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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.

KMT2A-rearranged acute lymphoblastic infant leukemia (KMT2A-r iALL) is associated with outsize risk of relapse and relapse mortality. We previously reported strong upregulation of the immediate early gene EGR3 in KMT2A::AFF1 iALL at relapse; now we provide analyses of the EGR3 regulome, which we assessed through binding and expression target analysis of an EGR3-overexpressing t(4;11) cell culture model. Our data identify EGR3 as a regulator of early B-lineage commitment. Principal component analysis of 50 KMT2A-r iALL patients at diagnosis and 18 at relapse provided strictly dichotomous separation of patients based on the expression of four B-lineage genes. Absence of B-lineage gene expression translates to more than two-fold poorer long-term event-free survival. In conclusion, our study presents four B-lineage genes with prognostic significance, suitable for gene expression-based risk stratification of KMT2A-r iALL patients.

Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the “gold standard” technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10−7 and transcripts down to 0.005 copies/ng.Implications: This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for MLL-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies MLL partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. Mol Cancer Res; 16(2); 279–85. ©2017 AACR.

Expression of the exon 10 fusion transcript in patient 17 and patient 32. Standard curve was established using patient 32 sample in a range from 25ng/µl to 0.1 ng/µl in 1 in 2 serial dilutions.

Comparison of MLL fusions detected by cytogenetics, fluorescent in situ hybridization (FISH), Long distance Inverse polymerase chain reaction (LDI-PCR) and Anchored multiplex PCR enrichment (AMP-E).

Purpose: Although the overall cure rate for pediatric acute lymphoblastic leukemia (ALL) approaches 90%, infants with ALL harboring translocations in the mixed-lineage leukemia (MLL) oncogene (infant MLL-ALL) experience shorter remission duration and lower survival rates (∼50%). Mutations in the p53 tumor-suppressor gene are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be beneficial. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenografts.Experimental Design: Eight MLL-ALL patient-derived xenografts were established in immune-deficient mice, and their molecular features compared with B-lineage ALL and T-ALL xenografts. The sensitivity of MLL-ALL xenografts to RG7112 was assessed in vitro and in vivo, and the ability of RG7112 to induce p53, cell-cycle arrest, and apoptosis in vivo was evaluated.Results: Gene-expression analysis revealed that MLL-ALL, B-lineage ALL, and T-ALL xenografts clustered according to subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA family, were significantly upregulated in MLL-ALL xenografts, confirming their ability to recapitulate the clinical disease. Exposure of MLL-ALL xenografts to RG7112 in vivo caused p53 upregulation, cell-cycle arrest, and apoptosis. RG7112 as a single agent induced significant regressions in infant MLL-ALL xenografts. Therapeutic enhancement was observed when RG7112 was assessed using combination treatment with an induction-type regimen (vincristine/dexamethasone/L-asparaginase) against an MLL-ALL xenograft.Conclusions: The utility of targeting the p53–MDM2 axis in combination with established drugs for the management of infant MLL-ALL warrants further investigation. Clin Cancer Res; 21(6); 1395–405. ©2015 AACR.

This file contains Supplementary Methods describing the Pediatric Preclinical Testing Program (PPTP) scoring method. Supplementary Figure S1: Engraftment rates and organ infiltration of MLL-ALL xenografts. Supplementary Figure S2: Comparison of expression of specific genes between MLL-ALL and BCP-ALL xenografts. Supplementary Figure S3: Comparison of HOXA gene expression between MLL-ALL and BCP-ALL xenografts. Supplementary Figure S4: RG7112 induces caspase-dependent cell death in RS4;11 cells. Supplementary Figure S5: In vivo efficacy of RG7112 against MLL-ALL xenografts. Supplementary Figure S6: Effects of RG7112 on mouse weight and haematological parameters. Supplementary Figure S7: Immunoblots of spleen-derived cells from replicate mice showing the effects of RG7112 on MLL-14 xenograft cells. Supplementary Figure S8: Relationships between TP53 and MDM2 gene expression and in vivo responses of ALL xenografts to RG7112. Supplementary Table S1: Rates of serial engraftment of MLL-ALL xenografts. Supplementary Table S2: The top 100 genes that distinguish MLL-rearranged ALL xenografts from BCP-ALL xenografts. Supplementary Table S3: Statistical analysis of genes identified by Armstrong et al (2002) between MLL-ALL and BCP-ALL xenografts. Supplementary Table S4: Details of individual mouse responses to in vivo treatment with RG7112. Supplementary Table S5: In vitro Combination Indices of RG7112 with established drugs used to treat ALL. Supplementary Table S6: In vivo efficacy of RG7112 in combination with a VXL induction-type regimen. Supplementary Table S7: Details of individual mouse responses to in vivo treatment with RG7112 combined with VXL against MLL-ALL xenografts.

IKZF1 deletions are associated with an increased risk of relapse in B-cell precursor acute lymphoblastic leukemia (B-ALL), and their accurate detection has great clinical impact. Here, we included four international cohorts of pediatric and adult patients with B-ALL, and reviewed literature to illustrate the recombination map of IKZF1 deletions, with a focus at non-recurrent deletions. We provide a substantial basis for the improvement of diagnostic methods based on MLPA and multiplex PCR for the identification of IKZF1 deletions, and also demonstrate that rare IKZF1 deletions increase the incidence of relapse in these patients. Of note, non-recurrent deletions comprised a wide range of alterations, but the majority were Δ1 and Δ1–3. They were often associated with reciprocal IKZF1 fusions. So far, a total of 23 IKZF1 gene fusions were identified in B-ALL. We also verified the occurrence of the heptamer sequence (E-value: 9.9 x 10− 9) and an enrichment of GC nucleotides (71% versus 56%; P value = 4.9 x 10− 3) exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote the majority of IKZF1 deletions, although rare types of alterations may be associated with other molecular mechanism of leukemogenesis, such as microhomology-mediated end joining.

About 25% of the patients with the translocation t(11;19)(q23;p13.3)/KMT2A-MLLT1 present three-way or more complex fusions, associated with a worse prognosis, suggesting that a particular mechanism creates functional KMT2A fusions for this condition. In this work, we show a cryptic three-way translocation t(9;11;19). Interestingly, long-distance inverse polymerase chain reaction sequencing revealed a KMT2A-MLLT1 and the yet unreported out-of-frame SEC16A-KMT2A fusion, associated with low SEC16A expression and KMT2A overexpression, in an infant with B-acute lymphoblastic leukemia presenting a poor prognosis. Our case illustrates the importance of molecular cytogenetic tests in selecting cases for further investigations, which could open perspectives regarding novel therapeutic approaches for poor prognosis childhood leukemias.

Therapy-related acute myeloid leukemia (t-AML) following treatment with topoisomerase-II inhibitors has been increasingly reported. These compounds (e.g. etoposide) promote DNA damage and are associated with KMT2A rearrangements. They are widely used as first-line treatment in hemophagocytic lymphohistiocytosis (HLH). Here we describe a newborn who developed t-AML after HLH treatment. We provide detailed clinical, cytogenetic, and molecular characteristics of this patient, including the identification of a novel gene fusion - KMT2A-SNX9 - in t-AML. Considering the dismal outcome of this case, we discuss the side-effects of etoposide administration during HLH treatment in infants.

PURPOSE Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of KMT2A gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with KMT2A-rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide). MATERIALS AND METHODS MRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged KMT2A, immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10−4), and high (≥ 5 × 10−4). RESULTS EOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively ( P = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively ( P < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively ( P < .0001), while for myeloid-style–treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9). CONCLUSION This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% v 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% v 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol.

The

Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq: PQ-2017#305529/2017-0 Deutsche Forschungsgemeinschaft, DFG: MA 1876/12-1 Alexander von Humboldt-Stiftung: 88881.136091/2017-01 RVO-VFN64165, 26/203.214/2017 2018.070.1 Associazione Italiana per la Ricerca sul Cancro, AIRC: IG2015, 17593 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES Cancer Australia: PdCCRS1128727 Cancerfonden Barncancerfonden VetenskapsrÃ¥det, VR Crafoordska Stiftelsen Knut och Alice Wallenbergs Stiftelse Lund University Medical Faculty Foundation Xiamen University, XMU 2014S06 17-74-30019 C7838/A15733 Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, SNSF: 31003A_140913 CNIB Institut National Du Cancer, INCa R01 NCI CA167824 National Institutes of Health, NIH: S10OD018522 2016/2017, 02R/2016 AU 525/1-1 Deutschen Konsortium für Translationale Krebsforschung, DKTK 70112951 Smithsonian Institution, SI Israel Science Foundation, ISF Austrian Science Fund, FWF: W1212 SFB-F06107, SFB-F06105 Acknowledgements BAL received a fellowship provided by CAPES and the Alexander von Humboldt Foundation (#88881.136091/2017-01). ME is supported by CNPq (PQ-2017#305529/2017-0) and FAPERJ-JCNE (#26/203.214/2017) research scholarships, and ZZ by grant RVO-VFN64165. GC is supported by the AIRC Investigator grant IG2015 grant no. 17593 and RS by Cancer Australia grant PdCCRS1128727. This work was supported by grants to RM from the “Georg und Franziska Speyer’sche Hochsschulstiftung”, the “Wilhelm Sander foundation” (grant 2018.070.1) and DFG grant MA 1876/12-1. Acknowledgements This work was supported by The Swedish Childhood Cancer Foundation, The Swedish Cancer Society, The Swedish Research Council, The Knut and Alice Wallenberg Foundation, BioCARE, The Crafoord Foundation, The Per-Eric and Ulla Schyberg Foundation, The Nilsson-Ehle Donations, The Wiberg Foundation, and Governmental Funding of Clinical Research within the National Health Service. Work performed at the Center for Translational Genomics, Lund University has been funded by Medical Faculty Lund University, Region Skåne and Science for Life Laboratory, Sweden. Acknowledgements This work was supported by the Fujian Provincial Natural Science Foundation 2016S016 China and Putian city Natural Science Foundation 2014S06(2), Fujian Province, China. Alexey Ste-panov and Alexander Gabibov were supported by Russian Scientific Foundation project No. 17-74-30019. Jinqi Huang was supported by a doctoral fellowship from Xiamen University, China. Acknowledgments This work was supported by the Swiss National Science Foundation (grant 31003A_140913; OH) and the Cancer Research UK Experimental Cancer Medicine Centre Network, Cardiff ECMCI, grant C7838/A15733. We thank N. Carpino for the Sts-1/2 double-KO mice. Acknowledgements This work was supported by the French National Cancer Institute (INCA) and the Fondation Française pour la Recherche contre le Myélome et les Gammapathies (FFMRG), the Intergroupe Francophone du Myélome (IFM), NCI R01 NCI CA167824 and a generous donation from Matthew Bell. This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. Research reported in this paper was supported by the Office of Research Infrastructure of the National Institutes of Health under award number S10OD018522. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank the Association des Malades du Myélome Multiple (AF3M) for their continued support and participation. Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization. We are indebted to all members of our groups for useful discussions and for their critical reading of the manuscript. Special thanks go to Silke Furlan, Friederike Opitz and Bianca Killing. F.A. is supported by the Deutsche For-schungsgemeinschaft (DFG, AU 525/1-1). J.H. has been supported by the German Children’s Cancer Foundation (Translational Oncology Program 70112951), the German Carreras Foundation (DJCLS 02R/2016), Kinderkrebsstiftung (2016/2017) and ERA PerMed GEPARD. Support by Israel Science Foundation, ERA-NET and Science Ministry (SI). A. B. is supported by the German Consortium of Translational Cancer Research, DKTK. We are grateful to the Jülich Supercomputing Centre at the Forschungszemtrum Jülich for granting computing time on the supercomputer JURECA (NIC project ID HKF7) and to the “Zentrum für Informations-und Medientechnologie” (ZIM) at the Heinrich Heine University Düsseldorf for providing computational support to H. G. The study was performed in the framework of COST action CA16223 “LEGEND”. Funding The work was supported by the Austrian Science Fund FWF grant SFB-F06105 to RM and SFB-F06107 to VS and FWF grant W1212 to VS.

Patients with childhood acute myeloid leukemia (AML) with complex karyotypes (CKs) have a dismal outcome. However, for patients with a KMT2A rearrangement (KMT2A-r), the prognosis appears to depend on the fusion partner gene rather than the karyotype structure. Thus, a precise characterization of KMT2A-r and the fusion partner genes, especially in CKs, is of interest for managing AML. We describe the clinical and molecular features of a child who presented with a large abdominal mass, AML, and a new CK, involving chromosomes 11, 16, and 19 leading to a KMT2A-MLLT1 fusion and 2 extra copies of the ELL gene, thus resulting in the concurrent overexpression of MLLT1 and ELL. Molecular cytogenetic studies defined the karyotype as 47,XY,der(11)t(11;16)(q23.3;p11.2),der(16)t(16;19)(p11.2;p13.3),der(19)t(11;19)(q23.3;p13.3),+der(19)t(16;19)(16pter→p11.2::19p13.3→19q11::19p11→19p13.3::16p11.2→16pter). Array CGH revealed a gain of 30.5 Mb in the 16p13.3p11.2 region and a gain of 18.1 Mb in the 19p13.3p12 region. LDI-PCR demonstrated the KMT2A-MLLT1 fusion. Reverse sequence analysis showed that the MLLT1 gene was fused to the 16p11.2 region. RT-qPCR quantification revealed that ELL and MLLT1 were overexpressed (4- and 10-fold, respectively). In summary, this is a pediatric case of AML presenting a novel complex t(11;16;19) variant with overexpression of ELL and MLLT1.

The detection of KMT2A gene rearrangements have an important impact on the prognosis and management of acute leukemias. These alterations most commonly involve reciprocal translocations at specific breakpoint regions within KMT2A. To date, more than 100 translocation partner genes of KMT2A have been identified, with different effects on risk stratification. We report the case of a mature plasmacytoid dendritic cells proliferation associated with B lymphoblasts harboring a KMT2A-ARHGEF12 fusion. This rare rearrangement, resulting from a cryptic deletion on the long arm of chromosome 11, is located outside the known major and minor breakpoint regions of KMT2A, not reported to date. The review of the few cases of KMT2A-ARHGEF12 reveals the tendency of this deletion to occur in therapy related hematologic neoplasm and confer unfavorable prognosis. This review sheds light into the rare KMT2A-ARHGEF12 fusion in leukemia. Reporting rare chimeras is essential to improve knowledge about the biological mechanism and associated clinical consequences

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence ofMRD was measured in 249 infants by DNA-based polymerase chain reaction of rearrangedEOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively (This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9%

We report on the Australian experience of blinatumomab for treatment of 24 children with relapsed/refractory precursor B-cell acute lymphoblastic leukaemia (B-ALL) and high-risk genetics, resulting in a minimal residual disease (MRD) response rate of 58%, 2-year progression-free survival (PFS) of 39% and 2-year overall survival of 63%. In total, 83% (n = 20/24) proceeded to haematopoietic stem cell transplant, directly after blinatumomab (n = 12) or following additional salvage therapy (n = 8). Four patients successfully received CD19-directed chimeric antigen receptor T-cell therapy despite prior blinatumomab exposure. Inferior 2-year PFS was associated with MRD positivity (20%, n = 15) and in KMT2A-rearranged infants (15%, n = 9). Our findings highlight that not all children with relapsed/refractory B-ALL respond to blinatumomab and factors such as blast genotype may affect prognosis.

We present a case of an infant who developed pro-B acute lymphoblastic leukemia with a rare and complex MLL-translocation. Cytogenetic analysis of bone marrow cells at diagnosis showed a 46,XY,t(X;11)(p11.2;q23)[13]/46,XY[7] karyotype. Fluorescence in situ hybridization analysis using a break apart specific probes showed a split in the MLL gene. Long distance inverse-PCR and next generation sequencing analysis depicted a complex rearrangement t(X;11;17)(p11.2;q23;q12) involving MLL, MLLT6 and the genomic region Xp11.23, 41 bases upstream of the WDR45 gene. WDR45 encodes a beta-propeller protein essential for autophagocytosis. MLL rearrangements with involvement of Xp have not been previously described.

Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the “gold standard” technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10−7 and transcripts down to 0.005 copies/ng. Implications: This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for MLL-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies MLL partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. Mol Cancer Res; 16(2); 279–85. ©2017 AACR.

Infant t(4;11) acute lymphoblastic leukemia (ALL) is associated with a high relapse rate with 4-year event-free survival (EFS) of only 36%. Relapse has been shown to be the major cause of death as 83% of relapsed infant t(4;11) ALL patients die within three years of diagnosis. Therefore, it is of utmost therapeutic interest to elucidate molecular mechanisms of relapse. Here we show that t(4;11) ALL cells upregulate the inducible T-cell costimulator ligand (ICOSLG) in an early growth response 3 (EGR3) dependent manner thereby promoting the development of regulatory T-cells (T regs). We propose that short EFS and high ICOSLG expression are causally linked, in that ICOSLG-mediated induction of T regs interferes with immune recognition of leukemia cells. According to that hypothesis, ICOSLG would not only be a novel and independent prognostic biomarker but a potential therapeutic target. We investigated the function of EGR3 in infant ALL since EGR3 has been described as an indirect target of the iroquois homeobox 1 (IRX1) transcription factor. For that purpose we created a stable sleeping beauty transposon-based SEM cell line expressing EGR3 in a Doxycycline-inducible manner. Gene expression and western blot analysis revealed strong upregulation of ICOSLG 48h after Doxycycline induction when compared to an empty vector control. Additionally, chromatin immunoprecipitation (ChIP) experiments depicted that EGR3 directly binds to the promoter of the ICOSLG gene. The expression of ICOSLG in mesenchymal stem cells has been shown to foster the induction of T regs and ICOSLG-mediated T reg expansion has been identified as a driver of acute myeloid leukemia and glioblastoma. In the bone marrow (BM) hematopoietic stem cells (HSC) colocalize with T regs which provide an immune privilege to the stem cell niche. Based on these observations, we hypothesize that ICOSLG expressing ALL cells could create an immune privileged niche appearing to be necessary for HSC maintenance and sanctuary from immune attack. This could create independence from the BM immune privilege enabling migration of the ALL cells. If so, this could contribute to minimal residual disease (MRD) formation after induction therapy and subsequently to higher probability of relapse. To evaluate our hypothesis, we cocultured the EGR3-SEM and control cells with primary human CD4 + and CD8 + T-cells. The T-lymphocytes were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors and stimulated with coated α-CD2, -CD3 and -CD28 beads. We observed 16% more CD4 +CD25 ++FOXP3 + T regs after 48h of coculture with EGR3-SEM cells compared to the control. The addition of a neutralizing monoclonal α-ICOSLG antibody to the coculture led to a reduction of the T reg frequency in EGR3-SEM coculture. Taken together, these results strongly suggest that ALL cells expressing EGR3 induce the formation of T regs via ICOSLG expression. To confirm our results with patient-derived material, we investigated the gene expression of 50 infant t(4;11) pro-B phenotypic ALL patients. Pearson correlation testing confirmed that ICOSLG expression strongly correlates with EGR3 and IRX1 expression. Furthermore, we were able to classify the patients considering their ICOSLG expression level into an ICOSLG-high (ICOSLG-hi) and an ICOSLG-low (ICOSLG-lo) group. Outcome data for a 5-year follow-up were available for 35 of 50 patients, n=5 ICOSLG-hi and n=30 ICOSLG-lo. 100% (5/5) of the ICOSLG-hi patients failed within 17 months of diagnosis whereas 53% (16/30) of the ICOSLG-lo patients failed within 56 months of diagnosis. The remaining 14 ICOSLG-lo patients were alive after 60 months. These data underscore the role of overexpressed ICOSLG in relapse development. However, a verification of these findings in a larger cohort is needed. In conclusion, our study identifies ICOSLG as a promising prognostic marker and novel therapeutic target in infant t(4;11) ALL. Furthermore, our findings implicate the interaction between T-cells and leukemia stem cells as contributory to disease progression. Disclosures Cario: Novartis: Other: Lecture Fee.

Chromosomal rearrangements of the KMT2A gene are associated with acute leukemias and myelodysplastic syndromes. The large number of known KMT2A fusions (>100) renders a precise diagnosis a demanding task. More than 50% of all KMT2A partner genes have been analyzed at the DCAL, including the novel partner genes BCAS4, FAM13A, RANBP3, and STK4. Even though all KMT2A rearrangements are associated with high-risk acute leukemia, the outcome (poor or very poor) is influenced by the partner gene. So far, we have analyzed more than 3,200 patients positive for a KMT2A rearrangement. The breakpoints of these cases are located mainly in the major breakpoint cluster region (bcr1) and to a small extent in the recently described minor bcr (bcr2). A small number of breakpoints were also found outside of these two bcrs. Most of these patients were analyzed by long distance inverse (LDI)- or multiplex-PCR which only cover bcr1. More recently, we used targeted KMT2A-NGS with whole gene coverage in over 450 patients, which was initially applied selectively in patients negative by LDI- and multiplex-PCR and then used more widely. Within the KMT2A-NGS group, 410 patients had bcr1 breakpoints mainly between the KMT2A exons 7 and 13, while 46 patients bcr2 breakpoints mainly between exons 20 and 24. Of note, five patients had their breakpoint outside of these two bcrs: three of them within intron 2 and no functional KMT2A rearrangement; the other two within intron 35 and intron 36, fusing almost the whole KMT2A gene in frame to the respective partner genes ARHGEF12 and MLLT4. These two breakpoints may define a third and rare bcr (bcr3), although further cases are needed to support this hypothesis. Interestingly, 70 patients displayed a 3'-KMT2A deletion, indicating that the number of terminal deletions is higher than described previously. Two patients had a 5'-KMT2A deletion. All deletions started or ended in bcr1 and bcr2. We also observed a striking difference in the distribution of partner genes between bcr1 and bcr2. The most frequent translocation partners fused to bcr1 sites are transcription factors, while the partner genes linked to bcr2 sites generally code for cytosolic proteins. In bcr1, the 4 most frequent partner genes AFF1, MLLT3, MLLT1, and MLLT10, found in 80% of cases, all code for transcription factors that are part of the super elongation complex (SEC). These fusions therefore all lead to disruption of the hematopoietic lineage commitment. In contrast in bcr2, 3 partner genes USP2, MLLT4, and USP8 account for 85% of the cases. USP2 and USP8 are ubiquitin specific peptidases involved in cell signaling and exclusively fused to bcr2 in KMT2A. While MLLT4 is found as a partner in bcr1, bcr2 and bcr3 fusions; unlike other recurrent KMT2A partners linked to bcr1, it is not a transcription factor and it exerts oncogenic potential via dimerization like other cytosolic partners. We hypothesize that the oncogenic properties of USP2 and USP8 are dependent on dimerization like MLLT4 and that the most frequent fusions involving at different bcrs favor different oncogenic mechanisms: bcr1 transactivation and bcr2 dimerization. Further studies are needed to explain why USP2 and USP8 are exclusively associated with bcr2, and why the most frequent partner genes AFF1 and MLLT3 of the bcr1 are less frequent in bcr2. In conclusion, targeted NGS combined with bioinformatic analysis has expanded our knowledge of the KMT2A recombinome to include more fusion partners and has generated new hypotheses for future research on oncogenic mechanisms. Disclosures No relevant conflicts of interest to declare.

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination.

SummaryThe translocation MLL-AF4 defines a high-risk subtype of acute leukaemia which, uniquely amongst MLL translocations, is almost exclusively associated with a pro-B lymphoid phenotype. However, the ability to switch lineage at relapse allowed interrogation of the cellular origin and lineage determinants of this leukaemia. The origin of MLL-AF4 lymphoid leukaemia was commonly within a multipotent precursor population. In contrast, relapse as MLL-AF4 AML occurs from either a multipotent progenitor or a committed pro-B leukaemic clone. Lineage switching results from the dysregulation of chromatin modifiers, including the nucleosome remodelling and deacetylation complex, NuRD, completely extinguishing the B lymphoid gene regulatory network, instead establishing a myeloid epigenotype. We propose that, in contrast to mixed phenotype acute leukaemias, lineage switch in MLL-AF4 leukemia is driven and maintained by a reorganised epigenome.

In pediatric acute leukemias, reciprocal chromosomal translocations frequently cause gene fusions involving the lysine (K)-specific methyltransferase 2A gene (KMT2A, also known as MLL). Specific KMT2A fusion partners are associated with the disease phenotype (lymphoblastic vs. myeloid), and the type of KMT2A rearrangement also has prognostic implications. However, the KMT2A partner gene cannot always be identified by banding karyotyping. We sought to identify such partner genes in 13 cases of childhood leukemia with uninformative karyotypes by combining molecular techniques, including multicolor banding FISH, reverse-transcriptase PCR, and long-distance inverse PCR. Of the KMT2A fusion partner genes, MLLT3 was present in five patients, all with acute lymphoblastic leukemia, MLLT1 in two patients, and MLLT10, MLLT4, MLLT11, and AFF1 in one patient each. Reciprocal reading by long-distance inverse PCR also disclosed KMT2A fusions with PITPNA in one patient, with LOC100132273 in another patient, and with DNA sequences not compatible with any gene in three patients. The most common KMT2A breakpoint region was intron/exon 9 (3/8 patients), followed by intron/exon 11 and 10. Finally, multicolor banding revealed breakpoints in other chromosomes whose biological and prognostic implications remain to be determined. We conclude that the combination of molecular techniques used in this study can efficiently identify KMT2A fusion partners in complex pediatric acute leukemia karyotypes. Copyright © 2016 John Wiley & Sons, Ltd.

In pediatric acute myeloid leukemia (AML), chromosomal abnormalities leading to a disruption of the lysine methyltransferase 2A (KMT2A) gene in 11q23 are the most frequent rearrangements. Here, we report on the identification of a novel cryptic insertion, ins(11;X)(q23;q28q12), resulting in a translocation of the KMT2A gene in 11q23, leading to a KMT2A-FLNA fusion in a 13-month-old boy with de novo acute myelomonocytic leukemia, who died 38 days after diagnosis. The patient presented a complex karyotype 48∼49,Y,del(X)(q12),+del(X)(q12),+8,ins(11;X)(q23; q28q12),+19. The identified fusion gene was predicted to be out-of-frame (fusion of portions of KMT2A exon 11 with FLNA exon 11). However, RT-PCR experiments demonstrated that a potentially functional transcript was generated by alternative splicing where KMT2A exon 10 was spliced in-frame to the truncated FLNA exon 11. This case report helps to better understand the rare but potentially severe impact of KMT2A- FLNA fusions in infants with AML to improve prognostic stratification of therapy and clinical management.

Patients with childhood acute myeloid leukemia (AML) with complex karyotypes (CKs) have a dismal outcome. However, for patients with a KMT2A rearrangement (KMT2A-r), the prognosis appears to depend on the fusion partner gene rather than the karyotype structure. Thus, a precise characterization of KMT2A-r and the fusion partner genes, especially in CKs, is of interest for managing AML. We describe the clinical and molecular features of a child who presented with a large abdominal mass, AML, and a new CK, involving chromosomes 11, 16, and 19 leading to a KMT2A-MLLT1 fusion and 2 extra copies of the ELL gene, thus resulting in the concurrent overexpression of MLLT1 and ELL. Molecular cytogenetic studies defined the karyotype as 47,XY,der(11)t(11;16)(q23.3;p11.2),der(16)t(16;19)(p11.2;p13.3),der(19)t(11;19)(q23.3;p13.3),+der(19)t(16;19)(16pter→p11.2::19p13.3→19q11::19p11→19p13.3::16p11.2→16pter). Array CGH revealed a gain of 30.5 Mb in the 16p13.3p11.2 region and a gain of 18.1 Mb in the 19p13.3p12 region. LDI-PCR demonstrated the KMT2A-MLLT1 fusion. Reverse sequence analysis showed that the MLLT1 gene was fused to the 16p11.2 region. RT-qPCR quantification revealed that ELL and MLLT1 were overexpressed (4- and 10-fold, respectively). In summary, this is a pediatric case of AML presenting a novel complex t(11;16;19) variant with overexpression of ELL and MLLT1.

Introduction Translocations involving the KMT2A gene (also known as MLL) are frequently diagnosed in pediatric acute leukemia cases with either lymphoblastic or myeloid origin. KMT2A is translocated to multiple partner genes, including MLLT10/AF10 localizing at chromosomal band 10p12. KMT2A-MLLT10 is one of the common chimeric genes diagnosed in acute leukemia with KMT2A rearrangement (8%), especially in acute myeloid leukemia (AML; 18%). MLLT10 is localized in very close proximity to two other KMT2A partner genes at 10p11-12-NEBL and ABI1, so they could not be distinguished by conventional cytogenetics. Methods In this work, we present a cohort of 28 patients enrolled into Russian Pediatric AML registration study carrying rearrangements between chromosomal regions 11q23.3 and 10p11-12. G-banding, FISH, reverse transcription PCR, and long-distance inverse PCR were used to characterize the KMT2A gene rearrangements in these patients. Results We demonstrate that 25 patients harbor the KMT2A-MLLT10 rearrangement, while three patients show the rare KMT2A rearrangements (2× KMT2A-NEBL; 1× KMT2A-ABI1). Conclusions Therefore, the combination of cytogenetic and molecular genetic methods is of high importance in diagnosing cases with t(10;11)(p11-12;q23.3).