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The prognosis for children with high-risk relapsed acute lymphoblastic leukemia (ALL) is poor. Here, we assessed the prognostic importance of response during induction and consolidation treatment prior to hematopoietic stem cell transplantation (HSCT) aiming to evaluate the best time to assess minimal residual disease (MRD) for intervention strategies and in future trials in high-risk ALL relapse patients. Included patients (n=125) were treated uniformly according to the ALL-REZ BFM (Berlin-Frankfurt-Münster) 2002 relapse trial (median follow-up time=4.8 years). Patients with MRD ⩾10(-3) after induction treatment (76/119, 64%) or immediately preceding HSCT (19/71, 27%) had a significantly worse probability of disease-free survival 10 years after relapse treatment begin, with 26% (±6%) or 23% (±7%), respectively, compared with 58% (±8%) or 48% (±7%) for patients with MRD <10(-3). Conventional intensive consolidation treatment reduced MRD to <10(-3) before HSCT in 63% of patients, whereas MRD remained high or increased in the rest of this patient group. Our data support that MRD after induction treatment can be used to quantify the activity of different induction treatment strategies in phase II trials. MRD persistence at ⩾10(-3) before HSCT reflects a disease highly resistant to conventional intensive chemotherapy and requiring prospective controlled investigation of new treatment strategies and drugs.

Despite risk-adapted treatment, survival of children with relapse of acute lymphoblastic leukemia (ALL) remains poor compared with that of patients with initial diagnosis of ALL. Leukemia-associated genetic alterations may provide novel prognostic factors to refine present relapse treatment strategies. Therefore, we investigated the clinical relevance of 13 recurrent genetic alterations in 204 children treated uniformly for relapsed B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. The most common alterations were deletions of CDKN2A/2B, IKZF1, PAX5, ETV6, fusion of ETV6-RUNX1 and deletions and/or mutations of TP53. Multivariate analysis identified IKZF1 deletion and TP53 alteration as independent predictors of inferior outcome (P=0.002 and P=0.001). Next, we investigated how both alterations can improve the established risk stratification in relapsed ALL. Intermediate-risk relapse patients with low minimal residual disease are currently considered to have a good prognosis. In this group, deletion of IKZF1 and alteration of TP53 identify patients with significantly inferior outcome (P<0.001). In high-risk relapse patients, deletion of IKZF1 is strongly predictive of a second relapse after stem cell transplantation (P<0.001). We conclude that IKZF1 and TP53 represent relevant prognostic factors that should be considered in future risk assessment of children with relapsed ALL to indicate treatment intensification or intervention.

In childhood acute lymphoblastic leukemia (ALL), persistence of leukemic blasts during therapy is of crucial prognostic significance. In the present study, we address molecular and cell biologic features of blasts persisting after 1 week of induction glucocorticoid therapy. Genome-wide gene expression analysis of leukemic samples from precursor B-cell ALL patients (n=18) identified a set of genes differentially expressed in blasts at diagnosis day 0 (d0) and persisting on day 8 (d8). Expression changes indicate a shift towards mature B cells, inhibition of cell cycling and increased expression of adhesion (CD11b/ITGAM) and cytokine (CD119/IFNGR1) receptors. A direct comparison with normal B cells, which are largely therapy resistant, confirmed the differentiation shift at the mRNA (n=10) and protein (n=109) levels. Flow cytometric analysis in independent cohorts of patients confirmed both a decreased proliferative activity (n=13) and the upregulation of CD11b and CD119 (n=29) in d8 blasts. The differentiation shift and low proliferative activity in d8 blasts may account for the persistence of blasts during therapy and affect their sensitivity to further therapeutic treatment. CD11b and CD119 are potential specific markers for d8 blast persistence and detection of minimal residual disease, which warrant further investigation.

Quantification of residual leukemic cells at early time points during therapy can reliably predict the outcome in children with acute lymphoblastic leukemia (ALL). Recently, semiquantitative minimal residual disease (MRD) detection assays such as dot-blot hybridization have been replaced by real-time quantitative PCR. We tested the flexibility of the two most used real-time PCR machines: the SDS 7700 or 'TaqMan' (TM) (Applied Biosystems) and the LightCycler (LC) (Roche) instruments. Clonal T-cell receptor and immunoglobulin gene rearrangements were used for MRD detection with germline hydrolyzation probes and clone-specific primers. Sensitivity tests for 65 clonal gene rearrangements and MRD quantification in 90 bone marrow samples during therapy of 49 children with ALL at diagnosis or relapse were performed with both machines. Both real-time PCR systems provided specific results for MRD quantification in all follow-up samples. In conclusion, we were able to demonstrate that TM and LC real-time PCR technologies produce similar MRD quantification results and that the quantification assays can be easily transferred from one detection system to the other. Using the same detection format, both techniques can be applied in combination in multicenter MRD studies.

Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.

Dysfunction of the p53/Bax/caspase-3 apoptosis signaling pathway has been shown to play a role in tumorigenesis and tumor progression, ie the development of acquired drug resistance. Low expression of the apoptosis inducer Bax correlates with poor response to therapy and shorter overall survival in solid tumors. In the present study, we analyzed the p53/Bax/caspase-3 pathway in a paired and an unpaired sample series of children with acute lymphoblastic leukemia (ALL) at initial diagnosis and relapse. The data demonstrate that both Bax expression levels and the Bax/Bcl-2 ratio are significantly lower in samples at relapse as compared with samples at initial diagnosis (P=0.013, Wilcoxon signed rank test (paired samples); P=0.0039, Mann-Whitney U test (unpaired samples)). The loss of Bax protein expression was not a consequence of Bax frameshift mutations of the G8 tract and could not be attributed to mutations of the p53 coding sequence (exons 5 to 8) which were detected to a similar extent in de novo ALL samples and at relapse. Analysis of the downstream effector caspase-3 showed loss of spontaneous caspase-3 processing at relapse. Whereas nine out of 14 (64%, paired samples) or 37 out of 77 (48%, unpaired samples) ALL patients at initial diagnosis displayed spontaneous in vivo processing of caspase-3, this was completely absent in patients at relapse (paired samples) or detected in only one out of 34 patients at relapse (2.9%, unpaired samples). We therefore conclude that in ALL relapse a severe disturbance of apoptotic pathways occurs, both at the level of Bax expression and caspase-3 activation.

A certain quantity of residual leukemic cells at several time points during chemotherapy of childhood acute lymphoblastic leukemia (ALL) was proved to predict outcome. Future childhood ALL treatment will take minimal residual disease (MRD) into consideration for stratification aiming at an individualization of chemotherapeutic regimens. Recently, the first quantitative real-time PCR assay for MRD detection was described using T cell receptor and immunoglobulin gene rearrangements as clonal markers. Quantitative real-time PCR was performed with TaqMan technology. Here, we present for the first time the potential of LightCycler real-time PCR technology to quantify MRD. We compare and assess different approaches for real-time PCR quantification of leukemic cells, based either on clone-specific primers and general fluorescence detection with SYBR Green, TaqMan probe or hybridization probes, or based on general PCR amplification and clone-specific detection with hybridization probes. MRD quantification with LightCycler real-time PCR technology is a sensitive, specific and incomparably rapid method that needs no post-PCR handling, hence eliminating contamination risk and saving time. Working towards the establishment of MRD quantification in routine diagnostics and towards treatment strategies based on these results, LightCycler quantitative PCR seems to be a promising new technique that makes results immediately available for treatment decisions.

A rapid and simple multiplex polymerase chain reaction (PCR) is described that is capable of identifying the six most frequent rearrangements of the T cell receptor (TCR)-delta gene segments in childhood acute lymphoblastic leukemia (ALL). The PCR products amplified in a single reaction are of different size for each TCR-delta gene rearrangement. Therefore, they are readily and unambiguously distinguished after agarose gel electrophoresis and assigned to a specific V-D-J gene rearrangement. There is no need for labor-intensive and time-consuming Southern blot hybridization or nested PCR. To evaluate the multiplex assay we chose 45 DNA samples of childhood ALL analyzed beforehand for TCR-delta gene rearrangements by Southern blot and single PCR of which 30 showed TCR-delta gene rearrangements. The multiplex PCR results corresponded to the Southern blot and single PCR analyses. The described multiplex PCR enables the detection of clonal markers in about 50% of patients in order to monitor minimal residual disease (MRD) in prospective studies with a high turnover of samples.

In order to investigate the role of T-cell receptor (TcR)-delta and TcR-gamma gene rearrangements and/or deletions in acute myeloid leukemia (AML) coexpressing T-cell-associated antigens (i.e. CD2 and/or CD4 and/or CD7), we examined blasts from a selected group of 56 AML cases (25 children, 31 adults) coexpressing either of these antigens without cytoplasmic CD3 expression. Forty-four typical AML cases (7 children, 37 adults) without T-cell associated antigens were further studied as controls. Germline configuration of the TcR-delta gene was observed in 91 out of the total of 100 AML cases investigated. Eight of nine cases with rearranged or deleted TcR-delta genes coexpressed T-cell-associated antigens. Blast cells of 7/9 cases were classified as FAB M1, two as FAB M2. In six of these cases TcR-gamma gene rearrangements were also detected. TcR-delta alterations were predominantly found in children whose blasts coexpressed T-lymphoid associated antigens (6/25, 24%), but were rarely detected in adult AML with or without coexpression of T-cell antigens (2/31 and 0/37, respectively).