[Show abstract][Hide abstract] ABSTRACT: The ETV6/RUNX1 gene fusion defines the largest genetic subgroup of childhood ALL with overall rapid treatment response. However, up to 15% of cases relapse. Because an impaired glucocorticoid pathway is implicated in disease recurrence we studied the impact of genetic alterations by SNP array analysis in 31 relapsed cases. In 58% of samples, we found deletions in various glucocorticoid signaling pathway-associated genes, but only NR3C1 and ETV6 deletions prevailed in minimal residual disease poor responding and subsequently relapsing cases (p<0.05). To prove the necessity of a functional glucocorticoid receptor, we reconstituted wild-type NR3C1 expression in mutant, glucocorticoid-resistant REH cells and studied the glucocorticoid response in vitro and in a xenograft mouse model. While these results prove that glucocorticoid receptor defects are crucial for glucocorticoid resistance in an experimental setting, they do not address the essential clinical situation where GC resistance at relapse is rather part of a global drug resistance.
[Show abstract][Hide abstract] ABSTRACT: Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.
Nature medicine 05/2015; 21(6). DOI:10.1038/nm.3840 · 27.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relative low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in RTK/Ras pathway and chromatin modifying genes. The relapse clones consisted of reappearing as well as new mutations and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18% to 30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.Leukemia accepted article preview online, 28 April 2015. doi:10.1038/leu.2015.107.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 04/2015; 29(8). DOI:10.1038/leu.2015.107 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 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 to 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.Leukemia accepted article preview online, 09 March 2015. doi:10.1038/leu.2015.59.
[Show abstract][Hide abstract] ABSTRACT: For most children who relapse with acute lymphoblastic leukaemia, the prognosis is poor and there is a need for novel therapies to improve outcome. We screened samples from children with B lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (ClinicalTrials.gov identifier: NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3 and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high risk features including early relapse, CNS involvement and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wildtype but using more sensitive allelic specific assays, low level mutated subpopulations were found in many cases, suggesting that they survived up front therapy and subsequently emerged at relapse. Preclinical evaluation of the MEK1/2 inhibitor, selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway mutated ALL compared to wild type cells both in vitro and in a orthotopic xenograft model engrafted with primary ALL and in the latter, reduced RAS mutated CNS leukaemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway mutated relapsed ALL.
[Show abstract][Hide abstract] ABSTRACT: Brief Summary Precursor T-cell acute lymphoblastic leukemia represents one of the major challenges of pediatric oncology, because relapses are frequently fatal and the molecular understanding of leukemogenesis and maintenance is limited in this entity. Here, we aimed at identifying novel leukemogenic driver mutations and first analyzed trios of bone marrow DNA of 5 patients with T-cell acute lymphoblastic leukemia obtained at initial diagnosis, remission and relapse by whole exome sequencing. We identified a recurrent, leukemia-specific mutation resulting in a N642H amino acid substitution in the SH2-domain of STAT5B. We subsequently validated these results by sequencing the entire STAT5B gene in two independent large cohorts of patients and identified this mutation in 17 of 301 patients (6.3%) with primary T-cell acute lymphoblastic leukemia recruited from the ALL-BFM 2000 trial and in 7 of 78 relapsed T-cell acute lymphoblastic leukemia patients (9.0%) from the BFM-ALL-REZ trials. The analysis of clinical data revealed that the presence of this mutation was associated with a significantly increased risk of relapse (RR 2.47, p=0.038). Cell based assays in transduced BaF3 cells and in xenografted leukemic cells showed this mutation to confer constitutive STAT5 phosphorylation, cytokine independent growth characteristics. We conclude that the STAT5B N642H mutation is common in pediatric T-cell acute lymphoblastic leukemia and results in an activation of oncogenic pathways, conferring an increased risk of relapse.
[Show abstract][Hide abstract] ABSTRACT: The clinical heterogeneity among first relapses of childhood ETV6/RUNX1-positive acute lymphoblastic leukemia indicates that further genetic altera-tions in leukemic cells might affect the course of salvage therapy and be of prognostic relevance. To assess the incidence and prognostic relevance of additional copy number alterations at relapse of the disease, we performed whole genome array comparative genomic hybridization of leukemic cell DNA from 51 patients with first ETV6/RUNX1-positive relapse enrolled in and treated according to the relapse trials ALL-REZ of the Berlin-Frankfurt-Munster Study Group. Within this largest cohort of relapsed ETV6/RUNX1-positive acute lymphoblastic leukemia analyzed for genome wide DNA copy number alterations to date, alterations were present in every ETV6/RUNX1-positive relapse and a high proportion of them occurred in recurrent overlap-ping chromosomal regions. Recurrent losses affected chromosomal regions 12p13, 6q21, 15q15.1, 9p21, 3p21, 5q and 3p14.2, whereas gains the re-gions 21q22 and 12p. Loss of 12p13 including CDKN1B was associated with a shorter remission duration (P=0.009) and a lower probability of event-free survival (P=0.001). Distribution of X-chromosomal copy number alterations was gender-specific: whole X-chromosome loss occurred exclusively in fe-males, gain of Xq only in males. Loss of the glucocorticoid receptor gene NR3C1 (5q31.3) conveyed a poor response to induction treatment (P=0.003) possibly accounting for the adverse prognosis of some of the ETV6/RUNX1-positive relapses.
[Show abstract][Hide abstract] ABSTRACT: Acute lymphoblastic leukemia (ALL) accounts for approximately 25% of pediatric malignancies. Of interest, the incidence of ALL is observed approximately 20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in approximately 30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P <0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P=0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared to girls.
Human Molecular Genetics 09/2013; 23(3). DOI:10.1093/hmg/ddt447 · 6.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PURPOSEIn 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.ResultsThe 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.