A.A. van de Loosdrecht

A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia

The MTT-colorimetric monocyte mediated cytotoxicity assay, based upon the ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) into formazan, was evaluated using leukemic cells from five representative human leukemic cell lines and from 28 patients with acute myeloid leukemia (AML). An excellent linearity between absorbance and leukemic cell number was observed up to 5 x 10(4) cells/well and 50 x 10(4) cells/well for all cell lines and patients samples tested, respectively, in a 96-wells microtiter culture system. A huge variability in the susceptibility of leukemic cells to purified and IFN-gamma-activated human monocytes could be observed at effector-to-target cell (E:T) ratios of 1. The mean signal-to-noise ratio of the MTT assay for monocyte-leukemic cell mixtures from patients was 2.69 +/- 0.39 at E:T 1. In conclusion, the MTT based monocyte mediated cytotoxicity assay should be useful for studying the susceptibility of a variety of leukemic cells from cell lines and from patients with AML to monocytes in a rapid, sensitive and semi-automated manner.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as ‘normal’, ‘suggestive of’, or ‘diagnostic of’ MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes—proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Vaccination of chronic myeloid leukemia patients with autologous in vitro cultured leukemic dendritic cells

Vaccination of chronic myeloid leukemia patients with autologous in vitro cultured leukemic dendritic cells

Validation of WHO classification-based Prognostic Scoring System (WPSS) for myelodysplastic syndromes and comparison with the revised International Prognostic Scoring System (IPSS-R). A study of the International Working Group for Prognosis in Myelodysplasia (IWG-PM)

A risk-adapted treatment strategy is mandatory for myelodysplastic syndromes (MDS). We refined the World Health Organization (WHO)-classification-based Prognostic Scoring System (WPSS) by determining the impact of the newer clinical and cytogenetic features, and we compared its prognostic power to that of the revised International Prognostic Scoring System (IPSS-R). A population of 5326 untreated MDS was considered. We analyzed single WPSS parameters and confirmed that the WHO classification and severe anemia provide important prognostic information in MDS. A strong correlation was found between the WPSS including the new cytogenetic risk stratification and WPSS adopting original criteria. We then compared WPSS with the IPSS-R prognostic system. A highly significant correlation was found between the WPSS and IPSS-R risk classifications. Discrepancies did occur among lower-risk patients in whom the number of dysplastic hematopoietic lineages as assessed by morphology did not reflect the severity of peripheral blood cytopenias and/or increased marrow blast count. Moreover, severe anemia has higher prognostic weight in the WPSS versus IPSS-R model. Overall, both systems well represent the prognostic risk of MDS patients defined by WHO morphologic criteria. This study provides relevant in formation for the implementation of risk-adapted strategies in MDS.

Treatment, trial participation and survival in adult acute myeloid leukemia: a population-based study in the Netherlands, 1989–2012

Large, comprehensive population-based studies in acute myeloid leukemia (AML) are scarce. We conducted a nationwide population-based study on treatment, trial participation and survival among all adult patients diagnosed with AML (n=12 032) and acute promyelocytic leukemia (APL; n=585) in the Netherlands between 1989–2012. Patients were categorized into four periods and four age groups (18–40, 41–60, 61–70 and >70 years). The application of allogeneic stem cell transplantation increased over time among AML patients up to age 70 years. For APL patients, the use of chemotherapy increased across all age groups. When a clinical trial was open for accrual in the Netherlands, the inclusion rates were 68%, 57%, 30% and 12% for AML patients in the four age groups, respectively (data for APL unavailable). Relative survival improved over time among AML (up to age 70 years) and APL patients. In the period 2007–2012, 5-year relative survival rates were 54%, 38%, 14% and 2% for AML patients and 84%, 75%, 54% and 37% for APL patients in the four age groups, respectively. As survival remained poor for older AML patients over the last two decades, clinical trials and active participation in those trials, are warranted that explore innovative treatment strategies for this elderly population.

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

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

High TRAIL-R3 expression on leukemic blasts is associated with poor outcome and induces apoptosis-resistance which can be overcome by targeting TRAIL-R2

Activation of the TNF-related apoptosis-inducing ligand (TRAIL) pathway can induce apoptosis in a broad range of human cancer cells. Four membrane-bound receptors have been identified. TRAIL-R1 and TRAIL-R2 contain a functional death domain; TRAIL-R3 and TRAIL-R4 lack a functional death domain and function as decoy receptors. Flow-cytometric analysis revealed that acute myeloid leukemic (AML) blasts expressed significantly more pro-apoptotic receptors compared to normal blasts. However, about 20% of AML patients highly expressed decoy receptor TRAIL-R3, which was strongly correlated to a shortened overall survival. TRAIL-R3 expression was also high on CD34+/CD38- cells, the compartment that harbors the leukemia initiating stem cell. Expression levels of pro-apoptotic TRAIL receptors were not correlated to the susceptibility for soluble TRAIL, which was generally low (mean level of cell death induction 14%). Cell death could be enhanced by down-modulation of TRAIL-R3, confirming its decoy function on AML blasts. Bypassing of TRAIL-R3 by treatment with antibodies directly targeting TRAIL-R2 resulted in higher rates of induced cell death (max. 80%). In conclusion, AML blasts do express pro-apoptotic TRAIL receptors. However, co-expression of decoy receptor TRAIL-R3 results in significant shortened overall survival. AML blasts could be targeted by anti-TRAIL-R2 antibodies, yielding a new therapeutic option for AML patients.

Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study

Kupffer cell (KC)-mediated cytotoxicity against tumor cells is of interest, since the liver is a major site of metastatic growth of primary colorectal cancer. KC isolation methods from rat livers, to study the tumoricidal properties of these cells, are based on perfusion of the liver and are therefore not suitable for human KC isolation from liver biopsies. In view of application to isolate KC from small wedge human liver biopsies, we have developed an isolation procedure for rat KC that does not require perfusion techniques. Liver tissue fragments were incubated with pronase with continuous pH registration and neutralization. KC were subsequently separated from other non-parenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. KC and other non-parenchymal cells were identified by immunophenotyping with a cytoplasmic monoclonal antibody ED1 and by ultrastructural analysis. About 3 x 10(6) KC per gram liver were isolated with a final purity of > 95% without loss of viability. To ensure that functionally competent KC were isolated, we assayed cytotoxicity against CC531 tumor cells in a recent developed cell-mediated MTT assay. Maximum cytotoxicity of KC was approximately 40% at an effector to target ratio of 10. In conclusion our approach seems to be a useful and simple method to isolate KC with good functional properties from rat livers, without the need for perfusion techniques.

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs

Massive parallel RNA sequencing of highly purified mesenchymal elements in low-risk MDS reveals tissue-context-dependent activation of inflammatory programs