Jutta Bradtke

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.

Prognostic impact of specific chromosomal aberrations in a large group of pediatric patients with acute myeloid leukemia treated uniformly according to trial AML-BFM 98.

PURPOSE Because cytogenetic data are essential for risk stratification of childhood acute myeloid leukemia (AML), the impact of chromosomal aberrations is crucial. PATIENTS AND METHODS Data of a large group of patients younger than 18 years treated according to study AML-Berlin-Frankfurt-Münster (BFM) 98 (n = 454), including their cytogenetics, were analyzed. RESULTS The favorable outcome in the subgroups of patients with t(8;21), inv(16), and t(15;17), with an overall survival of 91% (SE, 4%), 92% (SE, 6%), and 87% (SE, 5%), respectively, was confirmed. Within this group, the 5-year probability of event-free survival (pEFS) of all 17 children with t(8;21) and additional aberrations apart from del(9q) or -X/-Y was 100%. As expected, the cytogenetic finding of a complex karyotype (n = 35; pEFS, 33%; SE, 8%) or a monosomy 7 (n = 12; pEFS, 17%; SE, 11%) was associated with a poor outcome. Compared with remaining patients with cytogenetic data (pEFS, 48%; SE, 2%), prognosis in patients with an MLL rearrangement (n = 91) was inferior (pEFS, 34%; SE, 5%; P = .0005). Particularly, children with t(9;11) and additional aberrations (n = 13; pEFS, 31%; SE, 14%) and MLL rearrangements other than t(9;11) and t(11;19) (n = 41; pEFS, 24%; SE, 7%) had an unfavorable outcome. Nine patients with aberrations in 12p showed an adverse prognosis (pEFS, 11%; SE, 10%). The outcome of patients with aberrations of chromosome 5 (n = 13) was better than expected (pEFS, 50%; SE, 13%). CONCLUSION Because the prognostic value of rare recurrent chromosomal aberrations still has to be elucidated, these data will contribute to future risk stratification for the treatment of pediatric AML.

Acute leukaemias of ambiguous lineage in children: characterization, prognosis and therapy recommendations

Acute leukaemias of ambiguous lineage (ALAL) represent a rare type of leukaemia, expressing both myeloid and lymphoid markers. This study retrospectively analyzed data from 92 children (biphenotypic n = 78, bilineal n = 6, lineage switch n = 8) with ALAL registered in the Berlin‐Frankfürt‐Münster (BFM) acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) studies between 1998 and 2006 (2·4% of all cases with acute leukaemia). Our cohort of ALAL patients was characterized by comparatively high median age (8·9 years), high median white blood cell count (14·9 × 109/l), as well as frequent hyperleucocytosis (18·5%) and central nervous system involvement (24·1%). The most frequent cytogenetic abnormalities were ETV6/RUNX1 fusion (16%) and trisomy 8 (14·6%). Complete remission rate was significantly lower than in ALL‐BFM patients (91·8% vs. 99·1%, P < 0·001), but comparable to AML‐BFM patients (87·9%). Event‐free survival (EFS) and overall survival (OS) of ALAL patients were low, at 62 ± 5%. 5‐year probability of EFS was significantly worse than in ALL patients (80 ± 1%, P < 0·001), but better than for AML patients (49 ± 2%, P = 0·027). Our data suggest that an intensive therapy regimen including stem cell transplantation may be favourable for bilineal or lineage switch cases, whereas patients with ETV6/RUNX1 fusion, lymphoid morphology and patients with expression of cyCD22 and cyCD79a should be treated with an ALL‐directed therapy.

Treatment outcome of CRLF2-rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups.

The prognostic relevance of CRLF2 ‐rearrangements in childhood acute B‐cell precursor lymphoblastic leukaemia (ALL), was assessed by a comparative analysis of 114 non‐Down‐syndrome patients (99 P2RY8‐CRLF2+ , 15 IGH@‐CRLF2+ ), 76 from the AIEOP‐BFM ALL 2000 and 38 from the MRC ALL97 trials. The 6‐year cumulative relapse incidence of P2RY8‐CRLF2+ patients treated on the two trials was not statistically different: 0·37 ± 0·06 vs. 0·25 ± 0·08 (P = 0·194). In contrast, 0/9 IGH@‐CRLF2+ AIEOP‐BFM, but 5/6 ALL97 patients relapsed. Conclusively, P2RY8‐CRLF2+ patients had an intermediate protocol‐independent outcome while the different prognosis of IGH@‐CRLF2+ patients could be related to the different structures of the applied treatment protocols.

Distribution of NPM1‐ALK and X‐ALK fusion transcripts in paediatric anaplastic large cell lymphoma: a molecular–histological correlation

Anaplastic large cell lymphomas (ALCL) in children express anaplastic lymphoma kinase (ALK) fusion genes, most commonly NPM1‐ALK. The distribution of X‐ALK among 66 childhood ALCLs was analysed. One ALCL was ALK‐negative. Reverse transcription polymerase chain reaction detected NPM1‐ALK in 58 tumours, all showing nuclear and cytoplasmic ALK staining. The remaining seven ALCL stained for ALK in the cytoplasm only: two expressed TPM3‐ALK, one ATIC‐ALK, one MYH9‐ALK; three no TPM3‐, TFG‐, ATIC‐, CLTC‐ or MYH9‐ALK. Almost 90% of paediatric ALK‐positive ALCLs express NPM1‐ALK. There was complete concordance between ALK staining pattern and the presence of a typical/variant ALK fusion partner.

MicroRNAs Distinguish Cytogenetic Subgroups in Pediatric AML and Contribute to Complex Regulatory Networks in AML-Relevant Pathways

Background The role of microRNAs (miRNAs), important post-transcriptional regulators, in the pathogenesis of acute myeloid leukemia (AML) is just emerging and has been mainly studied in adults. First studies in children investigate single selected miRNAs, however, a comprehensive overview of miRNA expression and function in children and young adults is missing so far. Methodology/Principal Findings We here globally identified differentially expressed miRNAs between AML subtypes in a survey of 102 children and adolescent. Pediatric samples with core-binding factor AML and promyelocytic leukemia could be distinguished from each other and from MLL-rearranged AML subtypes by differentially expressed miRNAs including miR-126, -146a, -181a/b, -100, and miR-125b. Subsequently, we established a newly devised immunoprecipitation assay followed by rapid microarray detection for the isolation of Argonaute proteins, the hallmark of miRNA targeting complexes, from cell line models resembling core-binding factor and promyelocytic leukemia. Applying this method, we were able to identify Ago-associated miRNAs and their targeted mRNAs. Conclusions/Significance miRNAs as well as their mRNA-targets showed binding preferences for the different Argonaute proteins in a cell context-dependent manner. Bioinformatically-derived pathway analysis suggested a concerted action of all four Argonaute complexes in the regulation of AML-relevant pathways. For the first time, to our knowledge, a complete AML data set resulting from carefully devised biochemical isolation experiments and analysis of Ago-associated miRNAs and their target-mRNAs is now available.

Next-generation-sequencing-based risk stratification and identification of new genes involved in structural and sequence variations in near haploid lymphoblastic leukemia†‡

Near haploidy (23–29 chromosomes) is a numerical cytogenetic aberration in childhood acute lymphoblastic leukemia (ALL) associated with particularly poor outcome. In contrast, high hyperdiploidy (51–67 chromosomes) has a favorable prognosis. Correct classification and appropriate risk stratification of near haploidy is frequently hampered by the presence of apparently high hyperdiploid clones that arise by endoreduplication of the original near haploid clone. We evaluated next‐generation‐sequencing (NGS) to distinguish between “high hyperdiploid” leukemic clones of near haploid and true high hyperdiploid origin. Five high hyperdiploid ALL cases and the “high hyperdiploid” cell line MHH‐CALL‐2, derived from a near haploid clone, were tested for uniparental isodisomy. NGS showed that all disomic chromosomes of MHH‐CALL‐2, but none of the patients, were of uniparental origin, thus reliably discriminating these subtypes. Whole‐exome‐ and whole‐genome‐sequencing of MHH‐CALL‐2 revealed homozygous non‐synonymous coding mutations predicted to be deleterious for the protein function of 63 genes, among them known cancer‐associated genes, such as FANCA, NF1, TCF7L2, CARD11, EP400, histone demethylases, and transferases (KDM6B, KDM1A, PRDM11). Only eight of these were also, but heterozygously, mutated in the high hyperdiploid patients. Structural variations in MHH‐CALL‐2 include a homozygous deletion (MTAP/CDKN2A/CDKN2B/ANRIL), a homozygous inversion (NCKAP5), and an unbalanced translocation (FAM189A1). Together, the sequence variations provide MHH‐CALL‐2 with capabilities typically acquired during cancer development, e.g., loss of cell cycle control, enhanced proliferation, lack of DNA repair, cell death evasion, and disturbance of epigenetic gene regulation. Poorer prognosis of near haploid ALL most likely results from full penetrance of a large array of detrimental homozygous mutations.

PAX5-AUTS2: A recurrent fusion gene in childhood B-cell precursor acute lymphoblastic leukemia

PAX5 rearrangements resulting in the expression of fusion transcripts account for 2–3% of childhood B-cell precursor acute lymphoblastic leukemia. Most PAX5 fusions are rare and many of them have only been described in a couple of, or even only in single, cases. We have identified the third case with a PAX5-AUTS2 fusion, which results from unbalanced t(7;9)(q11.2;p13.2) rearrangements. Our findings substantiate that PAX5-AUTS2 is a recurrent fusion gene in pediatric B-cell precursor acute lymphoblastic leukemia, and we summarize the clinical characteristics of such patients.

Next-generation-sequencing of recurrent childhood high hyperdiploid acute lymphoblastic leukemia reveals mutations typically associated with high risk patients

20% of children suffering from high hyperdiploid acute lymphoblastic leukemia develop recurrent disease. The molecular mechanisms are largely unknown. Here, we analyzed the genetic landscape of five patients at relapse, who developed recurrent disease without prior high-risk indication using whole-exome- and whole-genome-sequencing. Oncogenic mutations of RAS pathway genes (NRAS, KRAS, FLT3, n=4) and deactivating mutations of major epigenetic regulators (CREBBP, EP300, each n=2 and ARID4B, EZH2, MACROD2, MLL2, each n=1) were prominent in these cases and virtually absent in non-recurrent cases (n=6) or other pediatric acute lymphoblastic leukemia cases (n=18). In relapse nucleotide variations were detected in cell fate determining transcription factors (GLIS1, AKNA). Structural genomic alterations affected genes regulating B-cell development (IKZF1, PBX1, RUNX1). Eleven novel translocations involved the genes ART4, C12orf60, MACROD2, TBL1XR1, LRRN4, KIAA1467, and ELMO1/MIR1200. Typically, patients harbored only single structural variations, except for one patient who displayed massive rearrangements in the context of a germline tumor suppressor TP53 mutation and a Li-Fraumeni syndrome-like family history. Another patient harbored a germline mutation in the DNA repair factor ATM. In summary, the relapse patients of our cohort were characterized by somatic mutations affecting the RAS pathway, epigenetic and developmental programs and germline mutations in DNA repair pathways.

Sequential karyotyping in Burkitt lymphoma reveals a linear clonal evolution with increase in karyotype complexity and a high frequency of recurrent secondary aberrations.

Typical Burkitt lymphoma is characterized by an IG‐MYC translocation and overall low genomic complexity. Clinically, Burkitt lymphoma has a favourable prognosis with very few relapses. However, the few patients experiencing disease progression and/or relapse have a dismal outcome. Here we report cytogenetic findings of seven cases of Burkitt lymphoma in which sequential karyotyping was performed at time of diagnosis and/or disease progression/relapse(s). After case selection, karyotype re‐review and additional molecular analyses were performed in six paediatric cases, treated in Berlin‐Frankfurt‐Münster‐Non‐Hodgkin lymphoma study group trials, and one additional adult patient. Moreover, we analysed 18 cases of Burkitt lymphoma from the Mitelman database in which sequential karyotyping was performed. Our findings show secondary karyotypes to have a significant increase in load of cytogenetic aberrations with a mean number of 2, 5 and 8 aberrations for primary, secondary and third investigations. Importantly, this increase in karyotype complexity seemed to result from recurrent secondary chromosomal changes involving mainly trisomy 21, gains of 1q and 7q, losses of 6q, 11q, 13q, and 17p. In addition, our findings indicate a linear clonal evolution to be the predominant manner of cytogenetic evolution. Our data may provide a biological framework for the dismal outcome of progressive and relapsing Burkitt lymphoma.