K. Hählen

Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia

Activating mutations in RAS and receptor tyrosine kinases such as KIT and FLT3 are hypothesized to cooperate with chimeric transcription factors in the pathogenesis of acute myeloid leukemia (AML). To test this hypothesis, we genotyped 150 pediatric AML samples for mutations in KIT (exons 8, 17), NRAS and KRAS (exons 1, 2) and FLT3/ITD. This is the largest cohort of pediatric AML patients reported thus far screened for all four mutations. Of the children with AML, 40% had a mutation in KIT (11.3%), RAS (18%) or FLT3/ITD (11.1%), and 70% of cases of core-binding factor (CBF) leukemia were associated with a mutation of KIT or RAS. Mutations in RAS or FLT3/ITD were frequently found in association with a normal karyotype. Patients with a FLT3/ITD mutation had a significantly worse clinical outcome. However, the presence of a KIT or RAS mutation did not significantly influence clinical outcome. We demonstrate that KIT exon 8 mutations result in constitutive ligand-independent kinase activation that can be inhibited by clinically relevant concentrations of imatinib. Our results demonstrate that abnormalities of signal transduction pathways are frequent in pediatric AML. Future clinical studies are needed to determine whether selective targeting of these abnormalities will improve treatment results.

Cross-lineage T cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease

A large series of 202 childhood precursor-B cell acute lymphoblastic leukemia (ALL) patients was analyzed by Southern blotting (SB) for cross-lineage rearrangements and/or deletions in the T cell receptor TCRB, TCRG and TCRD loci. In 93% (187/201) of the precursor-B-ALL patients one or more genes were rearranged and/or deleted. TCRB gene rearrangements were found in 35% (69/196), TCRG gene rearrangements in 59% (113/192), TCRD gene rearrangements in 55% (112/202), and isolated monoallelic or biallelic deletions of TCRD loci in 34% (68/202) of the cases. TCRB gene rearrangements involved exclusively the Jβ2 locus with complete V(D)Jβ2 joinings in 53% of gene rearrangements and incomplete Dβ-Jβ2 gene rearrangements in 33%. TCRG gene rearrangements frequently occurred on both alleles (65% of cases) and in approximately 70% concerned rearrangements to Jγ1 gene segments. Most rearranged TCRD alleles (80%) represented incomplete Vδ2-Dδ3 or Dδ2-Dδ3 gene rearrangements, while the remaining TCRD gene rearrangements remained unidentified. Subsequently, we evaluated, whether heteroduplex PCR analysis of rearranged TCRG and TCRD genes can be used for reliable identification of PCR targets for detection of minimal residual disease (MRD). The concordance between SB and heteroduplex PCR analysis for detection of the various types of clonal TCRG and TCRD gene rearrangements ranged between 78% and 87%. The discrepancies could be assigned to the presence of x91atypical TCRD gene rearrangements or translocations only detectable by SB, but also to efficient PCR-based detection of rearrangements derived from small subclones, which are difficult to detect with SB. Indications for oligoclonality were observed in 38% and 30% of patients with TCRG and TCRD gene rearrangements, respectively, which is comparable to the frequency of oligoclonality in IGH locus. Based on the combined data it was possible to reduce the broad panel of six TCRD and 12 TCRG primer combinations for MRD studies to two TCRD combinations (Vδ2-Dδ3 and Dδ2-Dδ3) and six TCRG combinations (VγI, VγII, VγIV family-specific primers with Jγ1.1/2.1 and Jγ1.3/2.3 primers) resulting in the detection of 80% and 97% of all TCRD and TCRG gene rearrangements, respectively. Finally, the heteroduplex PCR data indicate that MRD monitoring with TCRG and/or TCRD targets is possible in approximately 80% of childhood precursor-B-ALL patients; ∼55% of patients even have two TCRG and/or TCRD targets.

Obesity after successful treatment of acute lymphoblastic leukemia in childhood.

ABSTRACT: Early and late effects of treatment for acute lymphoblastic leukemia (ALL) on weight was retrospectively investigated in 113 children in continuous first remission. Weight was examined at diagnosis up to 10 y after cessation of treatment. There was an increased prevalence of overweight after treatment for ALL which persisted over time. All treatment regimens included corticosteroid therapy, and 52 patients received additional cranial irradiation. Patients treated with and without cranial irradiation did not differ in weight gain, indicating that not cranial irradiation but corticosteroid therapy might explain weight gain in children treated for ALL. Dexamethasone was associated with a significant increase of weight at cessation of treatment. Patients treated with a combination of prednisone and dexamethasone had as a late effect the highest prevalence of obesity (44%). Gender or age at diagnosis were not related to weight gain.

The Baxα:Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia

Bcl‐2 over‐expression has been shown to inhibit apoptosis induced by a variety of stimuli, whereas a predominance of Baxα to Bcl‐2 accelerates apoptosis upon apoptotic stimuli. We sought to study the relevance of these apoptotic regulating gene products in leukaemia. In a panel of leukaemia and lymphoma cell lines (HL60, DoHH2, CEM C7, L1210 and S49), the Baxα‐to‐Bcl‐2 ratio as assessed by Western‐blot analysis correlated with sensitivity to dexamethasone treatment. In addition, in HAbaxα‐transfected CEM C7 clones, a similar correlation was found for dexamethasone and thapsigargin sensitivity. In bone‐marrow aspirates from patients with childhood acute lymphoblastic or myelocytic leukaemia (ALL, n = 48; AML, n = 8), the Bcl‐2 and Baxα levels were highly variable, but well within the range found in the Baxα transfectants and in the established cell lines. Bcl‐2 levels were lower in T‐ than in B‐lineage ALL, which could be ascribed to simultaneous inverse relation between Bcl‐2 and WBC. By contrast, Baxα:Bcl‐2 was independent of any presenting feature and was largely dependent on Baxα levels. Results suggest that Baxα:Bcl‐2, rather than Bcl‐2 alone is important for the survival of drug‐induced apoptosis in leukemic cell lines and ALL. Int. J. Cancer 71: 959‐965, 1997.

Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR

Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex analysis at diagnosis, their stability at relapse, and their applicability in real-time quantitative PCR (RQ-PCR) analysis. In 77 selected children with precursor-B-ALL, Southern blotting detected 122 IGK-Kde rearrangements, 12 of which were derived from subclones in six patients (8%). PCR/heteroduplex analysis with BIOMED-1 Concerted Action primers identified 100 of the 110 major IGK-Kde rearrangements (91%). Comparison between diagnosis and relapse samples from 21 patients with PCR-detectable IGK-Kde rearrangements (using Southern blotting, PCR/heteroduplex analysis, and sequencing) demonstrated that 27 of the 32 rearrangements remained stable at relapse. When patients with oligoclonal IGK-Kde rearrangements were excluded, 25 of the 27 rearrangements remained stable at relapse and at least one stable rearrangement was present in 17 of the 18 patients. Subsequently, RQ-PCR analysis with allele-specific forward primers, a germline Kde TaqMan-probe, and a germline Kde reverse primer was evaluated for 18 IGK-Kde rearrangements. In 16 of the 18 targets (89%) a sensitivity of ⩽10−4 was reached. Analysis of MRD during follow-up of eight patients with IGK-Kde rearrangements showed comparable results between RQ-PCR data and classical dot-blot data. We conclude that the frequently occurring IGK-Kde rearrangements are generally detectable by PCR (90%) and are highly stable MRD-PCR targets, particularly where monoclonal rearrangements at diagnosis (95%) are concerned. Furthermore, most IGK-Kde rearrangements (90%) can be used for sensitive detection of MRD (⩽10−4) by RQ-PCR analysis.

Causes of death - other than progressive leukemia - in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience

We analyzed causes of death, other than resistant disease or relapse, in 875 children with acute lymphoblastic leukemia (ALL) and 229 with acute myeloid leukemia (AML), treated on three different Dutch Childhood Oncology Group (DCOG) ALL and three AML protocols. Overall, 23 (2.6%) ALL and 44 (19.2%) AML patients died. Early death (ED, before remission was reached) occurred in nine ALL (1%) and thirty AML (13.1.%) patients, including three and ten deaths before treatment was initiated. Chemotherapy-related mortality in remission (CRM) occurred in nine ALL (1.1%) and eight AML (4.4%) patients. For ALL, both ED and CRM declined over time, although this was not statistically significant. For AML a decrease in ED was observed (from 26% to approximately 10%), but counter-balanced by an increase in CRM (from 3 to 8%), maybe related to the scheduling of intensification blocks in AML-92/94. Including transplant-related mortality, death in CR rates in AML increased from 3 to 15% in the last study. The main cause of ED was hemorrhage, often associated with hyperleucocytosis, and infection for CRM. We conclude that mortality dropped favorably in ALL, but not in AML. Especially for AML, effective but less toxic therapy and better supportive care guidelines need to be developed.

Minimal residual disease levels in bone marrow and peripheral blood are comparable in children with T cell acute lymphoblastic leukemia (ALL), but not in precursor-B-ALL

Sensitive and quantitative detection of minimal residual disease (MRD) in bone marrow (BM) samples of children with acute lymphoblastic leukemia (ALL) is essential for evaluation of early treatment response. In this study, we evaluated whether the traumatic BM samplings can be replaced by peripheral blood (PB) samplings. MRD levels were analyzed in follow-up samples of 62 children with precursor-B-ALL (532 paired BM-PB samples) and 22 children with T-ALL (149 paired BM-PB samples) using real-time quantitative PCR (RQ-PCR) analysis of immunoglobulin and T cell receptor gene rearrangements with sensitivities of 10−3 to 10−5 (one ALL cell in 103 to 105 normal cells). In 14 of the 22 T-ALL patients, detectable MRD levels were found in 67 paired BM-PB samples: in 47 pairs MRD was detected both in BM and PB, whereas in the remaining pairs very low MRD levels were detected in BM (nu2009=u200911) or PB (nu2009=u20099) only. The MRD levels in the paired BM-PB samples were very comparable and strongly correlated (rsu2009=u20090.849). Comparable results were obtained earlier by immunophenotyping in 26 T-ALL patients (321 paired BM-PB samples), which also showed a strong correlation between MRD levels in paired BM and PB samples (rsu2009=u20090.822). In 39 of the 62 precursor-B-ALL patients, MRD was detected in 107 BM-PB pairs: in 48 pairs MRD was detected in both BM and PB, in 47 pairs MRD was solely detected in BM (at variable levels), and in 12 pairs only the PB sample was MRD-positive at very low levels (≤10−4). Furthermore, in the 48 double-positive pairs, MRD levels in BM and PB varied enormously with MRD levels in BM being up to 1000 times higher than in the corresponding PB samples. Consequently, BM samples cannot easily be replaced by PB sampling for MRD analysis in childhood precursor-B-ALL, in line with their BM origin. In T-ALL, which are of thymic origin, BM sampling might be replaced by PB sampling, because the dissemination of T-ALL cells to BM and PB appears to be comparable.

Heterogeneity in junctional regions of immunoglobulin kappa deleting element rearrangements in B cell leukemias: a new molecular target for detection of minimal residual disease.

Virtually all immunoglobulin kappa (IGK) gene deletions are mediated via rearrangements of the so-called kappa deleting element (Kde). Kde rearrangements occur either to Vκ gene segments (Vκ–Kde rearrangements) or to the heptamer recombination signal sequence in the Jκ–Cκ intron. Kde rearrangements were analyzed by the polymerase chain reaction (PCR) and heteroduplex analysis in 130 B-lineage leukemias: 63 precursor-B-acute lymphoblastic leukemias (ALL) and 67 chronic B cell leukemias. To obtain detailed information about Kde rearrangements, we sequenced 109 of the 189 detected junctional regions. Vκ gene family usage in the Vκ–Kde rearrangements in our series of B-lineage leukemias was comparable to Vκ gene family usage in functional Vκ–Jκ rearrangements in normal and malignant mature B cells, except for a higher frequency of Vκ II family usage in precursor-B-ALL. Junctional region sequencing of the Kde rearrangements in precursor-B-ALL revealed a mean insertion of 4.7 nucleotides and a mean deletion of 9.5 nucleotides, resulting in an extensive junctional diversity, whereas in chronic B cell leukemias the insertion (1.9) and deletion (6.0) were significantly lower. The relatively extensive junctional diversity of the Kde rearrangements in precursor-B-ALL allowed us to design leukemia/patient-specific oligonucleotide probes, which were proven to be useful for detection of minimal residual disease (MRD) with sensitivities of 10−4 to 10−5. Kde rearrangements occur in approximately 50% of precursor-B-ALL cases and are likely to remain stable during the disease course, because Kde rearrangements are assumed to be ‘end-stage’ rearrangements, which cannot easily be replaced by continuing rearrangement processes. These findings indicate that junctional regions of Kde rearrangements in precursor-B-ALL represent new valuable patient-specific PCR targets for detection of MRD.

Extramedullary infiltrates at diagnosis have no prognostic significance in children with acute myeloid leukaemia.

This retrospective study was designed to review the relative frequency and prognostic significance of extramedullary infiltrates in children with acute myeloid leukaemia (AML). The registration data and initial discharge summaries were reviewed for all children diagnosed with AML, and registered by the Dutch Childhood Leukaemia Study Group (DCLSG). Between 1972 and 1998, 477 children were diagnosed with AML. Of these patients, 120 (25.1%) had extramedullary leukaemia (EML) at diagnosis. Four categories of EML were found: skin, soft tissue or bone, gingival infiltration and central nervous system (CNS) involvement. Patients who presented with gingival infiltrates, were older than those without EML or those in the other EML subgroups, had a high initial WBC count and a high proportion of M4/M5 morphological variants. This type of presentation could indicate a special biological entity. Univariate analysis of prognostic factors in patients treated after 1985 with intensive protocols showed that initial WBC count and the presence of favourable cytogenetic findings were significant. The presence of EML at diagnosis had no significant effect on event-free survival. In a stepwise multiple regression analysis only favourable cytogenetic findings remained significant.

Gemtuzumab ozogamicin in pediatric CD33-positive acute lymphoblastic leukemia: first clinical experiences and relation with cellular sensitivity to single agent calicheamicin

Gemtuzumab ozogamicin in pediatric CD33-positive acute lymphoblastic leukemia: first clinical experiences and relation with cellular sensitivity to single agent calicheamicin