Aberrant DNA Methylation in Childhood Acute Lymphoblastic Leukemia as a Potential Biomarker Reflecting Disease Status.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2637-2637
Author(s):  
Seung Yeon Kwon ◽  
Sung Chul Won ◽  
Hyo Sun Kim ◽  
Hei-Cheul Jeung ◽  
Sun Young Rha ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Complete Resolution ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Disease Status ◽  
Childhood All ◽  
Remission Status ◽  
Potential Biomarker ◽  
Aberrant Dna Methylation

Abstract Abstract 2637 Poster Board II-613 Background: Aberrant DNA methylation of promoter-associated cystosine-guanine (CpG) islands is known to play an important role in leukemogenesis and has been reported to have association with poor prognosis in acute lymphoblastic leukemia (ALL). In adult, residual methylation at clinical remission status in patients with acute myeloid leukemia and Philadelphia chromosome-positive ALL showed significant association with poor prognosis in recent studies. We analyzed 47 patients with childhood ALL to evaluate the change of methylation status from the time of diagnosis to the time at morphologic remission. Methods: We analyzed the methylation status of CDH1, p16 and DAPK genes at the time of diagnosis and at the time of morphologic remission in 47 patients using methylation specific polymerase chain reaction method with genomic DNA extracted from bone marrow (BM) aspirates. All patients were treated with standard chemotherapy and after 4 weeks, every patient achieved morphologic complete remission. Methylation status of above three genes were also analyzed with the same method in 10 BM aspirate samples from healthy BM donor for comparison. Results: CDH1 was methylated in 30 (64%) patients, p16 in 2 (4%) patients and DAPK in 6 (13%) patients at the time of diagnosis. Thirty three (70%) patients had methylation at least one gene, and 5 (11%) had methylation of 2 genes. None of the healthy BM donors showed methylation of above genes. At the time of morphologic remission, all patients who had aberrant DNA methylation of any gene at the time of diagnosis had no detectable residual methylation showing complete resolution of aberrant DNA methylation of examined genes. Clinical prognostic factors including initial white blood cell count, immunophenotype and presence of specific translocations (TEL-AML1, BCR-ABL, E2A-PBX1) did not show any association with initial methylation status. Age was the only factor which showed correlation with methylation status; patients under 2 year old showed significantly low frequency of methylation of above three genes (p <0.001). Conclusion: Since aberrant DNA methylation of CDH1, p16 and DAPK genes were found in 70% of pretreatment ALL patients and none in healthy BM donor showing complete resolution of methylation at morphologic remission status, we cautiously suggest aberrant DNA methylation as a potential biomarker reflecting disease status in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The role of DNA methylation in ageing and cancer

2018 ◽  
Vol 77 (4) ◽  
pp. 412-422 ◽  
Author(s):  
A. E. Morgan ◽  
T. J. Davies ◽  
M. T. Mc Auley
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
Methylation Status ◽  
Electrochemical Techniques ◽  
Regulation Of Gene Expression ◽  
Homeobox Gene ◽  
Gene Promoter ◽  
Disease States ◽  
Potential Biomarker ◽  
Aberrant Dna Methylation

The aim of the present review paper is to survey the literature related to DNA methylation, and its association with cancer and ageing. The review will outline the key factors, including diet, which modulate DNA methylation. Our rationale for conducting this review is that ageing and diseases, including cancer, are often accompanied by aberrant DNA methylation, a key epigenetic process, which is crucial to the regulation of gene expression. Significantly, it has been observed that with age and certain disease states, DNA methylation status can become disrupted. For instance, a broad array of cancers are associated with promoter-specific hypermethylation and concomitant gene silencing. This review highlights that hypermethylation, and gene silencing, of the EN1 gene promoter, a crucial homeobox gene, has been detected in various forms of cancer. This has led to this region being proposed as a potential biomarker for diseases such as cancer. We conclude the review by describing a recently developed novel electrochemical method that can be used to quantify the level of methylation within the EN1 promoter and emphasise the growing trend in the use of electrochemical techniques for the detection of aberrant DNA methylation.

Detection of Aberrant DNA Methylation in Acute Lymphocytic Leukemia (ALL) Using a Real-Time Polymerase Chain Reaction (PCR) Assay.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 998-998
Author(s):  
Koyu Hoshino ◽  
Hui Yang ◽  
Blanca Sanchez-Gonzalez ◽  
Guillermo Garcia-Manero
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Target Gene ◽  
Methylation Status ◽  
Pcr Assay ◽  
Frequent Event ◽  
P73 Gene ◽  
Aberrant Dna Methylation

Abstract Aberrant DNA methylation of promoter-associated CpG islands is a frequent event in ALL. DNA methylation of specific subsets of genes is associated with poor prognosis and is stable in a majority of patients (pts) at the time of relapse (Clinical Cancer Res2002;8:1897), and therefore tracking these epigenetic marks during remission may predict for relapse risk. Most commonly used methods to detect DNA methylation exploit the availability of sodium bisulfite that transforms unmethylated C into A/T leaving methylated C as such. This has allowed the development of several techniques that use conventional PCR or sequencing to detect methylated alleles. Although there are significant differences in the sensitivity and specificity of these assays, with bisulfite sequencing considered the gold standard, most of them are labor intensive and require several days to be performed. To circumvent some of these problems, we have developed a real-time bisulfite PCR assay to detect methylation of p57KIP2, p73, and p15 in samples obtained from bone marrows in pts with ALL at remission. Methylation of these genes has been shown to be common in ALL and to predict for poor prognosis at initial presentation (Blood2003;101:4131). This method allows for the simultaneous analysis of multiple samples in less than 24 hours, is quantitative, and requires less than 0.2μg of DNA for each target gene. To amplify bisulfite treated DNA, we designed primer sets and probes for all three genes in regions known to be inversely correlated with gene expression. To quantify methylation density, we used the interferon γ (INFG) gene as an internal control because it has very rare CpG sites, is a single copy gene and has no homology with other known genes. Methylation density is defined as: Methylation (%) = 2CT of target gene / 2 CT of EFM x 100. CT is the number of cycles threshold, and EFM the estimated 100% fully methylation (EFM) of the target gene in control experiments. Using DNA artificially methylated with SssI in dilution experiments with unmethylated DNA, p57, p15 and p73 methylation density could be detected at dilutions of 0.2%, 1.2% and 0.1%, respectively. We then studied the methylation status of 30 cell lines (22 hematopoietic and 8 no-hematopoietic). Methylation of p57, p15 and p73 gene was detected in 17(57%), 19(63%) and 16(53%) cell lines respectively. Methylation of 3 genes, 2 genes and 1 gene was observed in 9(30%), 8(27%) and 7(23%) cell lines respectively. DNA methylation of p15 and p73 was not detected in the marrow from 6 healthy volunteers but p15 was detected (0.1% methylation) in 1 out of 6 of these specimens. Subsequently, we studied 50 pts with ALL at the time of remission. We found the frequencies of p57, p15 and p73 gene to be 2(4%), 26(52%) and 10(20%), respectively. There was a trend towards a better overall survival for pts with methylation of 0 or 1 gene (209 weeks) compared with those with methylation of 2 or more genes (71 weeks), p=0.1. In conclusion, the real-time bisulfite PCR described here allows for the rapid detection of aberrant DNA methylation in samples obtained at the time of remission in pts with ALL and may have a role in the development of techniques to assess minimal residual disease in this group of pts.

scholarly journals Poor prognosis of children with pre-B acute lymphoblastic leukemia is associated with the t(1;19)(q23;p13): a Pediatric Oncology Group study

Blood ◽  
1990 ◽  
Vol 76 (1) ◽  
pp. 117-122 ◽  
Author(s):  
WM Crist ◽  
AJ Carroll ◽  
JJ Shuster ◽  
FG Behm ◽  
M Whitehead ◽  
...  
Keyword(s):  
Treatment Outcome ◽  
Acute Lymphoblastic Leukemia ◽  
Pediatric Oncology ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Translocations ◽  
Oncology Group ◽  
Childhood All ◽  
Pediatric Oncology Group ◽  
Worse Prognosis

Abstract The prognostic significance of chromosomal translocations, particularly t(1;19) (q23;p13), was evaluated in children with pre-B and early pre-B acute lymphoblastic leukemia (ALL). Patients were treated on a risk- based protocol of the Pediatric Oncology Group (POG) between February 1986 and May 1989. An abnormal clone was detected in 46% (130 of 285) of pre-B cases and 56% (380 of 679) of early pre-B cases. Translocation of any type was associated with a worse treatment outcome than other karyotypic abnormalities: 15 of 66 versus 3 of 64 failed therapy in the pre-B group (P = .001), and 37 of 141 versus 23 of 239 failed in the early pre-B group (P less than .001). The t(1;19) (q23;p13) occurred significantly more often in cases of pre-B ALL with a clonal abnormality than in early pre-B ALL cases (29 of 130 v 5 of 380, P less than .001). Among the 285 pre-B cases in which bone marrow was studied cytogenetically, those with t(1;19) had a significantly worse treatment outcome than all others (11 of 29 v 27 of 256 have failed therapy, P less than .001). This difference is significant (P less than .001) after adjustment for leukocyte count, age, and other relevant features. Cases with the t(1;19) also had a worse prognosis than pre-B patients with other translocations (4 of 37 have failed, P less than .01) or with any other karyotypic abnormality (7 of 101 have failed, P less than .001). We conclude that chromosomal translocations confer a worse prognosis for non-T, non-B-cell childhood ALL, and that the t(1;19) is largely responsible for the poor prognosis of the pre-B subgroup.

scholarly journals In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3861-3868 ◽  
Author(s):  
E Klumper ◽  
R Pieters ◽  
AJ Veerman ◽  
DR Huismans ◽  
AH Loonen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Poor Prognosis ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Childhood All ◽  
Acquired Drug Resistance ◽  
The Poor ◽  
Response To Chemotherapy

Cellular drug resistance is thought to be an important cause of the poor prognosis for children with relapsed or refractory acute lymphoblastic leukemia (ALL), but it is unknown when, to which drugs, and to what extent resistance is present. We determined in vitro resistance to 13 drugs with the MTT assay. Compared with 141 children with initial ALL, cells from 137 children with relapsed ALL were significantly more resistant to glucocorticoids, L-asparaginase, anthracyclines, and thiopurines, but not to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Relapsed ALL cells expressed the highest level of resistance to glucocorticoids, with a median level 357- and >24-fold more resistant to prednisolone and dexamethasone, respectively, than initial ALL cells, whereas the resistance ratios for the other drugs differed from 0.8- to 1.9-fold, intraindividual comparisons between initial and relapsed samples from 16 children with ALL showed that both de novo and acquired drug resistance were involved. Specific in vitro drug-resistance profiles were associated with high-risk relapsed ALL groups. In vitro drug resistance was also related to the clinical response to chemotherapy in relapsed/refractory childhood ALL. We conclude that drug resistance may explain the poor prognosis for children with relapsed/refractory ALL. These day may be helpful to design alternative treatment regimens for relapsed childhood ALL.

scholarly journals DNA Methylation Adds Prognostic Value to Minimal Residual Disease Status in Pediatric T‐Cell Acute Lymphoblastic Leukemia

2016 ◽  
Vol 63 (7) ◽  
pp. 1185-1192 ◽  
Author(s):  
Magnus Borssén ◽  
Zahra Haider ◽  
Mattias Landfors ◽  
Ulrika Norén‐Nyström ◽  
Kjeld Schmiegelow ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Minimal Residual Disease ◽  
Prognostic Value ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Disease Status ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

Genome-Wide Identification of Aberrant Promoter Associated CpG Island Methylation in Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2127-2127
Author(s):  
Shao-qing Kuang ◽  
Weigang Tong ◽  
Hui Yang ◽  
Mathew K. Lee ◽  
Zhi-Hong Fang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
B Cell ◽  
Cell Lines ◽  
Cpg Island ◽  
Trichostatin A ◽  
Methylation Status ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Leukemia Cell Lines ◽  
Aberrant Dna Methylation

Abstract Aberrant DNA methylation is a common molecular feature of both pediatric and adult ALL. Specific methylation patterns predict for poor prognosis (Shen et al Blood 2004), and reactivation of epigenetically inactivated molecular pathways results in induction of leukemia cell death (Kuang et al. Oncogene 2007). Until now most studies of methylation in ALL have been based on arbitrary gene selection methods. To overcome this limitation and to study hundreds of promoter CpG islands simultaneously, we have developed a method that combines MCA (Methylated CpG Island Amplification) with either RDA (Representational Difference Analysis) or the Agilent Promoter Microarray platform. With these methods differentially methylated DNA treated with bisulfite is generated after mixing tester DNA (in our case DNA from de novo refractory Ph negative and MLL negative ALL patients) with driver DNA (normal B cell controls) and using specific restriction enzymes and several rounds of PCR. DNA fragments thus generated are either cloned (RDA) or labeled and spotted on the Agilent Array. Using this technology, that can potentially interrogate up to 17K promoters, we have identified 932 promoters targets of aberrant DNA methylation in poor risk ALL from patients that cannot be currently identified by standard molecular methods (Ph and MLL negative). The genes associated with these promoters are distributed through the human genome but an overrepresentation of methylated promoters located in chromosomes 3, 9, 11 and 19 was detected. Using molecular pathway clustering analysis, 404 of these genes are grouped together in 29 specific functional pathways. We have validated the methylation of 31 of these 923 genes by bisulfite pyrosequencing. Of these, 27 (87%) were confirmed to be hypermethylated in 23 human leukemia cell lines but not in normal controls (N=15). Methylation status analysis of these 27 genes allowed for the segregation of T cell versus B cell leukemia cell lines. Fifteen of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) were also frequently hypermethylated in primary ALL samples. Expression analysis of 6 of these genes (GIPC2, MAGI1, ADCY5, HSPA4L, OCLN and GNA14) in leukemia cell lines further confirmed methylation associated gene silencing. Treatment of methylated/silenced cell lines with 5′-aza-2′-deoxycytidine and trichostatin A resulted in gene re-expression, further confirming the role of DNA methylation in their silencing. In summary, we have identified in excess of 900 targets of aberrant DNA methylation in ALL. The study of the epigenetically suppressed pathways represented by these genes should allow us to further understand the molecular pathogenesis of ALL and develop new prognostic biomarkers for patients with Ph and MLL negative disease.

Dnmt3a Deletion and FLT3-ITD Cooperate in a Mouse Model of T-Lymphoblastic Leukemia (T-ALL).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2428-2428
Author(s):  
Liubin Yang ◽  
Min Luo ◽  
Mira Jeong ◽  
Choladda V. Curry ◽  
Grant Anthony Challen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Leukemic Transformation ◽  
Aberrant Dna Methylation

Abstract Abstract 2428 Aberrant DNA methylation repeatedly has been implicated in cancer development. DNA methyltransferase (DNMT) 3A, which mediates de novo DNA methylation, was found to be mutated in 20% of patients with acute myeloid leukemia and 10% of patients with myelodysplastic syndrome. Recently, mutations associated with myeloid malignancies such as DNMT3A and FLT3 have also been uncovered in patients with early T-cell precursor lymphoblastic leukemia (ETP-ALL) (Neumann et al., 2012; Van Vlierberghe et al., 2011; Zaremba et al., 2012). ETP-ALL is a type of very high-risk ALL associated with myeloid/stem cell gene expression signature and myeloid markers. We have demonstrated that Dnmt3a deletion in mouse causes increased self-renewal of hematopoietic stem cells and an impairment of differentiation (Challen et al., 2011). Dnmt3a loss also produces aberrant methylation associated with oncogenes and tumor suppressor genes. Yet, whether aberrant DNA methylation can drive leukemia remains unknown. As Dnmt3a deletion alone was insufficient for malignancy, secondary mutations are likely necessary for leukemic transformation. Because FLT3 internal tandem duplication (ITD) frequently co-exist with DNMT3A mutations in acute leukemias, we hypothesized that Dnmt3a-loss may cooperate with FLT3-ITD to promote leukemic transformation; and we established a mouse model to test this. Deletion of conditional Dnmt3a with Mx1-cre was induced by injections of pIpC. Subsequently, bone marrow from Dnmt3a-deleted (Dnmt3aKO) donor mice was transduced with MSCV-FLT3-ITD-GFP retrovirus or MSCV-GFP control and transplanted into lethally irradiated recipients. The mice were monitored monthly for development of malignancies by complete blood count and peripheral blood analysis by flow cytometry and followed for disease latency. Moribund mice were sacrificed and analyzed with peripheral blood smears, histology, and immunophenotyping. Dnmt3a deletion with overexpression of FLT3-ITD caused rapid onset T-ALL in 6/8 mice (n=6) with a median latency of 78 days compared to 121 days in WT mice (n=4) overexpressing FLT3-ITD (p&lt;0.0001 Log-rank Mantel-Cox Test) (See figure). Mice from both groups exhibited leukocytosis, splenomegaly, and thymomegaly with high GFP expression detected by FACS. Even after we transduced bone marrow cells enriched for myeloid progenitor and stem cells, Dnmt3a deletion again accelerated T-ALL with median survival of 89 days (n=9) versus 110 days in WT-FLT3-ITD (n=10) mice. T-ALL was observed in 2/4 WT-FLT3-ITD mice and 5/6 Dnmt3aKO-FLT3-ITD mice analyzed (p&lt;0.0001 Log-rank Mantel-Cox Test). By flow cytometry, two distinct types of T-ALL were observed in the bone marrow of Dnmt3a deleted leukemic mice: one was characterized by a double positive population (DP) of CD4+CD8+ lympoblasts (1/6) and another early immature T-cell-like type of CD4-CD8-CD44+CD25-CD11bloCD117+ lymphoblasts (4/6). Gene expression analysis by RT-PCR in the early immature T-ALL showed downregulation of Notch-pathway genes (such as Notch1, Notch 3, Deltex, Hes1) and upregulation of stem cell-associated genes Lyl1 and Scl1, suggesting an ETP-like T-ALL. The ETP-like ALL phenotype has not been seen in WT mice overexpressing FLT3-ITD. The opposite gene expression pattern was seen in the DP population with upregulation of Notch-pathway genes. Furthermore, the DP leukemia was transplantable to secondary recipients within 2 weeks. Whether ETP-like ALL can be transplanted is still under investigation. We are also currently studying the changes in global CpG methylation among the leukemias that have Dnmt3a loss, FLT3-ITD overexpression, and control and also anticipate data from transcriptome analysis by RNA-Seq. These data suggest that stem or progenitor bone marrow cells primed by early loss of Dnmt3a are transformed into DP T-ALL and ETP-like ALL fueled by the overexpression of the oncogene FLT3-ITD. The ETP-like ALL phenotype has not been seen previously in WT mice overexpressing FLT3-ITD, suggesting that Dnmt3a ablation is required. The Dnmt3a-deleted-FLT3-ITD mice with T-ALL is, to our knowledge, the first animal model of human immature T-cell leukemia. This model can enhance our understanding of the pathogenesis of ETP-like ALL with respect to aberrant DNA methylation and will serve as a powerful tool to test novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aberrant DNA methylation and epigenetic inactivation of Eph receptor tyrosine kinases and ephrin ligands in acute lymphoblastic leukemia

Blood ◽  
2010 ◽  
Vol 115 (12) ◽  
pp. 2412-2419 ◽  
Author(s):  
Shao-Qing Kuang ◽  
Hao Bai ◽  
Zhi-Hong Fang ◽  
Gonzalo Lopez ◽  
Hui Yang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
Cpg Island ◽  
Lymphoblastic Leukemia ◽  
Methylation Status ◽  
Eph Receptors ◽  
Raji Cells ◽  
Eph Receptor ◽  
Phosphorylated Akt ◽  
Ephrin Ligands

Eph receptors and their ephrin ligands are involved in normal hematopoietic development and tumorigenesis. Using methylated CpG island amplification/DNA promoter microarray, we identified several EPH receptor and EPHRIN genes as potential hypermethylation targets in acute lymphoblastic leukemia (ALL). We subsequently studied the DNA methylation status of the Eph/ephrin family by bisulfite pyrosequencing. Hypermethylation of EPHA2, -A4, -A5, -A6, -A7, -A10, EPHB1, -B2, -B3, -B4, EFNA1, -A3, -A5, and EFNB1 and -B2 genes was detected in leukemia cell lines and primary ALL bone marrow samples. Expression analysis of EPHB4, EFNB2, and EFNA5 genes demonstrated that DNA methylation was associated with gene silencing. We cloned the promoter region of EPHB4 and demonstrated that promoter hypermethylation can result in EPHB4 transcriptional silencing. Restoration of EPHB4 expression by lentiviral transduction resulted in reduced proliferation and apoptotic cell death in Raji cells in which EPHB4 is methylated and silenced. Finally, we demonstrated that phosphorylated Akt is down-regulated in Raji cells transduced with EPHB4. These results suggest that epigenetic silencing by hypermethylation of EPH/EPHRIN family genes contributes to ALL pathogenesis and that EPHB4 can function as a tumor suppressor in ALL.

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