Bijan Moshaver

In CD34(+) acute myeloid leukemia (AML), the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously, we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population, containing the CD34(+)CD38(-)CLL-1(+) cells, does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission, both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future.

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84–94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86–97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.

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 (FSC(high)) and high sideward scatter (SSC(high)), high CD38 expression, and usually with aberrant marker expression; (c) a more primitive FSC(low)/SSC(low), CD38(low), marker-negative myeloid fraction; and (d) a more primitive FSC(low)/SSC(low), CD38(low), 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 FSC(low)/SSC(low) 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.