Search

Contains fulltext : 289956.pdf (Publisher’s version ) (Open Access) The digital transformation of society raises high hopes for the improvement of the design and implementation of coproduction processes, which may lead to higher levels of acceptance, satisfaction, and trust with respect to public services. Yet and despite a growing number of studies on the role of digital technology on coproduction, our knowledge on how to assess the quality of digital coproduction is still very limited. The difficulty of defining quality indicators relates to three issues: the complexity of defining quality in public services, the lack of a clear understanding of quality in a coproduction process and the tendency to evaluate coproduction using the same approach as with other digitalised services. In this article, we adopt an interdisciplinary perspective to develop a conceptual model that builds on and bridges theoretical approaches commonly used to assess the quality of digital services, while adding elements that are specific to coproduction. 27 februari 2023 9 p.

We describe a new finding, the "excessive bright echoes" sign, for the diagnosis of hypertrophic pyloric stenosis (HPS). Portal venous gas and gastric wall pneumatosis were noted in 4 vomiting infants proven to have HPS. Portal venous gas can be concerning for ischemic bowel. Gastric wall pneumatosis can be seen in association with necrotizing enterocolitis and has been associated with increased gastric pressure from severe, usually proximal, bowel obstruction. Our HPS cases had prominent bright punctate echoes on sonography of the liver, portal vein lumen, and gastric wall. Knowledge of this excessive bright echoes sign suggests the need for sonography of the antropyloric area. One should consider HPS as a differential diagnostic possibility when the combination of bright echoes within the liver parenchyma, consistent with portal venous gas, and bright echoes in the gastric wall, consistent with gastric pneumatosis, are seen.

Based on a comparative design, the article shows that inter-ministerial working groups do not generally represent panaceas for coordination problems as is usually assumed by the literature. The analysis compares three inter-ministerial working groups in the German federal government. The article asks which factors influence the mode of coordination in interministerial working groups. The analysis reveals that it is affected by the organisational structure of these bodies and the negotiation mode for solving conflict among its members, which leads to variance in its capacity to establish positive coordination. Keywords: positive and negative coordination, interministerial working groups, German federal government ----- Interministerielle Arbeitsgruppen als Allheilmittel fur Koordinationsprobleme? Zusammenfassung Klimaanpassung, Zuwanderung und Rohstoffe in der deutschen Ministerialverwaltung Anhand einer Untersuchung von drei interministeriellen Arbeitsgruppen in der deutschen Ministerialverwaltung zeigt dieser Artikel, dass inter-ministerielle Arbeitsgruppen, anders als in der Literatur angenommen, kein Allheilmittel fur Koordinationsprobleme darstellen. Der Artikel fragt nach den Faktoren, welche den Koordinationsmodus in den interministeriellen Arbeitsgruppen pragen. Die Analyse zeigt, dass dieser von der organisationalen Struktur der Arbeitsgruppen und dem Verhandlungsmodus zwischen den vertretenen Ministerien beeinflusst wird. Dies fuhrt zu Varianz zwischen den interministeriellen Arbeitsgruppen und damit zu Unterschieden in der Fahigkeit, positive Koordination herzustellen. Schlagworte: Positive und negative Koordination, interministerielle Arbeitsgruppen, deutsche Ministerialverwaltung ----- Bibliographie: Radtke, Ina/Hustedt, Thurid/Klinnert, Anne: Inter-Ministerial Working Groups as a Panacea for Coordination Problems?, dms, 1-2016, S. 65-81. https://doi.org/10.3224/dms.v9i1.23641

This chapter analyses the creation of novel cross-sectoral and multi-level coordination arrangements inside the German federal bureaucracy during the recent refugee crisis. We argue that the refugee crisis can be considered as an administrative crisis that challenged organisational legitimacy. Various novel coordination actors and arenas were set up in order to enhance governance capacity. Yet, all of them have been selected from a well-known pool of administrative arrangements. As a consequence, those novel coordination arrangements did not replace but rather complement pre-existing patterns of executive coordination. Hence, the recent refugee crisis exemplifies how bureaucracies effectively adapt to changes in their surroundings via limited and temporary adjustments that coexist with existing organisational arrangements. Thus, the observed changes in coordination structures contribute to repairing organisational legitimacy by increasing governance capacity.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving subunits of the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n=87) and adult (n=78) samples, including cases with RUNX1-RUNX1T1 (n=85) or CBFB-MYH11 (n=80) rearrangements, by whole-genome or whole-exome sequencing. In addition to previously reported somatic mutations in the Ras signaling pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a recurrent and previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated a remarkably different spectrum of cooperating mutations as RUNX1-RUNX1T1 cases harbored recurrent somatic mutations in DHX15 and ZBTB7A, as well as an enrichment of somatic mutations in epigenetic regulators, including ASXL2, and in components of the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscape of cooperating mutations between these related AML subtypes.

We describe a new finding, the "excessive bright echoes" sign, for the diagnosis of hypertrophic pyloric stenosis (HPS). Portal venous gas and gastric wall pneumatosis were noted in 4 vomiting infants proven to have HPS. Portal venous gas can be concerning for ischemic bowel. Gastric wall pneumatosis can be seen in association with necrotizing enterocolitis and has been associated with increased gastric pressure from severe, usually proximal, bowel obstruction. Our HPS cases had prominent bright punctate echoes on sonography of the liver, portal vein lumen, and gastric wall. Knowledge of this excessive bright echoes sign suggests the need for sonography of the antropyloric area. One should consider HPS as a differential diagnostic possibility when the combination of bright echoes within the liver parenchyma, consistent with portal venous gas, and bright echoes in the gastric wall, consistent with gastric pneumatosis, are seen.

To define the mutation spectrum in non-Down syndrome acute megkaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL leukemia samples. Our analysis identified a cryptic chromosome 16 inversion [inv(16)(p13.3q24.3)] in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling, and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

To investigate the frequency of isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in pediatric acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), we sequenced these genes in diagnostic samples from 515 patients (227 AMLs and 288 ALLs). Somatic IDH1/IDH2 mutations were rare in ALL (N=1), but were more common in AML, occurring in 3.5% (IDH1 N=3 and IDH2 N=5), with the frequency higher in AMLs with a normal karyotype (9.8%). The identified IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=3) or histidine (N=1). By contrast, mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling of IDH2 suggested that codon 140 mutations disrupt the enzyme's ability to bind its substrate isocitrate. Accordingly, recombinant IDH2 R140Q/W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(−)-2-hydroxyglutarete (2-HG). Analysis of primary leukemic blasts confirmed high levels of 2-HG in AMLs with IDH1/IDH2 mutations. Interestingly, 3/5 AMLs with IDH2 mutations had FLT3 activating mutations, raising the possibility that these mutations cooperate in leukemogenesis.

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase II alpha-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

Pediatric de novo acute myeloid leukemia (AML) is an aggressive malignancy with current therapy resulting in cure rates of only 60%. To better understand the cause of the marked heterogeneity in therapeutic response and to identify new prognostic markers and therapeutic targets a comprehensive list of the genetic mutations that underlie the pathogenesis of AML is needed. To approach this goal, we examined diagnostic leukemic samples from a cohort of 111 children with de novo AML using single-nucleotide-polymorphism microarrays and candidate gene resequencing. Our data demonstrate that, in contrast to pediatric acute lymphoblastic leukemia (ALL), de novo AML is characterized by a very low burden of genomic alterations, with a mean of only 2.38 somatic copy-number alterations per leukemia, and less than 1 nonsynonymous point mutation per leukemia in the 25 genes analyzed. Even more surprising was the observation that 34% of the leukemias lacked any identifiable copy-number alterations, and 28% of the leukemias with recurrent translocations lacked any identifiable sequence or numerical abnormalities. The only exception to the presence of few mutations was acute megakaryocytic leukemias, with the majority of these leukemias being characterized by a high number of copy-number alterations but rare point mutations. Despite the low overall number of lesions across the patient cohort, novel recurring regions of genetic alteration were identified that harbor known, and potential new cancer genes. These data reflect a remarkably low burden of genomic alterations within pediatric de novo AML, which is in stark contrast to most other human malignancies.

Chromosomal aberrations are a hallmark of acute lymphoblastic leukaemia (ALL) but alone fail to induce leukaemia. To identify cooperating oncogenic lesions, we performed a genome-wide analysis of leukaemic cells from 242 paediatric ALL patients using high-resolution, single-nucleotide polymorphism arrays and genomic DNA sequencing. Our analyses revealed deletion, amplification, point mutation and structural rearrangement in genes encoding principal regulators of B lymphocyte development and differentiation in 40% of B-progenitor ALL cases. The PAX5 gene was the most frequent target of somatic mutation, being altered in 31.7% of cases. The identified PAX5 mutations resulted in reduced levels of PAX5 protein or the generation of hypomorphic alleles. Deletions were also detected in TCF3 (also known as E2A), EBF1, LEF1, IKZF1 (IKAROS) and IKZF3 (AIOLOS). These findings suggest that direct disruption of pathways controlling B-cell development and differentiation contributes to B-progenitor ALL pathogenesis. Moreover, these data demonstrate the power of high-resolution, genome-wide approaches to identify new molecular lesions in cancer.

Release of nitric oxide (NO) is of high importance for regulating endothelial cell functions during vasodilatation, vascular remodeling, and angiogenesis. Thus, a direct and reliable real-time method for NO detection that takes into account time-dependent variations of the NO concentration in the complex reaction within the diffusion zone above the cells is vital for obtaining information about the role of NO in intracellular endothelial signal transduction and its impact on the surrounding cells. In this study, the time course of vascular endothelial growth factor E (VEGF-E) stimulated NO release from transformed human umbilical vein endothelial cells (T-HUVEC) was investigated by means of metalloporphyrin-based NO sensors employed in an electrochemical robotic system. The NO sensor was obtained by electrochemically induced deposition of Ni(II) tetrakis(p-nitrophenylporphyrin) on a 50-microm diameter platinum disk electrode which was integrated, together with a 25-microm diameter platinum disk, in a double-barrel electrode arrangement. The second electrode was used as a guidance sensor for the automatic and highly reproducible positioning of the NO sensor at a known distance from a layer of adherently growing cells by using z-approach curves in the negative feedback mode of scanning electrochemical microscopy (SECM). The electrochemical robotic system allows the fully automated detection of NO with high sensitivity and selectivity to be performed in real time within 96-well microtiter plates. A functional cell assay was established to allow the standardized detection of NO released upon stimulation from T-HUVEC with a sensor positioned at a known distance above the endothelial cells. The overall system was evaluated by automatic detection of NO release from T-HUVEC upon stimulation with VEGF-E after incubation with a variety of drugs that are known to act on different sites in the complex signal-transduction pathway that finally invokes NO release.

Pt-disk electrodes (50 μm diameter) were modified with pyrrole-functionalised Mn(II) trimethoxycarboxyphenylporphyrin by means of an electrochemical pulse technique leading to stable and sensitive microsensors that displayed electrocatalytic activitiy suitable for the detection and quantification of nitric oxide (NO). In order to improve selectivity, a thin layer of Nafion® was deposited on top of the porphyrin-modified surface of the microsensor by means of a dip-coating procedure. These microelectrodes were successfully applied for the detection of the secretion of NO from a population of human umbilical vein endothelial cells (HUVEC) into a small volume of electrolyte.

Circadian rhythms, generated in the mouse suprachiasmatic nucleus (SCN), are synchronized to the environmental day–night changes by photic input. The activation of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) and cAMP response element-binding protein (CREB)-mediated transcription play a critical role in this photoentrainment. The small GTPase Ras is one of the major upstream regulators of the ERK1,2/CREB pathway. In contrast to the well-described role of Ras in structural and functional synaptic plasticity in the adult mouse brain, the physiological regulation of Ras by photic sensory input is yet unknown. Here, we describe for the first time a circadian rhythm of Ras activity in the mouse SCN. Using synRas transgenic mice, expressing constitutively activated V12-Ha-Ras selectively in neurons, we demonstrate that enhanced Ras activation causes shortening of the circadian period length. We found upregulated expression and decreased inhibitory phosphorylation of the circadian period length modulator, glycogen synthase kinase-3 beta (GSK3β), in the SCN of synRas mice. Conversely, downregulation of Ras activity by blocking its function with an antibody in oscillating cell cultures reduced protein levels and increased phosphorylation of GSK3β and lengthened the period of BMAL1 promoter-driven luciferase activity. Furthermore, enhanced Ras activity in synRas mice resulted in a potentiation of light-induced phase delays at early subjective night, and increased photic induction of pERK1,2/pCREB and c-Fos. In contrast, at late subjective night, photic activation of Ras/ERK1,2/CREB in synRas mice was not sufficient to stimulate c-Fos protein expression and phase advance the clock. Taken together, our results demonstrate that Ras activity fine tunes the period length and modulates photoentrainment of the circadian clock.

To identify genomic alterations in chronic lymphocytic leukemia (CLL), we performed single-nucleotide polymorphism–array analysis using Affymetrix Version 6.0 on 353 samples from untreated patients entered in the CLL8 treatment trial. Based on paired-sample analysis (n = 144), a mean of 1.8 copy number alterations per patient were identified; approximately 60% of patients carried no copy number alterations other than those detected by fluorescence in situ hybridization analysis. Copy-neutral loss-of-heterozygosity was detected in 6% of CLL patients and was found most frequently on 13q, 17p, and 11q. Minimally deleted regions were refined on 13q14 (deleted in 61% of patients) to the DLEU1 and DLEU2 genes, on 11q22.3 (27% of patients) to ATM, on 2p16.1-2p15 (gained in 7% of patients) to a 1.9-Mb fragment containing 9 genes, and on 8q24.21 (5% of patients) to a segment 486 kb proximal to the MYC locus. 13q deletions exhibited proximal and distal breakpoint cluster regions. Among the most common novel lesions were deletions at 15q15.1 (4% of patients), with the smallest deletion (70.48 kb) found in the MGA locus. Sequence analysis of MGA in 59 samples revealed a truncating mutation in one CLL patient lacking a 15q deletion. MNT at 17p13.3, which in addition to MGA and MYC encodes for the network of MAX-interacting proteins, was also deleted recurrently.

To identify cooperating lesions in core-binding factor acute myeloid leukemia, we performed single-nucleotide polymorphism-array analysis on 300 diagnostic and 41 relapse adult and pediatric leukemia samples. We identified a mean of 1.28 copy number alterations per case at diagnosis in both patient populations. Recurrent minimally deleted regions (MDRs) were identified at 7q36.1 (7.7%), 9q21.32 (5%), 11p13 (2.3%), and 17q11.2 (2%). Approximately one-half of the 7q deletions were detectable only by single-nucleotide polymorphism-array analysis because of their limited size. Sequence analysis of MLL3, contained within the 7q36.1 MDR, in 46 diagnostic samples revealed one truncating mutation in a leukemia lacking a 7q deletion. Recurrent focal gains were identified at 8q24.21 (4.7%) and 11q25 (1.7%), both containing a single noncoding RNA. Recurrent regions of copy-neutral loss-of-heterozygosity were identified at 1p (1%), 4q (0.7%), and 19p (0.7%), with known mutated cancer genes present in the minimally altered region of 1p (NRAS) and 4q (TET2). Analysis of relapse samples identified recurrent MDRs at 3q13.31 (12.2%), 5q (4.9%), and 17p (4.9%), with the 3q13.31 region containing only LSAMP, a putative tumor suppressor. Determining the role of these lesions in leukemogenesis and drug resistance should provide important insights into core-binding factor acute myeloid leukemia.

Recent advances in genome-wide single-nucleotide polymorphism (SNP) analyses have revealed previously unrecognized microdeletions and uniparental disomy (UPD) in a broad spectrum of human cancers. As acute myeloid leukemia (AML) represents a genetically heterogeneous disease, this technology might prove helpful, especially for cytogenetically normal AML (CN-AML) cases. Thus, we performed high-resolution SNP analyses in 157 adult cases of CN-AML. Regions of acquired UPDs were identified in 12% of cases and in the most frequently affected chromosomes, 6p, 11p and 13q. Notably, acquired UPD was invariably associated with mutations in nucleophosmin 1 (NPM1) or CCAAT/enhancer binding protein-alpha (CEBPA) that impair hematopoietic differentiation (P=0.008), suggesting that UPDs may preferentially target genes that are essential for proliferation and survival of hematopoietic progenitors. Acquired copy number alterations (CNAs) were detected in 49% of cases with losses found in two or more cases affecting, for example, chromosome bands 3p13-p14.1 and 12p13. Furthermore, we identified two cases with a cryptic t(6;11) as well as several non-recurrent aberrations pointing to leukemia-relevant regions. With regard to clinical outcome, there seemed to be an association between UPD 11p and UPD 13q cases with overall survival. These data show the potential of high-resolution SNP analysis for identifying genomic regions of potential pathogenic and clinical relevance in AML.

The genetic heterogeneity of cancer influences the trajectory of tumor progression and may underlie clinical variation in therapy response. To model such heterogeneity, we produced genetically and pathologically accurate mouse models of common forms of human acute myeloid leukemia (AML) and developed methods to mimic standard induction chemotherapy and efficiently monitor therapy response. We see that murine AMLs harboring two common human AML genotypes show remarkably diverse responses to conventional therapy that mirror clinical experience. Specifically, murine leukemias expressing the AML1/ETO fusion oncoprotein, associated with a favorable prognosis in patients, show a dramatic response to induction chemotherapy owing to robust activation of the p53 tumor suppressor network. Conversely, murine leukemias expressing MLL fusion proteins, associated with a dismal prognosis in patients, are drug-resistant due to an attenuated p53 response. Our studies highlight the importance of genetic information in guiding the treatment of human AML, functionally establish the p53 network as a central determinant of chemotherapy response in AML, and demonstrate that genetically engineered mouse models of human cancer can accurately predict therapy response in patients.

Despite best current therapy, up to 20% of pediatric patients with acute lymphoblastic leukemia (ALL) have a relapse. Recent genomewide analyses have identified a high frequency of DNA copy-number abnormalities in ALL, but the prognostic implications of these abnormalities have not been defined.We studied a cohort of 221 children with high-risk B-cell-progenitor ALL with the use of single-nucleotide-polymorphism microarrays, transcriptional profiling, and resequencing of samples obtained at diagnosis. Children with known very-high-risk ALL subtypes (i.e., BCR-ABL1-positive ALL, hypodiploid ALL, and ALL in infants) were excluded from this cohort. A copy-number abnormality was identified as a predictor of poor outcome, and it was then tested in an independent validation cohort of 258 patients with B-cell-progenitor ALL.More than 50 recurring copy-number abnormalities were identified, most commonly involving genes that encode regulators of B-cell development (in 66.8% of patients in the original cohort); PAX5 was involved in 31.7% and IKZF1 in 28.6% of patients. Using copy-number abnormalities, we identified a predictor of poor outcome that was validated in the independent validation cohort. This predictor was strongly associated with alteration of IKZF1, a gene that encodes the lymphoid transcription factor IKAROS. The gene-expression signature of the group of patients with a poor outcome revealed increased expression of hematopoietic stem-cell genes and reduced expression of B-cell-lineage genes, and it was similar to the signature of BCR-ABL1-positive ALL, another high-risk subtype of ALL with a high frequency of IKZF1 deletion.Genetic alteration of IKZF1 is associated with a very poor outcome in B-cell-progenitor ALL.

The Philadelphia chromosome, a chromosomal abnormality that encodes BCR-ABL1, is the defining lesion of chronic myelogenous leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). To define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed a genome-wide analysis of diagnostic leukaemia samples from 304 individuals with ALL, including 43 BCR-ABL1 B-progenitor ALLs and 23 CML cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 ALL, but not in chronic-phase CML. Deletion of IKZF1 was also identified as an acquired lesion at the time of transformation of CML to ALL (lymphoid blast crisis). The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant-negative Ikaros isoform, or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant RAG-mediated recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are an important event in the development of BCR-ABL1 ALL.

Somatic mutations in nucleophosmin (NPM1) occur in approximately 35% of adult acute myeloid leukemia (AML). To assess the frequency of NPM1 mutations in pediatric AML, we sequenced NPM1 in the diagnostic blasts from 93 pediatric AML patients. Six cases harbored NPM1 mutations, with each case lacking common cytogenetic abnormalities. To explore the phenotype of the AMLs with NPM1 mutations, gene expression profiles were obtained using Affymetrix U133A microarrays. NPM1 mutations were associated with increased expression of multiple homeobox genes including HOXA9, A10, B2, B6 and MEIS1. As dysregulated homeobox gene expression is also a feature of MLL-rearranged leukemia, the gene expression signatures of NPM1-mutated and MLL-rearranged leukemias were compared. Significant differences were identified between these leukemia subtypes including the expression of different HOX genes, with NPM1-mutated AML showing higher levels of expression of HOXB2, B3, B6 and D4. These results confirm recent reports of perturbed HOX expression in NPM1-mutated adult AML, and provide the first evidence that the NPM1-mutated signature is distinct from MLL-rearranged AML. These findings suggest that mutated NPM1 leads to dysregulated HOX expression via a different mechanism than MLL rearrangement.

Acute myeloid leukemia (AML) results from mutations that promote aberrant self-renewal, proliferation and differentiation. The specific combination of mutations not only influences the phenotype of the leukemia but also its response to therapy. The core-binding factor (CBF) leukemias represent one of the most common forms of AML and are defined by genetic lesions that target the RUNX1(AML1)/CBFβ transcription factor complex. These include t(8;21)[RUNX1-ETO], inv(16) and t(16;16)[CBFβ-MYH11], point mutations in RUNX1, and several other rarer translocations that target RUNX1. The AML1/CBFβ transcription factor complex functions as a master transcriptional regulator that is essential for the generation of definitive hematopoietic stem cells during development, and plays an important role in the differentiation of a number of hematopoietic lineages including T cells and megakaryocytes. Using a conditional Runx1-Eto murine knock-in model, we previously demonstrated that the expression of Runx1-Eto was insufficient to induce leukemia. To define the landscape of mutation that cooperate with RUNX1-ETO in leukemogenesis, the St. Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project performed whole genome DNA Sequencing (WGS) on a cohort of 17 pediatric CBF leukemia [7 t(8;21) and 10 inv(16) cases. Somatic mutations were identified using the PCGP analytical pipeline and all identified mutations were experimentally validated. The frequency of the identified mutations were then assessed by performing exome sequencing on a validation cohort consisting of 151 pediatric and adult CBF leukemias [79 t(8;21) and 72 inv(16)]. The combined analyses identified very few copy number alterations (CNAs) or structural alterations per case, and an average of only 12.5 somatic non-synonymous single nucleotide variations or insertions/deletions per case. Despite the low burden of mutations, recurrent activating mutations in the Kinase/RAS signaling pathway (NRAS, KIT, FLT3, KRAS, and PTPN11, and inactivating mutations in NF1) were collectively identified in >67% of cases An even more striking result was the identification of a high frequency of loss-of-function mutations in genes that encode key epigenetic regulatory proteins (28% including ASXL2, EZH2, UTX, SETD2, MLL2/3), and missense and loss of function mutations in genes that encode components of the cohesin complex (10% including SMC1A, SMC2, RAD21). Moreover, the frequency of both classes of mutations were significantly higher in the t(8;21) versus inv(16) containing cases (epigenetic regulatory mutations 48% vs 6.25% and cohesin mutations 20% vs 0%). No significant difference was noted in the frequency of cohesin mutations between pediatric and adult cases. The identified mutations targeted genes encoding core components of the cohesion complex required to generate the ring shaped DNA binding structure, SMC1A, SMC3, and RAD21, as well as a single case with a mutation in the SMC3 acetyl transferase ESCO2. In addition, loss of the X chromosome is a frequent somatic event in female patients with t(8;21) leukemia and SMC1A is located on Chr. X and is not subjected to X inactivation, suggesting that loss of Chr. X leads to haploinsufficiency. The majority of leukemias with cohesion mutations also contain activating mutation in the RAS/Kinase signaling pathway, suggesting that these mutations cooperate with AML1-ETO to induce leukemia. Collectively, these data provide an unprecedented view of the range of cooperating mutations that occur in the CBF leukemias and highlight fundamental differences between t(8;21) and inv(16) containing cases. Citation Format: Amanda Larson Gedman, Jinjun Cheng, Xiang Chen, Ina Radtke, Anna Andersson, Heather Mulder, Kristy Boggs, Bhavin Vadodaria, Donald Yergeau, Joy Nakitandwe, Lars Bullinger, Michael Kuehn, Hartmut Doehner, Konstanze Doehner, Sheila Shurtleff, John Easton, Richard Wilson, Jinghui Zhang, James R. Downing. The landscape of somatic mutations in the core-binding factor acute myeloid leukemias. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A15.

Acute myeloid leukemia (AML) can present with multiple concurrent subclones at diagnosis. Subclone-specific mutations may confer resistance to molecular-targeted drugs through loss of antigen expression or rewiring of intracellular signaling pathways, leading to relapse. Deep sequencing approaches improve the detection of rare subclones, but the ability to prospectively identify subclones (i.e. without relapse material) is limited, and the effects of subclone-specific mutations on phenotype are poorly understood. In the course of a broader study of oligoclonal pediatric AML patients, we focused investigation on a diagnosis bone marrow sample from a patient harboring 3 presumed subclones (two with distinct NRAS-G12D, NRAS-G13D mutations) as determined by whole-genome sequencing (WGS). We employed a combination of 31-parameter mass cytometry, deep sequencing, FACS sorting, and computational modeling to produce a detailed profile of the subclonal genotypes and phenotypes in this patient. The 3 anticipated subclones did not correlate with a clear subset of surface markers in the mass cytometry analysis. Instead, we observed a single continuous ‘differentiation trajectory’ from progenitor-like to monocyte-like blasts. To dissect this trajectory, 7 distinct myeloid/progenitor subsets were FACS-sorted, plus T/B cells as a non-leukemic control. For each subset we performed capture-based deep sequencing of 307 tumor-specific variants (Tier 1: 13; Tier 2: 33; Tier 3: 261; median depth: 1420x). Shifts in allele frequencies among the FACS-sorted subsets provided critical information to a Bayesian mixture modeling algorithm, allowing identification of 5 subclones, as opposed to the 3 subclones anticipated by WGS. The inferred subclonal genotypes were validated and further refined by targeted single-cell Sanger sequencing of multiple Tier 3 loci. Although all 5 subclones were present throughout the differentiation trajectory, some were enriched in certain phenotypic states or ‘reservoirs’. For example, the NRAS-G12D subclone was enriched in a progenitor-like subset, but its daughter subclone, which harbored an additional mutation in RAC2 (implicated in HSC engraftment), was enriched in the more differentiated subsets, suggesting an opposing effect. Notably, the T/B cell population harbored 2 tumor-specific Tier 1 mutations at >15% allele frequency, suggesting its presence in a preleukemic, multilineage-competent HSC. Taken together, subclone-specific mutations appear to skew cells toward either progenitor or mature phenotypes, but the developmental trajectory enforced by parental mutations is resistant to change. Furthermore, characterization of subclones is possible at diagnosis, and may improve the selection of targeted therapies. *Contributed equally: EFS, SCB & ALG Citation Format: Erin F. Simonds, Sean C. Bendall, Amanda L. Gedman, Jacob H. Levine, Kara L. Davis, Harris G. Fienberg, Astraea Jager, El-ad D. Amir, Ina Radtke, Wendy J. Fantl, Dana Pe'er, James R. Downing, Garry P. Nolan. Deep sequencing of immunophenotypically distinct subsets in acute myeloid leukemia reveals reservoirs of genetically distinct subclones along a conserved differentiation trajectory. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2013-3178

Abstract 2565 Acute myeloid leukemia (AML) encompasses a broad spectrum of cellular morphologies. This spectrum exists both within each patient in the form of a malignant developmental hierarchy, and across patients in the form of distinct disease classes with distinguishing mutational or pathological hallmarks. Flow cytometry has long been an essential tool for classifying AML surface markers, and has enabled improved diagnosis, minimal residual disease monitoring, and cell sorting for functional studies. Previous work by our laboratory and others (Irish et al., Cell 2004; Kornblau et al., Clin. Cancer Res. 2010) has shown that clinically relevant heterogeneity also exists at the level of single-cell intracellular signaling capabilities, particularly in response to ex vivo perturbations. Due to the constraints of fluorescent flow cytometry, the limited number of simultaneous parameters measured per cell in these studies was limited (e.g., 3–6). We hypothesized that there may exist additional layers of heterogeneity in both surface marker phenotypes and signaling responses which were invisible to the low-parameter systems used in these studies. To address this challenge, we used a next-generation 31-parameter ‘mass cytometry’ platform to survey the diversity of signaling responses and their relationship to subsets defined by surface marker patterns. We measured the simultaneous co-expression of 16 surface markers and 15 dynamic intracellular signaling epitopes (e.g. phosphorylated kinase substrates) in a cohort of pediatric AML diagnosis bone marrow samples (n=18) and healthy adult bone marrow controls (n=3). Signaling dynamics were measured under 18 stimulation conditions, including a battery of cytokines, chemical stimuli, and small molecule kinase inhibitors. The resulting single-cell data was overlaid and compared using a new, unsupervised flow cytometry analysis and visualization program–SPADE (Spanning-tree Progression Analysis of Density-Normalized Events)–which links rare, transitional, and abundant cell types alike along a branching tree organized by phenotypic progression. Thus, we distilled the diversity of cellular phenotypes, their relative frequencies, and the corresponding signaling dynamics of each cell population onto a single graph structure representing the distinct and overlapping expression patterns in the 16-dimensional “immunophenotypic space” explored by the 21 individuals. Striking signaling and immunophenotypic diversity was observed between the AML samples, particularly in response to pervanadate stimulation. Pervanadate liberates intracellular kinase cascades by broadly inhibiting tyrosine phosphatases, and thus reveals the cell's potential for kinase activity and the interplay of signaling circuits in different cell populations. Interestingly, two transcription factors implicated in AML pathogenesis – CREB and c-Cbl–often exhibited mutually exclusive phosphorylation patterns in different subsets of primitive (CD34+) cells. Specifically, CREB was readily phosphorylated at Ser133 upon pervanadate exposure in the CD34+CD123- cells, while c-Cbl phosphorylation at Tyr700 was restricted to CD34+CD123+ cells. As CD123 (IL-3Ra) has been implicated as a marker of AML stem cells, the finding that these cells are poised for c-Cbl signaling may suggest a potential therapeutic avenue. This work demonstrates the utility of 31-parameter mass cytometry for the simultaneous characterization of multiple cancer stem cell markers and comparison of their expression patterns to the normal tissue hierarchy. These results will inform future studies of AML signaling biology, prognostic biomarkers, and indicators of minimum residual disease. EFS and SCB contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Abstract 50 Genomic aberrations are important prognostic factors in chronic lymphocytic leukemia (CLL) [Döhner et al., 2000]. However, known genomic aberrations fail to fully explain the biologic and clinical heterogeneity of the disease. We sought to precisely map copy number alterations (CNA) and copy number neutral losses of heterozygocity (LOH) to better characterize known recurrent aberrations and to identify new genetic lesions. We used Affymetrix 6.0 single nucleotide polymorphism (SNP) array analyses on CD19 sorted CLL cells. Data were analyzed using dChipSNP, a modified array normalization algorithm guided by cytogenetic abnormalities and a circular binary segmentation. We studied samples from 346 patients enrolled on the CLL8 trial of the German CLL Study Group. Data of 145 samples were analyzed against intraindividual reference DNA (paired), data of 201 samples against a pool of reference DNA (unpaired). FISH data were available for all samples, the distribution of genomic aberrations was as follows: del(13q14) in 59.8%, del(11q23) in 26.3%, trisomy 12 in 11.6%, and del(17p13) in 8.4%. IGHV was mutated in 32.9%, unmutated in 63.3%, and unknown in 3.8%. In total, 261 tumor-specific CNA could be discovered among the 145 paired samples. Genomic aberrations were found in 85.5% of these cases. The average number of aberrations per case was 1.8; according to the hierarchical model of genomic aberrations, it was 3.5 in del(17p), 2.4 in del(11q23), 1.7 in del(13q14) single, and 0.5 in normal karyotype CLL. The minimally deleted region (MDR) on 13q14 was 277.25 kb in size and contained mir15a and mir16, DLEU1 and DLEU2; RFP2 was not part of the MDR. Deletions on 13q were highly heterogeneous in size, ranging from 294 kb to 68 Mb. On 11q23 the MDR only contained ATM, the smallest lesion of 78.5 kb being intragenic; in two of theses cases, the deletion size was too small to be detected by FISH analysis. TP53 was affected in all del(17p13) cases except two; one tumor-specific deletion of 635.7 kb was detected in cytoband 17p13.2 harboring 30 genes and a second deletion of 780 kb in 17p13.3 containing – among 15 other genes – MNT, a tumor suppressor acting as an antagonist of MYC. A partial trisomy on chromosome 12 was not detected. Of the 261 CNA, 95 were located in genomic regions that are not evaluated by our routine FISH probe panel; 17 regions were affected recurrently: del(1p35.3) [2/145], del(1q23.3) [2/145], del(1q42.12) [2/145], +2p [5/145], del(3p21.31) [2/145], del(6p25.3) [3/145], +(6p25.3) [2/145], del(6q) [11/145], del(7q23.1) [2/145], +(8q24.21) [3/145], del(9q13-q21.13) [2/145], del(10q24) [2/145], del(14q24.3) [2/145], del(14q12.3) [2/145], del(15q15.1) [2/145], +18 [3/145] and +19 [7/145]. The frequency of these CNA was subsequently evaluated within the cohort of 201 (unpaired) samples. Five of 17 regions were affected in more than 2% in the whole cohort: +2p, del(6q), +8q24.21, del(15q15.1), and +19. Gain of 2p was found in 6.9% of cases, the minimally amplified region was 1.9 Mb in size and contained e.g. BCL11A and REL. Del(6q) was detected in 6,4%, the deletions were heterogeneous, an MDR could not be identified. 16 cases had 8q24.21 gains, the minimally amplified region was delineated by three intragenic gains in MYC. 14 cases had loss in 15q15.1 focussing on MGA, a potential suppressor of transcriptional activation by MYC. 8 cases had total or partial gains of chromosome 19, among those two overlapping partial gains with a minimally amplified region of 2.17 Mb in 19p13.2. Tumor-specific LOH were identified in 6.0% (9/145) located on 13q in three cases and in one case each on 17p, 12q, 11p, 1p, 3 and 22q. The LOH on chromosomes one and three overlapped with recurrent losses in 1p35.3 and 3p21.31. Essential members of the ATR-pathway were located in these regions: ATRIP and RPA2. However, mutational analyses of the two candidate genes in 48 cases revealed no mutations. SNP array analysis is a reliable tool to identify and further characterize genomic aberrations in CLL. MDR on 13q14 was delineated to a 277.25 kb segment affecting mir15a, mir16, DLEU1 and DLEU2 but not RFP2; the MDR on 11q23 to a segment only containing ATM. Cases with del(11q23) and del(17p) showed a higher genomic complexity than those with normal karyotype or del(13q14) as single abnormality. Relatively few novel genetic lesions were identified. Although occurring at low frequency, they may lead to the discovery of new genes involved in CLL pathogenesis. Disclosures: Stilgenbauer: Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding.

Abstract 849 Core-binding-factor (CBF) acute myeloid leukemia (AML) defined by the presence of t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is associated with favorable outcome. However, about 30–40% of patients are not cured by current treatment approaches. Secondary genetic changes are believed to cause clinical heterogeneity. To identify new secondary genetic lesions, we performed high-resolution, genome-wide analysis of copy number aberrations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 300 adult and pediatric CBF AMLs; t(8;21), n=157 (adult, n=114; pediatric, n=43); and inv(16), n=143 (adult, n=104; pediatric, n=39). Germline control DNA from remission bone marrow or peripheral blood was available for paired analysis in 175 patients. In addition, for 42 patients matched relapse samples were analyzed. Data were processed using reference alignment, dChipSNP and circular binary segmentation. Paired analysis revealed a median of 1.28 somatic CNAs per case [t(8;21): 1.14, range: 0–5; inv(16): 1.45, range: 0–9], with deletions more common than gains [t(8;21): 0.94 losses/case vs. 0.2 gains/case; inv(16): 0.95 vs. 0.5]. Recurrent deletions were detected at chromosomal bands 7q36.1 (n=23), 9q21.13 (n=15), 11p13 (n=7), 17q11.2 (n=6), 10q24.32 (n=2), and at the chromosomal breakpoints of t(8;21) and inv(16) on 8q21.3 (n=9), 21q22 (n=16), 16p13.11 (n=28), and 16q22.11 (n=21). Deletions at 7q, 11p and 17q were validated using FISH analysis. Minimally deleted regions (MDR) less than 1.5 Mb were identified at 7q36.1 (647 Kb, 4 genes), 9q21.13 (1125 Kb, 9 genes), 11p13 (130 Kb, 1 gene), and 17q11.2 (902 Kb, 11 genes), with each region containing a putative tumor suppressor (e.g., MLL3 on 7q, FRMD3 on 9q, WT1 on 11p, and NF1 on 17q). Sequence analysis of MLL3 in 23 cases with del(7q), 1 case with 7q CN-LOH, and 23 randomly selected cases identified a MLL3 truncating mutation leading to a premature stop codon in a case that lacked a 7q alteration. The del(11p13) contained only WT1 and primarily affected inv(16)-cases (5 of 7). Sequence analysis of WT1 in four cases with del(11p) revealed an additional frame shift mutation in the remaining allele in one case. Sequence analysis of WT1 in an additional 103 inv(16)-containing cases revealed mutations in 10 (9%) of the cases. The MDR at 17q11.2 was exclusively identified in inv(16)-containing cases (n=6) and included the tumor supressor NF1. Sequence analysis of all coding exons in NF1 in 4 additional inv(16)-containing AMLs revealed no additional mutation. Recurrent gains were identified at 22q11.21-q13.33 (n=20; 32 Mb), 8q24.21 (n=14; 138 Kb), 13q21.1-q34 (n=6; 14 Mb), and 11q25 (n=5; 368 Kb). The smallest gains were identified at 8q24.21 and 11q25, both containing only a single non-coding RNA gene (CCDC26 and LOC283177, respectively). Somatic CN-LOH were uncommon and only found at 1p36.33-p12 (n=2), 4q (n=2), and 19p (n=2). In a comparative analysis of paired diagnostic and relapse samples, novel CNAs at the time of relapse were identified at 3q13.31 (n=5), 5q (n=2), 17p (n=2), and 17q (n=2). The MDR at 3q was only 46 kb in size and contains a single transcript that has been connected to LSAMP, a putative tumor suppressor located 404 Kb upstream of the deletion. In summary, our data provide a comprehensive profiling of copy number alterations in pediatric and adult CBF AML. These data demonstrate a very low number of CNAs, with no significant differences noted between pediatric and adult cases. Interestingly, a number of novel recurrent secondary genetic alterations are identified. Exploring the biological role of these lesions in leukemogenesis and drug resistance should provide important insights into the CBF leukemias. Disclosures: No relevant conflicts of interest to declare.

Expression of BCR-ABL1 is the hallmark of chronic myelogenous leukemia (CML) and a subset of de novo acute lymphoblastic leukemia (ALL), but the factors determining disease lineage, and progression of CML to myeloid or lymphoid blast crisis, are incompletely understood. We recently reported deletion of IKZF1 (encoding the lymphoid transcription factor Ikaros) in 85% of de novo pediatric and adult BCR-ABL1 ALL, and in lymphoid blast crisis in a small cohort of CML cases (Nature2008;453:110), suggesting that IKZF1 deletion is important in the pathogenesis of BCR-ABL1 lymphoid leukemia. To identify genetic determinants of disease stage and blast crisis lineage in CML, we have now performed high-resolution, genome wide analysis of DNA copy number abnormalities (CNA) and loss-of heterozygosity (LOH) and candidate gene resequencing in a cohort of 90 CML patients that included 64 samples obtained at chronic phase (CP), 15 samples at accelerated phase (AP), 9 lymphoid blast crisis (LBC) and 22 myeloid blast crisis (MBC) samples. Importantly, 25 patients had sequential samples (CP and/or AP, as well as blast crisis samples) enabling analysis of lesions acquired at progression to blast crisis. All blast crisis samples were flow sorted to at least 90% purity prior to DNA extraction. Germline samples for 28 cases obtained at remission or by flow sorting of blast crisis samples were also examined. Affymetrix SNP 6.0 arrays, interrogating over 1.87 million genomic loci, were used for 85 samples, and 500K arrays for the remainder. Identification of tumor-specific (somatic) copy number analysis was performed by directly comparing CML samples to matched germline samples were available, or by filtering results against databases of inherited copy number variants for samples lacking germline material. Genomic resequencing of IKZF1, PAX5 and TP53 was performed for all AP, LBC and MBC samples. There were few CNAs in CP-CML (mean 0.27 deletions and 0.07 gains per case), with no recurring lesions identified apart from deletions or gains at the chromosomal breakpoints of BCR and ABL1 (3 cases each). Notably, the size of these translocation associated deletions was highly variable, ranging from 6kb (one ABL1 deletion) and 15 kb (one BCR deletion) to deletions extending to the telomeres of chromosomes 9 and 22. No significant increase in lesion frequency was identified in AP cases (0.14 deletions and 0.9 gains per case), however the number and cumulative extent of genomic aberrations was significantly higher in both lymphoid and myeloid blast crisis samples. LBC cases had a mean of 8.1 deletions/case (P There were striking differences in the type of CNAs in MBC and LBC samples. Seven of 9 LBC cases had focal CNAs targeting genes regulating normal B-lymphoid development, including IKZF1 (6 cases, 2 homozygous), PAX5 (4 cases), and EBF1 (1 case with focal homozygous deletion restricted to the EBF1 locus). Thus, of these 7 cases, two had a single CNA in this pathway, three had two lesions, and two cases had three lesions. In contrast, only 4 of 22 MBC cases had lesions in this pathway, most commonly from whole or sub chromosomal deletions involving chromosomes 7 and 9. Deletion of the CDKN2A/B locus (encoding the tumor suppressors and cell cycle regulators INK4A, ARF and INK4B) was seen in 6 (67%) LBC samples, but only 2 (9%) MBC cases, and never in CP or AP CML. Other lesions commonly seen in de novo BCR-ABL1 ALL were also observed in LBC samples, including deletions of MEF2C, C20orf94, and the HBS1L gene immediately upstream of the oncogene MYB. Apart from acquisition of new or more complex abnormalities involving BCR and ABL1, the only recurring mutation observed in MBC was deletion (4 cases) or splice-site point mutations (2 cases) of TP53. These data demonstrate a lack of genomic instability with few genetic alterations in CP or AP CML. Lymphoid blast crisis samples have similar genetic alterations to those seen in de novo BCR-ABL1 ALL, whereas myeloid blast crisis displays completely distinct patterns of mutation, most commonly targeting P53. These results indicate that genomic abnormalities are important determinants of lineage and disease progression in BCR-ABL1 leukemia.

In genome-wide analyses of DNA copy number abnormalities (CNAs) and loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) microarrays, we previously reported a high frequency of genetic alterations of regulators of B lymphoid development and cell cycle in B-progenitor acute lymphoblastic leukemia (B-ALL), and a near-obligate deletion of the transcription factor Ikaros ( IKZF1 ) in BCR-ABL1 ALL (Nature2007;446:758 and Nature2008;453:110). To assess the prognostic significance of genetic alterations and their effects on the leukemic cell transcriptome, we have now extended these studies to a cohort of 221 NCI high risk pediatric ALL cases treated on the Children’s Oncology Group (COG) P9906 trial. The majority of these cases (N=170) lacked known cytogenetic abnormalities, and none were BCR-ABL1 positive. CNA and LOH data were generated using Affymetrix 500K SNP arrays, and gene expression data was available for 207 cases. Genomic resequencing was performed for PAX5 and IKZF1. These analyses revealed mutations in genes regulating B lymphoid development in 57.5% of cases, including PAX5 CNAs (31.7%), PAX5 sequence mutations (8.1%), IKZF1 deletions (24.9%) and IKZF1 sequence mutations (2.6%). In addition, recurring CNAs were detected in a number of other genes known to play roles in transformation and drug response including CDKN2A/B (45.7%) , RB1 (11.3%) , BTG1 (10.4%) , IL3RA (6.8%) , KRAS (6.3%), NRAS (2%) , NR3C1 (2%) , NR3C2 (3%) , and ERG (6.3%). To examine associations between genetic alterations and outcome in a genome-wide fashion, we used a semi-supervised principal components approach and identified a predictor of outcome driven by deletion/mutation of IKZF1 , EBF , and BTLA (hazard ratio 3.74, P =8.1×10 −5 ). IKZF1 lesions were most strongly associated with poor outcome (cumulative incidence of relapse 68.8% in IKZF1 -mutated ALL vs. 29.1% for IKZF1 wild type cases, P =0.002). This copy number predictor also predicted outcome in an independent cohort of 258 B-progenitor ALL cases treated at St Jude Children’s Research Hospital (HR 3.76, P =0.00023). Importantly, the St Jude cohort had a much higher proportion of cases with conventional recurring cytogenetic abnormalities including hyperdiploidy and translocations than the COG P9906 cohort (60.4% versus 23%, respectively), including BCR-ABL1 (N=21; 8.1%). Notably, the association of IKZF1 mutation and poor outcome was also observed in the cases lacking BCR-ABL1 . The presence of an IKZF1 mutation was also associated with a high-level of minimal residual disease following induction therapy in both cohorts. We next defined a gene expression profile of high risk ( IKZF1- mutated) ALL in each cohort. Using gene set enrichment analysis (GSEA), we found the high risk gene expression signature to be positively enriched for hematopoietic stem cell genes and genes associated with minimal residual disease, and negatively enriched for genes regulating cell cycle progression and genes upregulated during B cell maturation. Moreover, GSEA also demonstrated significant similarity of the expression signatures of high risk COG and St Jude ALL, a striking finding given the marked differences in sample composition of the two groups. As IKZF1 mutation is a near obligate lesion in BCR-ABL1 lymphoid leukemia (Nature2008;453:110) and in this study is a strong independent predictor of poor outcome in BCR-ABL1 negative ALL, we next examined similarity of the gene expression signatures of high risk ( IKZF1- mutated) BCR-ABL1 negative ALL and BCR-ABL1 positive ALL. Using GSEA and direct comparison of differentially expressed genes, there was highly significant similarity between the two signatures. These findings indicate that IKZF1 mutation is a key determinant of the gene expression signature of both BCR-ABL1 positive and negative ALL, and is thus likely to be central to the pathogenesis and poor outcome of both leukemia subtypes. Thus, IKZF1 deletion is a new prognostic factor in ALL, and is associated with a gene expression profile indicative of impaired maturation of the leukemic blasts, an observation compatible with the known role of IKZF1 in regulating early lymphoid development. These findings have important implications for risk stratification of ALL.

To define the complement of genetic lesions in pediatric AML, we performed high resolution genome-wide analysis on bone marrow blasts from 111 cases using Affymetrix 100K and 500K SNP microarrays and genomic resequencing. Cases included t(8;21) (N=20), inv(16) (N=16), t(15;17) (N=7), MLL rearranged (N=16), FAB-M7 (N=9), miscellaneous cytogenetic abnormalities (N=24) and normal karyotype (N=19). Germline DNA was available for 67 of the cases. The mean number of somatic copy number abnormalities (CNA) was 2.38 (range 0–45) with 1.32 gains (range 0–41) and 1.06 losses (range 0–12). Focal CNAs were detected at the breakpoints of known chromosomal translocations, most commonly in inv(16) cases (7/16) and t(8;21) cases (4/20). Based on this observation, we examined focal CNAs that affected the 5’ or 3’ regions of genes for their possible involvement in cryptic translocations. CNAs involving NUP98 and MLL led to the identification of two cases with t(5;11)[ NUP98-NSD1 ], and two with t(6;11)[ MLL-MLLT4 ] not evident on cytogenetic analysis. RT-PCR for these fusions identified two additional NUP98-NSD1 cases. Identification of focal CNAs are thus useful in identifying clinically significant translocations in AML. Other recurring somatic CNAs were uncommon. Twelve regions containing a single gene were identified, including amplification of EBF , PRDM5 , CCDC26* , GDF10 , ABCC4*, and deletion of FAM20C* , TUSC1 , GPC5 , A2BP1 , INSR, BCOR* ( * involved in N>2 cases). The most frequent abnormality was amplification of CCDC26 at 8q24.21 ( N =15; focal in 4 cases, broad in 11) which encodes a putative mediator of retinoic acid receptor signaling. The focal amplifications are predicted to abolish expression of normal transcripts. Seventeen additional recurring CNAs involving 2 to 25 genes were identified, many of which contain known or putative tumor suppressor genes or oncogenes, including deletions of regions at 7q36, 9p21 ( CDKN2A/B ), 11p14-p12 ( WT1 , WIT1 ), and amplification at 19p13 ( JUNB, LYL1 ). These regions showed significant correlation between copy number and local gene expression in an integrated analysis of CNA and Affymetrix U133A gene expression data. Copy-neutral LOH was uncommon and recurrent only with LOH of chromosome 13 in 3 cases. Sequence analysis has been completed for several genes involved in recurring CNAs ( CCDC26, TUSC1, FBXW7 ) and for genes known to be mutated in AML ( N/KRAS, PTPN11 , BRAF, SOS1 , FLT3 , CEPBA , NPM1, AML1, CKIT , GATA1 ). No mutations were identified in CCDC26 , TUSC1 , FBXW7 , BRAF , or SOS1 . Marked differences in the combination of CNA and sequence mutation were identified across the different genetic subtypes of AML. FAB M7 cases had the highest frequency of CNA (mean 9.3 lesions/case) but had sequence mutations limited to GATA1 . Mutations of FLT3 , CEPBA and NPM1 were most frequent in cases with no or miscellaneous cytogenetic abnormalities, where as RAS mutations were most frequent in t(8;21) and inv(16) cases. Importantly, approximately 30% of the cases with recurring translocations had no other sequence or numerical abnormalities. These data demonstrated that, in contrast to pediatric ALL, AML is characterized by relatively few recurring CNAs, and that spectrum of CNA and sequence mutation is significantly associated with disease subtype.

Failure of initial remission-induction therapy and disease recurrence remains a major problem in the management of acute leukemia, however the nature of the biologic factors promoting relapse are incompletely understood. To identify genomic abnormalities associated with relapse, we performed high-resolution, genome-wide analysis of DNA copy number abnormalities and loss-of heterozygosity using Affymetrix single nucleotide polymorphism (SNP) microarrays in 33 cases of relapsed acute leukemia. Sixteen ALL (2 ETV6-RUNX1, 2 MLL rearranged, 6 pseudodiploid or cytogenetically normal B-progenitor ALL, and 6 T-lineage) and 17 AML (two RUNX1-RUNX1T1 [AML1-ETO], two MLL-rearranged, one M7, and 12 with normal karyotype or miscellaneous cytogenetic abnormalities) were studied. Samples with less than 80% blasts were flow sorted to at least 90% purity prior to DNA extraction. Diagnostic samples were available for all cases, and germline samples for 22. DNA copy number and LOH analysis was performed using Affymetrix 250k Nsp and Sty arrays was performed for all samples. Data were analysed using a karyotype-guided normalization algorithm, dChipSNP, and circular binary segmentation. In a detailed comparative analysis of paired diagnostic and relapse samples, changes in DNA copy number abnormalities were identified in the relapse sample in 14 of 16 (87.5%) ALL cases. A striking finding was loss of copy number lesions present at diagnosis in 8/16 ALL relapse samples, and the acquisition of new copy number lesions in 4 of these 8 samples. In each case, the pattern of deletions at antigen receptor loci was comparable between relapse and diagnosis, suggesting the emergence of a related leukemic clone, rather than the development of a distinct second leukemia. An additional 8 ALL relapse samples retained the copy number lesions present at diagnosis, but 6 of these acquired additional copy number abnormalities at relapse, a finding further suggestive of significant clonal evolution. Of the newly acquired copy number abnormalities in the relapse samples, deletions (62.5% of cases) were more common than gains (12.5%). In constrast to ALL a more restricted range of copy number abnormalities was seen in AML, with new abnormalities at relapse seen in 5/17 (29.4%) cases, and deletions (29.4%) outnumbering gains (17.6%). The loss of lesions present at diagnosis was only observed in two AML relapse samples. Examining the entire cohort, the CDKN2A/B locus was most commonly involved (N=5), gains of 1q were noted in two cases, otherwise all observed copy number changes were noted in single cases only, and included focal deletions of ERG and RUNX1. Copy neutral loss of heterozygosity was uncommon, with the exception of three AML cases with UPD of the entire chromosome 13. These observations indicate that relapse is frequently the result of the emergence of a leukemic clone that shows significant genetic differences from the diagnostic clone. Whether these represent rare clones present at the time of diagnosis or are the emergence of new clones as the result of ongoing genomic abnormalities can now be determined using genomic probes specific for the newly acquired deletions.

In a previous gene expression profiling study of acute lymphoblastic leukemia (ALL), we identified a novel subtype of B-progenitor ALL (4.9% of 284 cases) with a unique gene expression profile, aberrant expression of CD2 and the absence of recurring cytogenetic abnormalities (Cancer Cell2002;1:133). Efforts to identify rearrangement or mutation of many of the top-ranked genes in the novel expression signature failed to identify a causative lesion. To further investigate the genetic basis of this subtype, we performed integrated genomic analysis of 277 ALL cases. Affymetrix 250k Sty and Nsp SNP microarrays were used in all cases, and Affymetrix U133A gene expression profiles were obtained on 183 of the cases. Unsupervised clustering of gene expression data identified 16 cases of the novel subtype, including all of the 14 previously defined cases. Remarkably, focal mono-allelic deletions of the ETS family member ERG (v-ets erythroblastosis virus E26 oncogene homolog) were detected by genome-wide copy number analysis in 11/16 (69%) of the novel cases, but not in any other ALL subtype. Extensive analysis failed to reveal evidence of translocations involving the altered ERG allele, indicating that these are intragenic deletions limited to ERG. The ERG deletions involved a subset of exons (ERG isoform 1 exons 3–7 or 3–9) resulting in the expression of internally deleted ERG transcripts with altered reading frames predicted to produce a prematurely truncated N-terminal protein fragment. However, using an alternative translational start site 5′ to exon 10, the transcripts also encode a ∼28kDa C-terminal ERG fragment that contains the entire C-terminal ETS DNA-binding and transactivation domains, but lacks all N-terminal domains. Importantly, western blot analysis of primary leukemic blasts revealed expression of only the 28kDa C-terminal ERG protein, along with full length ERG expressed from the retained wild type allele. Remarkably, the C-terminal ERG protein was also detected in 4 of 5 novel ALL cases that lacked detectable ERG deletions, but not in any other ALL subtype. In luciferase reporter assays, this aberrant ERG protein acts as a competitive inhibitor of wild type ERG. Analysis of a second cohort of 35 B-progenitor ALL cases lacking recurring cytogenetic abnormalities identified two cases with ERG deletions and a third expressing the aberrant ERG protein, all of which had the novel gene expression profile. Sequencing of ERG in 252 ALL cases identified only one case with an ERG mutation that resulted in a frameshift in the ETS domain. This case did not share the novel signature nor express the aberrant C-terminal ERG protein. Finally, in an analysis of 37 acute leukemia cell lines, the B-progenitor ALL line NALM-6 was found to harbor a focal, internally truncating ERG deletion, expressed the aberrant ERG protein, and shared the novel gene expression profile, thus identifying it as a model of this novel ALL subtype. These data establish focal ERG deletions as the genetic lesion underlying a novel subtype of ALL, and have expanded the genetic mechanisms that lead to the dysregulation of ERG from chromosomal translocations that result in enhanced transcriptional activity, to deletions that generate dominant negative forms of the transcription factor.

Using high-resolution SNP arrays and genomic resequencing, we recently reported deletions, translocations, and mutations involving regulators of B cell development in 40% of pediatric B-progenitor ALL (Nature2007;446:758). The most frequently involved genes were PAX5, EBF1, IKZF1 (Ikaros), IKZF3 (Aiolos) and LEF1. Ikaros is a transcription factor required for normal lymphoid development and acts as a tumor suppressor in mice. Focal deletions of IKZF1 were observed in 7 B-progenitor ALL cases, suggesting that the previously reported expression of dominant-negative, non-DNA binding Ikaros isoforms may be caused by genomic IKZF1 abnormalities. We have now extended the analysis to 283 pediatric ALL cases, including B-progenitor ALL with high hyperdiploidy (N=39), hypodiploidy (N=10), rearrangement of MLL (N=23), TCF3-PBX1 (N=17), ETV6-RUNX1 (N=48) and BCR-ABL1 (N=21), as well as cases with low hyperdiploid, normal or miscellaneous cytogenetics (N=75), and T-lineage ALL (N=50). We also examined 22 adult BCR-ABL1 positive ALL cases, 37 acute leukemia cell lines and 49 samples from 23 chronic myeloid leukemia (CML) cases at various stages of disease, including 15 with matched blast crisis samples (12 myeloid, 3 lymphoid). All samples were examined with 500,000 feature Affymetrix SNP arrays (250k Sty and Nsp). The 50k Hind and Xba arrays were also used in 252 ALL cases. Sixty-two (20.3%) ALL cases harboured IKZF1 deletions, including 36 of 43 (83.7%) BCR-ABL1 positive B-ALL cases (76.2% of 21 childhood cases, and 90.9% of 22 adult cases). The deletions were limited to IKZF1 in 25 BCR-ABL1 ALL cases, and in 19 cases deleted an internal subset of IKZF1 exons, most commonly 3–6 (Δ3–6). Remarkably, chronic phase CML samples lacked evidence of IKZF1 deletion, whereas four of 15 matched CML blast crisis samples (66% of lymphoid and 17% of myeloid) had acquired an IKZF1 deletion. The IKZF1 Δ3–6 deletion was also detected in the BCR-ABL1 B-progenitor cell lines BV173, OP1 and SUP-B15, the Δ1–6 deletion in the MYC-IGH/BCL2-IGH B-progenitor cell line 380, and Δ1–7 in the BCR-ABL1 B-progenitor cell line TOM-1. RT-PCR analysis for IKZF1 transcripts demonstrated complete concordance between the extent of IKZF1 deletion and the expression of aberrant Ikaros transcripts lacking internal exons. Importantly, on quantitative RT-PCR analysis and western blotting, expression of the dominant-negative Ik6 transcript and protein, which lacks exons 3–6, was exclusively observed in those cases with IKZF1 Δ3–6, demonstrating that the Ik6 transcript is the result of a specific genetic lesions and not alternative splicing of wild-type IKZF1. Lastly, sequence analysis of the IKZF1 Δ3–6 breakpoints indicated that the deletions arise from aberrant activity of RAG-mediated V(D)J recombination. Taken together, these data demonstrate that deletion of IKZF1, resulting in either haploinsufficiency or the expression of a dominant negative form of the transcription factor, is a central event in the pathogenesis of both pediatric and adult BCR-ABL1 B-progenitor ALL.

To obtain a comprehensive registry of oncogenic lesions in pediatric acute lymphoblastic leukemia (ALL), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of heterozygosity (LOH) in leukemic blasts and matched remission samples from 250 ALLs. We studied B-progenitor ALLs with high hyperdiploidy, n=39; ETV6-RUNX1, n=47; MLL rearranged, n=11; TCF3-PBX1, n=17; BCR-ABL1, n=9; low hyperdiploidy, n=23; hypodiploidy, n=10; unclassified cases, n=42; and 50 T-lineage ALLs. Four arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 loci at a mean inter-marker distance of 4.8 kb. Data was analyzed using dChipSNP and a modified array normalization algorithm using only SNPs from regions known to be diploid by routine karyotyping. Copy number abnormalities were confirmed by FISH and genomic quantitative PCR. Complementary methylation analysis and sequencing of candidate genes was performed. 84% of B-ALLs and 96% of T-ALLs had at least one region of somatic deletion, and excluding cases with high hyperdiploidy, 68% of B-ALLs and 50% of T-ALLs had at least one region of somatic amplification. These included previously identified abnormalities including chromosomal duplications in hyperdiploid B-ALL; 1q duplication in TCF3-PBX1 ALL; and deletions of 9p21 (harboring CDKN2A/B, 70% of T-ALLs, 34.5% of B-ALL), 12p13 (ETV6; 25.5% of B-ALLs, 10% of T-ALLs), 6q16 (22 cases) and 11q (15 cases). The resolution of the arrays enabled precise mapping of the minimal regions of deletion at 9p21 to CDKN2A, and at 12p13 to ETV6. Combined LOH and copy number analysis identified several patterns of 9p21 abnormality: focal hemizygous deletion with corresponding LOH; focal homozygous and flanking hemizygous loss with corresponding LOH, indicating two focal deletional events; and focal homozygous loss with LOH of all of 9p or chromosome 9, indicating loss of the normal 9 or 9p and duplication of the chromosome or chromosomal arm containing the focal deletion. Copy-neutral LOH without any focal deletion in the affected region was uncommon. Deletions involving other genes with potential roles in leukemogenesis were identified including BTG1 (17 cases), ERG (10), FHIT (14), mir-16/-15a (19), MYB (5), NF1 (9), the glucocorticoid receptor NR3C1 (11), PTEN (4), and RB1 (20). Furthermore, deletions, translocations, amplifications, and point mutations of genes that regulate B-cell development and differentiation, including EBF, PAX5, Ikaros and Aiolos, were identified in 40% of B-ALL. For each of the listed genes, cases were identified that contained focal deletions limited to the specific gene. Overall, 73.6% B-ALL and 88% T-ALLs harbored deletions of one of the common lesions listed above, with 48% of B-ALLs and 48.5% of T-ALLs having multiple common lesions. The average number of deletions per case was 3.8 and 5.7 for B and T-lineage ALLs respectively. By contrast, when hyperdiploid cases were excluded, it was rare to find more than 2 regions of amplification in a single case, and the majority of cases contained no amplifications. These findings show the power of high-resolution copy number analysis for the identification of new genetic lesions in cancer, and demonstrate that multiple genetic abnormalities contribute to leukemogenesis in pediatric ALL.

To identify a comprehensive registry of oncogenic lesions in pediatric acute myeloblastic leukemia (AML), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of-heterozygosity (LOH) in leukemic blasts from 112 cases of pediatric AML and corresponding remission samples from 63 of these cases. The analyzed cases included t(8;21)[AML1-ETO] (n=20), inv(16)[CBFβ-MYH11](n=16), t(15;17)[PML-RARα] (n=7), MLL rearranged (n=17), FAB M7 (n=9) and normal cytogenetics or miscellaneous cytogenetic abnormalities (n=43). Four SNP arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 markers at a mean inter-marker distance of 4.8 kb. Combined data were analyzed using dChipSNP and a modified array normalization algorithm using only those SNPs from regions known to be diploid by routine karyotyping. These analyses not only detected known whole or partial chromosomal losses or gains, but also detected numerous copy number abnormalities that were not evident by conventional cytogenetics. Somatic DNA copy number abnormalities were identified in 102 (91.1%) cases. The mean number of lesions per patient was 3.2 (range 1–12), with a mean of 2.13 deletions/whole chromosome losses and 1.02 amplifications/whole chromosome gains per patient. Deletions were detected in the leukemic blasts from over 90% of patients, whereas amplifications were only seen in the leukemic blasts from 54% of patients. The vast majority of deletions were focal (

Chromosomal aberrations are a hallmark of acute lymphoblastic leukemia (ALL) but alone fail to induce leukemia. To identify cooperating oncogenic lesions, we performed genome-wide analysis of leukemic blasts from 242 pediatric ALL patients using high-resolution 100K and 250K Affymetrix single nucleotide polymorphism arrays, and genomic DNA sequencing. Remarkably, our analyses identified deletion, amplification, point mutation and structural rearrangement in genes encoding regulators of B lymphocyte development in over 40% of B-progenitor ALL. PAX5, which encodes a transcription factor critical for B cell commitment and differentiation, was the most frequent target of somatic mutation, being altered in 31.7% of cases. The most frequent PAX5 mutations were copy number alterations with mono-allelic loss in 53 cases, biallelic loss in 3 cases, and an internal amplification in 1 case. PAX5 deletions and the amplification were confirmed by FISH and/or RT-PCR, and were present in over 90% of blasts. Twenty-five of the mono-allelic deletions were confined to PAX5, with the majority deleting only a subset of PAX5 exons. Thus, of the 57 cases with PAX5 copy number changes, the majority had haploinsufficiency of PAX5 (n=30), or generated hypomorphic alleles that produce proteins that lack the DNA-binding domain (n=20) or the transcriptional activation domain (n=7). Four cases contained cryptic PAX5 translocations: PAX5-ETV6 (n=2), PAX5-FOXP1 and PAX5-ZNF521 (1 case each). In addition to the structural alterations, sequencing identified 14 B-ALLs with PAX5 point mutations. Mutations were identified in the DNA-binding paired domain, homeodomain and transactivation domains. The mutations were somatically acquired, and present in a dominant clone. Gel-shift and transcriptional reporter assays demonstrated reduced DNA binding and/or transcriptional activity for each of the identified PAX5 fusion proteins and point mutants. In addition to the PAX5 mutations, deletions were also detected in the B cell regulatory genes Ikaros (20 cases), Aiolos (3), EBF (8), E2A (1), LEF1 (3), and RAG1/2 (11). Importantly, genomic sequencing of Ikaros and EBF revealed no point mutations. Although the high frequency of mutations in genes regulating B-cell development was unexpected, even more surprising was the marked difference in the frequency and type of mutations among the various genetic subtypes of ALL. Hypodiploid ALLs had alterations in B-cell development genes in 100% of cases, with broad deletions of one PAX5 allele, mutation of the other PAX5 allele in 50% of the cases, and mono-allelic deletion of other regulators of B-cell development, frequently with multiple genes affected within a single case. In contrast ETV6-RUNX1 cases exhibited a much more restricted pattern of mutations, with 27% showing focal PAX5 deletion without alterations of the retained allele. At the other end of the spectrum were hyperdiploid ALLs, with only rare cases harboring a deletion of one of these genes. These data demonstrate that disruption of pathways controlling B cell development and differentiation contributes to the pathogenesis of ALL. Moreover, the approach used provides a rational roadmap for the application of genome-wide approaches to the identification of new molecular lesions in cancer.