De onderzoeksinspanningen van de afdeling zijn te rubriceren in vier groepen:
a. Detectie en karakterisering van minimale residuale ziekte
bij myeloide leukemieën.
b. Karakterisering van gezonde hematopoietische en maligne
stamcellen en onderzoeknaar de interactie met beenmergstroma.
c. Ontwikkeling van leukemievaccins bij myeloide maligniteiten.
d. Patiëntgebonden onderzoeken onder andere op het
gebied van supportive care, antikankertherapie en
nucleaire detectietechnieken.
Immune escape mechanism of myeloid leukemic blasts
M.E.D. Chamuleau1, A.A. van de Loosdrecht1, S.M. van Ham2,
G.J. Ossenkoppele.
Depts. of Hematology, VU University Medical Center and 2Department of Immunopathology, Sanquin Research at CLB, Amsterdam
The aim of this study is to investigate the mechanism used by myeloid leukemic cells to escape immune surveillance in minimal residual disease status of acute myeloid leukemia patients. Topics are 1. insufficient recognition by T-cells, 2. insufficient apoptosis induction and 3. creation of a tolerogenic microenvironment.
Thus far, clinical correlations have been found between poor outcome and high expression of CLIP (which inhibits efficient antigen presentation by MHC class II molecules)1, and between poor outcome and high expression of TRAIL decoy receptors on the cell surface. Next steps will include the investigation of the role of high IDO expression, which can inhibit effector T cells.
Detection and eradication of minimal residual disease (MRD) in AML
N. Feller, G. Westra, A. Kelder, G.J.Ossenkoppele, G.J. Schuurhuis
Dept. of Hematology, VU University Medical Center, Amsterdam
Minimal residual disease (MRD) cells, which might be present in bone marrow (BM) and/or peripheral blood stem cells (PBSC) products of AML patients, are responsible for the emergence of relapses. These MRD cells can be detected by defining leukemia-associated phenotypes using immunophenotypical techniques on flow cytometer. In PBSC products the MRD% correlated with survival and based on this observation we defined a theoretical model for which patients purging of transplants are clinical useful. Next, purging techniques were studied that enabled to purge non-safe transplants. The purging efficacy of these methods was established using LAP monitoring. For this, chemical purging (ether lipid), physical purging (hyperthermia, freeze-thawing), immunological purging (CD34,CD133 and CD90 on AML) and combinations have been applied. Furthermore, we found that the MRD% in BM correlated with the normal CD34% in BM. Based on this finding we designed a model in which unfavourable pharmacokinetics overrules cellular resistance mechanisms and in which the unfavourable pharmacokinetics is responsible for the high relapse rates in a subgroup of patients.
publications
van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, van der Pol MA, Waisfisz Q, Ossenkoppele GJ, Schuurhuis GJ
High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual
disease and poor survival.
Clin Cancer Res 2005; 11: 6520-6527.
Feller N, van der Pol MA, Waaijman T, Weijers GWD, Westra G, Ossenkoppele GJ, Schuurhuis GJ.
Immunologic purging of autologous peripheral blood stem cell products based on CD34 and CD133 expression can effectively and safely be applied in half of the AML patients.
Clin Cancer Res 2005; 11: 4793-4801.
Schuurhuis GJ, Feller N, Huijgens PC, Ossenkoppele GJ.
Minimale restziekte in acute myeloïde leukemie.
Oncollectie 2005; 3: 2-9. Academic Pharmaceutical Productions bv.
Feller N, van der Weide M, van der Pol MA, Westra AH, Ossenkoppele GJ, Schuurhuis GJ.
Purging of peripheral blood stem cell transplants in AML: a predictive model based on minimal residual disease burden.
Exp Hematol 2005; 33: 120-130.
Establishing the nature of minimal residual disease in acute myeloid leukemia by gene expression profiling
C.J. Hess, F. Denkers, G.J. Ossenkoppele, Q. Waisfisz, G.J. Schuurhuis
Department of Hematology, VU University Medical Center, Amsterdam
High complete remission rates are currently achieved in patients with Acute Myeloid Leukemia (AML). However, many patients relapse due to low numbers of residual cells, Minimal Residual Disease (MRD). Detailed knowledge on the cause of emergence and characteristics of MRD cells is therefore obliged. By means of multi-parameter flow-cytometry we are able to detect MRD cells in >80% of the patients on bases of Leukemia Associated Phenotypes (LAP’s). For characterisation of these LAP bearing Minimal Residual Disease (MRD) cells in Acute Myeloid Leukemia (AML), we have validated the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) method for its applicability on low cell number. Further, to establish the clinical predictive importance of the target gene expression at diagnosis, using MLPA containing 35 apoptosis-related genes, we have evaluated apoptosis characteristics of viable (7AAD-/AnnexinV-) BM derived blasts (CD45dim) of 120 previously untreated AML patients. Gene expression signatures could be distilled from a limited number (2-3) of pro and anti apoptotic genes and enable the prediction of clinical outcome and risk stratification of patients with AML. In a second line of research we have started the characterization of apoptosis related gene expression in LAP bearing isolated MRD cells at different stages of disease. This approach will hopefully provide information that may guide the direction and timing of interventions additional to conventional chemotherapy, such as targeted treatment strategies.
Identification of differentially expressed genes during disease transformation in CML
J.J.W.M. Janssen, S.M. Klaver, Q. Waisfisz, G.J. Schuurhuis, G.J. Ossenkoppele
Department of Hematology, VU University Medical Center, Amsterdam
Previously, by using suppression subtractive hybridization, we identified a set of 28 genes differentially expressed at the three separate stages of CML (diagnosis, late chronic phase and blast crisis). One of these genes, GAS2, was significantly higher expressed in CML CD34+ cells than in normal CD34+ cells. As GAS2 has been shown to increase active beta-catenin levels in colon carcinoma cells, we are currently investigating the relationship between GAS2 and beta-catenin in CML by using GAS2 and GAS2-dominant negative transfectants of cell lines which have been established. Furthermore, the effect of GAS2 on proliferation and apoptosis sensitivity is being assessed.
publication
Janssen JJWM, Klaver SM, Waisfisz Q, Pasterkamp G, de Kleijn DP, Schuurhuis GJ,Ossenkoppele GJ.
Identification of genes potentially involved in disease transformation of CML.
Leukemia 2005; 19: 998-1004.
Myeloid leukemia r ole of the bone marrow microenvironment on cell survival in acute myeloid leukemia
B. Moshaver, M.A. der Pol, A.H. Westra, G.J. Ossenkoppele, S. Zweegman, G.J. Schuurhuis. Department of Hematology, VU University Medical Center, Amsterdam
Stromal cells have been found to support leukemic cell survival. However, the responsible mechanisms are still far from elucidated. Leukemic HL-60 cells and primary leukemic cells of patients with acute myeloid leukemia (AML) were either untreated or treated with cytarabine and subsequently cultured for 3-4 days, in the presence or absence of untreated or cytarabine-treated human fibroblastic stromal HS-5 or primary human bone marrow stromal cells. The effect on proliferation and apoptosis could be separately studied using an apoptosis model system and CFSE labeling by flow cytometry.
Stroma cells significantly stimulated spontaneous growth of HL-60 cells and protected them against cytarabine-induced reduction of cell number. This was mediated by an enhancement of proliferation and a delay in apoptosis. Cytarabine-treated stromal cells could still support AML cell survival but with decreasing protective effect already at low cytarabine concentration. Accordingly, stromal cells protected against loss of primary leukemic cells, however, in contrast to HL-60 cells, this was mediated by protection against spontaneous apoptosis via Bcl-2 upregulation. Stromal cells protected leukemic cells against drug effects both by decrease of proliferation inhibition and apoptosis. The loss of protective effect of stroma at clinically relevant cytarabine concentrations strongly suggests that stromal damage may be of high clinical importance. Ultimately targeting both the tumor cells and the bone marrow microenvironment may improve outcome of patients with AML. At present this research is particularly focussed on AML stem cells.
publications
van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, van der Pol MA, Waisfisz Q, Ossenkoppele GJ, Schuurhuis GJ.
High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.
Clin Cancer Res 2005; 11: 6520-6527.
van Stijn A, Feller N, Kok A, van der Pol MA, Ossenkoppele GJ, Schuurhuis GJ.
Minimal residual disease in AML is predicted by an apoptosis-resistant protein profile at diagnosis.
Clinical Cancer Res. 2005; 11: 2540-2546.
Role of signal transduction pathways and the microenvironment in the emergence of minimal residual disease in acute myeloid leukemia
M. A. van der Pol, W.J. Scholten, B. Moshaver, N. Feller, G.J. Ossenkoppele, S. Zweegman, G.J. Schuurhuis
Department of Hematology, VU University Medical Center, Amsterdam
Relapse is common in acute myeloid leukemia (AML) patients due to emergence and outgrowth of minimal residual disease (MRD). New treatment strategies are needed to effectively eradicate MRD in order to improve survival. Our research focuses on how aberrant signal transduction, e.g. constitutive phospho-AKT (pAKT) expression and Nuclear Factor kappa B (NFkB) activity, and the bone marrow microenvironment (BM-ME) contribute to the emergence of MRD and thereby effect prognosis. We will study AKT and NFkB activity status in AML cell subpopulations at diagnosis, in MRD and at relapse to investigate whether changes occur during/after therapy. AML cells are protected from apoptosis when attached to the BM-ME. By co-culture of AML blasts with BM-ME we want to elucidate whether AKT/ NFkB signaling contributes to this protective effect. A FACS assay was developed for the quanti-fication of phosphorylated protein expression in AML cell subsets. U937 cells, which constitutively express pAKT, was used for validation. Without LY294002, a specific inhibitor of PI3K-dependent AKT phosphorylation, pAKT expression in U937 cells (as mean ratio MFI Ab/isotype ±sd, n=5) was 2.2±0.4. With the inhibitor, pAKT expression was absent (1.0±0.2). When using sub-optimal LY294002 concen-trations a range of pAKT expressions were detectable with FACS, which was similar to results obtained with Western Blotting. Preliminary experiments with primary patient samples showed the presence of AML cell subsets at diagnosis with differential pAKT expression after LPS stimulation. When using fibronectin, as a simple model for the BM-ME, 20% of HL60 cells were attached after 45 min of incubation. An increase in NFkB activity (factor 1.6 higher) was found in the attached cells compared to the unattached ones. Whether pAKT expression is similarly upregulated after BM-ME contact and whether pAKT expression is preceding NFkB activation is currently under investigation. We anticipate that more insight into the contribution of AKT/ NFkB signaling in the interaction between leukemic cells and the BM-ME leading to the emergence of MRD will be obtaind. This might guide the development of new treatment strategies to prevent and eradicate MRD.
* financially supported by the Vanderes Foundation.
The nature of primitive clonogenic AML cells and the role in emergence of minimal residual disease in AML
A. van Rhenen, N. Feller, A. Kelder, AH. Westra, G.J. Ossenkoppele, G.J. Schuurhuis
Department of Hematology, VU University Medical Center, Amsterdam
The aim of this study is to identify putative primitive cell populations in AML that give rise to minimal residual disease (MRD) cells and to identify factors that contribute to the escape from the effects of chemotherapeutic treatment.
In vitro and in vivo experiments confirmed the stem cell characteristics of the CD34+CD38- population. The amount of stem cells was found to have major impact both on the level of MRD found after chemotherapeutic treatment and patient outcome: a high stem cell frequency, represented by a high percentage of CD34+CD38- cells, predicts high MRD and short survival. Next it was found that the CD34+CD38- cells at diagnosis display expression of the myeloid specific antigen CLL-1, which is not present on normal CD34+CD38- stem cells and which can therefore be used to study minimal residual stem disease after chemotherapy and in addition might be used in future to specifically target the malignant CD34+CD38- population.
Next to this antigen expression, other antibody combinations also offer the possibility to perform CD34+CD38- analysis in samples of patient material after treatment, and to quantify malignant cell populations, thereby identifying patients at risk for relapse.
Micro-array analysis is being performed on primary patient CD34+CD38- cells to identify stem cell specific characteristics, with a focus on new treatment targets, preferentially those involving production of proliferation stimulatory or inhibitory cytokines and growth factors as well as specific adhesion molecules.
publications
van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, van der Pol, MA, Waisfisz Q, Ossenkoppele GJ, Schuurhuis GJ.
High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival.
Clin Cancer Res 2005;11:6520-6527.
G.J. Schuurhuis, N. Feller, P.C. Huijgens, G.J. Ossenkoppele.
Minimale restziekte in acute myeloïde leukemie.
Oncollectie 2005; 3: 2-9. Academic Pharmaceutical Productions bv.
90Y-Zevalin myeloablative radioimmunotherapy and 89Zr-Zevalin PET in conditioning for stem cell transplantation in patients with aggressive B-cell NHL
O. Visser1, L.R. Perk2, J.M. Zijlstra1, G.A.M.S. van Dongen2, A.A. Lammertsma3, R. Boellaard3, P.C.Huijgens1
Depts. of 1Hematology, 2Otolaryngology/Head-Neck surgery and 3Nuclear Medicine / PET research, VU University Medical Center, Amsterdam
Background: Chemotherapy, followed by autologous stem cell transplantation (AuSCT), is the treatment of choice for patients (= 65 year) with aggressive B-cell non-Hodgkin’s Lymphoma (NHL) relapsing after or refractory to CHOP-like chemotherapy. Recent data show that ± 40% of patients do not meet transplantation eligibility. Zevalin (90Yttrium-ibritumomab tixuetan) is used at a fixed radioactivity dose, on the basis of patients’ body weight. The lack of g-radiation, however, does not allow imaging. Therefore, it is customary to use indium-111 (111In) as a g-emitting surrogate for SPECT tracing of 90Y. Unfortunately, SPECT has intrinsic limitations with respect to tracer quantification. PET is much better equipped for this. To enable PET with MAbs (immuno-PET), we introduced the coupling of the the long-lived positron emitter zirconium-89 (89Zr), and demonstrated for the first time ever the potential of 89Zr-immuno-PET in patients. Moreover, we demonstrated similar biodistribution of 89Zr-Zevalin and 90Y-Zevalin in NHL-bearing nude mice.
Aim of the project: The ultimate aim is to improve the efficacy of treatment of relapsed or refractory aggressive B cell NHL by adding high-dose 90Y-Zevalin to the standard conditioning regimen. As a first step, in this project we will demonstrate the feasibility and safety of adding escalated doses of 90Y-Zevalin. In contrast to previous studies, 90Y-Zevalin will be provided at fixed high doses (not individualized). Crucial in this approach is the use of 89Zr-immuno-PET, as it will keep RIT withhold from patients in case of unfavorable biodistribution.
Plan of investigation:High dose RIT will be implemented. Patients with any response after re-induction therapy will proceed to 90Y-Zevalin + BEAM + AuSCT. Zr-Zevalin will be added during step 1 and 2, for sequential PET imaging. PET images obtained during step 1 will be used to exclude patients with unfavorable biodistribution. For the next two cohorts of patients, dose escalation will be performed.
Response monitoring in non-Hodgkin’s lymphoma using Positron Emission Tomography
J.M. Zijlstra1, O.S. Hoekstra2, P.C. Huijgens1, A.A. Lammertsma3
Depts. of 1Hematology, 2Nuclear Medicine & PET Research and 3Clinical Epidemiology and Bio statistics, VU University Medical Center, Amsterdam
Positron Emission Tomography (PET) using 18F-fluorodeoxyglucose (18F-FDG) is a developing nuclear imaging technique, which has been introduced in the management of malignant lymphoma. Three research projects monitoring response in non-Hodgkin’s lymphoma (NHL) using PET are currently in progress.
In the first project, changes in 18F-FDG uptake are measured with PET during reinduction chemotherapy for relapsed NHL. PET scans are also performed after injecting H215O to compare perfusion with glucose metabolism as a prognostic marker. A larger prospective study aims to investigate the predictive value of PET using 18F-FDG for outcome after stem cell transplantation.
The second project, a multicenter prospective cohort study has been started in 2002 to assess the value of midtreatment FDG PET for early prediction of treatment outcome in terms of freedom from tumour progression in patients with aggressive NHL treated with CHOP chemotherapy. For this study, financial support of NWO has been achieved within the framework of “Doelmatigheidsbevordering van FDG PET-Diagnostiek”. The intended amount of patients (115) has been reached and the study will be analysed medio 2006.
The third project, in collaboration with the University Medical Center Groningen, is a project to develop a new prognostic classification combining FDG-PET with clinical risk scores to determine prognosis in relapsed lymphoma and improving patient selection for autologous stem cell transplantation. For this study, FDG PET and clinical data from 100 patients with relapsed lymphoma treated with chemotherapy and autologous transplantation, will be analysed.
Project fits in V-ICI program 4: therapy
Ongoing Project
Optimizing strategies in dendritic cell vaccination in AML
W. van den Ancker1, T.M. Westers1, T.D. de Gruijl2, G.J. Ossenkoppele1
A.A. van de Loosdrecht1
1 Department of Hematology/V-ICI, VU University Medical Center
2 Department of Medical Oncology/V-ICI, VU University Medical Center Funding: VUmc-V-ICI
Background: Therapeutic vaccination with dendritic cells (DCs) may be an option for the treatment of minimal residual disease in acute myeloid leukaemia (AML). Many sources of antigen (Ag) consisting of known and unknown leukaemia associated antigens (LAA) have been used for loading onto DCs in an attempt to optimize anti-tumour responses. These include apoptotic cells, necrotic cell lysates, eluted peptides, cellular DNA, or RNA as well as chemical or electrical fusion of whole cells with DCs. However, the optimal approach for inducing cell death/apoptosis or obtaining peptides that would lead to effective endocytosis and activation of DCs and thereby induction of a immune response against the leukaemic cells remains to be identified and poses a challenge in cancer immunotherapy protocols.
Methods: This study focuses on the analysis of the efficacy of whole tumour derived LAA-loaded DCs. We used monocyte derived DCs from healthy donors and loaded these with apoptotic AML cells (apo-DCs), with lysate from AML cells (lys-DCs) and compared these with unloaded MoDC or MoDCs loaded with lysate of lymphocytes (lympho-DCs). In co-culture with CD8+ T cells we studied T cell expansion, T cell receptor (TCR) V beta skewing, intracellular interferon-? (IFN?) and granzyme-B, leukaemia antigen specificity such as Wilms' tumour 1 (WT1), and cytotoxicity towards corresponding AML blasts. In co-culture experiments with DCs and CD4+ T cells we measured the percentage of FOXP3+CD25+++ Treg cells. Furthermore we examined the cytokine profiles of MoDC after overnight CD40 ligation.
Results: No differences were found in T cell proliferation after stimulation with apo- or lys-DC. Proliferation was seen most prominent by T cells stimulated with unloaded MoDCs. The decreased proliferation after stimulation with leukaemic cells was not explained by increased production of the suppressive cytokine IL-10. Cytokine IL-12 level was similar in all situations as well. In one co-culture (out of 4), apo-DCs induced a skewing of outgrowing CD8+ T cells from a naïve towards an effector CD45+CD27- phenotype. This was accompanied by outgrowth of a V-beta 16 positive T cell clone, and a higher percentage of intracellular IFN? and granzyme-B positive cells suggesting an outgrowth of cytotoxic T cells (CTLs) able to kill AML cells. Although low, we found in CD8+ T cells of a HLA-A2+ donor evidence of increased frequencies of WT1 tetramer positive T cells (2 fold) stimulated with lys-DCs as compared to lympho-DCs. In a second co-culture (out of 4), we found an increased WT1 tetramer positivity (5 fold) in the Apo-DC stimulated T cells as compared to the lys-DC and unloaded moDCs. WT1 positive cytotoxic T cells have previously been shown to induce killing of AML cells. Our preliminary data suggest that both lys-DCs and apo-DCs are able to induce tumour specific CTLs. Apo-DC and lys-DC stimulated T cells were able to induce lysis of the AML cells, in a partly MHC-I-restricted fashion. Finally, in co-culture experiments with CD4+ T cells we found a higher percentage of FOXp3+CD25+++ regulatory T cells (T regs) in the apo-DC stimulated group compared with the lys-DC group.
Conclusion: Our results point to differential effects of various loading strategies. Additional studies are needed to provide evidence for the DC formulation of choice for treatment of AML patients.
Project in programma 4: Therapy
Lopend+Nieuw project
Optimizing strategies in dendritic cell vaccination in myeloid leukaemia
T.M. Westers, W. van den Ancker, I. Houtenbos, G.J. Ossenkoppele, T.D. de Gruijl1, A.A. van de Loosdrecht. Department of Hematology and Department of Medical Oncology1, VU University Medical Center, V-ICI/CCA, Amsterdam, The Netherlands
Funding: V-ICI
Persistence of residual leukemia cells in acute and chronic myeloid leukemia will eventually lead to a relapse of the disease. Dendritic cell-based vaccines might constitute a therapeutic option for leukemia patients to control or eradicate minimal residual disease. However, intrinsic abnormalities in the leukemic cell that prevent differentiation towards DC or their aberrant function limit the feasibility of leukemia-derived DC-based immunotherapy. The aim of this program is to broaden the applicability of DC-based vaccines via alternative loading strategies such as tumor lysates. In addition, amplification of physiological mechanisms (e.g. co-stimulation) and reduction of inhibitory signals were analyzed. Addition of 4-1BBL to cocultures of AML-derived DC and T cells induced a preferential increase in the proliferation of CD8+ T cells and significantly increased IFN-? production in vitro. T cells primed by AML-DC in the presence of 4-1BBL were in part WT1-specific and showed cytolytic capacity towards leukaemic blasts. Furthermore, a novel bispecific diabody targeting CD28 and CD40 strengthened leukemic DC-induced T cell reactivity. Next to that, loading of whole CML lysates onto monocyte-derived DC induced WT1-specific T cells with anti-leukemia lytic potential towards CML progenitor cells. This research can be extended to loading of Ag corresponding to defined, specific subpopulations such as minimal residual disease cells. In addition, tumor cell lysates and apoptotic bodies of leukemic cells loaded onto monocyte-derived DC with or without TLR agonists are currently subject of investigations to further augment DC-induced T cell reactivity for specific immunisation strategies in AML.
Publications
(LE) Westers TM, Janssen JJWM, Snoijs NCL, van de Loosdrecht AA, Ossenkoppele GJ, Maintained immunogenicity of chronic myeloid leukemia-derived dendritic cells in the presence of Imatinib mesylate: Implication for vaccination regimens, Leukemia 2006; 20(1): 154-157.
(WI) Houtenbos I, Westers TM, Ossenkoppele GJ, van de Loosdrecht AA, Leukemia-derived dendritic cells: towards clinical vaccination protocols in acute myeloid leukemia. Haematologica 2006; 91(3): 348-355.
(LE) Houtenbos I, Westers TM, Ossenkoppele GJ, van de Loosdrecht AA. Leukaemic dendritic cell vaccination for patients with acute myeloid leukaemia. Br J Haematol 2006; 134(4): 445-446.
(WI) Westers TM, Houtenbos I, van de Loosdrecht AA, Ossenkoppele GJ. Principles of dendritic cell-based immunotherapy in myeloid leukemia. Immunobiology 2006; 211(6-8): 663-676.
(LE) Houtenbos I, Westers TM, Hess CJ, Waisfisz Q, Ossenkoppele GJ, van de Loosdrecht AA. Flt-3 internal tandem duplication hampers differentiation of AML blasts towards leukemic dendritic cells. Leukemia 2006; 20(10): 1892-1895.
(WI) Houtenbos I, Westers TM, Ossenkoppele GJ, van de Loosdrecht AA. Feasibility of clinical dendritic cell vaccination in acute myeloid leukemia. Immunobiology 2006; 211(6-8): 677-685.
(DI) Houtenbos I, Strategies to translate leukemia-derived dendritic cell vaccination into clinical practice. Vrije Universiteit Amsterdam, 24-11-2006, (Co-)promotores: prof. G.J. Ossenkoppele, dr. A.A. van de Loosdrecht, 2006, 168 p. 1e geldstroom (cat. A).
(WI) Houtenbos I, Westers TM, Dijkhuis A, de Gruijl TD, Ossenkoppele GJ, van de Loosdrecht AA. Leukemia-Specific T-Cell Reactivity Induced by Leukemic Dendritic Cells Is Augmented by 4-1BB Targeting. Clin Cancer Res 2007; 13: 307-315.
(WI) Westers TM, Ossenkoppele GJ, Loosdrecht AA van de. Dendritic cell based immunotherapy in acute and chronic myeloid leukemia. Biomedicine and Pharmacother 2007; 61:306-314.
(ED) Loosdrecht AA van de. The dendritic cell: the piano player in orchestrating the immune response in leukaemia. Leukemia and Lymphoma 2007; 48:217-218.
(WI) Houtenbos I, Westers TM, Santegoeds S, Waisfisz Q, Kipriyanov S, Denkers F, Gruijl TD de, Ossenkoppele GJ, Loosdrecht AA van de. Bispecific diabody targeting CD40 and CD28 strengthen leukemic dendritic cell induced T cell reactivity. Br J Haematol 2008 (in press).
(WI) Houtenbos I, Ossenkoppele GJ, Van de Loosdrecht AA. Active specific immunization by the use of leukemic dendritic cell vaccines. In: Innovative Leukemia and Lymphoma Therapy. Ed: GJ Kaspers, Coiffier B, Heinrich MC, Estey EH. Informa Healthcare USA Inc. 2008 (in press).
Project in program: Therapy
Ongoing project
CLIP expression on acute myeloid leukemia blasts adversely affects allorestricted CD4+ T cell recognition.
Marvin M. van Luijn1, Martine E.D. Chamuleau1, Theresia M. Westers1, James A. Thompson3, Suzanne Ostrand-Rosenberg3, Gert J. Ossenkoppele1, S. Marieke van Ham2, Arjan A. van de Loosdrecht1.
1Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Academical Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; 3Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, USA (VandeRes Funding no 125).
Previously we showed that high CLIP expression on AML blasts predicts a shortened disease-free survival, which points to an important role for HLA class-II antigen presentation in tumor immune surveillance. Therefore, an in vitro model was set up to investigate the potential effect of CLIP on CD4+ T cell functionality. Several AML cell lines were analysed for HLA-DR, CLIP and Invariant Chain (Ii) expression by flow cytometry. After selection of cell lines positive for both HLA-DR and CLIP, Ii expression was silenced with RNA interference to decrease relative CLIP amount. Wild type DR+CLIP+ and modulated DR+CLIP- cells were used in mixed leukocyte reactions to compare differences in CD4+ T cell proliferation. Kasumi-1 and THP-1 expressed extracellular HLA-DR (89%; MFI=31.3 and 91%; MFI=37.5 respectively) and CLIP (88%; MFI=37.2 and 91%; MFI=34.0 respectively). Treatment of these cell lines with Ii siRNA resulted in a significant decrease of intracellular Ii expression: MFI decreased 10.7-fold in Kasumi-1 and 2.3-fold in THP-1 cells. Moreover, extracellular relative CLIP amount (CLIP/DR MFI) declined 14.9-fold and 2.8-fold respectively. Modulated DR+CLIP- cells induced a strong increase in CD4+ T cell proliferation compared to wild type DR+CLIP+ cells, both in an HLA-DR-specific and stimulator to responder (S/R) dependent manner. At a S/R ratio of 1:10, mean CD4+ T cell proliferation increased 2.6-fold and 1.7-fold in the presence of DR+CLIP- Kasumi-1 and THP-1 cells respectively. These data support the hypothesis that CLIP expression on AML blasts plays an important role in immune surveillance. Expression of CLIP probably acts as a tumor escape mechanism by preventing HLA class-II presentation of AML-specific antigens to CD4+ T cells. Eventually, CLIP modulatory strategies might have an impact on cellular immunotherapy with dendritic cell-based vaccines for AML patients.
(WI) Chamuleau MED, Ossenkoppele GJ, Loosdrecht AA van de. MHC class II molecules in tumor immunology: prognostic marker and target for immune modulation. Immunobiology 2006:211:619-625.
(ED) Loosdrecht AA van de, Beelen RHJ. Emerging immunological concepts in macrophage and dendritic cell research and clinical implications. Immunobiology 2006;211:403-406.
Flow cytometry in myelodysplastic syndromes: a new tool in the classification and prognostication of low/intermediate-I risk MDS.
AA van de Loosdrecht1*, TM Westers1*, AH Westra1, AM Dräger1, VHJ van der Velden2, GJ Ossenkoppele1. 1Department of Haematology, VU University Medical Centre, Amsterdam, Netherlands and 2Department of Immunology, Erasmus University Medical Centre, Rotterdam, Netherlands. *contribute equally to the work.
Introduction: The WHO classification contribute to a more refined classification and prognostication of myelodysplastic syndromes (MDS). Flow cytometry (FC) may add diagnostic and prognostic criteria to discriminate RA from RCMD (+/- ringsideroblasts; (RS)). Methods: We developed a 4-colour FC procedure that recognises all differentiation stages of granulocytic and monocytic lineages in normal bone marrow (BM). Results: In 50 newly diagnosed MDS patients (RA, RARS, RCMD, RCMD-RS, MDS-U, RAEB-1/2) BM samples, aberrant expression of differentiation antigens were demonstrated in 1 or more lineages. FC identified aberrancies in granulopoiesis and monocytopoiesis in 94% of the cases. In the majority of cases abnormal relations between CD13, CD16, CD11b, CD15 and HLA-DR were prominent in the granulopoiesis. In 34% of the cases a striking monocytopenia and in 59% abnormal surface expression of CD14, CD36 was found. In 38% of the cases lineage infidelity antigen expression was detectable (co-expression of CD5, CD7, CD19 or CD56 on CD34+ myeloid blasts). In patients with RA+/-RS and MDS-U, additional FC aberrancies were identified including lineage infidelity Ag expression on myeloid blasts in 30% of the cases. A significant increase in the MDS dysplasia flow-score among WHO subgroups from RA+/-RS, RCMD+/-RS to RAEB1/2 was identified. No significant differences were seen between the MDS dysplasia flow-score and WHO-cytogenetic subgroups; within IPSS subgroups flow-scores were highly heterogeneous. A significant increased MDS dysplasia flow-score was observed between non-transfusion dependent patients and patients in progression to advanced MDS. In 1/19 non-transfusion dependent patients an infidelity marker on myeloid blasts could be observed. In contrast, 14/24 patients with transfusion-dependency and/or in progressive disease, infidelity markers on myeloid blasts were detected. Within the pure-RA+/-RS subgroup only patients with infidelity marker expression on myeloid blasts were transfusion dependent. Discussion: It is concluded that FC in MDS identifies aberrancies in the granulocytic and monocytic lineages and classifies patients with multilineage aberrancies not otherwise determined by cytology (WHO). FC may discriminate pure RA from FC-defined RCMD. FC identifies patients at risk for transfusion dependency and/or progression towards high risk MDS independent of known risk groups. The exact role of the MDS dysplasia flow-score on treatment decisions and as tool in disease monitoring have to be determined in ongoing prospective studies.
1. (WI) Valent P, Horny HP, Bennett JM, Fonatsch C, Germing U, Greenberg P, Haferlach T, Haase D, Kolb HJ, Krieger O, Loken M,
Loosdrecht AA van de, Ogata K, Orfao A, Pfeilstocker M, Ruter B, Sperr W, Stauder R, Wells D. Definitions and
standards in the diagnosis and treatment of myelodysplastic syndromes: consensus statements and report from a working conference. Leuk Res 2007;31:727-736.
2. (WI) Loken M, Loosdrecht AA van de, Ogata K, Orfao A, Wells D. Flow-cytometry in myelodysplastic syndromes. Leuk Res 2008:32:5-17.
3. (WI) Loosdrecht AA van de, Westers TM, Westra G, Drager A, Van der Velden VHJ, Ossenkoppele GJ. Identification of distinct
prognostic subgroups in low and intermediate-I risk myelodysplastic syndromes by flowcytometry. Blood 2008 (on-line Oct 30; 2007).
4. (ED) Loosdrecht AA van de, Westers TM, Ossenkoppele GJ. Flowcytometry in myelodysplastic syndromes; towards a new paradigm
in diagnosis and prognostication? Leuk Res 2008 (in press).
Project in program: Therapy
Ongoing project
Proteasome- and TAP-dependent HLA class-II expression points to 'reverse' cross-presentation and a potentially novel immunotherapeutic target in AML treatment.
Marvin M. van Luijn1,2, Maaike E. Ressing3, Emmanuel J. Wiertz3, Gert J. Ossenkoppele1, Arjan A. van de Loosdrecht1, S. Marieke van Ham2.
1Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Academical Medical Centre, University of Amsterdam, Amsterdam, The Netherlands; 3Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
Recent results showed that Invariant Chain (Ii) knock-down in acute myeloid leukemia (AML) blasts caused a decline in CLIP amount per HLA-DR. In these cell lines the absolute amount of HLA-DR expression was also lowered, in line with the need of Ii for exogenous antigen loading of HLA-DR. In other AML cell lines however, Ii knockdown did not affect HLA-DR expression. To study the potential involvement of endogenous antigens in early HLA class-II loading, KG-1 and Kasumi-1 AML cell lines were transduced with Invariant Chain (Ii) siRNA and UL49.5 or BNLF2a, viral proteins that block endogenous peptide transport into the ER by interfering with the function of TAP, the transporter associated with antigenic processing. Ii silencing in the Kasumi-1 cell line lead to a drastic decline in HLA-DR expression on the plasma membrane (13.9-fold decrease in MFI). Surprisingly, HLA-DR levels on KG-1 cells were hardly affected by Ii silencing, as MFI values differed only 0.9-fold compared to wild type cells. These results suggest that HLA-DR expression is not fully dependent on the Ii. In order to investigate this, supply of endogenously generated peptides into the ER was blocked by UL49.5. In line with previous findings, TAP inhibition in KG-1 cells by UL49.5 resulted in a significant down-regulation of HLA class-I (7.7-fold MFI decrease). Most interestingly, this modulation also lead to a clear HLA-DR- (52.3%; MFI=1.4) KG-1 population next to HLA-DR+ (36.5%; MFI=288.9) KG-1 cells. Upon sorting of both populations, the TAP-deficient HLA-DR- and HLA-DR+ cells contained a respectively lower (2.4-fold MFI decrease) and higher (3.0-fold MFI increase) amount of intracellular Ii as compared to wild type cells. Similar effects on HLA class-I, HLA-DR and Ii expression were obtained when using BNLF2a. Finally, specific inhibition of the proteasome also resulted in a marked down-regulation of HLA class-I and HLA-DR expression on KG-1 cells. At the most optimal concentration, MG-132 treatment caused a 2.1- and 2.7-fold MFI decrease respectively. These data demonstrate a second proteasome- and TAP-dependent pathway of HLA class-II expression in KG-1 AML cells, which points to 'reverse' cross-presentation of endogenous antigens by HLA class-II molecules.
