Intrahepatic cholangiocarcinomas with IDH1/2 mutation-associated hypermethylation at selective genes and their clinicopathological features

Abstract Intrahepatic cholangiocarcinoma (ICC) is a rare but fatal tumor. The isocitrate dehydrogenase 1 and 2 (IDH1/2) genes are known to be mutated in ICC. IDH1/2 mutations tend to be accompanied by enhanced hypermethylation at a subset of genomic loci. We sought to clarify the clinicopathological features, including prognostic value, of ICCs with IDH1/2 mutation-associated hypermethylation at a subset of genes. The mutation status of IDH1/2 and methylation status of 30 gene CpG island loci were analyzed in 172 cases of ICC using pyrosequencing and the MethyLight assay, respectively. The mutation status of IDH1/2 was correlated with clinicopathological features and the DNA methylation status at 30 gene loci. Then, the clinicopathological characteristics were analyzed regarding three-tiered methylation statuses in genes showing IDH1/2 mutation-associated methylation. IDH1/2 mutations were found in 9.3% of ICCs, and IDH1/2-mutated tumors were associated with the histological subtype, including the bile ductular type and small duct type, and poor differentiation. Eight DNA methylation markers showed associations with IDH1/2 mutations, and ICCs with > 5/8 methylated markers were associated with the bile ductular type or small duct type, absence of mucin production, absence of biliary intraepithelial neoplasia, and presence of chronic liver disease. > 5/8 methylated markers were an independent prognostic marker associated with better survival in both cancer-specific survival and recurrence-free survival. In summary, by analyzing the association between IDH1/2 mutations and DNA methylation in individual genes, we developed a panel of DNA methylation markers that were significantly associated with IDH1/2 mutations and were able to identify a subset of ICC with better clinical outcomes.

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DNA Methylation of Endoglin Pathway Genes in Pregnant Women With and Without Preeclampsia

Epigenetics Insights ◽

10.1177/2516865720959682 ◽

2020 ◽

Vol 13 ◽

pp. 251686572095968

Author(s):

Allison H Rietze ◽

Yvette P Conley ◽

Dianxu Ren ◽

Cindy M Anderson ◽

James M Roberts ◽

...

Keyword(s):

Dna Methylation ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Control Participant ◽

Sample Collection ◽

Promoter Regions ◽

Participant Group

Objective: We compared blood-based DNA methylation levels of endoglin ( ENG) and transforming growth factor beta receptor 2 ( TGFβR2) gene promoter regions between women with clinically-overt preeclampsia and women with uncomplicated, normotensive pregnancies. Methods: We used EpiTect Methyl II PCR Assays to evaluate DNA methylation of CpG islands located in promoter regions of ENG (CpG Island 114642) and TGFβR2 (CpG Island 110111). Preeclampsia was diagnosed based on blood pressure, protein, and uric acid criteria. N = 21 nulliparous preeclampsia case participants were 1:1 frequency matched to N = 21 nulliparous normotensive control participants on gestational age at sample collection (±2 weeks), smoking status, and labor status at sample collection. Methylation values were compared between case and control participant groups [( ENG subset: n = 20 (9 cases, 11 controls); TGFβR2 subset: n = 28 (15 cases, 13 controls)]. Results: The majority of the preeclampsia cases delivered at ⩾34 weeks’ gestation (83%). Average methylation levels for ENG ([M ± (SD)]; Case Participant Group = 6.54% ± 4.57 versus Control Participant group = 4.81% ± 5.08; P = .102) and TGFβR2 (Case Participant Group = 1.50% ± 1.37 vs Control Participant Group = 1.70% ± 1.40; P = .695) promoter CpG islands did not differ significantly between the participant groups. Removal of 2 extreme outliers in the ENG analytic subset revealed a trend between levels of ENG methylation and pregnancy outcome (Case Participant Group = 5.17% ± 2.16 vs Control Participant Group = 3.36% ± 1.73; P = .062). Conclusion: Additional epigenetic studies that include larger sample sizes, investigate preeclampsia subtypes, and capture methylation status of CpG island shores and shelves are needed to further inform us of the potential role that ENG and TGFβR2 DNA methylation plays in preeclampsia pathophysiology.

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Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

Cancers ◽

10.3390/cancers12030539 ◽

2020 ◽

Vol 12 (3) ◽

pp. 539 ◽

Cited By ~ 1

Author(s):

Alexei J. Stuckel ◽

Wei Zhang ◽

Xu Zhang ◽

Shuai Zeng ◽

Urszula Dougherty ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Cell Lines ◽

Promoter Methylation ◽

Cpg Island ◽

Methylation Status ◽

Cxcr4 Expression ◽

Normal Colonic Mucosa ◽

Gene Body ◽

Expression Levels

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4’s regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

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Methylation Profile of B-Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v106.11.2951.2951 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2951-2951

Author(s):

Jun Fan ◽

Asou Norio ◽

Masao Matsuoka

Keyword(s):

Dna Methylation ◽

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Mammalian Cells ◽

Mononuclear Cells ◽

Cpg Island ◽

Methylation Status ◽

Lymphocytic Leukemia ◽

Dna Hypomethylation ◽

Peripheral Blood Mononuclear

Abstract DNA methylation plays an important role in the development and aging of mammalian cells, and its dysregulation has been frequently observed in cancer cells. The purpose of this study is to investigate the involvement of aberrant DNA methylation in B chronic lymphocytic leukemia (B-CLL) cells. We compared methylation status of B-CLL cells isolated from patients with that of normal CD19+ cells isolated from health donors by methylated CpG island amplification/representative difference analysis method. 5 hypermethylated and 27 hypomethylated DNA regions were identified in B-CLL sample. Among the 27 hypomethylated regions, 5 located on chromosome 9q34, 3 on 10q25-26 and 4 on 19q13. Methylation status was confirmed by sequencing using sodium bisulfite-treated DNA samples. By comparing DNA samples from same patients at different clinical stages, we found that lower methylation density in these regions is linked with disease progression. Expression of 15 genes surrounding hypomethylated regions was studied by RT-PCR. Expression of laminin beta3 gene and melanotransferrin gene was found to be upregulated in all B-CLL cell lines as well as lymphoma cell lines comparing with normal CD19+ peripheral blood mononuclear cells. B-cell CLL/lymphoma 11b gene showed increased expression in only 2 B-CLL cell lines. For other genes, no transcriptional change was found regardless of changed DNA methylation. This study showed the predominance of DNA hypomethylation in B-CLL cells compared with hypermethylation. Hypomethylated regions clustered in a limited number of chromosomes and methylation density appeared to be inversely correlated with disease progress. Figure Figure

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

Blood ◽

10.1182/blood.v110.11.2127.2127 ◽

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.

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UTX, a Histone Demethylase, Is Inactivated through Homozygous Deletion, Mutation, and DNA Methylation in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.1798.1798 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1798-1798

Author(s):

Brian A Walker ◽

Paola E. Leone ◽

Nicholas J Dickens ◽

Kevin D Boyd ◽

David Gonzalez ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Copy Number ◽

Conflicts Of Interest ◽

Cpg Island ◽

Chromatin Condensation ◽

Methylation Status ◽

Significant Proportion ◽

Histone Demethylase ◽

Homozygous Deletions

Abstract Abstract 1798 Poster Board I-824 Histone modifications are known to mediate transcriptional regulation through changes in chromatin condensation and as such can lead to aberrant transcriptional patterns resulting in malignant transformation. Modulation of chromatin structure via histone modification is becoming recognised as an important pathogenic mechanism in myeloma and has been suggested by the over-expression of MMSET, a histone methyltransferase, by the t(4;14) chromosomal rearrangement. More recently inactivation of UTX, a histone demethylase, has also been suggested to have a role in myeloma pathogenesis and both UTX and MMSET are mediators of transcriptional repression. UTX is inactivated in a number of different cancer cell lines but importantly, mutations and deletions have been detected in myeloma cell lines and we wished to follow up on this observation in uniformly treated clinical cases. UTX is a large gene found on the X chromosome covering 240 kb of genomic DNA and consists of 29 exons encoding a protein with both JmjC-domains and tricopeptide repeats responsible for histone demethylation and polycomb protein interactions. Inactivation of UTX occurs through deletions of individual exons through to large whole gene deletions as well as by mutations scattered throughout the 29 exons. A further mechanism of UTX inactivation which has not been looked for to date is via DNA methylation of the CpG island upstream of the transcriptional start site. We set out to determine the status of UTX in our dataset which includes expression, mapping, and methylation array data from presenting myeloma samples entered into the MRC Myeloma IX clinical trial. The gene expression of UTX was measured on 272 samples using Affymetrix U133 Plus 2.0 arrays and showed that 80% of samples do not express UTX transcripts but using expression quartile analysis we could not detect an effect on overall survival. The mechanism underlying the abrogation of expression was investigated further using the Affymetrix 500K SNP mapping array on a subset of 114 samples to detect copy number alterations. UTX was hemizygously deleted in 21 (42%) female samples and was completely deleted in 1 male sample, at the resolution of the arrays. In order to determine if individual exons were deleted, at a resolution below that detectable by mapping arrays, we performed quantitative PCR coupled with high resolution melting (HRM) analysis using the Rotor-gene Q real-time cycler (Qiagen). Exons were amplified, over 40 cycles, to obtain products of ∼200 bp using LC Green Plus mastermix (Idaho Technologies) in a 10 μl reaction on the Rotor-gene Q with a final HRM step from 72-95 °C with increments of 0.1 °C. Amplification plots combined with the HRM step allows us to identify both homozygous deletions and mutations within the exons. We screened all 114 samples for micro-deletions and mutations and found homozygous deletions in ∼7% of samples and identified a significant proportion of mutations using the HRM method which accounted for a total of ∼10% of gene inactivation. In order to determine if methylation could be responsible for inactivation of the remaining allele we used the Illumina Infinium humanmethylation27 array to study the methylation status at the UTX locus. This array interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. The results of this analysis are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. In samples with a diploid copy number of UTX the methylation signals covered 2 ranges: hemi-methylated (0.35-0.55, n=7) and hyper-methylated (0.73-0.89, n=14). In samples with 1 copy of UTX, which includes all males, there were 3 ranges: hypomethylated (0.08-0.21, n=5), hemi-methylated (0.35-0.51, n=3), and hypermethylated (0.66-0.88, n=48). All of the hypomethylated samples with a single copy of UTX were male, and at least 1 of these samples contained an inactivating exonic deletion resulting in complete loss of function. These data indicate that methylation of the residual allele contributes significantly to the inactivation of UTX along with interstitial deletions and mutations. We will go on to present data on the interaction of UTX with variation at the UTY locus and how this modulates behaviour of the myeloma clone. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2009-05-222208 ◽

2010 ◽

Vol 115 (12) ◽

pp. 2412-2419 ◽

Cited By ~ 58

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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OS05.2.A MGMT promoter status in IDH1/2 mutant anaplastic astrocytoma patients assessed by DNA methylation profiling and qMS-PCR: a report from the EORTC Brain Tumor Group

Neuro-Oncology ◽

10.1093/neuonc/noab180.019 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii6-ii7

Author(s):

C M S Tesileanu ◽

P J French ◽

M Sanson ◽

A A Brandes ◽

W Wick ◽

...

Keyword(s):

Dna Methylation ◽

Anaplastic Astrocytoma ◽

Dna Methyltransferase ◽

Methylation Status ◽

High Grade Glioma ◽

Phase Iii ◽

Isocitrate Dehydrogenase 1 ◽

Kappa Score ◽

Dna Methylation Profiling ◽

Methylation Profiling

Abstract BACKGROUND Temozolomide (TMZ) efficacy in high-grade glioma is related to O 6-methylguanine DNA methyltransferase promoter (MGMTp) methylation. We compared the prognostic and predictive effect of MGMTp between DNA methylation profiling (the MGMT-STP27 model) and quantitative methylation specific polymerase chain reaction (qMS-PCR) in isocitrate dehydrogenase 1 and 2 (IDH1/2) mutant (mt) anaplastic astrocytoma patients. MATERIAL AND METHODS The 2x2 factorial design phase III CATNON trial randomized 751 adult patients with newly diagnosed 1p/19q non-codeleted anaplastic glioma to 59.4 Gy radiotherapy (RT), RT with concurrent TMZ, RT with 12 cycles of adjuvant TMZ, or RT with concurrent and adjuvant TMZ. MGMTp methylation status was assessed with the MGMT-STP27 model using 850k EPIC data, and qMS-PCR. IDH1/2 mutation status was determined with a next-generation sequencing panel. Overall survival (OS) was measured from date of randomization. RESULTS We identified 444 IDH1/2mt anaplastic astrocytoma patients of which MGMT-STP27 data was available for 440 patients (99.1%), qMS-PCR data for 361 patients (81.3%), and both for 357 patients (80.4%). MGMTp was methylated in 365 patients (83.0%) for the MGMT-STP27 model, and 168 patients (46.5%) for qMS-PCR. The agreement between the MGMT-STP27 model and qMS-PCR is 59.9% with a Cohen’s Kappa score of 0.229. At database lock, 289 patients with MGMT-STP27 data were still alive and 236 patients with qMS-PCR data. The median OS of MGMTp methylated glioma patients was 9.1 yrs [95 % confidence interval (CI) 7.5-not reached] for the MGMT-STP27 model, and not reached [95 % CI 9.1-not reached] for the qMS-PCR data. For MGMTp unmethylated glioma patients, the median OS was 6.9 yrs [95% CI 6.2-not reached] for the MGMT-STP27 model, and 6.8 yrs [95% CI 6.2–9.7] for the qMS-PCR data. The hazard ratio (HR) for OS based on MGMTp methylation was 0.88 [95% CI 0.58–1.31] for the MGMT-STP27 data, and 0.72 [95% CI 0.50–1.03]) for the qMS-PCR data. The HR for OS after RT with any TMZ vs RT alone for the MGMT-STP27 model was 0.53 [95% CI 0.37–0.78] for MGMTp methylated, and 0.54 [95% CI 0.25–1.18] for MGMTp unmethylated glioma patients; and for the MS-PCR data was 0.34 [95% CI 0.19–0.61] for MGMTp methylated, and 0.53 [95% CI 0.33–0.85] for MGMTp unmethylated glioma patients. CONCLUSION MGMTp methylation, regardless of assay, was neither prognostic nor predictive for outcome to temozolomide in IDH1/2mt anaplastic astrocytoma patients.

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Genome-Wide DNA Methylation Profile of CLL with 17p del Allowed Identification of Multiple Epigenetically Inactivated Molecular Pathways with Prognostic Value in Human Leukemia.

Blood ◽

10.1182/blood.v110.11.492.492 ◽

2007 ◽

Vol 110 (11) ◽

pp. 492-492

Author(s):

Wei-Gang Tong ◽

William G. Wierda ◽

Neby Bekele ◽

Shao-Qing Kuang ◽

Michael J. Keating ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Cpg Island ◽

Trichostatin A ◽

Methylation Status ◽

Cpg Islands ◽

Human Leukemia ◽

Dna Hypomethylation ◽

Line P ◽

Normal Controls

Abstract Aberrant DNA methylation of multiple promoter associated CpG islands is a very prevalent phenomenon in human leukemias. Data from our laboratory indicates that methylation profiling allows the identification of leukemia patients with different risk and prognosis. Despite the advances in the understanding of the molecular biology of CLL, few studies of DNA methylation have been performed in CLL. In the current study, we have developed a new assay combining MCA (Methylated CpG island Amplification) with the Agilent promoter CpG array to identify simultaneously hundreds of abnormally methylated CpG islands in CLL. To perform this, we compared DNA from two CLL patients with 17p del (tester) with that of CD19+ B cells from two age-matched controls (driver). We identified 280 promoter CpG islands differentially methylated in CLL compared to normal controls. Most of these genes are located on chromosomes 19 (16%), 16 (11%), 17 (10%) and 11 (9%). We also performed interaction pathway and functional analysis of these 280 genes using the online Ingenuity Pathway Analysis tools. The initial analysis divided these genes into 25 functional networks, with the majority of genes fall into top 10 networks. The major functions of genes in these interaction networks involve cancer, organ development, cell death, drug metabolism, DNA replication and repair. We validated 22 of these genes (ADCY5, R-spondin1, LHX1, GALGT2, TFAP2C, ING1, SOX11, SOX14, SALL1, LTBP2, APP, DXL1, DLX4, KLK10, BCL11B, NR2F2, FAM62T, HAND2, BNC1, SPOCK, Prima1 and MLL1) in samples from 78 CLL patients and 10 age-matched normal controls. The characteristics of the 78 patients are: median age 59 (range 39–79), male 70%, Rai stage 0–II/III–IV (83%/17%), IgVH unmutated 49%, ZAP-70 positive 33%. Our results indicate that most of the genes identified by the array are frequently hypermethylated in CLL patients compared with healthy controls. Methylation frequency ranged from 20%–100% in CLL patients. Expression analysis of four selected genes (LHX1, GALGT2, TFAP2C and Prima1) in human leukemia cell lines and CLL patient samples by real-time PCR further confirmed methylation associated gene silencing, and treatment of these cell lines with hypomethylating agent 5-aza-2′-deoxycitidine with or without the HDAC inhibitor Trichostatin A resulted in gene re-expression and induction of DNA hypomethylation. We also analyzed the association of methylation status of these genes with IgVH mutation status, ZAP70 expression and patient survival. Unmutated IgVH was associated with increased methylation levels of LINE (p<0.0001), which is a marker for global gene methylation and SALL1 (p=0.00008). Expression of ZAP-70 (>20%) was associated with increased methylation levels of LINE (p<0.00001), MLL (p=0.02) and SALL1 (p=0.048). Further analysis showed that methylation status of LINE (p=0.007), SALL1 (p=0.019), ADCY5 (p=0.021), R-spondin1 (p=0.002) and APP (p=0.002) correlated with survival. In conclusion, our studies indicate that MCA/promoter array technique allows the identification of large number of promoter CpG islands aberrantly methylated in CLL and also the identification of novel tumor suppressors and signaling pathways that could be important in the tumorigenesis of CLL and other hematological malignancies.

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Genome-Wide Epigenetic Analysis of Cancer Stem Cells (CSCs) In Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v116.21.3640.3640 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3640-3640 ◽

Cited By ~ 1

Author(s):

Jumpei Yamazaki ◽

Marcos R Estecio ◽

Jaroslav Jelinek ◽

David Graber ◽

Yue Lu ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Cell Population ◽

Progenitor Cell ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Significant Difference

Abstract Abstract 3640 Background & Aims: The hypothesis that cancer is driven by Cancer Stem Cells (CSCs or Cancer-Initiating Cell) has recently attracted a great deal of attention. Epigenetic mechanism such as DNA methylation and histone modification play an important role in cancer cells and also in normal stem cells. However, their role remains unclear in CSCs. We sought to determine if CSCs have distinct epigenetic patterns in acute myeloid leukemia (AML). Methods: Peripheral blood samples in AML patients were separated to obtain stem cells (CD34+CD38-) and progenitor cells (CD34+CD38+) by magnetic cell sorting (MACS®, Myltenyi biotec). To study DNA methylation in CSCs in AML, we performed genome wide screening using methylated CpG island microarray (MCAM), which detects 7202 promoter CpG islands, 1348 non-promoter CpG islands, and 632 non-CpG island promoter methylation. MCAM was performed on 4 AML patient samples Next, we evaluated the methylation status of 7 genes which showed apparent higher DNA methylation in stem cells or progenitor cells in MCAM analysis, using a quantitative bisulfite-pyrosequencing for each population of stem cell, progenitor cell, and mature cells (CD34-) from peripheral blood samples in 6 AML patients. For histone modification analysis, we used Chromation immuprecipitation followed by massively parallel sequencing (ChIP-Seq) for stem cell and progenitor cell populations for H3K4me3 which is known to be a marker for activated genes. Results: By MCAM, we found minimal differences between stem cells and progenitor cells present in 2 out of 4 AML patients. Those few genes (<1%) which were shown to have higher DNA methylation in stem or progenitor cells by MCAM analysis were likely false positives, as no significant difference was found when analyzed by quantitative bisulfite-pyrosequencing. DNA methylation status for stem cell-related gene (OCT4, SOX2, MYC, HOXB4, and KLF4) also showed no significant difference. By ChIP-seq analysis, we found differences in 2362 genes between stem cells and progenitor cells. In stem cells, H3K4me3 was enriched in genes (Bmi1, Notch1, Wnt1, and etc) which are known to be important for stem cell function, but they were not enriched in the progenitor cell population. In pathway analysis of the H3K4me3 data, Hypoxia-Inducible Factor signaling, NFkB signaling, and p53 signaling are found to be enriched specifically in the stem cell population whereas Cellular Growth and Cell Cycle, and DNA Damage Response signaling are found in the progenitor cell population. Conclusions: There is no significant difference in DNA methylation between stem cell, progenitor cell or mature cell populations in AML. DNA methylation of promoter CpG islands is unlikely to explain tumor hierarchy in AML. Rather, histone modifications seem to have a greater significance in this regard. Disclosures: No relevant conflicts of interest to declare.

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Aberrant CpG island methylation in acute myeloid leukemia is accentuated at relapse

Blood ◽

10.1182/blood-2007-11-126227 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1366-1373 ◽

Cited By ~ 103

Author(s):

Heike Kroeger ◽

Jaroslav Jelinek ◽

Marcos R. H. Estécio ◽

Rong He ◽

Kimie Kondo ◽

...

Keyword(s):

Dna Methylation ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cpg Island ◽

Methylation Status ◽

Cpg Islands ◽

Leukemia Cell ◽

Transcription Start Sites ◽

Remission Maintenance ◽

Acute Myeloid

AbstractDNA methylation of CpG islands around gene transcription start sites results in gene silencing and plays a role in leukemia pathophysiology. Its impact in leukemia progression is not fully understood. We performed genomewide screening for methylated CpG islands and identified 8 genes frequently methylated in leukemia cell lines and in patients with acute myeloid leukemia (AML): NOR1, CDH13, p15, NPM2, OLIG2, PGR, HIN1, and SLC26A4. We assessed the methylation status of these genes and of the repetitive element LINE-1 in 30 patients with AML, both at diagnosis and relapse. Abnormal methylation was found in 23% to 83% of patients at diagnosis and in 47% to 93% at relapse, with CDH13 being the most frequently methylated. We observed concordance in methylation of several genes, confirming the presence of a hypermethylator pathway in AML. DNA methylation levels increased at relapse in 25 of 30 (83%) patients with AML. These changes represent much larger epigenetic dysregulation, since methylation microarray analysis of 9008 autosomal genes in 4 patients showed hypermethylation ranging from 5.9% to 13.6% (median 8.3%) genes at diagnosis and 8.0% to 15.2% (median 10.6%) genes in relapse (P < .001). Our data suggest that DNA methylation is involved in AML progression and provide a rationale for the use of epigenetic agents in remission maintenance.

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