Comprehensive Methylation Analysis Research Articles

Comprehensive clinicopathological, molecular, and methylation analysis of mesenchymal tumors with NTRK and other kinase gene aberrations.

Alterations in kinase genes such as NTRK1/2/3, RET, and BRAF underlie infantile fibrosarcoma (IFS), the emerging entity 'NTRK-rearranged spindle cell neoplasms' included in the latest WHO classification, and a growing set of tumors with overlapping clinical and pathological features. In this study, we conducted a comprehensive clinicopathological and molecular analysis of 22 cases of IFS and other kinase gene-altered spindle cell neoplasms affecting both pediatric and adult patients. Follow-up periods for 16 patients ranged in length from 10 to 130 months (mean 38 months). Six patients were treated with targeted therapy, achieving a partial or complete response in five cases. Overall, three cases recurred and one metastasized. Eight patients were free of disease, five were alive with disease, and two patients died. All cases showed previously reported morphological patterns. Based on the cellularity and level of atypia, cases were divided into three morphological grade groups. S100 protein and CD34 were at least focally positive in 12/22 and 14/22 cases, respectively. Novel PWWP2A::RET, NUMA1::RET, ITSN1::RAF1, and CAPZA2::MET fusions, which we report herein in mesenchymal tumors for the first time, were detected by RNA sequencing. Additionally, the first uterine case with BRAF and EGFR mutations and CD34 and S100 co-expression is described. DNA sequencing performed in 13 cases uncovered very rare additional genetic aberrations. The CNV profiles showed that high-grade tumors demonstrate a significantly higher percentage of copy number gains and losses across the genome compared with low- and intermediate-grade tumors. Unsupervised clustering of the tumors' methylation profiles revealed that in 8/9 cases, the methylation profiles clustered with the IFS methylation class, irrespective of their clinicopathological or molecular features. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Integrated environmental, lifestyle, and epigenetic risk prediction of primary gastric neoplasia using the longitudinally monitored cohorts

DNA methylation accumulates in non-malignant gastric mucosa after exposure to pathogens. To elucidate how environmental, methylation, and lifestyle factors interplay to influence primary gastric neoplasia (GN) risk, we analyzed longitudinally monitored cohorts in Japan and Singapore. Asymptomatic subjects who underwent a gastric mucosal biopsy on the health check-up were enrolled. We analyzed the association between clinical factors and GN development using Cox hazard models. We further conducted comprehensive methylation analysis on selected tissues, including (i) mucosae from subjects developing GN later, (ii) mucosae from subjects not developing GN later, and (iii) GN tissues and surrounding mucosae. We also use the methylation data of mucosa collected in Singapore. The association between methylation and GN risk, as well as lifestyle and methylation, were analyzed. Among 4234 subjects, GN was developed in 77 subjects. GN incidence was correlated with age, drinking, smoking, and Helicobacter pylori (HP) status. Accumulation of methylation in biopsied gastric mucosae was predictive of higher future GN risk and shorter duration to GN incidence. Whereas methylation levels were associated with HP positivity, lifestyle, and morphological alterations, DNA methylation remained an independent GN risk factor through multivariable analyses. Pro-carcinogenic epigenetic alterations initiated by HP exposure were amplified by unfavorable but modifiable lifestyle choices. Adding DNA methylation to the model with clinical factors improved the predictive ability for the GN risk. The integration of environmental, lifestyle, and epigenetic information can provide increased resolution in the stratification of primary GN risk. The funds are listed in Acknowledgements section.

5mC DNA methylation modification-mediated regulation in tissue functional differentiation and important flavor substance synthesis of tea plant (Camellia sinensis L.).

In plants, 5mC DNA methylation is an important and conserved epistatic mark involving genomic stability, gene transcriptional regulation, developmental regulation, abiotic stress response, metabolite synthesis, etc. However, the roles of 5mC DNA methylation modification (5mC methylation) in tea plant growth and development (in pre-harvest processing) and flavor substance synthesis in pre- and post-harvest processing are unknown. We therefore conducted a comprehensive methylation analysis of four key pre-harvest tissues (root, leaf, flower, and fruit) and two processed leaves during oolong tea post-harvest processing. We found that differential 5mC methylation among four key tissues is closely related to tissue functional differentiation and that genes expressed tissue-specifically, responsible for tissue-specific functions, maintain relatively low 5mC methylation levels relative to non-tissue-specifically expressed genes. Importantly, hypomethylation modifications of CsAlaDC and TS/GS genes in roots provided the molecular basis for the dominant synthesis of theanine in roots. In addition, integration of 5mC DNA methylationomics, metabolomics, and transcriptomics of post-harvest leaves revealed that content changes in flavor metabolites during oolong tea processing were closely associated with transcription level changes in corresponding metabolite synthesis genes, and changes in transcript levels of these important synthesis genes were strictly regulated by 5mC methylation. We further report that some key genes during processing are regulated by 5mC methylation, which can effectively explain the content changes of important aroma metabolites, including α-farnesene, nerolidol, lipids, and taste substances such as catechins. Our results not only highlight the key roles of 5mC methylation in important flavor substance synthesis in pre- and post-harvest processing, but also provide epimutation-related gene targets for future improvement of tea quality or breeding of whole-tissue high-theanine varieties.

Methylation of CpG island promoters at ZNF625, LONRF2, SDC2 and WDR17 in a patient with numerous non-granular type laterally spreading tumors and colorectal cancer: A case report.

Patients with adenomatous polyposis syndromes such as familial adenomatous polyposis are at higher risk of colorectal cancer, hence continuous management is necessary. However, little is known about the etiology of patients with numerous laterally spreading tumors (LSTs), or how genetic alterations uniquely influence LSTs in colorectal carcinogenesis. The present case report investigated a woman with >150 non-granular type LSTs (LST-NG) and one sigmoid colon cancer. After subtotal colectomy via ileorectal anastomosis, genetic and epigenetic analyses were conducted by comparing the profiles of the patient's normal colonic mucosa, four LST-NG lesions and a cancer lesion. Using customized multigene panel testing, no pathogenic germline mutations were detected, including APC regulator of WNT signaling pathway, but identified a somatic pathogenic variant of APC in one LST-NG lesion, and both TP53 and F-box and WD repeat domain containing 7 somatic mutations in the cancer. Comprehensive genome-wide methylation analysis showed that CpG island promoters at zinc finger protein 625, LON peptidase N-terminal domain and ring finger 2, WD repeat domain 17 and syndecan 2 were methylated in both LST-NG and cancer, which may contribute to colorectal tumorigenesis as early as the LST-NG phase.

Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

Comprehensive Methylation Analysis in Pediatric Patients with Acute Myeloid Leukemia —the JCCG Study, JPLSG AML-05-

IntroductionThe genetic heterogeneity of acute myeloid leukemia (AML) makes it difficult to predict an appropriate prognosis of patients with AML. Currently, several gene mutations associated with the pathogenesis of AML have been identified owing to recent advances in massively parallel sequencing technologies. However, despite incorporating these molecular markers, the prognosis of many patients remains uncertain. Aberrant DNA methylation has frequently been observed in myeloid cells of patients with AML. Reportedly, DNA methylation patterns are associated with molecular features. Furthermore, a previous report has demonstrated that the methylation levels of polycomb target genes are associated with the outcome, implying that the definition of DNA methylation patterns of AML is expected to reveal clinical and molecular features and survival in patients with AML. However, there are few reports of DNA methylation in pediatric patients with AML. Thus, we analyzed genome-wide DNA methylation in pediatric patients with AML to reveal its association with clinical features, genetic alterations, and prognostic impact. Besides, our previous study revealed that the high expression of MECOM (also known as EVI1 ) or PRDM16 (also known as MEL1 ) was associated with inferior outcomes in patients with AML. We also investigated the correlation between DNA methylation and gene expression in pediatric patients with AML.MethodsBetween 2006 and 2010, 443 pediatric patients with de novo AML (0-17 years) participated in the Japanese AML-05 trial conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group. Of these, 369 samples were available, and 64 patients were enrolled in this study. The cytogenetic features of 64 patients were as follows: normal karyotype, 33; RUNX1-RUNX1T1 , 8; KMT2A rearrangement, 10; complex karyotype, 6; and other cytogenetics, 7. This cohort included 15 patients with FLT3 -internal tandem duplication (ITD), 8 with CEBPA biallelic mutations, 10 with high MECOM expression, and 30 with high PRDM16 expression. We performed comprehensive DNA methylation analysis using Infinium MethylationEPIC BeadChip (Illumina, Inc., San Diego, CA, USA) in 64 pediatric patients with AML to reveal the correlations among DNA methylation patterns, clinical features, genetic alterations, and prognostic impact in pediatric AML.Results and DiscussionUnsupervised hierarchical clustering based on methylation values of the 567 CpG sites, which showed most variable methylation values between 64 individuals, generated four clusters (clusters 1-4). The number of patients in clusters 1, 2, 3, and 4 was 23, 12, 13, and 16, respectively. Interestingly, these four clusters harbored their molecular features. Cluster 1 included patients with RUNX1/RUNX1T1 , normal karyotype with low PRDM16 expression, or KMT2A rearrangement with low MECOM expression. Cluster 2 included patients with FLT3 -ITD and/or normal karyotype with high PRDM16 expression. Cluster 3 included patients with normal karyotype with CEBPA biallelic mutations and cluster 4 patients with FLT3 -ITD, normal karyotype with high PRDM 16 expression, or KMT2A rearrangement with high MECOM expression. Differences in frequencies of common molecular aberrations among clusters were assessed with the Fisher-Freeman-Halton test. There was an unequal distribution of FLT3 -ITD and high PRDM16 expression (P = 0.003 for both), with concordantly high frequencies in clusters 2 and 4 and low in clusters 1 and 3. Clusters 2 and 4 tended to be correlated with older age, higher white blood cell counts at diagnosis, high relapse rate, and high mortality. These results suggest that DNA methylation can stratify the risk in pediatric AML. Conversely, no independent methylation marker was present to predict the adverse outcome, thereby indicating that an assortment of several methylation sites may be a useful marker for risk stratification.ConclusionWe investigated DNA methylation patterns in pediatric patients with AML and found four distinct clusters. Furthermore, our findings indicated that quantitative determination of DNA methylation percentage at specific CpG sites may be useful for risk stratification in pediatric AML. Considering the need of further analyses to reveal the significance of DNA methylation, we are analyzing the results currently and would like to discuss these data at the annual meeting. DisclosuresNo relevant conflicts of interest to declare.

Difference in potential DNA methylation impact on gene expression between fast- and slow-type myofibers.

Skeletal muscles are comprised of two major types of myofibers, fast and slow. It is hypothesized that once myofiber type is determined, muscle fiber-type specificity is maintained by an epigenetic mechanism, however, this remains poorly understood. To address this, we conducted a comprehensive CpG methylation analysis with a reduced representation of bisulfite sequencing (RRBS). Using GFP-myh7 mouse, we visually distinguished and separately pooled slow-type and myh7-negative fast-type fibers for analyses. A total of 31,967 and 26,274 CpGs were hypermethylated by ≥10% difference in the fast- and slow-type fibers, respectively. Notably, the number of promoter-hypermethylated genes with downregulated expression in the slow-type fibers was 3.5 times higher than that in the fast-type fibers. Gene bodies of the fast-type-specific myofibrillar genes Actn3, Tnnt3, Tnni2, Tnnc2, and Tpm1 were hypermethylated in the slow-type fibers, whereas those of the slow-type-specific genes Myh7, Tnnt1, and Tpm3 were hypermethylated in the fast-type fibers. Each of the instances of gene hypermethylation was associated with the respective downregulated expression. In particular, a relationship between CpG methylation sites and the transcription variant distribution of Tpm1 was observed, suggesting a regulation of Tpm1 alternative promoter usage by gene body CpG methylation. An association of hypermethylation with the regulation of gene expression was also observed in the transcription factors Sim2 and Tbx1. These results suggest not only a myofiber type-specific regulation of gene expression and alternative promoter usage by gene body CpG methylation but also a dominant effect of promoter-hypermethylation on the gene expressions in slow myofibers.

MADA: a web service for analysing DNA methylation array data

BackgroundDNA methylation in the human genome is acknowledged to be widely associated with biological processes and complex diseases. The Illumina Infinium methylation arrays have been approved as one of the most efficient and universal technologies to investigate the whole genome changes of methylation patterns. As methylation arrays may still be the dominant method for detecting methylation in the anticipated future, it is crucial to develop a reliable workflow to analysis methylation array data.ResultsIn this study, we develop a web service MADA for the whole process of methylation arrays data analysis, which includes the steps of a comprehensive differential methylation analysis pipeline: pre-processing (data loading, quality control, data filtering, and normalization), batch effect correction, differential methylation analysis, and downstream analysis. In addition, we provide the visualization of pre-processing, differentially methylated probes or regions, gene ontology, pathway and cluster analysis results. Moreover, a customization function for users to define their own workflow is also provided in MADA.ConclusionsWith the analysis of two case studies, we have shown that MADA can complete the whole procedure of methylation array data analysis. MADA provides a graphical user interface and enables users with no computational skills and limited bioinformatics background to carry on complicated methylation array data analysis. The web server is available at: http://120.24.94.89:8080/MADA

Changes in DNA methylation during epigenetic-associated sex reversal under low temperature in Takifugu rubripes.

DNA methylation has frequently been implicated in sex determination and differentiation in teleost species. In order to detect the DNA methylation patterns established during sexual differentiation in tiger pufferfish T. rubripes, we performed comprehensive whole genome methylation sequencing and analyses of the gonads of male, female, and pseudo male. We obtained a total of 33.12, 32.44, and 31.60 Gb clean data for male, female, and pseudo male, with a sequencing depth of 66.44×, 60.47× and 54.86×, respectively. The methylation level of cytosine (C) residues in the genomic DNA from gonads was 11.016%, 10.428%, and 11.083% in male, female, and pseudo male, respectively. More than 65% of C methylation was at CpG sites, and less than 1% was at CHG and CHH sites. In each regulatory element, there were low methylation levels on both sides of the transcription start site, and higher methylation levels in exons, introns, and downstream of genes. The highest mCpG was on chromosome 8 and the lowest mCpG was on chromosome 5. Comparisons of whole-genome DNA methylation between pairs of samples revealed that there were 3,173 differentially methylated regions (DMRs) between female and male, and 3,037 DMRs between male and pseudo male, corresponding to 0.232% and 0.223% of the length of the genome, respectively. There were only 1,635 DMRs between female and pseudo male, representing 0.127% of the length of the genome. A number of differentially methylated genes (DMGs) related to sex determination and differentiation were selected, such as amhr2 and pfcyp19a. After Bisulfite Sequencing PCR (BSP) verification, amhr2 was exhibited low methylation level in normal males and pseudo male, and high methylation level in normal females but pfcyp19a showed low methylation level in normal females and high methylation level in normal males and pseudo males. These results provide information about the molecular epigenetic mechanisms of DNA methylation during low-temperature induced masculinization of tiger pufferfish, and increase our understanding of the mechanisms of sex determination and differentiation in this important aquaculture fish species.

Comprehensive methylation analysis of imprinting-associated differentially methylated regions in colorectal cancer

BackgroundImprinted genes are regulated by DNA methylation at imprinting-associated differentially methylated regions (iDMRs). Abnormal expression of imprinted genes is implicated in imprinting disorders and tumors. In colorectal cancer (CRC), methylation and imprinting status of the IGF2/H19 domain have been studied. However, no comprehensive methylation analysis of iDMRs in CRC has been reported. Furthermore, the relationship between iDMR methylation status and other methylation-related issues, such as CpG island methylator phenotype (CIMP) and long interspersed element-1 (LINE-1) methylation, remains unclear.ResultsWe analyzed the methylation status of 38 iDMRs in 106 CRC patients. We also investigated CIMP, LINE-1 methylation, KRAS and BRAF gene mutations, and loss of imprinting (LOI) of IGF2. We further examined the relationship between these factors and clinicopathological factors. The overall trend in iDMR methylation was towards hypermethylation, and iDMRs could be grouped into three categories: susceptible, resistant, and intermediate-to-aberrant methylation. The susceptible and resistant iDMRs consisted of all types of iDMR (gametic and somatic, maternally and paternally methylated). Hypermethylation of multiple iDMRs (HyMiD)-positive status was statistically associated with CIMP-positive status, but not associated with mutations in the BRAF and KRAS genes. HyMiD-positive status was inversely associated with LINE-1 methylation. Among four iDMRs within the IGF2/H19 domain, IGF2-DMR0 hypomethylation occurred most frequently, but was not associated with IGF2 LOI. Finally, we statistically calculated predictive prognostic scores based on aberrant methylation status of three iDMRs.ConclusionIn CRC tissues, some iDMRs were susceptible to hypermethylation independent of the type of iDMR and genomic sequence. Although HyMiD-positive status was associated with CIMP-positive status, this was independent of the BRAF and KRAS pathways, which are responsible for CIMP. Since IGF2-DMR0 hypomethylation and aberrant methylation of other iDMRs within the IGF2/H19 domain were not associated with IGF2 LOI, dysfunction of any of the molecular components related to imprinting regulation may be involved in IGF2 LOI. The prognostic score calculated based on aberrant methylation of three iDMRs has potential clinical applications as a prognostic predictor in patients. Further study is required to understand the biological significance of, and mechanisms behind, aberrant methylation of iDMRs and IGF2 LOI in CRCs.

Modulation of Cellular CpG DNA Methylation by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is a gammaherpesvirus associated with several human malignancies. DNA methylation at CpG dinucleotides is an epigenetic mark dysregulated in many cancer types and in KSHV-infected cells. Several previous studies have analyzed in detail the CpG methylation of the KSHV episomal genomes, but little is known about the impact of KSHV on the human genome. Our knowledge of cellular CpG methylation in the context of KSHV infection is currently limited to four hypermethylated human gene promoters. Therefore, we undertook a comprehensive CpG methylation analysis of the human methylome in KSHV-infected cells and KSHV-associated primary effusion lymphoma (PEL). We performed Infinium HumanMethylation450K and MethylationEpic BeadChip arrays and identified panels of hyper- and hypomethylated cellular promoters in KSHV-infected cells. We combined our genome-wide methylation analysis with high-throughput RNA sequencing (RNA-seq) to add functional outcomes to the virally induced methylation changes. We were able to correlate many downregulated genes with promoter hypermethylation and upregulated genes with hypomethylation. In addition, we show that treating the cells with a demethylating agent leads to reexpression of these downregulated genes, indicating that, indeed, DNA methylation plays a role in the repression of these human genes. Comparison between de novo infection and PEL suggests that the virus induces initial hypermethylation followed by a slow increase in genome-wide hypomethylation. This study extends our understanding of the relationship between epigenetic changes induced by KSHV infection and tumorigenesis.IMPORTANCE In cancer cells, certain promoters become aberrantly methylated, contributing to the phenotype of the tumor. KSHV infection seems to modify cellular CpG methylation, but only a few methylated promoters have been identified in KSHV-infected cells. Here, we investigated the CpG methylation of the human genome in KSHV-associated primary effusion lymphoma (PEL) and KSHV-infected cells. We have identified many hyper- and hypomethylated gene promoters and correlated their methylation with cellular gene expression. These differentially methylated cellular promoters can distinguish KSHV-positive cells from uninfected cells and may serve as the foundation for the use of these differentially methylated regions as potential biomarkers for KSHV-associated malignancies. Drugs that reverse these cancerous methylation patterns have the potential to inhibit tumor growth. Here, we show that treating PEL cells with a demethylating drug (5-aza-2'-deoxycytidine) led to inhibition of cell growth, raising the possibility of testing this drug for the treatment of PEL.

Feasibility of quantifying SDC2 methylation in stool DNA for early detection of colorectal cancer

BackgroundColorectal cancer (CRC) screening is the most efficient strategy to reduce disease-related mortality. Frequent aberrant DNA methylation is known to occur in selected genes and early during CRC development, which has emerged as a new epigenetic biomarker for early detection of CRC. Previously, we reported that we identified that CpG sites of SDC2 were aberrantly methylated in tumor tissues of most CRC patients through comprehensive methylation analysis and demonstrated a high potential of quantification of SDC2 methylation in blood for early detection of colorectal cancer. In this study, we aim to investigate the feasibility of quantifying SDC2 methylation in stool DNA for the early detection of CRC. The objective of this study was to confirm a high frequency of SDC2 methylation in tumor tissues at various stages of CRC and investigate the feasibility of a quantitative test for SDC2 methylation in fecal DNA by highly sensitive and accurate real-time PCR for early detection of CRC.MethodsBisulfite-pyrosequencing assay was performed to measure the SDC2 methylation status in tissue samples. For methylation analysis in stool DNA, a highly sensitive and accurate method was applied which implements consecutive two rounds of PCR consisting of unidirectional linear target enrichment (LTE) of SDC2 and quantitative methylation-specific real time PCR (qMSP) for SDC2, named as meSDC2 LTE-qMSP assay. Its limit of detection was 0.1% methylation (corresponding to ~ 6 copies in total ~ 6200 genome copies).ResultsPositive SDC2 methylation was observed in 100% of primary tumors, 90.6% of adenomatous polyps, 94.1% of hyperplastic polyps, and 0% of normal tissues. SDC2 methylation level also significantly (P < 0.01) increased according to the severity of lesions. In stool DNA test for SDC2 methylation by LTE-qMSP comparing CRC patients with various stages (I to IV) (n = 50) and precancerous lesions (n = 21) with healthy subjects (n = 22), the overall sensitivity was 90.0% for detecting CRC and 33.3% for detecting small polyps, with a specificity of 90.9%.ConclusionsTaken together, our result indicates that stool DNA-based SDC2 methylation test by LTE-qMSP is a potential noninvasive diagnostic tool for early detection of CRC.

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs.

Cytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here, we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing. By using RNAs from mouse embryonic stem cells, we performed a comprehensive methylation analysis of mouse tRNAs, rRNAs, and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA. In addition, mRNAs were found to be very sparsely methylated or not methylated at all. Finally, the tRNA-specific activity of the DNMT2 methyltransferase could be resolved at single-base resolution, which provided important further validation. Our approach can be used to profile cytosine-5 RNA methylation patterns in many experimental contexts and will be important for understanding the function of cytosine-5 RNA methylation in RNA biology and in human disease.

Abstract 4887: Comprehensive molecular analysis of pediatric thalamic tumors

Abstract Childhood thalamic tumors are relatively rare cancers, accounting for 5% of all pediatric brain tumors and categorized as midline gliomas such as diffuse intrinsic pontine gliomas (DIPGs). We and others have shown that mutations in genes encoding for histone 3.3 (H3F3A), histone 3.2 (HIST2H3C) and histone 3.1 (HIST1H3B) along with their obligate partner mutations are the major driver mutations in DIPGs. Where recent studies have identified major histone partner mutations associated with DIPGs, more research is required to provide a clear landscape of genomic aberrations associated with thalamic tumors. We hypothesize that comprehensive whole genome sequence, methylation and proteome analysis of a large cohort of thalamic tumors will map differentially regulated pathways and identify potential novel driver and obligate partner mutations associated with thalamic gliomas. We have established a cohort (CNHS and CBTTC) of 128 thalamic specimens, including 56 pediatric and adolescent primary thalamic tumors with median age at diagnosis of 5.6 years (range 0-20 years); 40 normal controls with matched age and gender; and 32 midline tumors with potential thalamic involvement with median age at diagnosis of 7.6 years (range 0-19 years). Our cohort of primary thalamic tumors contained 31 (55.3%) and 19 (33.9%) tumors reviewed as high and low grade gliomas, respectively. From our midline tumors with potential thalamic involvement, 22 (68.7%) were classified as primary DIPG and 10 (31.2%) were other midline gliomas. Where available, MRI reviews and histopathological analysis were performed. Preliminary results showed that 7 of the extended tumors presented hypercellularity and positive histone 3 K27M staining, confirming that these tumors in fact extended to the thalamus as MRI showed. Additionally, Whole Exome Sequencing (WES) from one DIPG sample extending to the thalamus showed the same mutations found on the primary pons tumor: H3.1 K27M; ACVR1 G328V; PIK3CA H1047R; MAX R51Q and PTEN A126S. The remaining primary and extended thalamic tumors will be analyzed by WES to understand the molecular changes associated with this disease. In addition to our results, we analyzed the genomic landscape from pediatric thalamic tumors previously published (188), showing H3.3/H3.1 K27M (51%); BRAF (10.6%) and TP53 (8%) as the most frequent mutations among thalamic high and low grade astrocytomas. Further studies will allow us to compare comprehensive molecular analysis of thalamic tumors (primary and metastatic) and non-thalamic midline tumors (specimen and data already in hand) and identify similarities and differences in genomic, epigenomic and proteomic expression pattern which may guide a better characterization of thalamic tumor as a separate entity. Citation Format: Heloisa H. Moser, Susanne Yoon, Madhuri Kambhampati, Sridevi Yadavilli, Angela J. Waanders, Adam Resnick, Roger J. Packer, Javad Nazarian. Comprehensive molecular analysis of pediatric thalamic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4887. doi:10.1158/1538-7445.AM2017-4887

Prognostic methylation markers for overall survival in cytogenetically normal patients with acute myeloid leukemia treated on SWOG trials.

Aberrant DNA methylation is known to occur in patients with acute myeloid leukemia (AML), whereas methylation signatures and prognostic markers have been proposed. The objective of the current study was to evaluate all CpG sites of the genome and identify prognostic methylation markers for overall survival in patients with AML with normal karyotype (AML-NK). AML-NK samples from 7 SWOG trials were analyzed using a novel genome-wide approach called "CHARMcox" (comprehensive high-throughput array-based relative methylation analysis combined with the Cox proportional hazards model) controlling for known clinical covariates. CHARMcox was applied to a phase 1 discovery cohort (72 patients) to identify survival-associated methylation regions (SAMRs). Subsequently, using bisulfite pyrosequencing, SAMRs were studied in phase 2 model-building (65 patients) and phase 3 validation (65 patients) cohorts. An independent external cohort from The Cancer Genome Atlas (TCGA) AML study (LAML) was used for further validation (93 patients). Two SAMRs, located at the CpG island shores of leucine zipper tumor suppressor 2 (LZTS2) and nuclear receptor subfamily 6 group a member 1 (NR6A1), respectively, were identified. Multivariable analyses demonstrated that hypomethylation of either LZTS2 or NR6A1 was associated with worse overall survival in the SWOG cohort (P<.001). The prognosis was validated in patients with AML-NK from the TCGA-LAML cohort. Methylation values below the median at both markers predicted worse overall survival (SWOG: hazard ratio, 1.89 [P<.001]; and TCGA-LAML: hazard ratio, 2.08 [P=.006]). The C-statistic was 0.71 for both cohorts, and the impact was independent of the Fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) status. The 2 methylation markers, measurable by clinically applicable assays such as bisulfite pyrosequencing, are promising for risk stratification among patients with AML-NK. Cancer 2017;123:2472-81. © 2017 American Cancer Society.

Global DNA methylation profiling reveals new insights into epigenetically deregulated protein coding and long noncoding RNAs in CLL

BackgroundMethyl-CpG-binding domain protein enriched genome-wide sequencing (MBD-Seq) is a robust and powerful method for analyzing methylated CpG-rich regions with complete genome-wide coverage. In chronic lymphocytic leukemia (CLL), the role of CpG methylated regions associated with transcribed long noncoding RNAs (lncRNA) and repetitive genomic elements are poorly understood. Based on MBD-Seq, we characterized the global methylation profile of high CpG-rich regions in different CLL prognostic subgroups based on IGHV mutational status.ResultsOur study identified 5800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal controls. From cllDMGs, 40 % of hypermethylated and 60 % of hypomethylated genes were mapped to noncoding RNAs. In addition, we found that the major repetitive elements such as short interspersed elements (SINE) and long interspersed elements (LINE) have a high percentage of cllDMRs (differentially methylated regions) in IGHV subgroups compared to normal controls. Finally, two novel lncRNAs (hypermethylated CRNDE and hypomethylated AC012065.7) were validated in an independent CLL sample cohort (48 samples) compared with 6 normal sorted B cell samples using quantitative pyrosequencing analysis. The methylation levels showed an inverse correlation to gene expression levels analyzed by real-time quantitative PCR. Notably, survival analysis revealed that hypermethylation of CRNDE and hypomethylation of AC012065.7 correlated with an inferior outcome.ConclusionsThus, our comprehensive methylation analysis by MBD-Seq provided novel hyper and hypomethylated long noncoding RNAs, repetitive elements, along with protein coding genes as potential epigenetic-based CLL-signature genes involved in disease pathogenesis and prognosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0274-6) contains supplementary material, which is available to authorized users.

Prognostic Methylation Markers for Survival in Cytogenetically Normal AML Patients Treated on SWOG Trials

Purpose: Although abnormal DNA methylation is not limited to promoter CpG islands, studies to date have focused on identifying prognostic methylation markers in gene promoters. The current study aimed at identifying methylation markers for newly-diagnosed AML with normal cytogenetics (AML-NK) using a genome-wide approach.Patients and Methods: Pretreatment samples were from patients treated on SWOG studies S8600, S9333, S9031, S9500, S9918, S0106, and S0112 from 1986 to 2009. We combined comprehensive high-throughput array-based relative methylation analysis (CHARM) with the Cox proportional hazard model controlling for known AML prognostic factors, to identify 654 survival associated methylation regions (SAMRs) in a phase I discovery cohort (n=72). Top ranking SAMRs were then tested using quantitative bisulfite pyro-sequencing and, if validated, studied further in two additional cohorts: a phase II model building cohort (n=65) and a phase III validation cohort (n=65). In addition, an external cohort from the TCGA AML study (LAML) was used for further validation (n=93).Results: Two SAMRs, located in promoter CpG island shore of LZTS2 and NR6A1, respectively, were identified. Multivariate analyses considering age, performance status, white blood count, and platelet count, showed independent prognostic significance of LZTS2 in the phase II model building cohort (P = 0.0073) and a trend of significance in the phase III validation cohort (P = 0.076), with hypomethylation associated with shorter survival. When FLT3 -ITD status was considered in addition to the above factors, hypomethylation of NR6A1 was shown as an independent marker of shorter survival in both the 2nd and 3rd cohorts (P = 0.02 and 0.03, respectively). Kaplan-Meier analyses of SWOG patients from all three phases further demonstrated that hypomethylation of either LZTS2 or NR6A1 was associated with worse overall survival of AML-NK patients (P < 0.001). The prognosis was verified in AML-NK patients from the TCGA acute myeloid leukemia (LAML) cohort. Multivariate analysis showed that methylation values below median at both markers predicted significantly worse overall survival for both cohorts (SWOG cohort, HR 1.89, P <0.001; TCGA-LAML, HR 2.08, P = 0.006). The impact of the methylation was independent of the FLT3 -ITD status, with C-statistics of 0.71 for both studies. Both LZTS2 and NR6A1 function have been associated with cancer biology. LZTS2 encodes the leucine zipper putative tumor suppressor 2 protein. The over-expression of LZTS2 protein was shown to inhibit cancer cell growth in vitro. The NR6A1 gene encodes a nuclear receptor superfamily member. Increased NR6A1 expression was reported to associate with disease progression and aggressiveness in prostate cancer. The significance of NR6A1 in hematological malignancies has not been described.Conclusion: We identified two DNA methylation markers that are prognostic for overall survival of AML-NK patients. They warrant further validation in prospective clinical trials.This study was supported by HOPE Foundation Development Fund and by NIH/NCI National Clinical Trials Network (NCTN) grants CA180888 and CA180819. [Display omitted] [Display omitted] DisclosuresYan:EpigenDx, Inc.: Employment. Erba:Sunesis;Pfizer; Daiichi Sankyo; Ariad: Consultancy; Millennium/Takeda; Celator; Astellas: Research Funding; Seattle Genetics; Amgen: Consultancy, Research Funding; Novartis; Incyte; Celgene: Consultancy, Patents & Royalties; GlycoMimetics; Janssen: Other: Data Safety & Monitoring Committees. Fang:Affymetrix: Research Funding.

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.

GC-01 * ELUCIDATION OF PATHOGENESIS IN INTRACRANIAL GERM CELL TUMORS BASED ON A GENOME-WIDE METHYLATION STUDY

OBJECTIVE: Although “germ cell theory” that primordial germ cells (PGCs) might be a cell of origin of intracranial germ cell tumors (iGCTs) is now widely accepted, the nature of this heterogeneous tumor remains largely unknown. To elucidate the pathogenesis of iGCT, we are currently conducting a comprehensive methylation analysis. METHODS: Bisulfite-modified DNA of 58 iGCTs from frozen tissues (20 germinomas, and 38 NGGCTs), and 4 mixed-GCTs from FFPE tissues after microdissection (germinoma and the other histological component) were subjected to a genome-wide methylation analysis using HumanMethylation450 BeadChip, which were subclassified by an unsupervised hierarchical clustering. The DNA methylation levels of iGCTs were compared to those of mice PGCs in the published data. In mixed-GCTs after microdissection, mutation status of each component was analyzed by Sanger sequencing. RESULTS: According to the methylation profiles, iGCTs were subclassified into the following 3 groups; global lower methylation (GLM), partial lower methylation (PLM) and higher methylation (HM). Pure germinomas showed a prominent hypomethylation pattern clustered to GLM, which is a unique epigenomic feature distinguishable from NGGCTs. DNA methylation profiles in pure germinoma are similar to those in mice PGCs at E10.5. Mixed-GCTs show different methylation, but common mutation profiles between germinoma and the other histological component. CONCLUSION: We conclude that intracranial germinoma might originate from the migration phase of PGC, but the other subtypes of GCTs might not. We further propose that mutation is earlier event than methylation in the pathogenesis of iGCTs.

Cellular Reprogramming Erases Aberrant DNA Methylation and the Malignant Phenotype in Chronic Myeloid Leukemia

Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome deriving from the genetic translocation t(9;22)(q34;q11.2) that encodes for the BCR-ABL fusion gene. BCR-ABL is a constitutively active tyrosine kinase believed to be the primary genetic event driving CML development (Daley and Baltimore, 1988; Huettner et al., 2000). Tyrosine Kinase Inhibitors (TKI) targeting the BCR-ABL kinase have revolutionized CML therapy, however, they fail to fully eradicate the disease due to the presence of a drug-resistant stem cell pool that sustains continued growth of the malignant cells. Indeed, discontinuation of TKI results in relapse and/or disease progression.It has been shown that the emergence of leukemic clones resistant to TKI and responsible for CML evolution is correlated with aberrant DNA methylation (Machova Polakova et al., 2013). DNA methylation is a key epigenetic signature implicated in regulation of gene expression (Robertson, 2001), that occurs predominantly within CpG dinucleotides. CpG-rich regions (namely CpG islands) are frequently located within promoter regions (~70%) of human protein-coding genes (Illingworth et al., 2010). Methylation of CpG-rich promoters negatively correlates with gene expression levels and it is considered an important regulatory mechanism for long-term gene silencing (Herman and Baylin, 2003). Although numerous studies have established a link between aberrant promoter DNA methylation and cancer (Costello et al., 2000; Feinberg et al., 2006), the impact of DNA methylation in CML is still poorly understood (Yamazaki et al., 2012), particularly due to the lack of the specific animal models.In this study we tested the functional relevance of aberrant methylome in CML development and investigated the possibility that BCR-ABL oncogene triggers DNA methylation changes leading to an aggressive leukemic phenotype. To this end, we have combined cellular reprogramming (Amabile and Meissner, 2009; Carette et al., 2010; Kumano et al., 2012; Miyoshi et al., 2010; Takahashi et al., 2007), due to its ability to erase tissue-specific DNA methylation and to re-establish an embryonic stem-like DNA methylation state (Mikkelsen et al., 2008), with a previously developed BCR-ABL inducible murine model (Koschmieder et al., 2005). Using this approach, we demonstrate that the presence of a single genetic aberration is sufficient to trigger DNA methylation changes and leads to an aggressive leukemic phenotype. We show that by resetting the normal DNA methylation profile of primary human CML cells through cellular reprogramming, we are able to re-establish normal myeloid differentiation, despite the persistence of the native genetic lesion. Finally, by combining a comprehensive genome-scale methylation analysis with cellular reprogramming of leukemic cells, we elucidate the sequential events driving leukemogenesis and reveal the reciprocal interplay of genetic and epigenetic mechanisms during malignant transformation.In conclusion, these results dissect the role of DNA methylation alterations in CML development and warrant an application of demethylating agents such as 5-azacytidine as adjuvant treatment in therapeutic approaches to CML. DisclosuresMartinelli:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau.