Relationship between CD34/CD38 and side population (SP) defined leukemia stem cell compartments in acute myeloid leukemia.

Leukemic stem cells (LSCs), defined by CD34/CD38 expression, are believed to be essential for leukemia initiation and therapy resistance in acute myeloid leukemia. In addition, the side population (SP), characterized by high Hoechst 33342 efflux, reflecting therapy resistance, has leukemia initiating ability. The purpose of this study is, in both CD34-positive and CD34-negative AML, to integrate both types of LSC compartment into a new more restricted definition. Different CD34/CD38/SP defined putative LSC and normal hematopoietic compartments, with neoplastic or normal nature, respectively, were thus identified after cell sorting, and confirmed by FISH/PCR. Stem cell activity was assessed in the long-term liquid culture stem cell assay. SP fractions harbored the strongest functional stem cell activity in both normal and neoplastic cells in both CD34-positive and CD34-negative AML. Overall, inclusion of SP fraction decreased the size of the putative CD34/CD38 defined LSC compartment by a factor >500. For example, for the important CD34+CD38- LSC compartment, the median SP/CD34+CD38- frequency was 5.1 per million WBC (CD34-positive AML), and median SP/CD34-CD38+ frequency (CD34-negative AML) was 1796 per million WBC. Improved detection of LSC may enable identification of therapy resistant clones, and thereby identification of novel LSC specific, HSC sparing, therapies.

ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status.

The strongest prognostic factor in chronic B-cell lymphocytic leukemia (CLL) is the mutational status of the immunoglobulin heavy chain variable region (IGHV) genes. Determination of this mutational status is laborious and therefore not applied in routine diagnostics. A search for "surrogate markers" has been conducted over the past few years. One of the most promising surrogate markers is ZAP70, but standardization of the measurement of ZAP70 has proven to be difficult. Conventionally, ZAP70 expression in CLL cells is related to ZAP70 expression in T cells. We propose a new method in which ZAP70 expression in NK cells is used as reference (new NK-MFI method). We have measured ZAP70 expression in samples of 45 previously untreated CLL patients. ZAP70 in CLL cells related to ZAP70 in NK cells correlated better to cytogenetic risk profile and mutational status than the conventional methods. Negativity of both ZAP70 (new NK-MFI method) and CD38 resulted in a probability of 90% for mutated IGHV genes. In conclusion, ZAP70 expression in CLL cells related to ZAP70 expression in NK cells is a better surrogate marker for mutational status than the conventional T cell related methods.

ZAP70 in B-CLL cells related to the expression in NK cells is a surrogate marker for mutational status.

The strongest prognostic factor in chronic B-cell lymphocytic leukaemia (CLL) is the mutational status of the immunoglobulin heavy chain variable region (IGHV) genes. Determination of this mutational status is laborious and therefore not applied in routine diagnostics. A search for 'surrogate markers' has been conducted over the past few years. One of the most promising surrogate markers is ZAP70, but standardisation of the measurement of ZAP70 has proven to be difficult. Conventionally, ZAP70 expression in CLL cells is related to ZAP70 expression in T cells. We propose a new method in which ZAP70 expression in NK cells is used as reference (new NK-MFI method). We have measured ZAP70 expression in samples of 45 previously untreated CLL patients. ZAP70 in CLL cells related to ZAP70 in NK cells correlated better to cytogenetic risk profile and mutational status than the conventional methods. Negativity of both ZAP70 (new NK-MFI method) and CD38 resulted in a probability of 90% for mutated IGHV genes. In conclusion, ZAP70 expression in CLL cells related to ZAP70 expression in NK cells is a better surrogate marker for mutational status than the conventional T cell related methods. © 2013 Clinical Cytometry Society.

Immunophenotypic patterns of childhood acute leukemias in Indonesia.

BACKGROUND: Immunophenotyping, as suggested by WHO, may improve diagnosis of childhood leukemia since it offers a better classification of the hematopoietic lineage of malignant cells as compared to morphology. Therefore, we aimed to determine the proportion of the immunophenotypic subtypes of acute leukemia in Indonesian children. METHODS: Samples were obtained from patients (0-14 years of age) in 4 hospitals in Indonesia. We analyzed 541 suspected leukemia samples presented over a 4-year period (March 2006 - July 2010) by flow cytometry. Immunophenotyping allowed classification into acute myeloid leukemia (AML) and ALL (B-lineage and T-lineage ALL). RESULTS: Of 541 samples, 136 were tested using a single color method and 405 with a three-color method. Concordance with morphology was very good (?=0.82) using the three-color method with a panel of 15 monoclonal antibodies (n=387). A relatively high percentage of acute leukemia was classified as AML (23%). Of the ALL samples 83% were B-lineage ALL and 17% T- lineage ALL. Nine out of 239 morphological ALL were labeled AML, and 12/79 morphological AML were in fact ALL. CONCLUSION: Immunophenotyping in a multi-center study proved feasible and appears particularly important for prognostic assessment of childhood leukemia in low income countries such as Indonesia.

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.

Positive selection for CD90 as a purging option in acute myeloid leukemia stem cell transplants.

BACKGROUND: Several studies showed the benefit of purging of acute myeloid leukemia (AML) stem cell transplants. We reported previously that purging by positive selection of CD34+ and CD133+ cells resulted in a 3-4 log tumor cell reduction (TCR) in CD34- and/or CD133- AML, but has been shown to be potentially applicable in only about 50% of cases. Similar to CD34 and CD133, CD90 marks the hematopoietic CD34 positive stem cells capable of full hematopoietic recovery after myeloablative chemotherapy, and therefore, in the present study, we explored whether a similar purging approach is possible using CD90. METHODS: CD90 expression was established by flowcytometry in diagnosis AML on the clonogenic AML CD34+ blast population by flow cytometry. Positivity was defined as >3% CD90 (CD34+) expression on blasts. For the calculation of the efficacy of TCR by positive selection, AML blasts were recognized by either prelabeling diagnosis blasts with CD45-FITC in spiking model experiments or using expression of leukemia associated marker combinations both in spiking experiments and in real transplants. RESULTS: In 119 patients with AML and myelodysplastic syndrome, we found coexpression of CD34 and CD90 (>3%) in 42 cases (35%). In AML patients 60 years or younger, representing the patients who are eligible for transplantation, only 23% (16/69) of the patients showed CD90 expression. Positive selection for CD90 in transplants containing CD90 negative AML resulted in a 2.8-4 log TCR in the models used. CONCLUSIONS: Purging by positive selection using CD90 can potentially be applied effectively in the majority of AML patients 60 years or younger.

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.

Purging of peripheral blood stem cell transplants in AML: a predictive model based on minimal residual disease burden.

OBJECTIVE: Minimal residual disease (MRD) present in peripheral blood stem cell (PBSC) products of AML patients may contribute to relapse. Our goal was to 1) predict leukemia recurrence based on the frequency of MRD present in PBSC products, 2) establish the efficacy of different purging procedures, and 3) integrate this into a model that enables to predict whether or not to purge. METHODS: Minimal residual disease was measured with flow cytometry using leukemia-associated phenotypes as established at diagnosis. Toxicity of purging procedures was established using clonogenic assays. Purging procedures used were cryopreservation, hyperthermia, ether lipid ET-18-OCH3, and combinations. RESULTS: Minimal residual disease in PBSC products correlated significantly with relapse-free survival (n=24, p=0.003). At a cut-off value of 0.05% MRD the relative risk of relapse was 4.6 times lower in the group with less than 0.05% MRD. As measured in 54 PBSC products, the MRD level was less than 0.05% in 17 of 54 cases, between 0.05% and 0.5% in 19 of 54 cases, and higher than 0.5% in 18 of 54 cases. Based on the MRD cut-off of 0.05%, the log tumor reduction needed to achieve this threshold is zero for the 17 of 54 cases in which MRD was below 0.05%, less than or equal to 1 log in 19 of 54 cases, and greater than 1-2 log in 18 of 54 cases. When applying purging with 25 mug/mL ET-18-OCH3 combined with cryopreservation at 10% DMSO and hyperthermia at 42 degrees C combined with cryopreservation at 10% or 4% DMSO, there was greater than or equal to 1 log depletion of AML blasts. CONCLUSION: This study establishes (1) a threshold level for MRD above which prognosis is worse, (2) that stem cell products from 69% of patients have higher than this "safe" MRD level, and (3) that ET-18-OCH3 and hyperthermia may be used to purge products in part of these patients.

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

BACKGROUND AND OBJECTIVES: The percentages of CD34+ cells in the bone marrow of patients with acute myeloid leukemia (AML) vary widely. Especially in the low range (<5% CD34+ cells), the nature (normal or malignant) of the CD34+ cells is uncertain. Since only in a minority of cases are molecular techniques applicable, in this study we explored a multiparameter approach using phenotypic and functional characteristics to discriminate normal CD34+ cells from malignant ones. DESIGN AND METHODS: CD34+ cells from 24 AML patients with <5% CD34+ cells and from 3 patients with >50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants.

Novel multiparameter flow cytometry assay using Syto16 for the simultaneous detection of early apoptosis and apoptosis-corrected P-glycoprotein function in clinical samples.

BACKGROUND: The fluorescent probe Syto16 has been used successfully to measure P-glycoprotein (Pgp) function and, separately, early apoptosis and cell death. The present study was designed to evaluate whether the combined use of Syto16, the Pgp blocker PSC833, and 7-AAD allows simultaneous detection of all parameters, with emphasis on applications in acute myeloid leukemia (AML). METHODS: Pgp negative/positive KB cell lines treated with tumor necrosis factor alpha/hyperthermia and frozen-thawed AML samples were used as apoptosis/Pgp models. RESULTS: For the accurate assessment of apoptosis in samples with unknown Pgp status, it was essential to include a sample with PSC833: in such samples, viable cells always show a Syto16(high) and apoptotic cells a Syto16(low) fluorescence. Apoptotic cells loose their Pgp activity early on; in Pgp-positive cells, the Syto16(low) apoptotic cells then colocalize with the Syto16(low) viable cells in the situation minus PSC833. We have developed a gating strategy that, apart from quantifying apoptosis, allowed gating out these apoptotic cells for proper Pgp assessment. By using this strategy, no differences in Pgp activity were found in the treated versus the untreated samples (KB cells: P = 0.779, n = 10; AML cells: P = 0.525, n = 45). CONCLUSIONS: The use of the combination Syto16/PSC833/7-AAD provides a sensitive multiparameter flow cytometry method that enables accurate assessment of both apoptosis, cell death, and Pgp function in clinical samples.

Early apoptosis largely accounts for functional impairment of CD34+ cells in frozen-thawed stem cell grafts.

Quality assessment of stem cell grafts is usually performed by flow cytometric CD34(+) enumeration or assessment of clonogenic output of fresh material. Previously, we identified the occurrence of early apoptosis, not detectable with the permeability marker 7-amino actinomycin D (7-AAD), in purified frozen-thawed CD34(+) cells, using the vital stain Syto16. Syto(high)/7-AAD(-) cells were defined as viable, Syto16(low)/7-AAD(-) cells as early apoptotic and Syto16(low)/7-AAD(+) as dead. This was confirmed in a subsequent study using frozen-thawed transplants of lymphoma patients. In the present study on grafts from multiple myeloma and lymphoma patients, we investigated the functional consequences of the early apoptotic process. The mean Syto16-defined viability was 41 and 42%, respectively, for both graft groups, compared to 78% and 72%, respectively, using 7-AAD only. The established early apoptosis marker annexin V missed roughly 50% of the early apoptosis detected with Syto16. In contrast, viability of CD34(+) cells in nonmanipulated whole blood transplants from a matched group of lymphoma patients, after 72 h of storage at 4 degrees C, was more than 90%, even with the Syto16 assay. CFU recovery (median 26-33%) after cryopreservation matched CD34(+) recovery after Syto16, but not 7-AAD correction. In contrast, colony-forming unit (CFU) recovery in the whole blood transplant was close to 100%. Furthermore, early apoptotic CD34(+) cells had lost migratory ability toward stromal cell derived factor-1alpha (SDF-1alpha). The establishment of a Syto16(high)/7-AAD(-) proportion of CD34(+) cells offers a new approach for a more correct determination of the number of viable nonapoptotic CD34(+) cells in stem cell grafts. Further development of this assay should allow its incorporation into the routine CD34(+) assessment of post-thawed samples in clinical flow cytometry laboratories.