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Gene regulatory networks (GRNs) are often deregulated in tumor cells, resulting in altered transcriptional programs that facilitate tumor growth. These altered networks may make tumor cells vulnerable to the inhibition of specific regulatory proteins. Consequently, the reconstruction of GRNs in tumors is often proposed as a means to identify therapeutic targets. While there are examples of individual targets identified using GRNs, the extent to which GRNs can be used to predict sensitivity to targeted intervention in general remains unknown. Here we use the results of genome-wide CRISPR screens to systematically assess the ability of GRNs to predict sensitivity to gene inhibition in cancer cell lines. Using GRNs derived from multiple sources, including GRNs reconstructed from tumor transcriptomes and from curated databases, we infer regulatory gene activity in cancer cell lines from ten cancer types. We then ask, in each cancer type, if the inferred regulatory activity of each gene is predictive of sensitivity to CRISPR perturbation of that gene. We observe slight variation in the correlation between gene regulatory activity and gene sensitivity depending on the source of the GRN and the activity estimation method used. However, we find that there is consistently a stronger relationship between mRNA abundance and gene sensitivity than there is between regulatory gene activity and gene sensitivity. This is true both when gene sensitivity is treated as a binary and a quantitative property. Overall, our results suggest that gene sensitivity is better predicted by measured expression than by GRN-inferred activity.

It is essential to relate the biology of acute leukaemia to normal blood cell development. In this review, we discuss how modern models of haematopoiesis might inform approaches to diagnosis and management of immature leukaemias, with a specific focus on T-lymphoid and myeloid cases. In particular, we consider whether next-generation analytical tools could provide new perspectives that could improve our understanding of immature blood cancer biology.

The inv(16) acute myeloid leukemia-associated CBFβ-MYH11 fusion is proposed to block normal myeloid differentiation, but whether this subtype of leukemia cells is poised for a unique cell lineage remains unclear. Here, we surveyed the functional consequences of CBFβ-MYH11 in primary inv(16) patient blasts, upon expression during hematopoietic differentiation in vitro and upon knockdown in cell lines by multi-omics profiling. Our results reveal that primary inv(16) AML cells share common transcriptomic signatures and epigenetic determiners with megakaryocytes and erythrocytes. Using in vitro differentiation systems, we reveal that CBFβ-MYH11 knockdown interferes with normal megakaryocyte maturation. Two pivotal regulators, GATA2 and KLF1, are identified to complementally occupy RUNX1-binding sites upon fusion protein knockdown, and overexpression of GATA2 partly induces a gene program involved in megakaryocyte-directed differentiation. Together, our findings suggest that in inv(16) leukemia, the CBFβ-MYH11 fusion inhibits primed megakaryopoiesis by attenuating expression of GATA2/KLF1 and interfering with a balanced transcriptional program involving these two factors.

Classification of acute lymphoblastic and myeloid leukemias (ALL and AML) remains heavily based on phenotypic resemblance to normal hematopoietic precursors. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, and ignores recent advances in understanding of developmental multipotency of diverse normal hematopoietic progenitor populations that are identified by transcriptional signatures. We performed transcriptional analyses of a large series of acute myeloid and lymphoid leukemias and detected significant overlap in gene expression between cases in different diagnostic categories. Bioinformatic classification of leukemias along a continuum of hematopoietic differentiation identified leukemias at the myeloid/T-lymphoid interface, which shared gene expression programs with a series of multi or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a−CD7− subset of early thymic precursors. Within these interface acute leukemias (IALs), transcriptional resemblance to early lymphoid progenitor populations and biphenotypic leukemias was more evident in cases originally diagnosed as AML, rather than T-ALL. Further prognostic analyses revealed that expression of IAL transcriptional programs significantly correlated with poor outcome in independent AML patient cohorts. Our results suggest that traditional binary approaches to acute leukemia categorization are reductive, and that identification of IALs could allow better treatment allocation and evaluation of therapeutic options.

Summary: Mutations and deletions of polycomb repressive complex (PRC) components are increasingly recognized to affect tumor biology in a range of cancers. However, little is known about how genetic alterations of PRC-interacting molecules such as the core binding factor (CBF) complex influence polycomb activity. We report that the acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion oncoprotein physically interacts with the PRC1 complex and that these factors co-localize across the AML genome in an apparently PRC2-independent manner. Depletion of CBFβ-SMMHC caused substantial increases in genome-wide PRC1 binding and marked changes in the association between PRC1 and the CBF DNA-binding subunit RUNX1. PRC1 was more likely to be associated with actively transcribed genes in CBFβ-SMMHC-expressing cells. CBFβ-SMMHC depletion had heterogeneous effects on gene expression, including significant reductions in transcription of ribosomal loci occupied by PRC1. Our results provide evidence that CBFβ-SMMHC markedly and diversely affects polycomb recruitment and transcriptional regulation across the AML genome. : Cordonnier et al. report a physical and functional interaction between the leukemia-associated fusion protein CBFβ-SMMHC and polycomb repressive complex (PRC) 1. Their findings provide evidence that cancer-associated alterations in molecules that normally interact with epigenetic factors can lead to subversion of transcriptional regulation in malignant cells. Keywords: acute myeloid leukemia, core binding factor, oncogene, polycomb, epigenetic regulation

Comprehensive cataloguing of the acute myeloid leukaemia (AML) genome has revealed a high frequency of mutations and deletions in epigenetic factors that are frequently linked to treatment resistance and poor patient outcome. In this review, we discuss how the epigenetic mechanisms that underpin normal haematopoiesis are subverted in AML, and in particular how these processes are altered in childhood and adolescent leukaemias. We also provide a brief summary of the burgeoning field of epigenetic-based therapies, and how AML treatment might be improved through provision of better conceptual frameworks for understanding the pleiotropic molecular effects of epigenetic disruption.

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with chemoresistance and a high risk for relapse. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic 1,2. However, one issue with a targeted agent, such as ABT-199, is the development of acquired resistance. Interestingly, there have been numerous high impact papers connecting ABT-199 resistance to altered oxidative phosphorylation (OXPHOS) 3,4. While there are relatively few studies into T-ALL metabolism, there is evidence that aerobic glycolysis, the conversion of glucose to lactate, is greater in proliferating T-cells than in T-ALL and that NOTCH signalling can drive mitochondrial OXPHOS 5. A recent study showed that the transcription factor RUNX2 altered T-ALL metabolism, increasing both glycolysis and OXPHOS and enhancing leukemic cell migration 6. However, there has been relatively little research into the metabolic profile of T-ALL at distinct stages of differentiation. The aim of this study was to determine the role of ABT-199 resistance in altering metabolism and determine if that was due to the differentiation state of the T-ALL. ABT-199 R LOUCY cells were generated by chronic exposure to increasing concentrations of ABT-199 administered every two days. The EZH2 KO Jurkat cell lines were previously generated through CRISPR-Cas9 engineering 7. In order to assess the metabolic profile, cells were attached to a 96 well plate using CellTak and the extracellular acidification rate (ECAR) and oxidative phosphorylation (OXPHOS) was measured on a Seahorse Bioscience XF96 Extracellular Flux Analyzer. Anti-apoptotic dependence was measured using BH3 profiling and cell death by Annexin V/propidium iodide staining. The mitochondrial structure was visualized using transmission electron microscopy. Previously, we generated ABT-199 resistant ETP-ALL LOUCY cells (ABT-199 R) following continuous exposure to ABT-199 over a prolonged period of several months 8. The ABT-199 R cells showed dependence on BCL-XL for survival and sensitivity to the BCL-XL inhibitor WEHI 539. The ABT-199 R cells showed evidence of differentiation to a more mature T-cell. The ABT-199 R cells had increased surface CD3 (sCD3) expression and CD1A expression, along with increased expression of TAL1 and LMO2 genes compared to parental LOUCY cells. Interestingly, the ABT-199 R cells showed enhanced basal respiration, ATP production and max respiration compared to the parental cells. Indeed, analysis of the expression of OXPHOS complexes showed increased expression of complexes I-IV in the ABT-199 R cells, compared to the parental controls. Indeed, the parental LOUCY cells appeared to have reduced cristae number and length compared to the ABT-199R cells. Next, we assessed if inhibiting OXPHOS with a series of inhibitors (oligomycin, rotenone, antimycin) could sensitize the ABT-199 R LOUCY cells to ABT-199. However, we did not detect any changes to sensitivity of ABT-199. This led us to hypothesize that perhaps the changes in OXPHOS were due differentiation state of the LOUCY cells. We confirmed that more typical T-ALL cell lines (JURKAT and CEM-CCRF) had higher OXPHOS than the ETP-ALL cell line LOUCY and had higher expression of the OXPHOS complexes I-IV by Western blotting. To assess if de-differentiation of a more typical T-ALL cell line would cause a reduction in OXPHOS we turned to the EZH2 knockout (K/O) Jurkat cells 7. We found that EZH2 KO2 showed a reduction in the differentiation markers CD1A and CD3 on the cell surface and TAL1 gene expression, compared to WT control Jurkats. Next, we assessed the OXPHOS and found that the de-differentiated EZH2 cells had reduced OXPHOS compared to the parental controls, with altered mitochondrial structure. Suggesting, that de-differentiation of typical T-ALL cell line reduces OXPHOS. In this study we show that metabolic phenotype is linked to the maturation stage of T-ALL. We believe that the altered metabolism identified in ABT-199 resistance is linked to the selection of a more mature cell type. Highlighting, that altered metabolism may not be a driver of resistance to ABT-199 but a consequence of the maturation stage of the resistant cell. Disclosures Di Grande: Novartis: Current Employment. Leon: BenevolentAI: Current Employment. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy. Bond: Haematology Association of Ireland Award funded by Novartis: Research Funding. Ni Chonghaile: AbbVie: Research Funding.

T-lymphoblastic lymphoma (T-LBL) is by far the most common T-NHL in children and adolescents, typically presenting with a mediastinal mass and advanced disease. The molecular pathology of T-LBL is similar to T-acute lymphoblastic leukemia (T-ALL), with frequent activating mutations of the NOTCH1 pathway and signaling kinase molecules, and translocations of the T-receptor gene (TR) loci. There are several recognized differences with the T-ALL genotype, for example, differing patterns of loss of heterozygosity on chromosome 6q (6qLOH) that have been reported to predict prognosis. As in T-ALL, T-LBL can been categorized according to TR rearrangement status and transcriptional profiling. Current treatment efforts are based on therapy stratification according to disease risk, which is principally defined by genotype (i.e., NOTCH1/FBXW7 mutational status).

The prognostic implications of DNMT3A genotype in T-cell acute lymphoblastic leukemia are incompletely understood. We performed comprehensive genetic and clinico-biological analyses of T-cell acute lymphoblastic leukemia patients with DNMT3A mutations treated during the GRAALL-2003 and -2005 studies. Eighteen of 198 cases (9.1%) had DNMT3A alterations. Two patients also had DNMT3A mutations in non-leukemic cell DNA, providing the first potential evidence of age-related clonal hematopoiesis in T-cell acute lymphoblastic leukemia. DNMT3A mutation was associated with older age (median 43.9 years vs. 29.4 years, P

Introduction: Traditional classification of acute lymphoblastic and myeloid leukemias (ALLs and AMLs) remains heavily based on phenotypic resemblance to normal hematopoietic precursors of the respective lineages. This framework can provide diagnostic challenges for immunophenotypically heterogeneous immature leukemias, which often have poor responses to treatment. This system also takes little account of modern concepts of hematopoietic identity that are mainly based on transcriptional signature identification and functional assays. Recent advances in genome-wide analytical methods developed to reconstruct landscapes of normal differentiation now provide an opportunity to re-evaluate traditional binary approaches to myeloid and lymphoid lineage assignment in leukemia. Methods: We used novel computational tools, including the recently described Iterative Clustering and Guide Gene Selection (ICGS) method to perform transcriptional analyses of a series of 125 T-ALLs and AMLs, which comprised a high proportion of phenotypically immature cases (53.1% and 40.8% respectively). The leukemias were additionally characterized by targeted next generation sequencing (NGS). ICGS was also used to analyze independent adult and pediatric T-ALL cohorts. Results: There was significant overlap in gene expression between leukemias of different diagnostic categories. In contrast to traditional clustering methods, ICGS analysis permitted unbiased classification of acute leukemias along a continuum of hematopoietic differentiation, according to the expression of a limited number of lineage-discriminating guide genes that defined hematopoietic cell expression modules. While AMLs and T-ALLs at either end of the differentiation spectrum showed specific enrichment for transcriptional signatures of the corresponding lineage precursors, leukemias that were arrested at the myeloid/ T-lymphoid interface either showed no clear evidence of mature T-lymphoid or mature myeloid identity, or had incomplete Hematopoietic Stem and Progenitor Cell (HSPC) and mature myeloid cell profiles. NGS analysis revealed that the spectrum of differentiation arrest defined by ICGS is only partially paralleled by underlying mutational genotype. Notably, interface leukemias originally diagnosed as T-ALL were significantly more likely to have PTEN mutations than the rest of the T-ALL cohort (60% v 6.7%, p=0.0151), while RUNX1-mutated AMLs were restricted to interface clusters. We found that interface leukemias shared gene expression programs with a series of multi- or oligopotent hematopoietic progenitor populations, including the most immature CD34+CD1a-CD7- subset of early thymic precursors (ETPs). Within interface leukemias, enrichment for lymphoid progenitor population signatures including multi-lymphoid progenitors (MLPs), lymphoid-mono-dendritic progenitors (LMDPs), T-oriented CD127- and B-oriented CD127+ early lymphoid progenitors (ELPs) from an umbilical cord blood humanized mouse model and early B-cell progenitors, was more likely in cases that were originally diagnosed as AML, rather than T-ALL. In addition, transcriptional resemblance to both B/myeloid and T/myeloid mixed phenotype acute leukemias (MPALs) was primarily driven by AMLs within these interface clusters, suggesting that these cases demonstrate significant lymphoid transcriptional orientation. Conclusion: Our results suggest that traditional binary approaches to leukemia categorization are reductive, and that leukemias arrested at the T-lymphoid/ myeloid interface exhibit significant transcriptional heterogeneity. These data also provide evidence that a subset of leukemias originally diagnosed as AML may be more likely to arise from lymphoid-oriented progenitors and/or be arrested at an early stage of lymphoid orientation than is currently recognized. We believe that better identification of interface acute leukemias will allow improved evaluation of appropriate therapeutic options for these cases. Disclosures Boissel: NOVARTIS: Consultancy. Laurenti:GSK: Research Funding.

International audience; Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally a rise due to chromosomal rearrangements. Cis-splicing of adjacent genes (cis-SAGe) results in transcription of intergenically-spliced chimeric RNAs (ISCs) in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential cancer driver. We performed high-throughput RNA-sequencing of human T-acute lymphoblastic leukemia (T-ALL)samples, and used targeted analysis pipelines to detect fusion chimeras. We identified 55 candidate T-ALL-related ISCs, with a median of 4 per patient. We performed additional in-depth characterization of the NFATC3-PLA2G15 chimera, which was expressed at variable levels in primary T-ALL cases. Experimental analysis revealed that the fusion had lower activity than wild-type NFATC3 in vitro, and that T-ALL blasts with elevated NFATC3-PLA2G15 levels had reducedtranscription of canonical NFAT pathway genes in vivo. Strikingly, we found that high expression of the NFATC3-PLA2G15 chimera in leukemic blasts correlated with aggressive disease biology in murine patient-derived T-ALL xenografts,andpoor prognosis in human T-ALLpatients treated as part of the Francophone multinational GRAALL-2003 and -2005 studies. Our results suggest that ISCs are common in T-ALL, and that expression of specific ISCs may correlate with patient outcome.

Leukemias are frequently characterized by the expression of oncogenic fusion chimeras that normally arise due to chromosomal rearrangements. Intergenically spliced chimeric RNAs (ISC) are transcribed in the absence of structural genomic changes, and aberrant ISC expression is now recognized as a potential driver of cancer. To better understand these potential oncogenic drivers, high-throughput RNA sequencing was performed on T-acute lymphoblastic leukemia (T-ALL) patient specimens (

The core-binding factor (CBF) complex is a heterodimeric transcription factor comprising a CBFβ subunit and a variable DNA-binding RUNX subunit, usually RUNX1 in hematopoietic cells. Aside from its critical hematopoietic functions, CBF regulates the expression of ribosomal protein genes and ribosomal RNA (rRNA) in a cell context-dependent manner.1, 2, 3 Intriguingly, this function may have implications for the pathogenesis of acute myeloid leukemia (AML), as reduced ribosome biogenesis in RUNX1-deficient hematopoietic stem cells has recently been proposed to confer a survival advantage that favors outgrowth of preleukemic RUNX1-deficient clones.3 Furthermore, AML-associated fusion proteins that arise from translocations of CBF subunit genes have been shown to occupy nucleolar organizing regions at mitotic chromosomes,4, 5 suggesting that ribosomal homeostasis might be altered in CBF-AML.

International audience; no abstract

International audience; Unlabelled - Gene expression studies have consistently identified a HOXA-overexpressing cluster of T-cell acute lymphoblastic leukemias, but it is unclear whether these constitute a homogeneous clinical entity, and the biological consequences of HOXA overexpression have not been systematically examined. We characterized the biology and outcome of 55 HOXA-positive cases among 209 patients with adult T-cell acute lymphoblastic leukemia uniformly treated during the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. HOXA-positive patients had markedly higher rates of an early thymic precursor-like immunophenotype (40.8% versus 14.5%, P=0.0004), chemoresistance (59.3% versus 40.8%, P=0.026) and positivity for minimal residual disease (48.5% versus 23.5%, P=0.01) than the HOXA-negative group. These differences were due to particularly high frequencies of chemoresistant early thymic precursor-like acute lymphoblastic leukemia in HOXA-positive cases harboring fusion oncoproteins that transactivate HOXA Strikingly, the presence of an early thymic precursor-like immunophenotype was associated with marked outcome differences within the HOXA-positive group (5-year overall survival 31.2% in HOXA-positive early thymic precursor versus 66.7% in HOXA-positive non-early thymic precursor, P=0.03), but not in HOXA-negative cases (5-year overall survival 74.2% in HOXA-negative early thymic precursor versus 57.2% in HOXA-negative non-early thymic precursor, P=0.44). Multivariate analysis further revealed that HOXA positivity independently affected event-free survival (P=0.053) and relapse risk (P=0.039) of chemoresistant T-cell acute lymphoblastic leukemia. These results show that the underlying mechanism of HOXA deregulation dictates the clinico-biological phenotype, and that the negative prognosis of early thymic precursor acute lymphoblastic leukemia is exclusive to HOXA-positive patients, suggesting that early treatment intensification is currently suboptimal for therapeutic rescue of HOXA-positive chemoresistant adult early thymic precursor acute lymphoblastic leukemia. Trial registration - The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

Ikaros and Foxp1 are transcription factors that play key roles in normal lymphopoiesis and lymphoid malignancies. We describe a novel physical and functional interaction between the proteins, which requires the central zinc finger domain of Ikaros. The Ikaros-Foxp1 interaction is abolished by deletion of this region, which corresponds to the IK6 isoform that is commonly associated with high-risk acute lymphoblastic leukemia (ALL). We also identify the Gpr132 gene, which encodes the orphan G protein-coupled receptor G2A, as a novel target for Foxp1. Increased expression of Foxp1 enhanced Gpr132 transcription and caused cell cycle changes, including G2 arrest. Co-expression of wild-type Ikaros, but not IK6, displaced Foxp1 binding from the Gpr132 gene, reversed the increase in Gpr132 expression and inhibited G2 arrest. Analysis of primary ALL samples revealed a significant increase in GPR132 expression in IKZF1-deleted BCR-ABL negative patients, suggesting that levels of wild-type Ikaros may influence the regulation of G2A in B-ALL. Our results reveal a novel effect of Ikaros haploinsufficiency on Foxp1 functioning, and identify G2A as a potential modulator of the cell cycle in Ikaros-deleted B-ALL.

Introduction: Gene expression studies have consistently identified a HOXA positive (HOXAPos) subgroup of T-cell acute lymphoblastic leukemia (T-ALL) (Ferrando et al, Cancer Cell 2002, Soulier et al, Blood 2005, Homminga et al, Cancer Cell 2011). It is however unclear if HOXAPos T-ALL constitutes a distinct and homogeneous clinical entity, and the biological consequences of HOXA over-expression have not been systematically examined. Methods: We identified and characterized the biological characteristics and clinical outcome of 55 HOXAPos cases among a cohort of 209 adult T-ALL patients who were uniformly treated as part of the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL)-2003 and -2005 studies. Results: HOXAPos patients had higher rates of an early thymic precursor (ETP)-like immunophenotype (38% v 13.9%, p = 0.0008), early bone marrow chemoresistance (59.3% v 40.8%, p = 0.026) and positive minimal residual disease (MRD, 51.5% v 23.5%, p = 0.01) than the HOXANeg group. These differences were due to a particularly high frequency of chemoresistant ETP-ALL among HOXAPos cases harboring leukemic fusion proteins that trans-activate the HOXA locus (e.g. PICALM-MLLT10, SET-NUP214). Strikingly, the presence of an ETP-like immunophenotype conferred marked differences in outcome within the HOXAPos group (5 year event-free survival (EFS) 25% for HOXAPos ETP v 52.2% for HOXAPos non-ETP, p = 0.02), which were mirrored by corresponding increases in cumulative incidence of relapse (CIR, 57.1% v 25%, p = 0.01, Figure 1). In contrast, these survival differences were not seen in the HOXANeg patients, where ETP and non-ETP cases had similar 5 year EFS (54.9% v 50%, p = 0.73) and CIR (34.5% v 41.2%, p = 0.57). Multivariate analysis revealed that early bone marrow chemosensitivity was the clinico-biological covariate that had the strongest prognostic interaction with HOXA status. HOXA positivity conferred significant decreases in both the EFS and CIR of chemoresistant patients (p = 0.053 and 0.039 respectively), that was independent of white blood cell count (WCC), stem cell transplant (SCT), ETP phenotype, EGIL classification, and our recently reported risk classifier that integrates the prognostic effects of mutations of NOTCH1, FBXW7, RAS and PTEN (Trinquand et al, J Clin Oncol 2013). There were corresponding marked survival differences within the HOXAPos cohort between chemoresistant and chemosensitive cases. These disparities were not seen in the HOXANeg group, indicating that the prognostic value of chemosensitivity in adult T-ALL is specific to HOXAPos patients. Discussion: Our data show that clinico-biological phenotype is intimately linked to the underlying mechanism of HOXA locus deregulation, and we identify HOXA overexpression as a novel prognostic variable in ETP-ALL. Multivariate analysis suggests that this poor outcome is strongly related to intrinsic treatment resistance, and that this effect is exclusive to the HOXAPos cohort. Patients in the GRAALL-2003 and -2005 studies received enhanced induction and/ or salvage therapy in the event of poor early treatment response. Our results suggest that pediatric regimen-based intensification provides significant survival benefits for HOXANeg chemoresistant cases. In contrast, these modifications are inadequate for therapeutic rescue of the majority of HOXAPos chemoresistant ETP-ALL. The dramatically inferior prognosis of this group mandates consideration for alternative treatments in future clinical trials. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

X-linked neutropenia (XLN, OMIM #300299) is a rare form of severe congenital neutropenia. It was originally described in a three-generation family with five affected members that had an L270P mutation in the GTP-ase binding domain (GBD) of the Wiskott-Aldrich syndrome protein (WASP) [Devriendt et al (2001) Nature Genetics, Vol. 27, 313-317]. Here, we report and describe a large three-generation family with XLN, with 10 affected males and eight female carriers. A c.882T>C mutation was identified in the WAS gene, resulting in an I294T mutation. The infectious course is variable and mild in view of the profound neutropenia. In addition to the original description, low-normal IgA levels, low to low-normal platelet counts and reduced natural killer (NK)-cell counts also appear as consistent XLN features. However, inverted CD4/CD8 ratios were not found in this family, nor were cases identified with myelodysplastic syndrome or acute myeloid leukaemia. Female carriers exhibited a variable attenuated phenotype. Like L270P WASP, I294T WASP is constitutively active towards actin polymerization. In conclusion, this largest XLN kindred identified to date provides new independent genetic evidence that mutations disrupting the auto-inhibitory GBD of WASP are the cause of XLN. Reduced NK cells, low to low normal platelet counts and low to low-normal IgA levels are also features of XLN.

To the editor: Biological subclasses of T-cell acute lymphoblastic leukemia (T-ALL) can be defined by recurrent gene expression patterns, which typically segregate with specific chromosomal anomalies. The HOXA+ subgroup is characterized by deregulated homeobox A ( HOXA ) gene expression and is

‘Stroke-like syndrome’ caused by intrathecal methotrexate in patients treated during the UKALL 2003 trial

To the editor: Hereditary erythrocytosis can be caused by mutations in genes involved in the hypoxia-inducible factor (HIF) pathway.[1][1]–[3][2] For example, Chuvash polycythemia is caused by an R200W substitution in the von Hippel–Lindau protein (VHL).[1][1] There is increasing evidence

Within the human TCR-α/δ locus, ordered rearrangements requires RUNX1, which binds to the Dδ2-23RSS and interacts with RAG1 to enhance RAG1 deposition at this site. Absence of this RUNX1 binding site in the homologous murine Dδ1-23RSS offers an explanation for the lack of ordered TCR-δ gene assembly in mice., V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34+/CD1a−/CD7+dim stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymphoid differentiation between these species.

Purpose The Group for Research in Adult Acute Lymphoblastic Leukemia (GRAALL) recently reported a significantly better outcome in T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 (N/F) mutations compared with unmutated T-ALL. Despite this, one third of patients with N/F-mutated T-ALL experienced relapse. Patients and Methods In a series of 212 adult T-ALLs included in the multicenter randomized GRAALL-2003 and -2005 trials, we searched for additional N/K-RAS mutations and PTEN defects (mutations and gene deletion). Results N/F mutations were identified in 143 (67%) of 212 patients, and lack of N/F mutation was confirmed to be associated with a poor prognosis. K-RAS, N-RAS, and PTEN mutations/deletions were identified in three (1.6%) of 191, 17 (8.9%) of 191, and 21 (12%) of 175 patients, respectively. The favorable prognostic significance of N/F mutations was restricted to patients without RAS/PTEN abnormalities. These observations led us to propose a new T-ALL oncogenetic classifier defining low-risk patients as those with N/F mutation but no RAS/PTEN mutation (97 of 189 patients; 51%) and all other patients (49%; including 13% with N/F and RAS/PTEN mutations) as high-risk patients. In multivariable analysis, this oncogenetic classifier remained the only significant prognostic covariate (event-free survival: hazard ratio [HR], 3.2; 95% CI, 1.9 to 5.15; P < .001; and overall survival: HR, 3.2; 95% CI, 1.9 to 5.6; P < .001). Conclusion These data demonstrate that the presence of N/F mutations in the absence of RAS or PTEN abnormalities predicts good outcome in almost 50% of adult T-ALL. Conversely, the absence of N/F or presence of RAS/PTEN alterations identifies the remaining cohort of patients with poor prognosis.

Background. Core binding factor (CBF) acute myeloid leukemia (AML) includes AML with t(8;21) and inv(16) leading to RUNX1-RUNX1T1 or CBFB-MYH11 fusion genes. These recurrent genetic abnormalities are both associated with disruption of genes encoding subunits of the CBF, a heterodimeric transcription factor involved in hematopoiesis. Although the fusion proteins appear to be crucial for the leukemogenic process, considerable experimental evidence indicates that they are not sufficient to induce AML on their own. Due to their high sensitivity to chemotherapy with high complete remission rates and their relatively favorable outcome, CBF-AML is considered to have a good prognosis. Nonetheless, about 30-40% of these patients relapse after standard intensive chemotherapy. In this context, identification of additional genetic or molecular abnormalities could allow better understanding of CBF-AML leukemogenesis, prediction of clinical outcome and identification of novel therapeutic targets. Methods. This study focuses on 73 patients with CBF-AML [43 t(8;21) and 30 inv(16)-AML] enrolled in the pediatric trial ELAM02. Single nucleotide polymorphism array (SNP-A) was performed for all patients using Cytoscan® HD arrays according to the manufacturer instructions. In order to distinguish somatic from constitutional SNP-A lesions, we excluded known copy number abnormalities (CNA) if there was >50% overlap with variants from public database, except for breakpoints-related alterations. Interstitial uniparental disomies (UPD) Results. Among the 73 cases, 145 SNP-A lesions were found in 58 patients (81%) with a median of 2 lesions per case (range, 0-8). CNA was more frequent (84 losses, 47 gains) than UPD (n=14). No significant difference was noted between the number of CNA and UPD in inv(16) and t(8;21)-AML. Small lesions were common at breakpoints involved in the t(8;21) and inv(16) (respectively 4/43 and 6/30). Additional recurrent CNA mostly involved entire chromosomes, chromosomal arms or large chromosomal regions. Del(9q) and loss of sex chromosome were restricted to t(8;21)-AML (respectively 6/43 and 20/43). Trisomy 22 was restricted to inv(16)-AML (2/30). Other recurrent CNA included trisomy 8 (3/43 vs 1/30) and gains of 13q (2/43 vs 1/30) in both subtypes, gains of 1q and del(2q) in t(8;21)-AML (each 2/43). Del(7q) was among the most common aberrations regardless of subtype (7/43 and 7/30). The minimally deleted region of 7q contained 57 genes including MLL3 and EZH2. Additionally, we found focal deletions of IKZF1 in one patient, NF1 in another and 3 deletions of CCDC26. Except for known mutations (KIT, RAS, FLT3), NGS did not reveal any other alterations in inv(16)-AML. By contrast, t(8;21)-AML was marked by the frequency of mutations in ASXL1/2 (8%/24%) and cohesin genes SMC1A, SMC3, RAD21, STAG2, NIPBL (27% combined). Mutations were also detected in epigenetic-related genes EZH2 (5%), TET2 (8%), IDH1/2 (5%) and WT1(11%). Conclusions. SNP-A karyotyping of 73 pediatric CBF-AML revealed several recurrent alterations, with differing distribution between the 2 subgroups. Moreover, t(8;21) and inv(16)-AML appeared to have distinct mutational profiles, leading us to consider them separately for future studies. We recently reported high frequency of ASXL mutations in t(8;21)-AML and their absence in inv(16)-AML (Micol, Duployez and Boissel et al, Blood 2014). We now report high frequency of mutations in cohesin genes with the same distribution. Recent description of functional relations between cohesin and polycomb proteins, together with our results, suggest an important pathway in t(8;21) leukemogenesis. Concurrent ASXL and cohesin mutations were found in several patients, suggesting they could cooperate in some cases. Interestingly, ASXL mutations were exclusive of del(7q), suggesting that disruption of the ASXL-associated proteins MLL3 and EZH2 could be of great interest in the physiopathology of t(8;21)-AML. Finally, correlations with clinical outcome are in progress. Disclosures No relevant conflicts of interest to declare.

X-linked neutropenia (XLN, OMIM #300299) is a rare cause of severe congenital neutropenia and was first described in a three-generation Belgian family with 5 affected members. Reported features of XLN include severe congenital neutropenia and monocytopenia with recurrent bacterial infections, a decreased CD4/CD8 ratio and bone marrow maturation arrest at the promyelocyte/metamyelocyte stage. In the Belgian family, a L270P WAS mutation was identified, causing constitutive activity of the Wiskott-Aldrich-syndrome protein (WASP) toward actin polymerisation (Devriendt et al. Nat.Genet. 2001). Here, we report the clinical phenotype of a second large family with XLN and with a I294T WAS mutation. In this three-generation family, 10 affected males (7–45 y) and 8 female carriers were identified. Variable non-cyclic neutropenia is present in affected males (0.2–3.3*109/L in those not on G-CSF; 0.1–1.0*109/L on G-CSF). Five of 10 affected males in the I294T family have monocytopenia. A consistent feature in all cases is a reduced NK cell number. In fact, 3 of 3 tested L270P cases also had reduced NK cell counts. The severity of the clinical phenotype is variable without apparent correlation with the degree of neutropenia. Five of 10 affected males are receiving treatment with G-CSF because of recurrent infections. Four of 10 are reported healthy in the absence of G-CSF. One case with a borderline neutrophil count (2.4*109/L) is not on G-CSF, despite recurrent infections. In addition, two males with a history of recurrent infections, at least one of whom had neutropenia, died of infectious causes at age 5 and 18 years. Platelet counts are variably reduced in affected males, but with normal platelet volume. No consistent abnormalities in CD4/CD8 ratio are found. Available bone marrows have revealed no myelodysplastic features or cytogenetic abnormalities. Of note, female carriers show intermediate findings in neutrophil, platelet and NK cell counts. Only 1 female carrier is known with recurrent upper respiratory and ear infections and is treated with G-CSF. The T916C mutation we found in exon 9 of WAS, has recently been described (Ancliff et al. Blood online 2006) and results in a I294T mutation of the WASP GTPase-binding domain (GBD). The I294T GBD-VCA construct has a lower melting temperature (36°C) as measured by circular dichroism spectroscopy (78°C for wild-type). In the absence of Cdc42, I294T GBD-VCA is nearly completely active toward the Arp2/3 complex, contrary to wild-type GBD-VCA, but similar to the L270P construct. Thus, these data provide new and independent genetic evidence that mutations that disrupt the auto-inhibitory domain of WASP are the cause of XLN. In addition, based on this largest XLN kindred to date, reduced NK cell counts appear a consistent feature. None of the affected males presented with myelodysplasia. Finally, female carriers have moderately reduced neutrophil counts, mostly without clinical consequences. Thus, the presence of mild neutropenia in potential female carriers does not rule out the possibility of XLN in related males with neutropenia. Further investigation is needed to reveal how the L270P and the I294T WAS mutations lead to XLN.