Harnessing gene expression profiles for the identification of ex vivo drug response genes in pediatric acute myeloid leukemia

Novel treatment strategies are of paramount importance to improve clinical outcomes in pediatric AML. Since chemotherapy is likely to remain the cornerstone of curative treatment of AML, insights in the molecular mechanisms that determine its cytotoxic effects could aid further treatment optimization. To assess which genes and pathways are implicated in tumor drug resistance, we correlated ex vivo drug response data to genome-wide gene expression profiles of 73 primary pediatric AML samples obtained at initial diagnosis. Ex vivo response of primary AML blasts towards cytarabine (Ara C), daunorubicin (DNR), etoposide (VP16), and cladribine (2-CdA) was associated with the expression of 101, 345, 206, and 599 genes, respectively (p < 0.001, FDR 0.004–0.416). Microarray based expression of multiple genes was technically validated using qRT-PCR for a selection of genes. Moreover, expression levels of BRE, HIF1A, and CLEC7A were confirmed to be significantly (p < 0.05) associated with ex vivo drug response in an independent set of 48 primary pediatric AML patients. We present unique data that addresses transcriptomic analyses of the mechanisms underlying ex vivo drug response of primary tumor samples. Our data suggest that distinct gene expression profiles are associated with ex vivo drug response, and may confer a priori drug resistance in leukemic cells. The described associations represent a fundament for the development of interventions to overcome drug resistance in AML, and maximize the benefits of current chemotherapy for sensitive patients

Immunophenotypic measurable residual disease (MRD) in acute myeloid leukemia: Is multicentric MRD assessment feasible?

Flow-cytometric detection of now termed measurable residual disease (MRD) in acute myeloid leukemia (AML) has proven to have an independent prognostic impact. In a previous multicenter study we developed protocols to accurately define leukemia-associated immunophenotypes (LAIPs) at diagnosis. It has, however, not been demonstrated whether the use of the defined LAIPs in the same multicenter setting results in a high concordance between centers in MRD assessment. In the present paper we evaluated whether interpretation of list-mode data (LMD) files, obtained from MRD assessment of previously determined LAIPs during and after treatment, could reliably be performed in a multicenter setting. The percentage of MRD positive cells was simultaneously determined in totally 173 LMD files from 77 AML patients by six participating centers. The quantitative concordance between the six participating centers was meanly 84%, with slight variation of 75%–89%. In addition our data showed that the type and number of LAIPs were of influence on the performance outcome. The highest concordance was observed for LAIPs with cross-lineage expression, followed by LAIPs with an asynchronous antigen expression. Our results imply that immunophenotypic MRD assessment in AML will only be feasible when fully standardized methods are used for reliable multicenter assessment

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection

Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib

Mature circulating endothelial cell (CEC) as well as endothelial progenitor populations may reflect the activity of anti-angiogenic agents on tumor neovasculature or even constitute a target for anti-angiogenic therapy. We investigated the behavior of CECs in parallel with hematopoietic progenitor cells (HPCs) in the blood of renal cell cancer patients during sunitinib treatment. We analyzed the kinetics of a specific population of small VEGFR2-expressing CECs (CD45(neg)/CD34(bright)), HPCs (CD45(dim)/CD34(bright)), and monocytes in the blood of 24 renal cell cancer (RCC) patients receiving 50 mg/day of the multitargeted VEGF inhibitor sunitinib, on a 4-week-on/2-week-off schedule. Blood was taken before treatment (C1D1), on C1D14, C1D28, and on C2D1 before the start of cycle 2. Also plasma VEGF and erythropoietin (EPO) were determined. Remarkably, while CD34(bright) HPCs and monocytes decreased during treatment, CD34(bright) CECs increased from 69 cells/ml (C1D1) to 180 cells/ml (C1D14; P = 0.001) and remained high on C1D28. All cell populations recovered to near pre-treatment levels on C2D1. Plasma VEGF and EPO levels were increased on C1D14 and partly normalized to pre-treatment levels on C2D1. In conclusion, opposite kinetics of two circulating CD34(bright) cell populations, HPCs and small CECs, were observed in sunitinib-treated RCC patients. The increase in CECs is likely caused by sunitinib targeting of immature tumor vessel

Radiation damage to erythrocytes. Morphological and biochemical studies on membranes with special reference to proteins.

In chapter 1 of this thesis it is outlined how membrane research was started in the Centre for Medical Electron Microscopy and how an interest was developed for the action of ionizing radiation on cellular membranes, an interest shared with the Laboratory for Radio-pathology. Furthermore, it is explained why the human erythrocyte was chosen as an object for the irradiation experiments, including biochemical, cell biological and morphological aspects. ... See: Summary

MRD in AML: does it already guide therapy decision-making?

Prognostic factors determined at diagnosis are predictive for outcome whereas achievement of morphological complete remission (CR) is still an important end point during treatment. Residual disease after therapy may reflect the sum of all diagnosis and postdiagnosis resistance mechanisms/factors; its measurement could hypothetically be very instrumental for guiding treatment. The possibility of defining residual disease (minimal residual disease [MRD]) far below the level of 5% blast cells is changing the landscape of risk classification. In this manuscript, the various methods, all different in sensitivity, specificity, and phase of development, to assess MRD are discussed. Currently, the 2 methods mostly used are flow cytometry-based immune MRD (multiparameter flow cytometry [MPFC]) and molecular MRD assessed by real-time quantitative polymerase chain reaction. Both have advantages and disadvantages that are summarized in detail. Many studies in children as well as adults already demonstrated that MRD detection by MPFC or molecular MRD provides strong prognostic information in acute myeloid leukemia (AML) after both induction and consolidation. These studies are summarized in this review. The general conclusion of this review is that a better definition of disease burden than morphological CR is now emerging. MRD assessed by flow or molecular techniques should become standard in every clinical trial in AML. Harmonization of antibody panels, introduction of single-cell tube systems (for determination of residual leukemic stem cells), and standardized analytical programs will pave the way for individual risk assessment and become a surrogate end point for survival in studies investigating new drugs, hopefully resulting in faster drug approval in AML

Leukemic stem cells: identification and clinical application

Leukemic stem cells (LSCs) in acute myeloid leukemia (AML) represent a low-frequency subpopulation of leukemia cells that possess stem cell properties distinct from the bulk leukemia cells, including self-renewal capacity and drug resistance. Due to these properties, LSCs are supposed to facilitate the development of relapse. The existence of LSCs is demonstrated by the ability to engraft and initiate human AML in immune-compromised mouse models. Although several lines of evidence suggest the complex heterogeneity of phenotypes displayed by LSC, many studies consider the CD34+/CD38- compartment as the most relevant. To increase the understanding of the true LSC, techniques such as multicolor flow cytometry, side-population assay and ALDH assay are utilized in many laboratories and could aid in this. A better understanding of different LSC phenotypes is necessary to enhance risk group classification, guide clinical decision-making and to identify new therapeutic targets. These efforts to eliminate LSC should ultimately improve the dismal AML outcome by preventing relapse development

Immunophenotypic Detection of Measurable Residual (Stem Cell) Disease Using LAIP Approach in Acute Myeloid Leukemia

Half of the patients with acute myeloid leukemia (AML), who achieve complete remission after chemotherapy treatment, will ultimately experience a relapse. Measurable residual disease (MRD) is an important post-treatment risk factor in AML, because it gives additional information about the depth of the remission. Within MRD, the small population of leukemic stem cells (LSCs) is thought to be at the base of the actual relapse. In this protocol, the flow cytometric detection of MRD and LSCs herein is outlined. We give a detailed overview of the sampling procedures for optimal multiparameter flow cytometry assessment of both MRD and LSC, using leukemia associated immunophenotypes (LAIPs) and LSC markers. Moreover, an overview of the gating strategies to detect LAIPs and LSC markers is provided. This protocol serves as guidance for flow cytometric detection of measurable residual (stem cell) disease necessary for proper therapeutic decision making in AML patients