DNA Methylation Profiling of Childhood Acute Lymphoblastic Leukaemia Using Illumina Infinium DNA Methylation27 Bead Arrays Identifies a Distinct DNA Methylation Signature Associated with Leukaemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4650-4650
Author(s):  
Nicholas C. Wong ◽  
Zac Chatterton ◽  
Katrina Bell ◽  
Adam Kowalcyk ◽  
Justin Bedo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Significant Proportion ◽  
Chromosome Translocation ◽  
Support Vector ◽  
Childhood Leukaemia ◽  
Gene Promoters ◽  
Childhood All ◽  
Network Analyses

Abstract Abstract 4650 Acute Lymphoblastic Leukaemia (ALL) is the most common form of cancer in children. Although up to 80% of cases can be characterised by either abnormal chromosome number (hyper- or hypo-diploidy) or a specific chromosome translocation, a significant proportion of cases do no exhibit any major chromosome abnormality. Indeed, no common gene mutation has been found to be associated with childhood leukaemia. This suggests that epigenetic modifications, in particular DNA methylation, may also play a significant role in ALL pathogenesis. Understanding the specific epigenetic changes associated with this disease is therefore of paramount importance for predicting onset and disease outcome and understanding disease aetiology. From our cohort of over 450 cases of childhood leukaemia we analysed 12 cases of childhood ALL that carried the TEL/AML1 (RUNX1/ETV6) chromosome translocation using Illumina Infinium Human Methylation27K bead chip arrays. These interrogate 27,578 CpG methylation sites across the human genome covering 14,495 RefSeq gene promoters. We analysed matching leukaemia and remission bone marrow with the aim of identifying DNA methylation signatures associated with this leukaemia subtype. We used a number of computational methods to determine the DNA methylation signatures associated with childhood ALL including LIMMA, Centroid Analysis and Support Vector Machine Modelling. This identified 16 probes sufficient to define a leukaemia sample from a remission sample based on methylation level. We are currently validating our results using high throughput SEQUENOM and performing gene pathway and network analyses to look for commonly affected genes associated with leukaemia. These methylation changes we have identified may help refine the diagnosis of childhood ALL and identify cellular pathways subject to epigenetic disruption in ALL. Disclosures: No relevant conflicts of interest to declare.

Investigating the Effect of Hsp90 Inhibitors on Initiating Cells in Childhood Acute Lymphoblastic Leukaemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2952-2952
Author(s):  
Paraskevi Diamanti ◽  
Charlotte Victoria Cox ◽  
Allison Blair
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Cell Survival ◽  
Lymphoblastic Leukaemia ◽  
Conflicts Of Interest ◽  
Hsp90 Inhibitor ◽  
Childhood Leukaemia ◽  
Hsp90 Inhibitors ◽  
Childhood All ◽  
Childhood Acute Lymphoblastic Leukaemia ◽  
Haemopoietic Cells

Abstract Abstract 2952 Enhanced risk stratification protocols and intensified therapies have improved outcomes and reduced the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). Nevertheless, 20% of patients relapse, due to failure to eradicate the disease. Current chemotherapeutic regimens are designed around the properties of the bulk leukaemia cells, which might differ from those of the leukaemia initiating cell populations (LIC). Therefore, if drugs have no effect on LIC, these cells may expand and eventually cause relapse. Since several populations in childhood ALL have been shown to have LIC properties, developing therapies that are effective against all LIC populations may prevent relapse. We have previously shown that the NF-κB inhibitor parthenolide (PTL) could prevent proliferation and engraftment of unsorted cells and all LIC populations in NSG mice, in most cases examined. Heat shock protein (Hsp) 90 inhibitors are promising targets in cancer therapy. Targeting this protein has a combined impact on many oncogenic pathways involved in malignant progression. In order to investigate whether the ablation of LIC that we observed using PTL could be improved, we examined the effects of two Hsp90 inhibitors on childhood ALL cells in this study. 17-DMAG is used in preclinical and clinical phase I and II testing in breast cancers. It targets the binding site of ATP in Hsp90 and has low toxicity and high oral bioavailability. Celastrol is a novel Hsp90 inhibitor, which has recently been shown to eradicate LIC in AML by inhibiting NF-κB survival signals and inducing oxidative stress. Dose-response effects using 0.01–100nM of each Hsp90 inhibitor were assessed in primary T- and B-ALL cases and on normal haemopoietic cells at 24, 48 and 72h. Cells were stained with Annexin V and PI then viability and apoptosis were assessed by flow cytometry. An IC50 was observed in leukaemia samples using 1nM of both 17-DMAG and Celastrol after 72h. Increasing the dose of 17-DMAG to 100nM and reducing the incubation time to 48 hours for each drug further reduced ALL cell survival, without impacting normal cells. At these doses, 17-DMAG reduced the viability of T-ALL cells to 36±30% and B-ALL cells to 32±13%. In T-ALL cases, 43±15% survived treatment with Celastrol and similar results were observed with B-ALL cells (43±16%). Normal haemopoietic cells were relatively unaffected at these doses (17-DMAG: 81±2% and Celastrol: 86±36%). Subsequently, T-ALL cells were sorted for expression of CD34 and CD7 and B-ALL cells were sorted for CD34 and CD19. The subpopulations were treated with 1nM of each inhibitor and the results compared to those observed using untreated controls. However, at these concentrations the drugs had limited effects on the ALL subpopulations; 31–100% and 28–89% T-ALL subpopulations survived treatment with 17-DMAG and Celastrol respectively. Similar results were observed with B-ALL subpopulations, 9–86% survived treatment with 17-DMAG and 62–100% survived following Celastrol treatment. A number of studies have shown that a regulatory loop may exist between Hsp90 and NF-κB in that downregulation of NF-κB could lead to reduction in Hsp90 protein levels. Therefore, we investigated whether using the Hsp90 inhibitors in combination with a NF-κB inhibitor would be more effective. Samples were treated with 100nM 17-DMAG or 1nM Celastrol for 48h and 7.5mM PTL was added for the last 24 hours. In 3 T-ALL cases, PTL reduced the viability to 28±13%, 17-DMAG to 25±12% and Celastrol to 35±15%. When PTL was used in combination, cell survival was further reduced to only 18±9% (PTL + 17-DMAG) and to 19±10% (PTL + Celastrol). In 3 B-ALL cases, PTL treatment reduced viability to 57±9%, similar results were seen with 17-DMAG (59±6%), while Celastrol appeared to be the most effective of the 3 drugs (38±4%, P=0.02). When PTL was combined with the Hsp90 inhibitors the cell killing was increased by 2 fold compared to PTL or 17-DMAG alone (PTL + 17-DMAG: 31±6%, P=0.04 and PTL + Celastrol: 28±3% P=0.01). Results to date indicate a promising role for the use of Hsp90 inhibitors with PTL and data on the functional ability of treated LIC populations will provide further information on the potential of these drug combinations. In conclusion, these Hsp90 inhibitors were as effective as PTL against childhood leukaemia cells and when used in combination with PTL, cell survival was further reduced by up to 20%. Disclosures: No relevant conflicts of interest to declare.

DNA Methylation in Resistant Paediatric Acute Lymphoblastic Leukaemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1185-1185
Author(s):  
Georgia Kapatai ◽  
Stephen O Nyangoma ◽  
Hannah M Martin ◽  
Pamela R Kearns
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Hox Genes ◽  
Cpg Island ◽  
Methylation Status ◽  
Global Methylation ◽  
Childhood All ◽  
A Genome ◽  
Cpg Island Methylation

Abstract Aberrant DNA methylation is a frequent phenomenon in paediatric acute lymphoblastic leukaemia (ALL). Increased methylation has been identified as an independent factor of poor prognosis in paediatric ALL patients. This study investigates the evolution of epigenetic changes in resistant and relapsed paediatric ALL to increase our understanding of the mechanisms of treatment failure in these patients. The primary objective is to establish global DNA methylation profile in ALL and whether changes in this profile are associated with chemo-resistance and relapse. We performed a genome-wide analysis of CpG island methylation in leukaemic blasts on a cohort of 22 paediatric ALL patients; 11 at presentation and 11 at relapse. Global methylation profiles were investigated using the recently developed CpG island array technology, which includes 12,192 sequenced CpG-island clones obtained from the Wellcome Trust Sanger Institute (Cambridge, UK). These clones have undergone comprehensive sequence analysis which validated the array content and confirmed that 80% of the clones have usable sequences that can be aligned to genomic DNA. The majority of CpG-island loci are localised to known distal promoter regions or close to a transcriptional start site. We have validated the application of this technology to leukaemia cells using established ALL and AML cell lines. Pairwise Pearson correlation coefficient, used to compare log2 ratios between triplicate experiments, revealed high correlation values between replicates (median=0.85), demonstrating significant reproducibility. Bisulphite sequencing was performed to confirm that this technology could reliably depict the actual methylation status of the CpG islands. We have evaluated CpG island methylation profiling in childhood ALL both at presentation and relapse. We report that ALL specific CpG island methylation patterns are emerging. We have identified a significant number of genes, involved in cell growth and differentiation and cell cycle regulation that are consistently differentially methylated between paediatric ALL patients and healthy controls. Expressly, a number of Hox genes and Hox-genes regulators, involved in cell differentiation are differentially methylated in leukaemic blasts compared to controls. Comparisons between methylation profiles at presentation and relapse demonstrate that overall genes that are methylated at presentation remain methylated in relapse; however there is an important subset of genes that become differentially methylated. At relapse, there is a significant increase in methylation levels of a set of pro-apoptotic and tumour suppressor genes, as well as several genes involved in drug metabolism. Interestingly, a group of genes involved in ErbB signalling pathway are differentially methylated in relapse, suggesting that epigenetic regulation of this pathway might be implicated in resistant disease. Our results suggest altered methylation status within a defined subset of genes may be implicated in relapse of ALL.

Improved survival for acute lymphoblastic leukaemia in infancy: the experience of EORTC-Childhood Leukaemia Cooperative Group

1994 ◽  
Vol 86 (2) ◽  
pp. 284-290 ◽  
Author(s):  
Alina Ferster ◽  
Yves Bertrand ◽  
Yves Benoit ◽  
Annie Boilletot ◽  
Catherine Behar ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Childhood Leukaemia ◽  
Cooperative Group

Investigating the Expression of the MRD Marker CD58 on Leukaemia Initiating Cells in Childhood Acute Lymphoblastic Leukaemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1887-1887 ◽  
Author(s):  
Charlotte Victoria Cox ◽  
Paraskevi Diamanti ◽  
Allison Blair
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Model Systems ◽  
Childhood All ◽  
Therapy Assessment ◽  
Childhood Acute Lymphoblastic Leukaemia

Abstract Abstract 1887 Further improvements in outcome for childhood acute lymphoblastic leukaemia (ALL) will require a better understanding of the underlying biology of this disease and the fundamental mechanisms of drug resistance. The discoveries that a few populations can initiate leukemia in mouse models and that new populations of leukaemia initiating cells (LIC) can be detected following an initial round of transplantation in these models raises important questions about the biology of the leukaemias. If several cell populations have LIC properties, what are the relationships of these populations to each other and which populations are most important to target with therapy? It will also be important to determine whether there is any correlation in the biological properties of LIC identified in the model systems with the response of the patients to therapy. Assessment of minimal residual disease (MRD) levels provides a sensitive measurement of early treatment response and permits detection of the in vivo selected drug resistant population. CD58 (leucocyte function-associated antigen 3; LFA-3) is a useful marker in MRD tracking of B cell precursor (BCP) ALL. CD58 is over expressed in these cases permitting discrimination of leukaemia blasts from normal B cells. In this study we investigated whether CD58 is expressed on LIC populations in childhood ALL. Expression of CD58 and CD34 was assessed in a cohort of 12 diagnostic samples with mixed prognoses and compared to levels detected in 11 normal bone marrow (NBM) samples. Levels of CD58 were significantly higher in the ALL cases (57.4±37.7%) than on NBM cells (21.1±12.2%; p=0.007). Likewise, the CD34+/CD58+ population was larger in ALL cases than in normal cells (22.2±34.7% and 0.25±0.25%, respectively; p=0.05). Cells from eight of the 12 patients, were sorted on the basis of expression or lack of expression of these markers and the functional ability of the sorted subpopulations was assessed in vitro and in vivo. On sorting, the majority of cells were CD34−/CD58− (43.7±39.2%), 20.7±30.7% were CD34−/CD58+, 19±14.3% were CD34+/CD58+ and the CD34+/CD58− population accounted for 16.6±35.3%. Unsorted cells and all 4 sorted populations were set up in long-term culture to assess proliferative capability and the in vivo propagating potential was assessed in NSG mice. All 4 sorted subpopulations proliferated over the 6 week period but the highest levels of expansion were observed in the cultures of CD34+/CD58+ (6–420 fold) and CD34+/CD58− (3–24 fold) cells. Cytogenetic analyses confirmed that leukaemia cells were maintained in the culture system. Results from the in vivo analyses on 5 cases to date indicate that all 4 subpopulations contain LIC. In these cases, higher levels of engraftment were observed with CD34+/CD58+ (up to 20%) and with CD34−/CD58− subpopulations (6.1-98%). Serial transplantation studies will determine whether there are differences in the repopulating and self-renewal abilities of these LIC. These findings suggest that using CD58 alone or in combination with CD34 would be insufficient to track disease progression in ALL. Incorporating additional markers that are commonly used in MRD panels will provide valuable information on LIC populations and facilitate development of improved disease monitoring. Disclosures: No relevant conflicts of interest to declare.

Allelic Forms Of PRDM9 Associated With High Hyperdiploid Childhood Acute Lymphoblastic Leukaemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1351-1351
Author(s):  
Eleanor L Woodward ◽  
Martin L. Olsson ◽  
Bertil Johansson ◽  
Kajsa Paulsson
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Meiotic Recombination ◽  
Lymphoblastic Leukaemia ◽  
Conflicts Of Interest ◽  
Control Cohort ◽  
Childhood All ◽  
Significant Difference ◽  
The Family ◽  
Rare Alleles ◽  
Independent Cohort

Abstract Whilst a great deal is known about acquired somatic aberrations associated with the diagnosis and prognosis of acute leukaemias, relatively little is known about the effects of germline variation. A recent study reported a link between germline variations in the zinc finger (ZnF) binding domain array of the PR domain-containing 9 (PRDM9) gene, regulator of meiotic recombination, with the development of acute lymphoblastic leukaemia (ALL) in children. An excess of rare PRDM9 alleles that affect meiotic recombination events were seen in parents of children affected with B-cell precursor ALL (pre-B-ALL), and in an independent cohort of children with pre-B-ALL; in particular in aneuploid and infant ALL. In the present study, we carried out Sanger sequencing to investigate variation of PRDM9 alleles in a cohort of parents (n=59) with children diagnosed with high hyperdiploid ALL (HeH; 51-67 chromosomes) (n=31) and a control cohort of individuals (n=66) from the south of Sweden. Whereas similar numbers of rare PRDM9 alleles were observed in both groups, a larger number of rare alleles that affect recombination events were observed in the parents from the family cohort with children diagnosed with HeH ALL compared to individuals of the population control cohort (15.3% vs. 4.5%, P=0.0414). Two-thirds of the parents transmitted the rare allele to their affected children. A previously unreported ZnF repeat was also detected in two individuals of the control cohort but it was not detected in the family cohort. A statistically significant difference in frequency of rare alleles affecting recombination events between the two groups indicates a true association between PRDM9 allelic forms and HeH ALL in an independent cohort. Thus, our results confirm the previous findings that PRDM9 may play a role in the development of childhood ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals What Role for Angiogenesis in Childhood Acute Lymphoblastic Leukaemia?

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
P. Schneider ◽  
I. Dubus ◽  
F. Gouel ◽  
E. Legrand ◽  
J. P. Vannier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Prognostic Impact ◽  
Childhood All ◽  
Acute Myelogenous

The role of angiogenesis in acute leukaemia has been discussed since the cloning of the gene ofvascular endothelial growth factor(VEGF) from the acute myelogenous leukemia cell line (HL60) and, thereafter, when the first studies reported increased bone marrow vascularity and elevation of angiogenic cytokines in acute lymphoblastic leukaemia (ALL). VEGF andbasic fibroblast growth factor(bFGF) are the major proangiogenic cytokines that have been studied, and evaluation of their prognostic impact in childhood ALL has been reported in several studies, though with controversial results. The antiangiogenic response, contributing to the angiogenic balance, has scarcely been reported. The origin of the factors, their prognostic value, and their relevance as good markers of what really happens in the bone marrow are discussed in this paper. The place of antiangiogenic drugs in ALL has to be defined in the global treatment strategy.

scholarly journals Importance of genotyping of Thiopurine S-methyltransferase in children with acute lymphoblastic leukaemia during maintenance therapy

2008 ◽  
Vol 136 (11-12) ◽  
pp. 609-616 ◽  
Author(s):  
Lidija Dokmanovic ◽  
Dragana Janic ◽  
Nada Krstovski ◽  
Branka Zukic ◽  
Natasa Tosic ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
Tandem Repeats ◽  
Gene Mutations ◽  
Maintenance Phase ◽  
Variable Number ◽  
Childhood All ◽  
Dose Therapy ◽  
Significant Difference ◽  
Tpmt Gene

INTRODUCTION Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyses the inactivation of mercaptopurine (MP) which is widely used in the treatment of acute lymphoblastic leukaemia (ALL) of childhood. Potentially fatal myelotoxicity may develop after standard doses of MP in TPMT deficient patients. OBJECTIVES To establish if individually tailored doses of MP can reduce myelotoxicity in ALL patients carrying mutations in the TPMT gene. To establish if variable number of tandem repeats (VNTR) genotype influences the treatment effects of MP. METHOD Fifty randomly selected patients treated according to ALL IC-BFM 2002 protocol were tested for most frequent TPMT gene mutations using PCR based methods. VNTR genotype was determined in 20 children by PCR methods. During the maintenance phase, we recorded the number of weeks when therapy was applied in either full doses, reduced doses or when patients were without any therapy. RESULTS Fifty children were examined, 29 boys (58%) and 21 girls (42%); age ranged from 1.8-17.3 years (median 6.2 years). Four patients (8%) were heterozygous for TPMT mutations, all of them carrying the TPMT*3A variant. After 12, 14, 16 and 19 weeks of therapy with reduced doses of MP, the patients switched to full doses due to good tolerance. There was no therapy omission. Cumulative dose of MP was reduced for 7.8%, 7.4%, 11.2% and 16.6%, respectively, in patients with TPMT mutations. No significant difference was found between children with no mutations and TPMT heterozygotes regarding full dose therapy (53.6 vs. 55.7 weeks, respectively) and reduced dose therapy (19.9 vs. 15.2 weeks respectively). The number of detected VNTRs ranged from four to seven. The majority of patients had different number of VNTRs on homologous chromosomes. Most frequently detected polymorphism was VNTR*5. No correlation was found between TPMT and VNTR genotype inheritance. CONCLUSION Obeying pharmacogenetic principles in the treatment of childhood ALL may improve the tolerance of therapy with MP.

Acute Lymphoblastic Leukaemia Has a Poor Outcome in Children with Down Syndrome Due to Infective Death in Remission (Results of UK MRC ALL 97 Trial).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 873-873 ◽  
Author(s):  
Josu de la Fuente ◽  
Sue Richards ◽  
David K. Webb ◽  
Ian M. Hann ◽  
Christopher D. Mitchell ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Acute Lymphoblastic Leukaemia ◽  
Lymphoblastic Leukaemia ◽  
High Rate ◽  
Childhood All ◽  
Free Survival ◽  
Children With Down Syndrome ◽  
Medical Research Council Trial ◽  
The Uk

Abstract Acute lymphoblastic leukaemia (ALL) has a poorer outcome in children with Down syndrome (DS) which has been attributed in the past to a higher incidence of infective death in remission and late relapses. We report on the outcome of children with DS enrolled on the UK Medical Research Council trial for childhood ALL, MRC ALL 97, between January 1997 and June 2002. Thirty seven children had DS (2%), of whom three were treated on the high risk protocol (HR1). Thirty-three (89%) achieved complete remission at the end of induction, 3 died during induction (8%), and one died later without remission. The median follow up was 4.9 years (2.4–7.8). The 5-year event free survival (EFS: 48.0%, SD 8.9) was not an improvement on the previous MRC UKALL XI trial (57.9%, SD 8.0, p=0.2) and was significantly worse than for children without DS (p<0.00005). At the time of follow up, 46% of the children had died (n=17). Five patients suffered relapse, and the relapse rate was not significantly different from those without DS. One patient known to have cardiac disease died during maintenance due to arrhythmia and 8 died of infection, resulting in a significantly higher rate of death in remission (28%) than in children without DS (3%, p<0.00005). Infective deaths were associated with intensification therapy, except in one child who died during interim maintenance. It was possible to isolate a microorganism in 50% of the cases (two cases of Pseudomonas, one Staphylococcus aureus, one Staphylococcus epidermidis and yeast) plus Rhinovirus was found in a nasopharyngeal aspirate of a fifth case with clinical evidence of bacterial sepsis. For the randomised comparisons (prednisolone versus dexamethasone n=30, mercaptopurine versus thioguanine n=23), results within the DS patients were not significantly different from those in all patients, with benefit for dexamethasone. The increase in remission deaths with DS was greater with prednisolone, and with mercaptopurine (p for interaction = 0.0002, <0.00005, respectively). The revision of the trial in 1999 which adopted the template of CCG 1952 which improved EFS, did so for DS patients also, with no change in the DS remission death rate. In conclusion, children with DS may benefit from increased treatment intensity but still have an unacceptably high rate of infective death in remission.

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