Genome-wide Methylation Analysis Research Articles

PATH-52. DIFFERENCES IN GENE EXPRESSION, GENOME-WIDE METHYLATION AND METABOLITES IN ADULT DIFFUSE GLIOMAS

Abstract Metabolic reprogramming is a hallmark of cancer, including diffuse gliomas. IDH1 and IDH2 are key enzymes linking cellular metabolism to epigenetic regulation and redox states. IDH1/IDH2 enzymes catalyze the conversion of isocitrate to α-ketoglutarate, while the mutant enzymes increase the production of D-2-hydroxyglutarate, an oncometabolite that induces epigenetic changes, leading to gene expression alterations. Understanding how epigenetic alterations affect metabolic gene expression and metabolite levels in diffuse gliomas could help identify diagnostic biomarkers and therapeutic targets. We studied the expression of metabolism-related genes, metabolites, and genome-wide methylation status, which will facilitate an integral understanding of the metabolic changes in IDH-mutant and IDH-wildtype gliomas. Study design included 47 fresh frozen tissue samples. RNA was extracted and used for gene expression analysis using the NanoString platform (nCounter® Metabolic Pathways Panel), while extracted DNA was used for targeted sequencing with a panel interrogating 600 genes and for genome-wide methylation analysis using the Illumina Epic v2. In addition, unbiased metabolomic analysis by mass spectrometry was performed in 25 tissue and CSF samples out of 47 samples. Methylation status, gene expression and metabolite levels were compared between IDH-mutant and IDH-wildtype gliomas. Results showed significant differences in DNA methylation, gene expression and metabolites between IDH-mutant and IDH-wildtype gliomas. UPP1, an enzyme involved in uracil metabolism, was studied in depth to understand the interplay between DNA methylation, gene expression, and metabolic reprogramming in diffuse glioma. Epigenetic analysis revealed that the CpG sites within the UPP1 gene displayed reduced methylation in IDH wild-type compared to IDH-mutant tumors. IDH wild-type tumors showed higher UPP1 expression compared to their IDH-mutant counterparts. Consequently, we observed higher levels of uracil in tissue and CSF samples from patients with IDH wild-type gliomas. These data provide a framework to study the relationship between mutations, methylation, gene expression and metabolism in diffuse gliomas.

PATH-11. TECTAL GLIOMA: CLINICAL, PATHOLOGICAL, AND MOLECULAR FEATURES

Abstract Tectal glioma (TG) is a rare lower-grade glioma (LrGG) that occurs in the tectum, mainly affecting children. The clinical course is usually indolent, with common symptoms including headache, gait disturbance, and ataxia resulting from obstructive hydrocephalus. TG shares pathological similarities with pilocytic astrocytoma (PA), but recent genetic analyses have revealed distinct features, such as alterations in KRAS and BRAF. Here, we report the clinical, radiological, pathological, and molecular characteristics of TG cases in our hospital. We conducted a retrospective review of cases clinically diagnosed as TG and surgically treated at our institute between January 2005 and March 2023. Molecular analysis, including genome-wide DNA methylation profiling, was performed. Six cases were identified and the median age at diagnosis was 30.5 years (range: 6–45 years). The mean tumor diameter was 22.8 mm (range: 14.7–33.2 mm), with obstructive hydrocephalus observed in all cases. Contrast enhancement of the tumor was noted in four cases and cyst formation occurred in two cases. Four patients underwent biopsy or biopsy with endoscopic third ventriculostomy, and two patients underwent tumor resection. Postoperatively, three patients received radiotherapy and two were treated with chemotherapy. The median overall survival was 6.2 years (range: 1.4–7.3 years), and the median progression-free survival was 3.4 years (range: 1.4–7.3 years). Histological diagnoses included three cases of PA, one case of astrocytoma, and two cases of high-grade glioma. Among the three patients who underwent molecular evaluation, including genome-wide methylation analysis, two had KRAS mutation and one had H3-3A K27M mutation. Our results demonstrate the diverse histological and molecular characteristics of TG distinct from other LrGGs. Given the heterogeneous pathological background and the risk of pathological progression in TG, we emphasize the importance of comprehensive diagnosis, including molecular evaluation.

NIMG-27. RADIOMETHYLOMICS FOR NON-INVASIVE PREDICTION OF TUMOR PHENOTYPES AND SURVIVAL IN GLIOBLASTOMA

Abstract BACKGROUND DNA methylation profiling has become a pivotal tool in neuro-oncology. However, little is known about the impact of methylation profiling on radiological imaging in IDH-wildtype glioblastoma (GBM). This study explored the potential of radiomics to non-invasively predict molecular characteristics based on the methylation profiling of GBM. METHODS This study included a multi-sampling cohort of 32 patients with 113 samples and a single-sampling cohort of 87 patients with 87 samples, which underwent genome-wide methylation analysis and were classified as GBM by the DKFZ/Heidelberg CNS tumor classifier. We performed two different methylation-based deconvolution analyses. Deconvolution 1 estimated the fractions of tumor subtypes (RTK_I, RTK_II, MES_TYP, and MES_ATYP) and cell types in the microenvironment. Deconvolution 2 inferred the abundance of malignant cell states (stem-like and differentiated cell components) and cell types in the microenvironment. We derived 6084 radiomic features from preoperative multi-parametric MRI in each patient. RESULTS In the multi-sampling cohort, Deconvolution 1 and Deconvolution 2 demonstrated that tumor subclass components and immune components exhibited heterogeneous distribution across samples within patients; however, the stem-like to differentiated cell ratio was preserved across samples within patients. In the entire cohort, patients with tumors exhibiting a high proportion of the stem-like component, defined as stem-like tumors, had significantly shorter overall survival. Stem-like tumors exhibited significantly higher levels of the RTK_I component and lower proportions of the RTK_II component compared to non-stem-like tumors. A machine learning model that utilizes support vector machines to classify stem-like tumors using radiomic features achieved a mean AUC of 0.71 (95% confidence interval: 0.56–0.85) in nested cross-validation. CONCLUSIONS We showed that radiomethylomics can be used to predict tumor composition and patient survival from preoperative MRI in GBM. Based on these promising results we will increase our power with additional samples to facilitate a personalized approach to GBM management.

Genome-wide methylation and transcriptome differential analysis of skeletal muscle in broilers with valgus-varus deformity

ABSTRACT 1. Valgus-varus deformity (VVD) is a disease that severely affects leg function in broilers and for which there is no effective control method current available. Although DNA methylation has an important impact on most physiological and pathological processes, its involvement in skeletal muscle growth and development in VVD broilers is unknown. In this study, genome-wide DNA methylation was analysed in VVD-affected and normal broilers using whole genome resulphite sequencing. 2. The results showed that in the cytosine-phosphoric acid-guanine (CG) sequence environment there was a methylation rate of about 55% and 4,265 differentially methylated regions (DMRs) were found in the CG. Of these, 550 were located in the promoter, 547 in the exon region, and 1,718 in the intron region. 3. All differentially methylated genes (DMGs) were analysed for enrichment of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The GO was enriched in pathways related to protein degradation such as proteasome complex, endopeptidase complex and extracellular region. The KEGG pathways were enriched in signalling pathways related to protein degradation and catabolism such as proteasome, nitrogen metabolism, adherens junction and alanine. 4. Protein interactions analysis revealed that FOS, MYL9, and FRAS1 had a high degree of interactions, in which the DNA methylation level of the MYL9 promoter region was negatively correlated with mRNA expression level. Further studies showed that 5-azacytidine (5-AzaC) inhibited DNMT1 and DNMT3A gene expression and promoted MYL9 expression. 5. This study systematically investigated overall DNA methylation patterns in the leg muscle of VVD and normal broilers. It screened common differential genes in conjunction with transcriptomic data to further identify genes associated with muscle growth and development. This study provides new insights to better understand the pathogenesis of VVD from an epigenetic perspective.

DNA Methylation Signatures Characterize Gene Expression Modulation in Lung Cancer Patients Affected by Anorexia.

The pathophysiology of cancer anorexia is multifactorial and unclear. Transcriptomic analysis from PBMCs RNA showed diverse patterns of gene expression pathways in anorexic cancer patients. We assessed whether the different transcriptomic signatures are modulated by DNA methylation in lung cancer patients presenting with poor appetite. Lung cancer patients and controls were enrolled, and anorexia was assessed by the FAACT-score questionnaire. Genome-wide DNA methylation was determined by Human Infinium MethylationEPIC BeadChip Kit. Data from genome-wide methylation analysis were merged with those from gene expression analysis, previously obtained by RNA sequencing (NGS). Four groups of genes were identified for each comparison: hypermethylated repressed, hypermethylated induced, hypomethylated repressed, and hypomethylated induced. Cancer patients (n = 16) showed 382 differentially methylated genes when compared with controls (n = 8). Anorexic patients (n = 8) presented 586 hypomethylated and 174 hypermethylated genes compared with controls. In anorexic patients vs. non-anorexic (n = 8), 211 genes were identified as hypomethylated and 90 hypermethylated. When microarray methylation data were merged with transcriptomic data by RNA sequencing, we observed significant differences in anorexic patients vs. controls; a total of 42 genes resulted as hypomethylated and induced, 5 hypermethylated repressed, 10 hypermethylated induced, and 15 hypomethylated repressed. The CG sites analyzed by targeted bisulfite NGS in four genes of interest (FLNA, PGRMC1, GNL3L, and FHL1) resulting as hypomethylated in anorexic vs. controls allowed the validation of the data obtained from DNA methylation. Interestingly, the four genes resulted as hypomethylated in anorexic patients vs. non-anorexic patients and vs. controls (p < 0.0001). Our data support that methylation is implicated in cancer-associated anorexia and nutritional derangements among lung cancer patients.

Fetal programming by the parental microbiome of offspring behavior, and DNA methylation and gene expression within the hippocampus.

The microorganisms colonizing the gastrointestinal tract of animals, collectively referred to as the gut microbiome, affect numerous host behaviors dependent on the central nervous system (CNS). Studies comparing germ-free mice to normally colonized mice have demonstrated influences of the microbiome on anxiety-related behaviors, voluntary activity, and gene expression in the CNS. Additionally, there is epidemiologic evidence supporting an intergenerational influence of the maternal microbiome on neurodevelopment of offspring and behavior later in life. There is limited experimental evidence however directly linking the maternal microbiome to long-term neurodevelopmental outcomes, or knowledge regarding mechanisms responsible for such effects. Here we show that that the maternal microbiome has a dominant influence on several offspring phenotypes including anxiety-related behavior, voluntary activity, and body weight. Adverse outcomes in offspring were associated with features of the maternal microbiome including bile salt hydrolase activity gene expression ( bsh ), abundance of certain bile acids, and hepatic expression of Slc10a1 . In cross-foster experiments, offspring resembled their birth dam phenotypically, despite faithful colonization in the postnatal period with the surrogate dam microbiome. Genome-wide methylation analysis of hippocampal DNA identified microbiome- associated differences in methylation of 196 loci in total, 176 of which show conserved profiles between mother and offspring. Further, single-cell transcriptional analysis revealed accompanying differences in expression of several differentially methylated genes within certain hippocampal cell clusters, and vascular expression of genes associated with bile acid transport. Inferred cell-to-cell communication in the hippocampus based on coordinated ligand-receptor expression revealed differences in expression of neuropeptides associated with satiety. Collectively, these data provide proof-of-principle that the maternal gut microbiome has a dominant influence on the neurodevelopment underlying certain offspring behaviors and activities, and selectively affects genome methylation and gene expression in the offspring CNS in conjunction with that neurodevelopment.

A new insight of blood vs. buccal DNA methylation in the forensic identification of monozygotic triplets

The case of the monozygotic (MZ) twin as a suspect demonstrates a practical problem in forensic casework. As the MZ twins are genetically identical, they share the same short tandem repeat (STR) profile. Many studies showed that older MZ twins have significant differences in overall content and genomic distribution of methylation between them. However, studies addressing the investigation of epigenetic MZ triplet differentiation in various forensic reference materials are lacking. Here, one triplet set of Egyptian MZ twins was used as an analog to a forensic case. The genome-wide methylation analysis was performed via the new Human Methylation EPIC BeadChip array. Following normalization methods, potential differentially methylated positions (DMPs) were discovered. This resulted in the detection of 24 potential DMPs in reference-type blood DNA and 11 potential DMPs in reference-type buccal DNA. Then, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to show the associated biological functions. Our findings revealed that the 35 potential DMPs were enriched in 283 significant GO terms. These terms are mainly enriched in the immune system. Overall, this study demonstrates the general feasibility of epigenetic MZ triplet differentiation in the forensic context and highlights that some potential DMPs identified in blood DNA were not informative in buccal DNA. This is due to various reasons, including the tissue specificity of DNA methylation.

EBV infection alters DNA methylation in primary human colon cells: A path to inflammation and carcinogenesis?

Epstein-Barr Virus (EBV) is associated with several types of human cancers, and changes in DNA methylation are reported to contribute to viral-driven carcinogenesis, particularly in cancers of epithelial origin. In a previous study, we demonstrated that EBV infects human primary colonic cells (HCoEpC) and replicates within these cells, leading to pro-inflammatory and pro-tumorigenic effects. Notably, these effects were mostly prevented by inhibiting viral replication with PAA. Interestingly, the EBV-induced effects correlated with the upregulation of DNMT1 and were counteracted by pretreating cells with 5-AZA, suggesting a role for DNA hypermethylation.Building on this background, the current study investigates the methylation changes induced by EBV infection in HCoEpC, both in the presence and absence of PAA, or ERK1/2 and STAT3 inhibitors, pathways known to be activated by EBV and involved in the dysregulation of methylation in tumor cells. The genome-wide methylation analysis conducted in this study allowed us to identify several biological processes and genes affected by these epigenetic changes, providing insights into the possible underlying mechanisms leading to the pathological effects induced by EBV. Specifically, we found that the virus induced significant methylation changes, with hypermethylation being more prevalent than hypomethylation. Several genes involved in embryogenesis, carcinogenesis, and inflammation were affected.

Decoding the Root Lignification Mechanism of Angelica sinensis through Genome-wide Methylation Analysis.

Angelica sinensis is a traditional Chinese herbal medicine with significant economic and medicinal value. However, early bolting and flowering can occur during the second year of the vegetative growth period, rendering the roots unviable for medicinal use and resulting in substantial economic losses. Consequently, the growing interest in studying the molecular mechanisms underlying early bolting or increased root lignification in A. sinensis. Here, we conducted whole-genome bisulfite sequencing and observed an increase in whole-genome DNA methylation levels on chromosomes after bolting in A. sinensis. Comparative analysis revealed methylation patterns in the upstream, gene body, and downstream regions in the context of CG, CHG, and CHH, suggesting a possible association between CHH-type methylation of promoters and phenylpropanoid biosynthesis. Furthermore, joint analysis of transcriptomic and methylomics data revealed a positive correlation between DNA methylation and gene expression. We identified the hyperDMR gene in the CHH background within the promoter region; this gene is also a key gene (AsCOMT1), exhibiting dual catalytic activity and facilitating the synthesis of both ferulic acid and lignin. Enzyme kinetic analysis demonstrated that AsCOMT1 preferentially catalyzes the synthesis of lignin monomer precursors. These findings highlight the important regulatory role of DNA methylation in bolting and the synthesis of secondary metabolites in A. sinensis, providing valuable insights into the underlying molecular mechanisms. Therefore, as DNA methylation plays an important regulatory role in A. sinensis bolting and secondary metabolite synthesis, it has potential significance in the analysis of the underlying molecular mechanism.

DNA methylation changes in the genome of patients with hypogonadotropic hypogonadism

Although some Mendelian neurodevelopmental disorders have been shown to entail specific DNA methylation changes designated as epi-signatures, it remains unknown whether epi-signatures are consistent features of other genetic disorders. Here, we analyzed DNA methylation profiles of patients with hypogonadotropic hypogonadism (HH), a rare neuroendocrine disorder typically caused by monogenic or oligogenic mutations. First, we performed microarray-based genome-wide methylation analyses of nine patients with HH due to ANOS1, SOX2, or SOX10 variants and 12 control individuals. The results showed that 1118 probes were differentially methylated in one or more patients. The differentially methylated probes were highly variable among patients. No significant methylation changes were observed in genes functionally associated with ANOS1, SOX2, or SOX10. Then, we performed pyrosequencing of six selected CpG sites in the nine patients and 35 additional HH patients. The results of the patients were compared with those of 48 fertile men. There were no common methylation changes among these patients, with the exception of hypermethylation of two CpG sites in the ZNF245 promoter of three patients. Hypermethylation of the promoter has previously been reported as a very rare epigenetic polymorphism in the general population. These results indicate that genomes of HH patients have considerable DNA methylation changes; however, these changes are more likely to be physiological epigenetic variations than disease-specific epi-signatures. Our data suggest a possible association between hypermethylation of the ZNF254 promoter and HH, which needs to be examined in future studies.

A genome-wide methylation analysis of Chinese Han patients with chronic insomnia disorder.

As the most common sleep disorder, chronic insomnia disorder (CID) has become a global health burden to the public. However, it remains unclear about the pathogenesis of this disease. Epigenetic changes may provide important insights into the gene-environment interaction in CID. Therefore, this study was conducted to investigate the DNA methylation pattern in CID and reveal the epigenetic mechanism of this disease. In this study, whole blood DNA was extracted from 8 CID patients (the CID group) and 8 healthy controls (the control group), respectively. Besides, genome-wide DNA methylation was detected by Illumina Human Methylation 850K Beadchip. Moreover, the sleep quality and insomnia severity were evaluated by the Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), respectively. A total of 369 differentially methylated positions (DMPs) and 23 differentially methylated regions (DMRs) were identified between the CID and control groups. LHX6 was identified as the most important differentially methylated gene (DMG). The Gene Ontology (GO) analysis results corroborated that DMPs were significantly enriched in 105 GO terms, including cell signaling, homogenous cell adhesion of plasma membrane adhesion molecules, nervous system development, cell adhesion, and calcium ion binding. In addition, it was demonstrated that DMPs were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including the hippo signaling pathway, Ras signaling pathway, and vitamin B6 metabolism. The DMR-related GO analysis results revealed the positive regulation of protein kinase activities. DNA methylation plays a critical role in the development of CID, and LHX6 is validated to be an important DMG.

The effects of air and transportation noise pollution-related altered blood gene expression, DNA methylation, and protein abundance levels on gastrointestinal diseases risk

IntroductionAir pollution and transportation noise pollution has been linked to gastrointestinal (GI) diseases, but their relationship remains unclear. MethodsWe extracted the significantly modulated genes and CpG sites related to air pollution (PM2.5, PM10, and NOx) and transportation noise pollution (aircraft, railway, and traffic road noise) from previous published studies. Genome-wide methylation analysis and colocalization analysis with these CpG sites and GWAS data of GI diseases were performed to disentangle the relationship between pollution-related blood DNA methylation (DNAm) alterations and GI diseases risk. Summary-based Mendelian randomization (SMR) analysis assessed the impact of pollution-related genes on GI diseases risk across methylation, gene expression, and protein levels. Enrichment analysis investigated the implicated biological pathways and immune cell types. ResultsDNAm at cg00227781 [CD300A] (modulated by NOx exposure) and cg19215199 [ZMIZ1] (modulated by PM2.5 exposure) was significantly linked to increased noninfective enteritis and colitis risk, while cg08500171 [BAT2] (modulated by NOx exposure) is significantly associated with an increased gastroesophageal reflux disease (GERD) risk. Colocalization analysis provides strong evidence supporting a shared causal variant between these associations. Multi-omics levels SMR analysis revealed that pollution-modulated lower DNAm at 5 specific CpG sites were associated with increased expression of 4 genes (IL21R, EVPL, SYNGR1, and WDR46), subsequently increasing the risk of GERD, ulcerative colitis, and gastric ulcer. 7 circulating proteins coded by pollution-modulated genes were observed to be associated with 6 GI diseases risk. Enrichment analysis implicates immune and inflammatory responses, MAPK signaling, and telomere maintenance in these pollution-induced effects. ConclusionWe identified potential links between air and transportation noise pollution-related gene methylation, expression, and protein abundance with GI diseases risk, possibly revealing new therapeutic targets.

Genome-wide methylation and gene-expression analyses in thalassemia.

Thalassemia is the most common autosomal genetic disorder in humans. The pathogenesis of thalassemia is principally due to the deletion or mutation of globin genes that then leads to disorders in globin-chain synthesis, and its predominant clinical manifestations include chronic forms of hemolytic anemia. However, research on the epigenetics and underlying pathogenesis of thalassemia is in its nascency and not yet been systematically realized. In this study, we compared the results of RNA-seq and the whole-genome bisulfite sequencing (WGBS) on 22 peripheral blood samples from 14 thalassemic patients and eight healthy individuals revealed a genome-wide methylation landscape of differentially methylated regions (DMRs). And functional-enrichment analysis revealed the enriched biological pathways with respect to the differentially expressed genes (DEGs) and differentially methylated genes (DMGs) to include hematopoietic lineage, glucose metabolism, and ribosome. To further analyze the interaction between the transcriptome and methylome, we implemented a comprehensive analysis of overlaps between DEGs and DMGs, and observed that biological processes significantly enriched the immune-related genes (i.e., our hypermethylated and down-regulated gene group). Hypermethylated and hypomethylated regions of thalassemia-related genes exhibited different distribution patterns. We thus, further identified and validated thalassemia-associated DMGs and DEGs by multi-omics integrative analyses of DNA methylation and transcriptomics data, and provided a comprehensive genomic map of thalassemia that will facilitate the exploration of the epigenetics mechanisms and pathogenesis underlying thalassemia.

The missing link: ARID1B non-truncating variants causing Coffin-Siris syndrome due to protein aggregation

ARID1B is the most frequently mutated gene in Coffin-Siris syndrome (CSS). To date, the vast majority of causative variants reported in ARID1B are truncating, leading to nonsense-mediated mRNA decay. In the absence of experimental data, only few ARID1B amino acid substitutions have been classified as pathogenic, mainly based on clinical data and their de novo occurrence, while most others are currently interpreted as variants of unknown significance. The present study substantiates the pathogenesis of ARID1B non-truncating/NMD-escaping variants located in the SMARCA4-interacting EHD2 and DNA-binding ARID domains. Overexpression assays in cell lines revealed that the majority of EHD2 variants lead to protein misfolding and formation of cytoplasmic aggresomes surrounded by vimentin cage-like structures and co-localizing with the microtubule organisation center. ARID domain variants exhibited not only aggresomes, but also nuclear aggregates, demonstrating robust pathological effects. Protein levels were not compromised, as shown by quantitative western blot analysis. In silico structural analysis predicted the exposure of amylogenic segments in both domains due to the nearby variants, likely causing this aggregation. Genome-wide transcriptome and methylation analysis in affected individuals revealed expression and methylome patterns consistent with those of the pathogenic haploinsufficiency ARID1B alterations in CSS cases. These results further support pathogenicity and indicate two approaches for disambiguation of such variants in everyday practice. The few affected individuals harbouring EHD2 non-truncating variants described to date exhibit mild CSS clinical traits. In summary, this study paves the way for the re-evaluation of previously unclear ARID1B non-truncating variants and opens a new era in CSS genetic diagnosis.

ETMR-11. RECURRENT GENETIC ALTERATIONS IN EPIGENETICALLY DEFINED PINEOBLASTOMA SUBTYPES

Abstract INTRODUCTION Genome-wide methylation analyses recently revealed distinct epigenetic pineoblastoma (PBL) subtypes. The aim of the study was to shed further light onto the genetic characteristics underlying the pathogenesis of pineoblastoma subtypes. METHODS Cytogenetic alterations of tumor samples of 76 patients with a diagnosis of PBL confirmed by reference neuropathology and methylation-based subtyping were analyzed by high-resolution genome-wide molecular inversion probe analysis. Seventy-two cases were screened for mutations by next-generation DNA panel sequencing. Survival data of 49 patients were available. Patients ≥4y received postoperative RT followed by maintenance chemotherapy (n=42); infants (&amp;lt;4y) were primarily treated on RT-sparing regimens with intensified chemotherapy. RESULTS According to epigenetic consensus PBL subtypes (Liu et al., 2021), our cohort consisted of 48 PBL-miRNA1 (1A=40; 1B=8), 17 PBL-miRNA2, 4 PBL-MYC/FOXR2, and 7 PBL-RB1 samples. PBL-miRNA subtype tumors had characteristic alterations in microRNA-processing genes; DICER1 mutations (n=18) and homozygous deletions of the DROSHA locus (n=17) were most abundant, followed by DROSHA mutations (n=12). Most frequent cytogenetic aberrations in PBL-miRNA were whole chromosome (chr.) 7 gain (n=30) and chr. 14 loss (n=21). DICER1 mutations were significantly associated with chr. 14 loss (p&amp;lt;0.001). Twenty-one cases showed gains of chr. 14, 8 additional cases gains of the OTX2 oncogene (chr. 14q). In the miRNA subtypes we identified cases with polyploid cytogenetics (n=15). Of note, only one of the 8 patients with polyploid tumors and survival data died, while the remaining patients of the miRNA subtypes had a 5-year PFS/OS of 61.46±9.27/63.24±9.23% (14 of 35 died) after a median follow-up time of 5.52 years. However, this comparison did not reach statistical significance (p=0.25). CONCLUSION The epigenetically defined PBL-miRNA subtypes are characterized by distinct cytogenetic and mutational events. A possible prognostic role for a superior (e.g. polyploidy) or inferior outcome requires investigation in prospective clinical trials.

Genome-wide DNA methylation in relation to ARID1A deficiency in ovarian clear cell carcinoma

BackgroundThe poor chemo-response and high DNA methylation of ovarian clear cell carcinoma (OCCC) have attracted extensive attentions. Recently, we revealed the mutational landscape of the human kinome and additional cancer-related genes and found deleterious mutations in ARID1A, a component of the SWI/SNF chromatin-remodeling complex, in 46% of OCCC patients. The present study aims to comprehensively investigate whether ARID1A loss and genome-wide DNA methylation are co-regulated in OCCC and identify putative therapeutic targets epigenetically regulated by ARID1A.MethodsDNA methylation of ARID1Amt/ko and ARID1Awt OCCC tumors and cell lines were analyzed by Infinium MethylationEPIC BeadChip. The clustering of OCCC tumors in relation to clinical and mutational status of tumors were analyzed by hierarchical clustering analysis of genome-wide methylation. GEO expression profiles were used to identify differentially methylated (DM) genes and their expression level in ARID1Amt/ko vs ARID1Awt OCCCs. Combining three pre-ranked GSEAs, pathways and leading-edge genes epigenetically regulated by ARID1A were revealed. The leading-edge genes that passed the in-silico validation and showed consistent ARID1A-related methylation change in tumors and cell lines were regarded as candidate genes and finally verified by bisulfite sequencing and RT-qPCR.ResultsHierarchical clustering analysis of genome-wide methylation showed two clusters of OCCC tumors. Tumor stage, ARID1A/PIK3CA mutations and TP53 mutations were significantly different between the two clusters. ARID1A mutations in OCCC did not cause global DNA methylation changes but were related to DM promoter or gene-body CpG islands of 2004 genes. Three pre-ranked GSEAs collectively revealed the significant enrichment of EZH2- and H3K27me3-related gene-sets by the ARID1A-related DM genes. 13 Leading-edge DM genes extracted from the enriched gene-sets passed the expression-based in-silico validation and showed consistent ARID1A-related methylation change in tumors and cell lines. Bisulfite sequencing and RT-qPCR analysis showed promoter hypermethylation and lower expression of IRX1, TMEM101 and TRIP6 in ARID1Amt compared to ARID1Awt OCCC cells, which was reversed by 5-aza-2′-deoxycytidine treatment.ConclusionsOur study shows that ARID1A loss is related to the differential methylation of a number of genes in OCCC. ARID1A-dependent DM genes have been identified as key genes of many cancer-related pathways that may provide new candidates for OCCC targeted treatment.

Epigenetics in etiopathology of hyperprolactinemia

Aim: Epigenetic alterations have been reported in patients with pituitary tumors and those on antipsychotic drugs, which are also responsible for hyperprolactinemia. This suggests a possible role of epigenetics in the etiopathology of hyperprolactinemia. Methods: The study recruited 83 hyperprolactinemia cases with prolactin &gt; 100 ng/mL and 65 controls. Global DNA methylation status was studied by MethylFlash Methylated DNA Quantification Kit and genome-wide methylation analysis (GWMA) by Infinium Methylation EPIC BeadChip 850K array. Results: Hyperprolactinemia cases showed significant global DNA hypermethylation compared to controls. Around 66.67% of hypomethylated and 12.9% of hypermethylated cases were on antipsychotics. Gene enrichment analysis of 5-cytosine-phosphate-guanine-3 (CpG) site-associated genes demonstrated significantly enriched major histocompatibility complex (MHC)-related protein classes and cellular components. Conclusions: The study suggested the role of epigenetics in the etiopathology of hyperprolactinemia.

Genome-Wide Methylation Analysis in Two Wild-Type Non-Small Cell Lung Cancer Subgroups with Negative and High PD-L1 Expression.

We conducted a pilot study to analyze the differential methylation status of 20 primary acinar adenocarcinomas of the lungs. These adenocarcinomas had to be wild type in mutation analysis and had either high (TPS > 50%; n = 10) or negative (TPS < 1%; n = 10) PD-L1 status to be integrated into our study. To examine the methylation of 866,895 specific sites, we utilized the Illumina Infinium EPIC bead chip array. Both hypermethylation and hypomethylation play significant roles in tumor development, progression, and metastasis. They also impact the formation of the tumor microenvironment, which plays a decisive role in tumor differentiation, epigenetics, dissemination, and immune evasion. The gained methylation patterns were correlated with PD-L1 expression. Our analysis has identified distinct methylation patterns in lung adenocarcinomas with high and negative PD-L1 expression. After analyzing the correlation between the methylation results of genes and promoters with their pathobiology, we found that tumors with high expression of PD-L1 tend to exhibit oncogenic effects through hypermethylation. On the other hand, tumors with negative PD-L1 expression show loss of their suppressor functions through hypomethylation. The suppressor functions of hypermethylated genes and promoters are ineffective compared to simultaneously activated dominant oncogenic mechanisms. The tumor microenvironment supports tumor growth in both groups.

Abstract 3454: Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk

Abstract Cell-free DNA (cfDNA) epigenetic and fragmentomic profiling has emerged as prominent non-invasive approaches for early cancer detection and subtyping. However, owing to difficulties in observing the early development of human malignancies, most cancer biomarker and evolution studies to date have primarily examined biologics following a diagnosis. Utilizing cfDNA as a screening tool for early disease detection requires assessment of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers. Here, we leverage the Ontario Health Study (OHS), a prospective longitudinal population cohort, to assess cfDNA profiles in blood plasma collected from participants that developed breast (n=476), prostate (n=342) and pancreatic (n=32) cancers throughout the study follow up time between 2009 and 2019. We performed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) on baseline plasma samples from incident cancer cases up to eight years prior to diagnosis, in addition to matched controls (n=404) with no history of cancer through follow-up. Genome-wide plasma cfDNA differential methylation analysis revealed significantly hypermethylated regions, particularly among CpG island and shore regions, were predictive of early breast cancers, and overlapped hypermethylated regions in solid cancer tissues relative to adjacent normal tissues and peripheral blood leukocytes. Using a repeated cross-validation strategy we found that as few as 200 hypermethylated regions can identify individuals with breast cancer in blood up to seven years prior to diagnosis among discovery set samples (AUC=0.725) and are highly generalizable to samples processed in separate batches (AUC = 0.650). Remarkably, integrating biomarker hypermethylated regions with fragmentomic features including fragment length and 5’ tetranucleotide motif proportions further increased accuracy for detecting individuals with early prostate cancers (AUC=0.804). In addition, we observed shared genome-wide cfDNA methylome changes associated with aging and heavy alcohol consumption in cfDNA. We identified increased shedding of tissue-specific methylation markers from liver and pancreatic tissue among individuals consuming more than three drinks a week relative to non-drinkers, reflective of potential signatures of tissue damage. Citation Format: Nicholas Cheng, Kimberly Skead, Tom Oullette, Scott Bratman, Daniel De Carvalho, David Soave, Philip Awadalla. Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3454.

Abstract 3496: EpigenPlot: A tool for the gene-level methylation analysis of colorectal tumors

Abstract Introduction. Aberrant DNA methylation patterns are frequent in colorectal adenocarcinoma (CRC) tissues. The investigation of this layer of epigenetic regulation holds the promise of a better understanding of tumour progression and the identification of novel biomarker candidates. Purpose. Our goal was to collect and analyse publicly available DNA methylation data of colorectal samples and create a user-friendly tool for exploring and visualizing the CRC methylome. Methods. We used Illumina Infinium Methylation Arrays, which is a popular platform for genome-wide methylation analysis of large cohorts. Gene Expression Omnibus database (GEO) and TCGA-COAD (The Cancer Genome Atlas Colon Adenocarcinoma) datasets including normal colorectal mucosa, adenoma, and adenocarcinoma samples were systematically collected and filtered. Differentially methylated regions were identified using the Kruskal-Wallis test. Genes with significant methylation changes were further analysed using ROC (Receiver Operator Characteristic) analysis. Finally, we established a web application using the shiny R package. Results. We assembled a database including methylation data of 2,586 samples. Adenoma and adenocarcinoma samples were globally hypomethylated. Hypermethylation (p &amp;lt; 0.05, Δβ &amp;gt;= 0.2) in adenocarcinoma samples was predominantly present in regions near proximal promoters. Out of the 74 previously proposed biomarkers examined, 50 had a cross-validated AUC of over 0.8. FDA-approved biomarkers BMP3, NDRG4, and SEPT9 have shown the highest performance in the TSS200 region (cvAUC = 0.8, 0.83, and 0.76, respectively). Genes with the highest performance were ITGA4 (5’UTR, first exon: cvAUC = 0.9), MDFI (5’UTR: cvAUC = 0.9), CNRIP1 (5’UTR, first exon: cvAUC = 0.89). The established web application can visualize methylation at CpG sites and gene regions, and KEGG pathway genes. The platform is available at https://epigenplot.com. Conclusion. We collected and analysed a sizeable database of colorectal tissue data. Gene regions with the best performance include previously proposed and novel diagnostic biomarker candidates. The web platform provides an tool for the efficient exploration of the assembled data. Citation Format: Dalma Müller, Balazs Gyorffy. EpigenPlot: A tool for the gene-level methylation analysis of colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3496.