Chimeric Fusion Gene Research Articles

Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants

'Mexican' lime (Citrus aurantifolia Swingle) was transformed with constructs that contained chimeric promoter-gus gene fusions of phloem-specific rolC promoter of Agrobacterium rhizogenes, Arabidopsis thaliana sucrose-H(+) symporter (AtSUC2) gene promoter of Arabidopsis thaliana, rice tungro bacilliform virus (RTBV) promoter and sucrose synthase l (RSs1) gene promoter of Oryza sativa (rice). Histochemical β-glucuronidase (GUS) analysis revealed vascular-specific expression of the GUS protein in citrus. The RTBV promoter was the most efficient promoter in this study while the RSs1 promoter could drive low levels of gus gene expression in citrus. These results were further validated by reverse transcription real-time polymerase chain reaction and northern blotting. Southern blot analysis confirmed stable transgene integration, which ranged from a single insertion to four copies per genome. The use of phloem-specific promoters in citrus will allow targeted transgene expression of antibacterial constructs designed to battle huanglongbing disease (HLB or citrus greening disease), associated with a phloem-limited Gram-negative bacterium.

Molecular methods for genomic analyses of variantPML-RARAor otherRARA-related chromosomal translocations in acute promyelocytic leukemia

TO THE EDITOR: We read an interesting paper by Palta et al. in a recent issue of the Korean Journal of Hematology titled, variant of acute promyelocytic leukemia with tuberculosis: a case report and review of literature [1]. We would like to add some comments to their article and suggest additional molecular methods to confirm variant translocations in acute promyelocytic leukemia (APL). Apart from its characteristic morphology, APL is strongly correlated with presence of the PML-RARA fusion gene due to a t(15;17) translocation. Patients displaying cryptic PML-RARA gene rearrangements or other translocations involving RARA are very rare. Since RARA generates chimeric fusion genes with various partners such as ZBTB16 at 11q23, NUMA1 at 11q13, NPM1 at 5q35, FIP1L1 at 4q12, and STAT5b at 17q11.2 (Fig. 1), confirmatory molecular methods to detect gene rearrangements are necessary when unique or unusual chromosomal translocations are found in APL cases. Palta et al. reported a case of APL accompanied by tuberculosis where cytogenetic study showed a variant chromosomal translocation, t(11;17)(q23;q21). First, although they describe this case as APL with t(11;17)(q23;q21) through bone marrow (BM), immunophenotyping, and cytogenetic studies, we noted an absence of results based on molecular methods. To demonstrate the existence of the ZBTB16-RARA fusion gene, a single specific reverse transcriptase-polymerase chain reaction (RT-PCR) with a specific primer to detect the gene rearrangement or multiplex RT-PCR (e.g. HemaVision) to detect ZBTB16-RARA could have provided an accurate molecular diagnostic result. Second, the authors' reference to a paper by Kang et al. was puzzling despite the fact that it was cited as a similar case with chromosomal translocations at a similar location, in cases of AML with chromosomal translocation t(11;17) [2]. In the first case, cytogenetic analysis from BM aspirate showed t(11;17)(q23;q21) in 18 out of 20 cells while the MLL probe in fluorescence in situ hybridization (FISH) showed split-out signals in 98.5% of interphase cells. The second case also showed t(11;17)(q23;q25) in the chromosome study while the FISH assay showed split-out signals in 79% of interphase cells. However, normal fluorescence patterns were found in 200 cells in additional FISH assays when a dual-color single-fusion PML-RARA probe was used for both cases, which led to the conclusion that the MLL gene was involved in the 11q23 breakpoint and RARA was not involved in the 17q breakpoint. Hence, the authors' citation of this study to explain the ZBTB16-RARA fusion gene appears inadequate, and a molecular study could unambiguously identify the involvement of ZBTB16 at 11q23 in their case. Fig. 1 Various RARA partner genes in acute promyelocytic leukemias. We suggest the authors conduct RT-PCR analysis of the ZBTB16-RARA fusion gene if any remnant RNA or cDNA samples from their APL patient are available. However, if only genomic DNA is available, analyzing the genomic breakpoints by multiplex long distance-polymerase chain reaction (LD-PCR) would be helpful. LD-PCR or long distance inverse (LDI)-PCR can identify known or unknown partner genes, monitor minimal residual disease (MRD), and identify multi-way chromosomal translocations such as three-way or four-way translocations [3]. We have recently used LD-PCR to confirm APL cases showing unusual karyotypes or discrepant results between conventional cytogenetics and molecular studies. More precisely, we identified a novel PML-ADAMTS17-RARA rearrangement and confirmed the genomic fusion breakpoints of cryptic PML-RARA rearrangement in 2 APL patients [3-5]. In conclusion, detection of leukemia-associated fusion genes is important due to their direct relevance to diagnosis, MRD assessment, and treatment response. Since APL patients with ZBTB16-RARA are resistant to all-trans-retinoic acid (ATRA), molecular methods with higher resolution than chromosome studies must be conducted to detect fusion genes and facilitate selection of the most appropriate treatment. We believe that molecular techniques such as FISH, RT-PCR, and LD-PCR should be used in addition to morphology and cytogenetic studies in APL diagnosis and evaluation so that atypical PML-RARA or RARA variant gene rearrangements are not missed.

A t(17;22)(q21;q12) with partial ETV4 deletion in a soft tissue Ewing sarcoma

Cytogenetic analysis of a lumbar soft tissue Ewing sarcoma (ES) in a 7-month-old female child showed a t(17;22)(q21;q12), a rare translocation leading to an EWSR1-ETV4 chimeric transcript. These findings were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. The breakpoints were characterized by direct sequencing of the chimeric fusion gene. Tumor genotyping using the Affymetrix Genome-Wide Human single nucleotide polymorphism (SNP) array 6.0 Genechip identified deletions of both chromosomal regions involved in the translocation, resulting in partial deletion of ETV4, but an uninvolved EWSR1 gene. The creation of a fusion between EWSR1 and an ETS family gene consecutive to a chromosomal translocation is characteristic of the Ewing family of tumors (EFT). This is the first report of a deletion involving the two breakpoints in an EWS-ETS translocation. To date, only two cases of t(17;22)(q21;q12) in Ewing sarcoma have been reported, with no associated deletion. Interestingly, both cases had also occurred in soft tissue tumors, which are less common than their bone-involving counterparts.

Src Activation Plays an Important Key Role in Lymphomagenesis Induced by FGFR1 Fusion Kinases

Chromosomal translocations and activation of the fibroblast growth factor (FGF) receptor 1 (FGFR1) are a feature of stem cell leukemia-lymphoma syndrome (SCLL), an aggressive malignancy characterized by rapid transformation to acute myeloid leukemia and lymphoblastic lymphoma. It has been suggested that FGFR1 proteins lose their ability to recruit Src kinase, an important mediator of FGFR1 signaling, as a result of the translocations that delete the extended FGFR substrate-2 (FRS2) interacting domain that Src binds. In this study, we report evidence that refutes this hypothesis and reinforces the notion that Src is a critical mediator of signaling from the FGFR1 chimeric fusion genes generated by translocation in SCLL. Src was constitutively active in BaF3 cells expressing exogenous FGFR1 chimeric kinases cultured in vitro as well as in T-cell or B-cell lymphomas they induced in vivo. Residual components of the FRS2-binding site retained in chimeric kinases that were generated by translocation were sufficient to interact with FRS2 and activate Src. The Src kinase inhibitor dasatinib killed transformed BaF3 cells and other established murine leukemia cell lines expressing chimeric FGFR1 kinases, significantly extending the survival of mice with SCLL syndrome. Our results indicated that Src kinase is pathogenically activated in lymphomagenesis induced by FGFR1 fusion genes, implying that Src kinase inhibitors may offer a useful option to treatment of FGFR1-associated myeloproliferative/lymphoma disorders.

Discovery of Novel Recurrent Mutations in Childhood Early T-Cell Precursor Acute Lymphoblastic Leukemia by Whole Genome Sequencing - a Report From the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project

Abstract 68Early T-cell precursor acute lymphoblastic leukemia (ETP ALL) is characterized by an immature T-lineage immunophenotype (cCD3+, CD1a-, CD8- and CD5dim) aberrant expression of myeloid and stem cell markers, a distinct gene expression profile and very poor outcome. The underlying genetic basis of this form of leukemia is unknown.Here we report results of whole genome sequencing (WGS) of tumor and normal DNA from 12 children with ETP ALL. Genomes were sequenced to 30-fold haploid coverage using the Illumina GAIIx platform, and all putative somatic sequence and structural variants were validated. The frequency of mutations in 43 genes was assessed in a recurrence cohort of 52 ETP and 42 non-ETP T-ALL samples from patients enrolled in St Jude, Children's Oncology Group and AEIOP trials. Transcriptomic resequencing was performed for two WGS cases, and whole exome sequencing for three ETP ALL cases in the recurrence cohort.We identified 44 interchromosomal translocations (mean 4 per patient, range 0–12), 32 intrachromosomal translocations (mean 3, 0–7), 53 deletions (mean 4, 0–10) and 16 insertions (mean 1, 0–5). Three cases exhibited a pattern of complex rearrangements suggestive of a single cellular catastrophe (“chromothripsis”), two of which had mutations targeting mismatch and DNA repair (MLH3 and DCLRE1C). While no single chromosomal alteration was present in all cases, 10 of 12 ETP ALLs harbored chromosomal rearrangements, several of which involved complex multichromosomal translocations and resulted in the expression of chimeric in-frame novel fusion genes disrupting hematopoietic regulators, including ETV6-INO80D, NAP1L1-MLLT10, RUNX1-EVX1 and NUP214-SQSTM1, each occurring in a single case. An additional ETP case with the ETV6-INO80D fusion was identified in the recurrence cohort. Additionally, 51% of structural variants had breakpoints in genes, including those with roles in hematopoiesis and leukemogenesis, and genes also targeted by mutation in other cases (MLH3, SUZ12, RUNX1).We identified a high frequency of activating mutations in genes regulating cytokine receptor and Ras signalling in ETP ALL (67.2% of ETP compared to 19% of non-ETP T-ALL) including NRAS (17%), FLT3 (14%), JAK3 (9%), SH2B3 (or LNK; 9%), IL7R (8%), JAK1 (8%), KRAS (3%), and BRAF (2%). Seven cases (5 ETP, 2 non-ETP) harbored in frame insertion mutations in the transmembrane domain of IL7R, which were transforming when expressed in the murine cell lines, and resulted in enhanced colony formation when expressed in primary murine hematopoietic cells. The IL7R mutations resulted in constitutive Jak-Stat activation in these cell lines and primary leukemic cells expressing these mutations.Fifty-eight percent of ETP cases (compared to 17% of non-ETP cases) harbored mutations known or predicted to disrupt hematopoietic and lymphoid development, including ETV6 (33%), RUNX1 (16%), IKZF1 (14%), GATA3 (10%), EP300 (5%) and GATA2 (2%). GATA3 regulates early T cell development, and mutations in this gene were observed exclusively in ETP ALL. The mutations were commonly biallelic, and were clustered at R276, a residue critical for binding of GATA3 to DNA. Strikingly, mutations disrupting chromatin modifying genes were also highly enriched in ETP ALL. Genes encoding the the polycomb repressor complex 2 (EZH2, SUZ12 and EED), that mediates histone 3 lysine 27 (H3K27) trimethylation were deleted or mutated in 42% of ETP ALL compared to 12% of non-ETP T-ALL. In addition, alterations of the H3K36 trimethylase SETD2 were observed in 5 ETP cases, but not in non-ETP ALL. We also identified recurrent mutations in genes that have not previously been implicated in hematopoietic malignancies including RELN, DNM2, ECT2L, HNRNPA1 and HNRNPR.Using gene set enrichment analysis we demonstrate that the gene expression profile of ETP ALL shares features not only with normal human hematopoietic stem cells, but also with leukemic initiating cells (LIC) purified from patients with acute myeloid leukemia (AML). These results indicate that mutations that drive proliferation, impair differentiation and disrupt histone modification cooperate to induce an aggressive leukemia with an aberrant immature phenotype. The similarity of the gene expression pattern with that observed in the LIC of AML raises the possibility that myeloid-directed therapies might improve the outcome of ETP ALL. Disclosures:Evans:St. Jude Children’s research Hospital: Employment, Patents & Royalties; NIH & NCI: Research Funding; Aldagen: Membership on an entity’s Board of Directors or advisory committees.

Expression of CAMKIIγ, HSP70 and HSP90 Genes Are Differentially Associated with the Mutational Status of Tirosine Kinase Domain in Chronic Myeloid Leukemia Resistant Patients

Abstract 4877Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by the reciprocal chromosomal translocation between chromosome 9 and 22, t(9;22)(q34;q11), known as Philadelphia chromosome. At molecular level, this cytogenetic aberration associates with a chimeric BCR-ABL fusion gene responsible for the pathogenesis of the disease. Targeting BCR-ABL with tyrosine kinase inhibitors (TKIs) has led to a rapid clinical response in most CML cases; however, harboring of point mutations within the kinase domain (KD) of BCR-ABL is the most common mechanism responsible for acquired resistance to therapy.The objective of this work was to evaluate the expression profiles of 4 different genes involved in proliferation, differentiation or cell survival, in order to study their association with acquired missense mutations within the KD of BCR-ABL.Due to its clear involvement in cell proliferation and apotosis the following genes were analyzed. CAMKIIγ (Ca2+ /calmodulin dependent protein kinase IIγ) is a critical regulator of multiple signalling networks regulating the proliferation in myeloid leukaemia. HSP70 (Heat Shock protein 70) is a chaperone protein that protects cells from apoptotic and necrotic stimuli. HSP90 (Heat Shock protein 90) is a chaperone that facilitates folding of client proteins such as BCR-ABL. Ki-67 is a nuclear antigen associated with cellular proliferation and ribosomal RNA transcription.Total RNA was extracted from 50 TKI-resistant CML-patient’s PBMC samples; by quantitative real time-PCR (QRT-PCR) (SybrGreen method, melting analysis and β2-microglobulin as an endogenous control reference gene) we measured the gene expression of CaMKIIγ, Ki67, HSP70, HSP90 and BCR-ABL. All patients were treated with TKIs (26 imatinib, 13 nilotinib, 11 dasatinib) and showed primary lack or loss of haematological and/or cytogenetic response according to the ELN (European Leukaemia Net) criteria.To characterize TKI-resistant mutations within the KD of the chimeric BCR-ABL gene, ABL exons 4, 5, 6 and 7 were subjected to automated DNA sequencing following a nested- BCR-ABL-specific PCR. Mutations were observed in 27/50 cases at 15 different residues: 1 L248V, 1 G250E, 1 Y253H, 5 E255K/V, 1 E279K, 1 V289F, 4 T315I, 2 F317L, 1 L348M, 3 M351T, 1 E355G, 1 N358S, 2 F359V/C, 2 L387M and 1 L389V. Remaining 23 CML cases did not show mutations (detection limit 10–20%).Our results showed a significant increase in the expression of CaMKIIγ and HSP70 and decrease of HSP90 in mutated patients (MT) with respect to cases without mutations (WT) (p<0.01). On the contrary, transcript levels of Ki67 and BCR-ABL did not show significant differences between MT and WT, likely due to the resistant status of both groups.Taking into account these results we design a score (TKI-MT) to estimate the likelihoods for a patient to harbor TKI-resistance mutations using the transcript normalised expression of CaMKIIγ, HSP70 and HSP90 as follows: [Log10(CaMKIIγ) + Log10(HSP70) – Log10(HSP90)]. TKI-MT scores from 27 and 23 patients from the MT and WT groups respectively were analysed by use of ROC curves in order to find an optimal cut-off value to classify new unstudied cases. We found that patients with TKI-MT scores over a cut-off value of −0.79 showed 4.8 times more probabilities to present TKI-resistance mutations than those below −0.79 (OR 4.8, CI95% 1.3–17.6, P<0.02).We concluded that the expression of CaMKIIγ, HSP70 and HSP90 allowed prediction of mutations in the ABL tyrosine kinase domain with 82% of specificity in CML patients treated with TKIs and associated with lack or loss of response. Disclosures:No relevant conflicts of interest to declare.

Sequential Acquisition of AML1-ETO and c-Kit Mutation Leads to Formation of Human t(8;21) Acute Myelogenous Leukemia Stem Cells,

Abstract 3584The 8;21 translocation, one of the most general chromosomal abnormalities in acute myelogenous leukemia (AML), encodes the AML1-ETO chimeric fusion gene. Because AML1-ETO can inhibit the CBF complex to transactivate myeloid-lineage genes in a dominant negative fashion, the high level expression of this gene plays a critical role in inhibiting differentiation of target cells, which leads to progression of AML. We, however, have reported that patients maintaining a long-term remission retain AML1-ETO expression at a very low level that can be detected by nested RT-PCR. The AML1-ETO transcripts in these patients were derived from a small fraction of t(8;21)+ hematopoietic stem cells (HSCs) capable of multilineage differentiation (PNAS 2000). In fact, previous data shown that AML1/ETO knock-in or AML1/ETO transgenic mice did not develop AML. These data suggest that acquisition of the AML1-ETO fusion is not sufficient to develop t(8;21) AML. Since t(8;21) AML cells frequently possess constitutive active mutation of c-Kit, we hypothesized that the c-Kit mutation may work as a second oncogenic hit in t(8;21)+ HSCs to transform into AML. To test the hypothesis, we extensively analyzed the existence of c-Kit mutation within AML1-ETO+ HSCs from patients maintaining remission for a long-term. CD34+CD38− HSCs were purified from the bone marrow of patients in long-term remission, and were cultured in vitro to form colonies. These HSC-derived colonies were picked up, and tested for the presence ofAML1-ETO and c-Kit mutation. Five t(8;21) AML patients with c-Kit mutation were enrolled in this study. All of 1020 blastic colonies at diagnosis were positive for both AML1-ETO and c-Kit mutation. In 7187 colonies formed in the culture of remission marrow, almost 1% (89 colonies) of these colonies expressed AML1-ETO. Surprisingly, none of these colonies possessed c-Kit mutation, indicating that AML1-ETO+ clones in remission are not identical to these in t(8;21) AML. Accordingly, it is highly likely that HSCs first acquire AML1-ETO, and a fraction of these cells additionally mutated c-Kit, resulting in transformation into AML stem cells. This is the first clear-cut evidence that human HSCs transform into AML via multi-step oncogenesis in vivo. Disclosures:No relevant conflicts of interest to declare.

Gene Mutations and Minimal Residual Disease (MRD) As Predictors of Remission Duration in Adults with Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) Treated with High-Dose Cytarabine (HDAC) - First Results of the Prospective French Intergroup CBF-2006 Trial

Abstract 410 Background.CBF-AML is a favorable AML subset cytogenetically defined by t(8;21) or inv(16)/t(16;16) rearrangements, respectively responsible for RUNX1-RUNX1T1 (CBFa) or CBFB-MYH11 (CBFb) gene fusion. Nonetheless, relapse incidence may reach 30–40% in these patients. Even if not yet prospectively validated, early MRD monitoring based on chimeric fusion gene transcript quantification by RQ-PCR may help to define patients at higher risk of relapse. Detection of activating KIT and FLT3 gene mutations has also been retrospectively associated with higher relapse rate in these patients. The French CBF-2006 trial (NCT00428558) aimed to prospectively evaluate both markers as predictors of remission duration in homogeneously treated patients. Methods.Patients (18–60y) with newly diagnosed CBF-AML were eligible. All were studied for KIT exon 8/17, FLT3-ITD, FLT3-D835, N-RAS and K-RAS mutations. They were randomized to receive timed-sequential or standard induction. Timed-sequential induction was cytarabine (AraC) 500 mg/m2 CI D1-3 and daunorubicine (DNR) 60 mg/m2 D1-3, followed by AraC 1 g/m2/12h D8-10 and DNR 35 mg/m2 D8-9. Standard arm was AraC 200 mg/m2 CI D1-7 and DNR 60 mg/m2 D1-3, followed by the second sequence at D16-18 only in case of persistent bone marrow (BM) blasts at D15. Patients in first complete remission (CR1) received 3 HDAC consolidation cycles with AraC 3 g/m2/12h D1/3/5. Fusion gene expression was quantified at baseline and before each HDAC cycle (MRD1, MRD2, MRD3) by the 100 × fusion gene/cABL ratio. Patients not reaching a 3-Log ratio reduction at MRD2 were offered allogeneic stem cell transplantation (SCT) in CR1 if they had an HLA-identical donor or could enter a single-agent dasatinib sub-study (see Boissel et al., this meeting). Results.Among the planned 200 patients randomized, 198 met eligibility criteria (96 CBFa and 102 CBFb, compared in Table 1). Overall, incidences of KIT, RAS, FLT3-D835 and FLT3-ITD mutations were 21%, 29%, 10% and 6%, respectively. Patients with KIT and FLT3-D835 mutations had higher WBC (P=0.04 and 0.02) and BM blast percentage (P=0.0004 and 0.05). Those with RAS mutations had lower BM blast percentage (P=0.01) and baseline transcript ratio (P=0.001). Overall, 196 patients reached CR1 and 55 relapsed. At 3 years, probabilities of durable remission, disease-free (DFS) and overall survival from CR were 64%, 61% and 84%, respectively, without differences between the CBF subsets or induction arms. Fifty-five patients did not reach the 3-Log MRD2 reduction (18 CBFa and 37 CBFb, P=0.006), 9 receiving SCT in CR1. Poor MRD2 response correlated with age (P=0.01), WBC (P=0.04), KIT mutations (P=0.04), and initial transcript ratio (P=0.004). Among the characteristics listed in Table 1, shorter remission duration was associated with high WBC (P=0.011), high BM blast percentage (P=0.062), del(9q) (P=0.041), KIT mutation (P=0.048), FLT3-D835 mutation (P=0.077), high initial transcript ratio (P=0.053), and <3-Log MRD2 reduction (P=0.006) in univariate analysis stratified by CBF subtype and treatment arm. In multivariate analysis, the three latter factors, but not KIT mutations, remained independently predictive of shorter remission duration and DFS (P= 0.014, 0.033, 0.002 and 0.001, 0.012, 0.005, respectively). Censoring patients receiving SCT in CR1 yielded similar results. Conclusions.This prospective study of 198 CBF-AML patients homogeneously treated with HDAC revealed significant correlations between initial characteristics and response to therapy. Prognostic analysis did not find that KIT mutations had independent impact on outcome, which was rather influenced by baseline fusion transcript ratio and post-consolidation MRD reduction (reminiscent of Schnittger et al. 2003), and FLT3-D835 mutation. Ongoing pan-genomic analyses might help to further dissect CBF-AML heterogeneity.Table 1Patient characteristicsCBFa (N = 96)CBFb (N = 102)P valuesMedian age42y42y0.37Median WBC, G/L1122<0.001Median BM blast percentage52570.10Therapy-related AML1020.016Loss of Y chromosome*330<0.001Tri 22*018<0.001Tri 8*2150.002Del(9q)*141<0.001Del(7q)/–7*450.99KIT exon 8/17/both mutation**5/16/112/6/00.38FLT3 D835/ITD/both mutation**4/6/015/4/10.11N/K-RAS mutation**13/228/110.001Median baseline transcript ratio30389<0.001>3-Log ratio reduction at MRD2***71580.006*196,**194, and***184 patients studied. Disclosures:No relevant conflicts of interest to declare.

Neoplastic MiR‐17∼92 deregulation at a dna fragility motif (SIDD)

Chromosomal or mutational activation of BCL6 (at 3q27) typifies diffuse large B-cell lymphoma (DLBCL) which in the germinal center subtype may be accompanied by focal amplification of chromosome band 13q31 effecting upregulation of miR-17~92. Using long distance inverse-polymerase chain reaction, we mapped and sequenced six breakpoints of a complex BCL6 rearrangement t(3;13)(q27;q31)t(12;13)(p11;q31) in DLBCL cells, which places miR-17~92 antisense within the resulting ITPR2-BCL6 chimeric fusion gene rearrangement. MiR-17~92 members were upregulated ~15-fold over controls in a copy number independent manner consistent with structural deregulation. MIR17HG and ITPR2-BCL6 were, despite their close configuration, independently expressed, discounting antisense regulation. MIR17HG in t(3;13)t(12;13) cells proved highly responsive to treatment with histone deacetylase inhibitors implicating epigenetic deregulation, consistent with which increased histone-H3 acetylation was detected by chromatin immunoprecipitation near the upstream MIR17HG breakpoint. Remarkably, 5/6 DNA breaks in the t(3;13)t(12;13) precisely cut at stress-induced DNA duplex destabilization (SIDD) peaks reminiscent of chromosomal fragile sites, while the sixth lay 150 bp distant. Extended SIDD profiling showed that additional oncomiRs also map to SIDD peaks. Fluorescence in situ hybridization analysis showed that 11 of 52 (21%) leukemia-lymphoma (L-L) cell lines with 13q31 involvement bore structural rearrangements at/near MIR17HG associated with upregulation. As well as fueling genome instability, SIDD peaks mark regulatory nuclear-scaffold matrix attachment regions open to nucleosomal acetylation. Collectively, our data indict a specific DNA instability motif (SIDD) in chromosome rearrangement, specifically alterations activating miR-17~92 epigenetically via promoter hyperacetylation, and supply a model for the clustering of oncomiRs near cancer breakpoints.

Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

BackgroundBioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors.ResultsThe tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth.ConclusionLentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi-cassette vectors in combination with various inserts of interest.

Tomato polyphenol oxidase B is spatially and temporally regulated during development and in response to ethylene.

Plant polyphenol oxidases (PPOs) are ubiquitous plastid-localized enzymes. A precise analysis of PPO function in plants has been complicated by the presence of several family members with immunological cross reactivity. Previously we reported the isolation of genomic clones coding for the seven members of the tomato (Solanum lycopersicum) PPO family (A, A’, B, C, D, E, and F). Here we report the complex spatial and temporal expression of one of the members, PPO B. The PPO B promoter was sequenced and subjected to homology analysis. Sequence similarities were found to nucleotide sequences of genes encoding enzymes/proteins active in the following systems: phenylpropanoid biosynthesis, signal transduction and responsiveness to hormones and stresses, fruit and seed proteins/enzymes, and photosynthesis. Chimeric gene fusions were constructed linking PPO B 5′ flanking regions to the reporter gene, β-glucuronidase (GUS). The resultant transgenic plants were histochemically analyzed for GUS activity in various vegetative and reproductive tissues, and evaluated for PPO B responsiveness to ethylene induction. It was shown that PPO B expression was tissue specific, developmentally regulated, ethylene induced, and localized predominantly to mitotic or apoptotic tissues.

DEK/NUP214 Fusion Gene Predicts Good Outcome In Acute Myeloid Leukemia In Complete Remission After Allogeneic Hematopoietic Stem Cell Transplantation

AbstractAbstract 2386 IntroductionAcute myeloid leukemia (AML) with chromosomal translocation (6;9)(p23;q34) results in a chimeric fusion gene between DEK/NUP214 (formerly DEK/CAN), and is a rare disease, accounting for approximately 1% of cases. This entity is considered to be a good indication for allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to its poor prognosis and non-responsiveness to chemotherapy. However, whether the prognosis of patients with exclusive DEK/NUP214 abnormality is improved by allo-HSCT has not been demonstrated. We conducted a retrospective study to examine the outcomes of allo-HSCT in AML patients with the DEK/NUP214 gene using the databases of the Japan Society for Hematopoietic Cell Transplantation (JSHCT) and the Japan Cord Blood Bank Network (JCBBN). MethodsWe identified de novo AML patients who underwent transplant between January 1996 and December 2007 with no prior treatment by allo-HSCT. Clinical features and outcomes of the patients with DEK/NUP214 abnormality and 3 different control groups (patients with chromosome 7 abnormalities, complex karyotype or normal karyotype) were compared. ResultsOf 4517 AML cases meeting the above criteria, the number of patients with normal karyotype, chromosome 7 abnormalities, complex karyotype and DEK/NUP214 abnormality was 2227, 139, 416 and 64, respectively. The median age of each group was 41, 37, 41 and 34 years, respectively; patients with the DEC/NUP214 abnormality were significantly younger than those with the complex karyotype, and patients with normal chromosomes. Gender was not biased among the groups. Overall survival (OS) of the patients with DEC/NUP214 abnormality at 5 years was 49%, which was superior to the OS of the patients with normal karyotype, chromosome 7 abnormalities and complex karyotype (44%, 30% and 27%, respectively; P < 0.0001; Figure). This trend was significant in HSCT grafts with bone marrow and peripheral blood stem cells. In patients with DEK/NUP214 abnormality, none of the following factors improved survival; combination of gender between patient-donor, dose of total body irradiation prior to HSCT; and use of prophylactic immunosuppressants for graft-versus-host disease. However, the status at HSCT had a significant impact on outcome (5-year OS: 66% in patients in complete remission (CR) at HSCT vs. 31% in non-CR patients; P < 0.001). Analysis exclusively for the patients in CR at HSCT showed similar results as above. [Display omitted] ConclusionOur results suggest that patients with the DEK/NUP214 abnormality are more likely to respond to allo-HSCT. Allo-HSCT should be particularly recommended for patients experiencing CR after prior remission-induction chemotherapy. Disclosures:No relevant conflicts of interest to declare.

TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia.

AbstractAbstract 3624 Introduction:The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aimof our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods:the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results:The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions:in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures:No relevant conflicts of interest to declare.

Establishment and Characterization of a Novel Human Myeloid Leukemia Cell Line, AMU-AML1, Carrying t(12;22)(p13;q11) with No Fusion of MN1-TEL

AbstractAbstract 4375Leukemia cell lines are ubiquitous powerful research tools that are available to many investigators. In balanced chromosomal aberration in leukemia, a chimeric fusion gene formed by genes existing on breakpoints is frequently related to leukemogenesis. Cytogenetic abnormalities of chromosome band 12p13 are detected non-randomly in various hematological malignancies and usually involved TEL, which encodes a protein of the ETS transcription factor family. Chromosome band 22q11-12 is one of partners of translocation 12p13 and t(12;22)(p13;q11-12) results in fusion of TEL and MN1 or in just the partial inactivation of TEL. It is important to analyze precisely the breakpoint in a non-random translocation such as t(12;22)(p13;q11-12) and in addition it contributes to the better understanding of the molecular pathogenesis of leukemogenesis.In this study, we established a novel human myeloid leukemia cell line, AMU-AML1, having t(12;22) from a patient with acute myeloid leukemia with multilineage dysplasia and analyzed its characters. Mononuclear cells were isolated by Ficoll-Hypaque sedimentation from patient's bone marrow before initiation of chemotherapy and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS). After 3 months, cell proliferation became continuous. The cell line, named AMU-AML1, was established. In AMU-AML1, the following pathogens were negative for EBV, CMV, HBV, HCV, HIV-1, HTLV-1 and mycoplasma. A doubling time of AMU-AML1 cells was about 96 hours. Proliferation of the cells was stimulated by rhG-CSF (10 ng/ml), rhGM-CSF (10 ng/ml), M-CSF (50 ng/ml), rhIL-3 (10 ng/ml) and rhSCF (100 ng/ml) but not by IL-5 (10 ng/ml), rhIL-6 (10 ng/ml), and rhEPO (5 U/ml). AMU-AML1 was positive for CD13, CD33, CD117 and HLA-DR, negative for CD3, CD4, CD8 and CD56 by flow cytometry analysis. G-banding combined with SKY analysis of AMU-AML1 cells showed single structural abnormality; 46, XY, t(12;22)(p13;q11.2). Double-color FISH using PAC/BAC clones listed in NCBI website and array CGH analyses indicated that the breakpoint in 12p13 was within TEL or telomeric to TEL and it of 22q11 was centromeric to MN1. A chimeric MN1-TEL transcript and fusion protein of MN1-TEL could not be detected by RT-PCR and western blot analysis. The wild type of MN1 protein was strongly expressed in AMU-AML1 compared with other leukemic cell lines with t(12;22), MUTZ-3 and UCSD/AML1.Our data suggest that AMU-AML1 had a t(12;22)(p13;q11.2) without fusion of MN1-TEL and the expression level of MN1 protein was relatively high, which might have some effects on leukemogenesis. In conclusion, AMU-AML1 is a useful cell line to analyze the biological consequences of the leukemic cells with t(12;22)(p13;q11.2) but no fusion of MN1-TEL. Disclosures:No relevant conflicts of interest to declare.

Expression of chemokine receptor CXCR3, CXCR4, and CXCR7 and their respective ligands in rhabdomyosarcoma

Rhabdomyosarcomas (RMS) are the most common malignant soft-tissue tumors of childhood, and nearly 15% of cases develop metastatic disease. They are histologically divided into two main subtypes: embryonal rhabdomyosarcomas (eRMS), which usually gain extra copies of whole chromosomes, and alveolar rhabdomyosarcomas (aRMS), more aggressive, with characteristic reciprocal translocations in about 80% of the tumors. The resulting chimeric fusion genes involve PAX3 or PAX7 and FKHR, and encode fusion proteins with enhanced transcriptional activity.

Massive Ascites Associated with All-trans Retinoic Acid Treatment in Therapy-Related Acute Promyelocytic Leukemia

A 77-year-old man who developed pancytopenia was administered granulocyte colony-stimulating factor (G-CSF) by another doctor, and referred to us for the evaluation of pancytopenia. He had hepatocellular carcinoma and was treated with transcatheter arterial chemoembolization (TACE) containg epirubicin (total dose: 300 mg over the last two years). Bone marrow aspiration smear demonstrated hypercellular marrow with promyelocytes. Cytogenetic analysis demonstrated del(7), t(15;17)(q22;q12), and a fluorescence in-situ hybridization (FISH) study demonstrated chimeric fusion genes of PML-RAR-alpha. He was diagnosed with therapy-related acute promyelocytic leukemia (APL), and treated with all trans-retinoic acid (ATRA). He showed the progressive accumulation of ascites with liver damage, without pulmonary symptoms of ATRA differentiation syndrome. After 60 days of ATRA treatment, complete hematological and cytogenetic responses were achieved. However, the patient died of septic circulatory failure.

BCR-ABL DERIVED PEPTIDE VACCINES FOR CHRONIC MYELOID LEUKAEMIA

Chronic Myeloid Leukemia (CML) is a myeloproliferative pluripotent stem cell disorder characterized by the presence of a cytogenetic hallmark, the Philadelphia (Ph) chromosome, and accounts for 15% of adult leukemias. The disease progresses from a chronic phase through an accelerated phase to a blast phase and its natural course accounts for a median 4 years survival1. The Ph chromosome is derived by a reciprocal translocation termed t(9;22) in which the c-abl oncogene has moved from chromosome 9 into the breakpoint cluster region (bcr), within the bcr gene on chromosome 22, resulting in a chimeric bcr-abl fusion gene that encodes a 210 KD protein (p210) with constitutive tyrosine kinase activity. Two major alternative chimeric p210 can result from this fusion gene: p210-b2a2 where the junction occurs between bcr exon 2 (b2) and abl exon 2 (a2) and p210-b3a2 where the the junction occurs between bcr exon 3 (b3) and abl exon 2 (a2. About 40% of CML patients harbor the p210-b2a2 and about 60% of them show the p210-b3a2.

Amplification of Histone Methyltransferase, Mixed-Lineage Leukemia (MLL), Perturbs Normal Hematopoiesis and Induces Hematological Disorder.

Abstract 1965Poster Board I-988 Introduction:The Mixed-Lineage Leukemia Gene, MLL, encoding a SET domain with an H3K4 methyltransferase, is frequently mutated in a wide range of hematological malignancies. Apart from formation of chimeric fusion genes, MLL is amplified (MLLn) or partially tandem duplicated (MLL-PTD) in approximately 1% of acute myeloid leukemia (AML) and 4-7% of cytogenetically normal AML patients, respectively. MLLn is also observed in myelodysplastic syndrome and rare cases of pre-B ALL. While the roles of MLL fusion proteins in leukemogenesis are relatively well defined, very little is known about MLLn and MLL-PTD. In contrast to MLL fusions, MLL-PTD appears to be a weak oncoprotein, which could perturb normal hematopoiesis but was insufficient to induce leukemia in a murine model. MLLn is the only reported MLL aberration resulting in structural amplification of the SET domain, but also is the least characterized one with no animal models available. Its role and the underlying mechanisms for transformation are completely unknown. Materials and Methods:Although animal models have been established for MLL fusions using retroviral transduction/transplantation approaches, it has not been possible to model MLLn using similar approaches due to the size of the cDNA (>10kb). Similarly, knockin approach used for MLL-PTD will not be feasible for introducing any significant change of number copy. To this end, we have adopted a transgenic approach where human MLL cDNA under the control of SCL promoter/enhancer protected by globin insulator elements (MLLn). The employed SCL elements could drive expression of the transgene in HSCs and early progenitors, while the insulators could ensure proper transgene expression and protect any interference due to positional effect of random integration. MLL-PTD mice generated by a knockin approach were also analyzed and compared side-by-side with the MLLn mice. Results:Two independent MLLn mice with 10-15 and 80-100 copies of MLL transgene, which are in line with the corresponding ranges identified in AML patients, were obtained and characterized. Over-expression of MLL in various hematological organs including bone marrow, spleen and thymus positively correlated with the copy number of the transgene, confirming the molecular features of the models. Although these mice exhibited normal numbers and ratios of phenotypically defined hematopoietic compartments including HSCs and early progenitors (CMP, CLP, GMP, MEP) at young ages, their functional properties were clearly perturbed. In both myeloid and lymphoid CFC assays, a significant increase in colonies was observed in bone marrow cells and splenocytes from both MLLn and MLL-PTD mice. An enhanced replating ability was also observed in splenocytes although only MLL-PTD cells could form colonies after the third round. Bone marrow cells from both models exhibited a higher cell proliferation rate, and had an increased proportion of cycling cells in S phase. In contrast to MLL-PTD mice, a decrease in cellularity was found in the spleen of MLLn mice, which had however an expansion of Mac1+Gr1+ myeloid cells. Similar to MLL-PTD mice, most of the MLLn mice did not develop leukemia after 18 months of observation. However, about 10% of MLL(80-100) mice developed tumor(s) at about 12 months, while none were found in the littermate controls or MLL-PTD mice. The tumors were hematopoietic in origin and composed of mostly lymphoid (80%) and some myeloid (20%) cells. Most of the B-cells were in the pre-B fraction with oligo-clonal IgH rearrangement, suggesting a lymphoproliferative disorder (LPD). Conclusions:The initial discovery of enzymatic activity associated with oncogenic transcription factors has fuelled the enthusiasm of developing inhibitors for these classically untraceable targets, however the functional significance of H3K4 methyltransferase activity in MLL mediated pathogenesis remains elusive. We have now developed novel MLLn disease models where full-length MLL, including the catalytic SET domain, was amplified between 10-100 times. MLL amplification triggered various degrees of perturbation in different hematopoietic tissues, promoted cell growth and enhanced replating ability in vitro. Also in vivo, a small percentage of mice went on to develop a LPD. Together, these results reveal a critical function of MLLn and potentially its associated H3K4 methyltransferase activity in disease progression. Disclosures:No relevant conflicts of interest to declare.

Complexity of Clonal Architecture in Childhood Acute Lymphoblastic Leukaemia

Abstract LBA-5▪▪This icon denotes an abstract that is clinically relevant.Intra-clonal, mutational complexity is a hallmark feature of cancer and provides the substrate for sub-clonal selection, progression and therapeutic resistance. There is limited insight however into detailed sub-clonal, genetic architecture in cancers and their propagating 'stem' cells. Childhood acute lymphoblastic leukaemia (ALL) genotypes are characterized by chimeric fusion genes (or hyperdiploidy) coupled with recurrent, copy number alterations (CNA), primarily in genes regulating cell cycle or differentiation. For ETV6-RUNX1-positive cases of B precursor ALL, the temporal sequence of events is known with the fusion gene usually arising as a pre-natal, initiating event. Recurrent CNA, presumed to be functional or 'driver' mutations, are secondary to gene fusion and probably post-natal in origin. We have interrogated clonal architecture by multiplexed FISH analysis of single cells using probes labelled with three or four distinct fluorochromes. We pre-selected diagnostic samples from 30 ETV6-RUNX1-positive cases that we found to have deletion of ETV6 and CDKN2A or PAX5 or combinations of these. The application of multiple FISH probes allowed us to score all cells (200 per patient sample) for up to six genetic lesions: ETV6-RUNX1 fusion (and multiple copies of the fusion), deletion of the unrearranged ETV6 allele, extra copies of chromosome 21q (via RUNX1 signals), and one or two copy deletions of PAX5 and CDKN2A. The genetic classification of individual cells using this method allowed a designation of sub-clones and the assembly of putative ancestral, or evolutionary, trees. Although sub-clones were previously known to exist at diagnosis of ALL, the extent of sub-clonal diversity revealed by this study was unanticipated and very marked. Sub-clones, of varying sizes (1–90% of total cells), numbered 3 to 14 per case. This must be an under-estimate of genetic complexity as only selected mutations were included in the screen. The common CNA (ETV6, CDKN2A and PAX5 deletions, chromosome 21q gains) arose independently and recurrently in sub-clones and in no preferential order. Matched relapse and diagnosis pairs were available on 5 patients. In all of these, the clones at relapse could be matched to individual sub-clones at diagnosis, with relapse originating from a major or minor subclone at diagnosis. Importantly, the relapse clone often diversified further, reiterating the evolution pathway of the primary clones. These data indicate that sub-clonal diversification does not arise via linear, clonal succession but rather has a complex, branching architecture reminiscent of Charles Darwin's 1837 evolutionary speciation model. The ancestral trees so revealed in ALL are single time point snapshots and therefore disguise temporal or sequential dynamics. We show that sub-clonal architecture changes in the months leading up to a diagnosis of ALL and is re-ordered by treatment and subsequent relapse. An important prediction derived from this pattern of sub-clonal architecture is that leukaemia propagating or 'stem' cells in ALL (and other cancers) should themselves be genetically diverse. Preliminary transplantation experiments in NOD/SCID/IL2Rgamma(null) mice show that this is the case, since multiple, genetically distinct sub-clones regenerated in vivo (Figure1). The complexity of genetic architecture in ALL has substantial implications for the cancer stem cell concept and for the efficacy of generic or targeted treatments. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

Interphase FISH on TEL/AML1 positive acute lymphoblastic leukemia relapses – analysis of clinical relevance of additional TEL and AML1 copy number changes

TEL/AML1 (ETV6/RUNX1) fusion resulting from the translocation t(12;21)(p13;q22) constitutes the most common chimeric fusion gene in initial childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) (19-27%) and has been associated with good prognosis. Three secondary aberrations in TEL/AML1 positive ALL have been suspected to negatively influence outcome: deletion of the second TEL allele (T), gain of the second AML1 allele (A) and duplication of the derivative chromosome 21 (der(21), TA). Many studies have explored such aberrations in initial disease, while only few reports have investigated them in relapses. In this study, bone marrow samples from 38 children with relapsed TEL/AML1 RT-PCR positive and negative BCP-ALL were analyzed for these mutations by interphase fluorescence in situ hybridization and results were compared with published data. In children with TEL/AML1 positive ALL relapse, additional (a) TEL loss, (b) combined AML1 and der(21) gain, (c) combined TEL loss and AML1 gain as well as (d) the occurrence of a subpopulation with the signal pattern 1T/3A/1TA appear to be related to higher peripheral blast counts (PBCs) at relapse diagnosis (a and d) or a tendency towards the occurrence of a subsequent relapse (b and c) (P-values <0.05). Our data together with published results on TEL/AML1 positive ALL suggest that frequencies of additional TEL and AML1 mutations are, with the exception of loss of untranslocated TEL, higher in first relapses than in initial disease. They also show that it is important to consider combined mutations in the analysis of this leukemia entity.