MLL Gene Rearrangement Research Articles

Etoposideによると思われるt (9;11) とMLL遺伝子の再構成が認められた二次性白血病の1例

Etoposideによると思われるt (9;11) とMLL遺伝子の再構成が認められた二次性白血病の1例

Feasibility of gene-immunotherapy using WT1-specific T-cell receptor gene transfer for infant acute lymphoblastic leukemia with MLL gene rearrangement

Feasibility of gene-immunotherapy using WT1-specific T-cell receptor gene transfer for infant acute lymphoblastic leukemia with MLL gene rearrangement

Analysis of the Role of Hematopoietic Stem-Cell Transplantation in Infants With Acute Lymphoblastic Leukemia in First Remission and MLL Gene Rearrangements: A Report From the Children's Oncology Group

Analysis of the Role of Hematopoietic Stem-Cell Transplantation in Infants With Acute Lymphoblastic Leukemia in First Remission and MLL Gene Rearrangements: A Report From the Children's Oncology Group

Analysis of the Role of Hematopoietic Stem-Cell Transplantation in Infants With Acute Lymphoblastic Leukemia in First Remission and MLL Gene Rearrangements: A Report From the Children's Oncology Group

Although the majority of children with acute lymphoblastic leukemia (ALL) are cured with current therapy, the event-free survival (EFS) of infants with ALL, particularly those with mixed lineage leukemia (MLL) gene rearrangements, is only 30% to 40%. Relapse has been the major source of treatment failure for these patients. The parallel Children's Cancer Group (CCG) 1953 and Pediatric Oncology Group (POG) 9407 studies were designed to test the hypothesis that more intensive therapy, including dose intensification of chemotherapy, and hematopoietic stem-cell transplantation (HSCT) would improve the outcome for this group of patients. One hundred eighty-nine infants (CCG 1953, n = 115; POG 9407, n = 74) were enrolled between October 1996 and August 2000. For infants with the MLL gene rearrangement and an appropriate donor, HSCT was the preferred treatment on CCG 1953 and investigator option on POG 9407 after completion of the second phase of therapy. Fifty-three infants underwent HSCT. The 5-year EFS rate was 48.8% (95% CI, 33.9% to 63.7%) in patients who received HSCT and 48.7% (95% CI, 33.8% to 63.6%) in patients treated with chemotherapy alone (P = .60). Transplantation outcomes were not affected by the preparatory regimen or donor source. Our data suggest that routine use of HSCT for infants with MLL-rearranged ALL is not indicated. However, limited by small numbers, this study should not be considered the definitive answer to this question.

Early Phase Administration of Hematopoietic Stem Cell Transplantation to Prevent Early Relapse for Infants with Acute Lymphoblastic Leukemia and MLL Gene Rearrangement; a Report From the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) Trial MLL03

AbstractAbstract 529 Background:Infants with acute lymphoblastic leukemia (ALL) and MLL gene rearrangement (MLL-R) confer poor prognosis with approximately 40% probability of long term event-free survival (EFS). The major cause of treatment failure is leukemia relapse which usually occur very early after achieving the first remission (1CR). Prevention of early relapse is a key strategy to improve the outcome of MLL-R infants with ALL. On the basis of promising results in the previous MLL98 study with post-transplant EFS rate of 64.4% among the 29/44 patients transplanted in 1CR, we conducted a multi-center non-randomized trial for infants with MLL-R ALL to test an efficacy of intensive chemotherapy followed by early phase (≤ 4 months after achieving 1CR) hematopoietic stem cell transplantation (HSCT). Patients and Methods:Infants with ALL aged less than 12 months were registered in JPLSG MLL03 study if MLL gene rearrangements were confirmed by Southern Blot analysis. After 7-day prednisolone monotherapy, all patients received induction therapy consisted of dexamethasone, vincristine, doxorubicin, cyclophosphamide, cytarabine, etoposide, and triple intrathecal injections, followed by two intensification courses including high-dose methotrexate and high-dose cytarabine. L-asparaginase was not included. If 1CR was achieved, HSCT either from HLA ≥5/6 phenotypically matched related donor or ≥4/6 matched unrelated cord blood donor was carried out. Conditioning regimen consisted of busulfan, which was dose-adjusted based on individual pharmacokinetics, etoposide, and cyclophosphamide. Results:A total of 63 infants with ALL and MLL-R were registered in MLL03 study between Feb/2004 and Jan/2009. Patient characteristics showed high proportion of young infants; 22 (35 %) aged 0 – 90 days, 21 (33 %) aged 91 – 180 days, and 20 (32 %) aged 181 days or higher. Remission status was evaluated after 2 chemotherapy courses; 49 achieved 1CR, 4 failed to achieve 1CR, and 3 died of infectious complications. Seven patients were removed from the study; 2 for lineage switch to myeloid leukemia, 1 for guardians' refusal, 2 for severe adverse events (AEs), and 2 for protocol violations. Grade 3 and 4 non-hematological AEs during induction phase, such as infections (73 %), tumor lysis syndrome (TLS, 40 %), and liver transaminase elevation (44 %) were relatively common, especially in young infants. Forty-four out of 49 cases who achieved remission were able to undergo HSCT in 1CR; 32 cases from unrelated cord blood and 11 cases from related donor (1 unknown). All the recipients achieved stable engraftment. Ten patients developed grade II – IV acute GVHD and 8 developed chronic GVHD (only 1 with extensive type). Most of the post-HSCT events were leukemia relapses (18 post-HSCT relapses), and two non-relapse death were observed; one of transfusion-related acute lung injury (TRALI) and one of sudden death for unknown cause. Finally, probability of EFS in 18 months was 54.5 % (95 % confidence interval [CI], 41.3 % – 66.0 %) and overall survival in 18 months was 80.8 % (95 % CI, 68.7 % - 88.6 %). Young age (< 90 days old) and CNS leukemia at diagnosis were poor prognostic factors. Conclusions:The strategy of treating MLL-R ALL infants with intensive chemotherapy followed by early phase HSCT was feasible and prevention of early relapse was successful. However, significant improvement in EFS is unlikely compared with that of the previous MLL98 study. This may be due to, 1) high proportion of younger infants in the present study who are both high risk of relapse and severe treatment-related toxicities, 2) lack of sufficient intensity of pre-transplant chemotherapy in the present study to eradicate leukemia burden, and 3) limited efficacy of the strategy to treat MLL-R infant ALL with conventional chemotherapy and HSCT. Disclosures:No relevant conflicts of interest to declare.

Central Nervous System (CNS) Involvement In Pediatric Relapsed Acute Myeloid Leukemia: Results and Lessons From Study Relapsed AML 2001/01

AbstractAbstract 184Acute Myeloid Leukemia - Therapy, excluding Transplantation: Pediatric and Adult AML Therapy Introduction:With improving initial antileukemic therapy, CNS disease might become more important in AML. We therefore evaluated the incidence of CNS involvement in a large series of children with first relapsed AML. In addition, clinical and biological features of children with and without CNS involvement at relapse were compared, and finally the prognostic significance of CNS involvement at relapse was studied. Materials and Methods:Patients were selected from those registered in the setting of study Relapsed AML 2001/01 (ISRCTN code 94206677), based on having first relapsed AML and precise information on the location of the relapse, and on the type of CNS involvement. The latter was distinguished in asymptomatic and symptomatic disease. The protocol prescribed intensive intrathecal triple chemotherapy in case of CNS disease: first dose immediately before start of reinduction course 1. Second and subsequent doses every 7 days until 1 week after complete blast clearance of the CSF or disappearance of radiological abnormalities. Then, 2 more doses were given, one immediately before the start of the second reinduction course, and the other at the start of consolidation treatment. Cranial radiotherapy was not generally recommended and was actually used in 18% of patients with CNS relapse. Systemic therapy consisted of FLAG with or without liposomal daunorubicin (1:1 randomisation), followed by FLAG and allogeneic stem cell transplantation. This clinical study also enrolled patients with a combined relapse, or an isolated extramedullary relapse, or a bone marow relapse (isolated or combined) with <20% blasts in the BM, and these patients were also eligible for randomisation. The study was closed for enrollment in April 2009, and median follow-up for patients at risk is well above 3 years. Results:Out of 477 patients, 45 (9.4%) had CNS relapse, which percentage did not differ significantly between individual study groups. The far majority (41/45) of these patients had a combined relapse, only 4 patients had isolated CNS relapse. All 4 isolated relapses concerned early relapse, as compared to 66% of patients with a combined relapse including the CNS and to 48% of patients with first relapsed AML not involving the CNS (p-trend = 0.004). For further analyses, isolated and combined relapses involving the CNS were combined. Patients with CNS relapse were younger at relapse, mean 5.6 (SD 5.2) vs 9.7 (SD 5.3) years, p<0.0001. There was a trend for a higher percentage of males among those with CNS involvement, 71 vs 57%, p=0.07, and a trend for a higher WBC (mean 22.1 vs 13.4 × 109/l, p=0.09). Patients with CNS relapse more often had AML FAB type M5 (48 vs 16%, p<0.001), and more often MLL gene rearrangements (26 vs 11%, p=0.049) than patients with non-CNS relapse. Clinical outcome did not differ significantly between both groups, and patients with relapsed AML and CNS involvement had a 58% CR2 rate (vs 64% for the remaining patients), and a 4-years pOS of 32% (SE 8%) vs 37% (SE 2%) for the remaining patients. The majority of events within patients with relapsed AML and CNS involvement concerned refractory disease (12/45 patients), while early death occurred in 7 patients, death in CR in 3 patients, and 9 patients developed a 2nd relapse (in 3/9 patients again involving the CNS). Relapsed patients with CNS involvement had a higher rate of second complete remission when treated with liposomal daunorubicin, than if treated with FLAG only (82 v 38%, p=0.019), but overall survival was not significantly different between both treatment arms in this subgroup of patients. Conclusion:Isolated CNS relapse is rare in pediatric AML, but CNS involvement is observed in more than 9% of patients with first relapsed AML. CNS involvement is more frequent in younger patients, males and those with a high WBC at relapse. Biologically, patients with CNS involvement at relapse more frequently have acute monoblastic leukemia and MLL gene rearrangement, than patients with non-CNS relapse. Despite CNS disease at relapse, second complete remission was achieved in more than half of patients, and long-term survival was achieved in about one third of patients. Thus, intensive salvage treatment of pediatric patients with relapsed AML and CNS involvement is justified. Prevention of CNS relapse by improved therapy for newly diagnosed AML is warranted, with special attention to several subgroups which seem at higher risk for CNS relapse. Disclosures:Off Label Use: Liposomal daunorubicin in pediatric relapsed AML.

Myeloperoxidase-Dependent Oxidation of Etoposide in Human Myeloid Progenitor CD34+Cells

Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34(+) myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34(+) cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34(+) cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34(+) cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.

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.

Differential Gene and MicroRNA Expression In a HOXA9-MEIS1 Model of Leukemia

AbstractAbstract 64Altered expression of the HOX-MEIS axis is associated with leukemia, particularly AML, where it plays a role in increased proliferation and impaired differentiation. Particular focus has been placed on the role of HOX-MEIS in leukemia with MLL gene rearrangements and MLL-fusion induced models that represent this poor prognostic subgroup. However more recently, we and others have identified altered expression of HOX-MEIS in AML with normal karyotype (AML-NK) that accounts for the vast majority of patients with this disease. The prognostic outcome of this large subgroup of patients is varied and may depend on other molecular aberrations such as NPM-1 mutation, FLT3-ITD or CEBPB status. A murine model of leukemia using co-overexpression of HOXA9-MEIS1 was developed to investigate their role in an AML-NK setting. Expression levels of the collaborating oncogenes was limited by combination of a bicistronic ires targeting vector, low titre retroviral infection and limiting dilution assay (LDA). HOXA9 and MEIS1 expression was measured using quantitiative PCR (qPCR) and levels obtained in the mouse were similar to those observed for AML-NK patients. The resultant leukemia was transplantable leading to premature death in non-irradiated recipient mice within 30 days (104 cells transplanted). Observed pathology included circulating blasts in the periphery, splenomegaly, tissue infiltration of blast cells (kidney, lung, liver) and enlarged lymph nodes, indicative of leukemia. Blast cells were identified as Kit+, Gr-1+, Mac-1+, CD150lo consistent with a myeloid progenitor phenotype. To evaluate downstream events associated with the leukaemic phenotype TaqMan-based Low Density Arrays (TLDAs) and Open Arrays (Biotrove) were used to obtain gene and microRNA (mIR) expression profiles. The majority of genes assayed 699/821 were either not expressed or showed no appreciable change in expression (ΔΔCT < 2) between the leukaemic and normal bone marrow control samples. Of those genes that showed a measurable change in expression, 111/122 assessed in the pluripotent stem cell, transcription factor and immune arrays were downregulated in the HOXA9-MEIS1 induced leukemia samples compared to control mice (n=4 per group). Individual assays were designed for a subset of candidate genes and validated against the TLDA results. Several genes identified as consistently downregulated in the leukaemic cells, including Smad1 and E2f7, were associated with negative regulation of cell proliferation using DAVID Bioinformatics Resources 6.7. Consistent increased expression of HOXA9 and MEIS1 was confirmed by individual assays as was elevated Elf5, Cd34 and Cd28 levels. mIRs regulate the expression of protein-coding genes in part through cleavage of targeted transcripts by partial or complete base pairing. A growing number of mIRs are differentially regulated in normal and malignant hematopoiesis, where increased mIR levels are associated with downregulation of target gene subsets. mIR expression profiles were compared between leukemic and control mice to investigate their association with the global downregulation of gene expression observed in the leukaemic model. Approximately 16% (48/303) of the mIRs showed measurable changes in expression levels (ΔΔCT >2) between the two models, with >70% (34/48) demonstrating increased expression, up to 360-fold, in the leukemia samples. Candidate mIRs were further examined using the mirDB and/or miRecords target prediction databases. Several of the leukaemia-associated upreglated mIRs putatively target members of the validated subset of downregulated genes e.g. mIR-466c and mIR-181c are predicted to target Smad1 and E2F7 respectively. This study suggests that HOXA9-MEIS1 leukemogenesis occurs in part due to increased expression of a subset of mIRs that are predicted to target genes associated with inhibition of proliferation in an AML-NK model. Disclosures:No relevant conflicts of interest to declare.

Asparaginase-Associated Myelosuppression and Effects on Dosing of Other Chemotherapeutic Agents In Children and Adolescents with Acute Lymphoblastic Leukemia

AbstractAbstract 3249 Background:L-asparaginase (ASP) is a universal component of treatment for childhood acute lymphoblastic leukemia (ALL). It is associated with multiple toxicities, including allergy, pancreatitis, and thrombosis. It is not thought to have a significant direct myelosuppressive effect, but its impact on the ability to tolerate other chemotherapeutic agents has not been well-characterized. Patients and Methods:Between 2005–2010, 61 patients (pts) were diagnosed with Standard Risk (SR) ALL at DFCI/Children's Hospital Boston and treated on DFCI ALL Consortium Protocol 05-01. SR pts met all of the following criteria: 1 to <10 years of age; highest pretreatment WBC < 50,000/mm3; absence of central nervous system leukemia (ie, CNS-1 or -2, only); absence of T-cell markers; absence of t(9;22), MLL gene rearrangements, and hypodiploidy; and low minimal residual disease (MRD) levels at the end of the first 4 weeks of treatment. Beginning at week 7, all SR pts began a 30-week (wk) Consolidation phase, consisting of every 21-day cycles of vincristine (day 1), dexamethasone (days 1–5), 6-mercaptopurine (6MP) (days 1–14), and methotrexate (MTX) (days 1, 8 and 15), and also including 30 consecutive wks of ASP (either weekly IM E.coli ASP 25000 IU/m2 or every 2-wk IV PEG ASP 2500 IU/m2). The Continuation phase followed Consolidation, and consisted of identical systemic and intrathecal (IT) chemotherapy, except without any ASP. Starting dose of 6MP was 50 mg/m2/day and of MTX was 30 mg/m2/dose. Doses were adjusted every 21 days to maintain absolute phagocyte (APC) nadirs > 500/mm3 and platelet nadirs > 75,000. Chemotherapy cycles were delayed until APC was ≥ 1000/mm3 or platelets were ≥ 100,000. Medical records of the 61 SR pts were reviewed to ascertain the dose modifications of MTX and 6MP made due to low or high blood counts (APC and/or platelets) and/or delays in starting cycles (defined as ≥25 day interval between cycles). 27 pts were randomized to receive IM E. coli ASP, 29 to IV PEG ASP, and 5 declined randomization (directly assigned to IM E.coli ASP). A total of 1780 chemotherapy cycles were evaluated longitudinally using generalized estimating equations, 498 during Consolidation (with ASP) and 1282 during Continuation (No ASP). Results:Median age was 3.9 years and 57% of pts were female. There was a higher proportion of cycles modified for low blood counts (ie, dose reduction of MTX and/or 6MP) during Consolidation (ASP given) compared to Continuation (No ASP) (24% vs 8%, Odds Ratio (OR) = 2.35 [95% CI 1.41, 3.94]; p < 0.01). There was a lower proportion of cycles modified due to high blood counts (ie, dose escalation of MTX and/or 6MP) during Consolidation (ASP given) compared to Continuation (No ASP) (20% vs 33%, OR =0.20 [95% CI 0.13, 0.30]; p < 0.01). Figures 1 and 2 summarize the dosing of MTX and 6-MP during Consolidation and Continuation. During Consolidation, the proportion of cycles in which pts received escalated doses of MTX (above the starting dose) was 21% compared to 62% during Continuation. Similarly, the proportion of cycles in which pts received escalated doses of 6-MP (above starting dose) was 10% in Consolidation compared to 59% in Continuation. 17% of Consolidation cycles were delayed because starting criteria were not met compared to 9% of Continuation cycles when pts were not receiving ASP (OR =2.08, 95% CI 1.18, 3.64; p=0.01). No difference in delays or dose modification for counts was detected between ASP randomization arms in either the Consolidation or Continuation phases. [Display omitted] [Display omitted] Conclusion:During Consolidation, when SR pts received ASP, blood counts were lower than during Continuation (no ASP), despite an otherwise identical schedule of other systemic and IT agents during these two phases. Because of low blood counts, pts received lower doses of MTX and 6MP when they were also receiving ASP, and delays in beginning chemotherapy cycles were more common. This suggests that ASP has a myelosuppressive effect, either direct (due to ASP) or secondary to ASP-induced changes in the levels or metabolism of other chemotherapeutic agents; further investigation is necessary to elucidate the precise mechanism. The myelosuppressive effect of ASP should be taken into consideration when designing regimens for pts with ALL which combine ASP with other agents to avoid excessive dose reductions and/or treatment delays. Disclosures:Sallan:Enzon Inc.: Honoraria, Research Funding. Silverman:EUSA Pharmaceuticals: Consultancy, Honoraria; Enzon Pharmaceuticals: Consultancy, Honoraria.

Af9 Regulates gata2 Expression During Early Hemangioblast Specification and Vascular Pattern Formation In Zebrafish.

AbstractAbstract 2600 BackgroundAF9 is a transcription factor that plays an essential role in hematopoiesis and embryonic development. The alteration of AF9 is principally associated in acute myeloid leukemia as fusion partner of human MLL (mixed-lineage leukemia) gene rearrangements. Zebrafish is an excellent model organism to study embryonic development and hematopoiesis. We have previously shown that zebrafish af9 is expressed within the intermediate cell mass (ICM), a site of primitive hematopoiesis in zebrafish. Here we study the loss of af9 in zebrafish development to further understand how af9 modulates early hematopoietic and embryonic development. Methods and resultsTwo morpholino antisense oligos (MOs), designed to block translation and inhibit pre-mRNA splicing of af9, were co-injected in embryos at 1–2 cell stage. To control for off-target effects, two morpholino mismatch oligos were designed and co-injected. Efficacy of MOs was demonstrated by Western blot analysis and RT-PCR in controls and MO-injected embryos (morphants). In vivo monitoring of both morphants and control embryos was carried out by microscopy. Effects of af9 depletion on vasculature and erythropoiesis were evaluated in Tg(fli1:eGFP) and Tg(gata1:DsRed) transgenic lines, respectively. Whole-mount in situ hybridization of known hematopoietic markers was used to decipher the developmental time-points in which af9 regulates blood development. Following injection of two MOs at 1–2 cell stage, we compared the morphological features of the morphants with control embryos at about 24 hours post-fertilization (hpf). The af9 morphants showed small head and eyes, disruption of tail development and pronounced swelling in the posterior ICM. Circulating blood cells were reduced from 26 hpf to later stages of development. At 48 hpf the heart was enlarged, showed a paucity of blood-cells and pericardial edema. Decreased number of blood cells in morphant embryos was further confirmed by o-dianisidine staining at 48 hpf and 72 hpf and in living af9-knockdown gata1:DsRed transgenic animals, suggesting that the differentiation of erythroblasts remains insufficient or impaired. Concordant with this observation, we examined the expression of specific markers for early hematopoiesis (scl, lmo2 and gata2) and primitive erythropoiesis (gata1, hbbe, and band3) using whole-mount in situ hybridization (WISH). At the 5-somite stage, the early hematopoietic precursor marker gata2 was markedly increased while scl and lmo2 remained unaffected in af9 morphants. Interestingly, by 24 hpf gata2 was found to be specifically over-expressed in ICM while no change was observed for scl and lmo2 markers. Besides, the erythroid progenitors and mature erythrocyte markers gata1, band3 and hbbe displayed nearly normal expression. To further confirm the role af9 in early hematopoiesis, we examined its expression in moonshine, a mutant zebrafish with defects in erythroid maturation due to deficiency of tif1γ, a key regulator of hematopoietic gene expression. WISH analysis in moonshine showed loss of af9 expression in the ICM at 24 hpf, suggesting that af9 functions genetically downstream of tif1γ in normal erythroid cell development. To determine the effect of af9 on endothelial and vascular development, we performed knockdown of af9 in fli1:eGFP transgenic line. By 24 hpf, these morphants showed significant increase of fluorescence intensity in the posterior ICM and a clear perturbation in the inter-segmental vessels (ISV) of the trunk at 30 hpf, indicating that af9 is required for early steps in hemangioblast specification and vascular pattern formation in zebrafish. Conclusionaf9 regulates gata2 expression during early hemangioblast specification and vascular pattern formation in zebrafish. af9 may also be involved in caudal segment morphogenesis. Taken together, these data provide the initial framework of a pathway that can be used to further integrate the molecular events regulating hemangioblast differentiation. Disclosures:No relevant conflicts of interest to declare.

CBL Mutations In Therapy-Related Leukemia and Infant Leukemia

AbstractAbstract 2149Recent reports of somatic mutations of the CBL proto-oncogene in myeloid neoplasms are intriguing, because these CBL mutations were shown to results in aberrant tyrosine kinase signaling, which would lead also to activation of RAS signaling pathways. We and others reported that CBL mutations occurred in a variety of myeloid neoplasms, including de novo acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and MDS/myeloproliferative neoplasm, especially in chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML). The importance of CBL mutations concerning leukemogenesis is substantially increased. We investigated CBL mutations in 20 therapy-related leukemia/MDS (t-Leuk/MDS) cases and 26 infant leukemia cases. Homozygous mutation of theCBL gene (P417R), which was located in the RING finger domain, was identified in one out of 20 (5%) t-Leuk/MDS cases. This patient was a 5 year-old boy, whose biopsied specimen of the buccal lymph node showed malignant lymphoma (diffuse large T cell type, MT1(+), MB1(-), UCHL1(+)). No pathogenic nucleotide changes were identified in the CBL gene in the initial sample. Subsequently, the patient was treated with chemotherapy including VP-16 (200 mg/m2) given twice weekly. Nineteen months after initial diagnosis, he was diagnosed as having therapy-related leukemia with t(5;21) and MLL gene rearrangement due to VP-16. Furthermore, CBL gene mutations were found in 3 of 26 (12%) infant leukemia cases with 11q23 translocation/MLL gene rearrangement. CBL gene mutations were located in splice site in intron 8 and the RING finger domain (Y371H, in 2 cases), SNP array analysis (Affymetrix, GeneChip) of these cases with mutated CBL gene disclosed 11q-acquired uniparental disomy in all cases, but not in cases with wild-type CBL. CBL mutation has not been reported in acute leukemia with 11q23 translocation/MLL gene rearrangement. To our knowledge, these are the first t-Leuk/MDS case and infant cases with 11q23 translocation/MLL rearrangement, suggesting that CBL is mutated in a unique subset of t-Leuk/MDS and infant leukemia which is considered to play a pathogenic role in the development of t-Leuk/MDS and infant leukemia. Further accumulation of cases with t-Leuk/MDS and infant leukemia having the CBL mutation is needed. Disclosures:No relevant conflicts of interest to declare.

Prognostic Value of Immunophenotype In Infant ALL – Results of the INTERFANT'99 Study

AbstractAbstract 2700 Objective:Infant acute lymphoblastic leukemia (ALL) is characterized by distinct biological and clinical features. In this study, we aimed at detailed evaluation of infant ALL immunophenotype based on the large cohort of patients treated homogeneously on INTERFANT'99 protocol. Patients and Methods:We were able to obtain sufficient flow cytometry based immunophenotypic data from 286 (130 boys and 156 girls) of 483 infant ALL cases treated on INTERFANT'99 protocols. The distribution of patient and leukemia characteristics as well as clinical outcome were very similar to the entire study cohort (Pieters et al, Lancet 2007; 370: 240–250). The positivity for a certain antigen was defined at the cut-off value of >20% of cells expressing this antigen. In B-cell precursor ALL (BCP-ALL), evaluated antigens included CD10, CD20, CD34, myeloid markers (CD13, CD33, CD15 and/or CD65) and NG2 antigen assessed with 7.1 antibody. The strength of association between characteristics (patient's age and gender, initial white blood count [WBC], CNS involvement, presence of MLL gene rearrangements, prednisone response [PR]) and antigen expression was analyzed with Fisher exact test adjusted for multiple comparisons. Univariate analysis of outcome by immunophenotype was based on event free survival (EFS) and compared by log-rank test. Multivariate analysis was performed using Cox model in MLL-rearranged cases. P values < 0.05 were considered to be significant. Results:Altogether, 272 cases of infant BCP-ALL and 14 of infant T-ALL were analyzed. Most BCP-ALL cases were CD10 negative (66%), CD20 negative (88%), CD34 positive (60%), and NG2 positive (58%). Co-expression of myeloid antigens was less frequent and concerned CD13 in 8%, CD33 in 17%, CD15 in 31%, and CD65 in 28%. CD10 negativity and NG2 expression were significantly more frequent in younger infants, patients with initial high leukocytosis and with MLL rearrangements. CD34 expression was observed more frequently in younger infants, while CD20 positivity was significantly associated with lower initial leukocytosis and absence of MLL rearrangement. For myeloid antigen expression, the only significant association was more frequent CNS involvement in infants with CD13+ and/or CD33+ BCP-ALL. Analysis of outcome showed significantly better prognosis for CD10 positivity (4-year EFS of 56.3 as compared to 37.2 for CD10 negative BCP-ALL), absence of NG2 expression (4-year EFS of 63.9 as compared to 34.9 for NG2 positive BCP-ALL) and CD33 negativity (4-year EFS of 47.6 as compared to 27.7 for CD33 positive BCP-ALL). There was also a trend towards better outcome in CD20 positive infant BCP-ALL. While CD10 negativity and NG2 positivity were associated with well-known high-risk features, prognostic significance of CD33 expression is somewhat more difficult to explain. Of note, CD10 negativity was not associated to a worst outcome in MLL-rearranged patients. Moreover, in this subset, multivariate analysis showed that none of the markers analyzed had independent prognostic significance, after adjustment for sex, age at diagnosis, WBC and PR. In rare infant T-ALL cases, we observed equal distribution to different immunophenotypic subgroups as defined by EGIL classification. Conclusion:Infant BCP-ALL has distinct immunophenotypic features. However, immunophenotype has no independent prognostic relevance when MLL, age, WBC, PR and other factors are included. Disclosures:No relevant conflicts of interest to declare.

Multiplex Reverse Transcription-Polymerase Chain Reaction as Diagnostic Molecular Screening of 4 Common Fusion Chimeric Genes in Taiwanese Children With Acute Lymphoblastic Leukemia

The classification of B-lineage acute lymphoblastic leukemia (ALL) by specific chromosomal translocations has prognostic implications for risk-directed therapy. Reverse transcription-polymerase chain reaction (RT-PCR) assay is a useful tool for detecting fusion transcripts from common chromosomal translocations of ALL cells. Multiplex RT-PCR and nested-PCR assays were used to detect ALL-type BCR-ABL1 transcripts of the t(9;22), TCF-PBX1 transcripts of t(1;19), the MLL-AF4 transcripts of t(4;11), and 2 variants of ETV6-RUNX1 of the cryptic t(12;21) in 148 leukemic samples upon diagnosis. The patients received risk-directed protocols of the Taiwan Pediatric Oncology Group-ALL-2002 that consisted of multiple chemotherapeutic agents of different intensities. Event-free survival (EFS) and overall survival (OS) rates were analyzed for genetic abnormalities detected by multiplex PCR and conventional cytogenetic analysis by the Kaplan-Meier method, and compared with the Mantel-Haenszel test. The Cox proportional hazards model was implemented to identify independent prognostic factors for EFS and OS. In this cohort of Taiwanese children, the relative frequencies of the 4 translocations of B-lineage ALL were 8% with ALL-type t(9;22)/BCR-ABL1, 4% with (1;19)/TCF-PBX1, 2% with t(4;11)/MLL-AF4, and 17.6% with t(12;21)/ETV6-RUNX1. Patients with t(12;21)/ETV6-RUNX1 fusion, hyperdiploidy, and t(1;19)/TCF-PBX1 fusion had the most favorable outcomes, whereas those with the t(9;22)/BCR-ABL1 fusion or t(4;11) and other MLL gene rearrangement had poor prognosis (P<0.001 for EFS and OS). BCR-ABL1, MLL gene rearrangement, and very high-risk group were independent prognostic factors after Cox regression analysis. The biological factors of leukemia cells are associated with treatment outcomes in childhood ALL. Multiplex RT-PCR assay is an efficient and sensitive diagnostic tool that may improve the ability to accurately and rapidly risk-stratify children with ALL.

T(4;11) leukemias display addiction to MLL-AF4 but not to AF4-MLL

The most frequent MLL-gene rearrangement found in leukemia is a reciprocal translocation with AF4 on chromosome 4 resulting in the formation of the MLL-AF4 and the AF4-MLL fusion genes. The oncogenic role of MLL-AF4 is documented but the significance of the reciprocal product – AF4-MLL in leukemia is less clear. In the human leukemia cell lines – RS4;11 and SEMK2-M1, both of which express MLL-AF4 and AF4-MLL, we knocked down the expression of AF4-MLL using siRNA. Loss of AF4-MLL had no effect on the growth of either RS4;11 or SEMK2-M1 cells. Furthermore, in SEMK2-M1 cells there were no changes in cell cycle or apoptosis with loss of AF4-MLL. In contrast, knockdown of MLL-AF4 significantly inhibited growth of both RS4;11 and SEMK2-M1. Additionally, in SEMK2-M1 cells, loss of MLL-AF4 led to G2/M cell cycle arrest and increased apoptosis. Overall, these results demonstrate that in t(4;11) leukemia, the MLL-AF4 fusion protein is critical for leukemia cell proliferation and survival while the AF4-MLL fusion product is dispensable.

A Case of Therapy-related ALL with MLL Gene Rearrangement Following Treatment of Breast Cancer

ALL with MLL gene rearrangement secondary to chemotherapy has been rarely reported. We report a case of therapy-related ALL (t-ALL) with MLL gene rearrangement in a patient who had undergone treatment for breast cancer. A 60-yr-old woman with breast cancer underwent breast-conserving surgery followed by 6 cycles of adjuvant chemotherapy (cyclophosphamide, epirubicin, and fluorouracil) and radiation therapy (dose, 5,040 cGy to the left breast and a 1,000 cGy boost to the tumor bed). A follow-up examination performed 14 months after the chemotherapy revealed no evidence of breast malignancy. However, the patient's complete blood cell count indicated acute leukemia: white blood cell count, 174.1 x 10(9)/L with 88% blasts; Hb level, 12.5 g/dL; and platelet count, 103.0 x 10(9)/L. Examination of the bone marrow aspirate smear revealed a high percentage of blasts (85.1% of all nucleated cells); the blasts showed a pro-B immunophenotype and were positive for CD19, CD79a, HLA-DR, CD34, and terminal deoxynucleotidyl transferase (TdT). Cytogenetic and FISH analyses revealed t(4;11)(q21;q23) and MLL gene rearrangement, respectively. The patient received induction chemotherapy with cyclophosphamide, vincristine, doxorubicin, and dexamethasone and achieved complete remission. Following consolidation chemotherapy, she underwent allogenic peripheral blood stem cell transplantation and has been clinically stable. To our knowledge, this is the first reported case of t-ALL with MLL gene rearrangement following treatment of breast cancer in Korea.

Abstract 3498: The cytotoxicity and genotoxicity of etoposide and other phenolic and non-phenolic agents in human myeloid leukemia HL-60 cells: role of myeloperoxidase (MPO)

Abstract The clinical efficacy of the anticancer agent etoposide (VP-16) is compromised by its ability to cause acute myeloid leukemias (t-AML) associated with MLL gene rearrangements. We proposed previously that myeloperoxidase (MPO) found in myeloid precursors converts the phenolic drug VP-16 to its phenoxyl radical (VP-16-O[[Unable to Display Character: &amp;#8729;]]), which redox cycles, leading to the generation of reactive oxygen species, oxidative DNA damage linked to DNA topoisomerase II (topo II)-mediated strand cleavage, and resultant recombination events causal for t-AML. After knockdown with an shRNA for MPO in myeloid leukemia HL-60 cells, MPO activity was reduced to 13% of shRNA controls. In these MPO knockdown cells, VP-16-O[[Unable to Display Character: &amp;#8729;]] formation and VP-16-induced topo II/DNA covalent complexes were reduced compared to shRNA controls demonstrating that VP-16 activity was, in part, dependent on MPO. MPO can also oxidize VP-16 to its ortho-quinone metabolite which is subsequently inactivated by conjugation with glutathione. The cytotoxicities of VP-16, curcumin (a phenolic agent), and a curcumin analog (FLLL-10) were increased in MPO knockdown cells compared to shRNA controls. Podophyllotoxin, the non-phenolic VP-16 parental compound, was equally cytotoxic in MPO knockdown and control cells. Additionally, the cytotoxicity of hydrogen peroxide, an MPO co-substrate, was unaffected by MPO levels. Results indicate that MPO converts VP-16 to a pro-oxidant, which leads to genotoxicity, while simultaneously protecting cells from drug-induced cytotoxicity likely by glutathione conjugation to VP-16 ortho-quinone. The combined results suggest a mechanism by which MPO-containing myeloid progenitor cells can be relatively protected against VP-16-induced apoptosis/cytotoxicity while suffering increased genotoxic and recombinogenic insults responsible for leukemogenesis. Support: NIH CA090787. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3498.

Segmental amplification of MLL gene associated with high expression of AURKA and AURKB genes in a case of acute monoblastic leukemia with complex karyotype

We report a case of acute monoblastic leukemia showing a jumping translocation with the MLL gene in a 17-year-old male. Classic cytogenetic and spectral karyotyping revealed a complex karyotype, and fluorescence in situ hybridization (FISH) demonstrated amplification of the MLL gene followed by translocation to chromosomes 15q, 17q, and 19q. In addition, molecular analyses showed a high expression of AURKA and AURKB genes. It is already known that overexpression of Aurora kinases is associated with chromosomal instability and poor prognosis. The formation of jumping translocations is a rare cytogenetic event and there is evidence pointing toward preferential involvement of the heterochromatin region of donor chromosomes and the telomere ends of recipient chromosomes. Jumping translocation with the MLL gene rearrangement is an uncommon phenomenon reported in leukemia cytogenetics.

A partial nontandem duplication of the MLL gene in four patients with acute myeloid leukemia

Unusual MLL gene rearrangements were found in bone marrow cells of four patients with acute myeloid leukemia. A combination of conventional and molecular cytogenetic methods were used to describe translocations t(9;12;11)(p22;p13;q23), t(11;19)(q23;p13.3), and t(10;11)(p12;23) and inverted insertion ins(10;11)(p12;q23.3q23.1). Partial nontandem duplication of the MLL gene was identified by reverse transcriptase–polymerase chain reaction in all cases. The duplication, which included MLL exons 2 through 8–9, was interrupted by a cryptic insertion of one or two exons from the respective MLL partner gene: MLLT10, MLLT3, or MLLT1.

Promoter Hypermethylation in MLL-r Leukemia: Biology and Therapeutic Targeting.

Abstract 3472Poster Board III-409 BackgroundTumor suppressor silencing via promoter CpG island hypermethylation is important in oncogenesis. DNA methyltransferase (DNMT) maintains promoter hypermethylation, and DNMT inhibitors (DNMTis) are of proven benefit for MDS and are in trials for AML and CML. Infant ALL is a rare, aggressive and distinctive subset of childhood ALL with 80% of cases harboring MLL gene rearrangements (MLL-r). Cooperating events leading to leukemia in these cases are largely unknown and recent studies suggest that oncogenesis is related to neither copy number alterations nor mutations in the tyrosine kinome. MLL has multiple domains with epigenetic activity, and silencing of several single tumor suppressor genes in MLL-r infant ALL cases has been described. We explored the role of genome-wide CpG island hypermethylation in MLL-r infant ALL, with a focus on its potential role as a novel molecular target for therapy. MethodsHELP (HpaII-tiny fragment Enrichment by Ligation-mediated PCR), which accurately measures the abundance of DNA methylation at ∼30,000 CpG sites covering ∼13,000 promoter regions, was used to examine a cohorts of MLL-r infant ALL primary samples (n=5), other childhood ALL primary samples (TEL-AML1+, n=2 and hyperdiploid, n=3) and normal controls (CD34-selected- (n=2) and CD19-selected- (n=3) cord blood). To correlate HELP results with gene expression, we performed qRT-PCR on cDNA from the same samples for a set of 10 “genes of interest”, which were selected due to differential methylation in the HELP assay (n=6; DAPK1, DAXX, CASP9, LIFR, CCR6, HRK), or due to known biological significance (n=4; FLT3, HOXA9, MEIS1, FHIT). GAPDH and ABL were used as housekeeping genes. To explore the therapeutic potential of DNMTi in infant ALL, we treated MLL-r cell lines (n=3; SEMK2, KOPN8, HB1119) and an MLL-wt cell line (n=1; NALM6) with various concentrations (0, 0.5, 1, 2, 4 mM) of decitabine over multiple durations (8, 24, 48 and 72 hours), then performed MTT assays and qRT-PCR on the genes of interest for each concentration/time point combination. To validate the HELP assay findings and to determine whether the effects of decitabine on re-expression of silenced genes was due to DNMT activity, we also performed methylation specific PCR (MSP) for 3 of the genes of interest (DAPK1, CCR6, HRK) on the primary samples and the cell lines. ResultsUnsupervised analysis of the HELP assay showed tight clustering of samples into their known biological groups, indicating large differences in global methylation patterns, and the ability of these patterns to accurately sub-classify ALL samples. Global hypermethylation was seen in the MLL-r cohort compared to both the normals and the other ALLs, with ratios of significantly (p<0.01) hyper- to hypomethylated CpG's of 2.1 and 2.2, respectively.Of the 10 genes assayed by qRT-PCR, 4 (FLT3, MEIS1, FHIT, and DAPK1) showed marked and significant (p<0.05) differences in expression by cohort. As expected, FLT3 and MEIS1 were over-expressed in the MLL-r cohort, while FHIT and DAPK1 were under-expressed. LIFR was silenced in all samples. CCR6 and HRK were relatively under-expressed in the MLL-r groupIn the cell line treatment assays with decitabine, MLL-r cell lines showed dose and time dependent cytotoxicity and re-expression of 3 of the 4 silenced or under-expressed genes[DAPK1, CCR6, HRK]. These phenomena were not seen in the MLL-wt cell line. For the 3 re-expressed genes, MSP confirmed preferential promoter methylation in MLL-r samples at baseline, and a relative decrease in methylation after treatment with decitabine. ConclusionsMLL-r infant ALL samples demonstrate global hypermethylation. The HELP assay can reliably identify individual hypermethylated genes that can be confirmed to be hypermethylated by MSP and can be shown to be silenced or under-expressed at the transcript level. Treatment with DNMTi demonstrates selective cytotoxicity for MLL-r cell lines in a dose and time dependent manner that correlates with baseline hypermethylation/silencing of key genes, and correlates temporally with demethylation and re-expression of these same hypermethylated genes. DNMTi may represent a novel molecularly targeted therapy for infant ALL. Disclosures:No relevant conflicts of interest to declare.