Renate Panzer-Grümayer

Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study

The Associazione Italiana di Ematologia Oncologia Pediatrica and the Berlin-Frankfurt-Münster Acute Lymphoblastic Leukemia (AIEOP-BFM ALL 2000) study has for the first time introduced standardized quantitative assessment of minimal residual disease (MRD) based on immunoglobulin and T-cell receptor gene rearrangements as polymerase chain reaction targets (PCR-MRD), at 2 time points (TPs), to stratify patients in a large prospective study. Patients with precursor B (pB) ALL (n = 3184) were considered MRD standard risk (MRD-SR) if MRD was already negative at day 33 (analyzed by 2 markers, with a sensitivity of at least 10(-4)); MRD high risk (MRD-HR) if 10(-3) or more at day 78 and MRD intermediate risk (MRD-IR): others. MRD-SR patients were 42% (1348): 5-year event-free survival (EFS, standard error) is 92.3% (0.9). Fifty-two percent (1647) were MRD-IR: EFS 77.6% (1.3). Six percent of patients (189) were MRD-HR: EFS 50.1% (4.1; P < .001). PCR-MRD discriminated prognosis even on top of white blood cell count, age, early response to prednisone, and genotype. MRD response detected by sensitive quantitative PCR at 2 predefined TPs is highly predictive for relapse in childhood pB-ALL. The study is registered at http://clinicaltrials.gov: NCT00430118 for BFM and NCT00613457 for AIEOP.

New insights to the MLL recombinome of acute leukemias

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

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

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 ⩾10−3 at 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.

Glucocorticoid-regulated microRNAs and mirtrons in acute lymphoblastic leukemia

Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lymphoblastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are ∼22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15∼16 clusters. In an in vitro model, overexpression of miR15b∼16 mimics increased and silencing by miR15b∼16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner.

Minimal Residual Disease Values Discriminate Between Low and High Relapse Risk in Children With B-Cell Precursor Acute Lymphoblastic Leukemia and an Intrachromosomal Amplification of Chromosome 21: The Austrian and German Acute Lymphoblastic Leukemia Berlin-Frankfurt- Munster (ALL-BFM) Trials

PURPOSE We aimed to identify relapse predictors in children with a B-cell precursor acute lymphoblastic leukemia (ALL) and an intrachromosomal amplification of chromosome 21 (iAMP21), a novel genetic entity associated with poor outcome. PATIENTS AND METHODS We screened 1,625 patients who were enrolled onto the Austrian and German ALL-Berlin-Frankfurt-Münster (ALL-BFM) trials 86, 90, 95, and 2000 with ETV6/RUNX1-specific fluorescent in situ hybridization probes, and we identified 29 patient cases (2%) who had an iAMP21. Minimal residual disease (MRD) was quantified with clone-specific immunoglobulin and T-cell receptor gene rearrangements. RESULTS Twenty-five patients were good responders to prednisone, and all achieved remission after induction therapy. Eleven patients experienced relapse, which included eight who experienced relapse after cessation of front-line therapy. Six-year event-free and overall survival rates were 37% +/- 14% and 66% +/- 11%, respectively. Results of MRD analysis were available in 24 (83%) of 29 patients: nine (37.5%) belonged to the low-risk, 14 (58.5%) to the intermediate-risk, and one (4%) to the high-risk group. MRD results were available in 8 of 11 patients who experienced a relapse. Seven occurred among the 14 intermediate-risk patients, and one occurred in the high-risk patient. CONCLUSION The overall and early relapse rates in the BFM study were lower than that in a previous United Kingdom Medical Research Council/Childhood Leukemia Working Party study (38% v 61% and 27% v 47%, respectively), which might result from more intensive induction and early reintensification therapy in the ALL-BFM protocols. MRD values were the only reliable parameter to discriminate between a low and high risk of relapse (P = .02).

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

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.

Use of Allogeneic Hematopoietic Stem-Cell Transplantation Based on Minimal Residual Disease Response Improves Outcomes for Children With Relapsed Acute Lymphoblastic Leukemia in the Intermediate-Risk Group

PURPOSE In children with intermediate risk of relapse of acute lymphoblastic leukemia (ALL), it is essential to identify patients in need of treatment intensification. We hypothesized that the prognosis of patients with unsatisfactory reduction of minimal residual disease (MRD) can be improved by allogeneic hematopoietic stem-cell transplantation (HSCT). PATIENTS AND METHODS In the Acute Lymphoblastic Leukemia-Relapse Study of the Berlin-Frankfurt-Münster Group (ALL-REZ BFM) 2002, patients with an MRD level of ≥ 10(-3) (n = 99) at the end of induction therapy were allocated to HSCT, whereas those with an MRD level less than 10(-3) (n = 109) continued to receive chemotherapy. MRD was quantified by real-time polymerase chain reaction for clone-specific T-cell receptor/immunoglobulin gene rearrangements. RESULTS The probability of event-free survival for patients with MRD ≥ 10(-3) was 64% ± 5% in ALL-REZ BFM 2002 compared with 18% ± 7% in the predecessor study ALL-REZ BFM P95/96 (P < .001). This was mainly achieved by reducing the cumulative incidence of subsequent relapse (CIR) at 8 years from 59% ± 9% to 27% ± 5% (P < .001). The favorable prognosis of patients with MRD less than 10(-3) could be confirmed in those with a late combined or isolated bone marrow B-cell precursor (BCP) -ALL relapse (CIR, 20% ± 5%), whereas patients with an early combined BCP-ALL relapse had an unfavorable outcome (CIR, 63% ± 13%; P < .001). CONCLUSION Allogeneic HSCT markedly improved the prognosis of patients with intermediate risk of relapse of ALL and unsatisfactory MRD response. As a result, outcomes in this group approximated those of patients with favorable MRD response. Patients with early combined relapse require treatment intensification even in case of favorable MRD response, demonstrating the prognostic impact of time to relapse.

Acute lymphoblastic leukemia in children with Down syndrome: a retrospective analysis from the Ponte di Legno study group

Children with Down syndrome (DS) have an increased risk of B-cell precursor (BCP) acute lymphoblastic leukemia (ALL). The prognostic factors and outcome of DS-ALL patients treated in contemporary protocols are uncertain. We studied 653 DS-ALL patients enrolled in 16 international trials from 1995 to 2004. Non-DS BCP-ALL patients from the Dutch Child Oncology Group and Berlin-Frankfurt-Münster were reference cohorts. DS-ALL patients had a higher 8-year cumulative incidence of relapse (26% ± 2% vs 15% ± 1%, P < .001) and 2-year treatment-related mortality (TRM) (7% ± 1% vs 2.0% ± <1%, P < .0001) than non-DS patients, resulting in lower 8-year event-free survival (EFS) (64% ± 2% vs 81% ± 2%, P < .0001) and overall survival (74% ± 2% vs 89% ± 1%, P < .0001). Independent favorable prognostic factors include age <6 years (hazard ratio [HR] = 0.58, P = .002), white blood cell (WBC) count <10 × 10(9)/L (HR = 0.60, P = .005), and ETV6-RUNX1 (HR = 0.14, P = .006) for EFS and age (HR = 0.48, P < .001), ETV6-RUNX1 (HR = 0.1, P = .016) and high hyperdiploidy (HeH) (HR = 0.29, P = .04) for relapse-free survival. TRM was the major cause of death in ETV6-RUNX1 and HeH DS-ALLs. Thus, while relapse is the main contributor to poorer survival in DS-ALL, infection-associated TRM was increased in all protocol elements, unrelated to treatment phase or regimen. Future strategies to improve outcome in DS-ALL should include improved supportive care throughout therapy and reduction of therapy in newly identified good-prognosis subgroups.

TEL Deletion Analysis Supports a Novel View of Relapse in Childhood Acute Lymphoblastic Leukemia

Purpose: TEL (ETV6)-AML1 (RUNX1) chimeric gene fusions are frequent genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). They often arise prenatally as early events or initiating events and are complemented by secondary postnatal genetic events of which deletion of the non-rearranged, second TEL allele is the most common. This consistent sequence of molecular pathogenesis facilitates an analysis of the clonal origins of relapse in this leukemia, which has some unusual clinical features. Experimental Design: We compared the boundaries, by microsatellite mapping, of TEL deletions at relapse versus diagnosis in 15 informative patients. Moreover, we compared the relatedness of diagnostic and relapse clones using immunoglobulin and T-cell receptor genes rearrangements and clonotypic TEL-AML1 genomic fusion. Results: Five patients retained the apparent same size TEL deletion, seven had larger deletions, and three had smaller deletions at relapse. In all of the cases evaluated, the clonal relatedness of diagnostic and relapse cells was confirmed by the retention of clonotypic TEL-AML1 genomic sequence and/or at least one identical immunoreceptor gene rearrangement. Conclusions: These data provide further evidence that TEL deletions are secondary to TEL-AML1 fusions in ALL. They are compatible with the novel idea that in at least some cases of childhood ALL, remission occurs with persistence of a preleukemic “fetal” clone, and subsequent relapse reflects the emergence of a new subclone from this reservoir after an independent “second hit,” i.e., independent TEL deletion. To our knowledge, the study is the most extensive and comprehensive analysis of the relationship between diagnostic and relapse clones in childhood ALL presented thus far.

CREBBP HAT domain mutations prevail in relapse cases of high hyperdiploid childhood acute lymphoblastic leukemia

Despite their apparently good prognosis ∼15% of high hyperdiploid (HD) childhood acute lymphoblastic leukemia (ALL) cases relapse. To search for responsible risk factors we determined copy number aberrations as well as copy neutral loss of heterozygosity (LOH) in 13 matched diagnosis and relapse samples and added the data of the only three available cases from the literature. Deletions and copy neutral LOH in 3 and 2 of the 16 cases directed us to the histone-modifying CREB-binding protein (CREBBP) gene, whose functional impairment is implicated in drug resistance. We therefore screened all samples for mutations in this gene and discovered 9 acquired sequence mutations in 7/16 cases, leading to an overall frequency of somatic CREBBP aberrations in HD ALL relapse cases of 63% that is considerably higher than that of the reported, mainly non-HD ALL (18.3%). Moreover, mutations in HD cases occur almost exclusively in the HAT domain (8/9; 89%). Hot spot mutations are present at diagnosis in 18.8% of relapsing HD ALL cases but in none of 40 respective cases remaining in long-term remission. Thus, the particular high incidence of CREBBP mutations in relapse-prone HD ALL cases could eventually be exploited for refined risk stratification and customized treatment in this genetic subgroup.