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 ؊ stemcell 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 IntroductionDespite high-dose chemotherapy, only 30% to 40% of patients with acute myeloid leukemia (AML) survive, which is due mainly to relapse of the disease. 1 AML is generally regarded as a stem-cell disease. However, there is debate whether normal stem cells undergoing leukemogenic mutations is the explanation for leukemogenesis. Alternatively, leukemogenic mutations occurring at a later developmental stage, resulting in stem cell-like behavior, might be an alternative or additional option. [2][3][4] For CD34 ϩ AML, several authors have shown that leukemic stem cells are present in the CD34 ϩ CD38 Ϫ compartment. 5,6 It has been proven in vitro that these stem cells are more resistant to chemotherapy, compared with the progenitor CD34 ϩ CD38 ϩ cells. 7 In vivo, after chemotherapy, the residual malignant CD34 ϩ CD38 Ϫ cells are thought to differentiate to a limited extent, producing leukemic cells with an immunophenotype, which usually reflects that at diagnosis. Sensitive techniques allow early detection of small numbers of these differentiated leukemic cells, called minimal residual disease (MRD), which eventually causes relapse of the disease. 8 Since in this concept the stem cell is the origin of MRD and relapse, stem cell-targeted therapy would be of potentially high benefit for AML patients. Moreover, early detection of leukemic stem cells after chemotherapeutic treatment might offer prognostic value in predicting relapse of the disease. Different options for stem-cell identification and/or targeted therapy have been described such as anti-CD123, anti-CD44, and anti-CD33, but all have some (potential) disadvantages, including expression on normal stem cells and/or nonhematologic tissues. [9][10][11] Since the bone marrow of a (chemotherapy-) treated patient cannot be considered normal, it is extremely important to study whether after treatment nor...

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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 þ CD38À 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 þ CD38À cells led us to investigate whether they can be detected under MRD conditions. Various markers were identified to be aberrantly expressed on the CD34 þ CD38À 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 þ CD38À 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 þ CD38À cells in complete remission, indicating the presence of malignant CD34 þ CD38À cells. Thus, by identifying residual malignant CD34 þ CD38À 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.

C urrent 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.