease (MRD). Yet many mechanisms and properties are still insufficiently understood, which also represents a great potential for this form of therapy. Therefore, targeted therapy as immunotherapy could turn into an efficient tool to clear residual disease, improve the outcome of AML patients and reduce the relapse risk. In this review, established but also emerging immunotherapeutic approaches for AML patients will be discussed.