Angelika M. Dräger

High Prognostic Impact of Flow Cytometric Minimal Residual Disease Detection in Acute Myeloid Leukemia: Data From the HOVON/SAKK AML 42A Study

PURPOSE Half the patients with acute myeloid leukemia (AML) who achieve complete remission (CR), ultimately relapse. Residual treatment-surviving leukemia is considered responsible for the outgrowth of AML. In many retrospective studies, detection of minimal residual disease (MRD) has been shown to enable identification of these poor-outcome patients by showing its independent prognostic impact. Most studies focus on molecular markers or analyze data in retrospect. This study establishes the value of immunophenotypically assessed MRD in the context of a multicenter clinical trial in adult AML with sample collection and analysis performed in a few specialized centers. PATIENTS AND METHODS In adults (younger than age 60 years) with AML enrolled onto the Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research Acute Myeloid Leukemia 42A study, MRD was evaluated in bone marrow samples in CR (164 after induction cycle 1, 183 after cycle 2, 124 after consolidation therapy). RESULTS After all courses of therapy, low MRD values distinguished patients with relatively favorable outcome from those with high relapse rate and adverse relapse-free and overall survival. In the whole patient group and in the subgroup with intermediate-risk cytogenetics, MRD was an independent prognostic factor. Multivariate analysis after cycle 2, when decisions about consolidation treatment have to be made, confirmed that high MRD values (> 0.1% of WBC) were associated with a higher risk of relapse after adjustment for consolidation treatment time-dependent covariate risk score and early or later CR. CONCLUSION In future treatment studies, risk stratification should be based not only on risk estimation assessed at diagnosis but also on MRD as a therapy-dependent prognostic factor.

Standardization of flow cytometry in myelodysplastic syndromes: report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes

This article decribes the results of the first European LeukemiaNet working conference on flow cytometry immunophenotyping in myelodysplastic syndrome. This report is a very comprehensive analysis of the topic, and provides detailed information on what is currently known in the field. See related perspective article on page 1041. The myelodysplastic syndromes are a group of clonal hematopoietic stem cell diseases characterized by cytopenia(s), dysplasia in one or more cell lineages and increased risk of evolution to acute myeloid leukemia (AML). Recent advances in immunophenotyping of hematopoietic progenitor and maturing cells in dysplastic bone marrow point to a useful role for multiparameter flow cytometry (FCM) in the diagnosis and prognostication of myelodysplastic syndromes. In March 2008, representatives from 18 European institutes participated in a European LeukemiaNet (ELN) workshop held in Amsterdam as a first step towards standardization of FCM in myelodysplastic syndromes. Consensus was reached regarding standard methods for cell sampling, handling and processing. The group also defined minimal combinations of antibodies to analyze aberrant immunophenotypes and thus dysplasia. Examples are altered numbers of CD34+ precursors, aberrant expression of markers on myeloblasts, maturing myeloid cells, monocytes or erythroid precursors and the expression of lineage infidelity markers. When applied in practice, aberrant FCM patterns correlate well with morphology, the subclassification of myelodysplastic syndromes, and prognostic scoring systems. However, the group also concluded that despite strong evidence for an impact of FCM in myelodysplastic syndromes, further (prospective) validation of markers and immunophenotypic patterns are required against control patient groups as well as further standardization in multi-center studies. Standardization of FCM in myelodysplastic syndromes may thus contribute to improved diagnosis and prognostication of myelodysplastic syndromes in the future.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group

Abstract An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Rationale for the clinical application of flow cytometry in patients with myelodysplastic syndromes: position paper of an International Consortium and the European LeukemiaNet Working Group.

Abstract An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

Regulation of megakaryocytopoiesis in an in vitro stroma model: Preferential adhesion of megakaryocytic progenitors and subsequent inhibition of maturation

OBJECTIVE Studies of megakaryocytic progenitor cell interactions have focused on single receptor-ligand interactions using isolated components of the extracellular matrix. To approach a physiologic condition, we studied megakaryocytic development of human progenitor cells cultured on two stromal cell lines and on human bone marrow stroma. MATERIALS AND METHODS Human CD34(+) cells were cocultured with stromal layers in the presence of thrombopoietin. Megakaryocytes were quantified by monoclonal antibodies against glycoprotein (GP) IIb/IIIa (CD41) and GPIX (CD42a). Megakaryocytic clonogenic capacity (burst-forming unit-megakaryocyte and colony-forming unit-megakaryocyte) was determined using fibrin clot assays. RESULTS After 6 days, a higher percentage of megakaryocytes and more megakaryocytic colonies were recovered from the adherent cell fraction compared to the nonadherent cell fraction. In contrast, significantly more granulocytic and erythroid colonies were recovered from the nonadherent cell fraction. Repeated replating of nonadherent cells onto fresh stroma showed a decline in megakaryocytic recovery of the remaining adherent cells, pointing toward selective adhesion of megakaryocytic progenitors. This was supported further by the finding that burst-forming unit and colony-forming unit megakaryocytes were preferentially recovered from the adherent cell fraction at 24 hours. No effect of blocking the beta(1) integrins VLA-4 and VLA-5 on human progenitor cells was observed. A higher expression of CD42a antigen and a higher percentage of morphologically recognizable polyploid megakaryocytes were found when cells were grown in noncontact cultures compared to when grown adhered to stroma. CONCLUSION In contrast to granulocytic and erythroid progenitors, both very early and more mature megakaryocytic progenitors are preferentially located in the adherent fraction in an in vitro stromal model, leading to inhibition of maturation of megakaryocytes. This suggests that the presence of stroma components in ex vivo expansion cultures, aimed at preservation and expansion of megakaryocytic progenitors, might be a prerequisite.

Acute leukemias of ambiguous lineage: diagnostic consequences of the WHO2008 classification

Acute leukemias of ambiguous lineage: diagnostic consequences of the WHO2008 classification

Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: report from the Dutch Working Party on Flow Cytometry in MDS

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005-2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

Storage of unprocessed G-CSF-mobilized whole blood in a modified Leibovitz's L15 medium preserves clonogenic capacity for at least 7 days.

Autologous stem cell transplantation using unprocessed, G-CSF-mobilized whole blood (WB) is a simple, cost-reducing procedure and supports high-dose chemotherapy regimens not exceeding 72 h. Thereafter, clonogenic capacity rapidly decreases if routine anticoagulants are used for storage. In order to increase clinical applicability, we investigated the requirements for optimal preservation of unprocessed WB for 7 days. During storage at 22°C in CPDA-1, a decrease in pH was noted, which was at least partially responsible for the low recovery of clonogenic cells. Subsequently, WB cells were stored in various cell culture media (RPMI 1640, α-MEM, X-VIVO15, CellGro SCGM and Leibovitzs L15 medium) containing either serum, serum-free substitutes or no additives. Leibovitzs L15 showed significantly better CFU-GM recoveries than the other media. Using a calcium-free modification of L15 medium (added 3:10 to WB), 94 ± 24% of CD34+ cells, 41 ± 14% of BFU-E, 56 ± 17% CFU-GM and 90 ± 14% of LTC-IC were preserved during storage for 7 days at 22°C. Storage at 4°C was also feasible, but showed less optimal recoveries of 52 ± 29% (CD34), 32 ± 10% (BFU-E), 13 ± 7% (CFU-GM) and 58 ± 9% (LTC-IC). The expression of CD38, Thy-1, c-kit, AC133, L-selectin and CXCR4 on CD34-positive cells remained unchanged. In conclusion, a modified Leibovitzs L15 medium better meets the metabolic requirements of a high-density cell culture and allows safe storage of G-CSF mobilized WB for at least 7 days. The results encourage further exploration of WB transplants stored for 7 days for clinical use. Bone Marrow Transplantation (2001) 28, 145–155.

CELL CYCLE DEPENDENCY OF 67GALLIUM UPTAKE AND CYTOTOXICITY IN HUMAN CELL LINES OF HEMATOLOGICAL MALIGNANCIES

67Gallium (67Ga) is a radionuclide which accumulates in hematological malignancies and is used for diagnostic imaging. We investigated in this in vitro study the cell cycle dependency of cellular uptake and cytotoxicity of 67Ga. Cell cycle synchronization of cells was achieved by counterflow centrifugal elutriation and the use of cytostatic drugs. The human lymphoma cell lines U-937 and U-715 were used and in elutriation experiments we also used the leukemic cell line HL-60. The transferrin receptor (CD71) expression, 67Ga uptake and cell proliferation inhibition were the parameters measured. We also studied cytotoxicity in various schedules for combination of 67Ga and drugs and the residual proliferative capacity was measured. The CD71 expression in the three cell lines increased from 106-177% on S phase cells and from 118-233% on G2M cells, as compared to the G0/G1 cell fraction. The 67Ga uptake varied from 108-127% for S cells and 128-139% for G2M cells. The drugs chosen induced cell cycle phase accumulation in S and/or G2M phase during preincubation. 67Ga preincubation induced accumulation in the G2M phase. Almost all combinations of 67Ga and drugs resulted in a non-interactive effect, except for methotrexate which resulted in an antagonistic effect. No preferential effect of any of the incubation schemes was seen. CD71 expression and 67Ga uptake were increased in S and G2M cells. Combination of 67Ga with drugs which arrest cells in these cell cycle phases did not result in a change in cytotoxicity. However, these results implicate that 67Ga and the cytostatic drugs tested except for methotrexate might be used together or sequentially in therapy.

High-dose melphalan with re-infusion of unprocessed, G-CFS-primed whole blood is effective and non-toxic therapy in multiple myeloma

Abstract In order to shorten the pancytopenic period following high-dose melphalan 140 mg m us2 (HDM) treatment of multiple myeloma patients, we studied the effects of re-infusing granulocyte colony stimulating factor (G-CSF) [Filgrastim, Neupogen®]-primed unprocessed whole blood. 30 patients with multiple myeloma were treated with HDM. One litre of blood after 5 or 6 days stimulation with G-CSF (10 μg/kg) was drawn, kept unprocessed for 1 day and re-infused 24 h after chemotherapy. Time to granulocyte recovery ( > 0.5 × 10 9 1 ) and platelet recovery ( > 20 × 10 9 1 ) were assessed as well as length of hospital stay, number of transfusions and antibiotic use. These 30 patients were compared with 20 historical control patients who were similarly treated but without stem cell support. The response rate was 75% (21/28) including a complete remission (CR) rate of 29% (8/28). Two early deaths due to Aspergillus pneumonia were observed. The median overall survival after HDM has not been reached after a median follow-up of 14 months. 10 patients showed progression at a median of 7 months. Currently, 23 patients are alive with a median follow-up time of 14 months. Haematological recovery was significantly faster in the study group as compared to the historical control group. The neutrophil count reached 0.5 × 10 9 1 at a median of 14 days after infusion of 1 litre of unprocessed whole blood compared with 38 days in the historical control group. A platelet count of 20 × 10 9 1 was reached at a median of 26 days compared with 36 days in the historical control group. Length of hospital stay decreased from a median of 43 to 18.5 days. The number of days with antibiotics was reduced from a median of 21 to 6 days. HDM is effective therapy for multiple myeloma. Toxicity of the regimen is considerably reduced by the use of G-CSF-stimulated unprocessed whole blood, an easy to perform and cheap technique to mobilise and collect stem cells.