MLL-ENL Fusion Research Articles

A Case of Concordant Twins with Infant ALL and Discordant Clinical Outcome – Part I: the genetic basis – identification of DSC2 as a gene with prognostic impact in infant MLL-rearranged ALL

Infant acute lymphoblastic leukemia (ALL) is associated with an unfavorable prognosis due to a high proportion of early relapses. We report on a pair of monozygotic twin sisters diagnosed with ALL (MLL-ENL fusion) in early infancy. Despite good initial treatment responses in both twins, one sister developed very early relapse (Twin A) and succumbed to the disease. The other sister went into continuous complete remission (Twin B) and is alive after 9 years. We performed exome sequencing experiments to identify additional diverging mutations in the twins that may be connected to the different clinical outcomes. Results obtained were further complemented by sequencing analyses of xenografted twin leukemias from different disease time points providing insights into clonal evolution in this twin model of infant leukemia. Apart from an overall paucity of mutations, genetic alterations predicted to be relevant were detected only in leukemic cells of the survivor, Twin B. In particular, we show evidence for a heterozygous desmocollin 2 (DSC2) mutation which is absent in Twin A. Notably, high gene expression of DSC2 in a cohort of infant ALL patients harboring MLL-ENL and MLL-AF9 fusions conferred a poor prognosis characterized by a significantly increased relapse rate. Our data suggest that high levels of DSC2 expression in residual cells may be a pathogenic mechanism in relapsing infant ALL.

Analyses of a Pair of Concordant Twins with Infant ALL and Discordant Clinical Outcome Reveals Immunoescape As a Mechanism of Disease Persistence in MLL-Rearranged Leukemia

MLL-fusion is the most common genetic abnormality in acute lymphoblastic leukemia (ALL) of infancy and occurs in approximately 80% of the cases. Infant ALL represents a biologically distinctive entity with a highly immature pro-B immunophenotype associated with a particularly unfavorable prognosis due to a high proportion of early relapses. This may be due to the survival of dormant residual disease protected by the bone marrow niche.We have characterized a pair of monozygotic twin sisters diagnosed with ALL in early infancy. Tumor cells from both children carried the t(11;19) translocation (MLL-ENL fusion) and both patients were treated according to the ALL-BFM 2000 protocol. Despite good initial treatment responses in both twins, one sister developed very early relapse (twin A) and succumbed to the disease. The other went into continuous complete remission (twin B) and, as of today, is alive after 9 years. The clinical data of the 2 patients are presented in Table 1.Diagnostic bone marrow (BM) aspirates of both sisters were injected into the femoral bones of NOD SCID gamma (NSG) mice. Survival of xenografted mice bearing twin A was significantly longer due to a lower proliferative capacity of twin A as compared to twin B cells. In vivo Bromodeoxyuridine (BrdU) assays revealed that twin B cells were proliferating equally fast in BM and spleen, while in twin A cells, the BM markedly suppressed entry into S-phase. Flow cytometry from xenograft BM identified a large CD34+ population in twin A cells that was almost absent in twin B cells. Furthermore, BM of twin A xenografts showed a CD34+/CD38-/CD19- stem cell like population undetectable in twin B animals. Most interestingly, injection of minimal residual disease (MRD) negative remission bone marrow (PCR for Ig-gene rearrangements and the MLL-fusion gene) of both sisters into NSG mice resulted in a full-blown xenograft leukemia in animals bearing twin A but not twin B. Taken together, these data suggest that fatal relapse in twin A may be due to a quiescent stem cell like population kept in check by unknown mechanisms.Next, we performed gene expression analyses on xenografted leukemias from twin A (initial, leukemia amplified from remission BM, relapse) and from twin B (initial). STRING analysis of gene expression differences revealed that, amongst other findings related to cell cycle regulation and DNA-repair, twin A cells downregulated genes indicating a reduced interferon pathway activity (CXCL10, IFI30, TRAIL, STAT1, OAS1, MX1) and several genes connected to TYRO protein kinase binding protein (TYROBP) shown previously to regulate the activity of natural killer (NK) cells. This suggests that twin A cells may evade immunosurveillance as a mechanism of disease persistence. 51Chromium-release assays with IL-2 stimulated allogeneic NK cells from two healthy donors and the xenografted twin cells showed that twin A cells were significantly less sensitive to NK cell mediated lysis as compared to twin B cells. Whether the tumor cells also display differential susceptibility to antibody-dependent cell-mediated cytotoxicity is currently investigated.While the extensive genomic characterization of the twin leukemias is under way, we show evidence from xenograft experiments, gene expression profiles and functional in vitro experiments that dormant residual cells in MLL-rearranged ALL can evade immunosurveillance. These findings serve as a rationale to employ immunotherapeutic approaches in infant ALL patients in order to improve their dismal prognosis. Finally, we report the first monozygotic twin pair with MLL-rearranged ALL and discordant clinical outcomes. This provides a unique opportunity and a model to gain insight into the clonal composition of infant leukemia and the origins of leukemia relapse.Table 1:Patient characteristics of the MLL-ENL positive twin pair. WBC, white blood cells; BM, bone marrow; CNS, central nervous system; MRD, minimal residual disease.ParameterTwin ATwin BAge at diagnosis (days)98147WBC (initial)/µl334.000103.000Blasts (Peripheral Blood) %9791Blasts (BM) %9897CNS involvementNot determinedNegativeImmunophenotypePro-BPro-BCytogeneticst(11;19)t(11;19)Prednisone-ResponsePoorGoodBlasts (BM), day 15 %321MRD day 33NegativeNegativeMRD day 78Negative<10-4 *Event-free survival227 days>9 yearsTime to relapse227 daysn/aOverall survival390 days>9 years*low positive, not quantifiable DisclosuresNo relevant conflicts of interest to declare.

FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina.Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting.Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis.Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066).Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. DisclosuresNo relevant conflicts of interest to declare.

Enhanced Autophagy Promotes Survival of Peripheral Blast Cells from Murine Acute Myeloid Leukemia

Intensive chemotherapy for the treatment of leukemias inevitably provides cellular and metabolic stress to leukemic cells, leading to programmed cell death. In addition, anti-apoptotic property of leukemic cells could easily induce autophagy in response to anti-leukemic treatments and upregulation of autophagy is likely to contribute to the survival of leukemic cells by driving therapy resistance. Given that bone marrow (BM) supports leukemic cell proliferation by various types of stimuli from stromal cells or leukemic cells themselves, it is supposed that BM-occupying leukemic cells and circulating peripheral leukemic cells would have distinct difference in autophagic activity. However, comprehensive understanding of autophagic activity in leukemic cells has not been achieved so far. Here in this study, autophagic activities of leukemic cells in BM and peripheral blood (PB) from murine acute myeloid leukemia (AML) model driven by MLL-ENL fusion gene were evaluated. In the PB and BM from MLL-ENL AML mice, both mature (CD11b+c-Kit-) and immature (CD11b+c-Kit+) MLL-ENL+ leukemic cells showed no difference in apoptotic status by Annexin-V/DAPI staining regardless of c-Kit expression. By contrast, from cell cycle analysis, c-Kit- leukemic cells in the BM were found to have higher frequency of S/G2 phase than PB counterparts, indicating the proliferative potential of BM leukemic cells. When molecules of intracellular signaling pathways regarding proliferation and survival were assessed, components of MAPK and PI3K-mTOR signaling pathways such as Erk1/2, Akt, S6K, and S6 were highly phosphorylated in c-Kit- BM AML cells compared to c-Kit- PB AML cells, implying the importance of activated cytokine signaling in the BM of MLL-ENL AML. For the autophagic evaluation of PB and BM AML cells, MLL-ENL fusion gene was introduced into autophagy sensor mice, GFP-LC3 transgenic mice, enabling us to check autophagy by GFP. PB AML cells from these mice showed decreased GFP intensity compared to BM counterparts, which meant more activated degradation of autophagosome in peripheral AML cells. By western blotting analysis, PB AML cells had the enhanced conversion of LC3A-I to LC3A-II, indicating activation of autophagy. Furthermore, activation of stress responsive pathways such as nuclear localization of Foxo3a and enhanced phosphorylation of eIF2a was observed in c-Kit- PB AML cells. Interpretation of microarray data using public database (GSE9476 and GSE34577) comparing PB and BM cells from clinical AML samples revealed that PB AML cells have significantly higher expression of amino acid transporters. From these results, it is strongly suggested that peripheral AML cells have enhanced autophagic activity in vivo. To elucidate the functional role of activated autophagy in PB AML cells, MLL-ENL fusion gene was introduced into conditional Atg5 or Atg7 knockout mice (Atg5flox/flox or Atg7flox/flox), both of which are essential for autophagy. Interestingly, genetic deletion of Atg5 or Atg7 in irradiated recipients transplanted with Atg5flox/flox or Atg7flox/flox AML cells caused significant increase of apoptotic cells in peripheral leukemic cells and significant decrease of peripheral white blood cell (WBC) counts and donor cell engraftment in PB, whereas Atg5Δ/Δ or Atg7Δ/Δ MLL-ENL AML mice had neither prolonged survival nor reduced leukemia-initiating capacity in serial transplants. Similar to MLL-ENL AML model, genetic deletion of Atg7 in advanced phase of chronic myeloid leukemia (CML) induced by BCR-ABL plus NUP98-HOXA9 fusion genes caused decreased WBC counts and increased apoptosis in peripheral leukemic cells, indicating that circulating leukemic cells favor autophagy for their survival. It is of note that Atg7 deletion had no impact on apoptosis in normal CD11b+ myeloid fraction, implying that loss of Atg7 could spare normal myeloid cells. It remains to be seen what soluble factors could support the survival of AML cells in BM by suppressing autophagy, but our preclinical data provides the therapeutic potential of autophagy inhibition in the treatment of excessive peripheral leukocytosis. It is also highly expected that inhibition of autophagy could be more effective for BM AML cells when combined with chemotherapeutic agent to activate autophagy. DisclosuresSumitomo:Kyowa Hakko Kirin Co., Ltd.: Employment. Kurokawa:Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; SHIONOGI & CO., Ltd.: Consultancy; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Celgene K.K.: Consultancy, Speakers Bureau; Bristol-Myers Squibb Company: Consultancy, Research Funding, Speakers Bureau; Sanofi K.K.: Consultancy; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Astellas Pharma Inc., : Research Funding, Speakers Bureau; Dainippon Sumitomo Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Asahi Kasei Co.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; MSD K.K.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Co.,Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; Teijin Pharma Limited: Research Funding; Alexion Pharmaceuticals K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; GlaxoSmithKline K.K.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; Yakult Pharmaceutical Industry Co., Ltd.: Speakers Bureau; Ono Pharmaceutical Co.,Ltd.: Speakers Bureau; Miraca Holdings Inc.: Speakers Bureau; CSL Behring K.K.: Speakers Bureau.

Identifying Methylation Changes Driving Evolution of Relapse in MLL-Rearranged Acute Lymphoblastic Leukemias

IntroductionThe MLL gene is rearranged (MLL-r) in 80% of infants with B-lymphoblastic leukemia (B-ALL). MLL-r infant B-ALL has a poor prognosis, with 4-year event-free survival less than 45%. The typical pattern of failure in MLL-r infant ALL is successful remission induction followed by early relapse, suggesting rapid emergence and/or selection of one or more chemoresistant subclones. The markedly lower observed remission induction rates at relapse (<40%) vs. diagnosis (>90%) are consistent with this hypothesis. Genomic studies of MLL-r B-ALL have revealed a striking paucity of cooperating genomic abnormalities compared to other subsets of B-ALL, which suggests heritable epigenetic changes may drive leukemogenesis, chemoresistance and evolution of relapse in MLL-r B-ALL. MLL, its fusion partners and various components of its large complexes have functional domains with known or suspected epigenetic activity. Thus, MLL rearrangement in B-ALL may trigger chromatin modifications and DNA CpG methylation changes that interplay to disrupt normal gene transcription and expression. We hypothesized that infant MLL-r B-ALL cells dynamically acquire heritable DNA CpG methylation changes, some of which may contribute to chemoresistance and evolution of relapse. To test this hypothesis we performed whole-genome bisulfite sequencing (WGBS) using paired diagnosis-relapse (DX-RL) samples to identify methylation changes that may drive relapse.MethodsWe evaluated paired DX-RL specimens with >95% leukemic blasts from two infants with B-ALL harboring MLL-ENL fusions: A 4 month-old female who relapsed after 1 year (case a), and an 8 month-old male who relapsed after 5 months (case b). We prepared WGBS libraries and ran paired-end sequencing (2x100bp) using Illumina HiSeq at average 25X coverage. We used Bismark for aligning reads and calling CpG methylation states. We developed an analysis pipeline to convert CpG states into CpG haplotypes, detect methylation changes at the haplotype level between DX and RL, annotate methylation changes to genes and promoters, and perform gene set enrichment analysis (GSEA) on these genes.ResultsFirst, we filtered reads falling into repeated DNA. We then obtained methylation calls at 15.6/14.3 (DX/RL) million CpGs in case (a) and 15.5/15.6 (DX/RL) million CpGs in case (b). We extracted uniquely aligned reads covering 4 contiguous CpGs, which defined a “4 CpG-site”, or “site”. We required that each site in both DX and RL is covered by at least 4 reads, which yielded 26,747 and 85,174 sites for (a) and (b). We compared the methylation level at each site in DX versus RL (e.g. a “1-0-1-1” 4 CpG-site has a methylation level of 0.75). For (a), 165 sites showed a 50% increase in methylation level at relapse (e.g., from 0.2 to 0.7), and 468 showed a 50% decrease in methylation level at relapse. For (b) there were 605 increased and 57 decreased.The 633 methylation changes in (a) mapped to 175 genes (2kb upstream to transcription end site) and the 662 changes in (b) mapped to 135 genes. Some of these genes are known to be involved in MLL-r ALL [e.g., IKZF1, in (a) and TLX2 in (b)]. GSEA was performed independently using the 175 and 135 genes in (a) and (b), respectively. Interestingly, there was substantial overlap: The top 3 gene sets for (b) (FDR q-value ≤ 2.9E-8) were also within the top 9 sets for (a) (FDR ≤ 1.1E-5). Strikingly, all 3 of these sets involved components of the polycomb repressive complex 2 (PRC2) (1. EED target genes, 2. H3K27me3 bound genes and 3. Genes increased after EZH2 knockdown). Also in common was the KEGG gene set for antigen processing and presentation [FDR 3.4E-6 in (a) and 1.7E-4 in (b)].ConclusionWGBS allows an in-depth look at the epigenetic state of leukemic cell populations. While >98% of the methylome remained relatively stable between diagnosis and relapse (< 50% change), the hundreds of differentially methylated sites may be sufficient to influence expression of key genes and drive selection during the evolution of relapse. In addition, the ~15 million CpG methylation states are stably inherited yet variable, and comprise a rich source of information that can be used to identify evolving subclones, particularily in MLL-r ALL given its silent genomic landscape. Validation of the functional significance of the methylation changes using RNA-seq and identification of functionally important epigenetically-defined subclones is underway. DisclosuresNo relevant conflicts of interest to declare.

Sequential Gene Targeting to Make Chimeric Tumor Models with De Novo Chromosomal Abnormalities

The discovery of chromosomal translocations in leukemia/lymphoma and sarcomas presaged a widespread discovery in epithelial tumors. With the advent of new-generation whole-genome sequencing, many consistent chromosomal abnormalities have been described together with putative driver and passenger mutations. The multiple genetic changes required in mouse models to assess the interrelationship of abnormalities and other mutations are severe limitations. Here, we show that sequential gene targeting of embryonic stem cells can be used to yield progenitor cells to generate chimeric offspring carrying all the genetic changes needed for cell-specific cancer. Illustrating the technology, we show that MLL-ENL fusion is sufficient for lethal leukocytosis and proof of genome integrity comes from germline transmission of the sequentially targeted alleles. This accelerated technology leads to a reduction in mouse numbers (contributing significantly to the 3Rs), allows fluorescence tagging of cancer-initiating cells, and provides a flexible platform for interrogating the interaction of chromosomal abnormalities with mutations.

A Novel Conditional Mouse Model For MLL-ENL Induced Acute Leukemia

The t(11;19) translocation leading to the MLL-ENL fusion is recurrently found in pediatric and adult de novo and therapy related mixed-lineage acute leukemia and is often associated with a poor prognosis. Previous studies have shown that (retroviral) overexpression of MLL-ENL potently immortalizes bone marrow cells in vitro and induces a lethal acute myeloid leukemia (AML) in mice. To establish a mouse model that phenocopies more closely the human disease, we generated conditional transgenic mice in which the expression of MLL-ENL is controlled by doxycycline (DOX) through a stably integrated reverse tet-responsive transactivator (rtTA). Induction of MLL-ENL expression in newborn or adult mice resulted in a leukemic phenotype that phenocopied pre-B- and myeloid mixed lineage leukemia as observed in most patients with MLL-ENL. The diseased mice displayed excessive splenomegaly, massive lymph node as well as multiple organ infiltration by two co-existing types of blasts mostly expressing higher or lower levels of B220 and Gr1/Mac1 and similar levels of c-kit. Expression of the fusion gene and disease induction was DOX dosage dependent and reversible upon DOX removal. Despite significantly lower fusion gene expression levels as we observed in retroviral systems the median latency for the development of the disease in this model (104.3±16.9 days) was comparable to them (62±10.4 days) and much shorter than any of the previously reported MLL-ENL knock-in mouse models (> 1 year). Continuous ex vivo expression of MLL-ENL provided bone marrow and fetal liver hematopoietic cells with a strong self-renewal capacity and caused the accumulation of immature blast-like cells upon serial replating in methylcellulose cultures. In the presence of factors favoring myelopoiesis, like IL-3, DOX removal resulted in a complete differentiation towards the granulocytic-monocytic, lineages expressing high levels of Mac1/Gr-1, whereas IL-7 favored differentiation towards the B-cell lineage characterized by the expression of high levels of B220. In addition, MLL-ENL induced a DOX dependent aberrant self renewal capacity and a differentiation block in methylcellulose cultures of hematopoietic stem cells (Lin- c-kit+ Sca-1+, LSK) and various progenitors including common lymphoid progenitor (CLP) and granulocyte-macrophage progenitor (GMP) cells. Interestingly, MLL-ENL expression preferentially expanded LSK- rather than GMP-derived cells as assayed by growth curves in long-term (> 1 month) liquid cultures in the presence of cytokines in vitro. In line with this observation, in vivo, expression of MLL-ENL in long-term hematopoietic stem cells (LT-HSC) induced an aggressive mixed lineage leukemia characterized by the presence of two distinguishable populations of blasts, whereas induction in GMPs never induced a disease. These data suggest that MLL-ENL preferentially transforms hematopoietic stem cells rather than more differentiated progenitors. Thus, this novel transgenic mouse model for MLL-ENL induced acute leukemia closely recapitulates the human disease and combines the advantages of the existing knock-in and retroviral models. This model allowed us to demonstrate that in contrast to the MLL-AF9 fusion, that preferentially immortalizes GMPs, MLL-ENL preferentially transforms HSCs. We anticipate that our model will be a valuable tool to study the cellular origin and to search for and/or validate novel therapeutic targets for MLL-ENL induced acute leukemia. Disclosures:No relevant conflicts of interest to declare.

DNA Damage Response and Inflammatory Signaling Limit the MLL-ENL-Induced Leukemogenesis In Vivo

Activation of the MLL-ENL-ERtm oncogene initiates aberrant proliferation of myeloid progenitors. Here, we show induction of a fail-safe mechanism mediated by the DNA damage response (DDR) machinery that results in activation of the ATR/ATM-Chk1/Chk2-p53/p21(CIP1) checkpoint and cellular senescence at early stages of cellular transformation caused by a regulatable MLL-ENL-ERtm in mice. Furthermore, we identified the transcription program underlying this intrinsic anticancer barrier, and DDR-induced inflammatory regulators that fine-tune the signaling toward senescence, thereby modulating the fate of MLL-ENL-immortalized cells in a tissue-environment-dependent manner. Our results indicate that DDR is a rate-limiting event for acquisition of stem cell-like properties in MLL-ENL-ERtm-mediated transformation, as experimental inhibition of the barrier accelerated the transition to immature cell states and acute leukemia development.

Comparing Human Fetal Liver, Cord Blood and Adult Bone Marrow Stem/Progenitor Hematopoietic Cells As Cells of Origin of Human MLL Leukemias

Abstract 2956Acute leukemias in newborns, children, adults and elderly share the same name, but their genetic, biology and prognosis are significantly different. These differences may be explained by many factors including the nature of the recurrent genetic abnormalities, tolerance to chemotherapy, co-morbidities, etc. One noteworthy potential difference is the cell of origin in which the initial genetic event occurs, which can be a fetal liver, cord blood or adult bone marrow stem/progenitor cell. Genetically engineered mouse models of leukemias and gene expression studies on human acute lymphoblastic leukemias (ALL) suggest that leukemias developing in utero (i.e. in an early ontogenic cell of origin) are biologically distinct from child and adult leukemias. However, direct comparison of human hematopoietic stem/progenitor cells at different stages of ontogeny has not been tested experimentally.Over 70% of infant acute leukemias and 5–10% of adult leukemias exhibit rearrangements of the mixed-lineage leukemia gene (MLL). Therefore, MLL translocations are highly relevant oncogenes to investigate the leukemic potential of the different cells of origin. The MLL-ENL (MLL-MLLT1) fusion gene, from the t(11;19) (q23;p13.3), is one of the most frequent MLL fusion and can occur in infants and adults. It is found in both acute myeloid leukemias (AML) and ALL (both B-cell and T-cell ALL) and we have previously shown that it generates human B-ALL in immunodeficient mice when introduced into lineage depleted human cord blood cells using a retrovirus (Barabé et al, Science 2007).The same experimental design was used to study the cells of origin; CD34+ cells from human fetal liver (FL), cord blood (CB) and adult bone marrow (BM) were infected with either a retrovirus encoding MLL-ENL and an enhanced green fluorescent protein (EGFP) marker gene, or a control retrovirus encoding EGFP only, then injected into sub-lethally irradiated immunodeficient mice. After transduction with MLL-ENL, FL cells generated leukemias in 8/10 mice, CB cells in 6/6 mice and adult BM cells in only 2/7 mice, although four times more cells were transduced and injected. Adult BM cells were very difficult to infect with retroviruses and were clearly not as potent to reconstitute a human graft in immunodeficient mice in comparison to CB cells. All generated leukemias were B-ALL, regardless of the cell of origin. The leukemic phenotype (CD19+, HLA-DR+, CD20-, IgM-, IgG-, CD34 variable) was similar in all three cell types. Mice injected with MLL-ENL-transduced FL and adult BM cells that were not leukemic had a normal human graft but no EGFP cells. The frequency of leukemia-initiating cells (L-IC) was evaluated at 0.2–1% by limiting dilution in secondary mice and no significant differences were observed between the three cell types. Leukemic cells were harvested from the BM of primary mice and cultured in vitro under conditions supportive of both B-lymphoid and myeloid cells. All leukemias were able to grow in vitro and at least one culture derived from each cell type (FL, CB and adult BM) switched to a myeloid phenotype (monocytic morphology with CD33+ and loss of CD19), demonstrating that leukemias derived from all 3 cell types still have myeloid potential.Thus, our results suggest that MLL-ENL can generate leukemia in FL, CB and adult BM CD34+ cells. The lower penetrance of leukemia with adult BM cells is probably due to the difficulty to infect these cells and their limited reconstitution ability. MLL-ENL induces a leukemic program in all three cell types with similar clinical features, cell surface phenotype, frequency of L-IC and conserved bi-lineage potential (B and myeloid). Gene expression profiling and clonality studies are currently performed on leukemias generated from the 3 different cell types. So far, no significant differences between FL, CB and adult BM as cells of origin of MLL leukemias have been identified, suggesting that the nature of the oncogenic hit is probably more important than the ontogenic stage of the cell of origin. Disclosures:No relevant conflicts of interest to declare.

The first case of blastic plasmacytoid dendritic cell neoplasm with MLL-ENL rearrangement

Blastic plasmacytoid dendritic cell neoplasm (BPDCN), previusly referred to as blastic NK-cell lymphoma or CD4+/CD56+ ematodermic neoplasm, is an extremely rare hematologic maligancy which is characterized by co-expression of CD4 and CD56 ithout any other lineage-specific markers [1]. The disease was ategorized as a type of NK-cell lymphoma, but is now classified as rare subgroup of acute myeloid leukemia (AML) and related preursor myeloid neoplasms in the WHO classification 4th edition, ecause the precursor of plasmacytoid dendritic cells (pDCs) were dentified as the origin of the malignancy [2]. MLL gene, located at chromosome 11 band q23, is one of the most ommon targets for translocations associated with various types of dult and pediatric acute leukemia. MLL gene rearrangements are ound in 2.8% of AML patients and are relatively common in infant cute lymphoblastic leukemia and secondary AML. AML with MLL earrangement carries a relatively worse outcome [3]. Here we report a patient with BPDCN with MLL-ENL fusion gene. o our knowledge, this is the first report of BPDCN with MLL-ENL earrangement. A 45-year-old male without remarkable medical history visted our hospital because of multiple disseminated purpuric skin odules over the trunk, the limbs, and the face. Lymphadenopahy was not noted. Laboratory examinations revealed neutropenia Neutrophils, 0.492 × 109/L) and anemia (hemoglobin concentraion, 11.2 g/dL). Histological examination of the skin lesion showed iffuse infiltration of medium to large sized agranular blastic cells nto the dermis and the subcutaneous adipose tissue (Fig. 1). Bone arrow and peripheral blood infiltration were also observed. The eoplastic cells were negative for peroxidase, and flow cytomery revealed that the neoplastic cells were positive for CD4, CD11c, D33, CD45RA, CD56, CD68, CD117, CD123, HLA-DR and negative or CD3, CD5, CD7, CD8, CD10, CD11b, CD13, CD19, CD20, CD34,

Genome Wide Location Analysis Reveals Deregulated MicroRNA Genes In MLL Rearragned Leukemic Genome

AbstractAbstract 2507The MLL (mixed-lineage leukemia) gene encodes a histone methyltransferase that is critical in maintaining gene expression during hematopoiesis. Chromosomal translocations disrupting MLL often leads to the creation of MLL fusion genes that act as potent drivers of acute leukemia. MLL fusion proteins are oncogenic transcription factors that activate the expression of downstream target genes. Expression profiling on patient primary samples and established mouse models has revealed hundreds of protein coding genes which are either up-regulated or down-regulated in MLL associated leukemias. Persistent coexpression of two of those genes, HoxA9 and Meis1, is essential for the initiation and maintenance of MLL leukemia. Our studies have also shown strong association of a microRNA (miRNA) expression signature with MLL- rearranged leukemia, and the expression of several miRNAs were under the control of MLL wild type and fusion proteins. Although profiling of miRNA expression has been reported, the mechanisms underlying deregulated miRNA expression in MLL associated leukemia are poorly understood. Given the role of miRNA as a global suppressor of mRNA gene expression, we hypothesized that the expression of miRNAs could be directly activated by MLL fusion and/or wild type proteins upon MLL gene rearrangement and subsequently down-regulate pertinent target mRNAs to contribute to leukemogenesis. To test our hypothesis in a systematic way, we examined an inducible MLL-ENL-ER transformed mouse cell line, which grow as myeloblastic cells in the presence of MLL-ENL, and differentiate into neutrophils upon inactivation of the fusion protein. Using chromatin immunoprecipitation assay followed by next generation sequencing (ChIP-Seq), we determined whole genome MLL binding pattern in this cellular model. Upon activation of MLL-ENL, 24 miRNAs showed a significant increase in the level of MLL binding (FDR<0.25), suggesting that those genes are directly bound by the MLL-ENL fusion protein. To explore the impact of MLL fusion protein on miRNA and mRNA gene regulation, we performed whole genome expression analysis using Affymetrix mouse microarray in the presence or absence of MLL-ENL. Upon induction of MLL-ENL, the expression levels of 38 miRNAs (out of 609 tiled on the array) were increased, and 57 of 7858 expressed protein-coding genes were down-regulated. An integrative analysis of MLL binding and mRNA/miRNA expression profiling data showed that transcription of three miRNAs were activated upon binding of MLL-ENL, and ten protein coding genes are potential targets of these miRNAs. We are currently exploring the role of these three miRNAs and their respective mRNA target genes in leukemogenesis using in vitro and in vivo models. Taken together, our data suggest that MLL fusion protein may play an important role in leukemogenesis by promoting miRNA transcription, which subsequently inhibit the expression of critical mRNA target genes. Our study provides a basis to further explore the regulatory network involving MLL fusion protein and its key miRNA target genes in the leukemic genome. Disclosures:No relevant conflicts of interest to declare.

C-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis

Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear. Here we have demonstrated that c-Myb is recruited to the MLL histone methyl transferase complex by menin, a protein important for MLL-associated leukemic transformation, and that it contributes substantially to MLL-mediated methylation of histone H3 at lysine 4 (H3K4). Silencing MYB in human leukemic cell lines and primary patient material evoked a global decrease in H3K4 methylation, an unexpected decrease in HOXA9 and MEIS1 gene expression, and decreased MLL and menin occupancy in the HOXA9 gene locus. This decreased occupancy was associated with a diminished ability of an MLL-ENL fusion protein to transform normal mouse hematopoietic cells. Previous studies have shown that MYB expression is regulated by Hoxa9 and Meis1, indicating the existence of an autoregulatory feedback loop. The finding that c-Myb has the ability to direct epigenetic marks, along with its participation in an autoregulatory feedback loop with genes known to transform hematopoietic cells, lends mechanistic and translationally relevant insight into its role in MLL-associated leukemogenesis.

Effects of the Flt3 ITD on response to chemotherapy in a murine model of acute myeloid leukemia

7060 Background: Acute myeloid leukemia (AML) is an aggressive, genetically heterogeneous malignancy. This heterogeneity is thought to underlie the divergent responses to treatment observed and contribute to relapse. The Flt3 receptor tyrosine kinase containing an internal tandem duplication (Flt3 ITD) is a common mutation in AML and associated with a poor prognosis; however, its effect on chemotherapy response is currently unknown. Methods: Murine AML was generated by retroviral transduction of an MLL-ENL fusion protein into fetal liver cells and subsequent transplantation into syngeneic mice. Blasts were harvested from moribund animals and myeloid lineage confirmed by immunophenotyping. Flt3 ITD sequence was inserted into a GFP tagged vector. Blasts were infected with Flt3 ITD or control virus. For competition assays mixtures of infected and uninfected blasts were exposed to chemotherapy for 72 hours. Among the remaining viable cells, percentages of infected blasts were determined by flow cytometry. For in vivo competition assays animals were injected with mixtures of blasts, treated with chemotherapy and percentages of Flt3 ITD positive blasts as well as total leukemic burden in femoral bone marrow was assessed. Results: Presence of the Flt3 ITD confers an advantage to doxorubicin (dox) in vitro. After exposure to dox at 5ng/mL more infected blasts remained viable when compared to controls (p = 0.0001). Presence of the Flt3 ITD increased sensitivity to cytarabine (Ara-C). After exposure to 50 nM Ara-C fewer Flt3 ITD blasts remained viable (p = 0.0293). Flt3 ITD confers sensitivity to Ara-C in vivo. Bl6 mice were injected with 1x106 blasts, 10% containing Flt3 ITD. After 12 days mice were treated for 5 days with either Ara-C at 200mg/kg, dox at 3mg/kg or observation. Ara-C treated animals had fewer Flt3 ITD blasts than controls (p = 0.0030) or dox treated animals (p &lt; 0.0001) and lower leukemic burden (p &lt; 0.0001). In contrast there was no difference in leukemic burden between Ara-C treated, dox treated or control animals in AML without Flt3 ITD (p = 0.2833). Conclusions: The Flt3 ITD confers resistance to doxorubicin in vitro and sensitivity to Ara-C in vitro and in vivo. These results suggest AML patients with Flt3 ITD may benefit more from high-dose cytarabine regimens then anthracyclines. No significant financial relationships to disclose.

Myeloid or mixed-lineage leukemia-ENL fusion protein (MLL-ENL fusion protein)

Myeloid or mixed-lineage leukemia-ENL fusion protein (MLL-ENL fusion protein)

Collaboration of the Meningioma 1 (MN1) Oncogene with MLL-Fusions in Pediatric Leukemia

Expression of meningioma 1 (MN1) has been proposed to be a negative prognostic marker in adult acute myeloid leukemia (AML). In pediatric leukemia, we found overexpression of MN1 in 53 of 88 cases: whereas no MN1 expression was detected in T-cell acute lymphoblastic leukemia (T-ALL), significant amounts of MN1 were found in immature B-cell ALL and most cases of infant leukemia. Interestingly, 17 of 19 cases harboring fusion genes involving the mixed-lineage leukemia (MLL-X) gene showed elevated MN1 expression. Lentiviral siRNA mediated MN1 knock-down resulted in cell cycle arrest and impaired clonogenic growth of 3 MLL-X-positive human leukemia cell lines overexpressing MN1 (THP-1, RS4;11, MOLM-13). In a mouse model of MLL-ENL-induced leukemia we found MN1 to be overexpressed as a consequence of provirus integration. Strikingly co-expression of MN1 with MLL-ENL resulted in significantly reduced latency for induction of an AML phenotype in mice suggesting functional cooperation. Immunophenotyping and secondary transplant experiments suggested that MN1 overexpression seems to expand the L-GMP cell population targeted by the MLL-ENL fusion. Gene expression profiling allowed defining a number of potential MN1 hematopoietic targets. Upregulation of CD34, FLT3, HLF, or DLK1 was validated in bone marrow transiently overexpressing MN1, in MN1-induced mouse acute myeloid leukemia, as well as in pediatric leukemias with elevated MN1 levels. Our work shows that MN1 is overexpressed in a significant fraction of pediatric acute leukemia, is essential for growth of leukemic cells, and that MN1 can act as a cooperating oncogene with MLL-ENL most probably through modification of a distinct gene expression program that leads to expansion of a leukemic progenitor population targeted by MLL-fusion genes.

Discordance of MLL-Rearranged (MLL-R) Infant ALL in Monozygotic Twins with Spontaneous Clearance of Preleukemic Clone in Unaffected Twin

The concordance rate for acute leukemia in infant monozygotic twins is estimated to be between 50 and 100%. The leukemic clone is thought to develop in one twin and transfer to the other twin via placental anastamoses. This hypothesis is supported by studies documenting identical, clonal, non-constitutional fusion sequences in concordant leukemias of monozygotic twins. For the ~80% of infant leukemias that are MLL-R+, the twin concordance rate is thought to be 100%, with a very short latency period between the diagnosis of the two twins. This suggests that either the MLL fusion is sufficient to cause leukemia, or it promotes the rapid acquisition of additional mutations required for the development of overt disease.We report a case of discordance in an infant monozygotic twin pair in which twin A presented with MLL-ENL+ ALL and twin B, who was transiently RT-PCR+ for MLL-ENL in bone marrow (BM) and blood, remains healthy 2.5 years later. Twin A was diagnosed at age 9 months (T=0) with CD10(−) pre-B ALL, cytogenetics t(11;19)(q23;p13.3), FISH+ for MLL-R, RT-PCR+ for MLL-ENL fusion transcript. Twin B was healthy with normal CBC and physical exam (PE). At T+7wks, twin B's CBC and PE remained normal, but RT-PCR on peripheral blood mononuclear cells (PBMNC) was MLL-ENL+. Three weeks later (T+10wks), BM aspirate was also RT-PCR+ for MLL-ENL. Morphology, FISH and cytogenetics were normal, as were CBC and PE. Two weeks later (T+12wks), twin B developed neutropenia (WBC= 2,880/ml; ANC=461) and thrombocytopenia (29,000/ml) one week after a viral URI (fever, rhinorrhea, cough, diffuse erythematous rash). He had no lymphadenopathy or hepatosplenomegaly on PE. Review of his PB smear confirmed the cytopenias and revealed atypical lymphocytosis and activated monocytes compatible with a viral process, but no lymphoblasts. Large platelets were noted, suggesting a diagnosis of acute ITP. No treatment was given. Repeat CBC 2 days later showed recovery of counts with WBC of 6,390/ml (ANC=1150) and platelets of 117,000/ml. RT-PCR of PBMNC was negative for MLL-ENL; BM aspirate was not performed. One week later, CBC had normalized. Over the next 4 months, monthly CBC and PE were normal, and monthly RT-PCR of PBMNC were negative. Twin B remains healthy with no clinical or laboratory evidence of leukemia at T+2.5yrs.Twin A was treated according to COG protocol P9407. End-induction BM showed morphologic remission, normal cytogenetics and negative FISH, although RT-PCR remained MLL-ENL+. End-consolidation BM showed continued remission, and was negative for MLL-ENL by cytogenetics, FISH and RT-PCR. At the end of P9407 therapy, an additional 1 year of maintenance therapy was given (total duration of therapy = 2 years). Two months after completion of therapy (26 months from diagnosis), twin A experienced an isolated testicular relapse of CD10(−) pre-B ALL, MLL-ENL+. He received reinduction chemotherapy and testicular radiation and is currently receiving post-induction chemotherapy.To our knowledge, this is the first report of discordant MLL-R leukemia in infant monozygotic twins, and the first report of documented spontaneous clearance of a detectable preleukemic clone in a healthy “twin B”. There are reports of twins discordant for MLL-R leukemia, but placental status was either unknown, dichorionic, or the leukemia developed in the first twin at 5 years of age and was likely postnatally acquired. The temporal association of the disappearance of the preleukemic clone in twin B with an episode of viral-induced, self-limited thrombocytopenia and neutropenia suggests the possibility that the immune process that mediated the cytopenias may also have been responsible for clearance of the preleukemic clone.This report confirms prior reports that clonal MLL-R cells are shared between monozygotic twins. However, this case presents evidence against the hypothesis that MLL rearrangements (specifically the MLL-ENL fusion) are sufficient for the development of overt leukemia. It is possible that a viral-induced autoimmune reaction may have aborted the progression of twin B's MLL-ENL+ preleukemic clone to overt leukemia by eradicating the clone prior to the acquisition of additional mutations. It is also possible that twin B's preleukemic clone persists at a level beneath the sensitivity of our RT-PCR assay. A less speculative conclusion is that short latency concordance of infant MLL-R leukemia in monozygotic twins is not universal.

Acute Myeloid Leukemia Induced by MLL-ENL Is Cured by Oncogene Ablation despite Acquisition of Complex Genetic Abnormalities

Chromosomal translocations involving the Mixed-Lineage-Leukemia (MLL) gene on chromosome 11q23 are frequent in infant acute leukemia and give rise to the formation of MLL-fusion genes. Several studies have addressed the importance of MLL-fusion activity for the initiation and maintenance of hematopoietic transformation. However, the dependence of established leukemias on MLL-fusion activity has not been previously addressed. We have developed a model for conditional expression of MLL-ENL in hematopoietic progenitor cells, in which expression of the fusion oncogene is turned off by doxycycline. In this study, immortalized myeloid cells conditionally or constitutively expressing the MLL-ENL fusion gene were used to induce acute myeloid leukemia (AML) in vivo. Primary recipients developed AML with a mean latency of 81.4 (±4.8) days. Secondary recipients developed AML with much shorter latencies than primary recipients regardless of whether the leukemic cells were freshly transplanted (26.8 (±6.8) days) or cultured in vitro for one month prior to transplantation (18 (±3.9) days). Genetic analysis revealed that some leukemic cells had acquired gross chromosomal abnormalities such as trisomy 6 or gains and losses of chromosome regions, which were not detected in the immortalised cells from which they were derived. Despite the acquisition of additional genetic abnormalities, the leukemic cells remained dependent upon MLL-ENL expression in vitro and in vivo. The leukemic cells terminally differentiated into neutrophils upon doxycycline treatment in vitro and established leukemias regressed following administration of doxycycline to recipient mice in their drinking water. Leukemic regression was accompanied by the complete loss of leukemic cells from the peripheral blood and differentiation of leukemic cells in the spleen. In 7 out of 34 doxycycline treated mice, remission was not sustained and the leukemias relapsed. However, most of these were shown to have acquired constitutive expression of MLL-ENL. This study demonstrates that leukemic cells are addicted to MLL-ENL expression and suggests that targeting the transcriptional/signalling networks established by MLL-fusion oncogenes in patients with 11q23 rearrangements would be a major therapeutic advance.

Modeling Chromosomal Translocations Using Conditional Alleles to Recapitulate Initiating Events in Human Leukemias

Recurrent reciprocal chromosomal translocations are present in more than 50% of leukemias. A deeper understanding of how they affect cancer initiation is essential for evaluating the origins of cancer and the potential for therapy based on the translocation products. Mouse models of chromosomal translocations are required for this. Here we summarize three methodologies developed in our laboratory to model chromosomal translocations (knock-in, translocator, and invertor methods). We have used these models to study leukemias caused by fusions of the mixed lineage leukemia (MLL) gene and the Ews-ERG fusion gene to evaluate oncogenicity and elucidate some general principles about translocation products. We show that MLL fusions have the capacity to cause hematopoietic tumors only if expressed in permissive cells and that the Mll-Enl fusion can cause lineage reassignment if the chromosomal translocation occurs in lineage noncommitted progenitors. The leukemia-initiating cells generated by Mll fusions or by Ews-ERG fusion can be committed cells within the hematopoietic pathway. Our translocation mimic models are applicable to any human reciprocal chromosomal translocation.

A role for the MLL fusion partner ENL in transcriptional elongation and chromatin modification

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

The MLL Recombinome of Adult ALL - Results from the GMALL Study Group.

The pro B and the CD10-negative pre B immunophenotypes are found in approximately 12% and 2–3% of adult acute lymphoblastic leukemia cases, respectively. Both are characterized by a high prevalence of MLL aberrations. MLL fusion genes define a high risk group of ALL patients and they have proven useful as target structures for assessing minimal residual disease. It has been a matter of debate whether MLL aberrations are of prognostic significance within the group of pro B ALL patients. To better characterize the MLL recombinome in adult ALL we have prospectively and retrospectively investigated BCR-ABL-negative pro B (N=152) and CD10-negative pre-B ALL (N=31) patient samples obtained within the framework of the GMALL study group between 2001 and 2007. We used RT-PCR to detect MLL-AF4, MLL-ENL and MLL-AF9 and other MLL fusion genes. An inverse long PCR method was used to identify the exact chromosomal breakpoints in the MLL gene or unknown MLL fusions.Results: RT-PCR analysis revealed a MLL-AF4 fusion in 20/31 (64.5%) and a MLL-ENL fusion in 2/31 (6.5%) of CD10-negative pre-B cases. A MLL-AF4 fusion was detected in 101/151 (67.3%) and a MLL-ENL fusion in 9/151 (6.0%) of pro B cases. Additionally, two pro B patients showed a MLL-AF9 and MLL-CXXC6 fusion, respectively. The relative frequency of MLL aberrations was higher (50/68 = 73.5%) in those pro B patients with immunophenotypic coexpression of myeloid markers than in those without myeloid coexpression (41/84 = 48.8%). The chromosomal breakpoints in the MLL gene and the respective partner gene were determined on the genomic level in 91 patients. The distribution of chromosomal breakpoints in the MLL gene was the following: 1 in intron 7, 1 in intron 8, 33 in exon/intron 9, 27 in exon/intron 10, 28 in intron/exon 11, 1 in intron 12. These data illustrate the spectrum and relative frequency of MLL fusion genes in adult pro B and CD10-negative pre B ALL. The chromosomal breakpoint data provide insight into the molecular mechanisms leading to MLL aberrations in adult ALL. Chromosomal breakpoint sequences are powerful molecular markers for assessing MRD in individual patients as has been shown in the context of the GMALL MRD evaluation trials.