Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R) and 20 older children (MLL-R cases) with leukemia. Our data demonstrated infant MLL-R ALL to have one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite the paucity of mutations, activating mutations in kinase/PI3K/RAS signaling pathways were detected in 47%. Surprisingly, however, these mutations were often sub-clonal and frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (a mean of 6.5/case versus 1.3/case, P=7.15×10−5) and contained frequent mutations (45%) in epigenetic regulators, a category of genes that with the exception of MLL was rarely mutated in infant MLL-R ALL.

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (∼90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements.

Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the ratelimiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor A-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors. [Cancer Res 2008;68(23):9735-45]

Key Points• Chromosomal abnormalities predict outcome after relapse in BCP-ALL, and high-risk cytogenetics takes precedence over clinical risk factors. • Patients with mutations or deletions targeting TP53, NR3C1, BTG1, and NRAS were associated with clinical high risk and an inferior outcome.Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P 5 .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P 5 .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312. (Blood. 2016;128(7):911-922)

Relapse following remission induction chemotherapy remains a barrier to survival in approximately 20% of children suffering from acute lymphoblastic leukemia (ALL). To investigate the mechanism of relapse, 27 matched diagnosis and relapse ALL samples were analyzed for clonal populations using polymerase chain reaction (PCR)-based detection of multiple antigen receptor gene rearrangements. These clonal markers revealed the emergence of apparently new populations at relapse in 13 patients. More sensitive clone-specific PCR revealed that, in 8 cases, these "relapse clones" were present at diagnosis and a significant relationship existed between presence of the relapse clone at diagnosis and time to first relapse (P < .007). Furthermore, in cases where the relapse clone could be quantified, time to first relapse was dependent on the amount of the relapse clone at diagnosis (r ‫؍‬ ؊0.84; P ‫؍‬ .018). This observation, together with demonstrated differential chemosensitivity between subclones at diagnosis, argues against therapy-induced acquired resistance as the mechanism of relapse in the informative patients. Instead these data indicate that relapse in ALL patients may commonly involve selection of a minor intrinsically resistant subclone that is undetectable by routine PCR-based methods. Relapse prediction may be improved with strategies to detect minor potentially resistant subclones early during treatment, hence allowing intensification of therapy.

We used the genomic breakpoint between and genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of deletion, we observed very good correlation for the methods in a majority of patients; however,>20% of children (25% [8/32] with minor and 12.5% [1/8] with major- variants in the consecutive cohorts) had significantly (>1 log) higher levels of fusion than Ig/TCR rearrangements and/or deletion. We performed cell sorting of the diagnostic material and assessed the frequency of -positive cells in various hematopoietic subpopulations; 12% to 83% of non-ALL B lymphocytes, T cells, and/or myeloid cells harbored the fusion in patients with discrepant MRD results. The multilineage involvement of the -positive clone demonstrates that in some patients diagnosed with-positive ALL, a multipotent hematopoietic progenitor is affected by the fusion. These patients have-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)-like disease manifesting in "lymphoid blast crisis." The biological heterogeneity of -positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like-positive ALL.

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