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G J Schuurhuis

Publications and source records attributed to G J Schuurhuis.

At least 19 recordsLinked to original sources
PubMed2006

Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples.

In acute myeloid leukemia (AML), activating mutations in the fms-like tyrosine kinase 3 (FLT3) gene predict poor prognosis. We determined FLT3 internal tandem duplications (FLT3/ITD) and D835 point mutations in paired initial and relapse samples from 80 pediatric and adult AML patients. One D835 point mutation was found in an initial pediatric AML sample. Fms-like tyrosine kinase 3/ITDs were present in 21 initial and 22 relapse samples (26.3 and 27.5%, respectively). Interestingly, FLT3/ITD positivity was related to a significantly shorter time to relapse, most pronounced when the ITD-positive status was found at relapse (P<0.001). However, FLT3/ITD status changed between diagnosis and relapse in 14 cases. In four patients, the FLT3/ITD became undetectable at relapse in five patients FLT3/ITDs were only detected at relapse, and in five patients the length or number of FLT3/ITDs changed. Gain of FLT3/ITDs may suggest oligoclonality with selective outgrowth of the FLT3/ITD-positive clone, whereas losses may reflect ITDs in the more mature leukemic cells rather than in the leukemic stem cell, or, alternatively, that other genetic aberrations provided a greater selective advantage. Studying FLT3/ITD kinetics in minimal residual disease setting may provide some answers for the changes we observed. Fms-like tyrosine kinase 3/ITD is a relevant marker for prognosis, and remains an important target for therapeutic inhibition.

Adolescent↗
PubMed2005

Identification of genes potentially involved in disease transformation of CML.

In patients with chronic myeloid leukemia (CML) who do not reach a (near) complete cytogenetic response, the disease progresses over several years from an indolent, chronic phase into a rapidly fatal blast crisis. Events that are responsible for this transformation process are largely unknown. To identify changes in gene expression that occurred during the course of the disease, we performed cDNA subtraction on sequentially stored peripheral blood mononuclear cell pellets, collected throughout the course of disease of a single CML patient. In total, 32 differentially expressed sequences were identified, of which 27 corresponded to known genes. On quantitative PCR, eight of these genes, YWHAZ, GAS2, IL8, IL6, PBEF1, CCL4, SAT and MMRN, showed comparable differential expression in additional CML patient samples. This set of genes can be considered as a starting point for further research on causes of disease transformation in CML and may lead to new targets in the treatment of resistant CML.

Apoptosis↗
PubMed2004

MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in acute myeloid leukaemia.

Outgrowth of minimal residual disease (MRD) in acute myeloid leukaemia (AML) is responsible for the occurrence of relapses. MRD can be quantified by immunophenotyping on a flow cytometer using the expression of leukaemia-associated phenotypes. MRD was monitored in follow-up samples taken from bone marrow (BM) of 72 patients after three different cycles of chemotherapy and from autologous peripheral blood stem cell (PBSC) products. The MRD% in BM after the first cycle (n=51), second cycle (n=52) and third cycle (n=30), as well as in PBSC products (n=39) strongly correlated with relapse-free survival. At a cutoff level of 1% after the first cycle and median cutoff levels of 0.14% after the second, 0.11% after the third cycle and 0.13% for PBSC products, the relative risk of relapse was a factor 6.1, 3.4, 7.2 and 5.7, respectively, higher for patients in the high MRD group. Also, absolute MRD cell number/ml was highly predictive of the clinical outcome. After the treatment has ended, an increase of MRD% predicted forthcoming relapses, with MRD assessment intervals of < or =3 months. In conclusion, MRD parameter assessment at different stages of disease is highly reliable in predicting survival and forthcoming relapses in AML.

Acute Disease↗
PubMed2004

Gene expression profiling of minimal residual disease in acute myeloid leukaemia by novel multiplex-PCR-based method.

In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.

Acute Disease↗
PubMed2003

High percentage of CD34-positive cells in autologous AML peripheral blood stem cell products reflects inadequate in vivo purging and low chemotherapeutic toxicity in a subgroup of patients with poor clinical outcome.

In this study, a high CD34% in autologous peripheral blood stem cell (PBSC) products from 71 AML patients was associated directly with a high relapse rate (P = 0.006) and inversely with disease-free survival (P = 0.003), irrespective whether patients were transplanted or not. The relapse rate at 12 months was 67% in a group with >0.8% CD34+ cells and 34% in a group with < or = 0.8% CD34+ cells. Although the percentage of malignant CD34+ cells in the CD34+ compartment in the relapses of the first group was not high (median 8%), the total number of malignant cells as a percentage of WBC was about 13 times higher than for the patients remaining >12 months in remission. When all patients evaluable were taken together, this frequency of malignant cells correlated strongly with disease-free survival (P < 0.001). Both this massive mobilization of normal CD34+ cells and high frequency of malignant cells in the subgroup of patients with CD34 >0.8% and relapse within 12 months indicate an insufficient in vivo purging, as well as low chemotherapeutic bone marrow toxicity. This was confirmed by an inverse correlation between hypoplasia period after the induction therapy and CD34% in PBSC products (P < 0.002). It is concluded that a subgroup of patients has been identified that might benefit from a more intensive chemotherapeutic treatment.

Acute Disease↗
PubMed2003

A flow cytometric method to detect apoptosis-related protein expression in minimal residual disease in acute myeloid leukemia.

Minimal residual disease (MRD) cells are thought to be responsible for the persistence and relapse of acute myeloid leukemia (AML). Flow cytometric MRD detection by the establishment of a leukemia-associated phenotype (LAP) at diagnosis can be used in 80% of AML patients, allowing detection and functional characterization of MRD in follow-up bone marrow. One of the mechanisms contributing to inefficient chemotherapy is apoptosis resistance. Measuring apoptosis parameters in MRD cells will help to unravel the importance of apoptosis resistance in AML. We therefore developed a four-color flow cytometry method that enables establishment of apoptosis-related protein expression such as Bcl-2, Bcl-x(L), Mcl-1 and Bax at diagnosis and in MRD. Firstly, validation of this assay using Western blot analysis in five leukemia cell lines showed a significant correlation (R=0.70: P<0.0001). Secondly, the influence of the permeabilization procedure on LAP expression was investigated in 38 AML samples at diagnosis and in 42 MRD samples. Quantification of the frequency of LAP+ cells with and without permeabilization showed no significant differences (diagnosis: P= 0.57, follow-up: P= 0.43). The flow cytometric protocol thus enables analysis of apoptosis-related proteins at different stages of the disease, which will lead to a better understanding of the role of apoptosis resistance in the emergence of MRD in AML.

Acute Disease↗
PubMed2002

Extensive early apoptosis in frozen-thawed CD34-positive stem cells decreases threshold doses for haematological recovery after autologous peripheral blood progenitor cell transplantation.

Stem cell doses necessary for engraftment after myelo-ablative therapy as defined for fresh transplants vary largely. Loss of CD34+ cell quality after cryopreservation might contribute to this variation. With a new early apoptosis assay including the vital stain Syto16, together with the permeability marker 7-AAD, CD34+ cell viability in leucapheresis samples of 49 lymphoma patients receiving a BEAM regimen was analysed. After freeze-thawing large numbers of non-viable, early apoptotic cells appeared, leading to only 42% viability compared to 72% using 7-AAD only. Based on this Syto16 staining in the frozen-thawed grafts, threshold numbers for adequate haematological recovery of 2.8-3.0 x 10(6) CD34+ cells/kg body weight determined for fresh grafts, now decreased to 1.2-1.3 x 10(6) CD34+ cells/kg. In whole blood transplantation of lymphoma patients (n = 45) receiving a BEAM-like regimen, low doses of CD34+ cells were sufficient for recovery (0.3-0.4 x 10(6)CD34+ cells/kg). In contrast to freeze-thawing of leucapheresis material, a high viability of CD34+ cells was preserved during storage for 3 days at 4 degrees C, leaving threshold doses for recovery unchanged. In conclusion, the Syto16 assay reveals the presence of many more non-functional stem cells in frozen-thawed transplants than presumed thus far. This led to a factor 2.3-fold adjustment downward of viable CD34+ threshold doses for haematological recovery.

Antigens, CD34↗
PubMed2001

Monocyte-chemoattractant-protein-1-mediated migration of human monocytes towards blasts from patients with acute myeloid leukemia.

PURPOSE: In the present study the possible clinical relevance of monocyte chemoattractant protein (MCP)-1 in patients with acute myeloid leukemia (AML) was established. METHODS: The pattern of migration of human monocytes towards the supernatants of blasts from 15 patients with AML was studied and the role of MCP-1, produced by these blasts, was assessed. RESULTS: In 4 patients (group 1) the amount of monocyte migration was low and not inhibited by the addition of anti-hMCP-1. In 11 patients, the amount of monocyte migration was high; after addition of anti-hMCP-1, monocyte migration was either completely (8 patients, group 2), or partly or not (3 patients, group 3) inhibited to the level of chemokinesis. In groups 1 and 2, there was a good correlation (r = 0.67) between the concentration of MCP-1 in the supernatants and the amount of monocyte migration. In group 3, such a correlation was not evident, suggesting that another chemokine might be involved or MCP-1 function was impaired by an unknown substance. Finally, measurements of MCP-1 during culture of AML blasts showed that the time at which maximal amounts of MCP-1 are produced differs between the AML samples. CONCLUSIONS: AML blasts produce different amounts of MCP-1, which plays an important role in monocyte migration towards most AML blasts. Therefore, in the context of adoptive immunotherapy, MCP-1 might be involved in future tumor vaccination programmes using autologous MCP-1-transfected irradiated AML blasts.

Cancer Vaccines↗
PubMed2001

Large populations of non-clonogenic early apoptotic CD34-positive cells are present in frozen-thawed peripheral blood stem cell transplants.

Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain SytoR 16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34(+) peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols. In a cell line model as well as CD34(+) progenitor cells, different subpopulations with decreased SytoR 16 fluorescence (SytoR 16int or SytoR 16low, compared with the normal SytoR 16high) appeared which are not, or only partly, apoptotic using conventional techniques including morphology or 7-AAD staining: eg percentages of SytoR 16(int)/7-AAD(-) and SytoR 16(low)/7-AAD(-) may amount to the majority of cells present in a particular CD34(+) sample. Second, upon further incubation these subpopulations become late apoptotic/secondary necrotic much faster than the unmodified SytoR 16high population, as determined with 7-AAD staining and morphology. Third, these cells have strongly or completely reduced clonogenic capacity for committed (CFU-GM) and early (LTC-IC, determined only for CD34(+) cells) progenitors. This technique needs the inclusion of a blocker of P-glycoprotein, which is highly active in CD34(+) progenitor cells. This prevents the interference of the detection of SytoR16(low) apoptotic cells by SytoR 16low cells resulting from P-glycoprotein activity. By comparison with other apoptosis markers we found that early apoptotic subpopulations were detected in the order SytoR 16 > annexin V > 7-AAD. In conclusion, the combination of SytoR 16 and 7-AAD detects apoptotic events earlier than conventional apoptosis techniques or annexin V. Compared to the presently available viability tests, it allows a much better estimation of the number of viable clonogenic CD34(+) cells after freeze/thawing.

Annexin A5↗
PubMed2001

High-dose melphalan with G-CSF-stimulated whole blood rescue followed by stem cell harvesting and busulphan/cyclophosphamide with autologous stem cell transplantation in multiple myeloma.

In 90 consecutive patients with multiple myeloma, we investigated the feasibility of administering a tandem high-dose therapy regimen, using whole blood for rescue after the first and leucapheresis harvested between the two high doses, for rescue after the second high dose. After 5 days of G-CSF 1 litre of whole blood (WB) was obtained, left undisturbed at 4 degrees C and reinfused 24 h after HDM (140 mg/m(2)). Patients not in progression after 3-6 months were again mobilised, leucapheresed and treated with busulphan 16 mg/kg and cyclophosphamide 120 mg/kg (Bu/Cy) and reinfusion. In 90 patients, WB contained a mean (range) of 0.57 (0.02-3.22) x 10(6)/kg CD34(+) cells. Recovery after HDM was in 13 days for granulocytes and in 18 days for platelets, with 11 patients not recovering within 3 months. There were three toxic deaths. Sixty-six patients qualified for harvesting after HDM. In the first 11, marrow was harvested. The subsequent 55 patients were mobilised and in 45 the preset minimum of 1.5 x 10(6) CD34(+) cells was obtained. Forty-nine patients actually received Bu/Cy. Recovery after Bu/Cy and marrow reinfusion was in 35 days for granulocytes and 20 days for platelets, with two of five patients not recovering after 3 months. After Bu/Cy and leucapheresis reinfusion, recovery was in 17 days for granulocytes and in 34 days for platelets. Nine patients did not recover within 3 months. There were four toxic deaths. The median overall survival from diagnosis for patients receiving HDM was 49 months and for patients also receiving Bu/Cy, 84 months. We conclude that WB rescue after HDM followed by leucapheresis and a second transplant is feasible in the majority of patients. Better mobilisation techniques are required to increase the number of patients who can receive the second transplant.

Adult↗
PubMed2001

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 degrees 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, alpha-MEM, X-VIVO15, CellGro SCGM and Leibovitz's L15 medium) containing either serum, serum-free substitutes or no additives. Leibovitz's 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 degrees C. Storage at 4 degrees 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 Leibovitz's 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.

Adenine↗
PubMed2001

Functional characterization of minimal residual disease for P-glycoprotein and multidrug resistance protein activity in acute myeloid leukemia.

Relapse is common in acute myeloid leukemia (AML) due to persistence of residual leukemia cells: minimal residual disease (MRD). In 102 out of 127 patients (80%), cells at diagnosis displayed one or more leukemia-associated phenotypes (LAP), ie combinations of cell surface markers which are absent in normal cells and can thus be used to detect MRD at follow-up. Functional characterization of MRD cells for P-glycoprotein (Pgp) and multidrug resistance protein (MRP) activity is essential to investigate the role of these drug transport proteins in multidrug resistance in AML. A fluorescent probe assay using Syto16/PSC833 and calcein-AM/probenecid as substrate/modulator of the Pgp and MRP pump, respectively, and subsequent labeling of cells with monoclonal antibodies for LAP detection allowed simultaneous detection of LAP and Pgp or MRP activity. Validation of this assay is shown for 30 newly diagnosed AML and 11 MRD situations. In addition, no significant differences were found when comparing fresh and cryopreserved de novo AML for LAP expression (n = 43), Pgp (n = 30) and MRP (n = 24) function and for MRD samples for simultaneous LAP expression and Pgp/MRP activity (n = 10). This approach enables longitudinal and multicenter studies on the detection, quantification and functional characterisation of MRD cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

A single-step colony-forming unit assay for unseparated mobilized peripheral blood, cord blood, and bone marrow.

The colony-forming unit (CFU) assay is exposed to a lot of variation, part of which is introduced by several enrichment strategies that are routinely performed before assessment of clonogenic capacity in mobilized peripheral blood (PB), bone marrow (BM), or cord blood (CB). We investigated the possibility to perform a single-step CFU assay by direct plating of PB, BM, or CB into CFU culture medium to obtain more reproducible results than after a standard Ficoll or lysis procedure. Direct plating implies the presence of red blood cells (RBC), white blood cells (WBC), and plasma in the CFU assay, which could possibly influence the outcome of the assay. Of all components, only the RBC was found to negatively influence CFU-GM growth if a concentration of > 0.02 x 10(9)/ml was present in the CFU culture medium. Subsequently, depending on the RBC concentration PB, BM, and CB samples were prediluted in triplicate or quadruplicate and plated into CFU medium. Lysis and/or Ficoll procedures were also performed in triplicate or quadruplicate on the same samples, and the mean colony number and coefficient of variation (CV) of the three techniques were compared. Significantly smaller CV values were found using the direct plating technique (all assays, mean 7.5%, range 1.6-15.6%) than after Ficoll separation (mean 18.0%, range 2.2-62.5%). Intermediate results were obtained with the lysis method (mean CV 11.6%, range 3.3-29%). In most samples, and especially in those with a very low number of clonogenic cells per milliliter, more colonies were detected with the direct plating method than with either the lysis or Ficoll method. In conclusion, the single-step direct plating method significantly enhances reproducibility of the CFU assay for PB, BM, and CB samples in comparison with standard techniques by circumvention of loss of colony formation and by decreasing variability. Furthermore, the direct plating technique is a timesaving assay.

Blood Cells↗