Journal articles on the topic 'Leukaemia; microarray; gene expression'
To see the other types of publications on this topic, follow the link: Leukaemia; microarray; gene expression.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Leukaemia; microarray; gene expression.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Noman, Helal Mohammed Mohammed Ahmed, Yahya Saleh Al-Matary, Subbaiah Chary Nimmagadda, Pradeep Kumar Patnana, Longlong Liu, Lanying Wei, Daria Frank, Georg Lenz, and Cyrus Khandanpour. "Leukaemia Cells Induced Metabolic Alterations in AML Associated Mesenchymal Stem Cells Via Notch Signalling." Blood 138, Supplement 1 (November 5, 2021): 4347. http://dx.doi.org/10.1182/blood-2021-144468.
Full textAbstract:
Abstract Introduction: Acute myeloid leukaemia (AML) is a haematological malignancy with a high relapse rate and poor prognosis. Leukaemia cell proliferation is dependent on its interaction with the bone marrow (BM) microenvironment. AML associated mesenchymal stem cells (AML-MSCs) supported the proliferation of leukaemia cells and contributed to disease progression. Stromal microenvironment promoted a metabolic switch but precise underlying molecular mechanisms are poorly understood. Previous studies have demonstrated transfer of functional mitochondria from AML-MSCs to AML blasts facilitating energy requirements. To further improve our understanding of the crosstalk between leukaemia and AML-MSCs, we sought to determine contribution of AML-MSCs and signalling cascades regulating metabolic processes. Methods: Sorted MSCs from non-leukaemic and MLL-AF9 leukaemic mice were isolated, and gene expression profiling was performed using RNA microarray. Additionally sorted MSCs from long-term cultures were cultured alone or with MLL-AF9 leukaemia cells and analysed by RNA-sequencing. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML-MSCs. We further cocultured murine wild type BM-MSCs alone or together with murine AML cells (C1498 and MLL-AF9) or the control lineage negative cells (Lin -). Metabolic alterations, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analysed by Agilent Seahorse XFe96 analyser. Additionally, glucose consumption, lactate secretion and mitochondrial DNA copy number were measured. Results: Microarray analysis in sorted MSCs from leukaemic and non-leukaemic mice have identified hallmark oxidative phosphorylation (p<0.01, NES=-1.6) and glycolysis (p<0.01, NES=-1.3) gene sets to be negatively enriched in AML-MSCs. Interestingly, both the gene sets were also negatively enriched in sorted AML-MSCs when cocultured with leukaemia but not control cells. To validate these findings, we analysed OCR and EACR in WT-MSCs in an identical setting. The oxidative phosphorylation was significantly decreased in MSCs cocultured with C1498 (p<0.0001) and MLL-AF9 (p<0.005) but not with Lin - cells. Interestingly, glycolysis rate, glucose consumption, lactate secretion were significantly decreased in MSCs cocultured with leukaemia cells. Mitochondrial DNA copy number were significantly decreased in MSCs cocultured with C1498 (p<0.001) or MLL-AF9 (p<0.005) but not with control cells. Recent evidence from the lab has demonstrated an essential role for Notch signalling in the leukaemia and AML-MSCs interaction. To functionally determine the crosstalk of leukaemia-MSC interaction and subsequent Notch signalling, we ectopically expressed the Notch intracellular domain (Notch-ICN1) to mimic Notch activation in a murine stromal cell line, MS-5. Confirming Notch activation, Hes1 mRNA expression (encoding a transcriptional target of Notch signalling) was significantly increased in these cells. Underscoring a role for Notch signalling and activation, Notch-ICN1 overexpression in MS-5 cells demonstrated less oxidative phosphorylation and glycolysis rates as compared to MS-5 cells transduced with empty vector. Conclusion: In line with our microarray and GSEA analysis, our findings confirmed that leukaemia cells indeed induced metabolic alterations decreasing oxidative phosphorylation and glycolysis, and thereby potentially altering AML-MSCs function. At the molecular level, Notch signalling (via upregulated Notch1 and 2 expressions and Notch-ICN) in AML-MSCs contributed to metabolic alterations. Therefore, therapeutically interfering this pathway could target the bidirectional interaction between leukaemia and AML-MSCs improving therapeutic efficacy of AML. Disclosures Khandanpour: GSK: Honoraria; Takeda: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria, Research Funding; Pfizer: Honoraria; Sanofi: Honoraria, Research Funding; BMS/Celgene: Honoraria.
2
Mills, Ken I., Torsten Haferlach, Jesus M. Hernandez, Wolf-Karsten Hofmann, Alexander Kohlmann, Mickey Williams, and Lothar Wieczorek. "An International Multi-Center Microarray Study for the Molecular Classification of Leukemia Identifies Novel Sub-Groupings in MDS Overlapping with AML." Blood 108, no. 11 (November 16, 2006): 852. http://dx.doi.org/10.1182/blood.v108.11.852.852.
Full textAbstract:
Abstract Microarrays can identify robust gene expression signatures associated with distinct sub-classes of paediatric and adult leukemias. Recently, the MILE (Microarray Innovations in LEukemia) study has analysed 1901 expression profiles from retrospective samples in 11 centres (ELN: 7, USA: 3, Singapore: 1). MILE has compared the microarray classification accuracy of 16 acute and chronic leukaemia subclasses, MDS, and non-leukaemia as control group, to routine diagnostic workup. The achieved cross-validation accuracy was very high for the leukaemia subclasses: ~96%. Included in the study were 175 samples diagnosed as MDS, however, only 49.1% of these samples were correctly called as MDS from their underlying gene expression profiles. The remainder were approximately equally split between a call of “non-leukaemia” (24%) and “AML” (24.6%). A further sub-division of MDS samples called “AML”: 81% called as “AML with normal or other cytogenetics”; and the remainder as “AML with complex cytogenetics”. MDS is a heterogeneous group of disorders with a wide range in blast cell count, cytogenetics and number of cytopenias. Our analysis showed that neither study centre nor age were a factor in differentiating between “MDS”, “AML” or “non-leukaemia”. However, WHO classification was highly correlated with the microarray classification result; specifically RAEB(I or II) was associated with “AML” call (p < 0.0001). RA/RARS was highly correlated with “MDS” or “non-leukaemia” calls. Furthermore, IPSS was significantly correlated with call (p>0.0001): 65% of patients with an IPSS score of Int-2 or above were classified as “AML”. Examination of the individual components of the IPSS showed that two patients classified as “AML” had a blast count of >20%, under the WHO definition these would be defined as AML and were excluded. Individually, the blast, karyotype and cytopenia contributions were highly significant (p<0.0001, <0.013 and <0.0001 respectively) when comparing “AML”, “MDS” and “non-leukemia” calls. All the “non-leukemia” patients had <10% blasts, with 85% (34/40) having a cytogenetic score of 0 (Normal or Good (1 of: -Y, del(5q), del(20q))) and 82.5% having only 0 or 1 cytopenias. In contrast, 45% of the “AML” samples had between 11 & 20% blasts, 32% with intermediate (0.5) or poor/complex (1) cytogenetic score and 79% had 2 or 3 cytopenias. Furthermore, survival data was available for 122 of the diagnosed MDS patients and showed that MDS patients called “AML” had a trend towards shorter survival (2P=0.2) than those called “MDS” or “non-leukaemia”. These analyses, in combination with gene expression signatures, may contribute to a redefinition of MDS classifications.
3
Guo, Dachuan, Alex Fong, Andy Lail, Maree O’Sullivan, Glenn Stone, Harri Kiiveri, Michael Henry, Dietrich Stephan, Luce Dalla-Pozza, and Daniel R. Catchpoole. "Simplifying Complex Microarray Data To Derive Gene Expression Profiles Which Identify Childhood Acute Lymphoblastic Leukaemia Patients at Risk of Relapse." Blood 106, no. 11 (November 16, 2005): 4506. http://dx.doi.org/10.1182/blood.v106.11.4506.4506.
Full textAbstract:
Abstract The optimal treatment of patients with childhood acute lymphoblastic leukaemia (ALL) depends on establishing accurate diagnosis. Our investigations seek to strategically develop the application of microarray gene expression profiling to identify ALL patients with clinically homogenous presentations but which may respond differently to established treatment regimens. We have determined the gene expression profiles of ALL bone marrow (BM) samples taken from patients at diagnosis. Data analysis has focussed on the use of a novel and innovative statistical technology, Gene-RaVE. This series of patent protected algorithms builds a multinomial regression model using Bayesian variable selection. Gene-RaVE leads to the generation of a parsimonious and simple diagnostic gene expression signature, but which provides increased predictive ability over current analysis approaches. We describe our analysis of both Affymetrix (HU133A) and cDNA (10.5K) microarray gene expression profiles generated from diagnostic BM from >100 ALL patients covering the broad ALL subtypes including T and B lineage as well as T cell lymphoma leukaemia. Comparison of gene expression data failed to identify clearly distinguishing profiles between patients identified as ‘standard risk’ from ‘medium risk’ according to BFM95 clinical criteria. Gene expression profiles from a cohort of ALL patients, identified as ‘standard risk’ at diagnosis, were compared on the basis of their overall clinical outcome: relapse within 2 yrs vs non-relapse. Using a range of analyses including t-test, Gene-RaVE, discriminant analysis approaches and principle component analysis, we have discovered that small subsets of genes (<10), all of which included Nedd4BP3 and Ribosomal Protein L38 (RPL38), can be used to distinguish the two outcome groups. Subsequent validation using real time PCR supports the increase in Nedd4BP3 expression in standard risk patients which do not respond well to established treatment regimens. The Gene-RaVE algorithm also provides a generic framework for survival analysis. This approach indicates that the expression of these Nedd4BP3, RPL38 and inositol 1, 4, 5-triphosphate receptor, type 2 can be used to build a survival ‘index’ which correlates with the time to a relapse event in standard risk childhood ALL patients. Our results are suggestive of a way forward in the development of an informative, yet efficient diagnostic tool for this childhood malignancy using microarray gene expression analysis technology.
4
Zhao, Pan, Yuhuan Zheng, and Ting Niu. "SLC2A5 Overexpression in Childhood Philadelphia Chromosome Positive Acute Lymphoblastic Leukaemia." Blood 132, Supplement 1 (November 29, 2018): 5286. http://dx.doi.org/10.1182/blood-2018-99-118875.
Full textAbstract:
Abstract To study glycolysis/glycogenesis-related genes expression in childhood B cell acute lymphoblastic leukaemia (B-ALL), we performed a microarray-based analysis using published gene expression profiles. We found that gene SLC2A5, which encoded fructose transporter GLUT5 that facilitated cell fructose uptake, was up-regulated in Philadelphia chromosome positive ALL (Ph+ALL). Microarray-based analyses also suggested that SLC2A5 expression was significantly down-regulated in childhood B-ALL with t(1;19) or 11q23 mutation. High SLC2A5 expression was found in the patients who had recurrence within 3 years, early relapse, shortened complete remission duration, and positive minimal residue disease (MRD) status after treatment. The overexpression of SLC2A5 at both mRNA level and protein level in Ph+ALL was confirmed in a validation cohort of childhood B-ALL. We also validated the correlation of SLC2A5 expression and MRD status. In a mechanistic study using a human Ph+ALL cell line, we found that BCR-ABL kinase might regulate GLUT5 expression via c-myc. The tyrosine kinase inhibitors imatinib and dasatinib repressed GLUT5 expression and the cell uptake of fructose. Fructose protected the tumour cells from nutrition deficiency and drug-induced cell death. Overall, our findings showed that SLC2A5 was up-regulated in childhood Ph+ALL. The expression of SLC2A5 correlated with childhood B-ALL clinical factors, such as MRD status. Since TKI was able to inhibit GLUT5 expression, repression of fructose utility after TKI treatment contributes to TKI-induced Ph+ALL cytotoxicity. Targeting GLUT5 might be promising in B-ALL treatment, especially for Ph+ALL patients with high expression of SLC2A5. Disclosures No relevant conflicts of interest to declare.
5
Chen, Xiao Zhou. "LTSA Algorithm for Dimension Reduction of Microarray Data." Advanced Materials Research 645 (January 2013): 192–95. http://dx.doi.org/10.4028/www.scientific.net/amr.645.192.
Full textAbstract:
Dimension reduction is an important issue to understand microarray data. In this study, we proposed a efficient approach for dimensionality reduction of microarray data. Our method allows to apply the manifold learning algorithm to analyses dimensionality reduction of microarray data. The intra-/inter-category distances were used as the criteria to quantitatively evaluate the effects of data dimensionality reduction. Colon cancer and leukaemia gene expression datasets are selected for our investigation. When the neighborhood parameter was effectivly set, all the intrinsic dimension numbers of data sets were low. Therefore, manifold learning is used to study microarray data in the low-dimensional projection space. Our results indicate that Manifold learning method possesses better effects than the linear methods in analysis of microarray data, which is suitable for clinical diagnosis and other medical applications.
6
Ichim, Christine V., Mahadeo A. Sukhai, J. Brandwein, Mark D. Minden, Aaron D. Schimmer, Andre C. Schuh, Suzanne Kamel-Reid, Norman N. Iscove, and Richard A. Wells. "EAR-2: Identification of a Gene Involved in Maintenance of Clonogenicity in Haematopoiesis." Blood 104, no. 11 (November 16, 2004): 3226. http://dx.doi.org/10.1182/blood.v104.11.3226.3226.
Full textAbstract:
Abstract Primary acute myelogenous leukaemia (AML) samples are heterogeneous in clonogenicity, both among patients and within the leukaemic cell population of a single patient. To explain this heterogeneity the leukaemia stem cell model postulates that leukaemic hematopoiesis is organized in a hierarchy, sustained by leukaemia stem cells that may either self-renew or differentiate aberrantly to give rise to blasts that can no longer proliferate. This process is akin to the irreversible growth arrest entered by terminally differentiating normal blood cells. We wished to identify genes associated with clonogenicity in AML. To obtain pure populations of cells of defined growth abilities, we analyzed low passage cultures of the cell line OCI-AML4. This cell line resembles primary AML cells in several important respects; it is growth factor-dependent, contains a low proportion of clonogenic cells, and has a relatively simple karyotype. Clones consisting of four cells were micromanipulated so that a single cell was sampled for global RT-PCR while its three clonal siblings served as reporters of clonogenicity. By microarray analysis we found the orphan nuclear receptor EAR-2 to be expressed four-fold lower in leukemia single cells that spontaneously lose proliferative ability, compared to single cells with greater proliferative capacity. EAR-2 is a member of the COUP transcription factor family, which play roles in various developmental processes through interactions with nuclear receptors and other transcription factors. We assessed expression of EAR-2 in monoblastic leukaemia U937 cells induced to differentiate with a variety of induction agents. Treatment with dimethylsulfoxide, phorbol ester, vitamin D3, and all trans retinoic acid (ATRA) all induced significant decreases in EAR-2 expression. This phenomenon was also seen in a mouse model of acute promyelocytic leukaemia (APL). When primary bone marrow cultures of hCG-NuMA-RAR transgenic mice were induced to differentiate with ATRA, an average decrement in EAR-2 expression of 5.58 fold was observed (p<0.005). Since aberrant differentiation is an invariant feature of AML, we hypothesized that the overall expression of EAR-2 would be greater in AML patients relative to healthy controls. Analysis by quantitative RT-PCR of 15 AML, 10 CMML, 12 MDS and 16 normal bone marrow samples showed that EAR-2 is overexpressed in all three disease categories (p<0.0009 AML, 0.03 CMML, 0.0003 MDS). To characterize the effect of forced expression of EAR-2 on clonogenicity we transduced U937 cells with a retrovirus encoding either EAR-2 (U937-EAR2) or EGFP (U937-GFP). Analysis of FACS-purified U937-EAR2 and U937-GFP cultures showed that forced expression of EAR-2 reduces the doubling time of these populations (U937-EAR2 = 24h; U937-GFP = 34h; p<< 0.001), while no significant difference was observed in cell cycle profile. The decrease in doubling time of U937-EAR2 cells may reflect a decrease in the rate of cell loss in the population, consistent with the hypothesis that EAR-2 functions as a repressor of terminal differentiation. We have observed that expression of the orphan nuclear receptor EAR-2 is positively associated with maintenance of proliferative capacity and negatively associated with differentiation. These observations establish the importance of EAR-2 in the regulation of clonogenicity and terminal differention.
7
Greiner, Jochen, Elliott Brown, Lars Bullinger, Robert K. Hills, Vanessa Morris, Hartmut Döhner, Ken I. Mills, and Barbara-ann Guinn. "Survivin’ Acute Myeloid Leukaemia—A Personalised Target for inv(16) Patients." International Journal of Molecular Sciences 22, no. 19 (September 28, 2021): 10482. http://dx.doi.org/10.3390/ijms221910482.
Full textAbstract:
Despite recent advances in therapies including immunotherapy, patients with acute myeloid leukaemia (AML) still experience relatively poor survival rates. The Inhibition of Apoptosis (IAP) family member, survivin, also known by its gene and protein name, Baculoviral IAP Repeat Containing 5 (BIRC5), remains one of the most frequently expressed antigens across AML subtypes. To better understand its potential to act as a target for immunotherapy and a biomarker for AML survival, we examined the protein and pathways that BIRC5 interacts with using the Kyoto Encyclopedia of Genes and Genomes (KEGG), search tool for recurring instances of neighbouring genes (STRING), WEB-based Gene Set Analysis Toolkit, Bloodspot and performed a comprehensive literature review. We then analysed data from gene expression studies. These included 312 AML samples in the Microarray Innovations In Leukemia (MILE) dataset. We found a trend between above median levels of BIRC5 being associated with improved overall survival (OS) but this did not reach statistical significance (p = 0.077, Log-Rank). There was some evidence of a beneficial effect in adjusted analyses where above median levels of BIRC5 were shown to be associated with improved OS (p = 0.001) including in Core Binding Factor (CBF) patients (p = 0.03). Above median levels of BIRC5 transcript were associated with improved relapse free survival (p < 0.0001). Utilisation of a second large cDNA microarray dataset including 306 AML cases, again showed no correlation between BIRC5 levels and OS, but high expression levels of BIRC5 correlated with worse survival in inv(16) patients (p = 0.077) which was highly significant when datasets A and B were combined (p = 0.001). In addition, decreased BIRC5 expression was associated with better clinical outcome (p = 0.004) in AML patients exhibiting CBF mainly due to patients with inv(16) (p = 0.007). This study has shown that BIRC5 expression plays a role in the survival of AML patients, this association is not apparent when we examine CBF patients as a cohort, but when those with inv(16) independently indicating that those patients with inv(16) would provide interesting candidates for immunotherapies that target BIRC5.
8
Osuji, Nnenna, Ilaria Del Giudice, Tim Dexter, Estella Matutes, Vasantha Brito-Babapulle, David Gonzalez, Brian A. Walker, and Daniel Catovsky. "Gene Expression Reveals Two Distinct Biological Groups within T-Cell Prolymphocytic Leukaemia." Blood 106, no. 11 (November 16, 2005): 4366. http://dx.doi.org/10.1182/blood.v106.11.4366.4366.
Full textAbstract:
Abstract T-cell prolymphocytic leukemia (T-PLL) is rare and presents with widespread disease. Indolent presentations are seen but eventually progress. The disease shows marked chemoresistance and is best treated with the monoclonal anti-CD52 antibody (CAMPATH). Prolymphocytes show a post-thymic phenotype and are CD4+CD8− (65%), CD4−CD8+ (10%) or CD4+CD8+ (25%). This double positive phenotype, raises questions about the putative ontology of T-PLL. Morphological heterogeneity, with typical (75%), small cell (20%) and cerebriform/sezary-like variants (5%) is described. Inversions or reciprocal translocations of chromosome 14 involving breakpoints at q11 (TCR a/d) and q32.1 (TCL1 and TCL1b) are seen (~ 80%). Other common abnormalities involve chromosome 8, translocation (X;14)(q28;q11) and, ATM (11q23). We investigated the clinico-pathological heterogeneity in T-PLL, at the level of the transcriptome and evaluated the ability of gene expression profiling to sub-classify T-PLL. Total RNA was extracted from blood prolymphocytes (>92% purity) of 22 patients. cDNA synthesis followed by biotin-labelled cRNA synthesis was carried out as per Affymetrix protocols. Fragmented cRNA was hybridized to the Human U133 PLUS2 GeneChip array (54K probes). Microarray services were provided by MRC geneservice (UK HGMP Resource Centre). Hierarchical clustering of samples was performed using a filtered gene set (12,456) and >4 different algorithims. Prediction analysis for micoarray (PAM) and significance analysis of microarray (SAM) were used to evaluate class performance, and partition genes using pre-defined labels of immunophenotype, karyotype, response and morphology. Validation was performed by RT-PCR in a subset of genes.Unsupervised analysis robustly and reproducibly partitioned samples into 2 groups; A (n=8) and B (n=14). SAM analysis identified 4487 differentially expressed transcripts (false discovery rates <1%), >40% of which showed >2-fold difference in expression between the groups. There was no statistical difference in age, immunophenotype or karyotype betweeen groups, however, differential response to CAMPATH was seen. PAM analysis refined a sub-group of ~123 genes which most efficiently differentiated these groups. Group A showed significantly higher rates of non-response and progressive disease as compared to group B (n=14, p=0.036). Key differences related to apoptosis and cell-cycle associated gene expression. Down regulation of caspases (CASP1, CASP2,CASP4, CARD8 and CASP8AP2), cyclins (CCNC, CCND2, CCND3, CCNG1, CCNI, CCNT2), bcl-2, HDAC1, HIPK2, IL6R and ATM were frequent in group A with upregulation of genes implicated in NF-kB (TRAF4, SQSTM1) and TNF pathways (LMNA, ARTS-1), as well as transcription factors such as ATF-3. CD52 expression was ~2-fold higher in group B and may explain in part, differential responses to CAMPATH. RT-PCR validated gene expression data for LMNA and ATF-3. Despite the small numbers, algorithim-independent segregation into 2 consistent groups, in conjunction with the magnitude of gene differences, presence of many mutually exclusive divisions, and low prediciton errors, imply that the 2 identified profiles arise from fundamental differences at a regulatory level and thus likely represent a generalisable classification for T-PLL. Differential responses to CAMPATH may be a sub-feature of this grouping.
9
Saha, Sujay, Anupam Ghosh, Dibyendu Bikash Seal, and Kashi Nath Dey. "An Improved Fuzzy Based Missing Value Estimation in DNA Microarray Validated by Gene Ranking." Advances in Fuzzy Systems 2016 (2016): 1–19. http://dx.doi.org/10.1155/2016/6134736.
Full textAbstract:
Most of the gene expression data analysis algorithms require the entire gene expression matrix without any missing values. Hence, it is necessary to devise methods which would impute missing data values accurately. There exist a number of imputation algorithms to estimate those missing values. This work starts with a microarray dataset containing multiple missing values. We first apply the modified version of the fuzzy theory based existing method LRFDVImpute to impute multiple missing values of time series gene expression data and then validate the result of imputation by genetic algorithm (GA) based gene ranking methodology along with some regular statistical validation techniques, like RMSE method. Gene ranking, as far as our knowledge, has not been used yet to validate the result of missing value estimation. Firstly, the proposed method has been tested on the very popular Spellman dataset and results show that error margins have been drastically reduced compared to some previous works, which indirectly validates the statistical significance of the proposed method. Then it has been applied on four other 2-class benchmark datasets, like Colorectal Cancer tumours dataset (GDS4382), Breast Cancer dataset (GSE349-350), Prostate Cancer dataset, and DLBCL-FL (Leukaemia) for both missing value estimation and ranking the genes, and the results show that the proposed method can reach 100% classification accuracy with very few dominant genes, which indirectly validates the biological significance of the proposed method.
10
Ede, Ben Christopher, Paraskevi Diamanti, Charlotte V. Cox, and Allison Blair. "A Novel Combination Therapy for Paediatric T Cell Acute Lymphoblastic Leukaemia." Blood 126, no. 23 (December 3, 2015): 3767. http://dx.doi.org/10.1182/blood.v126.23.3767.3767.
Full textAbstract:
Abstract T cell acute lymphoblastic leukaemia (T-ALL) is a rare form of leukaemia that accounts for approximately 15% of paediatric ALL cases. Unfortunately, approximately 20% of patients do not achieve long term remission as a result of failure of therapy to eradicate the disease. T-ALL is a highly heterogeneous disease that displays a spectrum of immunophenotypes, chromosomal aberrations and gene expression profiles. This heterogeneity has prompted research into more targeted therapies, with the aim of overcoming drug resistance often found with standard chemotherapeutic regimens. Here, we build upon use of the drug Parthenolide (PTL), which has shown promise in treatment of T-ALL and other leukaemias such as BCP-ALL and AML, in combination with ABT-263, a BCL-2 family antagonising agent. Bone marrow samples from 10 T-ALL cases, taken at diagnosis, were treated with PTL in vitro for 24 hours then viability was assessed using the annexin V / PI flow cytometric assay. Variable cytotoxic effects were observed in samples treated with PTL (1-10µM), with half maximal inhibitory concentrations ranging from 2.6-10 µM. At the highest dose tested, the proportion of surviving cells ranged from 5.79-56% (median 35.33%). BM from 5 of these samples was used for whole genome microarray (WGA) analysis. We compared gene expression in bulk ALL and in specific subpopulations, known to have leukaemia initiating capacity in vivo; CD34+/CD7+, CD34+/CD7-, CD34-/CD7+ and CD34-/CD7- cells. WGA data demonstrated that CD34+/CD7- was the only subpopulation to express significantly lower levels (5.38 fold) of the pro-apoptotic gene Bcl-2L11 (BIM) compared to the unsorted bulk T-ALL cells, p=0.006. Interestingly, we have previously shown that CD34+/CD7- cells from a few patients were resistant to PTL treatment in vivo compared to unsorted cells. To validate these results, mRNA and relative protein quantification was performed by qPCR and western blotting in bulk material from 8 of the 10 samples, 3 of which had been analysed by microarray for BIM expression. We found that the gene and protein expression levels of BIM were negatively correlated with PTL resistance in vitro, p≤0.0001 and p=0.049 respectively. This suggests that reduced BIM expression is related to PTL resistance. We next evaluated the effects of combining PTL and ABT-263 on T-ALL cells in vitro. ABT-263 is a BH3 protein mimetic, like BIM it promotes apoptosis by blocking the inhibitory effects that BCL-2 anti-apoptotic proteins have on pro-apoptotic proteins. The effects of combining the drugs were assessed in 7 of the original 10 samples. Unsorted ALL cells were incubated with PTL and ABT-263 for 24 hours, before viability was analysed by flow cytometry and drug synergy was calculated via the Chou Talalay method. This drug combination showed enhanced cytotoxicity to T-ALL cells compared to PTL (p=0.0282) or ABT-263 (p=0.0358) alone. Moreover, the highest combined dose tested (2.5µM PTL with 0.25µM ABT-263) killed 86.1±9% cells cf 71.8±18% with ABT-263 alone and only 21.7±11% with PTL alone. The combination also showed synergism with a combination index value below 1 in all doses tested. Previous findings in our laboratory have shown that in vivo PTL treatment eliminated childhood leukaemia in NOD/LtSz-scid IL-2Rγc null (NSG) mice, in most cases tested. It may be possible to further enhance this toxicity using ABT-263 alone or in combination with PTL. NSG mice were inoculated with unsorted T-ALL cells and leukaemia was allowed to establish until levels in peripheral blood (PB) exceeded 0.1%. NSG mice were subsequently treated orally for 21 days with 100mg/kg of ABT-263 or placebo and leukaemia burden was monitored weekly in PB aspirates. Twenty-eight days following commencement of treatment, leukaemia burden in the placebo treated group was 80.73±2.94% and the animals were electively culled. In contrast, disease burden was significantly lower in the treated animals at this stage (35.2±2.1%, p=0.004). ABT-263 treatment has significantly improved survival of all xenografts to date, (P<0.014). In summary, we have shown that PTL resistance is related to the expression of BIM. By combining PTL with ABT-263, which mimics the pro-apoptotic action of BIM, the drugs work synergistically to enhance T-ALL cytotoxicity in vitro. Ongoing in vivo studies will assess the full potential of this combination therapy for paediatric T-ALL. Disclosures No relevant conflicts of interest to declare.
11
Tonks, Alex, Lorna Pearn, Amanda Tonks, Ken I. Mills, Alan K. Burnett, and Richard L. Darley. "Expression of RUNX1-RUNX1T1 Alone Has No Effect on the Intrinsic Susceptibility to Cytotoxic Chemicals." Blood 108, no. 11 (November 16, 2006): 2602. http://dx.doi.org/10.1182/blood.v108.11.2602.2602.
Full textAbstract:
Abstract The RUNX1 gene (aka AML1 on chromosome 21) encodes the alpha component of the Core Binding Factor (CBF) complex. This heterodimeric transcription factor is central to haematopoietic development and has been shown to be involved in the regulation of a number of haematopoietic-specific genes including IL-3, MPO and GM-CSF. RUNX1 is one of the most frequently disrupted genes in acute myeloid leukaemia (AML); and is particularly associated with chromosomal translocations. The t(8;21) encodes the RUNX1-RUNX1T1 (aka AML1-ETO) fusion protein which promotes self-renewal of haematopoietic cells and also inhibits their subsequent differentiation. Leukaemias expressing this abnormality are generally associated with a good prognosis in terms of complete remission, relapse risk and overall survival compared with other subtypes and tends to respond favourably to chemotherapeutic agents. However it is not currently known why patients expressing the t(8;21) have a good prognosis. It has been suggested that in AML patients expressing RUNX1-RUNX1T1, the fusion gene may promote the expression of p-glycoprotein (encoded by MDR-1) in mediating drug resistance. We therefore tested this hypothesis directly by expressing the RUNX1-RUNX1T1 fusion as a single abnormality in human haematopoietic cell subsets and performed Affymetrix microarray analysis to determine whether this fusion had any effect on the transcription of MDR genes. Using this approach we generated independent replicate sets of data from control and RUNX1-RUNX1T1 matched CD34+ cultures as well as matched sets constituting granulocytic (CD14lo, CD36lo, CD15hi) and monocytic (CD14hi) unilineage populations (isolated from day 6 cultures by immunomagnetic sorting). cRNA was prepared from each sample and hybridised to Affymetrix human 133A oligonucleotide arrays which allowed the simultaneous analysis of 6 MDR family gene members. In each of these populations, the expression of MDR genes was not significantly different from controls. We could therefore find no evidence that RUNX1-RUNX1T1 expression directly influences MDR gene expression as a single abnormality. We next addressed the issue of whether the t(8;21) abnormality directly influences the susceptibility to chemotherapeutic agents. We therefore assessed the sensitivity of CD34+ cells expressing RUNX1-RUNX1T1 to a number drugs commonly used to treat AML (Daunorubicin, Cytarabine, Fludarabine, Idarubicin or Etoposide) in comparison with matched controls. Remarkably, none of these agents differentially affected the growth of RUNX1-RUNX1T1 transduced cells. Since treatment of AML commonly involves multiple drugs, we also determined the effect of combining two or more of these chemotherapeutic agents. Again, we observed little difference in the in vitro growth response of RUNX1-RUNX1T1 expressing cells compared to controls. Taken together, these data suggest that expression of RUNX1-RUNX1T1 itself has no effect on the intrinsic susceptibility to cytotoxic chemicals. This raises the alternative hypothesis that RUNX1-RUNX1T1 moderates the influence of secondary abnormalities which are required for RUNX1-RUNX1T1 expressing cells to undergo leukaemic transformation.
12
Tholouli, Eleni, Dolores Di Vizio, Fionnuala O’Connell, Massimo Loda, David Twomey, Todd Golub, Richard Levenson, Judith A. Hoyland, John A. L. Yin, and Richard Byers. "Quantum Dot Based Duplex In Situ Hybridisation for Gene Expression Profiling." Blood 106, no. 11 (November 16, 2005): 3265. http://dx.doi.org/10.1182/blood.v106.11.3265.3265.
Full textAbstract:
Abstract Quantum dots (QDs) are fluorescent semiconductor nanocrystals (2–10-nm core diameter) possessing the unique properties of extremely high fluorescence efficiency, lack of photobleaching and long fluorescence lifetime, making them an ideal tool for bioimaging. We have developed a novel technique for in situ hybridisation (ISH) using biotinylated oligonucleotides conjugated to streptavidin coated QD, and used them in this study to label bone marrow trephine samples. 50-mer long oligonucleotide probes were conjugated to QDs prior to ISH and conjugation efficiency was demonstrated by gel electrophopresis. ISH conditions and molar ratio of QDs to probe were optimised using a polyT probe. Images were captured using a CRI Nuance spectral imaging system and signal intensity was semi-quantitated using IPLab software. Specific oligonucleotide hybridisation was demonstrated using a probe for myeloperoxidase (MPO) in 40 bone marrow sections infiltrated by acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) as well as reactive bone marrow. In each case hybridisation signal was consistent with the distribution of MPO by standard immunohistochemistry - MPO was strongly expressed by myeloid blasts and absent in lymphoid blasts; in CML, most, but not all, cells were positive for MPO, in comparison to many fewer positive cells in reactive marrow. Duplex ISH was demonstrated using a probe for bcl-2 together with MPO in 5 bone marrow sections infiltrated by follicular lymphoma (FL). Strong hybridisation signal for bcl-2 was detected in all cells of the paratrabecular aggregates of FL but showed only scattered positivity in the remainder of the bone marrow. Conversely, MPO was absent in the paratrabecular aggregates and present in the myeloid cells in the remainder of the marrow. This pattern was present in both single and duplex ISH for MPO and bcl-2 in the marrow infiltrated by FL. Duplex ISH was performed both by sequential hybridisation with bcl-2 followed by MPO, and simultaneously with a mix of bcl-2 and MPO probes. As negative controls, scrambled oligonucleotide probes for the corresponding genes were used in each case and did not show hybridisation. In summary, we have developed a generic method for QD labelling and semi-quantitative detection of oligonucleotide ISH in routinely processed clinical tissue samples. Although, in this study we primarily used bone marrow trephine samples, this technique can be applied to any tissue. It has the potential to facilitate transfer of microarray-identified gene signatures to clinical research and diagnostics, whilst the ability of spectral imaging to resolve multiple signals offers the possibility of multiplexed probe detection.
13
Zhang, Xiang-Zhong, Ai-Hua Yin, Dong-Jun Lin, Xiao-Yu Zhu, Qian Ding, Chun-Huai Wang, and Yun-Xian Chen. "Analyzing Gene Expression Profile in K562 Cells Exposed to Sodium Valproate Using Microarray Combined with the Connectivity Map Database." Journal of Biomedicine and Biotechnology 2012 (2012): 1–8. http://dx.doi.org/10.1155/2012/654291.
Full textAbstract:
To explore the mechanism underlying antileukaemia effect of sodium valproate, the growth and survival of the K562 cell line were investigated. Global profiles of gene expression in K562 cells exposed to sodium valproate were assessed and validated. The differentially expressed genes identified were further used to query the connectivity map database to retrieve a ranked list of compounds that act on the same intracellular targets as sodium valproate. A significant increase in cell apoptosis and a change in gene expression profile were observed in valproate-exposed K562 cells. The significant enrichment analysis of gene ontology terms for the differentially expressed genes showed that these genes were involved in many important biological processes. Eight differentially expressed genes involved in apoptosis were verified by quantitative real-time PCR. The connectivity map analysis showed gene expression profile in K562 cells exposed to sodium valproate was most similar to that of HDACi and PI3K inhibitors, suggesting that sodium valproate might exert antileukaemic action by inhibiting HDAC as well as inhibiting PI3K pathway. In conclusion, our data might provide clues to elucidate the molecular and therapeutic potential of VPA in leukaemia treatment, and the connectivity map is a useful tool for exploring the molecular mechanism of drug action.
14
Sakhinia, Ebrahim, Maboubeh Faranghpour, John A. Liu Yin, Gerard Brady, Judith A. Hoyland, and Richard J. Byers. "Routine expression profiling of microarray gene signatures in acute leukaemia by real-time PCR of human bone marrow*." British Journal of Haematology 130, no. 2 (July 2005): 233–48. http://dx.doi.org/10.1111/j.1365-2141.2005.05594.x.
Full text
15
Tambaro, Francesco Paolo, Ilaria Lepore, Carmela Dell Aversana, Floriana De Bellis, Marco Miceli, Vincenzo Carafa, Angela Nebbioso, Felicetto Ferrara, Guillermo garcia Manero, and Lucia Altucci. "BARD1: a New Target In Leukemia." Blood 116, no. 21 (November 19, 2010): 4642. http://dx.doi.org/10.1182/blood.v116.21.4642.4642.
Full textAbstract:
Abstract Abstract 4642 Background BARD1 (BRCA1-associated RING domain protein 1) was first described as a BRCA1 partner and tumour suppressor protein, muted in most cases of breast and ovarian cancer. BARD1 functions are BRCA1-dependent, requiring formation of a stable heterodimer through interaction of the RING finger domains. BRCA1-independent functions for BARD1 have recently been described, making it an interesting and important molecule for study. BARD1 is expressed in almost all human tissues, including haematological cells, testis and breast tissue, but it is over-expressed in leukaemias, sarcomas and testis cancer, implicating BARD1 in development of cancer. Different BARD1 isoforms are up-regulated in breast, ovarian and uterine cancers but markedly down-regulated or absent in healthy tissues. Therefore, the presence of these isoforms might be a risk factor or causal event in the cancer pathogenesis. We investigated the role of BARD1 isoforms in leukaemia, through the study of the epigenetic mechanisms involved in regulation of its expression and function. As such, we determined the expression of a specific BARD1 isoform in different human leukaemia cell lines the effects of the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid, Vorinostat) of expression of BARD1. Results. Four human leukaemia cell lines (U937, NB4, K562 and HL60) express the BARD1 isoform of interest. Treatment of these cell lines with SAHA at 5 μ M resulted in decreased expression of this isoform (Fig. 1). Decreased expression of BARD1 by SAHA was also observed in in human breast cancer MCF7 cells, the usual model for BARD1 experiments and in the human neuroblastoma cell line Kelly (Fig. 2), but not in HeLa cells or an human epithelial carcinoma cell line (Fig. 2). The reduced expression of BARD1 was attributed to the action of 2 specific micro-RNAs (miRNAs), that directly bind its 3′untranslated region (UTR). SAHA-induced expression of miR-19a and miR-19b in primary human leukemia cells as demonstrated using miRNA microarray expression analysis and confirmed by Real-Time PCR (Fig. 3). These 2 miRNA were up-regulated after SAHA stimulation in human leukaemia cells. BARD1 is the predicted target for miR-19a and miR-19b based on miRBase database analyses. Transient transfection in NB4 cells with mimic miR-19a and mimic miR-19b confirmed expression of these miRNAs was associated with BARD1 down-regulation (Fig. 4 5.). The BARD1 3′UTR was cloned into a pGL3 vector with a downstream the luciferase gene reporting gene (Fig. 6). The plasmid was co-transfected in HeLa cells with each mimic miRNA and a luciferase assay was performed. We demonstrate that expression of miR-19a and miR-19b can directly bind BARD1 3′UTR, reducing luciferase activity in this system (Fig. 7), thereby confirming that BARD1 is a target of these miRNAs. These findings support our hypothesis that BARD1 is a promising target in leukemia and should be further investigated for its diagnostic and prognostic features. Disclosures: No relevant conflicts of interest to declare.
16
Pulford, Karen, Linden Lyne, Giovanna Roncador, Ryo Kominami, and Alison H. Banham. "Human BCL11B Is Expressed in Normal T Cells and Differentially Expressed in T-Cell Malignancies." Blood 106, no. 11 (November 16, 2005): 4393. http://dx.doi.org/10.1182/blood.v106.11.4393.4393.
Full textAbstract:
Abstract B-cell lymphomaleukaemia 11B (BCL11B), the human homologue of murine Bcl11b/Rit-1/CTIP2, was originally identified as a novel tumor suppressor gene in a study of gamma-radiation induced thymic lymphomas in mice. Later studies, however, showed Bcl11b to have a vital role in T cell development and survival, with BCL11B translocations involving a variety of partner genes being reported in both T-cell and myeloid leukemias. The human BCL11B gene, located at 14q32.2, encodes an 832aa Kruppel C2H2 zinc finger protein that is functionally uncharacterized but likely to act as a transcriptional regulator. Analysis of publically available normal tissue Affymetrix microarray expression data indicates an expression pattern restricted to hematopoietic cells, with high levels of BCL11B transcripts being present only in peripheral blood T cells, NK cells, thymus and tonsil. We have used two rabbit polyclonal anti-BCL11B antibodies to study the distribution of BCL11B protein in both normal and neoplastic human cells. These reagents, raised against two distinct regions of the murine Bcl11b protein (zinc finger and C-terminus), recognized the human BCL11B protein. While neither antibody stained B-cells in tonsil, one was crossreactive with the highly homologous BCL11AXL protein by Western blotting. In normal tissues, BCL11B protein expression was confined to the nuclei of the vast majority of T cells in thymus (foetal and adult) and tonsil. High levels of BCL11B were detected in T-cell lines, including the Molt-4, CCRF-CEM and Jurkat T-cell acute lymphoblastic leukaemia (T-ALL) derived cell lines. No expression was detected in any B-cell derived (pre-B to plasma cell stage) or myeloid cell lines studied. These results are consistent with the microarray gene expression data. In T-cell malignancies, BCL11B protein was only detected in a proportion of tumors, including 5/6 T-ALLs (one being weakly stained) and 2/8 peripheral T-cell lymphomas (weak cytoplasmic staining only). Interestingly, no expression was detected in ALK-positive anaplastic large cell lymphoma lines or tumors. Further studies of a larger series of T-cell malignancies are in progress. All other tumors studied, including B-ALL, chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, follicular lymphoma, myeloma and Hodgkin’s lymphoma, were unlabelled. In conclusion, the distribution pattern of the BCL11B protein in a wide range of both normal and neoplastic tissues is described for the first time. The study of BCL11B expression is an invaluable first step towards elucidating the role of this protein in T-cell biology and the significance of its differential expression in T-cell malignancies.
17
TERAMOTO, H., H. MIWA, V. PATEL, N. LETWIN, M. D. CASTELLONE, N. IMAI, M. SHIKAMI, et al. "Gene expression changes in a patient presenting nonleukaemic nasal granulocytic sarcoma to acute myelogenous leukaemia using 40 K cDNA microarray." Clinical & Laboratory Haematology 28, no. 4 (July 18, 2006): 262–66. http://dx.doi.org/10.1111/j.1365-2257.2006.00803.x.
Full text
18
Gómez-Castañeda, Eduardo, Lisa Hopcroft, Simon Rogers, Chinmay Munje, Joana Bittencourt-Silvestre, Mhairi Copland, David Vetrie, Tessa Holyoake, and Heather Jørgensen. "Tyrosine Kinase Inhibitor Independent Gene Expression Signature in CML Offers New Targets for LSPC Eradication Therapy." Cancers 14, no. 21 (October 26, 2022): 5253. http://dx.doi.org/10.3390/cancers14215253.
Full textAbstract:
Tyrosine kinase inhibitors (TKI) have revolutionised the treatment of CML. However, TKI do not eliminate the leukaemia stem cells (LSC), which can re-initiate the disease. Thus, finding new therapeutic targets in CML LSC is key to finding a curative treatment. Using microarray datasets, we defined a list of 227 genes that were differentially expressed in CML LSC compared to the healthy controls but were not affected by TKI in vitro. Two of them, CD33 and PPIF, are targeted by gemtuzumab–ozogamicin and cyclosporin A, respectively. We treated CML and the control CD34+ cells with either drug with or without imatinib to investigate the therapeutic potential of the TKI-independent gene expression programme. Cyclosporine A, in combination with imatinib, reduced the number of CML CFC compared with non-CML controls, but only at supra-therapeutic concentrations. Gemtuzumab–ozogamicin showed an EC50 of 146 ng/mL, below the plasma peak concentration of 630 ng/mL observed in the AML patients and below the EC50 of 3247 ng/mL observed in the non-CML cells. Interestingly, gemtuzumab–ozogamicin seems to promote cell cycle progression in CML CD34+ cells and demonstrated activation of the RUNX1 pathway in an RNAseq experiment. This suggests that targeting the TKI-independent genes in CML LSC could be exploited for the development of new therapies in CML.
19
Qureshi, Moosa, Wajid Jawaid, Fernando J. Calero-Nieto, Rebecca Hannah, Sarah J. Kinston, Evangelia Diamanti, Winnie W. Lau, Isabel Jimenez-Madrid, Chiara Cossetti, and Berthold Gottgens. "Cellular Model of CEBPA N321D Captures Gene Expression Profile in the Transition to Pre-Leukaemic Status." Blood 128, no. 22 (December 2, 2016): 3922. http://dx.doi.org/10.1182/blood.v128.22.3922.3922.
Full textAbstract:
Abstract Background C/EBPα plays a pivotal role in myeloid differentiation at the CMP to GMP transition point, where it interacts with other transcription factors (TFs) implicated in haematopoiesis. CEBPA mutations are common in acute myeloid leukaemia (AML), predominantly in patients with M1 and M2 French-American-British (FAB) morphological classifications, but relatively little is understood about the pre-leukaemic alterations caused by mutated CEBPA. Murine models have established N321D as a particularly potent CEBPA mutation which causes AML with high mortality (Togami et al, Experimental Hematology, 2015). We aimed to develop an inducible expression system for CEBPA N321D in a cellular model which replicates early haematopoietic progenitors, to study the effects of this mutation on gene expression profiles relevant for malignant haematopoiesis. Methods We constructed a Piggy-bac Tet-on inducible expression system which has a 2A peptide mechanism enabling simultaneous expression of both N321D and mCherry fluorescent protein from the same transcript (Fig. 1). We also constructed a control with inducible expression of mCherry. These two plasmids were then transfected into the mouse progenitor cell line Hoxb8-FL (Redeckeet al, Nature Methods, 2013), which is conditionally immortalized and models multipotent myelo-lymphoid progenitors. Single cell clones were established and selected for analysis on the basis of cell growth and mCherry fluorescence on induction. RNA was collected post-induction and without induction at 24, 48 and 72 hours in two replicates each from the N321D clone and from the empty control vector. RNA-seq data was aligned to the mouse genome using STAR aligner, processed to generate high throughput sequencing counts, and finally differential expression analysis was performed between N321D and the control. Results Differential expression analysis identified 172 downregulated and 60 upregulated genes after N321D induction. Further analysis of the 172 downregulated genes against online published datasets of gene expression (Gene Expression Commons, https://gexc.stanford.edu), revealed that 19 of these genes are normally upregulated at the CMP to GMP transition. These include genes such as Hck, Met, Hdac8 and Kdm7a which have been previously implicated in haematological malignancy and which may provide novel insights into the leukaemic process fostered by the CEBPA N321D mutation. To further validate our data, we performed unsupervised hierarchical clustering of previously published microarray data from a large collection of over 400 AML expression profiles (Verhaaket al, Haematologica, 2009) using the genes identified in our study, and found that patient samples who had predominantly FAB classifications M1 and M2 clustered together (Fig. 2A,B), as would be expected in CEBPA-mutated AML. Conclusions Our inducible expression system has the potential to provide novel insights into altered gene expression caused by induction of mutated CEBPA. In particular, our cellular model replicates an early stage of haematopoiesis, and implicates genes which were not previously known to interact with CEBPA. The importance of these genes in CEBPA N321D-mediated re-configuration of the myeloid transcriptional regulatory network requires further analysis. Disclosures No relevant conflicts of interest to declare.
20
Albertioni, Freidoun, Alan Fotoohi, Jamileh Hashemi, Elham Yektaei, and Catharina Larsson. "RNA Interference Targeting Thiopurine Methyl Transferase Gene Alters the Cytotoxic Effect of the 6-Mercaptopurine In Human Leukaemia Cells." Blood 116, no. 21 (November 19, 2010): 2891. http://dx.doi.org/10.1182/blood.v116.21.2891.2891.
Full textAbstract:
Abstract Abstract 2891 Thiopurines; mercaptopurine (6-MP) and 6-thioguanine (6-TG) are important drugs in treatment of paediatric cancer patients. The activity of these drugs depends on the activity of several common enzymes in the metabolism pathways such as thiopurine methyl transferase (TPMT) and guanine monphosphate synthetase (GMPS). In the present study the efficacy of thiopurines was investigated upon inhibition of TPMT and GMPS gene expression by RNA interference (siRNA). Treatment of the MOLT4 human T-cell leukemia cells with TPMT and GMPS siRNA resulted in decreased mRNA expression as determined by Real-Time PCR by 60% and 70% respectively. When reducing TPMT mRNA, the MOLT-4 cells were 70% less sensitive to 6-MP while the sensitivity to 6-TG was unchanged. When down-regulating GMPS using siRNA the sensitivity was unchanged upon treatment with both drugs. A microarray experiment was conducted using wild type on MOLT-4, 6-MP and 6-TG resistant MOLT-4 variants. The aim was to identify affected genes in response to the resistance induction. The mRNA levels of several nucleoside transporter genes were down-regulated in both thiopurine-resistant sub-lines; concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). The expression of genes encoding the purine de novo synthesis enzymes was reduced to the same extent in both resistant cell lines as well as the expression of GMPS (>40% in both resistant sub-lines) which indicates defected purine metabolism. The activity of TPMT and other enzymes in metabolism pathway of 6-MP and 6-TG was unchanged in resistant cells. Our results suggest that the contribution of TPMT activity in cytotoxicity of 6-MP is greater than for 6-TG, probably due to inhibition of de novo purine synthesis by methylated nucleotides, whereas TPMT inactivates 6-TG by methylation. Disclosures: No relevant conflicts of interest to declare.
21
Winteringham, Louise N., Raelene Endersby, Ian J. Majewski, Jennifer Beaumont, Simon Kobelke, Jean-Philippe Lalonde, and Svend Peter Klinken. "Genes Identified in a Hemopoietic Lineage Switch Influence Transcription." Blood 110, no. 11 (November 16, 2007): 1250. http://dx.doi.org/10.1182/blood.v110.11.1250.1250.
Full textAbstract:
Abstract The J2E erythroblastoid cell line responds to erythropoietin (Epo) by morphological maturation and hemoglobin synthesis. However, on rare occasions, these cells have undergone a spontaneous lineage switch and display features of monoblastoid cells which do not respond to Epo. Amongst the genes up-regulated in the monoblastoid variants were Hemopoietic lineage switch (Hls) 5 and 7. Hls5 is a recently identified member of the RING finger, B Box, Coiled coil (RBCC) or tripartite motif (TRIM) family, which includes PML, a gene involved in acute promyelocytic leukaemia. Hls7 is the murine orthologue of Myeloid leukaemia factor 1 (Mlf1), a gene involved in a t(3;5), associated with acute myeloid leukaemia. We have shown previously that Hls7/Mlf1 imposes a dramatic phenotypic change upon the erythroid cells, rendering them monoblastoid (Williams, J. et al EMBO 1999). We have studied the role of Hls5 and Mlf1 in erythroid commitment and differentiation. Ectopic expression of Hls5 inhibits globin production in erythroid cells and suppresses development of B-FUE and C-FUE. A yeast-two-hybrid screen identified FOG-1 as a binding partner of Hls5. Significantly, FOG-1 is a transcriptional co-regulator of GATA-1, which controls globin gene expression. While Hls5 is able to enhance the repression of GATA-1 activity imposed by FOG-1, it is also able to repress GATA-1 transcriptional activity in the absence of FOG-1. Using electrophoretic mobility shift assay we have shown that Hls5 is able to reduce GATA-1 binding to DNA in a dose dependant manner. This observation that Hls5 reduces GATA-1 binding to promoter elements is mirrored by chromatin immunoprecipitation assays. Expression of MLF1 is highest in CD34+ cells, but is markedly down regulated during erythroid differentiation. Microarray analysis identified a number of known transcriptional regulators differentially expressed in the presence of Mlf1 including ets1, Myc intron binding protein and Tbr2. Mlf1 is able to bind DNA and luciferase reporter assays demonstrated that Mlf1 is able to affect transcription. In addition, Mlf1 interacts with a novel member of the hnRNP family viz Mlf1 associated nuclear protein (Manp). Manp binds to DNA, is able to influence the subcellular localisation of Mlf1 by translocating Mlf1 from the cytoplasm to the nucleus. Importantly, Manp also has an affect on transcription. These data demonstrate that both Hls5 and Mlf1 affect transcription of genes associated with erythroid differentiation.
22
Etienne, Gabriel, Maryse Dupouy, Patricia Costaglioli, Claudine Chollet, Valérie Lagarde, Jean-Max Pasquet, Josy Reiffers, Bertrand Garbay, François-Xavier Mahon, and Béatrice Turcq. "α-Defensin 1-3 And α-Defensin 4 as Predictive Markers of Imatinib Resistance and Relapse in CML Patients." Disease Markers 30, no. 5 (2011): 221–27. http://dx.doi.org/10.1155/2011/287690.
Full textAbstract:
Objective: Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukaemia. Despite a remarkable effectiveness, treatment failure cases have been reported in 20 percent of CML patients. The identification of biomarkers which can predict the response to imatinib is our point of interest.Methods: Gene expression profiling microarray was carried out on secondary imatinib resistant patients. Longitudinal studies were performed on imatinib treated responder/resistant patients. Then, Q-RT/PCR studies were realized on patients prior imatinib initiation.Results: For imatinib responder patients, we observed a strong and lasting decrease of α-defensin 1-3 and α-defensin 4 expression. For relapse patients, we observed a dramatic increase of α-defensin 1-3 and α-defensin 4 expression before BCR-ABL transcript increase. Moreover, before imatinib initiation, α-defensin 1-3 and α-defensin 4 expression was significantly lower in the resistant group than in the responder group.ConclusionThe variation of expression of α-defensin 1-3 and α-defensin 4 in peripheral blood is associated with imatinib resistance and may reflect an adequate immune control of the disease. Monitoring of α-defensin 1-3 and α-defensin 4 could be helpful to predict the patients who are not going to respond to the treatment.
23
Kohlmann, Alexander, Thomas J. Kipps, Laura Z. Rassenti, James R. Downing, Sheila A. Shurtleff, Ken I. Mills, Amanda F. Gilkes, et al. "An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase." British Journal of Haematology 142, no. 5 (September 2008): 802–7. http://dx.doi.org/10.1111/j.1365-2141.2008.07261.x.
Full text
24
Messa, Francesca, Monica Pradotto, Francesca Arruga, Roberto Bernardoni, Enrico Bracco, Chiara Maffè, Emanuela Messa, et al. "Genome-Wide Screening for Dominant Modifiers in Drosophila Identified New Cluster of Genes Involved in BCR-ABL Signalling and CML Progression." Blood 114, no. 22 (November 20, 2009): 2186. http://dx.doi.org/10.1182/blood.v114.22.2186.2186.
Full textAbstract:
Abstract Abstract 2186 Poster Board II-163 Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukaemia (CML) is well established, the mechanisms responsible for CML progression remain largely unknown. The aim of the study was to perform a genome-wide screening to identify new genes and pathways leading to CML progression. We performed a genome-wide genetic screening using our set-up model of human p210 Bcr-Abl transgenic Drosophila melanogaster (Dm) in which the expression of hBcr-Abl in a tissue specific manner is able to induce a severe eye-glazed phenotype or the formation of melanotic tumors, (clusters of hemocytes) when expressed into the fly lymph gland which represent the Dm hematopoietic system. A wide modifier screening of the whole fly genome containing approx 14.000 genes was performed using 278 fly stocks commercially available and carrying well characterized chromosome deletions. The resulting progeny was screened using the eye phenotype as first read-out system. Furthermore each deletion responsible for phenotype changes was analyzed either by expressing it into lymph gland as second read-out system, in order to analyze their function into a haematopoietic background and to exclude genes involved in eye development, such as genes able to modify the eye phenotype even without being directly involved in Bcr-Abl oncogenic signalling (false positives). Data obtained from primary and secondary screen were first analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified candidate genes were tested and validated analyzing either BM or PB samples from CML patients and healthy donors. 14.000 Dm genes were analyzed for their capability to genetically interact with hBcr-Abl in the fly model. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screening crosses, shows a first group of flies (38%) displaying a more aggressive phenotype since they lack genes encoding for hBcr-Abl negative regulators and a second group (32%) showing a mild phenotype due to the absence of genes involved in the oncogenic signalling. We found that 42% of the 4000 Dm genes mapping in the deleted regions able to modify Bcr-Abl phenotype, displayed a known human counterpart. GeneOntology profiles of these genes included oncogenes, tumor suppression genes and human genes encoding proteins involved in the regulation of transcriptions, signal transduction, proliferation and cell growth, differentiation, apoptosis and splicing processes. Moreover, a computational comparison of our results with gene expression signatures of CML from Microarray data, showed only a partially overlap between genes identified in fly screen and genes obtained from Microarray analysis. The 72% of identified genes in fact was not known to be involved in human leukaemia. However, further confirmation of our findings into fly comes from the validation in human samples in which 1250 genes were found to be significantly associated with human CML; among these genes, we found not only an alteration of their expression profiles in CML patients with respect to the healthy donors, but also protein alterations, such as expression of different splicing forms or misplaced proteins, suggesting that Dm screening is a valid approach able to identify not only differentially expressed genes but also specific pathways and genes otherwise altered by hBcr-Abl. In conclusion, the identification of these genes allows identifying of the changes occurring in CML at the genomic level and gives deeper insights into the molecular basis of the disease; moreover this study point to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression Disclosures: No relevant conflicts of interest to declare.
25
Kochethu, Geothy, Andy Bell, Mike Griffiths, Gulnaz Begum, Farooq Wandroo, and Paul A. H. Moss. "A Broad Distribution of ZAP-70 Expression Is Demonstrated in Adult Acute Lymphoblastic Leukaemia by Quantitative PCR Analysis." Blood 108, no. 11 (November 16, 2006): 4309. http://dx.doi.org/10.1182/blood.v108.11.4309.4309.
Full textAbstract:
Abstract The ZAP-70 tyrosine kinase plays a critical role in signal transduction in T cells and NK cells but has limited expression in primary human B cells. However ZAP-70 is expressed in many cases of B cell chronic lymphocytic leukemia and correlates with a poor prognosis. Microarray analysis has recently identified ZAP-70 expression in a range of other B cell malignancies, including acute lymphoblastic leukaemia (ALL), and ZAP-70 expression may carry prognostic value in these diseases. Microarray analysis may not offer the most accurate method for quantitation of small changes in mRNA expression and this technology may therefore limit the potential information to be derived from ZAP-70 expression. We developed a real time quantitative PCR assay for ZAP-70 and used this to determine ZAP-70 expression in 76 adult patients with ALL (64 B lineage and 12 T lineage). RNA was extracted from diagnostic bone marrow specimens taken from 76 patients aged over 18 years presenting with ALL. Quantitative PCR was optimised using Jurkat T cells and utilized GAPDH as the internal control. ZAP-70 and GAPDH cDNA were co-amplified in a multiplex PCR reaction with five-fold serial dilutions of Jurkat cDNA ranging from 25ng to 40pg, and this data was used to construct the calibration curve. ZAP-70 expression within tumours was determined relative to expression within the T cell line Jurkat, defined as having an arbitrary value of 1.0. A wide distribution of ZAP-70 expression was seen in individual ALL cases with a range between 0.002 and 5.3. The average ZAP-70 expression level within ALL was below that of Jurkat with a mean of 0.332 and median of 0.185. Expression of ZAP-70 showed a relatively continuous pattern of expression across the cohort apart from six samples (8%) with a level of ZAP-70 expression above that of the Jurkat T cell line. Statistical analysis of a potential association between ZAP-70 expression and cytogenetic subgroup was performed by group comparison using either a two-sample test or ANOVA. No significant association between ZAP-70 expression and cytogenetic subgroup was found. Only 4 cases of t(1,19) translocation encoding the E2A/PBX1 gene fusion were seen in the cohort and all had ZAP-70 levels above the median level. Within the T-ALL subgroup, 11 of the 12 patients showed ZAP-70 expression above the median with a mean of 0.26. Low expression was observed in 4 of the 5 cases in the b3a2/b2a2 p210 BCR/ABL subgroup. Study of a potential correlation between ZAP-70 expression and clinical prognosis is continuing but the three patients with the highest levels of ZAP-70 failed to achieve remission with induction therapy and died of refractory disease. These data confirm that ZAP-70 expression is seen in the majority of cases of ALL and demonstrate the wide range of expression between individual cases. Large cohort studies are likely to be required to confirm an association between ZAP-70 expression and specific cytogenetic subgroup and to confirm the potential prognostic value of ZAP 70 expression.
26
Franiak-Pietryga, Ida, Ewelina Ziółkowska, Barbara Ziemba, Dietmar Appelhans, Henryk Maciejewski, Brigitte Voit, Aleksandra Kaczmarek, et al. "Glycodendrimer PPI as a Potential Drug in Chronic Lymphocytic Leukaemia. The Influence of Glycodendrimer on Apoptosis in In Vitro B-CLL Cells Defined by Microarrays." Anti-Cancer Agents in Medicinal Chemistry 17, no. 1 (January 2017): 102–14. http://dx.doi.org/10.2174/1871520616666160622092947.
Full textAbstract:
Background: Chronic lymphocytic leukaemia (CLL) cells are characterized by failures in the apoptosis pathway and increased proliferation, resulting in the progressive accumulation of B-lymphocytes in blood. Despite the wide range of antileukaemic drugs, CLL remains an incurable disease. However, a breakthrough is expected which will allow more effective treatment. Objective: The study investigates the influence of poly(propyleneimine) (PPI) dendrimer with peripheral amino groups, 30% of which were coated with maltotriose (PPI-G4-OS-Mal-III), on CLL cells, and demonstrates that it acts through the induction of the apoptotic mechanism. It is important to note that the dendrimer was used as a drug itself and not as a drug carrier. Method: CLL and normal lymphocytes were treated in vitro with the dendrimer, either alone or in combination with fludarabine (FA). The percentages of apoptotic and necrotic cells, and the protein expression, were checked using a flow cytometer. Gene expression was screened using a two-colour microarray with 60-mer probes. Results: The results confirm that PPI-G4-OS-Mal-III influences the viability of CLL cells in vitro and does not exert any significant harmful effect on normal lymphocytes. The dendrimer appears to significantly influence gene and protein expression in CLL cells. Conclusion: Since dendrimers can be specifically targeted, they may be very effective in CLL therapy, especially since in vitro PPI-G4-OS-Mal-III has been found to have stronger effect than fludarabine.
27
Al-Asadi, Mazin Gh, Marcos Castellanos, Sean T. May, Nigel H. Russell, Claire H. Seedhouse, and Monica Pallis. "Molecular Signature of Dormancy in CD34+CD38- Acute Myeloid Leukaemia Cells." Blood 128, no. 22 (December 2, 2016): 1660. http://dx.doi.org/10.1182/blood.v128.22.1660.1660.
Full textAbstract:
Abstract Chemotherapy drugs tend to spare cells in a state of dormancy (G0 phase of the cell cycle). Relapse of acute myeloid leukaemia (AML) is likely in part due to dormant cells evading remission-induction chemotherapy. Dormant AML cells have been identified in the bone marrow endosteal region which is characterised by an excess of TGFβ1 and a shortage of nutrients. We developed and characterized an in-vitro model of AML cell dormancy by exploiting these features. Following preliminary investigation of several cell lines, the CD34+CD38- line TF1a was selected for in depth investigation. TF1a cells showed 72% inhibition of proliferation (p<0.001), with features of dormancy, in response to 72 hours TGFβ1+mTOR inhibitor treatment (mTOR pathway inhibition mimics major effects of nutrient scarcity).This treatment caused loss of Ki-67, as well as upregulating ALDH and CD34 and causing nuclear translocation of FOXO3a. In contrast to conventional serum-withdrawal assays for dormancy, the treatment had no impact on cell viability, assessed by Annexin V assay. Nor did the treatment lead to cell differentiation, assessed by CD11b staining and morphology. Using whole human genome expression microarray and by intersecting differentially regulated genes in dormancy-induced TF1a cells in comparison to their proliferating (untreated) counterparts, we identified 240 genes which are significantly up-regulated >2 fold including genes involved in stemness, chemoresistance and tumour suppressor genes in addition to genes involved in canonical cell cycle regulation. There was striking upregulation of genes involved in adhesion and migration: raised expression levels of SPP1 (the gene coding for osteopontin), ITGB3, ITGB4, ITGA3 and CD44 in dormant cells were confirmed by real-time PCR. The most upregulated gene was SPP1/osteopontin (16 fold). Immunocytochemistry of biopsy material from AML patients confirmed high levels of osteopontin in the cytoplasm of blasts near the paratrabecular bone marrow. Osteopontin and other genes identified in this model, including well-characterised genes (e.g. CD44, CD47, CD123, ABBC3 and CDKN2B) as well as little-known ones (e.g. PTPRU, ITGB3 and BTG2), are potential therapeutic targets in dormant AML cells. Disclosures No relevant conflicts of interest to declare.
28
Craddock, Charles F., Farhat Khanim, Julie Arrazi, Bryan Turner, and Christopher Bunce. "Identification of Candidate Genes Associated with Sensitivity and Resistance to the Histone Deacetylase Inhibitor Valproic Acid in Patients with Acute Myeloid Leukemia." Blood 110, no. 11 (November 16, 2007): 810. http://dx.doi.org/10.1182/blood.v110.11.810.810.
Full textAbstract:
Abstract Histone deacetylases inhibitors (HDIs), such as valproic acid (VPA), demonstrate significant clinical activity in a proportion of patients with high risk acute myeloid leukemia (AML). However the mechanism by which HDIs selectively induce apoptosis in leukemic blasts remains unknown. We have therefore correlated the impact of VPA exposure on gene expression in leukemic cell lines and primary AML blasts with its ability to induce cell death in vitro and induce clinical responses in vivo. 14 hemato-lymphoid cell lines were tested for their sensitivity (IC50) to apoptotic cell killing by VPA. Gene expression array analyses using HGMP chip 6500 gene arrays were performed on the same cell lines prior to HDI exposure. A bioinformatics approach combined the array and IC50 data to generate a score for each gene identifying those whose elevated expression correlated with sensitivity or resistance to VPA in vitro. In a concurrent Phase I/II clinical trial 24 patients with high risk AML (relapsed n=11, newly diagnosed n=6, primary refractory n=3) with a median age of 64 yrs (41–83 yrs) received combination treatment with VPA, all trans-retinoic acid (ATRA) and theophylline. Changes in histone acetylation and expression of HDI responsive genes was measured in leukaemic blasts before and after commencement of VPA therapy. Expression of genes associated with VPA sensitivity in vitro were re-analysed with respect to their expression in pre-treatment blasts from non-responding and responding trial patients. By combining LC50 values to VPA and microarray data generated from pre-treatment RNA in 14 hematolymphoid cell lines we were able to identify candidate genes and signalling networks mediating sensitivity and resistance to VPA in vitro. Genes whose higher expression conferred sensitivity included IL-1β and those associated with VPA resistance included PLOD2, cyclin B1 (CCNB1) and ACVR2A. In the clinical trial 5 patients, all with relapsed AML, achieved clinical responses by Cheson criteria (complete remission n=1, partial remission n=4). Comparison of gene expression, as defined by microarray studies, in responding and non-responding patients with the previously identified in vitro VPA signalling networks identified that similar networks to those defined in vitro appeared to be correlated with clinical response. This study demonstrates induction of pro-apoptotic gene expression by VPA in a clinical context and identifies potential mechanisms through which its anti-leukaemic effect may be mediated in vivo. Furthermore we have identified potential VPA-signalling networks containing novel sensitivity and resistance genes whose expression correlates with VPA-mediated tumour cell killing in vitro and may predict clinical activity.
29
Jim, Johanna H., Pawandeep Dhami, Amanda King, Jonathan Cooper, and Dave Vetrie. "Identification of Target Genes of an Erythroid Transcription Factor Complex Containing SCL (TAL1)." Blood 110, no. 11 (November 16, 2007): 4068. http://dx.doi.org/10.1182/blood.v110.11.4068.4068.
Full textAbstract:
Abstract Haematopoiesis is the process whereby haematopoietic stem cells give rise to mature blood cell lineages. The SCL (TAL1) gene, originally identified by chromosomal translocations associated with T-cell acute lymphocytic leukaemia, encodes a key transcription factor (TF) which is expressed in various blood lineages and is essential for haematopoietic development. It has been shown that the SCL protein forms a multi-protein complex during erythroid development with other TFs (GATA1, E2A, LDB1, and LMO2) which binds to a sequence-specific motif to regulate the expression of glycophorin A and c-kit. We have used two complementary approaches to identify novel target genes regulated by this TF complex during erythroid development. In the first approach, we have transfected short interfering RNAs (siRNAs) into the K562 cell line to knockdown transiently the TFs of the erythroid complex. For all members of the complex, a knockdown efficiency of at least 70% was confirmed at the mRNA and protein level within 48 hours after transfection. The consequences of the knockdown at the level of gene expression were observed using Affymetrix GeneChips in order to identify downstream events associated with the erythroid complex in transcriptional programmes. In the second approach, chromatin immunoprecipitation (ChIP) was performed for each member of the complex in the K562 cell line and the ChIP material hybridised to a human transcription factor promoter microarray. A number of novel target genes for the SCL erythroid complex have been identified and verified independently using both approaches. Our data shows that members of the erythroid complex are involved in auto-regulation and regulate genes which control chromatin structure and function. These findings demonstrate that the expression of this TF complex is tightly controlled and point to an important role for it in orchestrating fundamental biological processes which have profound effects on gene expression in erythroid development.
30
Tholouli, Eleni, Sara A. MacDermott, Judith A. Hoyland, Caroline Glennie, Ric Swindell, John Liu-Yin, and Richard J. Byers. "Multiplex Quantum Dot ISH Measurement of Gene Expression in Tissue Microarrays Identifies HOXA9 and DNMT3A as Markers of Poor Response to Chemotherapy in Patients with Acute Myeloid Leukaemia." Blood 112, no. 11 (November 16, 2008): 2530. http://dx.doi.org/10.1182/blood.v112.11.2530.2530.
Full textAbstract:
Abstract Microarray-based expression profiling has identified prognostic gene signatures for many cancers and validation is required in clinical samples. However, most clinical material is in the form of formalin fixed and paraffin embedded tissue (FFPET) in which gene expression analysis is problematic. We have developed a generic quantum dot (QD) based multiplexed in-situ hybridization (ISH) method enabling quantitative localization of multiple mRNA targets in FFPET. We expand on our previous work introducing a method for standardization of ISH signal, enabling comparative measurement of gene expression across multiple samples. This was applied to tissue microarrays (TMAs) using archived trephine biopsies from patients with acute myeloid leukaemia (AML) to identify prognostic genes. A total of 15 TMAs were prepared using FFPET samples from 240 patients with AML diagnosed and treated between 1994 and 2005 at Manchester Royal Infirmary (Manchester, UK). For the analysis, 192 patients were included as the remainder either died before, during or immediately after one course of chemotherapy or there was incomplete data collection. The median age was 52 years (range 17–77) and all patients received intensive chemotherapy according to standard UK MRC AML protocols. Three cores were taken from each sample for TMA preparation. A standard was prepared using a cell pellet obtained from whole blood white cells which was embedded, in triplicate, in each TMA. QD-ISH was performed for nine genes recognized to be of prognostic value in AML. Triplex QD-ISH using QD labeled anti-sense cDNA oligonucleotides was performed for the following targets: Bcl2, survivin and XIAP; DNMT1, DNMT3A and DNMT3B; HOXA4, HOXA9 and Meis1. Signal intensity for each gene was measured using spectral imaging. Scrambled sense cDNA oligonucleotides were used to measure the level of background staining for each gene in each core. Background noise was corrected for by dividing expression levels of anti-sense probes by that of the scrambled probe, for both samples and standards. This enabled direct comparison between TMAs as gene expression values of samples were normalized against the standard. The mean expression of each gene was calculated for each patient, divided into quartiles and correlated with clinical outcome data. Statistical analysis was performed using contingency tables, the chi-square test and Mann Whitney-U. Overall survival (OS) and disease free survival (DFS) were displayed using the Kaplan-Meier method and Cox regression was performed for univariate and multivariate analysis. The OS in this cohort of patients was 43% at 5 years with 80% achieving complete remission (CR) after induction chemotherapy. Patient age (<60 years), WCC (<100×109/l) at diagnosis, cytogenetics (good and intermediate risk) and low HOXA4 expression (median 577 [95%CI 325–828]) were all associated with improved OS (p<0.0001; p=0.02; p<0.0001; p=0.013) and DFS (p<0.0001; p=0.013; p<0.0001; p=0.025) on univariate and multivariate analysis. High expression of HOXA9 (median 0.843 [0.145–7.479]; p<0.0001) and DNMT3A (median 1.305 [0.073–5.477]; p=0.04) were associated with failure to achieve CR. High Meis1 expression was found to be of borderline significance for poor response to chemotherapy (median 0.716 [0.051–7.840]; p=0.05). Expression levels of the remaining 5 genes did not show any correlation with CR, DFS or OS. These findings are consistent with recently published data regarding the prognostic significance of various new markers. In line with others we have demonstrated low expression of HOXA4 is an independent good prognostic marker in adult AML. Although high expression of HOXA9, DNMT3A and Meis1 was associated with inferior CR rates in our study, OS and DFS were not adversely affected. This may be related to improvements and more aggressive clinical practice (eg stem cell transplantation) over recent years which can overcome potential deleterious gene effects. These results demonstrate that the application of a standardized, quantitative multiplex QD-ISH can be used for identification of prognostic markers in FFPET samples. The advantages of this method include its application to TMAs which allows high sample throughput, use of archived materials and its transferability across a spectrum of malignancies.
31
Banerji, Lolita, Blanca Scheijen, and James D. Griffin. "Identification of Common Target Genes of the BCR/ABL and FLT3-ITD Tyrosine Kinases by Microarray Analysis." Blood 104, no. 11 (November 16, 2004): 3551. http://dx.doi.org/10.1182/blood.v104.11.3551.3551.
Full textAbstract:
Abstract Human myeloid leukaemias are commonly associated with activating mutations in tyrosine kinases. The BCR-ABL oncogene causes CML and is generated by the Philadelphia (Ph) chromosome translocation t(9;22)(q34;q11). The FLT3 tyrosine kinase receptor is mutated in about 30 % of all cases of AML, most often through a mechanism that involves an internal tandem duplication (ITD) of a small number of amino acid residues in the juxtamembrane domain. Both BCR/ABL and FLT3 (ITD) produce a constitutively active tyrosine kinase that enhances proliferation and viability of myeloid cells, and induce a similar myeloproliferative disorder when transplanted into mice. In an effort to identify any common downstream targets of BCR/ABL and FLT3(ITD) we generated paired Ba/F3 cell lines in which either Bcr/Abl or FLT3-ITD could be induced under the control of a tetracycline-inducible promoter. Treatment of these cells with doxycycline rapidly induced expression of BCR/ABL or FLT3-ITD proteins, respectively proteins. The gene expression profile of these conditionally expressing BCR/ABL and FLT3-ITD BaF3cells was examined using the U430A gene chip (Affymetrix), which contains more than 34000 well substantiated genes. With a 2.5 fold cut-off, Bcr/Abl altered the expression of 336 genes and FLT3-ITD altered expression of 231 genes at 24 hours after induction. Fifty one genes were regulated by both Bcr/Abl and FLT3-ITD, including Cyclin G2, cyclin D2, CXCR4, osteopontin and FKBP12. Following induction of BCR/ABL and FLT3-ITD, cyclin D2, osteopontin and FKBP12 are strongly upregulated, whereas cyclin G2 and CXCR4 are downregulated. We validated the expression pattern of these genes by real-time PCR analysis. To further validate that these genes are directly regulated by the kinase activity of BCR/ABL and FLT3-ITD, cell lines were treated with specific small molecule kinase inhibitors, resulting in both cases in a significant decrease in cyclin D2, osteopontin, and FKBP12 and an increase in cyclin G2 and CXCR4 expression. The functional role of several of these genes is under investigation. For example, we found that the PI3K/AKT pathway is important for regulating osteopontin gene expression, and that osteopontin is involved in mediating the increase in cell growth caused by both BCR/ABL and FLT3-ITD. The identification of downstream targets, such as cyclins, osteopontin, and others, that are shared in common by several oncogenic tyrosine kinase oncogenes suggest that they could have value as therapeutic targets.
32
Kapatai, Georgia, Stephen O. Nyangoma, and Pamela Kearns. "A Study of Aberrant DNA Methylation in Refractory and Relapsed Childhood Acute Lymphoblastic Leukaemia." Blood 114, no. 22 (November 20, 2009): 3456. http://dx.doi.org/10.1182/blood.v114.22.3456.3456.
Full textAbstract:
Abstract Abstract 3456 Poster Board III-344 Refractory and relapsed Acute Lymphoblastic Leukaemia (ALL) raise difficult clinical challenges and represent a significant cause of death in childhood and adolescence. This study examines epigenetic changes in relapsed paediatric ALL to increase our understanding of the mechanisms of treatment failure in these patients. Aberrant DNA methylation, a frequent phenomenon in paediatric ALL, has been identified as an independent factor of poor prognosis. In this study, using a CGI microarray consisting of 12K clones representing 5411 unique chromosomal sequences spread throughout the genome, we analysed a cohort of 22 childhood B-precursor ALL patient samples, 11 diagnosis samples and 11 relapse samples (unmatched) from patients who experienced a hematologic relapse, and developed genome-wide methylation profiles of leukaemic cells, at diagnosis and relapse. Comparison of these profiles identified 227 genes with statistically significant methylation changes – increase and decrease of methylation levels - between the two patient groups. The methylation status and the methylation change of 5 genes (ECRG4, CDH5, IRX3, FOXD3 and ERBB4) were independently verified, using pyrosequencing, in 6 ALL patients, 3 diagnosis and 3 relapse samples. The relationship between methylation and expression of one of these genes (ECRG4) was examined in ALL cell lines (CEMC1 and Jurkat) before and after 5-Aza-2'-deoxycytidine treatment. More genes are currently under investigation. Methylation studies on the repetitive element LINE-1 showed a significant increase in methylation of repetitive sequences in relapse samples (mean 79% vs. 89%, respectively; p=0.003, Mann Whitney test). Chromosomal mapping of the 227 genes revealed significant clustering of differentially methylated targets in chromosomes 11, 13 and 15, chromosomal locations that were previously found to be associated with ALL prognosis. Functional gene relation analysis revealed a connection between 11 genes (FOXD3, HPGD, NR4A2, CAST, TBRG4, XRCC6, NAB2, SEZL6, CDH13, NR2F1, SP1) that show increased methylation in relapse ALL and NR3C1, the gene coding for glucocorticoid receptor. Currently we are investigating the role of methylation in glucocorticoid resistance in ALL cell lines. Our data indicate an underlying epigenetic mechanism responsible for the chemoresistance observed in relapsed paediatric ALL patients. Disclosures No relevant conflicts of interest to declare.
33
Graham, Susan M., Gerry J. Graham, and Tessa L. Holyoake. "Gene Expression Profiling in Quiescent Stem Cells from Normal and Chronic Myeloid Leukaemia Patients." Blood 104, no. 11 (November 16, 2004): 2962. http://dx.doi.org/10.1182/blood.v104.11.2962.2962.
Full textAbstract:
Abstract Earlier studies have shown that Ph+ quiescent cells exist in chronic myeloid leukaemia (CML) (Blood (1999)94:2056) and we have previously shown that these cells are primitive in that they express the stem cell marker CD34. We have also shown that quiescent CML stem cells are insensitive to the effects of imatinib (IM Novartis Pharma) (Blood (2002) 99:319) and may present a possible source for relapse. This quiescent population therefore represents a potentially significant clinical problem and thus studies aimed at developing methods for eradicating this population are timely. In an effort to identify molecular markers of this population that may allow it to be specifically targeted during therapy, we have set out to investigate the transcriptional differences between quiescent and cycling stem cells. To this end, we have used specific stem cell enrichment and sorting protocols. Leukapheresis products from CML patients (N=5) in chronic phase at diagnosis and mobilised peripheral blood from allogeneic donors (N=3), were selected for CD34+ cells. Hoechst 33342 and Pyronin Y were used to discriminate the quiescent (G0) cells identified as Hoechstlo/Pyroninlo from the cycling cells. In combination with propidium iodide for dead cell exclusion we were able to sort 4–9x105 viable, quiescent stem cells and 4–11x106 cycling cells, which were processed for microarrays. Affymetrix gene chips (U133A) were used for the analysis and the data obtained was analysed using GeneSpring. Number of Genes Changed in Each Comparison 3 Fold 4 Fold 5 Fold CML G0 V CML Div 37 21 10 Norm G0 V Norm Div 188 92 47 CML G0 V Norm G0 168 85 49 CML Div V Norm Div 49 27 8 Initial analysis indicates that the greatest differences in gene expression are between the normal quiescent cells (G0) and normal dividing cells (Div) and between the normal quiescent cells and CML quiescent cells. A large percentage of the genes differentially expressed between the quiescent and cycling normal cells encode regulators of the cell cycle confirming the success of the sorting strategy for quiescent and cycling cells A selection of Genes Up-Regulated in Normal Cycling Cells Compared to G0 Gene Fold Up-regulation PCNA 3 CDC2 8 CCNB2 5 CCN1 3.5 CDC20 6 CDC25A 3.5 MCM5 3 In addition, many of the genes identified in our analysis are consistent with other published expression profiles for haemopoietic cells. Curiously, we have identified unanticipated changes in expression of cell cycle genes in the CML quiescent cells, which merit further investigation. We have also identified a number of unexpected genes as being more than 5 fold changed in the quiescent cells compared to dividing cells for both normal and CML samples. Specifically, there is a large cohort of genes preferentially expressed in quiescent normal or CML cells, which encode members of the chemokine family of proteins. Work is ongoing to establish the relevance, if any, of these genes to stem cell quiescence.
34
Pulford, Karen, Alison H. Banham, Linden Lyne, Margaret Jones, Greg Ippolito, Hiu Liu, Phil W. Tucker, et al. "BCL11AXL Protein: Its Distribution in Normal and Malignant Tissues and Use as a Marker for Plasmacytoid Dendritic Cells." Blood 106, no. 11 (November 16, 2005): 4392. http://dx.doi.org/10.1182/blood.v106.11.4392.4392.
Full textAbstract:
Abstract The B-cell lymphoma/leukaemia 11A (BCL11A) gene was first identified in the rare t(2;14)(p16;q32.3) translocation involving the IGH locus in aggressive B-cell chronic lymphocytic leukaemia (CLL) and, together with REL, is a candidate for the 2p16 chromosomal amplifications in B-cell malignancies. BCL11A encodes a Kruppel zinc finger transcription factor essential for pre-B-cell development and thymocyte maturation. Alternative RNA splicing generates at least three BCL11A transcripts encoding proteins that share a common N-terminus with the BCL11AXL transcript being the most abundant BCL11A transcript in normal tissues. We have produced a murine monoclonal antibody (BCL11A/123, isotype IgG1) specific for the BCL11AXL protein and, using this reagent, provide the first description of BCL11AXL protein distribution in normal and malignant human tissues. Initial studies using another antibody to the common N-terminus indicated the BCL11AXL protein to be the dominant isoform. In lymph node, tonsil and spleen, BCL11AXL expression was detected in B-cell nuclei in the mantle zone, germinal centers, interfollicular areas, splenic marginal zones and in tonsillar epithelium, but not in plasma cells or T cells. The differential expression of BCL11AXL in B-cell populations suggests its downregulation might be a requirement for plasma cell differentiation and is consistent with reports that BCL11A is transcriptionally repressed by Blimp-1. A subpopulation of cells, mostly B-cells, in the thymic medulla and scattered cells in the cortex were BCL11AXL+. Labeling of a very small number of CD3+ T-cells in the cortex and epithelial cells around the cortical periphery was also observed. Labeling of B-cell follicles in rat spleen demonstrated the BCL11A/123 epitope to be conserved across species. Another important finding was the high level expression of BCL11AXL in CD74+CD68+CD123+ plasmacytoid dendritic cells (pDCs). Publicly available microarray expression data are consistent with these findings, showing BCL11A transcripts to be present at high levels in peripheral blood B cells, in tonsil, lymph node and fetal brain with the highest expression in pDCs. BCL11AXL therefore represents an additional marker for the identification of pDCs and may be important in the differentiation and/or functional activity of this cell type. BCL11AXL was commonly detected in B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia and a subgroup of Hodgkin’s lymphomas, with the exception of multiple myeloma. Tumor cells in a small number of T-cell lymphomas (3/29) also expressed BCL11AXL. In a tissue microarray, comprising 107 cases of de novo DLBCL at diagnosis, BCL11AXL expression correlated with that of its interaction partner BCL6 and showed a trend towards increased overall survival. The study of this molecule should provide additional invaluable information concerning the possible role of BCL11A in lymphomagenesis.
35
Starkova, Julia, Jozef Madzo, Gunnar Cario, Katerina Krejcikova, Ondrej Hrusak, and Jan Trka. "Identification of Genes Differentially Expressed after HDACi Treatment in TEL/AML1-Positive Leukaemic Cells." Blood 106, no. 11 (November 16, 2005): 4364. http://dx.doi.org/10.1182/blood.v106.11.4364.4364.
Full textAbstract:
Abstract The role of the TEL/AML1 fusion gene in leukaemogenesis is still not understood despite its frequent occurrence in children with acute lymphoblastic leukaemia (ALL; 25%). Both partner genes are important for the development and maintenance of normal haematopoiesis. The TEL part of the fusion protein contains domains interacting with the mSin3, N-CoR and HDAC-3 corepressors. A part of the AML1 gene involved in the fusion carries a DNA binding domain that controls the interaction with the cis-regulatory elements of the target genes. The TEL/AML1 protein seems to behave like a repressor blocking the expression of the genes originally transactivated by AML1. Target genes of AML1 protein are described in myeloid differentiation, but not in lymphopoiesis. We aim to identify these target genes. In our previous experiments we monitored the changes of immunophenotype after histone deacetylase inhibitors (HDACi; valproate (VPA), Trichostatin A (TSA)) administration of TEL/AML1[+] cells. Surviving TEL/AML1[+] cells showed drift in expression of CD10 and CD20 surface antigens and in intracellular level of RAG-1 and TdT proteins, thus reflecting a change in the differentiation stage. The protein expressions of RAG1 and TdT were confirmed to RNA level by RQ-RT-PCR (control(C) vs. TSA p<0.0001, C vs. VPA p=0.0002). We performed an expression profiling of treated vs. untreated TEL/AML1[+] cells and TEL/AML1[+] vs. other ALL patients. First we calculated a cut-off expression for each gene in ALL patients. According to the analysis of these cut-offs we selected genes specifically over- or under-expressed in TEL/AML1[+] patients. These genes were compared to genes which expression changed significantly after HDACi. Finally, we analysed interdigitated genes for their divergence between TEL/AML1[+] and other subgroups of ALL from publicly available expression profiling data. By this combined multi-step approach, we chose 5 genes which expression is i) specific for TEL/AML1[+] patients and ii) significantly changed after HDACi administration. Microarray data of chosen genes showed downregulation of JunD, ACK1, PAK1 and PDGFRB in TEL/AML1[+] patients as well as in our cell-line model with increased expression after HDACi treatment. TCF4 gene was upregulated in the studied group and the administration of HDACi lead to its downregulation. We confirmed changes of chosen genes expression levels by RQ-RT-PCR: JunD - C vs. TSA p=0.013, C vs. VPA p= 0.0008; PDGFRB - C vs. TSA p< 0.0001, C vs. VPA p=0.016; TCF4 - C vs. TSA p< 0.0001, C vs. VPA p=0.0002; ACK1 - C vs. VPA p=0.07. These genes have a fundamental role in cell cycle progression and proliferation, therefore their role in leukaemogenesis is presumptive. These data also support the hypothesis that the effect of HDACi on TEL/AML1[+] cells is directly related to the function of TEL/AML1 protein, and the treatment with HDACi is able to release cells from differentiation block caused by TEL/AML1 aberrant transcription factor. Supported by Zentiva,Inc.
36
Mills, Ken I., Alex Kohlmann, Mickey Williams, Wei-Min Liu, Rachel Li, David T. Bowen, Helmut Loeffler, et al. "Microarray Classification of Myelodysplastic Syndrome (MDS) Identifies Subgroups with Distinct Clinical Outcomes." Blood 110, no. 11 (November 16, 2007): 2426. http://dx.doi.org/10.1182/blood.v110.11.2426.2426.
Full textAbstract:
Abstract The MILE (Microarray Innovations in LEukemia) study has previously shown that gene expression signatures associated with initial leukaemia classifier (LCver7) give an overall cross-validation accuracy of >95% for distinct sub-classes of pediatric and adult leukemias. However, only 50% of the 174 MDS samples in the whole-genome microarray analysis (Stage 1) of the MILE study were correctly identified; the remainder showed AML-like or non-leukemia-like gene profiles. An external morphological review (DB & HL) according to FAB and WHO criteria, of the 174 slides was performed independently (blind) which resulted in 6 samples being reclassified as AML and 4 non-leukemia cases excluded from the study. A recently improved, hierarchical based algorithm correctly identified 100% of the confirmed MDS cases. In this study, using LCver7, the confirmed 164 samples had 50% MDS classifications (Class 17), 23.8% non-leukemia classifications (Class 18), and 22.6% AML classifications (Classes 13 or 14) with the remaining 3.7% having a classification tie between 2 or 3 Classes (due to low confidence). No 5q- syndrome patients had an AML call, whilst 68.3% of RAEB2 patients had an AML classification and none were Class 18. Similarly, 95.6% of Low IPSS patients were classified as Class 17 or 18, whilst all patients (n=5) with High IPSS had an AML call. The classification was independent of blast cells: 10.2% of Class 18 calls had >5% blasts; 28.2% of AML-like cases had <5% blasts. Outcome data (132 MDS patients) was correlated with Class: significant difference (p<0.028, (Kaplan-Meier)) was seen in overall survival; with p <0.004 if AML (Classes 13 & 14) was compared to “non AML” (Classes 17 & 18). Statistically significant differences were seen for time to transformation to AML between the classes (p<0.0001) and between AML and “non AML” (p<0.00007, Kaplan-Meier)) with a probability of transformation of 44% at 18 months for the AML group compared to <8% for the “non-AML” group. A further linear classifier has been used to discriminate patients who transform to AML within 18 months (poor prognosis) with patients with no transformation after >60 months (good prognostic group). Bioinformatic analysis of molecular mapped functions and canonical pathways showed that cell signalling processes were over-represented when comparing de novo AML (n=204) with MDS, from the MILE study, whilst signal transduction pathways were deregulated when comparing non-leukemia samples (n=71) with MDS. Similar pathways and functions were also deregulated when comparing the correctly classified MDS with Class 17 call against MDS with Class 18 call and MDS with AML Classes 13 or 14 calls. In conclusion, the use of microarrays within the initial study, solely intended for diagnostic purposes, has now evolved towards a position in which novel prognostic value may be gained from distinct gene expression signatures. This has also resulted in a better molecular understanding of the progression from non-leukemia, through MDS into full blown AML.
37
Skov, Vibe, Thomas Stauffer Larsen, Mads Thomassen, Caroline Riley, Morten Krogh Jensen, Ole Weis Bjerrum, Torben A. Kruse, and Hans Carl Hasselbalch. "Gene Expression Profiling with Principal Component Analysis Depicts the Biological Continuum From Essential Thrombocythemia Over Polycythemia Vera to Myelofibrosis." Blood 116, no. 21 (November 19, 2010): 4115. http://dx.doi.org/10.1182/blood.v116.21.4115.4115.
Full textAbstract:
Abstract Abstract 4115 Introduction: The classical Philadelphia-negative chronic myeloproliferative neoplasms (CMPNs) encompass polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). After several years, a proportion of patients with PV may develop increasing splenomegaly and bone marrow fibrosis to terminate in postpolycythemic myelofibrosis, which then has a very poor prognosis with median survival of only a few years. A high proportion of the ET-population has been shown to have prefibrotic PMF or a “PV-like” phenotype, which after a variable time may terminate in myelofibrosis with myeloid metaplasia (MMM). Although the concept of prefibrotic myelofibrosis as a separate disease entity to be distinguished from ET by distinct histopathological features has been questioned, it seems logical that a continuum exists from “early” myelofibrosis with no or minimal bone marrow fibrosis to the advanced stage of myelofibrosis with myeloid metaplasia. Accordingly, a new concept of these diseases as a biological continuum from ET over PV to PMF has emerged. Indeed, this biological continuum reflects the various clinical stages as also described for chronic myelogenous leukaemia (CML) – a chronic stable phase (ET-PV), an accelerated phase (transitional phase), and a terminal myelofibrosis or blast phase of the disease (PMF-BP). It is evident that the JAK2 V617F mutational load is an important determinant of phenotype, but other genetic and epigenetic events contribute to the phenotypic presentation. Several gene expression studies have identified genes which might be of pathogenetic relevance for the development of CMPNs. However, no study has applied an unsupervised method as Principal Component Analysis (PCA) to discover unknown trends in the data. We have carried out gene expression profiling with PCA to ascertain if this analysis might unravel distinct grouping of disease entities supporting the biological continuum from early ET over PV to the advanced myelofibrosis phase. Patients and Methods: Using HG-U133 Plus 2.0 microarray from Affymetrix, recognizing 54675 probe sets (38500 genes), gene expression microarray studies have been performed on control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). An unsupervised statistical method, PCA, which is an exploratory tool used to uncover unknown trends in the data based on gene expression profiles, was applied to the data. Results: The results from the PCA confirm the hypothesis of a biological continuum from control subjects over ET to PV and finally PMF (figure 1). The figure also reveals that patients with PV in a transitional stage (PVtrans) with pancytosis, huge splenomegaly, and bone marrow fibrosis have a gene profile between PV and PMF. Furthermore, single gene analysis revealed that several genes are highly dysregulated in patients with ET, PV, and PMF compared to control subjects even after correcting for multiple testing. Discussion and Conclusions: Using global gene expression profiling with PCA in patients with different CMPNs (ET, PV, PMF), we have yielded support to the concept of a biological continuum from early ET to the advanced myelofibrosis stage. Studies are in progress to identify single genes or clusters of genes accounting for the biological continuum and accordingly the progression from ET over PV to myelofibrosis. Disclosures: No relevant conflicts of interest to declare.
38
Thomas, Daniel, Jason A. Powell, Emma F. Barry, Luen Bik To, Angel Lopez, and Mark A. Guthridge. "A Molecular Signature for a Cytokine Receptor Survival Pathway in AML Identifies Unique Prognostic Indicators and Therapeutic Targets." Blood 112, no. 11 (November 16, 2008): 2893. http://dx.doi.org/10.1182/blood.v112.11.2893.2893.
Full textAbstract:
Abstract The IL-3 receptor regulates myeloid cell survival and proliferation and is overexpressed on AML blasts and CD34+CD38- AML “stem-like” cells suggesting it plays a role in leukemic cell survival that could be exploited by targetted therapy. We have previously shown that Serine 585 in the cytoplasmic domain of the beta subunit of the IL-3 receptor is phosphorylated in response to sub-picomolar concentrations of cytokine, binds 14-3-3 zeta adaptor and is required for cytokine mediated survival (Guthridge et al, 2006, EMBO). We wished to examine the phosphorylation status of this receptor in AML blasts from patients and the expression levels of downstream Ser585 target genes. Results: We show that Ser585 of the beta common subunit is constitutively phosphorylated in the majority of leukaemic patients regardless of Flt3 status or cytogenetics (18/21 patients). In contrast, tyrosine phosphorylation of the receptor is not deregulated and remains cytokine-dependent (20/21). We have identified a panel of 139 Ser585-regulated genes by microarray (WT receptor vs Ser to Gly mutant) and show that a specific subset of these genes is deregulated in AML blasts confirmed by RT-PCR compared to normal mononuclear cells consistent with constitutive activation of this pathway in AML. We have identified a 4-gene signature that is a molecular correlate of the Ser585-survival pathway in AML. We further identify Ser585-regulated genes that are prognostic indicators of clinical outcome in a cohort of adult patients AML (n=33) independent of WCC or cytogenetic abnormalities. Targetting Ser585 survival genes using siRNA or blocking mAbs induces apoptosis in AML blasts validating Ser585-regulated genes as potential targets for therapy. Conclusions: Given the emerging problem of resistance to tyrosine kinase inhibitors (TKI) and limited success of Flt3 inhibitors in AML, new strategies for therapy are needed. By interrogating a cytokine survival pathway that functions independently of tyrosine phosphorylation we have uncovered novel Ser585 gene targets with promising clinical utility in leukaemia.
39
Strefford, Jon C., Frederic van Delft, Manoj Raghavan, Hazel M. Robinson, Anthony V. Moorman, Bryan D. Young, Vaskar Saha, and Christine J. Harrison. "Molecular Characterization of AML1 (RUNX1) Amplification: A Poor Risk Chromosomal Marker in Acute Lymphoblastic Leukaemia (ALL)." Blood 104, no. 11 (November 16, 2004): 140. http://dx.doi.org/10.1182/blood.v104.11.140.140.
Full textAbstract:
Abstract We have recently defined a new recurrent chromosomal abnormality in patients with acute lymphoblastic leukemia (ALL) involving the duplication of chromosomal band 21q22 and amplification of the AML1 (RUNX1) gene. Currently, fluorescence in situ hybridization (FISH) with probes directed to the AML1 gene is the only reliable detection method. AML1 signals are seen as clusters in interphase cells and in tandem duplication on the long arm of chromosome 21 in metaphase. To date, we have identified 46 patients with this abnormality: 26 males and 20 females. The median age of the 44 children was 9 years (range 4–16 years): two adults were 20 and 30 years old. Their median white cell count was 3.7x109/L (range 1−80x109/L) and all had a common/pre-B immunophenotype. The 3 year event free survival for the 24 children treated on MRC ALL97 was 51% (95% confidence interval 27%–71%). Hence, this abnormality has been associated with older children with a low white cell count and, most significantly, a poor prognosis. The NCRI UK Childhood Leukaemia Working Party has categorized these children into a high-risk group and recommended that they be treated on a more intensive protocol, making their accurate detection vital. To achieve this goal, we have attempted to define the amplified region on chromosome 21 and to identify the genes involved, employing a range of molecular techniques. Microarray analysis (genomic and expression) in conjunction with FISH and quantitative PCR were used to investigate DNA and/or RNA samples from 11 patients. Comparative genomic hybridization (Spectral Genomics 1Mb) (aCGH) showed variable regions of amplification (common minimal region ~10Mb DNA). Complementary FISH, using a range of specific probes corresponding to the aCGH clones, confirmed the extent of the amplified region on chromosome 21, while indicating the precise copy number changes for each clone. FISH also revealed unexpected chromosomal rearrangements of the amplified chromosome, including loss of the 21q telomeric region. These observations confirm the chromosomal instability associated with this abnormality, as previously indicated from the variable chromosome morphology seen by cytogenetic analysis. Expression profiling (Affymetrix HG-U133) detected 60 over-expressed genes in these patients, of which six were located within the chromosome 21 amplicon. This study has defined the extent of the chromosome 21 amplified region surrounding AML1, and highlighted the over-expression of specific genes within the amplicon. This combined molecular approach will help to identify the most appropriate test for the detection of this high-risk abnormality.
40
Ichim, Christine V., Harold L. Atkins, Norman N. Iscove, and Richard A. Wells. "Characterization of a Hierarchy of Proliferative Ability in AML: Identification of a Role for NR2F6 in the Maintenance of the Undifferentiated State." Blood 108, no. 11 (November 16, 2006): 2539. http://dx.doi.org/10.1182/blood.v108.11.2539.2539.
Full textAbstract:
Abstract AML cells are heterogeneous in their capacity to proliferate and form colonies in vitro. Determination of the genes whose expression is necessary for clonogenicity has been hindered by the inability to isolate pure populations of AML cells with defined proliferative abilities. By analyzing the growth of clonal siblings we established that the hierarchical organization of AML can be resolved into distinct strata, permitting the use of clonal siblings as biological reporters of the transcriptional signature of single cells with defined proliferative abilities. We then analyzed low passage cultures of the cell line OCI-AML4. Clones consisting of four cells were micromanipulated so that a single cell was sampled for global RT-PCR while its three clonal siblings served as reporters of clonogenicity. By microarray analysis we found the orphan nuclear receptor NR2F6 to be expressed four-fold lower in leukaemia single cells that spontaneously lose proliferative ability, compared to single cells with greater proliferative capacity. NR2F6 is a poorly characterized member of the COUP transcription factor family. We observed that NR2F6 is overexpressed in AML, CMML and MDS relative to normal bone marrow (BM). We then assessed expression of NR2F6 in U937 leukaemia cells induced to differentiate with a variety of induction agents. All differentiation agents induced significant decreases in NR2F6 expression. To characterize the effect of forced expression of NR2F6 we transduced U937 cells with a retrovirus encoding either NR2F6 or EGFP. Forced expression of NR2F6 reduced the doubling time of these populations, and inhibited retinoic acid induction of U937 cell differentiation. NR2F6 overexpressing cells did not acquire expression of the maturation marker CD11b nor were capable of reducing nitroblue tetrazolium, indicating functional maturity. Assessment of DNA content and analysis of growth kinetics revealed that NR2F6 overexpressing cells did not undergo the proliferative arrest associated with terminally differentiating U937 cells. We then investigated the role of NR2F6 in normal haematopoiesis in the C57/BL6 and Balb/c strains of mice. In both strains we observed that overexpression of NR2F6 resulted in a significant reduction in BFU-E and CFU-GM colony numbers, and colony size. These data are consistent with the notion that NR2F6 inhibits maturation of normal BM. More specifically, analysis of the immunophenotype of BM cultured in suspension for 10 days showed that NR2F6 overexpression resulted in a significant reduction in monocytes, and an increase in mast cells. An excess of mast cells was also observed in cytospin preparations of CFU-GM colonies. Additionally, we have demonstrated that overexpression of NR2F6 augments the in vitro self-renewal ability of BM, using serial replating of semi-solid cultures. Competitive BM transplantation experiments were employed to confirm the role of NR2F6 on stem cell self-renewal and differentiation in vivo. While these investigations are in progress, progenitor assays and analysis of serial-replating in all animals examined have recapitulate our ex vivo observations. Overall, these data establish the importance of NR2F6 in the regulation of normal and leukaemic haematopoietic cell self-renewal and differentiation. Furthermore, our results establish that analysis of clonal siblings allows the elucidation of differences in gene expression within the AML hierarchy.
41
Hotinski, Anya K., Karen M. Lower, and Bryone J. Kuss. "Somatic MDC1 Mutation in Putative Pre-Leukaemic Stem Cell of a Biclonal Case of Chronic Lymphocytic Leukaemia." Blood 132, Supplement 1 (November 29, 2018): 5534. http://dx.doi.org/10.1182/blood-2018-99-115704.
Full textAbstract:
Abstract Chronic lymphocytic leukaemia (CLL) is an inherently heterogeneous disease in which founding lesions and cell of origin remain a subject of debate. This case of truly biclonal CLL provides evidence of the presence of a pre-leukaemic stem cell, supporting other lines of evidence that pathogenic genetic and epigenetic lesions in CLL arise early in haematopoiesis. An individual with trisomy 12 containing CLL, present at a frequency of 30%, was identified. Two distinct CD5/19-positive CLL clones were separated by flow cytometry, based on bimodal expression of CD49d. Each leukaemic clone was subjected to targeted immunoglobulin heavy chain variable region gene (IGHV) sequencing, whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray. CD5-positive T cells and a non-malignant superficial skin biopsy sample from the same individual were analysed as comparators. The CD49d-positive and CD49d-negative fractions harboured completely different IGHV rearrangements and mutational statuses (V4-34*02, hypermutated, and V3-21*02, unmutated, respectively) indicative of two unique leukaemic clones. Furthermore, trisomy 12, a relatively common aneuploidy in CLL, was present at high frequency (89.9%) in the CD49d-positive fraction but was absent in the CD49-negative and T-cell fractions. A mutation in the known CLL driver SF3B1 (c.1866G>T) was present in the CD49-negative, unmutated clone alone, along with a 17p deleted subclone in around 20% of this fraction. The presence of a 17p deletion is highly clinically relevant in this disease, and it is noteworthy that this subclone was not detected by routine fluorescence in situ hybridisation (FISH) at diagnosis. Two putative driver mutations in KMT2D and BCL11B were detected in the CD49d-positive, trisomy 12 clone alone (c.15256C>T and c.1387G>A respectively). A TET2 mutation (c.3609C>G) was found in all three fractions. The mutation in this epigenetic regulator has previously been described in clonal haematopoiesis of indeterminate potential (CHIP) and was hypothesised to have been acquired in a haematopoietic stem cell predisposing the individual to the development of haematological malignancy. However, the presence of this mutation was confirmed by Sanger sequencing of genomic DNA extracted from the skin sample, and is therefore of germline origin. The role of germline TET2 variants has not been described in lymphoid malignancy. Importantly, eight coding mutations were found common to both leukaemic clones but were absent from T-cells, implicating a common cell of origin prior to IGHV rearrangement but following B-lineage commitment (that is, a putative pre-leukaemic stem cell). One of these mutations was a disruptive 123 base pair deletion (c.4824_4946del) in MDC1 (mediator of DNA damage checkpoint 1) and was present at a variant allele frequency (VAF) of approximately 50% in both CLL clones, suggesting it was a founder mutation prior to subclone divergence. MDC1 is a mediator in the Ataxia Telangiectasia Mutated (ATM)-dependent DNA damage repair pathway and has not been previously implicated in CLL pathogenesis unlike ATM, which is frequently lost in del11q CLL. The proposed evolution of CLL in this case is presented in Figure 1. In summary, this highly unusual case of CLL provides insights into the evolution of CLL and adds another line of evidence to support the proposed early origins of CLL in haematopoiesis. The role that MDC1 may contribute to CLL pathogenesis, in particular its association with ATM dysregulation, remains to be elucidated. Further work to delineate critical pathways based on the discovered mutations is underway, and includes global transciptomic analysis of the purified leukaemic clones by RNAseq. Disclosures No relevant conflicts of interest to declare.
42
Tholouli, Eleni, Sarah MacDermott, Judith Hoyland, John Liu Yin, and Richard Byers. "Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia." Biochemical and Biophysical Research Communications 425, no. 2 (August 2012): 333–39. http://dx.doi.org/10.1016/j.bbrc.2012.07.092.
Full text
43
Grace, Colin, and Elisabeth P. Nacheva. "Are CML Lymphoid Blast Crisis and Ph Positive ALL Genomically Indistinguishable?" Blood 116, no. 21 (November 19, 2010): 4464. http://dx.doi.org/10.1182/blood.v116.21.4464.4464.
Full textAbstract:
Abstract Abstract 4464 Philadelphia positive malignant disorders are a clinically divergent group of hemoblastoses with a unique identifying feature, the BCR/ABL1 fusion gene, usually resulting from the chromosome rearrangement t(9;22)(q34;q11) or its variants, that leads to constitutive expression of an aberrant tyrosine kinase. These include chronic myeloid leukaemia (CML) and de novo acute leukaemia of both myeloid Ph(+)AML and lymphoid origin Ph(+)ALL. The latter two disorders are clinically aggressive and therapy challenging even in the era of the powerful tyrosine kinase inhibitors. CML is a multistage progressive disease, which if untreated, inevitably ends as fatal acute leukaemia, either myeloid or lymphoid. The latter is often thought to be indistinguishable from Ph(+)ALL, the most common type of ALL in adults. We have identified DNA sequences the imbalances of which appear to be significantly associated with the disease stage and lineage origin in CML and Ph(+)ALL samples. We used array CGH at a resolution of ~2kb to explore hot spot regions obtained from 102 patient samples comprising 92 CML and controls together with 10 Ph(+)ALL and show how Significance Analysis of Microarrays (1) can be used to identify differences in the genome profile of de novo Ph(+)ALL and lymphoid blast transformation of CML. We show that lymphoid blast crisis CML differs significantly from Ph(+)ALL not only due to the presence of 9p deletions but also due to genomic gains in other chromosomes. Furthermore we identify a sub group of Ph(+)ALL with a distinctive genomic profile. Having identified genome regions of potential interest, ranked in order of significance, out of the 100's of thousands of array results, it is then a challenge to design further experiments to evaluate their contribution to the biology of the BCR/ABL positive disease. 1 Tusher V, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98:5116-5121 (2001). Disclosures: No relevant conflicts of interest to declare.
44
Zibara, Kazem, Daniel Pearce, David Taussig, Spyros Skoulakis, Simon Tomlinson, Emilie Bureau, Bryan D. Young, and Dominique Bonnet. "Gene Expression Profiling Reveals Fundamental Differences between Leukemic Stem Cells (Lin-CD34+CD38−) and Mature Blasts (Lin−CD34+CD38+) of Acute Myeloid Leukaemia (AML) Patients." Blood 106, no. 11 (November 16, 2005): 1377. http://dx.doi.org/10.1182/blood.v106.11.1377.1377.
Full textAbstract:
Abstract The identification of LSC has important implications for future research as well as for the development of novel therapies. The phenotypic description of LSC now enables their purification and should facilitate the identification of genes that are preferentially expressed in these cells compared to normal HSC. However, gene-expression profiling is usually conducted on mononuclear cells of AML patients from either peripheral blood and/or bone marrow. These samples contain a mixture of blasts cells, normal hematopoietic cells and limited number of leukemic stem cells. Thus, this results in a composite profile that obscure differences between LSC and blasts cells with low proliferative potential. The aim of this study was to compare the gene expression profile of highly purified LSC versus leukemic blasts in order to identify genes that might have important roles in driving the leukemia. For this purpose, we analyzed the gene expression profiles of highly purified LSCs (Lin−CD34+CD38−) and more mature blast cells (Lin−CD34+CD38+) isolated from 7 adult AML patients. All samples were previously tested for the ability of the Lin−CD34+CD38− cells but not the Lin−CD34+CD38+ fraction to engraft using the non-obese diabetic/severe combined immuno-deficiency (NOD-SCID) repopulation assay. Affymetrix microarrays (U133A chip), containing 22,283 genes, were used for the analysis. Comparison of Lin-CD34+CD38- cell population to the Lin−CD34+CD38+ cell fraction showed 5421 genes to be expressed in both fractions. Comparative analysis of gene-expression profiles showed statistically significant differential expression of 133 genes between the 2 cell populations. Most of the genes were downregulated in the LSC-enriched fraction, compared to the more differentiated fraction. Gene ontology was used to determine the categories of the up-regulated transcripts. These transcripts, which are selectively expressed, include a number of known genes (e.g., receptors, signalling genes, proliferation and cell cycle genes and transcription factors). These genes play important roles in differentiation, self-renewal, migration and adhesion of HSCs. Among the genes showing the highest differences in expression levels were the following: ribonucleotide reductase M2 polypeptide, thymidylate synthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II, CDC20, nucleolar and spindle associated protein 1, Rac GTPase activating protein 1, leukocyte immunoglobulin-like receptor, proliferating cell nuclear antigen, myeloperoxidase, cyclin A1 (RRM2, TYMS, ZWINT, CTSG, AZU1, TOP2A, CDC20, NUSAP1, RACGAP1, LILRB2, PCNA, MPO, CCNA1). Some transcripts detected have not been implicated in HSC functions, and others have unknown function so far. This work identifies new genes that might play a role in leukemogenesis and cancer stem cells. It also leads to a better description and understanding of the molecular phenotypes of these 2 cell populations. Hence, in addition to being a more efficient way to further understand the biology of LSC, this should also provide a more efficient way of identifying new therapeutics and diagnostic targets.
45
Ford, Anthony M., Penny Cardus, and Mel F. Greaves. "Modelling Molecular Consequences of Leukemia Initiation by TEL-AML1 Fusion." Blood 104, no. 11 (November 16, 2004): 2056. http://dx.doi.org/10.1182/blood.v104.11.2056.2056.
Full textAbstract:
Abstract We have previously shown that TEL-AML1 (ETV6-RUNX1) fusions in acute lymphoblastic leukemia commonly arise pre-natally. The fusion protein initiates, presumably via transcriptional deregulation, a pre-leukemia condition that infrequently (~1%) converts over a 10 year period to overt leukaemia in the presence of post-natal, secondary genetic changes including TEL deletion. One challenge is to identify the molecular mechanisms by which TEL-AML1 elicits and sustains stable pre-leukemic clones which are then vulnerable to additional genetic alterations in the context of particular etiological exposures (e.g. infection). We have established cell lines of murine lymphoid progenitor cells that stably express a regulatory, truncated human progesterone receptor ligand-binding domain (pSwitch, Invitrogen) that undergoes a conformational change in the presence of the steroid agonist, mifepristone and conversion to an active form. The active binding domain is then able to bind to and activate transcription from an otherwise silent TEL-AML1 gene that we have also stably introduced into these cells. In the presence of sub-physiological levels of mifepristone, TEL-AML1 becomes abundantly expressed in the nucleus within a few hours. Microarray analysis comparing uninduced (time 0) cDNA with cDNA from both control and TEL-AML1 induced cells grown for 4 days in the presence of mifepristone showed changes in the expression pattern for over 250 genes. The majority of these genes were down regulated in the presence of TEL-AML1, in keeping with a repressive activity for the fusion gene. A number of apoptotic and cell cycle genes were down regulated, while anti-apoptotic genes were up regulated in the presence of the fusion protein. These genes are currently being examined by both in-silico and laboratory-based validation techniques and may provide key insights into the pathways that are deregulated by TEL-AML1 in ALL.
46
Gupta, Dikshat Gopal, Neelam Varma, Shano Naseem, Man Updesh Singh Sachdeva, Pankaj Malhotra, Sreejesh Sreedharanunni, Subhash Varma, et al. ""Detection of BCR-ABL1-like Subtype in Adult Acute Lymphoblastic Leukemia Using Digital Ncounter Nanostring Technology"." Blood 136, Supplement 1 (November 5, 2020): 13–14. http://dx.doi.org/10.1182/blood-2020-136890.
Full textAbstract:
Introduction A new provisional entity of "B-lymphoblastic leukaemia/lymphoma, BCR-ABL1-like" has been introduced in the 2017 revised edition of WHO classification of tumours of haematopoietic and lymphoid tissues. BCR-ABL1-like cases are negative for Ph chromosome or t(9:22)(q34;q11.2) translocation, do not express the fusion BCR-ABL1 RNA transcripts and proteins resulting from the Ph chromosome,and are characterized by similar gene expression profiles as that of BCR-ABL1-positive acute lymphoblastic leukemia (BCR-ABL1-positive ALL).There is no 'short-cut approach' for making an accurate diagnosis of BCR-ABL1-like ALL. Two approaches namely Gene expression profiling (GEP) or Next-generation sequencing (NGS) and TLDA (TaqMan low-density array) are used for the detection of BCR-ABL1-like ALL cases. NGS is very costly and data interpretation requires a lot of bioinformatics skills and TLDA is not commercially available in India. Aims We planned to study the whole transcriptome of BCR-ABL1-positive ALL cases using microarray GEP, followed by customizing targeted gene panel using nCounter NanoString technology, for the detection of BCR-ABL1-like cases. METHODS Flow cytometric immunophenotying (FCM-IP) and multiplex RT-PCR were performed on 200 B-ALL cases to detect BCR-ABL1 chimeric fusion transcripts. Further, 12 BCR-ABL1-positive cases were subjected to transcriptome profiling using Affymetrix microarray (Gene Chip Human Genome U133 Plus 2.0 Array). The results were analyzed using TAC 4.0 software. Finally, a targeted panel of 50 differentially expressed genes [including 5 Housekeeping genes (HKGs)] was constructed according to our microarray findings and previously published data (Harvey RC et al.ASH 2013). A total of 96 B-ALL cases (16 BCR-ABL1-positive cases & 80 BCR-ABL1-negative cases) were subjected to GEP using nCounter Platform. The results were analyzed using nSolver4.0 software. RESULTS In the study cohort of 200 adult B-ALL cases, BCR-ABL1 chimeric fusion transcripts were detected in 34% (b2a2 and b3a2=18.05% & e1a2=15.5%), as revealed by multiplex assay. Global transcriptome profiling of 12 BCR-ABL1 RNA transcripts revealed a total of 1574 as DE genes (460 genes in e1a2, 515 genes in b2a2 and 599 genes in b3a2). DE genes were further filtered through hierarchical clustering analysis and a total of 45 DE genes with 10- to -86-fold change were identified. These genes were further analyzed using nCounter NanoString. To further identify the best classifier genes, log2 normalized expression values were analyzed using penalized logistic regression. Based on previous literature and regression coefficient values, 15 genes were finally selected whose performance was individually analyzed using receiver operating characteristic curve (ROC) and area under the curve (AUC). Optimal thresholds for these genes were estimated as the values with maximum sensitivity and specificity. Out of 78 examined BCR-ABL1-negative cases, 33(42.30%) BCR-ABL1-negative cases were clustered together with 15 BCR-ABL1-positive cases and were attributed as BCR-ABL1-like ALL cases in principal component analysis. Further, we categorized CRLF2 in two categories; high CRLF2 cases 25/33 (75.75%) & low 8/33 (24.24%) in BCR-ABL1-like ALL cases. JAK2p.R683G mutation was screened in CRLF2 high cases and showed positivity in 19/24 (79.16%) by the Amplification Refractory Mutation (ARMS) PCR. In 25 cases, the average log fold change of -0.80 &-5.83 was seen in P2YR8 & CSF2RA respectively by qPCR. In CRLF2 low expressing cases, the average log fold change of 11 kinase genes showed -0.75 in CENPC, -0.66 FOXP1, -0.16 NUP153, 1.04 RCSD1, 1.50 PAX5, 1.12 FLT3, -5.65 EPOR, -4.03 ILR2B, -3.46 PDGRFB, -7.49 NTRK3 &-2.83 ZNF274 respectively. The average log fold change of IKZF1 in 80 BCR-ABL1-negative cases was found to be 1.07. DISCUSSION & CONCLUSION We have devised a method that includes 15 genes according to AUC/ROC for the detection of BCR-ABL1-like ALL cases, using nCounter NanoString technology for the first time in Indian patients. Furthermore, we are planning to validate this model in future, on 50 BCR-ABL1-positive and 150 BCR-ABL1-negative cases and devise a simple, efficient, cost-effective qPCR method. It is very important to detect BCR-ABL1-like ALL cases to start the desirable TKI therapy & aid in treatment stratification, prognostication, and improve the overall survival of these patients. Figure Disclosures No relevant conflicts of interest to declare.
47
Fabris, Sonia, Katia Todoerti, Laura Mosca, Luca Agnelli, Domenica Ronchetti, Daniela Intini, Marta Lionetti, et al. "Molecular and Transcriptional Characterization of the Novel 17p11.2-p12 Chromosome Amplification in Multiple Myeloma." Blood 110, no. 11 (November 16, 2007): 2486. http://dx.doi.org/10.1182/blood.v110.11.2486.2486.
Full textAbstract:
Abstract Multiple myeloma (MM) is a fatal malignancy of clonal bone marrow plasma cells characterized by a high genomic instability increasing with disease progression. We describe here a genomic amplification at 17p11.2–p12, an unstable chromosomal region characterized by a large number of low copy repeats which have been proven to mediate deletion and duplication in several genomic disorders and amplifications in solid tumors. An approximately 5 Mb 17p11.2–p12 amplified region was detected in KMS-26 myeloma cell line by SNP microarray analysis. Further FISH mapping showed two unidentified amplified chromosomes as well as a complex pattern of rearranged chromosomes 17. The analysis of transcriptional profiles in a proprietary data base of myeloma cell lines, identified 12 significantly overexpressed genes in KMS–26 amplified region, including TNFRSF13B/TACI, COPS3, and NCOR1. The evaluation of their expression levels in a data base including 11 monoclonal gammopathy of uncertain significance, 121 MM and 9 plasma cell leukaemia, showed a significant overexpression of at least one gene in 13 patients. FISH analyses of these patients identified one MM carrying a 3.8 Mb amplified region and two MMs with gains specifically involving the TACI locus. Interestingly, the complete inactivation of TP53 at 17p13.1 was found in KMS–26 whereas a monoallelic loss was identifiable in two of the three patients carrying gain/amplification. Our data suggest that, as in the case of solid tumors, amplification/gain of the 17p11.2–p12 in MM could be mediated by the presence of repeats located in this region and may provide insights for defining novel candidate myeloma-associated genes.
48
Hauer, Julia, Charles G. Mullighan, Estelle Morillon, Gary P. Wang, Julie Bruneau, Nicole Brousse, Arndt Borkhardt, et al. "Leukemia Prone B-Precursor Population in a p19ARF-/-RAG1-/- Mouse Model." Blood 114, no. 22 (November 20, 2009): 3971. http://dx.doi.org/10.1182/blood.v114.22.3971.3971.
Full textAbstract:
Abstract Abstract 3971 Poster Board III-907 Aberrant V(D)J-recombination may contribute to the oncogenic transformation of B-lymphoid precursor cells, suggesting that ongoing aberrant activity of recombinase activating gene (RAG) during lymphoid development is one of the potentially dangerous factors. Unexpectedly, deletions of RAG1 have been observed in a subset of cases of human B-progenitor acute lymphoblastic leukemia. Thus, the role of RAG1-mediated recombination in the pathogenesis of B-cell progenitor ALL remains unclear. The setting of RAG1-deficient severe combined immunodeficiency provides a unique opportunity to address this question. RAG1-deficient patients harbour an atypical CD34+CD19+ B-lymphoid precursor population in the bone marrow that is not present in healthy individuals. The role of these cells in the development of leukaemia has so far not been reported. To analyze the role of RAG1 deficiency in the pathogenesis of leukaemia, we analyzed mice that lacked expression of p19ARF (and were thus tumour prone) and RAG1. p19ARF-/- (A) mice develop predominantly solid tumours, and in a minority of cases, T-lineage leukemia/lymphoma. p19ARF-/-RAG1-/- (AR) mice spontaneously developed B-precursor leukaemia (CD19+IgM-) at an incidence of 30% within 52 weeks of follow up, indicating that the simultaneous loss of both p19ARF and RAG1 promotes the development of B-lineage leukemia. Array CGH analysis of the resulting leukemia using customized microarrays incorporating genome wide coverage and dense tiling of genes involved in B-cell development identified recurring aneuploidy of chromosomes 1, 5 and 14, but a notable absence of RAG1-mediated DNA copy number alterations that are observed in RAG-sufficient mouse models for ALL. In support of this, we identified a leukemia-prone Sca1+CD19+ precursor cell population in the bone marrow of AR mice, which was not present in A mice. Transplantation of AR haematopoietic precursor cells (Sca1+) into irradiated wild type recipients accelerated leukaemia incidence to 100% in contrast to transplantation of AR Sca1+CD19- population, which resulted in a decreased incidence of B-precursor cell leukaemia. The leukaemia prone AR Sca1+CD19+ population showed a high BrdU uptake with an abnormal cell cycle progression and seems to be committed to the B cell lineage, as colony forming assays demonstrated. Co-culture of the AR Sca1+CD19+ population with OP9delta1 cells failed to differentiate these cells into T-precursor cells, however ongoing proliferation of B-lineage cells was observed. Further characterisation of the RAG1-/- lymphoid precursor compartment revealed an equivalent Sca1+CD19+ population that was not leukaemia prone, and expressed p19ARF. This suggests that p19ARF is an important factor implied in tumour suppression in this B-precursor compartment. Here we show for the first time in a mouse model that RAG1 loss of function in combination with inactivation of p19ARF contributes to leucemogenesis via accumulation of a leukaemia prone B-precursor cell population in the bone marrow. Disclosures: No relevant conflicts of interest to declare.
49
Mead, Adam J., Shabnam Kharazi, Iain C. Macaulay, Debbie Atkinson, Stephen Loughran, Michael Lutteropp, Petter Woll, et al. "FLT3-ITDs Introduce a Myeloid Differentiation and Transformation Bias to Multipotent Lympho-Myeloid Progenitors." Blood 118, no. 21 (November 18, 2011): 1380. http://dx.doi.org/10.1182/blood.v118.21.1380.1380.
Full textAbstract:
Abstract Abstract 1380 Although the growth factor receptor (GFR) FLT3 has a crucial role in normal early B- and T-lymphoid development, constitutively activating internal tandem duplications (ITDs) of FLT3 are almost entirely restricted to patients with adverse-risk acute myeloid leukemia. We used a murine knock-in model of FLT3-ITD myeloproliferative disease (MPD) to gain a better understanding of the cellular and molecular basis for the myeloid-bias of FLT3-ITD-induced hematological malignancies. As Flt3 cell surface expression is lacking in homozygous Flt3-itd mice, we used CD48 and CD150 expression to investigate the distribution of multipotent progenitors (MPPs) and hematopoietic stem cells (HSCs) within the primitive Lin-Sca1+Kit+ (LSK) compartment. Notably, phenotypic (LSKCD150+CD48-) and functional HSCs were markedly reduced in adult Flt3-itd mice. Competitive transplantation experiments using fetal liver confirmed that HSC numbers were reduced (20-fold reduction) by Flt3-ITDs, in a cell-extrinsic manner. Rather, LSKCD48+150- cells (MPPs) were expanded 2.7-fold in Flt3-itd mice comprising >90% of LSK cells. Similarly to Flt3high wild type (WT) lymphoid-primed multipotent progenitors (LMPPs), nanofluidic gene-expression analysis demonstrated that WT MPPs and Flt3-itd MPPs were myeloid-primed (Csf1r, Csf2r, Cebpa, Mpo) with loss of megakaryocyte and erythroid (MkE) priming (Eklf, Epor, Vwf, Gata1). In contrast, the lymphoid (Il7r, Rag1, sIgH) transcriptional priming of WT MPPs was downregulated in Flt3-itd MPPs. In agreement with this, Flt3-itd MPPs sustained extensive GM potential in vitro, with no MkE potential and, unlike WT MPPs, considerably reduced lymphoid potential. Furthermore, microarray analysis demonstrated global upregulation of the myeloid program in Flt3-itd MPPs. These findings demonstrate that primitive lympho-myeloid MPPs, are expanded and biased towards myeloid development by Flt3-ITDs. In agreement with reduced lymphoid-priming of Flt3-itd MPPs, analysis of early thymic development demonstrated a 10-fold reduction of early thymic progenitors (DN1 Kit+) in Flt3-itd mice. Subsequent stages of thymic development were also reduced, as was overall thymic cellularity. Interestingly, expression of the chemokine receptor CCR9 was 5.5-fold reduced in Flt3-itd MPPs suggesting that thymic seeding progenitors in the bone marrow are suppressed by FLT3-ITDs. Previous studies have suggested that the earliest stage of B-cell development, pre-pro-B cells, retain both B-cell and myeloid potential. Lin-CD19-CD24-AA4.1+CD43+B220+ pre-pro-B cells were expanded 13.7-fold in Flt3-itd mice, whereas subsequent stages of CD19+ B-lymphopoiesis were all reduced. The expanded pre-pro-B cells in Flt3-itd mice were myeloid biased at the transcriptional level with markedly reduced expression of lymphoid genes. Pu1 is a master-regulator of myeloid commitment in early hematopoiesis and a STAT3 target gene. As FLT3-ITDs are known to activate STAT3, unlike WT FLT3, we therefore investigated Pu1 expression in Flt3-itd mice using a Pu1-YFP reporter. Expression of Pu1 was significantly increased in LSK cells (1.4 fold) and in pre-pro-B cells (2.6 fold) in Flt3-itd mice. Furthermore, other STAT3 target genes (Cish, Id1, Pim1, Socs1, Junb) were also upregulated in these cell populations in Flt3-itd mice. Moreover, gene-set enrichment analysis in MPPs demonstrated upregulation of Pu1 target genes in Flt3-itd mice, thus providing a link between aberrant ITD signaling and the observed myeloid bias. In order to determine the functional relevance of this myeloid-bias of Flt3-itd MPPs for disease transformation, we targeted a conditional Aml1-ETO fusion-gene to the earliest B-cell progenitors in Flt3-itd mice using Mb1-Cre. Expression of AML1-ETO in WT mice did not induce any phenotype. However, Mb1-Cre induced AML1-ETO expression in Flt3-itd mice led to a high-penetrance, short latency acute leukaemia. All leukaemias expressed myeloid markers (Mac1 and Gr1) but lacked CD19 and B220 expression. These data demonstrate that Flt3-ITDs expand primitive MPPs with a myeloid lineage bias at the molecular and cellular level, at the expense of HSCs and early lymphoid development. This provides insight into the mechanisms by which mutations resulting in activation of a GFR introduce a lineage bias of resulting hematological malignancies. Disclosures: No relevant conflicts of interest to declare.
50
Strefford, Jonathan C., Helen Parker, Anton Parker, Hazel Robinson, Tracy Chaplin, Xiao-He Chen, David Bunyan, Anne Gardiner, Bryan D. Young, and David Graham Oscier. "13q Deletion Size Predicts Disease Progression and Response to Treatment in Patients with Chronic Lymphocytic Leukaemia." Blood 114, no. 22 (November 20, 2009): 671. http://dx.doi.org/10.1182/blood.v114.22.671.671.
Full textAbstract:
Abstract Abstract 671 Chronic lymphocytic leukaemia (CLL) is a heterogeneous disease characterised by a variable clinical course. Chromosomal abnormalities involving 13q14 are the most common genetic alterations in CLL, occurring in ∼60% of cases and when identified as a sole abnormality, are associated with a favourable clinical outcome. A minimally deleted region (MDR), found in most cases, encompasses DLEU2, DLEU1, miR15a and miR16-1. However, recent SNP profiling data showed that 13q deletions extends beyond the MDR and exhibit considerable heterogeneity both in size and location (Ouillette et al, Cancer Res 2008 68(4);1012). Furthermore, this study showed that larger genomic deletions were associated with a higher Rai stage and were enriched in treated patients. To explore this association further, we performed an initial analysis on samples taken at diagnosis from 100 Stage A patients, of whom 50% had stable disease for over 10 years, and on pre-treatment samples from 100 patients enrolled on the LRF CLL4 trial who had either a complete or partial response to fludarabine and cyclophosphamide [n=200] using the Affymetrix SNP6.0 microarray platform. Deletions of 13q were identified in 99/200 patients (49.5%), the majority (94%) of which included a mono- or biallelic deletion of the MDR. The smallest deletion in this series was 130Kb, while the largest was an 82Mb terminal deletion (mean deletion size 7.25Mb). Furthermore, we identified 12 cases with acquired copy number neutral LOH affecting 13q, exclusive in the presence of a bi-allelic deletion of the MDR. In agreement with the previous study we demonstrated that the size and genomic location of the deletions were highly heterogeneous. Common deletion breakpoints within a 1.9Mb region on 13q between genomic locations 48.7 and 50.6Mb defined 42 cases with small deletions. The remaining 57 cases (58%) showed deletions extending further towards the telomere or centromere and defined those cases with a large deletion. The presence of a large 13q deletion at diagnosis was associated with disease progression (p<0.03) and in cases with unmutated VH genes, a median treatment free survival of 3 months compared to 18 months for cases with small deletions (p<0.003). In the CLL4 trial samples, large deletions were associated with partial rather than complete response to treatment (p<0.04). Interestingly, the deletion sub-types defined by Ouillette et al were not associated with disease progression or response to treatment in our series. To extend this observation we designed a series of custom 13q oligonucleotides for multiplex ligation-dependant probe amplification (MLPA) and are in the process of screening an unselected cohort of 500 CLL patients. Of the 50 patients currently screened with MLPA, five cases analyzed with both the SNP6.0 platform and MLPA showed 100% concordance. The remaining 45 cases had a 13q deletion identified by FISH and confirmed the association between 13q deletion size and clinical outcome (Figure, p<0.03). Furthermore, this analysis shows that this association is independent of VH gene mutational status and the presence of an 11q or 17p deletion. These findings suggest that whilst 13q abnormalities are generally associated with a favourable outcome, aberrant expression of gene(s) on 13q outside of the MDR adversely affect outcome. We are currently refining the 13q region associated with adverse outcome to identify causative gene loci. In conclusion, 13q deletion size represents a new biomarker for predicting outcome of CLL, whose target gene(s) could provide new therapeutic strategies.FigureKaplan-Meier survival analysis of time from diagnosis to first treatment for 13q deletion size in cases analyzed by SNP6.0 and MLPA analysis [n=113].Figure. Kaplan-Meier survival analysis of time from diagnosis to first treatment for 13q deletion size in cases analyzed by SNP6.0 and MLPA analysis [n=113]. Disclosures: No relevant conflicts of interest to declare.