Velden, V.H.J. van der

No significant prognostic value of normal precursor B-cell regeneration in paediatric acute myeloid leukaemia after induction treatment (Article)

2013-04-19T00:00:00Z

B-cell precursors (BCP) regeneration in bone marrow (BM) after induction chemotherapy is prognostic for good treatment response in adult acute myeloid leukaemia (AML). We detected BCP regeneration in 81% of 59 paediatric AML patients at first complete remission; this compared to 46% in an adult study. BCP regeneration did not correlate with outcome or minimal residual disease levels. In 36 healthy BM controls, BCP levels were significantly higher in children as compared to adults. Therefore, BCP regeneration does not reflect good response to treatment in paediatric AML, possibly due to the relatively high base-line levels of BCP in children.

Applicability of a reproducible flow cytometry scoring system in the diagnosis of refractory cytopenia of childhood (Article)

2013-03-15T00:00:00Z

Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia (Article)

2013-03-01T00:00:00Z

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics.

Telomere length and telomerase complex mutations in pediatric acute myeloid leukemia (Article)

2013-02-21T00:00:00Z

The novel calicheamicin-conjugated CD22 antibody inotuzumab ozogamicin (CMC-544) effectively kills primary pediatric acute lymphoblastic leukemia cells (Article)

2012-02-01T00:00:00Z

We investigated whether the newly developed antibody (Ab) -targeted therapy inotuzumab ozogamicin (CMC-544), consisting of a humanized CD22 Ab linked to calicheamicin, is effective in pediatric primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells in vitro, and analyzed which parameters determine its efficacy. CMC-544 induced dose-dependent cell kill in the majority of BCP-ALL cells, although IC 50 values varied substantially (median 4.8 ng/ml, range 0.1-1000 ng/ml at 48 h). The efficacy of CMC-544 was highly dependent on calicheamicin sensitivity and CD22/CMC-544 internalization capacity of BCP-ALL cells, but hardly on basal and renewed CD22 expression. Although CD22 expression was essential for uptake of CMC-544, a repetitive loop of CD22 saturation, CD22/CMC-544 internalization and renewed CD22 expression was not required to achieve intracellular threshold levels of calicheamicin sufficient for efficient CMC-544-induced apoptosis in BCP-ALL cells. This is in contrast to studies with the comparable CD33 immunotoxin gemtuzumab ozogamicin (Mylotarg) in acute myeloid leukemia (AML) patients, in which complete and prolonged CD33 saturation was required for apoptosis induction. These data suggest that CMC-544 treatment may result in higher response rates in ALL compared with response rates obtained in AML with Mylotarg, and that therefore clinical studies in ALL, preferably with multiple low CMC-544 dosages, are warranted.

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 (Article)

2011-10-06T00:00:00Z

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.

Targeted drug delivery by gemtuzumab ozogamicin: Mechanism-Based mathematical model for treatment strategy improvement and therapy individualization (Article)

2011-09-13T00:00:00Z

Gemtuzumab ozogamicin (GO) is a chemotherapy-conjugated anti-CD33 monoclonal antibody effective in some patients with acute myeloid leukemia (AML). The optimal treatment schedule and optimal timing of GO administration relative to other agents remains unknown. Conventional pharmacokinetic analysis has been of limited insight for the schedule optimization. We developed a mechanism-based mathematical model and employed it to analyze the time-course of free and GO-bound CD33 molecules on the lekemic blasts in individual AML patients treated with GO. We calculated expected intravascular drug exposure (I-AUC) as a surrogate marker for the response to the drug. A high CD33 production rate and low drug efflux were the most important determinants of high I-AUC, characterizing patients with favorable pharmacokinetic profile and, hence, improved response. I-AUC was insensitive to other studied parameters within biologically relevant ranges, including internalization rate and dissociation constant. Our computations suggested that even moderate blast burden reduction prior to drug administration enables lowering of GO doses without significantly compromising intracellular drug exposure. These findings indicate that GO may optimally be used after cyto-reductive chemotherapy, rather than before, or concomitantly with it, and that GO efficacy can be maintained by dose reduction to 6 mg/m2and a dosing interval of 7 days. Model predictions are validated by comparison with the results of EORTC-GIMEMA AML19 clinical trial, where two different GO schedules were administered. We suggest that incorporation of our results in clinical practice can serve identification of the subpopulation of elderly patients who can benefit most of the GO treatment and enable return of the currently suspended drug to clinic.

Late recurrence of childhood T-cell acute lymphoblastic leukemia frequently represents a second leukemia rather than a relapse: First evidence for genetic predisposition (Article)

2011-04-20T00:00:00Z

Purpose Relapse of childhood T-cell acute lymphoblastic leukemia (T-ALL) often occurs during treatment, but in some cases, leukemia re-emerges off therapy. On the basis of previous analyses of T-cell receptor (TCR) gene rearrangement patterns, we hypothesized that some late recurrences of T-ALL might in fact represent second leukemias. Patients and Methods In 22 patients with T-ALL who had late relapses (at least 2.5 years from diagnosis), we studied TCR gene rearrangement status at first and second presentation, NOTCH1 gene mutations, and the presence of the SIL-TAL1 gene fusion. We performed genome-wide copy number and homozygosity analysis by using oligonucleotide- and single nucleotide polymorphism (SNP) -based arrays. Results We found evidence of a common clonal origin between diagnosis and relapse in 14 patients (64%). This was based on concordant TCR gene rearrangements (12 patients) or concordant genetic aberrations, as revealed by genome-wide copy number analysis (two patients). In the remaining eight patients (36%), TCR gene rearrangement sequences had completely changed between diagnosis and relapse, and gene copy number analysis showed markedly different patterns of genomic aberrations, suggesting a second T-ALL rather than a resurgence of the original clone. Moreover, NOTCH1 mutation patterns were different at diagnosis and relapse in five of these eight patients. In one patient with a second T-ALL, SNP analysis revealed a germline del(11)(p12; p13), a known recurrent aberration in T-ALL. Conclusion More than one third of late T-ALL recurrences are, in fact, second leukemias. Germline genetic abnormalities might contribute to the susceptibility of some patients to develop T-ALL. Copyright

Long-term survival after significant treatment reduction in a patient with CBF-AML (Article)

2011-02-01T00:00:00Z

Integrated use of minimal residual disease classification and IKZF1 alteration status accurately predicts 79% of relapses in pediatric acute lymphoblastic leukemia (Article)

2010-11-19T00:00:00Z

Response to therapy as determined by minimal residual disease (MRD) is currently used for stratification in treatment protocols for pediatric acute lymphoblastic leukemia (ALL). However, the large MRD-based medium risk group (MRD-M; 50-60% of the patients) harbors many relapses. We analyzed MRD in 131 uniformly treated precursor-B-ALL patients and evaluated whether combined MRD and IKZF1 (Ikaros zinc finger-1) alteration status can improve risk stratification. We confirmed the strong prognostic significance of MRD classification, which was independent of IKZF1 alterations. Notably, 8 of the 11 relapsed cases in the large MRD-M group (n=81; 62%) harbored an IKZF1 alteration. Integration of both MRD and IKZF1 status resulted in a favorable outcome group (n=104; 5 relapses) and a poor outcome group (n=27; 19 relapses), and showed a stronger prognostic value than each of the established risk factors alone (hazard ratio (95%CI): 24.98 (8.29-75.31)). Importantly, whereas MRD and IKZF1 status alone identified only 46 and 54% of the relapses, respectively, their integrated use allowed prediction of 79% of all the relapses with 93% specificity. Because of the unprecedented sensitivity in upfront relapse prediction, the combined parameters have high potential for future risk stratification, particularly for patients originally classified as non-high risk, such as the large group of MRD-M patients.Leukemia advance online publication, 19 November 2010; doi:10.1038/leu.2010.275.

The potential use of basigin (CD147) as a prognostic marker in B-cell precursor acute lymphoblastic leukaemia (Article)

2010-09-01T00:00:00Z

Detection of fusion genes at the protein level in leukemia patients via the flow cytometric immunobead assay (Article)

2010-09-01T00:00:00Z

Nowadays, the presence of specific genetic aberrations is progressively used for classification and treatment stratification, because acute leukemias with the same oncogenetic aberration generally form a clinically and diagnostically homogenous disease entity with comparable prognosis. Many oncogenetic aberrations in acute leukemias result in a fusion gene, which is transcribed into fusion transcripts and translated into fusion proteins, which are assumed to play a critical role in the oncogenetic process. Fusion gene aberrations are detected by karyotyping, FISH, or RT-PCR analysis. However, these molecular genetic techniques are laborious and time consuming, which is in contrast to flow cytometric techniques. Therefore we developed a flow cytometric immunobead assay for detection of fusion proteins in lysates of leukemia cell samples by use of a bead-bound catching antibody against one side of the fusion protein and fluorochrome-conjugated detection antibody. So far, we have been able to design such fusion protein immunobead assays for BCR-ABL, PML-RARA, TEL-AML1, E2A-PBX1, MLL-AF4, AML1-ETO and CBFB-MYH11. The immunobead assay for detection of fusion proteins can be performed within 3 to 4 hours in a routine diagnostic setting, without the need of special equipment other than a flow cytometer. The novel immunobead assay will enable fast and easy classification of acute leukemia patients that express fusion proteins. Such patients can be included at an early stage in the right treatment protocols, much faster than by use of current molecular techniques. The immunobead assay can be run in parallel to routine immunophenotyping and is particularly attractive for clinical settings without direct access to molecular diagnostics.

Clinical significance of flowcytometric minimal residual disease detection in pediatric acute myeloid leukemia patients treated according to the DCOG ANLL97/MRC AML12 protocol (Article)

2010-09-01T00:00:00Z

Analysis of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) may predict for clinical outcome. MRD levels were assessed by flowcytometric immunophenotyping in 94 children with AML enrolled into a single trial (United Kingdom Medical Research Council AML12 and similar Dutch Childhood Oncology Group ANLL97). An aberrant immunophenotype could be detected in 94% of patients. MRD levels after the first course of chemotherapy predicted for clinical outcome: 3-year relapse-free survival was 85%8% (s.e.) for MRD-negative patients (MRD0.1%), 64%10% for MRD-low-positive patients (0.1%MRD0.5%) and only 149% for MRD-high-positive patients (MRD0.5%; P0.001), whereas overall survival was 95%5%, 70%10% and 40%13%, respectively, (P0.001). Multivariate analysis allowing for age, karyotype, FLT3-internal tandem duplications and white blood cell count at diagnosis showed that MRD after the first course of chemotherapy was an independent prognostic factor. Although comparison of paired diagnosis-relapse samples (n23) showed immunophenotypic shifts in 91% of cases, this did not hamper MRD analysis. In conclusion, flowcytometric MRD detection is possible in children with AML. The level of MRD after the first course of chemotherapy provides prognostic information that may be used to guide therapy.

Preemptive alloimmune intervention in high-risk pediatric acute lymphoblastic leukemia patients guided by minimal residual disease level before stem cell transplantation (Article)

2010-08-01T00:00:00Z

Relapse of pediatric acute lymphoblastic leukemia (ALL) remains the main cause of treatment failure after allogeneic stem cell transplantation (alloSCT). A high level of minimal residual disease (MRD) before alloSCT has been shown to predict these relapses. Patients at risk might benefit from a preemptive alloimmune intervention. In this first prospective, MRD-guided intervention study, 48 patients were stratified according to pre-SCT MRD level. Eighteen children with MRD level 1 × 10 4 were eligible for intervention, consisting of early cyclosporine A tapering followed by consecutive, incremental donor lymphocyte infusions (n1-4). The intervention was associated with graft versus host disease grade II in only 23% of patients. Event-free survival in the intervention group was 19%. However, in contrast with the usual early recurrence of leukemia, relapses were delayed up to 3 years after SCT. In addition, several relapses presented at unusual extramedullary sites suggesting that the immune intervention may have altered the pattern of leukemia recurrence. In 8 out of 11 evaluable patients, relapse was preceded by MRD recurrence (median 9 weeks, range 0-30). We conclude that in children with high-risk ALL, immunotherapy-based regimens after SCT are feasible and may need to be further intensified to achieve total eradication of residual leukemic cells.

IKZF1 deletions predict relapse in uniformly treated pediatric precursor B-ALL (Article)

2010-07-01T00:00:00Z

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL) and is often difficult to predict. To explore the prognostic impact of recurrent DNA copy number abnormalities on relapse, we performed high-resolution genomic profiling of 34 paired diagnosis and relapse ALL samples. Recurrent lesions detected at diagnosis, including PAX5, CDKN2A and EBF1, were frequently absent at relapse, indicating that they represent secondary events that may be absent in the relapse-prone therapy-resistant progenitor cell. In contrast, deletions and nonsense mutations in IKZF1 (IKAROS) were highly enriched and consistently preserved at the time of relapse. A targeted copy number screen in an unselected cohort of 131 precursor B-ALL cases, enrolled in the dexamethasone-based Dutch Childhood Oncology Group treatment protocol ALL9, revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Separate analysis of ALL9-treatment subgroups revealed that non-high-risk (NHR) patients with IKZF1 deletions exhibited a ∼12-fold higher relative relapse rate than those without IKZF1 deletions. Consequently, IKZF1 deletion status allowed the prospective identification of 53% of the relapse-prone NHR-classified patients within this subgroup and, therefore, serves as one of the strongest predictors of relapse at the time of diagnosis with high potential for future risk stratification.Leukemia advance online publication, 6 May 2010; doi:10.1038/leu.2010.87.

Bimodal distribution of genomic MLL breakpoints in infant acute lymphoblastic leukemia treatment (Article)

2010-04-01T00:00:00Z

Genome-wide expression analysis of paired diagnosis-relapse samples in ALL indicates involvement of pathways related to DNA replication, cell cycle and DNA repair, independent of immune phenotype (Article)

2010-03-01T00:00:00Z

Almost a quarter of pediatric patients with acute lymphoblastic leukemia (ALL) suffer from relapses. The biological mechanisms underlying therapy response and development of relapses have remained unclear. In an attempt to better understand this phenomenon, we have analyzed 41 matched diagnosis-relapse pairs of ALL patients using genome-wide expression arrays (82 arrays) on purified leukemic cells. In roughly half of the patients, very few differences between diagnosis and relapse samples were found (stable group), suggesting that mostly extra-leukemic factors (for example, drug distribution, drug metabolism, compliance) contributed to the relapse. Therefore, we focused our further analysis on 20 sample pairs with clear differences in gene expression (skewed group), reasoning that these would allow us to better study the biological mechanisms underlying relapsed ALL. After finding the differences between diagnosis and relapse pairs in this group, we identified four major gene clusters corresponding to several pathways associated with changes in cell cycle, DNA replication, recombination and repair, as well as B-cell developmental genes. We also identified cancer genes commonly associated with colon carcinomas and ubiquitination to be upregulated in relapsed ALL. Thus, about half of the relapses are due to the selection or emergence of a clone with deregulated expression of genes involved in pathways that regulate B-cell signaling, development, cell cycle, cellular division and replication.

Reduced versus intensive chemotherapy for childhood acute lymphoblastic leukemia: Impact on lymphocyte compartment composition (Article)

2010-01-01T00:00:00Z

Chemotherapy for childhood acute lymphoblastic leukemia may cause severe immune damage. The lymphocyte compartment of 140 patients during and after a new strongly reduced (standard risk (SR), n = 43) and intensive chemotherapy regimen (medium risk (MR), n = 97) was studied between 2006 and 2009. Transitional and naive B cells and IgG+/A+, IgM+ and IgM only memory B cells were significantly reduced during chemotherapy; significantly more in MR group. One year after treatment CD27+IgG+/A+, IgM+ and IgM only memory B cells had still not fully recovered, but this was not confined to the MR group. The T cell compartment was less but also significantly affected during chemotherapy and recovered to normal levels. In the MR group, NK cells had not fully recovered to normal levels 1 year after treatment. Thus, intensive chemotherapy regimens cause severe, mainly B cell memory damage that persists even 1 year after treatment.

Cluster analysis of genomic ETV6-RUNX1 (TEL-AML1) fusion sites in childhood acute lymphoblastic leukemia (Article)

2009-08-01T00:00:00Z

Fusion between ETV6 and RUNX1 defines the largest genetic subgroup in childhood ALL. The genomic fusion site, unique to individual patients and specific for the malignant clone, represents an ideal molecular marker for quantification of minimal residual disease. Sequencing of DNA breakpoints has been difficult due to the extended size of the respective breakpoint cluster regions. We therefore evaluated a specially designed multiplex long-range PCR assay in 65 diagnostic bone marrow samples for its suitability in routine use. Resulting fusion sites and breakpoints derived from previous studies were subject to cluster analysis to identify potential sequence motifs involved in translocation initiation.

Standardization of flow cytometry in myelodysplastic syndromes: Report from the first European LeukemiaNet working conference on flow cytometry in myelodysplastic syndromes (Article)

2009-08-01T00:00:00Z

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.

MRD detection in acute lymphoblastic leukemia patients using Ig/TCR gene rearrangements as targets for real-time quantitative PCR. (Article)

2009-05-26T00:00:00Z

Minimal residual disease (MRD) diagnostics has proven to be clinically relevant for evaluation of treatment effectiveness in patients with acute lymphoblastic leukemia (ALL). In most ALL treatment protocols, MRD diagnostics is performed by real-time quantitative PCR (RQ-PCR) analysis of the junctional regions of rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes.MRD diagnostics via Ig/TCR genes is broadly applicable (>95% of ALL patients) and can reach a good sensitivity (< or =10 (-4)). However, the technique is complex and requires extensive knowledge and experience, because the junctional regions of each leukemia have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal Ig/TCR gene rearrangements in ALL cells at diagnosis to the actual MRD measurements in clinical follow-up samples. This information aims at facilitating the PCR-based MRD diagnostics in ALL patients. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients (Article)

2009-04-24T00:00:00Z

BCR-ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR-ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR-ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR-ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR-ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR-ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR-ABL immunobead assay detects all types of BCR-ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within ∼4h, and can be run in parallel to routine immunophenotyping.

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: A European multicenter study (Article)

2009-03-27T00:00:00Z

A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.

Identification of new microRNA genes and aberrant microRNA profiles in childhood acute lymphoblastic leukemia (Article)

2009-02-19T00:00:00Z

MicroRNAs (miRNAs) control the expression of protein-coding genes in normal hematopoietic cells and, consequently, aberrant expression may contribute to leukemogenesis. To identify miRNAs relevant to pediatric acute lymphoblastic leukemia (ALL), we cloned 105 known and 8 new miRNA genes expressed in patients' leukemia cells. Instead of known miRNA genes, new miRNA genes were not evolutionarily conserved. Quantification of 19 selected miRNA genes revealed an aberrant expression in ALL as compared with normal CD34+ cells (P<0.02); both upregulated (14/19) and downregulated (5/19) expressions were observed. Eight miRNAs were differentially expressed between MLL and non-MLL precursor B-ALL cases (P<0.05). Most remarkably, miR-708 was 250- up to 6500-fold higher expressed in 57 TEL-AML1, BCR-ABL, E2A-PBX1, hyperdiploid and B-other cases than in 20 MLL-rearranged and 15 T-ALL cases (0.0001< P<0.01), whereas the expression of miR-196b was 500-fold higher in MLL-rearranged and 800-fold higher in 5 of 15 T-ALL cases as compared with the expression level in the remaining precursor B-ALL cases (P<0.001). The expression did not correlate with the maturation status of leukemia cells based on immunoglobulin and T-cell receptor rearrangements, immunophenotype or MLL-fusion partner. In conclusion, we identified new miRNA genes and showed that miRNA expression profiles are ALL subtype-specific rather than linked to the differentiation stadium associated with these subtypes.

Prognostic significance of minimal residual disease in infants with acute lymphoblastic leukemia treated within the Interfant-99 protocol (Article)

2009-02-13T00:00:00Z

Acute lymphoblastic leukemia (ALL) in infants younger than 1 year is a rare but relatively homogeneous disease (∼80% MLL gene rearranged, ∼70% CD10-negative) when compared with childhood and adult ALL. Several studies in children and adults with ALL have shown that minimal residual disease (MRD) status is a strong and independent prognostic factor. We therefore evaluated the prognostic significance of MRD in infant ALL. Ninety-nine infant patients treated according to the Interfant-99 protocol were included in this study. MRD was analyzed by real-time quantitative PCR analysis of rearranged immunoglobulin genes, T-cell receptor genes and MLL genes at various time points (TP) during therapy. Higher MRD levels at the end of induction (TP2) and consolidation (TP3) were significantly associated with lower disease-free survival. Combined MRD information at TP2 and TP3 allowed recognition of three patients groups that significantly differed in outcome. All MRD-high-risk patients (MRD levels ≥10-4 at TP3; 26% of patients) relapsed. MRD-low-risk patients (MRD level <10-4 at both TP2 and TP3) constituted 44% of patients and showed a relapse-rate of only 13%, whereas remaining patients (MRD-medium-risk patients; 30% of patients) had a relapse rate of 31%. Comparison between the current Interfant-06 stratification at diagnosis and the here presented MRD-based stratification showed that both stratifications recognized different subgroups of patients. These data indicate that MRD diagnostics has added value for recognition of risk groups in infant ALL and that MRD diagnostics can be used for treatment intervention in infant ALL as well.Leukemia advance online publication, 12 February 2009; doi:10.1038/leu.2009.17.

Chromosome 14 copy number-dependent IGH gene rearrangement patterns in high hyperdiploid childhood B-cell precursor ALL: implications for leukemia biology and minimal residual disease analysis (Article)

2009-01-16T00:00:00Z

Childhood B-cell precursor acute lymphoblastic leukemia (BCP ALL) is generally a clonal disease in which the number of IGH rearrangements per cell does not exceed the number of the IGH alleles on chromosome 14. Consequently, monoclonal high hyperdiploid (HeH) cases with a trisomy 14 can harbor three rearrangements, a pattern that otherwise may be misinterpreted to be oligoclonal. Oligoclonal IGH rearrangements, on the other hand, may be instable at relapse and should therefore not be used for minimal residual disease analysis. We thus investigated the association between IGH allele copy numbers and the IGH rearrangement patterns in 90 HeH BCP ALL with either two (13%) or three copies (87%) of chromosome 14. HeH cases (44%) had an oligoclonal IGH rearrangement pattern, but true oligoclonality-after correction for the respective copy number of IGH alleles-was only 16%. Monoclonal and oligoclonal HeH cases had predominantly VH to preexisting DJH recombinations, a finding that contrasts with oligoclonal cases of other major genetic BCP ALL subgroups in which VH replacements prevail. We conclude that for the precise assessment and correct interpretation of clonality patterns in BCP ALL, the IGH allele copy number has to be taken into consideration.Leukemia advance online publication, 15 January 2009; doi:10.1038/leu.2008.390.

Immunophenotyping of mast cells: A sensitive and specific diagnostic tool for systemic mastocytosis (Article)

2009-01-01T00:00:00Z

Introduction: The diagnosis of systemic mastocytosis (SM) is based on a combination of major and minor criteria. Flow cytometric detection of aberrant expression of CD2 and/or CD25 on CD117-positive mast cells is one of the minor criteria used. In the present study we examined the sensitivity and specificity of mast cell immunophenotyping in the diagnosis of SM. Material and methods: Patients were 36 persons with systemic mastocytosis diagnosed according to WHO criteria. Controls were 31 patients without SM. Immunophenotyping was performed according to published guidelines. Results: All patients with SM were positive for CD2 and/or CD25. All patients without SM, except one, were negative for these markers. The sensitivity for immunophenotyping was 100%, the specificity 91%. The positive and negative predictive values were 97% and I00% respectively. Conclusion: Immunophenotyping of bone marrow derived mast cells is not only a very sensitive but also a very specific method to diagnose SM with high positive and negative predictive value.

Improving minimal residual disease detection in precursor B-ALL based on immunoglobulin-κ and heavy-chain gene rearrangements (Article)

2008-07-18T00:00:00Z

Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia (Article)

2008-04-01T00:00:00Z

Detection of minimal residual disease (MRD) is the most sensitive method to evaluate treatment response and one of the strongest predictors of outcome in childhood acute lymphoblastic leukemia (ALL). The 10-year update on the I-BFM-SG MRD study 91 demonstrates stable results (event-free survival), that is, standard risk group (MRD-SR) 93%, intermediate risk group (MRD-IR) 74%, and high risk group (MRD-HR) 16%. In multicenter trial AIEOP-BFM ALL 2000, patients were stratified by MRD detection using quantitative PCR after induction (TP1) and consolidation treatment (TP2). From 1 July 2000 to 31 October 2004, PCR target identification was performed in 3341 patients: 2365 (71%) patients had two or more sensitive targets (≥10-4), 671 (20%) patients revealed only one sensitive target, 217 (6%) patients had targets with lower sensitivity, and 88 (3%) patients had no targets. MRD-based risk group assignment was feasible in 2594 (78%) patients: 40% were classified as MRD-SR (two sensitive targets, MRD negativity at both time points), 8% as MRD-HR (MRD ≤10-3at TP2), and 52% as MRD-IR. The remaining 823 patients were stratified according to clinical risk features: HR (n=108) and IR (n=715). In conclusion, MRD-PCR-based stratification using stringent criteria is feasible in almost 80% of patients in an international multicenter trial.

Non-specific amplification of patient-specific Ig/TCR gene rearrangements depends on the time point during therapy: Implications for minimal residual disease monitoring [5] (Article)

2008-03-01T00:00:00Z

Identification of distinct prognostic subgroups in low- and intermediate-1-risk myelodysplastic syndromes by flow cytometry (Article)

2008-02-12T00:00:00Z

The World Health Organization (WHO) classification contributes to refined classification and prognostication of myelodysplastic syndromes (MDSs). Flow cytometry might add significantly to diagnostic and prognostic criteria. Our analysis of bone marrow samples from 50 patients with MDS showed aberrant expression of differentiation antigens in the myelomonocytic lineage. This also accounted for refractory anemia (RA) with or without ringed sideroblasts (RS), indicating multilineage dysplasia. In 38% of patients, CD34+myeloid blasts expressed CD5, CD7, or CD56. Flow cytometry data were translated into a numerical MDS flow-score. Flow-scores increased significantly from RA with or without RS, refractory cytopenia with multilineage dysplasia (RCMD) with or without RS up to refractory anemia with excess of blasts-1 (RAEB-1) and RAEB-2. No significant differences were observed between WHO cytogenetic subgroups. Flow-scores were highly heterogeneous within International Prognostic Scoring System (IPSS) subgroups. Patients in progression to advanced MDS or acute myeloid leukemia had a significantly higher flow-score compared with non-transfusion-dependent patients. In 60% of patients with transfusion dependency or progressive disease, myeloid blasts expressed CD7 or CD56, in contrast to only 9% of non-transfusion-dependent patients. Moreover, all patients with pure RA with or without RS with aberrant myeloid blasts showed an adverse clinical course. In conclusion, flow cytometry in MDS identified aberrancies in the myelomonocytic lineage not otherwise determined by cytomorphology. In addition, flow cytometry identified patients at risk for transfusion dependency and/or progressive disease independent of known risk groups, which might have impact on treatment decisions and the prognostic scoring system in the near future.

Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: Towards new standards for gene expression measurements (Article)

2007-07-01T00:00:00Z

Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20°C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.

CD33 expression and P-glycoprotein-mediated drug efflux inversely correlate and predict clinical outcome in patients with acute myeloid leukemia treated with gemtuzumab ozogamicin monotherapy (Article)

2007-05-15T00:00:00Z

Gemtuzumab ozogamicin (GO) contains an anti-CD33 antibody to facilitate uptake of a toxic calicheamicin-γ1derivative. While recent in vitro data demonstrated a quantitative relationship between CD33 expression and GO cytotoxicity, previous correlative studies failed to identify a significant association between CD33 expression and clinical outcome. Studying patients undergoing GO monotherapy for relapsed acute myeloid leukemia (AML), we now find that AML blasts of responders have a significantly higher mean CD33 level and lower P-glycoprotein (Pgp) activity compared with nonresponders. CD33 expression and Pgp activity are inversely correlated. While both variables are associated with outcome, Pgp remains significantly associated with outcome even after adjusting for CD33, whereas CD33 does not show such an association after adjusting for Pgp. The inverse relationship between CD33 and Pgp suggests a maturation-stage-dependent expression of both proteins, and offers the rationale for using cell differentiation-promoting agents to enhance GO-induced cytotoxicity.

Acute lymphoblastic leukemia with t(4;11) in children 1 year and older: The 'big sister' of the infant disease? (Article)

2007-04-01T00:00:00Z

The t(4;11)-positive acute lymphoblastic leukemia (ALL) is a rare disease in children above the age of 1 year. We studied the clinical and biological characteristics in 32 consecutively diagnosed childhood cases (median age 10.0 years, range 1.0-17.1 years). Immunophenotyping revealed a pro-B and a pre-B stage in 24 and eight cases, respectively. IGH genes were rearranged in 84% of leukemias with a predominance of incomplete DJHjoints. Whereas IGK-Kde and TCRD rearrangements were rare, TCRG rearrangements were present in 50% of cases and involved mainly Vγ11 or Vγ9 together with a Jγ1.3./2.3 gene segment, an unusual combination among t(4;11)-negative B-cell precursor ALL. Oligoclonality was found in about 30% as assessed by heterogeneous IGH and TCRG rearrangements. Our data are in line with transformation of a precursor cell at an early stage of B-cell development but retaining the potential to differentiate to the pre-B cell stage in vivo. Although a distinct difference between infant and older childhood cases with t(4;11) became evident, no age-related biological features were found within the childhood age group. In contrast to infants with t(4;11)-positive ALL, childhood cases had a relatively low cumulative incidence of relapse of 25% at 3.5 years with BFM-based high-risk protocols.

Immunobiological diversity in infant acute lymphoblastic leukemia is related to the occurrence and type of MLL gene rearrangement (Article)

2007-04-01T00:00:00Z

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period.

A novel BCR-ABL fusion transcript (e18a2) in a child with chronic myeloid leukemia [13] (Article)

2007-04-01T00:00:00Z

Optimization of PCR-based minimal residual disease diagnostics for childhood acute lymphoblastic leukemia in a multi-center setting (Article)

2007-04-01T00:00:00Z

Minimal residual disease (MRD) diagnostics is used for treatment stratification in childhood acute lymphoblastic leukemia. We aimed to identify and solve potential problems in multicenter MRD studies to achieve and maintain consistent results between the AIEOP/BFM ALL-2000 MRD laboratories. As the dot-blot hybridization method was replaced by the real-time quantitative polymerase chain reaction (RQ-PCR) method during the treatment protocol, special attention was given to the comparison of MRD data obtained by both methods and to the reproducibility of RQ-PCR data. Evaluation of all key steps in molecular MRD diagnostics identified several pitfalls that resulted in discordant MRD results. In particular, guidelines for RQ-PCR data interpretation appeared to be crucial for obtaining concordant MRD results. The experimental variation of the RQ-PCR was generally less than three-fold, but logically became larger at low MRD levels below the reproducible sensitivity of the assay (<10-4). Finally, MRD data obtained by dot-blot hybridization were comparable to those obtained by RQ-PCR analysis (r2=0.74). In conclusion, MRD diagnostics using RQ-PCR analysis of immunoglobulin/T-cell receptor gene rearrangements is feasible in multicenter studies but requires standardization; particularly strict guidelines for interpretation of RQ-PCR data are required. We further recommend regular quality control for laboratories performing MRD diagnostics in international treatment protocols.

Analysis of minimal residual disease by Ig/TCR gene rearrangements: Guidelines for interpretation of real-time quantitative PCR data (Article)

2007-04-01T00:00:00Z

Most modern treatment protocols for acute lymphoblastic leukaemia (ALL) include the analysis of minimal residual disease (MRD). To ensure comparable MRD results between different MRD-polymerase chain reaction (PCR) laboratories, standardization and quality control are essential. The European Study Group on MRD detection in ALL (ESG-MRD-ALL), consisting of 30 MRD-PCR laboratories worldwide, has developed guidelines for the interpretation of real-time quantitative PCR-based MRD data. The application of these guidelines ensures identical interpretation of MRD data between different laboratories of the same MRD-based clinical protocol. Furthermore, the ESG-MRD-ALL guidelines will facilitate the comparison of MRD data obtained in different treatment protocols, including those with new drugs.

Clinically and genetically atypical T-cell prolymphocytic leukemia underlines the relevance of a multidisciplinary diagnostic approach. (Article)

2007-03-01T00:00:00Z

Vdelta2-Jalpha rearrangements are frequent in precursor-B-acute lymphoblastic leukemia but rare in normal lymphoid cells (Article)

2004-01-01T00:00:00Z

The frequently occurring T-cell receptor delta (TCRD) deletions in precursor-B-acute lymphoblastic leukemia (precursor-B-ALL) are assumed to be mainly caused by Vdelta2-Jalpha rearrangements. We designed a multiplex polymerase chain reaction tified clonal Vdelta2-Jalpha rearrangements in 141 of 339 (41%) childhood and 8 of 22 (36%) adult precursor-B-ALL. A significant proportion (44%) of Vdelta2-Jalpha rearrangements in childhood precursor-B-ALL were oligoclonal. Sequence analysis showed preferential usage of the Jalpha29 gene segment in 54% of rearrangements. The remaining Vdelta2-Jalpha rearrangements used 26 other Jalpha segments, which included 2 additional clusters, one involving the most upstream Jalpha segments (ie, Jalpha48 to Jalpha61; 23%) and the second cluster located around the Jalpha9 gene segment (7%). Real-time quantitative PCR studies of normal lymphoid cells showed that Vdelta2 rearrangements to upstream Jalpha segments occurred at low levels in the thymus (10(-2) to 10(-3)) and were rare (generally below 10(-3)) in B-cell precursors and mature T cells. Vdelta2-Jalpha29 rearrangements were virtually absent in normal lymphoid cells. The monoclonal Vdelta2-Jalpha rearrangements in precursor-B-ALL may serve as patient-specific targets for detection of minimal residual disease, because they show high sensitivity (10(-4) or less in most cases) and good stability (88% of rearrangements preserved at relapse).

Targeting of the CD33-calicheamicin immunoconjugate Mylotarg (CMA-676) in acute myeloid leukemia: in vivo and in vitro saturation and internalization by leukemic and normal myeloid cells (Article)

2001-01-01T00:00:00Z

Antibody-targeted chemotherapy is a promising therapy in patients with acute myeloid leukemia (AML). In a phase II study of Mylotarg (CMA-676, gemtuzumab ozogamicin), which consists of a CD33 antibody linked to calicheamicin, saturation and internalization by leukemic and normal myeloid cells were analyzed in 122 patients with relapsed AML. Peripheral blood samples were obtained just before and 3 and 6 hours after the start of the first and second Mylotarg treatment cycles. Within 3 to 6 hours after infusion, near complete saturation of CD33 antigenic sites by Mylotarg was reached for AML blasts, monocytes, and granulocytes, whereas Mylotarg did not bind to lymphocytes. Saturation levels prior to the start of the second Mylotarg treatment cycle were significantly increased compared with background levels before the start of the first cycle. This apparently was caused by remaining circulating Mylotarg from the first treatment cycle (approximately 2 weeks earlier). On binding of Mylotarg to the CD33 antigen, Mylotarg was rapidly internalized, as determined by the decrease in maximal surface membrane Mylotarg binding. Internalization of Mylotarg was also demonstrated in myeloid cells in vitro and was confirmed by confocal laser microscopy. In vitro studies using pulse labeling with Mylotarg showed a continuous renewed membrane expression of CD33 antigens, which can significantly increase the internalization process and thereby the intracellular accumulation of the drug. Finally, Mylotarg induced dose-dependent apoptosis in myeloid cells in vitro. These data indicate that Mylotarg is rapidly and specifically targeted to CD33(+) cells, followed by internalization and subsequent induction of cell death.

Bronchial Epithelial Cells and Peptidases: Modulation by cytokincs and glucocorticoids ill vitro and in asthma (Doctoral Thesis)

1998-06-24T00:00:00Z

The airways can be divided in the upper respiratory tract, including the nose, the pharynx, and the larynx. and the lower respiratory tract. consisting of the trachea, bronchi, bronchioles, and alveoli. This structure provides an enormous surface area where the exchange of oxygen and carbon dioxide. the function of the lungs, can take place. Respiratory diseases may affect onc or more of the different parts of the airways. For example, emphysema is characterized by a decreased number of alveoli which also have a reduced elasticity. On the other hand, asthma, the main focus of this thesis, is considered to be a disease affecting predominantly the bronchi and bronchioli.