Infinium 450K Research Articles

Screening and identifying of biomarkers in early colorectal cancer and adenoma based on genome-wide methylation profiles

BackgroundColorectal cancer is one of the most common malignant tumors worldwide with high morbidity and mortality. This study aimed to identify different methylation sites as new methylation markers in CRC and colorectal adenoma through tissue detection.MethodsDNA extraction and bisulfite modification as well as Infinium 450K methylation microarray detection were performed in 46 samples of sporadic colorectal cancer tissue, nine samples of colorectal adenoma, and 20 normal samples, and bioinformatic analysis was conducted involving genes enrichments of GO and KEGG. Pyrosequencing methylation detection was further performed in 68 sporadic colorectal cancer tissues, 31 samples of colorectal adenoma, and 49 normal colorectal mucosae adjacent to carcinoma to investigate the differentially methylated genes obtained from methylation microarray.ResultsThere were 65,535 differential methylation marker probes, among which 25,464 were hypermethylated markers and 40,071 were hypomethylated markers in the adenoma compared with the normal group, and 395,571 were differentially methylated markers in patients with sporadic colorectal cancer compared with the normal group, including 21,710 hypermethylated markers and 17,861 hypomethylated markers. Five hypermethylated genes including ZNF471, SND1, SPOCK1, FBLIM1, and OTX1 were detected and confirmed in 68 cases of colorectal cancer, 31 cases of adenoma, and 49 cases of normal control group.ConclusionsHypermethylated genes of ZNF471, SND1, SPOCK1, FBLIM1, and OTX1 were obtained from methylation chip detection and further confirm analysis in colorectal cancer and adenoma compared with normal tissue, which may be promising diagnostic markers of colorectal cancer and colorectal adenoma.

Genome-Wide DNA Methylation Profiles in Whole-Blood and Buccal Samples-Cross-Sectional, Longitudinal, and across Platforms.

The field of DNA methylation research is rapidly evolving, focusing on disease and phenotype changes over time using methylation measurements from diverse tissue sources and multiple array platforms. Consequently, identifying the extent of longitudinal, inter-tissue, and inter-platform variation in DNA methylation is crucial for future advancement. DNA methylation was measured in 375 individuals, with 197 of those having 2 blood sample measurements ~10 years apart. Whole-blood samples were measured on Illumina Infinium 450K and EPIC methylation arrays, and buccal samples from a subset of 58 participants were measured on EPIC array. The data were analyzed with the aims to examine the correlation between methylation levels in longitudinal blood samples in 197 individuals, examine the correlation between methylation levels in the blood and buccal samples in 58 individuals, and examine the correlation between blood methylation profiles assessed on the EPIC and 450K arrays in 83 individuals. We identified 136,833, 7674, and 96,891 CpGs significantly and strongly correlated (>0.50) longitudinally, across blood and buccal samples as well as array platforms, respectively. A total of 3674 of these CpGs were shared across all three sets. Analysis of these shared CpGs identified previously found associations with aging, ancestry, and 7016 mQTLs as well.

Epigenetic Regulation of Inflammatory Mechanisms and a Psychological Symptom Cluster in Patients Receiving Chemotherapy.

A psychological symptom cluster is the most common cluster identified in oncology patients. Although inflammatory mechanisms are hypothesized to underlie this cluster, epigenetic contributions are unknown. This study's purpose was to evaluate associations between the occurrence of a psychological symptom cluster and levels of DNA methylation for inflammatory genes in a heterogeneous sample of patients with cancer receiving chemotherapy. Prior to their second or third cycle of chemotherapy, 1,071 patients reported the occurrence of 38 symptoms using the Memorial Symptom Assessment Scale. A psychological cluster was identified using exploratory factor analysis. Differential methylation analyses were performed in two independent samples using Illumina Infinium 450K and EPIC microarrays. Expression-associated CpG (eCpG) loci in the promoter region of 114 inflammatory genes on the 450K and 112 genes on the EPIC microarray were evaluated for associations with the psychological cluster. Robust rank aggregation was used to identify differentially methylated genes across both samples. Significance was assessed using a false discovery rate of 0.05 under the Benjamini-Hochberg procedure. Cluster of differentiation 40 ( CD40 ) was differentially methylated across both samples. All six promoter eCpGs for CD40 that were identified across both samples were hypomethylated in the psychological cluster group. This study is the first to suggest associations between a psychological symptom cluster and differential DNA methylation of a gene involved in tissue inflammation and cell-mediated immunity. Our findings suggest that increased CD40 expression through hypomethylation of promoter eCpG loci is involved in the occurrence of a psychological symptom cluster in patients receiving chemotherapy. These findings suggest a direction for mechanistic studies.

Prenatal maternal anxiety promotes atopic dermatitis in offspring via placental DNA methylation changes.

Maternal anxiety during pregnancy has been previously reported to be associated with atopic dermatitis (AD) in offspring. The potential mechanism is not yet proven but epigenetic change may be suggested. We examined whether maternal anxiety during pregnancy may alter placental DNA methylation, then develop AD in the offspring. We evaluated maternal anxiety at 36 weeks of gestation by self-reported questionnaires, the State-Trait Anxiety Inventory-Trait subscale (STAI-T), in the Cohort for Childhood Origin of Asthma and Allergic Diseases (COCOA) study. AD was diagnosed at 6 months of age by pediatric allergists. We stratified the subjects into four groups according to the STAI score of mothers and diagnosis of AD in children. Placental genome-wide methylation microarray was analyzed using Infinium 450K BeadChip and selected genes were validated by pyrosequencing. From microarray, several differential methylation sites were identified in AD and healthy subjects and in total subjects, regarding to the STAI scores. Among differential methylation sites in microarray, six sites were selected for pyrosequencing. And site of matrix metalloproteinases 27 (MMP27) among 6 sites showed decreased methylation in AD infants with high STAI mothers compared to healthy infants with low STAI mothers. Epigenetic change in placenta can be a suggesting mechanism for the development of AD in offspring at 6 months of age associated with maternal anxiety during pregnancy and MMP27 may be a candidate gene.

What Happens In Utero Does Not Stay In Utero: a Review of Evidence for Prenatal Epigenetic Programming by Per- and Polyfluoroalkyl Substances (PFAS) in Infants, Children, and Adolescents.

Review human literature on the relationship between prenatal exposure to per- and polyfluoroalkyl substances (PFAS) and epigenetic modifications in infants, children, and adolescents < 18years of age. Eleven studies were identified, with study populations located in the U.S., Taiwan, Japan, and the Kingdom of Denmark. Many studies (n = 5) were cross-sectional, with PFAS exposure and epigenetic outcomes measured in the same tissue collected at delivery via cord blood or dried newborn blood spots. The other six studies were prospective, with prenatal PFAS measured on maternal blood during pregnancy and DNA methylation (DNAm) assessed in cord blood and childhood peripheral leukocytes (n = 1 study). Epigenetic marks of interest included global DNAm measures (LINE-1, Alu, and an ELISA-based method), candidate genes (IFG2, H19, and MEST), and epigenome-wide DNA methylation via array-based methods (Infinium 450K and EPIC). Two studies using array-based methods employed discovery and validation paradigms, in which a small subset of loci (n = 6 and n = 4) were replicated in the discovery population. One site (TNXB) was a hit in two independent studies. Collectively, loci associated with PFAS were in regions involved in growth and development, lipid metabolism, and nutrient metabolism. There is moderate human evidence supporting associations of prenatal PFAS exposure on DNAm at birth, with one study suggesting sustained effects into childhood. Future studies are warranted to link PFAS-associated DNAm to health outcomes, as well as to investigate the role of other epigenetic marks such as hydroxymethylation, miRNA expression, and histone modifications.

Adenocarcinoma of the stomach and esophagogastric junction with low DNA methylation show poor prognoses.

Gastric cancer (GC) is characterized by unique DNA methylation epigenotypes (MEs). However, MEs including adenocarcinomas of the esophagogastric junction (AEG) and background non-neoplastic columnar mucosae (NM) remain to be clarified. We analyzed the genome-wide DNA MEs of AEG, GC, and background NM using the Infinium 450k beadarray, followed by quantitative pyrosequencing validation. Large-scale data from The Cancer Genome Atlas (TCGA) were also reviewed. Unsupervised two-way hierarchical clustering using Infinium data of 21 AEG, 30 GC, and 11 NM revealed four DNA MEs: extremely high-ME (E-HME), high-ME (HME), low-ME (LME), and extremely low-ME (E-LME). Promoter methylation levels were validated by pyrosequencing in 146 samples. Non-inflammatory normal mucosae were clustered into E-LME, whereas gastric or esophagogastric junction mucosae with chronic inflammatory changes caused by either Helicobacter pylori infection or reflux esophagitis were clustered together into LME, suggesting that inflammation status determined DNA MEs regardless of the cause. Three cases of Barrett's-related adenocarcinoma were clustered into HME. Among 94 patients whose tumors could be clustered into one of four MEs, 11 patients with E-LME cancers showed significantly shorter overall survival than that in the other MEs, even with the multivariate Cox regression estimate. TCGA data also showed enrichment of AEG in HME and a poorer prognosis in E-LME. E-LME cases, newly confirmed in this study, form a unique subtype with poor prognosis that is not associated with inflammation-associated elevation of DNA methylation levels. LME could be acquired via chronic inflammation, regardless of the cause, and AEG might preferentially show HME.

Aclust2.0: a revamped unsupervised R tool for Infinium methylation beadchips data analyses.

A wide range of computational packages has been developed for regional DNA methylation analyses of Illumina's Infinium array data. Aclust, one of the first unsupervised algorithms, was originally designed to analyze regional methylation of Infinium's 27K and 450K arrays by clustering neighboring methylation sites prior to downstream analyses. However, Aclust relied on outdated packages that rendered it largely non-operational especially with the newer Infinium EPIC and mouse arrays. We have created Aclust2.0, a streamlined pipeline that involves five steps for the analyses of human (450K and EPIC) and mouse array data. Aclust2.0 provides a user-friendly pipeline and versatile for regional DNA methylation analyses for molecular epidemiological and mouse studies. Aclust2.0 is freely available on Github (https://github.com/OluwayioseOA/Alcust2.0.git).

DNA Methylation and Ischemic Stroke Risk: An Epigenome-Wide Association Study.

Ischemic stroke (IS) risk heritability is partly explained by genetics. Other heritable factors, such as epigenetics, could explain an unknown proportion of the IS risk. The objective of this study is to evaluate DNA methylation association with IS using epigenome-wide association studies (EWAS). We performed a two-stage EWAS comprising 1,156 subjects. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) were assessed using the Infinium 450K and EPIC BeadChip in the discovery cohort (252 IS and 43 controls). Significant DMPs were replicated in an independent cohort (618 IS and 243 controls). Stroke subtype associations were also evaluated. Differentially methylated cell-type (DMCT) was analyzed in the replicated CpG sites using EpiDISH. We additionally performed pathway enrichment analysis and causality analysis with Mendelian randomization for the replicated CpG sites. A total of 957 CpG sites were epigenome-wide-significant (p ≤ 10-7) in the discovery cohort, being CpG sites in the top signals (logFC = 0.058, p = 2.35 × 10-22; logFC = 0.035, p = 3.22 × 10-22, respectively). ZFHX3 and MAP3K1 were among the most significant DMRs. In addition, 697 CpG sites were replicated considering Bonferroni-corrected p-values (p < 5.22 × 10-5). All the replicated DMPs were associated with risk of cardioembolic, atherothrombotic, and undetermined stroke. The DMCT analysis demonstrated that the significant associations were driven by natural killer cells. The pathway enrichment analysis showed overrepresentation of genes belonging to certain pathways including oxidative stress. ZFHX3 and MAP3K1 methylation was causally associated with specific stroke-subtype risk. Specific DNA methylation pattern is causally associated with IS risk. These results could be useful for specifically predicting stroke occurrence and could potentially be evaluated as therapeutic targets.

Circulating tumor cell methylation profiles reveal the classification and evolution of non-small cell lung cancer.

BackgroundThe ability of circulating tumor cells (CTCs) to identify lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) could improve pathological diagnosis and the selection of treatments for non-small cell lung cancer (NSCLC). Previous studies have shown that deoxyribonucleic acid (DNA) methylation exhibits cell and tissue specificity. Thus, we aimed to explore the methylation status of CTCs in LUAD and LUSC and identify the potential biomarkers.MethodsWe first analyzed Infinium 450K methylation profiles obtained from The Cancer Genome Atlas and Gene Expression Omnibus. We then performed whole-genome sequencing of CTCs in tumor and matched normal lung tissues and white blood cells from 6 NSCLC patients.ResultsThe bioinformatics analysis revealed a NSCLC-specific DNA methylation marker panel, which could accurately distinguish between LUAD and LUSC with high diagnostic accuracy. The whole-genome sequencing of CTCs in NSCLC patients also showed 100% accuracy for distinguishing between LUAD and LUSC based on the CTC methylation profiles. To investigate the function of CTCs, we further analyzed similar and different methylation profiles between the CTCs and their primary tumors, and found very high similarities between the CTCs and their primary tumor tissues, indicating that these cells inherit information from primary tumors. However, the CTCs also displayed some characteristics that differed to those of primary tumor tissues, which suggest that CTCs acquire some unique characteristics after migrating from the primary tumor; these characteristics may partly explain the ability of tumor cells to evade immune surveillance.ConclusionsOur findings provide insights into the potential use of CTCs in the pathological classification of NSCLC patients. Our findings also show how CTC primary tumor inheritance and CTC evolution affect metastasis and immune escape.

Epigenetic Age Acceleration and Change in Frailty in MOBILIZE Boston.

Age-associated changes in DNA methylation have been implicated as 1 mechanism to explain the development of frailty; however, previous cross-sectional studies of epigenetic age acceleration (eAA) and frailty have had inconsistent findings. Few longitudinal studies have considered the association of eAA with change in frailty. We sought to determine the association between eAA and change in frailty in the MOBILIZE Boston cohort. Participants were assessed at 2 visits 12-18 months apart. Intrinsic, extrinsic, GrimAge, and PhenoAge eAA were assessed from whole-blood DNA methylation at baseline using the Infinium 450k array. Frailty was assessed by a continuous frailty score based on the frailty phenotype and by frailty index (FI). Analysis was by correlation and linear regression with adjustment for age, sex, smoking status, and body mass index. Three hundred and ninety-five participants with a frailty score and 431 with an FI had epigenetic and follow-up frailty measures. Mean (standard deviation) ages were 77.8 (5.49) and 77.9 (5.47) for the frailty score and the FI cohorts respectively, and 232 (58.7%) and 257 (59.6%) were female. All participants with epigenetic data identified as White. Baseline frailty score was not correlated with intrinsic or extrinsic eAA, but was correlated with PhenoAge and, even after adjustment for covariates, GrimAge. Baseline FI was correlated with extrinsic, GrimAge, and PhenoAge eAA with and without adjustment. No eAA measure was associated with change in frailty, with or without adjustment. Our results suggest that no eAA measure was associated with change in frailty. Further studies should consider longer periods of follow-up and repeated eAA measurement.

Multi-Omics of Adult T Cell Acute Lymphoblastic Leukemia

Introduction:Risk stratification and minimal residual disease (MRD) monitoring improved outcome for adult T-ALL patients, but a relevant percentage of adult patients, particularly high-risk subtypes, still relapse with then very limited therapeutic options. For improved individualized treatment strategies, better understanding of molecular inter-leukemic heterogeneity would be important. Increasing amounts of unbiased next generation sequencing study data are available for pediatric T-ALL patients, but comprehensive data for adult T-ALL patients are scarce.Patients and methods:Eighty-four adult T-ALL patients (age: median 32, range 17-59 years) with different immunophenotypes were studied: 19 early, 14 mature and 51 thymic T-ALL. For all samples, deep targeted DNA sequencing with a gene panel consisting of 206 genes was performed (HighSeq 1500, 100 bp, average ~800 reads/bp). We sequenced mRNA with ~30 million reads/sample (HighSeq 2000, 125 bp). DNA methylation status of all samples were addressed by Infinium 450k methylation bead arrays.Results:Unsupervised clustering of 600 most variant expressed genes in our cohort identified five stable clusters with aberrant expression of oncogenes: TLX1 - (n=21), TLX3 - (n=10), HOXA - (n=12), TAL / LMO - (n=21) deregulated subgroups and cases with an immature gene expression signature (n=16). While the TLX1 - (thymic), the TAL / LMO - (thymic/mature), and the immature (early/mature) clusters are highly correlated with immunophenotype, HOX-A- and TLX3-cases occurred in any immunophenotypically defined subgroup. Different gene fusions were found in respective subgroups: TAL1 / 2 (n=7, fusion partners (FP): STIL1, TRDC, TCF7), LMO1/2 (n=2, FP: CAPRIN, RIC3), TXL1 (n=7, FP: TCRA, TCRB), and in the HOXA-cluster SET-NUP214-fusions (n=7). Differential expression (DE) analysis revealed an enrichment of stem cell genes like MEF2C, MN1, IGFBP7, FLT3 in the immature subgroup (2879 DE genes, DEG). TLX1 (2198 DEGs) and LMO/TAL (2165 DEGs) clusters were associated with mature T cell development stage (FDR<0.0001). Interestingly, HOXA-cluster (422 DEGs) showed a marked enrichment for ribosomal proteins (FDR<.00001).On genomic level, we found a slightly lower rate of mutations in the HOXA-cluster (median mutations/patient: TAL/LMO 8, TLX1 9, TLX3 8.5, HOXA 6, immature 9). Most frequently mutated genes included NOTCH1 (51%), PHF6 (32%), DNM2 (18%), PTEN (18%), FBXW7 (16%), and JAK3 (14%). Noteworthy, multiple NOTCH1 mutations were present in many patients and frequently on a subclonal level pointing to a late event in leukemogenesis. Mutations of epigenetic modifiers including KMT2D, DNMT3A, SUZ12, and EP300 were enriched in the TLX3 and immature cluster. JAK/STAT pathway was affected in all subgroups with frequently two hits in individual patients, e.g. 6/7 (86%) JAK1 mutations co-occurred with JAK3 mutations.On level of epigenetics, principal component analysis of the most variable CG-sites revealed five distinct subgroups. These are highly concordant with the subgroups based on gene expression profiling, with the exception of the HOXA-cluster. The increase of genomic altered epigenetic modifiers in TLX3 and immature cluster is reflected by a common DNA methylation signature (cluster M1).Conclusion:We characterized molecular defined subgroups of adult T-ALL on a genomic, transcriptional and epigenetic level finding high concordance for all subgroups across the platforms. We identified subgroup specific mutations and fusion genes for adult T-ALL patients reflecting inter-leukemic heterogeneity with association of molecularly defined subgroups to enriched pathways. These results might build the basis for the identification of patients eligible for targeted therapy like JAK/STAT inhibitors or epigenetic modulators. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human methylome variation across Infinium 450K data on the Gene Expression Omnibus.

While DNA methylation (DNAm) is the most-studied epigenetic mark, few recent studies probe the breadth of publicly available DNAm array samples. We collectively analyzed 35 360 Illumina Infinium HumanMethylation450K DNAm array samples published on the Gene Expression Omnibus. We learned a controlled vocabulary of sample labels by applying regular expressions to metadata and used existing models to predict various sample properties including epigenetic age. We found approximately two-thirds of samples were from blood, one-quarter were from brain and one-third were from cancer patients. About 19% of samples failed at least one of Illumina’s 17 prescribed quality assessments; signal distributions across samples suggest modifying manufacturer-recommended thresholds for failure would make these assessments more informative. We further analyzed DNAm variances in seven tissues (adipose, nasal, blood, brain, buccal, sperm and liver) and characterized specific probes distinguishing them. Finally, we compiled DNAm array data and metadata, including our learned and predicted sample labels, into database files accessible via the recountmethylation R/Bioconductor companion package. Its vignettes walk the user through some analyses contained in this paper.

Molecular Subgroups of T Cell Acute Lymphoblastic Leukemia in Adults Treated According to GMALL Protocols

Molecular Subgroups of T Cell Acute Lymphoblastic Leukemia in Adults Treated According to GMALL Protocols

Abstract 154: Genetic and epigenetic analysis of HPV-positive and HPV-negative Oropharyngeal squamous cell carcinoma

Abstract Background: The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV). Whereas HPV-associated OPSCC generally correlates with better prognosis, approximately 20% of the patients do not show good prognosis or response to treatment. Appropriate molecular markers have not been available to stratify OPSCC into distinct molecular subtypes reflecting these different clinicopathological features, making treatment stratification difficult, while de-escalation strategies for better prognosis group are expected to decrease the long-term chemoradiation-related morbidities. Methods: We performed DNA methylome analysis using Infinium 450k for 89 OPSCC cases and targeted exon sequencing for 86 OPSCC cases, and analyzed the data together with 81 cases reported by The Cancer Genome Atlas. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. Association between clinicopathological factors and DNA methylation or mutation status was statistically analyzed. Results: OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(−) intermediate-methylation (OP3), and HPV(−) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(−) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(−) than in HPV(+) groups (P&amp;lt;0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis (P=0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(−) cases. Generally favorable HPV-associated OPSCC as well as generally unfavorable HPV-negative OPSCC can be successfully subdivided into two different epigenotypes each, reflecting significantly distinct prognosis. Conclusions: HPV(+) and HPV(−) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis. These findings will contribute to greater understanding of the molecular basis of OPSCC and de-escalation treatment strategies. Citation Format: Takuya Nakagawa, Keisuke Matsusaka, Kiyoshi Misawa, Masaki Fukuyo, Tomoya Kurokawa, Masato Mima, Hisahiro Matsubara, Toyoyuki Hanazawa, Atsushi Kaneda. Genetic and epigenetic analysis of HPV-positive and HPV-negative Oropharyngeal squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 154.

Abstract 163: DNA methyltransferase modulation in lymphoma cells involves functional redundancy and maintains hypermethylation of selected regions

Abstract Background. DNA methyltransferases (DNMTs) are key enzymes necessary to the cells in order to establish and maintain methylation marks across the genome during both embrional development and adult cell divisions. DNMT1 is the main target of hypomethylating drugs 5-azacytidine (5-AZA) and 5-aza-2'-deoxycytidine (decitabine). These drugs have been used for a long time even though some mechanisms of action are yet unknown. We previously demonstrated that 5AZA and decitabine markedly down-regulate DNMT1 in a dose-dependent fashion, activate PARP and casp-3/7. However, 5AZA does not affect the methylation of selected targets, namely KLF4, DAPK1 and SPG20, who had been shown to play a role in lymphoma malignancy. We also observed that human PBMCs display an unmethylated profile that is opposite to the lymphoma cells. Experimental procedures. Toledo (Germinal center-derived) and NU-DUL-1 (Activated B-cell like) diffuse large-B cell lymphoma (DLBCL) cell lines. Epitect methyl II methylation qPCR. Immunoblottings. Gene silencing through siRNAs. Illumina Infinium 450K methylation arrays. Bioinformatics analysis for CpG doublets identification. Results. In order to dissect the molecular contribution of the main DNMTs during methylation maintenance, we individually silenced DNMT1 and DNMT3a in DLBCL cell lines. We show that neither DNMT1-, DNMT3a- nor double-silencing significantly affects the promoter methylation of KLF4, DAPK1 and SPG20. Genome-wide methylation analysis on a panel of 45 B-cell lymphoma cell lines reveals that the SPG20 locus presents hypermethylated CpG doublets upstream of the transcription starting site (open sea regions) while is mainly hypomethylated within the first exon. An intronic CpG island maps within SPG20 locus and presents heterogeneous beta-values among CpG sites. After 7 days from transfection, surprisingly, DNMT1, 3a and 3b gene expressions increase above the controls in Toledo cells. Furthermore, DNMT3a-silencing leads to DNMT1 protein upregulation in Toledo and NU-DUL-1 cells, suggesting a feedback regulatory mechanism. These data support a mechanism through which the methylation maintaining capacity of the cells is preserved even under pharmacological pressure. Conclusions. DNMT1- and DNMT3a-silencing exert a different effect on DNMTs regulation in lymphoma cells and evidences a functional redundancy among family members. Our data overall suggest that DNMT3a exerts suppressive effect on DNMT1, possibly by participating to the formation of a multi-factorial repressor complex. Citation Format: Raffaele Frazzi, Vincenza Ylenia Cusenza, Luciano Cascione, Andrea Rinaldi, Francesco Bertoni, Mariaelena Pistoni, Francesco Merli. DNA methyltransferase modulation in lymphoma cells involves functional redundancy and maintains hypermethylation of selected regions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 163.

DNA methylation patterns of LINE-1 and Alu for pre-symptomatic dementia in type 2 diabetes.

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 μg/day) and vitamin B12 (500 μg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD.

1642-P: Colocalization Analyses of Genetic Associations of Type 2 Diabetes with DNA Methylation on Chromosome 11p in American Indians

Our recent genome-wide association study (GWAS) identified variants strongly associated with type 2 diabetes (T2D) on chromosome 11p15 in American Indians at KCNQ1 (index variant rs2237895, P=2.5×10-8) and TH (rs11564707, P=3.8×10-8). To assess whether these variants influence epigenetic variation we analyzed associations with DNA methylation. Participants included 7659 American Indians (33% with T2D) who had participated in the GWAS, 842 of whom had DNA methylation measured in peripheral blood. Methylation was measured with the Infinium 450k array (Illumina, San Diego, CA). To identify local CpG sites associated with index variants, we analyzed associations for sites within 1 Mb of each gene (2162 sites for KCNQ1, 2325 for TH). Strong associations were identified: the diabetes risk allele at rs2237895 associated with decreased methylation at cg14637411 (β= 1.35 SD per copy, P=2.6×10-169), and the risk allele at rs11564707 associated with increased methylation at cg19878200 (β=0.46 SD, P=1.1×10-21). We further conducted Bayesian functional fine-mapping association of T2D and statistical colocalization of association of methylation at these sites with T2D using CAVIAR- including 1943 variants for KCNQ1 and 2954 variants for TH. Potentially causal variants for T2D were identified for each gene (30 for KCNQ1 and 33 for TH with posterior probability of causality &amp;gt;0.01); all were in strong linkage disequilibrium with the index variant. Strong evidence for colocalization of the association of T2D with methylation at cg14637411 (colocalization posterior probability &amp;gt;0.01, suggesting both traits are influenced by the same causal variants) was seen with 4 KCNQ1 variants: rs60808706, rs2299620, rs2237896 and rs2237897. Evidence for colocalization was weak for cg19878200 and T2D at TH variants. These results support the notion that KCNQ1 variants may influence T2D risk via an epigenetic effect, and they identify candidate functional variants. Disclosure R.L. Hanson: None. S. Kobes: None. W. Hsueh: None. Y.L. Muller: None. W.C. Knowler: None. L. Baier: None. Funding American Diabetes Association (1-12-DNP-01 to R.L.H.)

Higher methylation subtype of malignant melanoma and its correlation with thicker progression and worse prognosis.

Malignant melanoma (MM) is the most life‐threatening disease among all skin malignancies, and recent genome‐wide studies reported BRAF, RAS, and NF1 as the most frequently mutated driver genes. While epigenetic aberrations are known to contribute to the oncogenic activity seen in various cancers, their role in MM has not been fully investigated. To investigate the role of epigenetic aberrations in MM, we performed genome‐wide DNA methylation analysis of 51 clinical MM samples using Infinium 450k beadarray. Hierarchical clustering analysis stratified MM into two DNA methylation epigenotypes: high‐ and low‐methylation subgroups. Tumor thickness was significantly greater in case of high‐methylation tumors than low‐methylation tumors (8.3 ± 5.3 mm vs 4.5 ± 2.9 mm, P = .003). Moreover, prognosis was significantly worse in high‐methylation cases (P = .03). Twenty‐seven genes were found to undergo significant and frequent hypermethylation in high‐methylation subgroup, where TFPI2 was identified as the most frequently hypermethylated gene. MM cases with lower expression levels of TFPI2 showed significantly worse prognosis (P = .001). Knockdown of TFPI2 in two MM cell lines, CHL‐1 and G361, resulted in significant increases of cell proliferation and invasion. These indicate that MM can be stratified into at least two different epigenetic subgroups, that the MM subgroup with higher DNA methylation shows a more progressive phenotype, and that methylation of TFPI2 may contribute to the tumor progression of MM.

Stratification of HPV-associated and HPV-negative oropharyngeal squamous cell carcinomas based on DNA methylation epigenotypes.

While the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV), stratification of OPSCC into molecular subgroups showing different clinicopathological features has not been fully investigated. We performed DNA methylome analysis using Infinium 450k for 170 OPSCC cases, including 89 cases in our cohort and 81 cases reported by The Cancer Genome Atlas, together with targeted exon sequencing analysis. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(-) intermediate-methylation (OP3) and HPV(-) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(-) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(-) than in HPV(+) groups (p < 0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis (p = 0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(-) cases. HPV(+) and HPV(-) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis.

Diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (DGONC) - a molecularly defined glioneuronal CNS tumour class displaying recurrent monosomy 14.

DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. DNA methylation profiling was performed using the Infinium MethylationEPIC or 450k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. Genome-wide DNA methylation data from >25000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.