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Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Childhood B-cell acute lymphoblastic leukaemia (B-ALL) is characterised by recurrent genetic abnormalities that drive risk-directed treatment strategies. Using current techniques, accurate detection of such aberrations can be challenging, due to the rapidly expanding list of key genetic abnormalities. Whole genome sequencing (WGS) has the potential to improve genetic testing, but requires comprehensive validation. We performed WGS on 210 childhood B-ALL samples annotated with clinical and genetic data. We devised a molecular classification system to subtype these patients based on identification of key genetic changes in tumour-normal and tumour-only analyses. This approach detected 294 subtype-defining genetic abnormalities in 96% (202/210) patients. Novel genetic variants, including fusions involving genes in the MAP kinase pathway, were identified. WGS results were concordant with standard-of-care methods and whole transcriptome sequencing (WTS). We expanded the catalogue of genetic profiles that reliably classify PAX5alt and ETV6::RUNX1-like subtypes. Our novel bioinformatic pipeline improved detection of DUX4 rearrangements (DUX4-r): a good-risk B-ALL subtype with high survival rates. Overall, we have validated that WGS provides a standalone, reliable genetic test to detect all subtype-defining genetic abnormalities in B-ALL, accurately classifying patients for the risk-directed treatment stratification, while simultaneously performing as a research tool to identify novel disease biomarkers.

Incorporating genetics into risk-stratification for treatment of childhood B-progenitor acute lymphoblastic leukaemia (B-ALL) has contributed significantly to improved survival. In about 30% B-ALL (B-other-ALL) without well-established chromosomal changes, new genetic subtypes have recently emerged, yet their true prognostic relevance largely remains unclear. We integrated next generation sequencing (NGS): whole genome sequencing (WGS) (n = 157) and bespoke targeted NGS (t-NGS) (n = 175) (overlap n = 36), with existing genetic annotation in a representative cohort of 351 B-other-ALL patients from the childhood ALL trail, UKALL2003. PAX5alt was most frequently observed (n = 91), whereas PAX5 P80R mutations (n = 11) defined a distinct PAX5 subtype. DUX4-r subtype (n = 80) was defined by DUX4 rearrangements and/or ERG deletions. These patients had a low relapse rate and excellent survival. ETV6::RUNX1-like subtype (n = 21) was characterised by multiple abnormalities of ETV6 and IKZF1, with no reported relapses or deaths, indicating their excellent prognosis in this trial. An inferior outcome for patients with ABL-class fusions (n = 25) was confirmed. Integration of NGS into genomic profiling of B-other-ALL within a single childhood ALL trial, UKALL2003, has shown the added clinical value of NGS-based approaches, through improved accuracy in detection and classification into the range of risk stratifying genetic subtypes, while validating their prognostic significance.

The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.Statement of SignificanceWe demonstrate diverse cellular origins of lineage switched relapse within MLL-AF4 pro-B acute leukaemia. Irrespective of the developmental origin of relapse, dysregulation of NuRD and/or other epigenetic machinery underpins fundamental lineage reprogramming with profound implications for the increasing use of epitope directed therapies in this high-risk leukaemia.

Chromosomal translocations are important drivers of haematological malignancies whereby proto-oncogenes are activated by juxtaposition with enhancers, often called enhancer hijacking. We analysed the epigenomic consequences of rearrangements between the super-enhancers of the immunoglobulin heavy locus (IGH) and proto-oncogene CCND1 that are common in B-cell malignancies. By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we characterised the normal chromatin landscape of the human IGH locus and its dynamics after pathological genomic rearrangement. We detected an H3K4me3 broad domain (BD) within the IGH locus of healthy B cells that was absent in samples with IGH-CCND1 translocations. The appearance of H3K4me3-BD over CCND1 in the latter was associated with overexpression and extensive chromatin accessibility of its gene body. We observed similar cancer-specific H3K4me3-BDs associated with hijacking of super-enhancers of other common oncogenes in B-cell (MAF, MYC and FGFR3/NSD2) and T-cell malignancies (LMO2, TLX3 and TAL1). Our analysis suggests that H3K4me3-BDs can be created by super-enhancers and supports the new concept of epigenomic translocation, where the relocation of H3K4me3-BDs from cell identity genes to oncogenes accompanies the translocation of super-enhancers.

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to 3D genome structure remains poorly understood. Here, we used the highly predictive heteromorphic polymer (HiP-HoP) model to predict chromatin conformations at the proto-oncogeneCCND1in healthy and malignant B cells. After confirming that the model gives good predictions of Hi-C data for the nonmalignant human B cell–derived cell line GM12878, we generated predictions for two cancer cell lines, U266 and Z-138. These possess genome rearrangements involvingCCND1and the immunoglobulin heavy locus (IGH), which we mapped using targeted genome sequencing. Our simulations showed that a rearrangement in U266 cells where a singleIGHsuper-enhancer is inserted next toCCND1leaves the local topologically associated domain (TAD) structure intact. We also observed extensive changes in enhancer-promoter interactions within the TAD, suggesting that it is the downstream chromatin remodeling which gives rise to the oncogene activation, rather than the presence of the inserted super-enhancer DNA sequence per se. Simulations of theIGH-CCND1reciprocal translocation in Z-138 cells revealed that an oncogenic fusion TAD is created, encompassingCCND1and theIGHsuper-enhancers. We predicted how the structure and expression ofCCND1changes in these different cell lines, validating this using qPCR and fluorescence in situ hybridization microscopy. Our work demonstrates the power of polymer simulations to predict differences in chromatin interactions and gene expression for different translocation breakpoints.

Leukaemia cells re-program their microenvironment to provide proliferation support and protection from standard chemotherapy, molecularly targeted therapies, and immunotherapy. Although much is becoming known about molecules that drive niche-dependent treatment resistance; means of targeting these in the clinics has remained a key obstacle. To address this challenge, we have developed human induced pluripotent stem cell engineered niches ex vivo to reveal insights into druggable cancer-niche dependencies. We show that mesenchymal (iMSC) and vascular niche-like (iANG) cells support ex vivo proliferation of patient-derived leukaemia cells, impact dormancy and mediate therapy resistance. iMSC protected both non-cycling and cycling blasts against dexamethasone treatment while iANG protected only dormant blasts. Leukaemia proliferation and protection from dexamethasone induced-apoptosis was dependent on direct cell-cell contact and mediated by CDH2. To explore the therapeutic potential of disrupting this cell-cell interaction, we tested the CDH2 antagonist ADH-1 (previously in phase I / II for solid tumours) in a very aggressive patient-derived xenograft leukaemia mouse model. ADH-1 showed high in vivo efficacy. ADH-1/ dexamethasone combination therapy was superior to dexamethasone alone with no ADH1 conferred additional toxicity. These findings provide a proof-of-concept starting point to develop novel, potentially safer therapeutics that target niche-mediated cancer cell dependencies in haematological malignancies.SummaryCDH2 mediated niche-dependent cancer proliferation and treatment resistance is clinically targetable via ADH-1, a low toxic agent that could be potentially repurposed for future clinical trials in acute leukaemia.

Genomic rearrangements are known to result in proto-oncogene deregulation in many cancers, but the link to genome 3D structure remains poorly understood. Here we used the highly-predictive heteromorphic polymer (HiP-HoP) model to predict chromatin conformations at the proto-oncogene CCND1 in healthy and malignant B-cells. After confirming that the model gives good predictions of Hi-C data for the non-malignant human B-cell derived cell line GM12878, we generated predictions for two cancer cell lines, U266 and Z-138. These possess genome rearrangements involving CCND1 and the immunoglobulin heavy chain locus (IGH), which we mapped using targeted genome sequencing. Our simulations showed that a rearrangement in U266 cells where a single IGH super-enhancer is inserted next to CCND1 leaves the local topologically associated domain (TAD) structure intact. We also observed extensive changes in enhancer-promoter interactions within the TAD, suggesting that it is the downstream chromatin remodelling which gives rise to the oncogene activation, rather than the presence of the inserted super-enhancer DNA sequence per se. Simulations of the IGH-CCND1 reciprocal translocation in Z-138 cells revealed that an oncogenic fusion TAD is created, encompassing CCND1 and the IGH super-enhancers. We predicted how the structure and expression of CCND1 changes in these different cell lines, validating this using qPCR and fluorescence in situ hybridization microscopy. Our work demonstrates the power of polymer simulations to predict differences in chromatin interactions and gene expression for different translocation break-points.

Comparison of intratumor genetic heterogeneity in cancer at diagnosis and relapse suggests that chemotherapy induces bottleneck selection of subclonal genotypes. However, evolutionary events subsequent to chemotherapy could also explain changes in clonal dominance seen at relapse. We therefore investigated the mechanisms of selection in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) during induction chemotherapy where maximal cytoreduction occurs. To distinguish stochastic versus deterministic events, individual leukemias were transplanted into multiple xenografts and chemotherapy administered. Analyses of the immediate post-treatment leukemic residuum at single-cell resolution revealed that chemotherapy has little impact on genetic heterogeneity. Rather, it acts on extensive, previously unappreciated, transcriptional and epigenetic heterogeneity in BCP-ALL, dramatically reducing the spectrum of cell states represented, leaving a genetically polyclonal but phenotypically uniform population, with hallmark signatures relating to developmental stage, cell cycle and metabolism. Hence, canalization of the cell state accounts for a significant component of bottleneck selection during induction chemotherapy. Enver and colleagues report that epigenetic cell state, rather than genetic diversity, drives bottleneck selection of subclonal genotypes during induction chemotherapy in childhood B-cell precursor acute lymphoblastic leukemia.

International audience; Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain–associated genes in healthy human thymic T cell populations and a collection of T cell acute lymphoblastic leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation, and their varying breadth allows the distinction between normal and neoplastic cells. Although broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain–associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we showed that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain–associated genes in preleukemic cells. Finally, an H3K4me3 demethylase inhibitor differentially targets T-ALL cell lines depending on the extent and number of broad domains. Our results show that the regulation of broad H3K4me3 domains is associated with leukemogenesis, and suggest that the presence of these structures might be used for epigenetic prioritization of cancer-relevant genes, including long noncoding RNAs.

Prevention of central nervous system (CNS) relapse is critical for cure of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Despite this, mechanisms of CNS infiltration are poorly understood, and the timing, frequency, and properties of BCP-ALL blasts entering the CNS compartment are unknown. We investigated the CNS-engrafting potential of BCP-ALL cells xenotransplanted into immunodeficient NOD.Cg- ITALIC! Prkdc (ITALIC! scid) ITALIC! Il2rg (ITALIC! tm1Wjl)/SzJ mice. CNS engraftment was seen in 23 of 29 diagnostic samples (79%): 2 of 2 from patients with overt CNS disease and 21 of 27 from patients thought to be CNS negative by diagnostic lumbar puncture. Histologic findings mimic human pathology and demonstrate that leukemic cells transit the blood-cerebrospinal fluid barrier situated close to the dural sinuses, the site of recently discovered CNS lymphatics. Retrieval of blasts from the CNS showed no evidence for chemokine receptor-mediated selective trafficking. The high frequency of infiltration and lack of selective trafficking led us to postulate that CNS tropism is a generic property of leukemic cells. To test this, we performed serial dilution experiments which showed CNS engraftment in 5 of 6 mice after transplant of as few as 10 leukemic cells. Clonal tracking techniques confirmed the polyclonal nature of CNS-infiltrating cells, with multiple clones engrafting in both the CNS and periphery. Overall, these findings suggest that subclinical seeding of the CNS is likely to be present in most BCP-ALL patients at original diagnosis, and efforts to prevent CNS relapse should concentrate on effective eradication of disease from this site rather than targeting entry mechanisms.

Intrachromosomal amplification of chromosome 21 is a heterogeneous chromosomal rearrangement occurring in 2% of cases of childhood precursor B-cell acute lymphoblastic leukemia. These abnormalities are too complex to engineer faithfully in animal models and are unrepresented in leukemia cell lines. As a resource for future functional and preclinical studies, we have created xenografts from the leukemic blasts of patients with intrachromosomal amplification of chromosome 21 and characterized them by in-vivo and ex-vivo luminescent imaging, flow immunophenotyping, and histological and ultrastructural analyses of bone marrow and the central nervous system. Investigation of up to three generations of xenografts revealed phenotypic evolution, branching genomic architecture and, compared with other B-cell acute lymphoblastic leukemia genetic subtypes, greater clonal diversity of leukemia-initiating cells. In support of intrachromosomal amplification of chromosome 21 as a primary genetic abnormality, it was always retained through generations of xenografts, although we also observed the first example of structural evolution of this rearrangement. Clonal segregation in xenografts revealed convergent evolution of different secondary genomic abnormalities implicating several known tumor suppressor genes and a region, containing the B-cell adaptor, PIK3AP1, and nuclear receptor co-repressor, LCOR, in the progression of B-cell acute lymphoblastic leukemia. Tracking of mutations in patients and derived xenografts provided evidence for co-operation between abnormalities activating the RAS pathway in B-cell acute lymphoblastic leukemia and for their aggressive clonal expansion in the xeno-environment. Bi-allelic loss of the CDKN2A/B locus was recurrently maintained or emergent in xenografts and also strongly selected as RNA sequencing demonstrated a complete absence of reads for genes associated with the deletions.

Purpose To determine the prevalence and prognostic association of immunoglobulin heavy chain (IGH@) translocations in acute lymphoblastic leukemia (ALL). Patients and Methods The cohort comprised 3,269 patients treated on either the UKALL2003 trial for children and adolescents (1 to 24 years old) or the UKALLXII trial for adolescents and adults (15 to 59 years old). High-throughput fluorescent in situ hybridization was used to detect IGH@ translocations. Results We identified IGH@ translocations in 5% of patients with ALL (159 of 3,269 patients), in patients with both B-cell (148 of 2,863 patients) and T-cell (11 of 408 patients) disease. Multiple partner genes were identified including CRLF2 (n = 35), five members of the CEPB gene family (n = 17), and ID4 (n = 11). The level of the IGH@-positive clone varied and indicated that some IGH@ translocations were primary events, whereas others were secondary aberrations often associated with other established aberrations. The age profile of patients with IGH@ translocations was distinctive, with a median age of 16 years and peak incidence of 11% among 20- to 24-year-old patients. Among patients with B-cell precursor ALL who were Philadelphia chromosome negative, those with an IGH@ translocation had an inferior overall survival compared with other patients (UKALL2003: hazard ratio, 2.37; 95% CI, 1.34 to 4.18; P = .003; UKALLXII: hazard ratio, 1.73; 95% CI, 1.22 to 2.47; P = .002). However, this adverse effect was not independent of age or minimal residual disease status and did not seem to be driven by an increased risk of relapse. Conclusion IGH@ translocations define a genetic feature that is frequent among adolescents and young adults with ALL. Although associated with an adverse outcome in adults, it is not an independent prognostic factor in children and adolescents.

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are recurrent but rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and various partner genes have been described. Here, we report a new series of 29 cases of BCP-ALL with IGH@ translocations. The partner gene was identified by fluorescence in situ hybridization and/or molecular cloning in 20 patients. Members of the CEBP gene family (n = 11), BCL2 (n = 3), ID4 (n = 3), EPOR (n = 2), and TRA/D@ (n = 1) were identified and demonstrated by quantitative real-time reverse transcriptase-polymerase chain reaction to be markedly up-regulated. The present cases, added to those already reported, confirm the diversity of the partner genes, which, apart from BCL2, are specific to BCP-ALL. Collectively, patients with IGH@ translocations may represent a novel sub-group of BCP-ALL occurring in adolescents and young adults.

Abstract 290 Multiplex Ligation-dependent Probe Amplification (MLPA) provides an accurate and reliable high throughput method to screen for copy number abnormalities (CNA) of the significant genes in BCP-ALL. We have screened a consecutive cohort of 1427 childhood BCP-ALL patients treated on UKALL97 and UKALL2003 using the P335 SALSA MLPA kit IKZF1 (MRC Holland, The Netherlands). We here report the association of CNA involving these genes with clinical features. The overall breakdown of CNA was CDKN2A/B 28% (395), ETV6 22% (312), PAX5 19% (272), IKZF1 14% (196), RB1 6% (92), BTG1 6% (89) and EBF1 2% (30), as well as the rearrangement, P2RY8-CRLF2 4% (63). The cohort comprised 762 (54%) males and 665 (46%) females. There was no shift in the gender balance according to CNA. The median age of the entire cohort was 5 years (range 1–23) with 24% of patients being ≥10 years. There was some correlation between the distribution of CNA and patient age. Patients with IKZF1 and CDKN2A/B deletions were significantly older at 7 years (p50×109/L (each p There is particular interest in IKZF1 and CRLF2 in relation to outcome in BCP-ALL. We have previously shown an intermediate outcome for patients with CRLF2 rearrangements. We here examined the prognostic effect of IKZF1 deletions in this patient cohort; but excluded Down syndrome and BCR-ABL1 positive patients from survival analyses. Among 469 ALL97 patients, 13% (63) had IKZF1 deletions and after a median follow-up time of 9.5 years, 49% (31) had relapsed and 40% (25) had died. In the NCI standard risk (SR) group, patients with IKZF1 deletions had a significantly inferior event free survival (EFS) at 5 years (61% v 85%, p=0.0006). However, the effect appeared to be largely confined to patients with intermediate risk cytogenetics (63% v 85%, p=0.025) rather than those with good risk cytogenetics (75% v 87%, p=0.2915). As expected the NCI high risk (HR) patients with IKZF1 deletions had an inferior EFS: 50% v 68%, p=0.0112. In ALL2003, 776 patients with a median follow-up time of 3.9 years were available for analysis. Among 11% (87) patients with IKZF1 deletions, 13% (11) had relapsed and 5% (4) died in remission, resulting in a significantly inferior 5 year EFS: 78% v 90% p=0.0025. Of note, 8/11 IKZF1 deleted patients who relapsed were MRD positive (>0.01%) at day 28 and only 1/22 patients with good risk cytogenetics and IKZF1 deletion relapsed. There was no evidence that the type of deletion (deletion of exons 4–7 vs others) was linked to outcome in either the ALL97 or ALL2003 cohort. In conclusion, we have shown that IKZF1, PAX5 and/or CDKN2A/B aberrations are significantly associated with NCI HR, which may point to an associated poor outcome. Indeed, we showed that patients with IKZF1 deletions had an inferior outcome overall. However, there was evidence to suggest that the effect was pleiotropic and, importantly, IKZF1 deleted patients treated on ALL2003 in general achieved a respectable EFS rate of 75% at 5 years. Although in most studies IKZF1 deletions have been associated with a poor prognosis in BCP-ALL, while the risk relating to CRLF2 has been variable, thus far these findings, including those from this study, do not support changes to treatment for patients with these abnormalities. Disclosures: No relevant conflicts of interest to declare.

Deregulated expression of CRLF2 (CRLF2-d) arises via its juxtaposition to the IGH@ enhancer or P2RY8 promoter. Among 865 BCP-ALL children treated on MRC ALL97, 52 (6%) had CRLF2-d, but it was more prevalent among Down syndrome patients (54%). P2RY8-CRLF2 (n = 43) was more frequent than IGH@-CRLF2 (n = 9). CRLF2-d was not associated with age, sex, or white cell count, but IGH@-CRLF2 patients were older than P2RY8-CRLF2 patients (median 8 vs 4 years, P = .0017). Patients with CRLF2-d were more likely to present with enlarged livers and spleens (38% vs 18%, P < .001). CRLF2-d was not seen in conjunction with established chromosomal translocations but 6 (12%) cases had high hyperdiploidy, and 5 (10%) had iAMP21. Univariate analysis suggested that CRLF2-d was associated with an inferior outcome: (event-free survival [EFS] hazard ratio 2.27 [95% confidence interval 1.48-3.47], P < .001; OS 3.69 [2.34-5.84], P < .001). However, multivariate analysis indicated that its effect was mediated by other risk factors such as cytogenetics and DS status (EFS 1.45 [0.88-2.39], P = .140; OS 1.90 [1.08-3.36], P = .027). Although the outcome of IGH@-CRLF2 patients appeared inferior compared with P2RY8-CRLF2 patients, the result was not significant (EFS 2.69 [1.15-6.31], P = .023; OS 2.86 [1.15-6.79], P = .021). Therefore, we concluded that patients with CRLF2-d should be classified into the intermediate cytogenetic risk group.

A novel translocation, t(14;19)(q32;p13), involving IGH@ and the cytokine receptor for erythropoietin

Subtle variation in the expression or function of a small group of transcription factors can drive leukemogenesis. The CEBPA protein is known to regulate the balance between cell proliferation and differentiation during early hematopoietic development and myeloid differentiation. In human myeloid leukemia, CEBPA is frequently inactivated by mutation and indirect and posttranslational mechanisms, in keeping with tumor suppressor properties. We report that CEBPA is activated by juxtaposition to the immunoglobulin gene enhancer upon its rearrangement with the immunoglobulin heavy-chain locus in precursor B-cell acute lymphoblastic leukemia harboring t(14;19)(q32;q13). Overexpression of apparently normal CEBPA RNA or protein was observed in 6 patients. These data indicate that CEBPA may exhibit oncogenic as well as tumor suppressor properties in human leukemogenesis.

Introduction: Incorporating cytogenetics into risk stratification for the treatment of childhood ALL has contributed to increased survival rates. However approximately 25% of patients, the B-other subgroup, harbour none of the known major chromosomal abnormalities. Within this group, Philadelphia-like (Ph-like) patients show a similar gene expression profile to those with the BCR-ABL1 fusion and share the same high risk of relapse. Ph-like ALL is genetically heterogeneous with some patients harbouring tyrosine kinase activating gene fusions, such as EBF1-PDGFRB, or activation of the JAK-STAT signalling pathway, making them amenable to targeted therapy with kinase inhibitors. Methods: We studied patients with B-other ALL entered to the MRC UKALL2003 treatment trial using a panel of break-apart FISH probes to identify rearrangements of PDGFRB/CSF1R , ABL1 , ABL2 and JAK2. Rearrangements of the CRLF2 gene were identified using break-apart FISH probes for CRLF2, P2RY8 and IGH and/or MLPA using the P335-ALL-IKZF1 kit (MRC Holland, The Netherlands). Where possible, partner genes were identified by cytogenetics and FISH. RNA samples from 135 of the B-other cohort were screened using a Taqman low density array (TLDA) to detect the Ph-like gene expression signature and P2RY8-CRLF2 gene fusion. The overlap between the two cohorts is shown in Figure 1. Results: Within this B-other cohort, rearrangement of CRLF2 was the most prevalent abnormality, occurring in 13% of patients (n=65/503), with two-thirds of them showing deletion of the PAR1 region, resulting in P2RY8-CRLF2 fusion (n=43) and the remaining third showing IGH-CRLF2 (n=22). Rearrangements of PDGFRB/CSF1R occurred in 3.4% of the cohort(n=17/491), while ABL1 (n=4/485), ABL2 (n=1/454) and JAK2 (n=2/481) rearrangements were rare, each occurring in less than 1% of B-other patients. Partner genes were identified in 17 cases with kinase rearrangements, with EBF1-PDGFRB being the most common (n=10). PAX5-JAK2 was identified in 2 patients and ATF7IP-PDGFRB, MEF2D-CSF1R, SSBP2-CSF1R, ETV6-ABL1 and ZC3HAV1-ABL2 in single cases. By TDLA, 33/135 (24%) patients tested positive for a Ph-like signature. In 9 patients the arrays indicated high expression of CRLF2 with 7 of them having a P2RY8-CRLF2 transcript by TLDA, which was confirmed by FISH in 5 cases and MLPA in another case. In the remaining patients, the mechanism of CRLF2 over-expression remains unknown as no P2RY8-CRLF2 transcript was detectedby TDLA and no material was available for FISH or MLPA. A single case negative for Ph-like signature tested positive for P2RY8-CRLF2 transcript. Seven patients showed an ABL-class rearrangement by cytogenetics and FISH (EBF1-PDGFRB, n=5; ETV6-ABL1, n=1; ABL1 rearrangement partner unknown, n=1). The genetics underlying the remaining cases is being further investigated by whole genome and transcriptome sequencing. Of the 102 patients negative for a Ph-like signature, 71 of them were tested by FISH for kinase fusions. In a single case positive for EBF1-PDGFRB by FISH, expression of the EBF1-PDGFRB transcript was confirmed by RT-PCR. The 5-year event free survival (EFS) of the entire B-other cohort was 82% with a relapse rate (RR) of 12%. Patients with rearrangements of PDGFRB/CSF1R (n=17)and those testing positive for a Ph-like expression signature (n=33) showed an inferior outcome, with EFS rates of 31% and 65% and RR of 64% and 28%, respectively (p Conclusion: This is the largest unbiased cohort of paediatric B-other ALL screened for ABL1, ABL2, PDGFRB/CSF1R and JAK2 fusions. We demonstrate that FISH can reliably detect the full spectrum of these genetic changes, most of which are cryptic by cytogenetic analysis. These abnormalities were mutually exclusive with an overall frequency in B-other ALL of ~15%. Patients with rearrangements of PDGFRB/CSF1R and those testing positive for a Ph-like gene expression had high rates of relapse. Given the efficacy of tyrosine kinase inhibitors in treatment of these patients, testing for these abnormalities should be integrated into future clinical trials. Disclosures Mullighan: Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding.

A new recurrent translocation t(11;14)(q24;q32) involving IGH@ and miR-125b-1 in B-cell progenitor acute lymphoblastic leukemia

In childhood B-cell precursor acute lymphoblastic leukemia, cytogenetics is important in diagnosis and as an indicator of response to therapy, thus playing a key role in risk stratification of patients for treatment. Little is known of the relationship between different cytogenetic subtypes in B-cell precursor acute lymphoblastic leukemia and the recently reported copy number abnormalities affecting significant leukemia associated genes. In a consecutive series of 1427 childhood B-cell precursor acute lymphoblastic leukemia patients, we have determined the incidence and type of copy number abnormalities using multiplex ligation-dependent probe amplification. We have shown strong links between certain deletions and cytogenetic subtypes, including the novel association between RB1 deletions and intrachromosomal amplification of chromosome 21. In this study, we characterized the different copy number abnormalities and show heterogeneity of PAX5 and IKZF1 deletions and the recurrent nature of RB1 deletions. Whole gene losses are often indicative of larger deletions, visible by conventional cytogenetics. An increased number of copy number abnormalities is associated with NCI high risk, specifically deletions of IKZF1 and CDKN2A/B, which occur more frequently among these patients. IKZF1 deletions and rearrangements of CRLF2 among patients with undefined karyotypes may point to the poor risk BCR-ABL1-like group. In conclusion, this study has demonstrated in a large representative cohort of children with B-cell precursor acute lymphoblastic leukemia that the pattern of copy number abnormalities is highly variable according to the primary genetic abnormality.

The P2RY8-CRLF2 fusion defines a particular relapse-prone subset of childhood acute lymphoblastic leukemia (ALL) in Italian Association of Pediatric Hematology and Oncology Berlin-Frankfurt-Münster (AIEOP-BFM) 2000 protocols. To investigate whether and to what extent different clone sizes influence disease and relapse development, we quantified the genomic P2RY8-CRLF2 fusion product and correlated it with the corresponding CRLF2 expression levels in patients enrolled in the BFM-ALL 2000 protocol in Austria. Of 268 cases without recurrent chromosomal translocations and high hyperdiploidy, representing approximately 50% of all cases, 67 (25%) were P2RY8-CRLF2 positive. The respective clone sizes were ≥ 20% in 27% and < 20% in 73% of them. The cumulative incidence of relapse of the entire fusion-positive group was clone size independent and significantly higher than that of the fusion-negative group (35% ± 8% vs 13% ± 3%, P = .008) and primarily confined to the non–high-risk group. Of 22 P2RY8-CRLF2–positive diagnosis/relapse pairs, only 4/8 had the fusion-positive dominant clone conserved at relapse, whereas none of the original 14 fusion-positive small clones reappeared as the dominant relapse clone. We conclude that the majority of P2RY8-CRLF2–positive clones are small at diagnosis and virtually never generate a dominant relapse clone. Our findings therefore suggest that P2RY8-CRLF2–positive clones do not have the necessary proliferative or selective advantage to evolve into a disease-relevant relapse clone.

Children with Down's syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL) and have a low frequency of established genetic aberrations. We aimed to determine which genetic abnormalities are involved in DS ALL. We studied the frequency and prognostic value of deletions in B-cell development genes and aberrations of janus kinase 2 (JAK2) and cytokine receptor-like factor 2 (CRLF2) using array-comparative genomic hybridization, and multiplex ligation-dependent probe amplification in a population-based cohort of 34 Dutch Childhood Oncology Group DS ALL samples. A population-based cohort of 88 DS samples from the UK trials was used to validate survival estimates for IKZF1 and CRLF2 abnormalities. In total, 50% of DS ALL patients had ≥1 deletion in the B-cell development genes: PAX5 (12%), VPREB1 (18%) and IKZF1 (35%). JAK2 was mutated in 15% of patients, genomic CRLF2 rearrangements in 62%. Outcome was significantly worse in patients with IKZF1 deletions (6-year event-free survival (EFS) 45 ± 16% vs 95 ± 4%; P=0.002), which was confirmed in the validation cohort (6-year EFS 21 ± 12% vs 58 ± 11%; P=0.002). This IKZF1 deletion was a strong independent predictor for outcome (hazard ratio EFS 3.05; P=0.001). Neither CRLF2 nor JAK2 were predictors for worse prognosis. If confirmed in prospective series, IKZF1 deletions may be used for risk-group stratification in DS ALL.

Deregulated expression of the type I cytokine receptor, cytokine receptor-like factor 2 (CRLF2 -d), is observed in 5% of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). It occurs via three known aberrations; a cryptic reciprocal translocation with the immunoglobulin heavy chain locus (IGH); an interstitial deletion within PAR1, resulting in the P2RY8-CRLF2 fusion; rare but recurrent CRLF2 mutation (F323C). All three result in overexpression of CRLF2, however alone it is insufficient to cause overt leukaemia. Mutations of the Janus kinase (JAK) family genes and the IL7 receptor, are recurrent (50% of CRLF2-d) and together with CRLF2 -d result in transformation of mouse BaF3 cells. We noted that outcome data from different study groups were inconsistent, with patients being classified as either high or intermediate risk. Thus, in this largest study to date, our aim was to acertain whether differences in clinical and/or genetic features between patients with CRLF2-d and involvement of either IGH or P2RY8 may define them as different disease entities and partly explain the outcome heterogeneity. Among 160 CRLF2-d patients, chromosomal analysis confirmed a high incidence of additional somatic copies of chromosomes X (39/88, 44%) and 21 (23/88, 26%) and identified that aneuploidy of chromosomes 9 (5/88, 6%) and 17 (7/88, 8%) were recurrent in both groups. From comparison of patients with IGH-CRLF2 and P2RY8-CRLF2, we noted a higher frequency of IKZF1 (79% v 37% p3 additional copy number alterations (CNA)) by MLPA (SALSA MLPA P335 kit, MRC Holland, The Netherlands) (p=0.037) among IGH-CRLF2 patients. We verified that (48/160) 30% of CRLF2 -d patients were Down syndrome (DS). In addition to CNA within the immunoglobulin loci and the genes investigated by MLPA, SNP6.0 arrays (Affymetrix, Santa Clara, USA) (n=25) and subsequent FISH screening identified recurrent CNA in the histone cluster at 6p22.2 (7/25, 28%), VPREB1 (6/25, 24%), ADD3 (14/58, 24%), BTLA (4/25, 16%), SLX4IP (12/43, 28%), SERP2 and TSC22D1 (6/53, 11%), PBX3 (16/54, 30%). A novel finding was an interstitial deletion at Xp11.4 fusing USP9X to DDX3X (9/50, 18%). A total of 137 somatic structural variants (SV) were detected by whole genome sequencing of 11 patients [tandem duplication- (n=6), deletion- (n=104), inversion- (n=14) and translocation-type (n=13)] creating an average of 12.7 validated SV per patient. Mutations were identified at an incidence of 12.8 per patient, including mutations in JAK1 and JAK2 (40%) and IL7R, CACNA1D and USH2A (n=2). Mutations in kinase genes were common and unique to all patients, a number have not been previously reported (e.g. ERBB4, TTBK1 and STK38L). Of interest, 7 patients had ≥1 point mutation in gene(s) involved in cell adhesion (e.g. NPNT, ADAM29, ITGB7, COL3A1 and USH2A). There was a significant difference in the median age and white blood cell count (WBC) between P2RY8-CRLF2 and IGH-CRLF2 (4yrs v 14yrs, p50x106 /L - 25% v 44%, p=0.021). Overall IGH-CRLF2 patients had a worse outcome compared to P2RY8-CRLF2 (p=0.191) with outcome heterogeneity in relation to gender (IGH females, HR 3.75, p=0.003), number of CNA (IGH In summary we have confirmed differences in age and WBC, involvement of kinase gene mutations and chromosomal gains in CRLF2-d ALL. Other findings included recurrent deletions involving TSC22D1 and PBX3, transcription factors involved in other human cancers; SLX4IP, an uncharacterized gene, frequently deleted in childhood ALL; the novel involvement of USP9X, a deubiquitinase involved in cancer development. We also identified recurrent mutations in cell adhesion genes. We showed that IGH-CRLF2 patients had a worse outcome, with heterogeneity seen between gender, number of gene deletions and treatment trial. We have shown that there are genetic and clinical differences dependent on whether CRLF2 is deregulated by IGH or P2RY8, suggesting that they should be regarded as different clincial entities in future studies. Disclosures Campbell: 14M genomics: Other: Co-founder and consultant.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is a heterogeneous disease in which patient outcome is influenced by genetic lesions. Cytogenetic classification has improved survival through risk stratification for treatment. Translocations involving the Immunoglobulin Heavy Chain Locus (IGH) comprise 5% of BCP-ALL and lead to overexpression of juxtaposed genes, due to the powerful IGH enhancer elements. Multiple IGH partner genes have been described in BCP-ALL, including five members of the Ccaat Enhancer-Binding Protein (CEBP) transcription factor family: CEBPA, CEBPB, CEBPD, CEBPE and CEBPG, which comprise 12% of the IGH-translocation subgroup. Patients with potential IGH-CEBP translocations were identified by mining cytogenetic data from UK ALL trials. The translocations were confirmed by fluorescence in situ hybridisation (FISH), followed by screening for commonly occurring BCP-ALL lesions using Multiplex Ligation-dependent Probe Amplification (MLPA) (SALSA P335 kit, MRC Holland), and detection of novel genetic lesions using SNP6.0 arrays. A total of 33 IGH-CEBP patients were identified; CEBPD (n=11, 34%); CEBPA (n=10, 30%); CEBPB (n=8, 24%); CEBPE (n=3, 9%); CEBPG (n=1, 3%). Cohort median age was 15 years (range, 2-65 years). Interestingly the majority of IGH-CEBPD patients were under the age of 10 years, while CEBPA and CEBPB patients were older, all over the age of 10 years (p=0.005). The median white blood cell (WBC) count of the cohort was low at 8.24x109/l (range, 0.9-430 109/l), the highest WBCs were observed in the CEBPB subgroup (p=0.04). Associated abnormalities were mixed: 5 Down syndrome (DS) patients were identified in the cohort all exclusive to the IGH-CEBPD subgroup, an observation which has been previously reported, BCR-ABL1 translocation (n=1), high hyperdiploidy (n=1) and hypodiploidy (n=1). Although numbers were small, some trends were observed: 27% (n=9) of patients were deceased: CEBPB (n=4, 44%), CEBPA (n=2, 22%), CEBPD (n=2, 22%) and CEBPE (n=1, 13%). Overall 15% (n=5) relapsed: CEBPD (n=3, 60%), CEBPA and CEBPB (each n=1, 20%). Minimum residual disease data (MRD) at day 28 were available for 8 (24%) patients, CEBPD (n=3, 38%), CEBPA (n=2, 25%), CEBPB, CEBPE and CEBPG (n=1, 13% each). Although numbers were low, all 3 MRD negative patients were IGH-CEBPD while the remaining patients, all MRD positive, comprised the remaining CEBP subgroups. MLPA screening of 28 patients identified that 25% had whole gene deletions of CDKN2A/CDKN2B (n=7): the most common copy number abnormality (CNA) observed in IGH-CEBP. IKZF1 deletions of exons 4-7, a deletion resulting in the formation of the dominant negative isoform IK6, were present in 21% (n=6) patients: CEBPB (n=4, 67%) and CEBPD (n=2, 33%) (P=0.04). Four CNAs of the PAX5 gene were observed: individual gains of exon 5 and exon 7 (n=1 each, 50%); single exon gains of PAX5 have been predicted to lead to altered protein activity, whole gene deletion (n=1), deletion of exon 1 along with gain of exons 7 and 8 (n=1). Using SNP6.0 arrays (n=13) a recurrent focal deletion of the ABL2 gene was discovered (n=4, 31%). These patients had a common region of deletion covering exon 2 of the gene. This deletion covers the Src Homology 2 domain (SH2), which allows for the recognition of phosphorylated tyrosine residues, vital for cell signalling. Patients spanned three subgroups; CEBPB (n=2, 50%), CEBPA (n=1, 25%) and CEBPD (n=1, 25%). In this study, we show that in the IGH-CEBP subgroup, the CEBP partner gene influences disease phenotype. CEBPB patients showed more aggressive BCP-ALL characteristics, with the highest WBC, death rate (50%), older age and highest levels of associated abnormalities, with all patients showing one or more genetic lesions. The CEBPD subgroup comprises the youngest patients, a statistically significant finding both with and without inclusion of DS status. This subgroup also had the highest number of relapses in the cohort, and was the only CEBP subgroup to be MRD negative at day 28, albeit based on low patient numbers. A focal deletion within ABL2, a paralog of the ABL1 gene, is an interesting new abnormality in this subgroup, which may contribute to disease development. Disclosures No relevant conflicts of interest to declare.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Genomic and expression analyses of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL) have uncovered several oncogenic events that contribute to the transformation of B-cell progenitors.1 Besides BCR-ABL1, mutations and rearrangements detected in BCP-ALL predominantly affect genes coding for transcriptional regulators involved in the control of early hematopoietic differentiation, B-cell development or both. Activating mutations in the JAK2 gene that targets the R683 residue2, 3, 4 have been recently suggested to cooperate with the transcriptional activation of the gene coding for the cytokine receptor chain gene TSLPR/CRLF2.5 TSLPR codes for a cytokine receptor chain related to γc/IL2Rc. Both chains are able to interact with IL7R to constitute the receptor for IL7 (γc+IL7R) or TSLP (TSLPR+IL7R). Signaling from the IL7 receptor is known to involve JAK1 and JAK3 whereas intracellular signaling by the TSLP receptor is poorly characterized.6 In BCP-ALL, aberrant expression of TSLPR itself occurs through either a cryptic chromosomal translocation between the TSLPR locus (Xp22/Yp11) and the immunoglobulin heavy-chain locus (14q32) or through a deletion of chromosome X or Y pseudoautosomal sequences juxtaposing the P2RY8 gene to TSLPR.5 Sox5 upregulation through juxtaposition to the P2RY8 promoter has been previously reported in lymphoma.7

Translocations involving the immunoglobulin heavy chain locus (IGH@) at chromosome band 14q32 are common in mature B-cell neoplasms, but are rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we report the translocation, t(6;14)(p22;q32), involving IGH@ as a novel recurrent translocation in 13 BCP-ALL patients. Fluorescence in situ hybridization and long-distance inverse polymerase chain reaction (PCR) identified ID4 as the partner gene. Breakpoints were scattered over a 19kb region centromeric of ID4. Quantitative real-time PCR showed up-regulation of ID4 mRNA. All patients had deletions of CDKN2A and PAX5 located on the short arm of chromosome 9, frequently as a result of an isochromosome, i(9)(q10) (9/13, 69%). This study defines a new subgroup of BCP-ALL characterized by ID4 over-expression and CDKN2A and PAX5 deletions. Preliminary survival data suggest that this subgroup may be associated with a good response to therapy.

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse–polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5′ or 3′ of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)–PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Insight into the role of recurrent genomic changes arising in leukemia has depended largely on the functional analysis of cell lines and transgenic animals. However not all acquired abnormalities are represented in available cell lines and a few are too complex to be faithfully engineered in animal models. One such abnormality is intrachromosomal amplification of chromosome 21 (iAMP21), a heterogeneous cytogenetic rearrangement, with a distinct clinical profile, occurring in 2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most highly amplified segment of iAMP21 varies in size and copy number but has always included a 5.1 Mb common region of amplification (CRA). Regions flanking the most highly amplified segment have profiles that may be step-like, or more complex combinations of lower level amplification, normal copy number and deletion. Abnormalities at other chromosomal locations such as deletions of IKZF1, CDKN2A/B or RB1 and rearrangements activating CRLF2 co-occur with iAMP21 but have never been shown to precede its formation. To investigate clonal evolution and to provide a resource for future functional studies, we established xenografts from three cases of iAMP21 BCP-ALL in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Signs of disease were seen between 6 and 8 months after intrafemoral injection and autopsy revealed marked splenomegaly in all cases. Cells isolated from the bone marrow and spleen, were positive for human B cell markers CD19 and CD10. Disease onset was more rapid (3-6 months) when primografts were transplanted and cells from these secondary mice were used to establish leukemia in a third generation. Whole body imaging of mice transplanted with xenograft cells, transduced with a lentivirus expressing luciferase, revealed the rapid spread of disease from the injection site to the contralateral femur, sternum, pelvis, ribs, spine and skull, with the spleen becoming the major site of engraftment at late stage disease. Comparison of SNP 6.0 array profiles of presentation and xenografts samples revealed several examples of post-transplant genomic progression which, with the exception of a biallelic deletion of the CDKN2A/B locus, had not previously been reported in iAMP21 patients. Additional copies of chromosomes 9 and 12 in patient samples were lost in xenografts resulting in copy number neutral loss of heterozygosity (CNN-LOH) for chromosome 12 but not 9. Analysis of further primary iAMP21 BCP-ALL cases identified two with CNN-LOH for part of the long arm of chromosome 12, suggesting involvement of an imprinted locus in this region. iAMP21 chromosomes were retained in all xenografts, supporting evidence that it is a primary abnormality. However, as these rearrangements have been considered to be stable once formed, we were surprised to observe acquisition of a non–contiguous deletion of chromosome 21 in one secondary mouse and all of its tertiary recipients. The deletion was proximal to the CRA and resulted in extension of pre-existing haploinsufficient regions by 6.6 Mb. Evolution of this iAMP21 chromosome was confirmed by fluorescence in-situ hybridisation (FISH) using probes targeting the deletion, with loss of signal observed in all cells from mice carrying the deletion but in no cells from the presentation sample or unaffected mice. Although we cannot rule out that the deletion arose in response to adaptive pressure specific to the xeno-environment, opportunities to observe similar progression in patients have been limited because FISH analysis has generally targeted only the CRA and few paired presentation/relapse samples have been analysed by genomic array. While it remains likely that overrepresentation of oncogenes within the CRA acts as a primary driver of iAMP21 BCP-ALL, the additional deletion may highlight the position of tumour suppressor genes involved in leukemia progression. As these patients are treated as a single clinical entity, an important implication of these data is that the position and extent of deletions in iAMP21 rearrangements may influence response to therapy. In conclusion, xenografts of iAMP21 BCP-ALL closely resemble human primary disease and will make valuable pre-clinical models for basic and translational research. As models for the study of clonal evolution they support previous evidence that iAMP21 is a primary abnormality and have revealed novel forms of clonal progression. Disclosures: No relevant conflicts of interest to declare.

Abstract 1274 Translocations involving the IGH@ locus have recently been characterised as a novel cytogenetic subgroup in BCP-ALL, involving unique partner genes including: CEBP family of transcription factors; cytokine receptors, EPOR and CRLF2; and the inhibitory transcription factor, ID4. As for the mature B-cell malignancies, their expression is always deregulated by juxtaposition of transcriptional enhancers within the locus. We have developed a high throughput FISH screening approach to ascertain the true incidence of IGH@ translocations in BCP-ALL. We screened approximately 80% (2603/3194) of patients entered to the childhood clinical trial (UKALL2003) using an IGH@ break-apart rearrangement probe with automated scanning and capture (CytoVision scanning system, Leica Microsystems). We identified IGH@ translocations in 4% (104/2603) of patients tested. This included an incidence of 4% (96/2286) in BCP-ALL and the novel observation of 2% (8/317) in T-ALL patients. The median age at diagnosis of BCP- and T-ALL IGH@ translocation patients was significantly higher at 12 and 14 years, respectively, compared to the median age of 5 years for the whole trial. The median white cell count (WCC) was low in IGH@ positive BCP-ALL (median 9.05×109/L) and higher in T-ALL (median 72.8×109/L). This study confirmed CRLF2 to be the most frequent IGH@ partner gene, observed in 20% (22/104) of BCP-ALL patients. ID4 and the CEBP family were found in 8% (8/104) and 9% (10/104), respectively. CEBPD was most common (n=6), while CEBPA (n=2), CEBPE (n=1) and CEBPG (n=1) were rare. Single patients were identified with involvement of BCL2, IGF2BP1, IGK@ and the TCRA/D locus. Novel involvement of TAL1 was identified in one T-ALL patient. In 57% of children (59/104: BCP-ALL n=52, T-ALL n=7) the IGH@ translocation partner gene remains unknown. Patients with a known partner gene did not differ from those with an unknown partner gene with respect to gender, age, WCC and National Cancer Institute (NCI) risk. However, patients with a known partner gene were strongly associated with an intermediate cytogenetic risk group while patients with an unknown partner gene were mixed (good risk, 7% v 19%, intermediate risk, 93% v 69% and poor risk, 0% v 12% p=0.01). There was no difference seen for gender, age, WCC, cytogenetic risk and NCI risk when each partner gene was investigated independently, apart from those with CRLF2 involvement who had a higher WCC (50×109/l 41% v 12%, p=0.005) and were solely within the intermediate cytogenetic risk group (100% v 74%, p=0.03). We investigated copy number aberrations of genes commonly altered in BCP-ALL by Multiplex Ligation-dependent Probe Amplification (MLPA) using the Salsa P335-A1 IKZF1 kit (MRC Holland) in 60 IGH@ translocation patients. The genes investigated were IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, as well as the rearrangement, P2RY8-CRLF2. Deletions of none (31%), one (18%), two (17%), three (22%) or four (8%) genes were found. Deletions of CDKN2A, CDKN2B and IKZF1 were the most frequent, in 18%, 21% and 20%, respectively. Whilst the incidence of CDKN2A/B deletions was similar to that seen in childhood BCP-ALL, IKZF1 deletions were more prevalent (20% v 14%) and PAX5 deletions occurred at a lower incidence in IGH@ rearranged patients (13% v 19%). In the two T-ALL patients with DNA available, both showed deletions of CDKN2A with one patient also showing deletion of CDKN2B. In conclusion, IGH@ translocations are present in 4% of childhood ALL with involvement in both B- and T-cell disease. Patients belong to the intermediate cytogenetic risk group with an increased incidence of IKZF1 deletions. This differs from our recent report on adult BCP-ALL, where patients with an IGH@ translocation were associated with a worse outcome, although the increased incidence of IKZF1 deletions remained consistent across all age ranges. In over 50% of IGH@ positive children, the translocation partner remains to be elucidated, indicating the presence of as yet unidentified cryptic rearrangements. Characterisation of these partners may identify additional oncogenes involved in leukemogenesis or provide potential novel therapeutic targets, as recently demonstrated for CRLF2 in BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

The aim of this study is to identify the underlying molecular genetic mechanisms of radiation induced breast carcinogenesis. Studies have shown that women exposed to ionising radiation at a young age are more at risk of developing breast cancer than older women exposed to the same level of radiation. Higher levels of estrogen are present in young women and estrogen has a known transforming effect on breast epithelial cells. One hypothesis suggests that radiation and estrogen synergise to drive breast epithelial cell transformation. We have developed an in vitro model of radiation-induced breast epithelial cell transformation in order to investigate genetic alterations associated with breast cell transformation. The immortalised, non-transformed breast epithelial cell line MCF-10A was exposed to fractionated doses of X-rays in the presence or absence of exogenous estrogen. We have shown that radiation treated cells display evidence of transformation, including loss of contact inhibition, increased cell invasion, disrupted acini formation and tumor formation in immunocompromised Rag2−/− γ−/− mice. Analysis of radiation treated cells by SNP array karyotyping identified focal gene deletion (EP300 and OCT-1) and amplification (c-MYC) which may be linked to radiation-induced breast cell transformation. Gene deletion and amplification was confirmed by Fluorescent In Situ Hybridization analysis and alterations in expression have been confirmed by western analysis. EP300 is a transcriptional co-activator and a putative tumor suppressor. Somatic mutations in EP300 have been found in solid tumours and the gene is located in a region of chromosome 22 affected by loss of heterozygosity in numerous cancers. Oct-1 encodes an octamer binding transcription factor that has been associated with regulating DNA damage response through interactions with BRCA1 and GADD45. c-MYC is a known proto-oncogene that encodes a transcription factor involved in cell proliferation, cell cycle regulation and apoptosis. Analysis of our in vitro model of radiation-induced breast epithelial cell transformation has identified genetic alterations that have established roles in mediating cellular response to DNA damage and which might be key events in the development of radiogenic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5093. doi:10.1158/1538-7445.AM2011-5093

Abstract 581 Patients with iAMP21 (intrachromosomal amplification of chromosome 21) represent a distinct cytogenetic subgroup of childhood B cell precursor ALL (BCP-ALL; incidence ∼2%) with a poor prognosis on standard therapy (29% EFS at 5 years). From cytogenetic studies the amplification of chromosome 21 appears to be the primary genetic change, however, whether this or associated abnormalities provide the initiating event of leukaemogenesis remains to be elucidated. Currently fluorescence in situ hybridisation (FISH) with probes directed to the RUNX1 gene provides the only reliable detection method. Knowledge of the underlying genetic mechanism will lead to improved diagnosis and determine those patients at high risk of relapse who require more intensive therapy. Our previous study using genomic arrays (1Mb aCGH, n=10; NimbleGen chromosome 21 custom oligonucleotide array, n=5) identified a common region of amplification (CRA) on 21q of 6.6 Mb and a common region of deletion (CRD) of 3.3 Mb within the telomeric region in 100% and 70% of patients, respectively. However, expression profiling (Affymetrix U133A, n=8) indicated that no important genes within the CRA or CRD were differentially expressed. We now have clinical, cytogenetic and FISH data on 94 iAMP21 patients (44 F, 50 M; median age 9.5y, range 2-30); the largest iAMP21 cohort investigated to date. Higher resolution analysis of 17/94 diagnostic samples by aCGH (Agilent 185K platform) have refined these regions to 5.1 Mb and 2.5 Mb in 100% and 88% of patients, respectively. Recent genome mapping has highlighted the presence of the microRNA miR-802 within the CRA and shown this region overlaps with the Down Syndrome Critical Region at 21q22.3 encompassing the genes DSCR1, DSCR3 and DYRK1. Detailed analysis of chromosome 21 identified recurrent breakpoints centromeric of the CRD within three genes: PDE9A (n=2), COL6A2 (n=2) and DSCAM (n=2). The role of these genes in the initiation of the chromosomal instability associated with iAMP21 is currently under investigation. Global analysis of the iAMP21 genome identified an average of 13 copy number alterations (CNA) per case with recurrent deletions of the leukaemia associated genes PAX5 (n=2), IKZF1 (n=4) and CDKN2A (n=4). To determine the incidences of PAX5, IKZF1 and CDKN2A deletions among the cohort of 94 iAMP21 patients, FISH using probes targeting these genes was carried out on availabel samples. IKZF1 (exons 3-6) was deleted at the highest incidence of 21% (13/62), while CDKN2A and PAX5 were deleted at a lower incidence of 17% (12/71) and 8% (4/48), respectively. The high incidence of IKZF1 deletions found in this patient group correlates with their association with high risk ALL. We have recently reported a deletion within the PAR1 region of the sex chromosomes, centromeric of the CRLF2 gene involving CSF2RA and IL3RA, which leads to deregulated expression of CRLF2. This deletion occurred at an incidence of 23% (17/73), as determined by FISH. Among pairs of matched diagnostic and relapse samples; one had this deletion at both diagnosis and relapse while the second had a deletion in the diagnostic sample only. Gain of an additional × chromosome was identified in 28% (18/65). Of note is that the 3 aberrations: abnormalities of chromosome 21, gain of the × chromosome and high incidence of deletions centromeric of CRLF2 in this patient group mirror the findings in Down Syndrome (DS) ALL, although we have not identified any mutations of the JAK2 kinase domain among this cohort of iAMP21 patients to date. This genome-wide study provides further support for the proposal that amplification of genes within the CRA on chromosome 21 is the primary event driving leukaemogenesis in iAMP21 patients. It has also highlighted that the 3 abnormalities in common with DS ALL and/or deletions of IKZF1, which all occur at a high incidence, contribute towards the progression of this high risk subtype of childhood BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

We have recently described recurrent IGH@ translocations in BCP-ALL, which result in deregulated expression of the translocated oncogene. We carried out screening for novel IGH@ translocations in BCP-ALL by fluorescence in situ hybridization (FISH) and identified two cryptic translocations involving PAR1 of either sex chromosomes in 29 patients: t(X;14)(p22;q32) (n=18) and t(Y;14)(p11;q32) (n=7) (X or Y unknown, n= 4) and 2 BCP-ALL cell lines with t(Y;14)(p11;q32). The median age of the patients was 18 years (range 4–76 years) and median WBC was 70 ×109/l (range 1–342). Follow-up data was available for 16 patients. Among 5 children diagnosed 1996–2001, 1 failed to remit, 1 suffered an early death and 3 relapsed (16m, 54m, and 64m post diagnosis). To date, all 5 children diagnosed 2004–2005 are in first continuing complete remission (CCR) after 33–46 months, suggesting an improved outcome on the current childhood ALL treatment trial. All 6 adults were diagnosed 2004–2006: 2 died within a year (1 in remission and 1 post transplant); 2 relapsed and died within 1 year and 2 are in 1st CCR after 20 and 36 months. These observations indicate a variable outcome among adults. Translocation breakpoint cloning from IGHJ by long distance inverse PCR and subsequent FISH mapping identified CRLF2, also known as thymic stromal-derived lymphopoietin receptor, at Xp22 and Yp11, to be juxtaposed to IGH@ in these translocations. Breakpoints were cloned in 11 patients and clustered between 2–27kb centromeric of the 3′ UTR of CRLF2. Expression levels of CRLF2, measured by qRT-PCR in 4 patients and the cell lines, was 500–6,500 fold higher than the control cohort of BCP-ALL patients without the translocation. This overexpression confirmed CRLF2 to be the target gene. CRLF2 and IL2RGC bind to IL7RA to form functional receptors for TSLP and IL7, respectively. However, neither IL2RGC nor IL7RA were aberrantly expressed. The presence of associated genomic copy number alterations was shown by array-based comparative genomic hybridization and FISH. As described for BCP-ALL in general, the significant changes included deletions of the B cell differentiation genes: PAX5 and IKAROS, as well as the cell cycle control gene, CDKN2A. The cell lines revealed some biochemical insights into the functional consequences of this translocation involving PAR1. Cell surface expression of CRLF2 was demonstrated, together with constitutive tyrosine phosphorylation of JAK2 and STAT5, the signaling intermediates of the classical JAK-STAT pathway. Further evidence of increased STAT5 activation came from retroviral infection of normal mouse fetal liver cells with human CRLF2, which sustained the proliferation of B-cell precursors in vitro. Conversely, initial studies of CRLF2 knockdown in the cell lines using shRNA demonstrated decreased proliferation. Together, these results show for the first time, the involvement of both sex chromosomes in an IGH@ translocation in leukemia, resulting in deregulated expression of CRLF2 and constitutive JAK-STAT activation. These data suggest CRLF2 as a potential therapeutic target in this subset of patients. Laboratories of MJSD, RS and CJH contributed equally to this work.

Chromosomal translocations involving the IGH locus on 14q32 are a hallmark of B-cell malignancies. These translocations are particularly frequent in non-hyperdiploid (NHD) multiple myeloma (MM), representing approximately 50% of myeloma cases. MM-associated primary t(14q32) target at least 7 partner genes including cyclins D (CCND1, −D3), MAF transcription factors (CMAF, MAF B and A) and MMSET/FGFR3. Some of the translocations are predictive of clinical outcome. Recently, we identified a novel interstitial del(14q) involving IGH and recurrent in chronic lymphocytic leukaemia and MM (Pospisilova et al, Leukemia, 2007). In spite of extensive studies, the mechanism(s) and molecular consequences of del(14q) remain unknown. We report here 34 cases of plasma cell (PC) malignancies with del(14q) involving IGH, as proven by FISH. Cases were collected in UK and Belgium. The estimated incidence of these aberrations in PC malignancies was 1.4%. There were 13 female and 21 male patients ranging in age from 49 to 86 years (average 68). Twenty seven patients had MM, one had SMM and 6 had MGUS. In almost all cases, the del(14q) was detected at diagnosis. Clinical data of the reported cases have been collected. The del(14q) were roughly mapped by FISH and grouped into 3 categories according to the proximal breakpoint: del(14)(q24.1q32.33) involving the ZFP36L1 region (11 cases); deletions proximal to ZFP36L1 (14 cases) and deletions distal to ZFP36L1 (7 cases). The size of del(14q) was not determined in 2 cases. Biallelic deletion of TRAF3/14q32.33 recurrently occurring in MM, was detected by FISH in 1 out of 9 analyzed cases. Additional FISH analysis showed that the del(14q) was predominantly associated with NHD tumors (62% vs 38% with hyperdiploid karyotypes) and frequently (60%) accompanied by del(13q), regarded as a poor prognostic factor. All reported cases were negative for t(4;14) and t(11;14); they also showed a normal status of CCND3, CMAF, MAFB and CMYC, when examined. A gain of 1q/CKS1B was found in 57% (8/14) of analyzed cases. The expression pattern of cyclin D1−D3 has been examined.

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are rare in BCP-ALL (comprising ~3% of cases) and involve different breakpoints from those found in lymphoma and myeloma. Cytogenetics, metaphase and interphase FISH, using a split-signal probe specific for IGH@, identified four translocations: t(14;19)(q32;q13) (n = 10), t(8;14)(q11;q32) (n = 8), t(14;14)(q11;q32) (n =3) and t(14;20)(q32;q13) (n=2), among a series of BCP-ALL patients. Sequential FISH mapping of these translocations showed that four different members of the CEBP gene family flanked these breakpoint regions. Long-distance inverse PCR with primers specific for the IGHJ6 gene segment allowed molecular cloning of many of these breakpoints and confirmed the involvement of the CEBPA (19q13, n= 4), CEBPD (8q11, n=6), CEBPE (14q11, n =2) genes. Breakpoints were located within the 3′ UTR of CEBPA and either 3′ UTR or the 5′ region of CEBPE, whereas breakpoints in 8q11 were ~30kb centromeric of CEBPD. Over-expression of the respective target genes was shown by QRT-PCR. Evidence that the IGH@ locus directly contributed to gene deregulation was demonstrated by lower expression of CEBPA in 9/17 derived BCP-ALL cell lines without t(14;19)(q32;q13). CEBP proteins have been implicated in the differentiation of myeloid progenitors and CEBPA mutations have been detected in a subset of sporadic and familial acute myeloid leukemia, suggesting that CEBPA may act as a tumor suppressor gene in these malignancies. However, no mutations within the 5′ region of CEBPA comparable to those seen in AML were detected in any cases with t(14;19)(q32;q13), although one case did exhibit deletion of six nucleotides resulting in the loss of 2 amino acids. Moreover, the deregulated expression of CEBPA and other CEBP genes in a subset of BCP-ALL indicates that they may act as dominant oncogenes in B-cell precursors. Constitutive expression of CEBPA and CEBPB in normal lymphoid progenitors has previously been shown to induce myeloid differentiation whilst CEBPE is involved in the terminal differentiation of neutrophils. We showed that expression of full-length CEBPE in the IL3-dependent B-cell precursor cell line Ba/F3 did not result in enhanced proliferation or protection from a variety of apoptotic stimuli, but instead resulted in expression of myeloid differentiation antigens. Most CEBP genes are intronless, different protein isoforms with differing functions being generated translationally. 2-D western blot analysis of nuclear extracts from the SEM BCP-ALL cell line showed selective expression of the 32kd CEBPA protein isoform only, an isoform that lacks the anti-mitotic activity of full-length CEBPA. These data implicate deregulation of the CEBP gene family via juxtaposition to the IGH@ in the pathogenesis of a subset of BCP-ALL. They demonstrate for the first time, the involvement of four members of the same gene family in a single subtype of hematological disease. The transformation of B-cell progenitors may occur through the selective expression of shorter CEBP protein isoforms that lack the ability to induce myeloid differentiation.

Abstract 880 Background The prognosis of childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) has improved enormously due to risk-adapted therapeutic stratification of patients. Still, most relapses occur in the group that is supposed to have an intermediate relapse risk. To overcome this problem, new prognostic markers are needed. Recently, new high-risk molecular features were identified in BCP-ALL: the BCR-ABL1-like signature, IKZF1 deletions and high CRLF2 expression (CRLF2-high). These features can occur together; however, whether they confer independent prognostic value is currently unknown. In this study we determined the frequency, co-occurrence and prognostic relevance of these new molecular features in four independent cohorts of children with ALL. Methods BCR-ABL1-like gene signature (110-gene probe classifier), IKZF1 deletions (Multiplex Ligation-dependent Probe Amplification) and CRLF2 expression levels (Affymetrix U133 plus 2.0) were determined in leukemic cells (>90% blasts) from 1128 children with newly diagnosed ALL enrolled in three consecutive Dutch Childhood Oncology Group trials (DCOG ALL-8/-9/-10) and in the German COALL-97/03 trial. The cumulative incidence of relapse (CIR) was calculated using death as a competing event. Results BCR-ABL1-like, IKZF1-deleted and CRLF2-high cases were found in 16%, 17% and 10% of the BCP-ALL cases, respectively. Collectively, these three features constitute 34% of BCP-ALL cases for which 4% of those had all three, 29% had any two and 67% had only one these features. The BCR-ABL1-like signature and IKZF1-deletions co-occurred the most frequent of these three features (22%). The BCR-ABL1-like signature was only found in B-other cases negative for the genetic abnormalities ETV6-RUNX1, BCR-ABL1, hyperdiploid, TCF3- and MLL-rearrangement, whereas IKZF1-deleted and CRLF2-high cases were also found in other subtypes of BCP-ALL. The frequency of IKZF1- deletions was highest in BCR-ABL1- positive (70%) and BCR-ABL1-like (40%) cases. The BCR-ABL1-like signature, IKZF1 deletions and CLRF2-high were associated with prognosis in four, three and one out of 4 studied treatment protocols, respectively. Protocol-stratified analysis revealed that five-year CIR was higher in BCR-ABL1-like (32%, p Conclusions High CRLF2 expression is the least predictive, whereas the BCR-ABL1-like signature and IKZF1- deletions –both individually and combined- are strong independent prognostic factors and have additional prognostic value on top of intermediate minimal residual disease levels in children with BCP-ALL. These findings demonstrate that these two molecular features are important clinically for identifying high-risk patients who may require more intensive therapy. Disclosures: No relevant conflicts of interest to declare.