Bijan Moshaver

Aberrant marker expression patterns on the CD34+CD38− stem cell compartment in acute myeloid leukemia allows to distinguish the malignant from the normal stem cell compartment both at diagnosis and in remission

Acute myeloid leukemia (AML) is generally regarded as a stem cell disease. In CD34-positive AML, the leukemic stem cell has been recognized as CD38 negative. This CD34+CD38− population survives chemotherapy and is most probable the cause of minimal residual disease (MRD). The outgrowth of MRD causes relapse and MRD can therefore serve as a prognostic marker. The key role of leukemogenic CD34+CD38− cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34+CD38− population in AML and high-risk MDS samples at diagnosis, including C-type lectin-like molecule-1 and several lineage markers/marker-combinations. Fluorescent in situ hybridization analysis revealed that marker-positive cells were indeed of malignant origin. The markers were neither expressed on normal CD34+CD38− cells in steady-state bone marrow (BM) nor in BM after chemotherapy. We found that these markers were indeed expressed in part of the patients on malignant CD34+CD38− cells in complete remission, indicating the presence of malignant CD34+CD38− cells. Thus, by identifying residual malignant CD34+CD38− cells after chemotherapy, MRD detection at the stem cell level turned out to be possible. This might facilitate characterization of these chemotherapy-resistant leukemogenic cells, thereby being of help to identify new targets for therapy.

Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia.

In acute myeloid leukemia (AML), apart from the CD34+CD38− compartment, the side population (SP) compartment contains leukemic stem cells (LSCs). We have previously shown that CD34+CD38− LSCs can be identified using stem cell‐associated cell surface markers, including C‐type lectin‐like molecule‐1 (CLL‐1), and lineage markers, such as CD7, CD19, and CD56. A similar study was performed for AML SP to further characterize the SP cells with the aim of narrowing down the putatively very low stem cell fraction. Fluorescence‐activated cell sorting (FACS) analysis of 48 bone marrow and peripheral blood samples at diagnosis showed SP cells in 41 of 48 cases that were partly or completely positive for the markers, including CD123. SP cells in normal bone marrow (NBM) were completely negative for markers, except CD123. Further analysis revealed that the SP fraction contains different subpopulations: (a) three small lymphoid subpopulations (with T‐, B‐, or natural killer‐cell markers); (b) a differentiated myeloid population with high forward scatter (FSChigh) and high sideward scatter (SSChigh), high CD38 expression, and usually with aberrant marker expression; (c) a more primitive FSClow/SSClow, CD38low, marker‐negative myeloid fraction; and (d) a more primitive FSClow/SSClow, CD38low, marker‐positive myeloid fraction. NBM contained the first three populations, although the aberrant markers were absent in the second population. Suspension culture assay showed that FSClow/SSClow SP cells were highly enriched for primitive cells. Fluorescence in situ hybridization (FISH) analyses showed that cytogenetically abnormal colonies originated from sorted marker positive cells, whereas the cytogenetically normal colonies originated from sorted marker‐negative cells. In conclusion, AML SP cells could be discriminated from normal SP cells at diagnosis on the basis of expression of CLL‐1 and lineage markers. This reveals the presence of a low‐frequency (median, 0.0016%) SP subfraction as a likely candidate to be enriched for leukemia stem cells.

Chemotherapeutic treatment of bone marrow stromal cells strongly affects their protective effect on acute myeloid leukemia cell survival

Bone marrow stromal cells (BMSCs) have been found to support leukemic cell survival; however, the mechanisms responsible are far from elucidated yet. Therefore, the effect of BMSCs on both proliferation and apoptosis characteristics of acute myeloid leukaemia (AML) cells was investigated as well as the effect of BMSCs exposure to chemotherapy on the stromal supportive capacity. Leukemic HL-60 and primary AML cells were either untreated or treated with cytarabine and subsequently cultured for 3 – 4 days, in the presence or absence of untreated or cytarabine-treated BMSCs. The effect on proliferation and apoptosis was investigated with flow cytometry using CFSE labeling and Syto16 and 7AAD staining. BMSCs were found to maintain cytarabine-exposed primary AML cells by protection against spontaneous apoptosis. Accordingly, an increase in phosphorylated-AKT and Bcl-2 expression was found. Concomitant exposure of BMSCs to cytarabine resulted in a dose-dependent decrease of protective capacity of BMSCs. Thus, inhibition of spontaneous apoptosis of leukemic cells mediated by phosphorylation of AKT/Bcl-2 pathway results in protection of leukemic cells by BMSCs, which decreases after BMSCs exposure to chemotherapy. Targeting both the tumor cells and intervening in their interaction with the bone marrow microenvironment may thus affect clinical outcome in AML.

New approaches for the detection of minimal residual disease in acute myeloid leukemia.

The detection of minimal residual disease (MRD) in patients with acute leukemia has been studied for about 15 years by different groups in both the United States and Europe. It has been found that MRD detection can be performed using molecular and immunophenotypic aberrancies that are present in the leukemic clone at diagnosis and not in normal bone marrow. When performing MRD assessments after chemotherapy, it is possible to identify patients at risk for relapse. This review is not an overview of all MRD studies, but rather discusses the possibilities for optimizing MRD detection, the use of flow cytometry versus polymerase chain reaction techniques, and the implications for future patient treatment. When informative, we compare literature on MRD in acute myeloid leukemia (AML) with information from MRD studies in acute lymphoblastic leukemia. Finally, we address the promising detection of AML stem cells, the likely cells of origin in AML, for prediction of clinical outcome and guidance of future therapies.