Myeloid Lymphoid Leukemia Research Articles

Abstract 3924: Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite the current pediatric treatment protocols have resulted in 5-year overall survival rates of up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) Chromatin Immunoprecipitation Sequencing (ChIP-Seq) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements as the T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generated mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes growing on OP9-DL4 pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To better understand the genes regulated by TLX3 on driving a mixed phenotype leukemia we performed ChiP-seq of D1 cell line ectopically expressing mutIL7R and TLX3. We identified RUNX as a possible key mediator of the mixed-phenotype-leukemia induced by TLX3 in combination with mutIL7R. Additionally, scRNAseq showed upregulation of PU.1 in mice leukemia, suggesting PU.1 as a potential therapeutic target. To assess whether targeted therapy could successfully treat mutIL7R/TLX3-mouse leukemia, we treated mice that had been injected with the thymocytes transduced with mutIL-7R+TLX3 genes. We found that PU.1 inhibition reduced spleen weights, indicating decreasing of leukemia engraftment. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies. Citation Format: Gisele Rodrigues. Cooperation between mutant IL7R and TLX3 generates mixed myeloid lymphoid leukemia from thymocyte progenitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3924.

First Description of Upfront Target Therapy Treating AML and Bcll Presented Simultaneously As First Line

INTRODUCTION: The occurrence of acute myeloid leukemia (AML) and chronic lymphoid leukemia (B-CLL) simultaneously is rarely described. We describe a case report of AML and B-CLL, diagnosed simultaneously, without any previous treatment for any of the hematological neoplasms [1]. The patient received low-dose cytarabine (ARA-C) and Venetoclax, which is medically indicated on label for both hematological neoplasms. CASE REPORT: A 75-year-old male, presented with edema and joint pain one month before hospital admission, showing pancytopenia on a complete blood count and presence of blasts in peripheral blood. The patient was then referred to the hematology service. The morphological analysis of the bone marrow aspirate showed 67.7% of myeloblasts, compatible with AML. Bone marrow immunophenotyping was performed, which identified 34.10% of myeloblasts, compatible with AML and 50.48% of monoclonal B lymphocytes (chronic B-cell lymphoproliferative disease). In flow cytometry there were two distinct populations of myeloblasts. Type 1 myeloblasts labeling CD7 +, CD13 +, CD34 ++, CD38 ++, CD45 ++, CD56 ++, CD117 ++, CD123 +, HLA-DR +++ and MPO + / ++. The second population marked CD13 + / ++, CD34 + / ++, CD38 ++, CD45 +, CD117 ++, CD123 +, HLA-DR ++ / +++ and MPO + (30%). Monoclonal lymphocytes showed CD11c + / ++ (70%), CD19 ++, CD20 + / ++ (84%), CD22 + (39%), CD23 + / ++, CD25 +, CD31 +/-, CD43 ++, CD45 ++ / +++, CD81 + (38%), CD200 + (85%) and Lambda +. The molecular study was negative for genetic abnormalities: FLT3, KIT and NPM, configuring the patient as an intermediate risk for AML. In the cytogenetic analysis there was no growth of metaphases. Patient received simultaneous diagnosis of AML and B-CLL. As he was ineligle to intensive chemotherapy (IC), we started original protocol Subcutaneous Cytarabin+venetoclax(VIALE C). The patient had grade 2-3 AE(neutropenia managed with GCSF) ending the fourth cycle in July 2020. The evolution of hematimetric parameters and diseases are described in graphics. DISCUSSION: This is the first described case in our knowledge treated upfront with bcl2-inh target therapy for two absolutely different hematological neoplasms: AML and BCLL. Nowadays we are experiencing a new therapeutic model in oncohematology, in which the targeted therapy is gaining ground in relation to IC with excellent results. In this way, the importance of comprehension of the pathophysiological mechanism of the neoplasms and the way we can stop the disease proliferation is progressively guiding the new protocols. Elderly patients are more likely to have early treatment-related death and exhibit therapeutic resistance, limiting alternatives. We decided to start first-line treatment with ARA-C and Venetoclax [2]. Venetoclax associated with ARA-C has a manageable safety profile, producing quick and durable remissions in elderly people with AML ineligible for IC, as well as in B CLL, being the best therapeutic alternative for the case, in our opinion. Venetoclax belongs to a group of drugs called Bcl-2 inhibitors, an anti-apoptotic protein, which works by blocking this protein in the body, causing apoptosis of both neoplastic cells. The high rate of remission and low early mortality, combined with fast and durable remission, make Venetoclax and ARA-C a new and attractive treatment for the elderly [2]. In our case, the intention of the product in the first line was not B-CLL, but it would certainly be a good option for this profile of elderly patients. CONCLUSION: We report the first description of simultaneous diagnosis of AML and B CLL treated with a Bcl-2 inhibitor, demonstrating that antitumor mechanisms can be extremely effective in completely different diseases. We have a long way to go in the search for full knowledge of oncohematological diseases and targeted therapies. However, this case report shows that we are on the right track.

Abstract 3064: Epigenetic manipulation can sensitize AML cells to differentiate with ATRA

Abstract Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on two MLL-driven AML cell lines, MV4;11 and THP-1, showing translocations t(4;11) and t(9;11), respectively, and one non-MLL related AML cell line, U937. MV4;11, THP-1, and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, N-acetyl-dinaline (CI-994), a general histone deacetylase inhibitor, and 3-deazaneplanocin A (DZNep), a S-adenosylhomocystein hydrolase inhibitor that depletes EZH2 and thus its associated H3K27me3 activity. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), myeloid-related nuclear morphological changes noted with cytospin analysis of cells, and decreased AML-associated gene expression with qPCR. All three drugs seemed to sensitize U937 and THP-1 cells to differentiate when treated with ATRA. MV4;11 cells were sensitized to differentiate by CI-994 and DZNep with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with or without ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to ATRA differentiation therapy, but since the two MLL cell lines responded differently the response may still be dependent on the specific MLL partner gene driving the AML. Citation Format: Kalsi Heimdal, Bikalpa Ghimire, Edjay Ralph Hernandez, Heidi J. Gill Super. Epigenetic manipulation can sensitize AML cells to differentiate with ATRA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3064.

Abstract 870: Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy

Abstract Human acute myeloid leukemia (AML), the most common type of acute leukemia, has approximately eight subtypes, many of which have poor prognosis. Most of these subtypes are associated with specific, recurrent chromosome translocations. These translocations result in fusion genes, which encode oncoproteins that block differentiation and promote proliferation of immature cells. The Myeloid Lymphoid Leukemia gene (MLL) is frequently involved in these translocations, and is considered a driver of the AML. Differentiation promoting drugs, such as all-trans-retinoic acid (ATRA) are an attractive alternative to cytotoxic chemotherapy, but few types of AML, other than acute promyelocytic leukemia (APL), respond to ATRA. We hypothesize that specific genes must be activated or inhibited in AML for drugs like ATRA to induce differentiation, and that gene activation or inhibition may be the result of specific epigenetic modification. We also hypothesize that AML with different genetic alterations may respond differently to specific epigenetic inhibitors. Our initial studies have focused on one AML cell line, MV4;11, with alterations in the MLL gene, and one non-MLL related AML cell line, U937. MV4;11 and U937 were treated with specific epigenetic modifiers, including tranylcypromine (TCP), an inhibitor of histone demethylase KMD1A/LSD1, and CI-994, a general histone deacetylase inhibitor. Evidence for differentiation was noted in a variety of assays, including reduced cell proliferation as measured directly and by MTT assay, upregulation of myeloid-specific cell surface markers such as CD11b as measured by fluorescence-activated cell sorting (FACS), and myeloid-related nuclear morphological changes noted with cytospin analysis of cells. Both CI-994 and TCP seemed to sensitize U937 cells to differentiate when treated with ATRA as measured by all indicators of differentiation. MV4;11 cells were sensitized to differentiation by CI-994 with or without ATRA, although there was no indication of CD11b upregulation. However, MV4;11 was not sensitized by TCP to differentiate with ATRA. These experiments suggest epigenetic inhibitors may increase sensitivity to differentiation therapy, but that the response may still be dependent on the specific genetic alteration driving the AML. Our studies suggest that in MLL-driven leukemia, the MLL fusion oncoprotein may override genetic manipulation resulting from treatment with epigenetic inhibitors, preventing differentiation by ATRA. Citation Format: Kalsi K. Heimdal, Edjay Ralph A. Hernandez, Heidi J. Gill Super. Effects of epigenetic modifier inhibitors on AML cell sensitivity to differentiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 870.

High expression of FLT3 is a risk factor in leukemia.

Several studies have shown that internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) can result in the failure of leukemia treatment and contribute to a poor prognosis. However, the role of the overexpression of FLT3 in leukemia remains to be fully elucidated. By mining public database, the present study first identified that the expression of FLT3 in leukemia was markedly higher, compared with that in other types of tumor and cell lines, indicating that FLT3 is important in leukemia. In leukemia, FLT3 was found to be significantly upregulated in acute myeloid leukemia and acute lymphoblastic leukemia, and a high expression of FLT3 contributed to reduced survival rates. By analyzing Gene Expression Omnibus and The Cancer Genome Atlas data, it was found that genetic alterations and modification of DNA methylation increased the expression of FLT3 in leukemia. FLT3-ITD and FLT3 tyrosine kinase domain point mutations increased the expression of FLT3 in four independent datasets. In addition, the status of FLT3 gene methylation was negatively correlated with the expression of FLT3, and haploinsufficiency of DNA methyltransferase 1 increased the expression of Flt3 in mouse leukemia cells. By analyzing the enrichment of differentially-expressed genes in chemical and genetic perturbation datasets, it was found that genes, which were upregulated in the FLT3 high expression group had myeloid lymphoid leukemia- and nucleophosmin 1-like signatures, indicating that the overexpression of FLT3 may use the same mechanism to promote leukemia. Collectively, the results of the present study showed that the overexpression of FLT3 is a potential risk factor in leukemia.

Surveying Mutation of FLT3 Genes in Children with Acute Leukemia

Background and purpose: Mutation of FMS like tyrosine kinase (flt3) gene causes uncontrolled proliferation of leukemic cells and a bad prognosis. The present study is aimed at implementing molecular tests to diagnose and screen the mutations in acute leukemia patients. Methodology: Totally, 91 children with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) were examined as to flt3 mutation, internal tandem duplication (ITD) mutation, and point mutation in exon 17 (e17). ITD mutation in flt3 receptor was carried out in exon 12&12 and intron 11. As to point mutation in e17, PCR products of the subjects after PCR on genomic DNA of them were examined using restriction enzyme (ECORV) and restriction fragment length polymorphism (RFLP). As to ITD positive, sequencing method was used. Findings: ITD mutation was observed in seven (7.7%) of acute leukemia patients and two (2.2%) patients were diagnosed with point mutation D835. Distribution in different subgroups of FAB was not identical. Conclusion: FLT3 mutation was highly prevalent in children with acute leukemia. Therefore, molecular diagnosis of these mutation, regardless of FAB categorization and before initiation of intervention, can be used to make better decision about therapeutic protocol.

A rare case of blastic plasmacytoid dendritic cell neoplasm with deletion 7q.31, in the setting of heavy pre-treatment with alkylating chemotherapy

Blastic plasmacytoid dendritic cell neoplasm is rare myeloid malignancy clinically characterized by non-pruritic, violaceous and papulo-nodular skin lesions, together with bone marrow and lymph node involvement. Histologically, there is infiltration of dermis by neoplastic mono-nuclear CD4, CD56, CD123 co-expressing cells with epidermal sparing. Most commonly blastic plasmacytoid dendritic cell neoplasm presents as a de-novo condition, and treatment-related blastic plasmacytoid dendritic cell neoplasm is a rare phenomenon. Due to rarity of the disease, there is no established standard of care treatment. Both acute myeloid leukemia and acute lymphoid leukemia type induction regimens have been used for treatment of blastic plasmacytoid dendritic cell neoplasm, with initial response rate of 50%-80%. We present a rare case of therapy-associated blastic plasmacytoid dendritic cell neoplasm in a patient with remote history alkylating agent systemic therapy. A lag period of five to seven years and presence of deletion 7q.31 seen in bone marrow biopsy specimen in our patient are consistent with a likely therapy-associated etiology of his blastic plasmacytoid dendritic cell neoplasm.

Abstract PR01: A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells

Abstract The Mixed Lineage Leukemia (MLL) gene on chromosome 11q23 is fused by reciprocal translocation to a diverse group of partner genes in both acute myeloid and acute lymphoid leukemia (AML, ALL). As a result of the t(4;11)(q21;q23), MLL fuses to AF4 (AFF1), one of the most common MLL fusion partner proteins. Unlike several other MLL fusions that are frequently found in both AML and ALL, MLL-AF4 is almost exclusively associated with B-cell ALL with a pro-B immunophenotype. It accounts for 10-15% of ALL cases and confers a poor prognosis. Although many MLL-fusion leukemia models have been established, it has not been possible to generate a t(4;11) pro-B leukemia model that accurately recapitulates the human disease, hampering research into the molecular mechanisms that underlie the development of this subtype of leukemia. Here we present a faithful model of t(4;11) pro-B-ALL that fully recapitulates the immunophenotypic and molecular aspects of the human disease. Human hematopoietic CD34+ cells transduced with a modified MLL-AF4 fusion gene successfully initiate ALL in NSG mice with high penetrance. The leukemia cells have a CD19+CD34+ pro-B immunophenotype and are CD10(-), a common feature in MLL-AF4 patients. The effect of the oncogene is species-specific, as retroviral transduction and transplantation of murine hematopoietic cells results in only AML using either lymphoid or myeloid conditions. An MLL-AF4 specific gene signature derived from patients is significantly enriched in our model cells, as shown by RNA-Seq, and the model samples group tightly with MLL-AF4 patients, even when compared with other MLL-fusion leukemia samples in unsupervised hierarchical clustering analysis. Interestingly, using gene profiles of normal pro-B and pre-B cells as reference, our MLL-AF4 leukemia cells show strong enrichment for pro-B genes, while instead, pre-B but not pro-B genes are overrepresented in our MLL-AF9 B-ALL leukemia cells. This differential developmental stage blockage of MLL-fusions is also reflected in patient samples. More strikingly, in accordance with the distinct lineage bias of MLL-fusions observed in the clinic, human cells expressing MLL-AF4 have a strong predilection for the lymphoid lineage and a demonstrated resistance to lineage redirection in response to myeloid signals compared to human cells expressing MLL-AF9. This difference in lineage predisposition of MLL-AF4 compared to MLL-AF9 can be attributed to differential effects on lineage-specific gene expression. Under myeloid-priming conditions, phenotypically (CD33+CD19-) and morphologically myeloid MLL-AF4 cells are still able to initiate pro-B ALL in immunodeficient mice, while only AML is generated by MLL-AF9 myeloid cells. Accordingly, an active lymphoid molecular program with lower expression of critical myeloid genes is observed in MLL-AF4 myeloid cells compared to MLL-AF9 myeloid cells. Interestingly, we find that the polycomb gene BMI1, which was reported to be critical to prevent lymphoid priming in normal hematopoiesis, is expressed at significantly lower levels in MLL-AF4 than in MLL-AF9 myeloid cells. Reintroduction of BMI1 into MLL-AF4 cells enables AML generation with variable penetrance, while control vector transduced cells always result in B-ALL. Our results demonstrate that lineage fate in response to MLL-fusion protein expression involves a complex interplay of oncogene, intra- and extra-cellular microenvironmental cues. In addition, our data clearly demonstrate the species specificity associated with the t(4;11). This in vivo model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis. This abstract is also presented as Poster A06. Citation Format: Shan Lin, Roger T. Luo, Mark Wunderlich, Joseph J. Kaberlein, Ahmad Rayes, John Anastasi, Maureen M. O'Brien, James C. Mulloy, Michael J. Thirman. A novel MLL-AF4 in vivo model phenocopies t(4;11) pro-B ALL and reveals a lymphoid lineage bias of the fusion protein in human cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR01.

Cis-regulatory elements are harbored in Intron5 of the RUNX1gene

BackgroundHuman RUNX1 gene is one of the most frequent target for chromosomal translocations associated with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). The highest prevalence in AML is noted with (8; 21) translocation; which represents 12 to 15% of all AML cases. Interestingly, all the breakpoints mapped to date in t(8;21) are clustered in intron 5 of the RUNX1 gene and intron 1 of the ETO gene. No homologous sequences have been found at the recombination regions; but DNase I hypersensitive sites (DHS) have been mapped to the areas of the genes involved in t(8;21). Presence of DHS sites is commonly associated with regulatory elements such as promoters, enhancers and silencers, among others.ResultsIn this study we used a combination of comparative genomics, cloning and transfection assays to evaluate potential regulatory elements located in intron 5 of the RUNX1 gene. Our genomic analysis identified nine conserved non-coding sequences that are evolutionarily conserved among rat, mouse and human. We cloned two of these regions in pGL-3 Promoter plasmid in order to analyze their transcriptional regulatory activity. Our results demonstrate that the identified regions can indeed regulate transcription of a reporter gene in a distance and position independent manner; moreover, their transcriptional effect is cell type specific.ConclusionsWe have identified nine conserved non coding sequence that are harbored in intron 5 of the RUNX1 gene. We have also demonstrated that two of these regions can regulate transcriptional activity in vitro. Taken together our results suggest that intron 5 of the RUNX1 gene contains multiple potential cis-regulatory elements.

Abstract LB-246: MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Abstract Classification of acute leukemia (AL) is based on commitment of the leukemic cells to either the myeloid or the lymphoid lineage. However, a small percentage of AL cases display features of both hematopoietic lineages and lacks immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). The lack of a clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have shown to be able to accurately discriminate AL of the lymphoid lineage from those of the myeloid lineage. Here, we compared the microRNA expression profiles of acute leukemias of ambiguous lineage with those of B-ALL, T-ALL and AML. MicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed. Unsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between cases of the myeloid and lymphoid lineages. The top differentially expressed microRNAs between AML and ALL were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. Subsequently, we compared the general miRNA expression profiles of AL of ambiguous lineage with those of the AML, B-ALL and T-ALL cases thereby showing that these leukemias of ambiguous lineage do not segregate as a separate entity. Moreover, unsupervised clustering of all the AL samples using the top 10 percent of most variable microRNAs resulted in clustering of leukemias of ambiguous lineage with either AML, B-ALL or T-ALL. Furthermor, qRT-PCR expression analysis of the five most discriminative microRNAs between ALL and AML was able to define AL of ambiguous lineage as either AML or ALL. Thus, our results indicate the presence of a lymphoid or myeloid lineage specific microRNA expression profile in AL cases of ambiguous lineage, despite their mixed immunophenotype. At diagnosis, the classification of AL of ambiguous lineage by microRNA expression analysis might be of additional value in therapeutic decision making. Citation Format: Dave C. de Leeuw, Willemijn van den Ancker, Fedor Denkers, Rene X. Menezes, Theresia M. Westers, Gert J. Ossenkoppele, Arjan A. van de Loosdrecht, Linda Smit. MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-246. doi:10.1158/1538-7445.AM2013-LB-246

Molecular diagnosis of lymphoblastic leukemia

The mixed lineage leukemia (MLL) gene at chromosome band 11q23 is commonly involved in reciprocal translocations that is detected in acute leukemia. The MLL gene, commonly known as mixed lineage leukemia or myeloid lymphoid leukemia, has been independently identified and cloned from the 11q23 breakpoint of acute leukemia. We describe a patient with acute lymphoblastic leukemia whose cells had shown reciprocal translocation between short arm (p21) of chromosome 2 and long arm (q23) of chromosome number 11 [t(2;11) (p21;q23)] by cytogenetic analysis. Fluorescence in situ hybridization analysis (FISH) was also performed for reconfirmation with a probe for MLL which showed split signals, hybridizing to both the derivative 2 and 11 chromosomes. Our study confirmed FISH as the most suitable assay for detecting MLL rearrangements because of its sensitivity and speed. It recommended that FISH should be used as complementary to conventional cytogenetic analysis. In conclusion, evaluation of the t(2;11)(p21;q23) was done by molecular clarification and flow cytometry.

Rearrangement of the Myeloid/Lymphoid Leukemia Gene in Therapy-Related Myelodysplastic Syndrome in Patients Previously Treated with Agents Targeting DNA Topoisomerase II

Background: Therapy-related acute myeloid leukemias (t-AML), with balanced translocations affecting the 11q23 point in the myeloid/lymphoid leukemia (MLL) gene, are one of the most serious complications of treatments with topoisomerase II inhibitors. However, only a few reports of t-AML exist. We aimed to study if these translocations are cumulative-dose-dependent, their frequency in therapy-related myelodysplastic syndrome and the relationship between their presence, the type of therapy and the response criteria. Methods: This retrospective study included 120 patients with various malignancies (108 non-Hodgkin’s lymphoma, 8 Hodgkin’s disease and 4 neuroblastoma) in remission, being treated with topoisomerase 2 inhibitors; 74 had been diagnosed with therapy-related myelodysplasia and 46 did not have dysplasia. All bone marrow biopsy samples were evaluated by fluorescence in situ hybridization for 11q23 point breakage in the MLL gene. Results: MLL gene rearrangement frequency was 6% in dysplastic versus 2% in nondysplastic groups; p < 0.001. It was associated with a worse overall survival (mean 13 ± 2 vs. 39 ± 3 months, log-rank p value <0.0001). It was dose-dependent with a cut-off value of 290 mg/kg of topoisomerase II inhibitors as assessed by ROC curve (area under the curve 0.84 ± 0.05, p < 0.0001). Conclusions: It is proposed that the MLL gene is etiopathogenetically relevant for hematological neoplasias transformation and survival.

Diagnostic Usefulness of Genomic Breakpoint Analysis of Various Gene Rearrangements in Acute Leukemias: A Perspective of Long Distance– or Long Distance Inverse-PCR–based Approaches

Diagnostic Usefulness of Genomic Breakpoint Analysis of Various Gene Rearrangements in Acute Leukemias: A Perspective of Long Distance– or Long Distance Inverse-PCR–based Approaches

Abstract 1245: Characterization of human leukemic cell lines and changes of the characteristics according to passage number

Abstract Background: Human leukemic cell lines are valuable tools for the studies on the biology of hematologic cancers and new drug development. We investigated the baseline characteristics of human leukemic cell lines and changes of the properties according to passage number. Materials and methods: A total of 11 human leukemic cell lines were investigated in this study, including myeloid leukemia cell lines (HL60, NB4, Molm-13, MV4-11, KG-1, K562) and lymphoid leukemia cell lines (CCRFCEM, SR, U937, Molt4, Jurkat). Genetic mutations (N-Ras, K-Ras, B-Raf, FLT3-ITD, FLT3-TKD, MLL) and polymorphisms (MDR3435CT and GST genes [A, M, T]) were detected using appropriate methods such as PCR, RFLP or DNA sequencing. MDR activities were measured using Rhodamine 123 efflux assay. Establishment of xenograft model for each cell line was tried with subcutaneous injection of leukemic cells mixed with matrigel matrix into Balb/c nude mouse. Cell lines were maintained with two times of passage per week. Western blot assay was done for changes of expression of intracellular signaling pathway proteins such as ERK, p-ERK, AKT, p-AKT, and HR23B according to the passage number. Summary of results: N-Ras mutation was found in HL60 (Gln61Leu) and Molt4 (Gly13Val), K-Ras mutation in CCRFCEM (Gly12Asp) and NB4 (Ala18Asp), B-Raf mutation in none, FLT3-ITD and MLL rearrangements in Molm-13 and MV4-11, and FLT3-TKD in none. High MDR activity was noted in KG-1 (92.5%) and other cell lines showed less than 30% of MDR activities. Mouse xenograft model was established with 7 leukemic cell lines (HL60, NB4, MV4-11, Molm-13, K562, Molt4 and CCRFCEM). ERK phosphorylation was observed in most cell lines except CCRFCEM, but AKT phosphorylation was not found in any of the cell lines. As increasing the passage number, the levels of p-ERK in K562 and HL60 significantly decreased whereas those of p-AKT in Molm-13 were elevated. Conclusion: Our study results provide genetic and biologic data for human leukemic cell lines and show the importance of passage number of the leukemic cell lines in designing experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1245. doi:10.1158/1538-7445.AM2011-1245

THE SIGNIFICANCE OF DETECTING WT1 EXPRESSION IN CHILDHOOD ACUTE LEUKEMIAS

WT1 (Wilms' tumor gene 1) overexpression is implicated in the prognosis of acute leukemia. The purpose of this study was to investigate WT1 expression and its clinical implication in childhood acute leukemia (AL) in Chinese population. Bone marrow specimen from 200 children at different stages of acute leukemia and from 21 children without leukemia were studied. The WT1 expression at diagnostic marrow specimen in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) was higher than control group, whereas WT1 expression in AML was higher than in ALL, and WT1 expression level in relapse in ALL increased more significantly than in AML. The WT1 expression level showed positive correlation with the hypodiploidy and BCR-ABL fusion gene in acute leukemia. A rapidly decrease of WT1 expression level predicted a good response to the induction therapy and low expression of WT1 correlates with remission status. This study suggested that WT1 expression levels in acute leukemia can potentially be a marker for evaluating therapeutic efficacy, correlating with monitoring minimal residue disease, and predicting hematological relapse in children acute leukemia.

Cytogenetic manifestation of chromosome 11 duplication/amplification in acute myeloid leukemia

Gene amplification is a frequent genetic abnormality in solid tumors, and many oncogenes are activated in this way. In acute myeloid leukemia (AML), a frequent target of gene amplification is chromosome 11, particularly chromosome region 11q23, including the MLL (myeloid/lymphoid leukemia) gene. However, the number of other amplicons from the long arm of chromosome 11 has also been described. Duplication/amplification of chromosome 11 was found by cytogenetic methods in 10 of 119 newly diagnosed patients with AML. The amplification was presented as: amplification including only the 5′ segment of the MLL gene (1 patient), trisomy 11 (3 patients), partial trisomy 11q (2 patients), isochromosome 11q (1 patient), and multiple amplification of specific regions (3 patients). In two cases, amplification involved parts of not only long arm but also of short arm of the chromosome 11: 11p15 and 11p11.1 to 11p13.

Abstract 2990: Analysis of chromatin structure in the Myeloid-Lymphoid Leukemia gene translocation breakpoint cluster region

Abstract The Myeloid-Lymphoid Leukemia (MLL) gene fuses to &amp;gt;50 different loci as the result of reciprocal translocations associated with several subtypes of human acute leukemia. In every case, the fusion point in MLL is within the same 8 kilobase pair (kbp) region, called the MLL breakpoint cluster region (bcr). Despite this conserved breakpoint, no translocation mechanism has been definitively described. Recently, electrophoretic mobility shift assay (EMSA) was used to determine protein binding characteristics in the MLL bcr. Proteins were noted to bind at the extreme 5’ and 3’ boundaries of the MLL bcr, but not in an internal region near the 3’ end of the MLL bcr which includes the translocation breakpoint hot spot in infant and therapy-related acute myeloid leukemia patients. The limited mobility observed with the protein-DNA complexes indicate a large protein or multiple proteins may bind at the 5’ and 3’ ends of the MLL bcr. Binding occurred with nuclear proteins from hematopoetic cells, but not with nuclear proteins from fibroblast cells and was sequence-specific as shown by competitor assays. Analysis of the most 5’ region of the MLL bcr showed several discontinuous regions are important for protein binding. Our results suggest that specific protein-DNA interactions may define the limits of the MLL bcr, and that the previously described internal hotspot does not bind the same protein complex. Binding of proteins at the boundaries of the MLL bcr prompted a study of chromatin organization within the same regions of the MLL bcr. Our initial hypothesis was that the MLL bcr regions which bound the protein complex would be relatively nucleosome depleted. Using chromatin immunoprecipitation assay (ChIP), we consistently showed a lack of histone H3 at the extreme 5’ end of the MLL bcr in Hela cells, and in the B-cell leukemia cell line, REH, suggesting a nucleosome-depleted region. In repeated experiments the 3’ MLL boundary showed minimal histone H3 binding, suggesting this area is also relatively free of nucleosomes. The internal breakage hot spot showed prominent H3 binding consistent with the presence of nucleosomes. The chromatin study suggests that the MLL bcr boundary regions are more exposed, especially in the 5’ region. This further supports our EMSA findings, that the MLL bcr may be bordered by large DNA-protein complexes. The project described was supported by NIH Grant Number P20 RR016741 from the North Dakota INBRE Program of the National Center for Research Resource, and by a Minot State University institutional research grant. 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 2990.

5′ MLL Gene Deletion in a Case with Childhood Acute Lymphoblastic Leukemia

Myeloid/lymphoid leukemia (MLL) gene rearrangements are high risk cytogenetic characteristics of acute lymphoblastic leukemia (ALL). Translocations of this gene are well defined, and their impact on the patient’s prognosis is well known, but deletions of the same region are rare, and little is known about their prognostic significance and the significance of their accompanying translocations. Here we present a case of childhood ALL with a deletion of the 5’region of the MLL gene detected by fluorescence in situ hybridization (FISH) analysis. This result also confirmed the sensitivity and efficiency of FISH analysis.

HOX expression patterns identify a common signature for favorable AML

Deregulated HOX expression, by chromosomal translocations and myeloid-lymphoid leukemia (MLL) rearrangements, is causal in some types of leukemia. Using real-time reverse transcription-PCR, we examined the expression of 43 clustered HOX, polycomb, MLL and FLT3 genes in 119 newly diagnosed adult acute myeloid leukemias (AMLs) selected from all major cytogenetic groups. Downregulated HOX expression was a consistent feature of favorable AMLs and, among these cases, inv(16) cases had a distinct expression profile. Using a 17-gene predictor in 44 additional samples, we observed a 94.7% specificity for classifying favorable vs intermediate/unfavorable cytogenetic groups. Among other AMLs, HOX overexpression was associated with nucleophosmin (NPM) mutations and we also identified a phenotypically similar subset with wt-NPM. In many unfavorable and other intermediate cytogenetic AMLs, HOX levels resembled those in normal CD34+ cells, except that the homogeneity characteristic of normal samples was not present. We also observed that HOXA9 levels were significantly inversely correlated with survival and that BMI-1 was overexpressed in cases with 11q23 rearrangements, suggesting that p19(ARF) suppression may be involved in MLL-associated leukemia. These results underscore the close relationship between HOX expression patterns and certain forms of AML and emphasize the need to determine whether these differences play a role in the disease process.

Eena Promotes Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation in Zebrafish

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena- deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, whereas eena lacking the Src homology 3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of the myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.