Nicole Feller

High Stem Cell Frequency in Acute Myeloid Leukemia at Diagnosis Predicts High Minimal Residual Disease and Poor Survival

Purpose: In CD34-positive acute myeloid leukemia (AML), the leukemia-initiating event originates from the CD34+CD38− stem cell compartment. Survival of these cells after chemotherapy may lead to minimal residual disease (MRD) and subsequently to relapse. Therefore, the prognostic impact of stem cell frequency in CD34-positive AML was investigated. Experimental Design: First, the leukemogenic potential of unpurified CD34+CD38− cells, present among other cells, was investigated in vivo using nonobese diabetic/severe combined immunodeficient mice transplantation experiments. Second, we analyzed whether the CD34+CD38− compartment at diagnosis correlates with MRD frequency after chemotherapy and clinical outcome in 92 AML patients. Results:In vivo data showed that engraftment of AML blasts in nonobese diabetic/severe combined immunodeficient mice directly correlated with stem cell frequency of the graft. In patients, a high percentage of CD34+CD38− stem cells at diagnosis significantly correlated with a high MRD frequency, especially after the third course of chemotherapy. Also, it directly correlated with poor survival. In contrast, total CD34+ percentage showed no such correlations. Conclusions: Both in vivo data, as well as the correlation studies, show that AML stem cell frequency at diagnosis offers a new prognostic factor. From our data, it is tempting to hypothesize that a large CD34+CD38− population at diagnosis reflects a higher percentage of chemotherapy-resistant cells that will lead to the outgrowth of MRD, thereby affecting clinical outcome. Ultimately, future therapies should be directed toward malignant stem cells.

MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia

Outgrowth of minimal residual disease (MRD) in acute myeloid leukaemia (AML) is responsible for the occurrence of relapses. MRD can be quantified by immunophenotyping on a flow cytometer using the expression of leukaemia-associated phenotypes. MRD was monitored in follow-up samples taken from bone marrow (BM) of 72 patients after three different cycles of chemotherapy and from autologous peripheral blood stem cell (PBSC) products. The MRD% in BM after the first cycle (n=51), second cycle (n=52) and third cycle (n=30), as well as in PBSC products (n=39) strongly correlated with relapse-free survival. At a cutoff level of 1% after the first cycle and median cutoff levels of 0.14% after the second, 0.11% after the third cycle and 0.13% for PBSC products, the relative risk of relapse was a factor 6.1, 3.4, 7.2 and 5.7, respectively, higher for patients in the high MRD group. Also, absolute MRD cell number/ml was highly predictive of the clinical outcome. After the treatment has ended, an increase of MRD% predicted forthcoming relapses, with MRD assessment intervals of ⩽3 months. In conclusion, MRD parameter assessment at different stages of disease is highly reliable in predicting survival and forthcoming relapses in AML.

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.

C-Type Lectin-Like Molecule-1 A Novel Myeloid Cell Surface Marker Associated with Acute Myeloid Leukemia

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.

High percentage of CD34-positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome.

In this study, a high CD34% in autologous peripheral blood stem cell (PBSC) products from 71 AML patients was associated directly with a high relapse rate (P = 0.006) and inversely with disease-free survival (P = 0.003), irrespective whether patients were transplanted or not. The relapse rate at 12 months was 67% in a group with >0.8% CD34+ cells and 34% in a group with ⩽0.8% CD34+ cells. Although the percentage of malignant CD34+ cells in the CD34+ compartment in the relapses of the first group was not high (median 8%), the total number of malignant cells as a percentage of WBC was about 13 times higher than for the patients remaining >12 months in remission. When all patients evaluable were taken together, this frequency of malignant cells correlated strongly with disease-free survival (P < 0.001). Both this massive mobilization of normal CD34+ cells and high frequency of malignant cells in the subgroup of patients with CD34 >0.8% and relapse within 12 months indicate an insufficient in vivo purging, as well as low chemotherapeutic bone marrow toxicity. This was confirmed by an inverse correlation between hypoplasia period after the induction therapy and CD34% in PBSC products (P < 0.002). It is concluded that a subgroup of patients has been identified that might benefit from a more intensive chemotherapeutic treatment.

Interleukin-2 receptor alpha-chain (CD25) expression on leukaemic blasts is predictive for outcome and level of residual disease in AML

We investigated the role of CD25 as a prognostic marker in acute myeloid leukaemia (AML). Seventy-two newly diagnosed patients < or =60 years were retrospectively analysed by flow cytometry for CD25 positivity of AML blasts. Patients with CD25 expression of >10%, when compared to < or =10%, had a significantly shorter overall survival (OS, p=0.0005) and relapse-free survival (RFS, p=0.005). In multivariate analysis CD25 expression is an independent adverse factor for OS and RFS. High CD25 combined with FLT3-ITD positivity resulted in the poorest OS and RFS (p=0.001 and p=0.003, respectively). CD25 expression remained prognostic within the intermediate cytogenetic risk group. In addition, after the first cycle of chemotherapy, a significantly higher MRD frequency was found in patients expressing CD25 above cut-off (p=0.003). Our results show that CD25 expression is an independent adverse prognostic marker in AML patients < or =60 and correlates with MRD.

Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia

Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD). In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up. Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML. A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/ modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity. Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations. In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10). This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.

Immunologic purging of autologous peripheral blood stem cell products based on CD34 and CD133 expression can be effectively and safely applied in half of the acute myeloid leukemia patients

Purpose: Several studies have shown survival benefit by autologous stem cell transplantation in acute myeloid leukemia (AML) after purging of grafts. This has, however, not been confirmed in randomized studies due to high toxicity of purging modalities for normal progenitor/stem cells. In this study, we investigated whether positive selection for CD34+ and/or CD133+ cells, which results in high recovery of normal progenitor/stem cells, is applicable for purging AML grafts. Experimental Design: Positive selections of normal stem cells using CD34 and/or CD133 can be done if one or both markers are absent or have dim expression and remain so during the course of the disease. Marker expressions in newly diagnosed AML were measured with flow cytometry using a cutoff value for positivity of 1%. Stability of marker expression was studied by pairwise comparison of material at diagnosis and relapse. Leukemia associated phenotype expression was used to measure the efficacy of tumor cell reduction. Results: In newly diagnosed AML (n = 165), we found no CD34 and/or CD133 expression in 32% of the cases and dim expression in 20% of the cases. No increase in the percentage of CD34+ cells (n = 44) and CD133+ cells (n = 29) was found in corresponding relapses. Positive selection using grafts contaminated with AML blasts, showing either no or dim expression of CD34 or CD133, resulted in a 3 to 4 log tumor cell reduction (n = 11) with median 50% recovery of normal stem cells. Conclusions: Purging by positive selection of CD34+ and/or CD133+ cells can safely, effectively, and reproducibly be applied in about 50% of AML cases.

A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia *

Minimal residual disease (MRD) cells are thought to be responsible for the persistence and relapse of acute myeloid leukemia (AML). Flow cytometric MRD detection by the establishment of a leukemia-associated phenotype (LAP) at diagnosis can be used in 80% of AML patients, allowing detection and functional characterization of MRD in follow-up bone marrow. One of the mechanisms contributing to inefficient chemotherapy is apoptosis resistance. Measuring apoptosis parameters in MRD cells will help to unravel the importance of apoptosis resistance in AML. We therefore developed a four-color flow cytometry method that enables establishment of apoptosis-related protein expression such as Bcl-2, Bcl-xL, Mcl-1 and Bax at diagnosis and in MRD. Firstly, validation of this assay using Western blot analysis in five leukemia cell lines showed a significant correlation (R=0.70: P<0.0001). Secondly, the influence of the permeabilization procedure on LAP expression was investigated in 38 AML samples at diagnosis and in 42 MRD samples. Quantification of the frequency of LAP+ cells with and without permeabilization showed no significant differences (diagnosis: P= 0.57, follow-up: P= 0.43). The flow cytometric protocol thus enables analysis of apoptosis-related proteins at different stages of the disease, which will lead to a better understanding of the role of apoptosis resistance in the emergence of MRD in AML.

Minimal Residual Disease in Acute Myeloid Leukemia Is Predicted by an Apoptosis-Resistant Protein Profile at Diagnosis

Purpose: Apoptosis is an important mechanism regulating survival of acute myeloid leukemia cells. The apoptosis-related protein profile at diagnosis is important for achieving complete remission thereby affecting survival variables such as disease-free survival (DFS) and overall survival (OS). To investigate the role of the apoptosis protein profile in further response to therapy and outgrowth of disease. Experimental Design: We studied whether Bcl-2, Bcl-xL, Mcl-1, Bax as well as the Bcl-2/Bax ratio and a combination of all (antiapoptosis index, AAI) are related to the frequency of malignant cells surviving the chemotherapy (i.e., minimal residual disease, MRD). MRD cells were identified by leukemia-associated aberrant phenotypes established at diagnosis by flow cytometry. Results: We found that Bcl-2 (R = 0.55, P = 0.002), Bcl-2/Bax (R = 0.42, P = 0.02), and AAI (R = 0.47, P = 0.01) at diagnosis directly correlated with MRD after the first cycle of chemotherapy. In turn, MRD frequency after first cycle correlated with DFS (P = 0.04). Taken together, these results directly explain why Bcl-2/Bax and especially AAI (P = 0.007) at diagnosis correlate with DFS. Conclusion: Our results show that apoptosis resistance plays an important role in the first stage of the therapy (i.e., to eliminate the bulk of malignant cells), in terms of achievement of complete remission and frequency of MRD after first cycle of therapy.