C-type Lectin-like Molecule-1 Research Articles

Identification of Primitive Subpopulations of Acute Myeloid Leukemia Side Population (SP) Stem Cells Defined by Differentiation Status and Malignant Character.

Acute myeloid leukemia (AML) is generally regarded as a stem cells disease. A large proportion of remission patients ultimately relapses, indicating ineffectiveness of current therapies in eradicating leukemia stem cells (LSCs). Additional to the CD34+CD38− compartment, the so-called side population (SP) exists. In order to trace residual SP stem cells under remission conditions, allowing adapted risk stratification or more accurate prediction of relapses, we sought for leukemia stem cell specific immunophenotypic markers i.e. not staining the normal SP stem cells. We used C-type Lectin-like molecule-1 (CLL-1, van Rhenen, Blood 106: 6a, 2005) and IL-3 receptor α-chain CD123, previously shown to be stem cell markers. Lastly, several markers making up the so-called leukemia associated phenotypes (LAP), used for MRD detection (Feller, Leukemia 18:1380, 2004), were tested for their ability to stain the AML SP stem cell as well. Using Hoechst 33342 dye, PI and monoclonal antibodies against CD7, CD19, CD56, CD123 and CLL-1, SP immunophenotyping using FACS analysis was performed on bone marrow (BM) samples of 17 AML patients at diagnosis, 8 healthy donors (nBM) and 4 relevant donors with BM regenerating after chemotherapy (rBM). SP cells were detected in 13 of 17 AML patients with median frequency of 0.01% (% of WBC, range 0.00002-0.17%). In all 13 cases, SP cells were partly or completely positive for CLL-1 and/or CD123. In 10/13 cases SP cells were partly or completely positive for LAP markers These results strongly suggest that the majority of SP cells are malignant, which was confirmed by FISH analysis [n=2; t(8;21)]. Marker positive SP cells as a fraction of total SP was 0.66 (range 0.22–1.0). Furthermore, SP cells from control nBM and rBM were completely negative for CLL-1 and LAP markers, but not for CD123 (40%-82%). Remarkably, 11/13 AML samples revealed two subpopulations within the SP, in terms of sideward scatter (SSC): high side scatter (HSSC) SP cells, with median frequency of 46 % (% of whole SP compartment, range 21–86%) and low side scatter (LSSC) SP cells (median frequency 54 %, range 14–79%). In the 2 remaining cases only LSSC SP was present. HSSC and LSSC populations were also seen in 5/8 nBM SP cells, with only LSSC SP present in the remaining 3 cases. HSSC AML SP cells had a more differentiated phenotype (high SSC, high CD38 expression) and were all malignant in terms of LAP marker positivity and CLL-1 positivity. LSSC SP cells were more primitive (low SSC, mostly CD38 negative or low), only partly expressing CLL-1 or LAP. FISH analysis for a patient with t(8;21) confirmed the malignant nature of both the HSSC and LSSC LAP marker (CD19 in this case) positive cells and the normal character of the CD19 negative LSSC cells. Marker positive, primitive LSSC SP cells (n=13) had median frequency of 14.2% of total SP (range 4.6–48.9%). The presumed median frequency of these LSSC SP cells at diagnosis is thereby close to 1: 105, (% of WBC) which is close to the presumed AML stem cell frequency in diagnosis AML. In conclusion, diagnosis AML SP cells can be discriminated from normal SP cells based on expression of CLL-1 and LAP markers. SP cells show a primitive low-frequency sub fraction as a likely candidate to contain the leukemia initiating cell. Future studies include functional characterization of this LSSC SP subfraction for purposes of risk stratification and therapeutic intervention.

Acute Myeloid Leukemia Remission Bone Marrow Reveals the Presence of Malignant and Normal Side Population (SP) Stem Cells Whose Frequencies and Ratios Predict Clinical Outcome.

A high relapse rate in AML suggests that present therapies are ineffective in eliminating leukemic stem cells. These may be present in the side population (SP) cells, which are able initiate leukemia in NOD/SCID mice. LSC detection may offer opportunities for detection of stem cells under conditions of minimal residual disease (MRD). Separately (Moshaver, this meeting) we show that leukemia associated immunophenotypes (LAP; used for MRD detection, Feller, Leukemia 18:1380, 2004) and CD34+CD38- stem cell marker C-type Lectin-like molecule-1 (CLL-1, van Rhenen, Blood 106: 6a, 2005), and Il-3 receptor α-chain CD123, are able to discriminate between normal and leukemic SP stem cells at diagnosis. In addition, LAP marker and CLL-1 expression, but not CD123 expression, on SP cells in control bone marrows (n=12) was lacking. SP cells could be detected in 13/17 diagnosis AML patients. The contribution of the AML SP to total SP was calculated based on expression of above-mentioned AML markers and checked with FISH analysis. We further use “AML SP frequency” (AML SP as % of WBC) and “AML SP fraction” (AML SP as fraction of total SP). We obtained 12 follow-up samples of 6 of these patients. At diagnosis median AML SP frequency was 0.015 and median AML SP fraction was 0.7. At follow-up, median total SP frequency was 0.01% (0.0007%–2.0 %). AML SP frequency herein was median 0.004% (<0.00001 – 1.05). AML SP frequency detection was compared with MRD detection method, known to predict prognosis (Feller, 2004). In 5/6 patients with low AML SP frequencies (< 0.004%) MRD was undetectable (≤0.01%). In 4/6 patients with high frequencies, MRD>0.01% was found. 3/6 patients showed a remarkable phenomenon: AML SP frequency was relatively high (0.006%, 0.007% and 0.014%, respectively) with apparently non-matching, relatively low MRD frequencies (0.02%, 0.02% and 0.01%, resp.), but with a drastic increase of MRD frequency at the next sampling time point (to 0.17%, 0.11% and 0.21%, resp). Such increases as well as the absolute percentages (>0.1%) inevitably lead to relapse (Feller 2004), which at present short follow up time already occurred for one patient. In addition, AML SP fraction was compared with MRD: 5/5 with low AML SP fraction (<0.21) had no detectable MRD (MRD≤0.01%). For high AML SP fraction (≥0.21) 4/6 had MRD>0.01%. AML SP fraction identified the same 3 special patients as did AML SP frequency: the AML SP fraction was high (0.33, 0.64 and 1.0, resp.) with the low MRD frequencies that subsequently strongly increased. Both stem cell parameters thus add additional prognostic value to MRD assessment. In conclusion, AML and normal stem cells can be detected with great specificity in AML remission bone marrow using LAP markers and CLL-1, that specifically stain AML SP stem cells and not normal SP stem cells. Additional predictive power for forthcoming relapses comes from the selective sparing of AML SP stem cells after therapy compared to both the more mature AML cells and the normal stem cells. The introduction of such stem cell parameters may thus contribute to MRD based risk stratification during treatment, offer guidelines for future clinical intervention time points, while studying cellular characteristics of both AML and normal stem cells may allow to design stem cell directed therapies that are effective for eradication of AML stem cells with minimal toxicity towards normal stem cells.

The Novel AML Stem Cell Associated Antigen CLL-1 Discriminates between Normal and Leukemic Stem Cells.

In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event likely takes place in the CD34+CD38- stem cell compartment. Survival of these cells after chemotherapy hypothetically leads to minimal residual disease (MRD) and relapse. We have previously shown that a high CD34+CD38- frequency correlates with both MRD frequency, especially after the third course of chemotherapy and poor survival (Clin Cancer Res, in press). Furthermore, we have shown that a monoclonal antibody against the novel cell surface marker C-type lectin-like molecule-1 (CLL-1), directed against myeloid cells, stains 92% of diagnosis AML (Bakker et al., Cancer Res. 64:8443, 2004). In the present study we investigated whether this antibody can be used to identify AML stem cells in remission bone marrow. Such would offer opportunities for MRD stem cell detection and stem cell-directed therapy.We found that anti-CLL-1 antibody homogeneously stained the CD34+CD38- compartment in 77/89 cases (median expression of 33.3% in all 89 cases, range 0–100%). The median stem cell expression of CLL-1 in control bone marrow was 0% ranging from 0–11% (n=11). Furthermore, CLL-1 expression on AML stem cells is highly stable: no differences between paired diagnosis and relapse samples (p=0.9, n=12). Like most antigens CLL-1 is expressed on part of the CD34+CD38+ compartment, but expression is absent on megakaryocytic precursors, which for therapeutic use would circumvent delayed platelet recovery. For antibody-mediated therapy it is crucial that normal stem cells remain negative throughout treatment of the disease. Therefore we tested bone marrow regenerating after high dose chemotherapy, obtained from either non-AML hematological patients or CD34 negative or CLL-1 negative AML patients. In those patients complete absence of CLL-1 expression was found in CD34+CD38− cells (n=4).Under MRD-conditions CLL-1 staining thus enables to accurately discriminate between normal and malignant CD34+CD38− stem cells. In agreement with this, the different ratios of AML and normal stem cells that were found in a number of patients, paralleled clinical outcome in terms of probability of relapse.For comparison, the stem cell marker CD123 was studied. Although anti-CD123 antibody homogeneously stained CD34+CD38− cells with high intensity in almost all AML samples studied (35/36 cases) with also no differences between diagnosis and relapse (p=0.6, n=6) and with low expression in normal bone marrow (median 14.9%, range 0–18.8%, n=5), a high expression was found in regenerating bone marrow (median 60%, range 53–84%, n=4). The latter suggests that anti-CD123 antibody is not AML stem cell specific under all conditions of disease.In conclusion, our data provide strong evidence that a large CD34+CD38− population at diagnosis reflects a higher percentage of chemotherapy-resistant cells, which, in remission, will lead to the outgrowth of MRD, thereby affecting clinical outcome. The specificity of anti-CLL-1 antibody under all conditions of disease enables both reliable detection and quantification of the stem cell compartment for prognostic use under MRD conditions, as well as characterization. Moreover, it shows that AML stem cell targeting using antibody treatment at different stages of disease has now become an option in the treatment of AML patients.

C-Type Lectin-Like Molecule-1

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34(+)/CD38(-) or CD34(+)/CD33(-) stem cells and present on subsets of CD34(+)/CD38(+) or CD34(+)/CD33(+) progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33(-) AMLs expressed CLL-1. CLL-1 showed variable expression (10-60%) in CD34(+) cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.