HpaII Tiny Fragment Enrichment By Ligation-mediated PCR Research Articles

Methylation status and long-fragment cell-free DNA are prognostic biomarkers for gastric cancer.

BackgroundCirculating tumor DNA (ctDNA) detected before surgery disappears after complete surgical resection of the cancer. Residual ctDNA indicates minimal residual disease (MRD), which is a cause of recurrence. The presence of long‐fragment circulating cell‐free DNA (cfDNA) or methylated cfDNA also implies the presence of cancer. In this study, we evaluated the prognostic value of cfDNA methylation and long‐fragment cfDNA concentration in gastric cancer patients undergoing curative surgeryMethodsNinety‐nine gastric cancer patients were included. Peripheral blood samples were collected before and 1 month after surgery. In patients administered chemotherapy, samples were collected before starting chemotherapy. qPCR was performed to detect long‐ and short‐fragment LINE‐1. A plasma HELP (HpaII tiny fragment Enrichment by Ligation‐mediated PCR) assay to determine the concentration of HpaII small fragments was performed using ligation‐mediated PCR and HpaII was quantified as the HpaII:MspI ratio to detect methylation levels of cfDNA.ResultsOverall survival (OS) of patients with low methylation levels before starting treatment was significantly worse than that of patients with high methylation levels (P = 0.006). In the 90 patients who underwent curative surgery, recurrence‐free survival (RFS) and OS of patients with low methylation levels before surgery were worse than those with high methylation levels (P=0.08 and P = 0.11, respectively). RFS and OS of patients with high concentrations of long‐fragment LINE‐1 after surgery were significantly worse than those with low concentrations of long‐fragment LINE‐1 (P = 0.009, P = 0.04).ConclusionsPre‐surgical low methylation levels of LINE‐1 are a negative prognostic factor. Post‐surgical high concentrations of long‐fragment LINE‐1 indicate MRD and a high risk of recurrence.

Simulated microgravity attenuates myogenic differentiation via epigenetic regulations

The molecular mechanisms involved in myogenic differentiation are relatively well-known. Myogenic differentiation is regulated by the sequential activation of the basic helix-loop-helix myogenic regulatory transcription factors (MRFs), and biomechanical signals play an important role in the regulation of myogenesis. In this study, we sought to determine whether simulated microgravity culture using Gravite® may affect myoblast differentiation and expression of MRF genes. Although rat myoblasts, L6 cells were differentiated to myotubes in an incubation period-dependent manner, myogenesis of L6 cells was significantly attenuated under simulated microgravity (10-3G) conditions. Real-time Reverse transcription polymerase chain reaction (RT-PCR) showed that expressions of Myog, Myf6, Mef2c, Des, and Ckm under 1 G conditions increase in an incubation period-dependent manner, and that Myod1 expression was specifically observed to increase transiently in the early phase. However, expressions of Myod1 and Myog were significantly inhibited under simulated microgravity conditions. To clarify the molecular mechanisms, L6 cells were treated with 5-AzaC, and further incubated with differentiation medium under 1 G or 10−3 G conditions. The results showed differences in expression levels of Myod1, Myog, and, as well as those of myotube thickness between 1 G and 10−3 G conditions, completely disappeared in this experimental condition. Modified HpaII tiny fragment enrichment by ligation-mediated PCR (HELP)-assay showed that kinetic changes of DNA methylation status were attenuated in simulated microgravity conditions. These results indicate that microgravity regulates myogenesis and Myod1 expression by controlling DNA methylation.

The HELP-Based DNA Methylation Assays.

Restriction enzymes have been valuable tools for representing the genome for DNA methylation assays, whether by using methylation-dependent enzymes or by sampling a reduced representation of the genome using a methylation-insensitive enzyme. These survey assays have remained mainstays of genome-wide approaches even with the development of more comprehensive shotgun genome-wide bisulphite sequencing-based assays, as they are significantly more affordable. DNA methylation survey assays are numerous and include reduced representation bisulphite sequencing (RRBS), the Illumina HumanMethylation450K and EPIC microarray system, and our evolving series of HELP-based assays. The HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assays initially involved microarray-based reporting of DNA methylation, but have now migrated to the use of massively parallel sequencing. In this chapter, we describe the latest HELP-tagging assay that uses Illumina Tru-Seq adapters, and mention the extension of the HELP-tagging assay to quantify 5-hydroxymethylation using the HELP-GT assay.

Amnion as a surrogate tissue reporter of the effects of maternal preeclampsia on the fetus.

BackgroundPreeclampsia, traditionally characterized by high blood pressure and proteinuria, is a common pregnancy complication, which affects 2–8 % of all pregnancies. Although children born to women with preeclampsia have a higher risk of hypertension in later life, the mechanism of this increased risk is unknown. DNA methylation is an epigenetic modification that has been studied as a mediator of cellular memory of adverse exposures in utero. Since each cell type in the body has a unique DNA profile, cell subtype composition is a major confounding factor in studies of tissues with heterogeneous cell types. The best way to avoid this confounding effect is by using purified cell types. However, using purified cell types in large cohort translational studies is difficult. The amnion, the inner layer of the fetal membranes of the placenta, is derived from the epiblast and consists of two cell types, which are easy to isolate from the delivered placenta. In this study, we demonstrate the value of using amnion samples for DNA methylation studies, revealing distinctive patterns between fetuses exposed to proteinuria or hypertension and fetuses from normal pregnancies.ResultsWe performed a genome-wide DNA methylation analysis, HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP)-tagging, on 62 amnion samples from the placentas of uncomplicated, normal pregnancies and from those with complications of preeclampsia or hypertension. Using a regression model approach, we found 123, 85, and 99 loci with high-confidence hypertension-associated, proteinuria-associated, and hypertension- and proteinuria-associated DNA methylation changes, respectively. A gene ontology analysis showed DNA methylation changes to be selecting genes with different biological processes in exposure status. We also found that these differentially methylated regions overlap loci previously reported as differentially methylated regions in preeclampsia.ConclusionsOur findings support prior observations that preeclampsia is associated with changes of DNA methylation near genes that have previously been found to be dysregulated in preeclampsia. We propose that amniotic membranes represent a valuable surrogate fetal tissue on which to perform epigenome-wide association studies of adverse intrauterine conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0234-1) contains supplementary material, which is available to authorized users.

Myelodysplastic Syndrome Marrow Stroma Shows Widespread Aberrant Hypermethylation That Is Abrogated By Treatment with Dnmt Inhibitors

The marrow microenvironment contributes to the pathogenesis of ineffective hematopoiesis in Myelodysplastic Syndromes (MDS). Since mutations and cytogenetic alterations seen in the hematopoietic compartment are generally not present in marrow stromal cells, we hypothesized that epigenetic alterations may be responsible for altered stroma function in MDS. To elucidate the epigenome of MDS microenvironment, we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to study cytosine methylation patterns of 50,000 CpGs loci covering 14,000 promoters in primary stromal cells. Global DNA methylation of 6 MDS marrow-derived stroma was analyzed by HELP assay and compared to 3 healthy controls. MDS stroma showed aberrant hypermethylation compared with controls (3626 hypermethylated vs 306 hypomethylated loci in untreated MDS stromal cells) that preferentially occurred outside of CpG islands (Test of Proportions, P value <0.01) and involved important signaling pathways and binding sites for transcription factors (Sp1, NFY, MYB,). Aberrant genomic methylation was seen to be significantly enriched at chromosomal regions chr12q15, chr5q32, chr4q21, chr7q31, chr3q13, chr2p12 and chr8q24 when compared with the genomic distribution of all HpaII fragments from the HELP array (P value <0.05; MDSig Program). Comparison with stroma derived from 5-Azacytidine (5-Aza) treated MDS patients revealed abrogation of aberrant methylation in treated samples. Integrative expression analysis revealed that the WNT pathway was epigenetically dysregulated in MDS stroma, and the WNT antagonists FRZB and SFRP1 were aberrantly hypermethylated and underexpressed. These epigenetic changes were validated in a co-culture model of stroma (HS27) and leukemic cells (Kg1a) and analyzed at a single base pair resolution using next gen sequencing based HELP-Tagging assay. Epigenetic changes in MDS stroma involving the WNT antagonists FRZB/SFRP1 were also validated at the protein level by IHC in an independent set of MDS stroma samples .After demonstrating aberrant hypermethylation in MDS stroma, we next examined the functional role of these alterations by treatment with DNMT inhibitors. 5-Aza treatment of MDS stroma enhanced hematopoietic activity and erythroid differentiation from co-cultured healthy CD34+ cells. Significantly increased erythroid differentiation was seen in CD34+ cells cocultured with aza pretreated MDS stroma as shown by their glycophorin A positivity.Conclusions: These results reveal widespread aberrant epigenetic changes in the MDS marrow microenvironmentthat affect many important pathways. A recent report had shown that activation of beta-catenin in the bone marrow niche can trigger MDS/AML in vivo. Our results demonstrate that epigenetic silencing of WNT antagonists can potentially lead to activation of the WNT pathway in MDS stroma. Finally, we demonstrate that DNA methyl transferase inhibitors alter the epigenomic profiles of stromal cells and it raises the possibility that epigenetic modulation of the stroma by these drugs contributing to their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Genome-Wide DNA Methylation Analysis Reveals Biological and Clinical Insights In Relapsed Childhood Acute Lymphoblastic Leukemia: A Report From The COG ALL Target Project

▪Although survival of children with B-cell acute lymphoblastic leukemia (B-ALL) has improved substantially over time, 15% to 20% of patients will relapse, and most of those who experience a bone marrow relapse will die. A better understanding of genetic and epigenetic aberrations in relapsed ALL will facilitate new strategies for risk stratification and targeted therapy. In this collaborative study with the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project, we performed high resolution genome-wide DNA methylation profiling using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) array on a total of 178 (110 diagnosis, 68 relapse) leukemia samples from 111 patients with childhood B-ALL enrolled on the Children's Oncology Group (COG) clinical trials who experienced relapsed, and 12 normal preB samples isolated from the bone marrows of 12 healthy individuals. The HELP array covers 117,521 CpG sites, annotated to ∼22,000 gene promoters. For eight diagnosis/relapse pairs, base-pair resolution DNA methylation using the eRRBS (enhanced Reduced Representation Bisulfite Sequencing) method was also performed on Illumina HiSeq2000.The median relapse time for the 111 patients was 21.8 months (range 2.1 to 56.2). Unsupervised clustering analysis using the HELP data revealed seven clusters: one cluster contained only the 12 normal preB samples; four clusters were enriched with MLLr, ETV6/RUNX1, Trisomy 4+10, and TCF3/PBX1 samples, respectively. The sixth cluster was not enriched for specific cytogenetic cases, but interestingly, all cases in this cluster were NCI High Risk (age>10 years or WBC>=50,000; p<0.0001, Fisher's Exact test) while the seventh cluster has a mixture of other cases.Supervised analysis of HELP profiles between paired relapse/diagnosis samples (n=67) revealed a markedly aberrant DNA methylation signature (1011 probesets, 888 genes, FDR<0.01 and methylation difference dx >25%, paired t-test), with 70% of the genes hyper- and 30% hypo-methylated in relapse samples. Using a Bayesian predictor and leave-one-out cross validation, this methylation signature could predict a sample as diagnosis or relapse with 95.3% accuracy. When comparing early (<36 months; n=50) versus late relapses (>=36 months; n=18), we detected a profound hypermethylation signature in early relapse (96.6% of the 610 probesets, 544 genes, FDR<0.01, dx >25%).Finally, we identified 1800 probesets (1658 genes) as differentially methylated within all cytogenetic subtypes described above compared to the normal preB samples (Dunnett's test with normal preB as reference, FDR<0.01, dx>25%). Again the majority (70%) of those genes were hypermethylated in relapse as compared to diagnostic and normal preB.The base-pair resolution and more comprehensive eRRBS methylation analysis for the eight pairs of samples identified 39,679 CpG sites as differentially methylated (dx >25%, FDR<0.01), with 78.2% CpG sites hyper- and 21.2% hypo-methylated in relapse samples. Remarkably, the hypermethylated CpGs are primarily in promoter regions (50%, defined as +/-1kb to TSS), followed by intergenic (26%), then intragenic (14%), and exonic (10%) regions. In contrast, the hypomethylated CpGs are mainly in intragenic (48%), followed by intergenic (31%), exonic (14%) and promoter (7%) regions. The hypermethylated CpGs were mainly in CpG islands (86%) or CpG shores (10%), while hypomethylated CpGs were not (CpG islands: 8%, CpG shores: 27%). We further identified 3040 differentially methylated regions (DMRs) with a median size 426 bp. 78.4% of those DMRs were hyper- (1362 gene promoters) and 21.6% hypo-methylated (98 promoters) in relapse compared to diagnostic samples. Gene set enrichment and Ingenuity pathway analysis showed epigenetically disrupted pathways that are highly involved in cell signaling, and embryonic and organismal development.Taken together, our genome-wide high resolution DNA methylation analysis on a large cohort of relapsed childhood B-ALL from the COG trial identified unique methylation signatures that correlated with relapse and with specific genetic subsets. Those methylation signatures featured prevailing promoter hypermethylation and to a lesser extent, intrageneic hypomethylation. Epigenetically dysregulated gene networks in those relapse samples involved cell signaling, and embryonic and organismal development. Disclosures:No relevant conflicts of interest to declare.

Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

Lineage, Fusion Partner and Age Differences in the Methylome of MLL-r Leukemias

AbstractAbstract 3506 Background:MLL gene rearrangements (MLL-r) are seen in all ages in both acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). The most common fusion partners are AF4, AF9 and ENL/ELL. Murine models suggest that the expression of MLL fusion proteins is necessary but not sufficient for leukemogenesis, but true cooperating lesions have been difficult to identify. Our lab and others have previously shown that MLL-r infant ALL can be defined by a unique signature characterized by genome-wide hypermethylated CpG islands leading to tumor suppressor gene silencing. In this study we aimed to discover the driving mechanism of MLL-r infant ALL epigenetic signature and whether that signature is intrinsic to the MLL-r or possibly one of the elusive cooperating leukemogenic events. Methods:We measured DNA methylation levels at roughly 225,000 CpG sites covering over 19,000 genes using the HELP (HpaII-tiny fragment Enrichment by Ligation-mediated PCR) assay to evaluate 102 leukemia samples and 12 normal controls. Gene expression using Illumina HT-12 arrays were concurrently performed for future paired expression-methylation analyses. Leukemia samples were primary diagnostic patient samples representing ages infancy to adulthood and included 60 ALL samples (36 MLL-r and 24 MLL-wt) and 42 AML samples (29 MLL-r and 13 MLL-wt). We used unsupervised hierarchical clustering and principal component analysis (PCA) to evaluate whether the methylation patterns in the 102 samples discriminate between lineage (ALL vs. AML), MLL fusion status (MLL-r vs. MLL-wt), fusion partners, and age (infant, pediatric, adult). Results:Hierarchical clustering, using the top 50% of genes that show highest variance in methylation level across all 102 samples, discriminated leukemia samples into distinct lineage specific groups (ALL vs. AML). Within the ALL cluster, there were distinct sub-clusters separating T- and B- cell disease. In addition, In B- cell disease only, MLL-r and MLL-wt formed distinct sub-clusters while no MLL-r vs MLL-wt subclustering was noticed in AML.PCA recapitulated descriptive findings. Regression analysis of the first principal component of all 102 samples showed significant linear separation of AML vs. ALL (p<0.001), but not by age or MLL-r status. However, analysis of the second principal component (diminishing the effect of lineage) showed that there was a secondary MLL-r specific signature among all samples (p<0.001). Regression analysis of the first principal component in the ALL-only samples showed significant linear separation of MLL-r vs. MLL-wt (p<0.001) and showed distinct separation by fusion partner group defined as AF4, AF9 and all other MLL-r (p<0.01). In addition, PCA showed significant distinction between infant ALL and childhood ALL (p<0.01) and trended towards significance when compared to adult disease (p=0.07). These patterns were not duplicated in AML where PCA showed no significant linear separation of MLL-r vs. MLL-wt (p=0.42) or separation by age(p>0.4). While there was no intrinsic MLL-r specific signature in AML, there was a weaker but significant clustering of MLL-r samples into two distinct groups; one consisting of AF9 samples and the other containing all other MLL-r fusion partners (p<0.03). Finally, we explored linage differences controlling for fusion partner, using false discovery rate analysis. Enumerating regions with q-values of <0.05, 29,736 probes showed differential methylation patterns when comparing infant MLL-AF9 ALL and infant MLL-AF9 AML while there were 0 probes were isolated when comparing infant MLL-AF9 AML to adult MLL-AF9 AML. Conclusions:Methylation patterns in MLL-r ALL and AML are distinct. The primary determinant of methylation signatures in MLL-r leukemia is lineage, not MLL status, even when controlling for fusion partners, suggesting that the MLL-r does not drive methylation patterns observed. However, within lineages MLL status is an important contributor to methylation patterns, but this influence is different in ALL and AML. In B-ALL, MLL-r defines a unique methylation signature, subgrouped by fusion partner and additionally, but less so, by age. In AML, MLL fusions AF9 and other MLL-r cluster together in restricted analyses but there is no intrinsic MLL-r signature. However, while lineage is the principle driver of methylation, MLL status does define a secondary significant signature irrespective of lineage. Disclosures:No relevant conflicts of interest to declare.

A Phase I/II Study of Vorinostat (SAHA), Cladribine (2-CdA), and Rituximab Shows Significant Activity in Previously Untreated Mantle Cell Lymphoma

Abstract 441Background: Despite significant progress in the treatment of mantle cell lymphoma (MCL), relapse remains the norm and additional therapies are needed especially for patients who are not candidates for aggressive treatment approaches. Increasingly, it has become evident that epigenetic modifications, including DNA hypomethylation and histone deacetylase inhibition, are critical to the pathogenesis and treatment of hematologic malignancies; important to cancer biology; and may be essential to the development of treatment resistance in B-cell malignancies. Further development and understanding of new and effective treatment regimens that target the epigenome are needed.2-CdA has activity in a variety of B and T cell malignancies. In addition to its cytotoxic effects, our preliminary work shows that 2-CdA has hypomethylating properties in lymphoid malignancies. When primary MCL and CLL cells -before and 96 hours after cladribine treatment-were analyzed by HELP (HpaII tiny fragment Enrichment by Ligation mediated PCR), an array based genome-wide methylation assay, 2-CdA affected DNA hypomethylation. One of the genes hypomethylated was identified as DUSP2, a dual specificity phosphatase gene that is a p53 target gene. DUSP2 dephosphorylates phosphoserine/threonine and phosphotyrosine residues, negatively regulating mitogen-activated protein (MAP) kinases ERK1 and ERK2, which are associated with cellular proliferation and differentiation in B-NHL.Vorinostat (SAHA) is a histone deacetylase inhibitor (HDACi), which has shown modest single agent activity in lymphoma and is FDA approved for use in cutaneous T cell lymphoma (CTCL). MCL cell lines treated with cladribine activated DUSP2 mRNA and when treated with the HDAC inhibitor SAHA synergistically increased transcription of DUSP mRNA. Furthermore, MCL treated with cladribine in vitro showed inhibition of global histone methylation. Our hypothesis is that cladribine and vorinostat synergistically activate silenced genes such as but not limited to DUSP 1 and 2 that are important for tumor cell death. The mechanism of rapid tumor cell death is under investigation, and does not appear to involve the classical apoptosis pathway. Given the need for novel therapies and the potential synergy seen with 2-CdA and SAHA, we initiated a Phase I/II trial combining SAHA, 2-CdA, and rituximab (SCR) for the treatment of B-cell non-Hodgkin's Lymphoma (NHL). The Phase I portion has been completed while Phase II is actively enrolling patients including those with newly diagnosed MCL.Methods: Phase I enrolled 10 patients with relapsed/refractory NHL. The MTD of vorinostat for the Phase I was 400 mg (D 1–14) combined with 2-CdA 5mg/m2 IV (D 1–5), and R 375 mg/m2 IV (weekly × 4 for cycle 1 and 1x/month) every 28 days for up to 6 cycles. Phase II eligibility includes relapsed NHL as well as previously untreated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). Primary outcome is response rate (ORR). Scientific correlatives include analysis of CD20 expression, histone acetylation, gene microarray and HELP methylation analysis, ERK phosphorylation, and Q-PCR of potential target genes.Results: 52 patients (Phase I/II) have been enrolled and 45 patients have been treated. The ORR in evaluable relapsed patients (3 DLBCL, 10 MCL, 1 FL, 1 MZL, 7 CLL) is 32% (7/22). Among these relapsed patients, complete remissions (CR) have been observed in MCL as well as follicular and marginal zone lymphomas. Of the 20 previously untreated MCL patients, 19 have completed ≥ 2 cycles and are evaluable for response. ORR is 100% (19/19) with 79% (15/19) CR. Toxicities by CTCAE 3.0 criteria have primarily included reversible myelosuppression, fatigue, dehydration, 1 gr. 4 thrombo-embolic event (probably related), and 1 grade 5 pulmonary hemorrhage in a patient with relapsed pulmonary lymphoma. One previously untreated mantle cell lymphoma patient has ongoing Gr. 3 thrombocytopenia six weeks after completing therapy. Preliminary analysis of ongoing correlative studies is available in 1 MCL patient and shows DUSP2 upregulation.Conclusions: The SCR regimen shows activity across a number of B-cell malignancies and shows particular therapeutic promise in patients with previously untreated mantle cell lymphoma. Correlative studies are ongoing and will be presented. Future studies should continue to explore this regimen in previously untreated mantle cell lymphoma. Disclosures:Off Label Use: vorinostat (SAHA) is not FDA approved for the treatment of B cell lymphomas. Okada:Merck: Speakers Bureau. Epner:Merck: Speakers Bureau.

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in “chromatin context”.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

High-resolution methylome analysis of renal cell carcinoma and identification of aberrant epigenetic changes in gene bodies.

e15000 Background: Metastatic renal cell carcinoma (RCC) has a very poor prognosis with limited therapeutic options. Despite this, there have been very few global genetic and epigenetic studies of RCC. DNA methylation is an epigenetic change that has been extensively studied in cancer, but most studies have focused on changes in promoters and CpG islands. We utilized a novel high resolution, high throughput assay, the HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) assay, to determine DNA methylation in RCC. The HELP assay examines CpG methylation at 1.3 million loci in the genome, including promoters, gene bodies and intergenic regions, making it the most comprehensive study of the RCC methylome available. Methods: We performed the HELP assay on 13 clear cell RCC and 13 control samples. The HELP assay compares restriction enzyme digestion with a methylation-sensitive enzyme (HpaII) to a methylation-insensitive enzyme (MspI) at CCGG sequences in the genome. These digestion fragments are amplified by PCR, fluorescently labeled, and co-hybridized onto a custom microarray. Methylation can be determined at each loci by comparison of the HpaII to MspI signal. Results: Unsupervised clustering showed that the RCC samples were epigenetically distinct and clustered completely separately from controls. Next, we determined common epigenetic alterations in all samples of RCC and observed that these tumors are characterized by a dramatic hypermethylation of a large number of loci in the gene bodies when compared to controls. Ingenuity pathway analysis of genes that were methylated and underexpressed in RCC revealed the TGFβ1 pathway to be the dominant pathway affected by epigenetic silencing, while cell death and cancer were two major biological functions influenced by epigenetic methylation. Conclusions: The HELP assay revealed widespread novel epigenetic changes and identified differentially regulated genes in RCC. This is the first study of DNA methylation changes in gene bodies in RCC. We hope to correlate methylation profiles with clinical response to uncover novel signatures of prognosis in RCC and identify new therapeutic targets within its epigenome.

Abstract 4820: Whole methylome explorations of paired tumor and non-tumor clinical samples using HELP

Abstract Introduction: Gene silencing by promoter methylation is common in carcinogenesis. In lung cancer, hypermethylation of specific gene promoters has been found to occur during the earliest histologic stages of the cancer. Thus far surveys of methylation in lung tumors have focused on specific gene panels (5 – 10 genes). A few large scale studies have reported selection of longer gene lists or focused on larger sets of CpG islands. Methods: We have adapted an approach to survey genome-wide methylation events in tumor and adjacent non-tumor tissue from clinical lung resection samples using the HELP (HpaII tiny fragment Enrichment by Ligation mediated PCR) assay. This assay, based on the generation of restriction enzyme libraries generated by methylation sensitive (HpaII) and methylation insensitive (MspI) isoschizomers yields information on 1.2 million HpaII fragments throughout the genome. Each HpaII fragment corresponds to two CpG sites. The assay lends itself to low starting amounts of DNA (3 ug) and robust assessment of methylation status by comparing ratios of HpaII- generated- fragments to MspI- generated fragments co-hybridized to a Nimbelgen custom high-density microarray. Summary of results: In a pilot set of three paired tumor and non-tumor tissue samples, we identified 1504 fragments (corresponding to two CpG sites each) that were hypermethylated in tumor versus non-tumor in all three pairs. Of these, 165 fragments were located within gene promoters. In the opposite direction, 2907 fragments were found to be hypermethylated in non-tumor versus tumor. Of these, 81 were located within gene promoters. We could identify both known and novel regions of the genome as well as specific gene-promoters that are hypermethylated in tumor versus non-tumor. We will discuss selected methylation events in further detail. Conclusion: The HELP assay is a powerful approach to interrogate methylation status of individual HpaII fragments across the whole genome, offering a large sample size of 1.2 million HpaII fragments. We are able to discover individual methylation events common across different clinical specimens and are currently expanding our sample set, and planning correlations with transcriptome-wide studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4820. doi:10.1158/1538-7445.AM2011-4820

Epigenomic Profiling of Multiple Myeloma Shows Widespread Stage Specific Alterations In DNA Methylation That Occur Early During Myelomagenesis

AbstractAbstract 784Gene expression is a tightly regulated process and is influenced by aberrant epigenetic changes that can lead to carcinogenesis. We used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to perform an unbiased genome-wide analysis of DNA methylation in 11 MGUS, 16 newly diagnosed myeloma, 17 relapsed myeloma, and 8 healthy control samples. The HELP assay uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling, and co-hybridization to quantitatively determine individual promoter methylation of 25,626 loci. The methylome analysis was performed using CD 138+ sorted bone marrow plasma cells in all cases. We observed extensive DNA methylation changes in myeloma that were seen even in MGUS cases when compared to normal plasma cells. Unsupervised clustering of all samples showed that MGUS samples were distinct but epigenetically closer to normal plasma cells than to cases of newly diagnosed or relapsed Myeloma. MGUS cases were characterized predominantly by aberrant hypomethylation, with 456 hypomethylated probes versus 130 hypermethylated probes (cutoff criteria were defined as a difference of means > 1.5 and significance of this difference with p < 0.001, Figure 1) that affected pathways regulated by NF-kb transcription factor. Untreated newly diagnosed myeloma samples were also predominantly hypomethylated (461 hypomethylated probes, 83 hypermethylated probes) when compared to controls. In addition to NF-kB, the MAP kinase and PI3 kinase regulated pathways were affected by hypomethylated genes. In contrast, cases of relapsed myeloma showed predominantly hypermethylated loci (221 hypomethylated, 560 hypermethylated probes) when compared to controls and involved the TNF and retinoblastoma pathways. A large number of important genes including the tumor suppressors CDKN2A and CDKN2B were aberrantly hypermethylated in this cohort. [Display omitted] Analysis of our differentially methylated targets using the Molecular Signatures Database (MSigDB, Tamayo, et al. 2005, PNAS 102, 15545–15550), showed significant overlap for hypomethylated genes in MGUS and new multiple myeloma (MM). These gene sets contain genes with promoter regions around transcription start site containing motifs which match annotation for transcription factors SP1 and TCF3 as well as enrichment for genes with gene ontology annotations for plasma membrane proteins. Hypermethylated genes in new MM show significant overlap with genes in the neighborhood of RAD23A, CTBP1, G22P1 and SMC1, suggesting impairment of DNA repair, as well as enrichment for genes associated with apoptosis and programmed cell death.In conclusion, genome-wide DNA methylation analysis is able to clearly differentiate between normal bone marrow plasma cells, MGUS as well as new MM and relapsed MM cells. Correlation of significantly differentially methylated genes with published gene sets reveals enrichment for genes involved in DNA repair, cell-cell signaling, cell death, apoptosis and cell cycle regulation. Disclosures:Mehta:Celgene: Consultancy, Speakers Bureau; Takeda/Millennium: Speakers Bureau; Onyx: Research Funding. Singhal:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding; Celgene: Speakers Bureau.

Experimental Intrauterine Growth Restriction Induces Alterations in DNA Methylation and Gene Expression in Pancreatic Islets of Rats

Intrauterine growth restriction (IUGR) increases susceptibility to age-related diseases, including type 2 diabetes (T2DM), and is associated with permanent and progressive changes in gene expression. Our study was designed to test whether epigenomic dysregulation mediates the cellular memory of this intrauterine event. To test this hypothesis, we isolated pancreatic islets from control and IUGR (induced by bilateral uterine artery ligation at day 18 of fetal life) animals at 7 weeks of age. Using the HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) assay, we generated the first DNA methylation map at almost 1 million unique sites throughout the rat genome in normal pancreatic islet cells, allowing us to identify the changes that occur as a consequence of IUGR. We validated candidate dysregulated loci with quantitative assays of cytosine methylation and gene expression. IUGR changes cytosine methylation at approximately 1,400 loci (false discovery rate of 4.2%) in male rats at 7 weeks of age, preceding the development of diabetes and thus representing candidate loci for mediating the pathogenesis of metabolic disease that occurs later in life. Epigenetic dysregulation occurred preferentially at conserved intergenic sequences, frequently near genes regulating processes known to be abnormal in IUGR islets, such as vascularization, beta-cell proliferation, insulin secretion, and cell death, associated with concordant changes in mRNA expression. These results demonstrate that epigenetic dysregulation is a strong candidate for propagating the cellular memory of intrauterine events, causing changes in expression of nearby genes and long term susceptibility to type 2 diabetes.

Abstract 168: Genome-wide survey of CpG methylation across the spectrum of B-cell lymphomas

Abstract Introduction: An international consortium was established to create comprehensive methylomes of B-cell lymphoma cell lines representing multiple stages of B-cell differentiation. One of the consortium's long-term goals is to identify genes and pathways that are perturbed by methylation in these cell lines either individually, or wholly, so that this information may be utilized to identify novel targets of aberrant methylation in patient samples. Experimental procedures: The B-cell derived tumor cell lines included in this study represent the spectrum of B-cell development: chronic lymphocytic leukemia (Mec1); mantle cell lymphoma (Granta); diffuse large B-cell lymphoma (Val and Ly10); follicular lymphoma (RL); Burkitt's lymphoma (Raji); and multiple myeloma (U266B1). Three methods were employed in this study: 1) Methylated CpG island amplification (MCA) and Agilent CpG island arrays; 2) HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) and Agilent CpG island arrays; and 3) Bisulfite treatment in conjunction with the Illumina Infinium Methylation BeadChip. Data summary: Comprehensive genome-wide methylation profiles were constructed for each tumor cell line using the data generated from three different methods. The number of detected differentially methylated probes, genes and gene promoters differed for each method. The number of unique methylated genes identified ranged from 25 for Granta to more than 200 for U266B1. Recurrent aberrant methylation was detected in ∼100 genes in all seven cell lines included in this study. Methylation of key miRNAs was identified in each of the cell lines. Gene lists that included genes with promoter methylation were submitted to the database for annotation, visualization and integrated discovery (DAVID) to identify enriched functional-related gene groups. Cell differentiation, transcription factor activity and HOX were among the top three functionally enriched gene groups in all cell lines. Genes associated with the Wnt, MAPK and Hedgehog pathways were also enriched in select cell lines. Conclusions: All approaches suffer from limitations in their ability to detect methylated sequences. The probes assayed on the Agilent CpG island array are limited to sequences that are contained within the restriction fragments produced in the MCA and HELP assays. The MCA method relies on the co-hybridization of a sample of interest to a “normal control” which may introduce biases if the “proper” control is not used. Finally, the Illumina Infinium BeadChip assays, on average, only 2 probes per gene and is the least informative of the detection assays. Despite these shortcomings, the combination of the data generated by each method has allowed the identification of novel genes and pathways that can be used to design screening assays for large numbers of patient samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 168.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.

Two Different EVI1 Expressing Poor-Risk AML Subgroups with Distinct Epigenetic Signatures Uncovered by Genome Wide DNA Methylation Profiling

Although the clinical outcome for patients with acute myeloid leukemia (AML) has improved over the years, failure to maintain complete remission remains a major problem with current standard treatments. The development of individually tailored and patient-specific therapy could potentially significantly improve therapeutic efficacy. In particular we are interested in better understanding the biological features associated with aberrant expression of the EVI1 oncogene, which we previously showed is associated with a poor prognosis. Two different EVI1 transcripts have been identified, i.e. a short form (E) and a long form called MDS1-EVI1 (ME) encoding respectively, a 140 kDa and 170 kDa protein. In EVI1 positive AMLs a distinction can be made between patients that express both EVI1 transcripts (E+/ME+) and cases that express the short form solely (E+), since the latter group is exclusively associated with 3q26 chromosomal abnormalities. EVI1 is a nuclear zinc-finger transcriptional repressor oncoprotein that is known to interact with several epigenetic regulators, e.g. HDACs, CtBPs, histone methyl transferases and MBD3. Since EVI1 presumably mediates its effects through aberrant transcriptional repression, we hypothesize that its aberrant expression results in aberrant epigenetic programming of leukemia cells, which might provide an opportunity for epigenetic-targeted therapy in these patients. In order to determine whether EVI1 over-expressing (EVI1+) AMLs display aberrant epigenetic programming we performed HELP (HpaII tiny fragment enrichment by ligation-mediated PCR) DNA methylation assays in 26 EVI1+ AMLs and 8 CD34+ normal bone marrow controls (NBM). Our HELP assay measured the abundance of DNA methylation at ~50,000 CpG sites covering ~13,000 promoter regions. Single locus validation assays using Sequenom Epityping showed that HELP was >95% accurate in quantifying CpG methylation. We found that unsupervised analysis using hierarchical clustering (Pearson correlation distance with Ward's clustering method) readily separated the EVI1+ AMLs from NBMs. Supervised analysis comparing EVI1+ to NBM identified 303 promoter sequences as being differently methylated (P<0.001 and methylation change >1.5). Remarkably, 80% of these genes were hypermethylated in EVI1+ patients, while only 20% of genes were hypomethylated. The hypermethylated profile included genes associated with cell death (Caspase-2, MAD1L1) and cell cycle (TNF, JARID1B). The 26 EVI1+ leukemias further segregated into two distinct subgroups in unsupervised analysis: one cluster (n=14) was highly enriched for E+ AML cases carrying 3q26 abnormalities (n=7) while the other one (n=12) mainly harbored the E+/ME+ AMLs (n=10). Supervised analysis of these two EVI1+ clusters revealed that the 3q26-enriched group featured 122-gene signature (P<0.001 and methylation change >1.5) consisting entirely of hypermethylated genes. When each of the individual EVI1 clusters was independently compared to the NBM samples using supervised analysis we found that the 3q26-enriched group contained a significantly more methylated gene signature containing 429 hypermethylated and 47 hypomethylated HpaII fragments (P<0.001 and methylation change >1.5). Pathway analysis of the promoter regions differentially methylated in the 3q26-enriched AML group included genes involved in protein degradation and cellular response to therapeutics. In contrast, the E+/ME+ enriched group showed a more balanced distribution of differential methylation when compared to the NBMs (226 hypermethylated and 158 hypomethylated genes). Taken together, our data show that EVI1 overexpression is associated with specific alterations in epigenetic programming vs. normal CD34+ cells. Even more remarkably, we showed that EVI1+ AMLs form two epigenetically distinct AML subtypes. Specifically, the 3q26 subgroup, short EVI1+ isoform AMLs display marked hypermethylation vs. the MDS1-EVI1 expressing patients, involving aberrant methylation of different pathways. This shows that the two forms of EVI1+ AMLs become aberrantly programmed in different ways and are biologically distinct entities, and further suggest distinct mechanisms of action for the different EVI1 isoforms. The marked hypermethylation profile of the short EVI1 isoform AMLs suggests that these patients might benefit from treatment with DNA methyltransferase inhibitors.

DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

Myelodysplastic Syndrome (MDS) Displays Profound and Functionally Significant Epigenetic Deregulation Compared to Acute Myeloid Leukemia (AML) and Normal Bone Marrow Cells.

MDS are a clinically heterogeneous group of clonal disorders, which share a high frequency of progression to secondary AML (MDS-AML). In contrast to de novo AML, MDS and MDS-AML are uniformly resistant to conventional chemotherapy. Among the few active drugs in MDS are the nucleoside analog DNA methyltransferase inhibitors (MTIs) 5-azacytidine and decitabine. Since tumor suppressor genes such as CDKN2B can be silenced by DNA methylation in MDS, it is believed that reversal of DNA methylation by MTIs might contribute to their anti-tumor effects. However, it is not clear whether methylation-dependent silencing of specific genes are predictive of response or even correlate with response to MTIs. Like MDS, AML also presents epigenetic silencing of CDKN2B and other genes; yet appear to not be as sensitive to MTIs. Given the particular sensitivity of MDS to MTIs and its resistance to standard AML chemotherapy, we hypothesized that MDS is a biologically distinct disease from AML due largely to extensive epigenetic deregulation, which is missed by single locus studies. In order to test this we studied DNA methylation levels at 24,000 gene promoters in 13 MDS pts. and 16 de novo normal karyotype AML cases, and compared and contrasted these to CD34+ bone marrow cells from 8 healthy donors. For this we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, a robust method for detection of whole-genome DNA methylation, and using MassArray quantitative methylation for single locus validation. Remarkably, MDS was found to have a far greater number of hypermethylated genes than AML or normal CD34+ cells, while AML and CD34+ cells had similar number of methylated genes (MDS vs. AML: 6303 vs. 4177 promoters, p=0.026; MDS vs. normal CD34+ cells: 6303 vs. 4296 promoters, p=0.056). Using a moderated T test, an aberrant methylation signature of 736 genes (p<0.0000001) was identified in MDS vs. normal CD34+ cells, reflecting extensive epigenetic deregulation in this disease, including p16, CEBPZ, MSH2, CHES1, AKT1, Caspase2, BMP3, DAP and MYOD1. A comparison between MDS and de novo AML identified 498 genes (p<0.00005) differentially methylated, with a clear predominance of hypermethylated promoters in MDS vs. de novo AML. These genes included RUNX2 and 3, GFI1, DAPK2, MDM2, TGFA, CEBPZ and SHARP. Finally, a comparison between normal CD34+ cells and de novo AML demonstrated an aberrant pattern of methylation in 341 genes (p<0.00001), including CXCL1 and 5, PPARD, Caspase 2 HOXA4 and HOXA10, GFI1 and the MLL translocation partner Septin11. 7 of the 13 MDS cases were also examined for gene expression using the Affymetrix Hgu133plus2 array. A significant proportion of the genes that had been found to be methylated were also found to be underexpressed, including p16, DAP, BMP3, HOXA2 and HOXB8. Taken together, our data show that MDS is a unique and distinct biological entity than de novo AML featuring profound and functionally significant genome wide epigenetic deregulation. While the de novo AML methylation profile was clearly different from normal CD34+ cells, it was not as severely altered as MDS. These data also suggest that MTIs are most likely uniquely active in MDS due to their DNA methyltransferase activity.