EPIC Array Research Articles

BIOM-41. CITRULLINATED HISTONE H3 ELISA REVEALS NETOSIS AS A SOURCE OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA

Abstract BACKGROUND We have previously shown that higher concentrations of plasma cell free DNA (cfDNA), both at baseline and following chemoradiotherapy, are associated with worse survival in GBM. Because only scant amounts of GBM-derived cfDNA shed into the peripheral circulation, we sought to determine the predominant source of prognostic plasma cfDNA in these patients and hypothesized that this may help unravel the mechanism underlying the association of high cfDNA and survival. METHODS Plasma was obtained for 126 patients prior to resection of newly diagnosed GBM. A subset of 46 plasma samples had ccfDNA extracted, enzymatically converted, whole genome amplified, and analyzed on the Illumina Epic array for deconvolution of cfDNA methylation patterns. Based on these results demonstrating neutrophils as the predominant cfDNA source, we performed ELISA for citrullinated-histone 3 (citH3) in plasma from 113 of the patients as a specific marker of NETosis, a pro-tumor process of programmed neutrophil cell death during which DNA is released in web-like structures called neutrophil extracellular traps (NETs). RESULTS Deconvolution of cfDNA methylation patterns revealed that neutrophils were the dominant source of cfDNA (median percentage of cfDNA derived from neutrophils = 46% , range 17-67%) and correlated with baseline plasma cfDNA concentration (n = 46, r = 0.35, p = 0.017). Additionally, cfDNA concentration was significantly correlated with the concentration of citH3 (n = 113, r = 0.60, p < 0.0001), which was higher in GBM patients compared to 26 healthy controls (p < 0.0001). CONCLUSION Our findings suggest that NETosis may be the dominant source of plasma cfDNA, which we have previously shown to be highly prognostic in patients with GBM. Additional studies interrogating the tumor microenvironment for NETosis in GBM tumors are ongoing. These results lay the foundation for further exploration of NETosis as a therapeutic target and underscore the emerging importance of neutrophil biology in GBM.

EPCO-02. DNA METHYLATION PROFILING OF BRAIN METASTASIS REVEALS UNDERLING TUMOR RECURRENCE SIGNAL AND ASSOCIATED PATHWAYS

Abstract INTRODUCTION Brain metastases, the most common form of intracranial neoplasms, carry a poor survival rate, mostly attributed to their high recurrence rates. Currently there are no reliable methods to determine which patients will progress. DNA methylation profiling (DNAmp) has become a useful tool in the diagnosis and stratification of intracranial neoplasms, and has the potential to provide clues on the epigenetic mechanisms that govern tumor behavior. METHODS A cohort of 58 BM tumor samples were selected for analysis. The cohort was composed of three groups: N=21 primary metastatic tumors (PMT), N=21 matched paired recurrent tumors (RMT), and a cohort of N= 16 primary metastatic tumors without any evidence of recurrence (NRMT). All tumor samples underwent DNAmp on the Illumina Infinium EPIC array to determine their methylation status at 850,000 CpG sites. Tumors were profiled via unsupervised hierarchical clustering, pathway enrichment analysis, and cell deconvolution analysis. RESULTS Differential methylation analysis revealed over 32617 differentially methylated CpG sites between grouped PMT and RMT samples, and 835424 sites between PMT and NRMT samples. When comparing PMT with NRMT samples, we revealed a distinct DNAmp that was upheld upon hierarchical clustering analysis. NRMT samples show a significant downregulation of pathways involved in DNA-binding and transcriptional regulation. Paired analysis of PMT and RMT revealed a relative paucity of differentially methylated CpG sites shared across paired samples: a total of 257 probes were differentially methylated across 14/19 samples. These probes however showed enrichment primarily for DNA-binding and transcription factor regulation, in reverse from the non-recurrent samples. CONCLUSION Our data suggests that DNAmp may be capable of differentiating tumors destined to progress from those with less aggressive features, and that DNAmp appear to remain stable through to recurrence. This work demonstrates the potential for DNA methylation to be utilized to uncover pathways associated with BM recurrence.

EPCO-26. INTRATUMORAL HETEROGENEITY OF GBM IDENTIFIED AND CHARACTERIZED BY A MULTISAMPLING APPROACH AND METHYLATION PROFILING

Abstract Intratumoral heterogeneity in glioblastoma is known and is considered a key feature of GBM’s clinical aggressiveness. To more fully characterize this phenomenon, we conducted methylation profiling on the EPIC array for 79 regions from 20 glioblastomas. Using the Heidelberg classifier, each of the 20 tumors had at least 1 region with a match to a GBM subtype with >0.84 score. Among these, 6 tumors (30%) showed homogeneous-high score matches to the same GBM subclass, while the remaining 14 tumors (70%) showed heterogeneity in GBM subclass, with either lower scores, or disparate matches to different glioma subtypes. Comparing these subclass-homogeneous versus subclass-heterogeneous groups of tumors using methylCIBERSORT deconvolution, the subclass-heterogeneous cases showed a significantly higher proportions of CD8+ T cells and microglia, and significantly lower proportions of CD4+ T cells, B cells and monocytes. While low tumor purity can be a cause of lower confidence scores in the methylation classifier, we did not find that the subclass-heterogeneous tumors had lower tumor purity compared to subclass-homogeneous cases, suggesting that this heterogeneity was due to factors other than tumor purity. Within the subclass-heterogeneous tumors we identified the common intratumorally most variable probes and examined their characteristics. Compared to the overall distribution of probes on the array, these intratumoral-variable probes were significantly more likely to be intergenic and located on enhancers. Conversely, they were significantly less likely to be on gene promoters and CpG islands. Gene ontology analysis showed that genes associated with these probes were enriched for pathways involved in DNA repair. Overall, our multisampling methylation profiling analysis identifies specific biologic correlates of intratumoral heterogeneity that is not simply due to tumor purity. Methylation changes associated with intratumoral heterogeneity converge on specific genomic patterns that warrant further study.

NIMG-83. ELUCIDATING INTRA-TUMORAL HETEROGENEITY AND DISTINCT RADIOMIC FEATURES IN GLIOBLASTOMA METHYLATION SUBCLASSES

Abstract Glioblastoma, IDH-wildtype (GBM), is characterized by the inter- and intra-tumoral molecular heterogeneity. Genome-wide DNA methylation profiling has recently emerged as a promising tool offering complementary value to the classification of central nervous system tumors. In adult GBM patients, the most common methylation subclasses comprise RTK I, RTK II, and MES. We hypothesize that quantification of intricate and spatially complex radiomic features extracted from multi-parametric MRI (mpMRI) is informative to non-invasively determining characteristics of GBM methylation subclasses and their heterogeneity. In this study, mpMRI scans (T1, T1-Gd, T2, T2-FLAIR, DSC, DTI) of newly diagnosed 23 GBM patients were retrospectively collected. We derived 6339 radiomic features, including histograms, morphologic and textural descriptors. For each case, 3-4 multiple samplings from different parts of the tumor were conducted. DNA was extracted from FFPE for each of sample and analyzed for genome-wide DNA methylation patterns using the Illumina EPIC array. Classification of methylation subclasses was performed using the MNP brain tumor classifier v12b6 of the DKFZ. Analysis of variance (ANOVA) with Bonferroni correction was employed to extract subclass-specific radiomic features. Of the 23 patients, 7 indicated intra-tumoral heterogeneity in methylation subclasses. We identified 68 radiomic features (p< 0.05) and 12 radiomic features (p< 0.01) that showed statistically significant differences among the methylation subclasses as determined by ANOVA with Bonferroni correction. A heatmap illustrating the relationships between subclasses and extracted radiomic features displayed distinct trends among subclasses and between heterogeneous and homogeneous cases within the same subclass. Our findings suggest the presence of heterogeneity within methylation subclasses and distinctive imaging patterns for each subclass. These findings potentially pave the way for a more personalized approach to glioblastoma management, highlighting the importance of further validation through the analysis of additional cases.

Abstract 17374: Epigenetic Age Acceleration in Adult Congenital Heart Disease

Introduction: DNA methylation (DNAm) changes reflect alterations in the immune response to cardiac surgery in older adults. However, whether adults with congenital heart disease (ACHD) who underwent palliative cardiac surgery during childhood experience epigenetic age acceleration (EAA) is unknown. Hypothesis: ACHD patients with Fontan(F) procedures would have advanced EAA compared to healthy adults. Methods: A case-control study in young adults (aged 20-30 years) with Fontan circulation at a tertiary ACHD hospital with age-matched healthy adults. DNAm were measured by the EPIC Array. EAAs were calculated by extracting residuals by regressing epigenetic ages on chronological age in linear regression models for four epigenetic clocks. DNAm-based cell type proportions and EAA differences between the two groups were tested by robust linear models (rlm) adjusted for age, sex, and self-reported ethnicity. Results: Thirteen Fontan patients (6 female: 7 male) were compared with 20 age-matched controls (15 female). The median age of the F group was 23 years, compared to 27 years in control. Both subgroups had a predominant white race and non-Hispanic ethnicity. The F group has lower B memory cells and naïve CD4+ cells than controls (adjusted p<0.05). The F group has statistically significant higher Grimage and Phenoage EAA than the controls (p<0.01). Conclusions: Patients who underwent the Fontan procedure experience advanced EAA at a young age. Future studies evaluating the key predictors that drive EAA difference is needed to understand the impact of chronic alteration of physiology.

Abstract 17447: Effects of Inflammatory Biomarkers on Epigenetic Age Acceleration in Adult Congenital Heart Disease

Introduction: DNA methylation (DNAm) changes reflect alterations in the immune response to cardiac surgery in older adults. However, the role of inflammation on epigenetic age acceleration (EAA) in adults with congenital heart disease (ACHD) who underwent palliative cardiac surgery during childhood is unknown. Hypothesis: ACHD patients with Fontan(F) procedures would have advanced EAA due to higher DNAm predicted IL-6 and CRP levels compared to healthy adults or ACHD patients who underwent single surgery as an infant, likely capturing chronic inflammation associated with the abnormal pathophysiologic state. Methods: A case-control study in young adults (aged 20-30 years) with Fontan circulation or single surgery (SS) as an infant at a tertiary ACHD hospital. DNAm were measured by the EPIC Array. DNAm predicted IL-6 and CRP level differences were tested by robust linear model (rlm) adjusted for age, sex, cell types, and self-reported ethnicity. EAAs were calculated by extracting residuals by regressing epigenetic ages on chronological age in linear regression models. EAA differences were tested by rlm adjusted for the same covariates. Contribution analysis was performed to determine contribution of IL-6 and CRP to EAA. Results: Thirteen Fontan patients (6 female: 7 male, median age: 23) and 5 SS patients (3 female: 2 male, median age: 23) were compared with 20 age-matched controls (15 female: 5 male, median age: 27). Each subgroup had a predominant white race and non-Hispanic ethnicity. The F group has a statistically higher predicted IL-6 and CRP level (p<0.001). Also, the F group had significantly higher EAA for mortality predictor Grimage (p<0.001) and PhenoAge (p<0.001). Conclusions: Patients who underwent Fontan procedure experience advanced EAA contributed by inflammatory biomarkers at a young age. Future studies evaluating the key predictors that drive EAA difference is needed to understand the impact of chronic alteration of physiology.

EPIC Array Study in Liquid Biopsy Identifies a Non-invasive Epigenetic Signature for Early Diagnosis in Non-small Cell Lung Cancer

EPIC Array Study in Liquid Biopsy Identifies a Non-invasive Epigenetic Signature for Early Diagnosis in Non-small Cell Lung Cancer

Identification of a robust DNA methylation signature for Fanconi anemia

Fanconi anemia (FA) is a clinically variable and genetically heterogeneous cancer-predisposing disorder representing the most common bone marrow failure syndrome. It is caused by inactivating predominantly biallelic mutations involving >20 genes encoding proteins with roles in the FA/BRCA DNA repair pathway. Molecular diagnosis of FA is challenging due to the wide spectrum of the contributing gene mutations and structural rearrangements. The assessment of chromosomal fragility after exposure to DNA cross-linking agents is generally required to definitively confirm diagnosis. We assessed peripheral blood genome-wide DNA methylation (DNAm) profiles in 25 subjects with molecularly confirmed clinical diagnosis of FA (FANCA complementation group) using Illumina's Infinium EPIC array. We identified 82 differentially methylated CpG sites that allow to distinguish subjects with FA from healthy individuals and subjects with other genetic disorders, defining an FA-specific DNAm signature. The episignature was validated using a second cohort of subjects with FA involving different complementation groups, documenting broader genetic sensitivity and demonstrating its specificity using the EpiSign Knowledge Database. The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating robustness. Using the selected probes, we trained a machine-learning model able to classify EPIC DNAm profiles in molecularly unsolved cases. Finally, we show that the generated episignature includes CpG sites that do not undergo functional selective pressure, allowing diagnosis of FA in individuals with reverted phenotype due to gene conversion. These findings provide a tool to accelerate diagnostic testing in FA and broaden the clinical utility of DNAm profiling in the diagnostic setting.

Mesonephric-like adenocarcinoma harbours characteristic copy number variations and a distinct DNA methylation signature closely related to mesonephric adenocarcinoma of the cervix.

Mesonephric-like adenocarcinoma (MLA) of the female genital tract is an uncommon histotype that can arise in both the endometrium and the ovary. The exact cell of origin and histogenesis currently remain unknown. Here, we investigated whole genome DNA methylation patterns and copy number variations (CNVs) in a series of MLAs in the context of a large cohort of various gynaecological carcinoma types. CNV analysis of 19 MLAs uncovered gains of chromosomes 1q (18/19, 95%), 10 (15/19, 79%), 12 (14/19, 74%), and 2 (10/19, 53%), as well as loss of chromosome 1p (7/19, 37%). Gains of chromosomes 1q, 10, and 12 were also identified in the majority of mesonephric adenocarcinomas of the uterine cervix (MAs) as well as subsets of endometrioid carcinomas (ECs) and low-grade serous carcinomas of the ovary (LGSCs) but only in a minority of serous carcinomas of the uterine corpus (USCs), clear cell carcinomas (CCCs), and tubo-ovarian high-grade serous carcinomas (HGSCs). While losses of chromosome 1p together with gains of chromosome 1q were also identified in both MA and LGSC, gains of chromosome 2 were almost exclusively identified in MLA and MA. Unsupervised hierarchical clustering and t-SNE analysis of DNA methylation data (Illumina EPIC array) identified a co-clustering for MLAs and MAs, which was distinct from clusters of ECs, USCs, CCCs, LGSCs, and HGSCs. Group-wise comparisons confirmed a close epigenetic relationship between MLA and MA. These findings, in conjunction with the established histological and immunophenotypical overlap, suggest bona fide mesonephric differentiation, and support a more precise terminology of mesonephric-type adenocarcinoma instead of MLA in these tumours. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Epigenomic signature of major congenital heart defects in newborns with Down syndrome

BackgroundCongenital heart defects (CHDs) affect approximately half of individuals with Down syndrome (DS), but the molecular reasons for incomplete penetrance are unknown. Previous studies have largely focused on identifying genetic risk factors associated with CHDs in individuals with DS, but comprehensive studies of the contribution of epigenetic marks are lacking. We aimed to identify and characterize DNA methylation differences from newborn dried blood spots (NDBS) of DS individuals with major CHDs compared to DS individuals without CHDs.MethodsWe used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: (1) 45 DS-CHD (27 female, 18 male) and (2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD versus DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell-type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS versus typical development (TD) WGBS NDBS samples.ResultsWe found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS versus TD samples.ConclusionsA sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.

SAT587 Methylation-based Machine Learning Classifiers Discriminate Sellar Tumors By Lobe Origin Using Liquid Biopsy Or Surgical Specimens

Abstract Disclosure: G.A. Herrgott: None. K.P. Asmaro: None. M. Wells: None. K. Nelson: None. B. Thomas: None. L.A. Hasselbach: None. A. Transou: None. S. Cazacu: None. K.M. Tundo: None. S. Nadimidla: None. L. Scarpace: None. J. Barnholtz-Sloan: None. A.E. Sloan: None. W.R. Selman: None. A.C. deCarvalho: None. A. Mukherjee: None. A.M. Robin: None. I.Y. Lee: None. J. Craig: None. S. Kalkanis: None. J. Snyder: None. T. Walbert: None. J. Rock: None. H. Noushmehr: None. A.B. Castro: None. Background: The differential diagnosis of challenging sellar tumor cases can be inconclusive through imaging features and could benefit from noninvasive diagnostic approaches, such as liquid biopsy (LB). Similar to tissue, LB specimens carry tumor-specific DNA methylation signatures amenable to the construction of accurate machine learning models able to discriminate CNS tumors. We aimed to develop methylation-based classifiers which classify sellar tumors by lobe of origin, using either LB or tumor tissue specimens. Methodology: We analyzed the DNA methylome (EPIC array) of tumor tissue (T) and LB specimens from adult patients with tumors representing each of the three pituitary lobes (Anterior: T=177; LB=37; Intermediate: T= 7; LB: 10 and Posterior: T=44, LB=2 cases). Using the most variably methylated CpG probes derived from the unsupervised variance-based analyses across tumors from different lobes, we applied multi-class linear discriminant analysis to construct machine learning models to classify sellar tumor tissue and/or LB specimens. Results: We generated classifiers based on lobe-specific methylation signatures that were able to discriminate across sellar tumors either using tissue and/or LB specimens (500 and 600 CpGs, respectively) with observed accuracies of ∼99% across independent validation. DISCUSSION/CONCLUSION: Our findings suggest that methylation-based classifiers constitute an accurate diagnostic approach to discriminate sellar tumors according to the lobe origin, either pre-surgically through a blood draw or through surgical tumor specimens. These classifiers are objective approaches that could complement imaging and pathology reports for an accurate diagnosis of inconclusive cases, ultimately leading to optimal management of the patients with these diseases. Presentation: Saturday, June 17, 2023

THU494 VHL Protein Deficiency Alters The Immune Microenvironment Of Pancreatic Neuroendocrine Neoplasms

Abstract Disclosure: A. Chmelnik: None. A. Telerman: None. A. Tirosh: None. Introduction - Pancreatic neuroendocrine tumors (PanNENs) may develop sporadically or as part of an inherited disease, such as von Hippel-Lindau (VHL). VHL disease is caused by a germline pathogenic variant in the VHL gene encoding VHL protein (pVHL). Hypoxia inducible factor (HIF) is responsible for cellular oxygen supply. Its degradation is mediated by pVHL via ubiquitination in normoxic states. In pVHL-deficient state leads to HIF overexpression and pseudohypoxia. Several studies suggested immunomodulatory role for HIF in kidney cancer. Aims- To assess the impact of pseudohypoxia on PanNEN immune tumor microenvironment (iTME). Methods- A total of 16 vPanNEN and 23 sPanNEN were processed. Whole genome DNA methylation was performed by Illumine Infinium EPIC Array and analyzed using ChAMP on RStudio. Immune cells composition was compared using methylation-based deconvolution algorithms (MethylResolver, Robust Partial Correlations, Constrained Projection and Cibersort), and gene set enrichment analysis identified pathways affected in each group based on promoter methylation analysis. Results- Unsupervised hierarchical analysis of immune-related gene promoter methylation demonstrated separation of vPanNEN vs. sPanNEN. In GSEA analysis methylation was altered in immune memory-related genes in vPanNEN, and in T cells receptors-related genes in sPanNEN. Deconvolution algorithms revealed a lower fraction of CD4 and NK cells (p<0.05) and a lower PD-L1 expression in vPanNENs (p=0.037). Conclusion- Pseudohypoxic vPanNEN have distinct iTME than sPanNEN, in immune cells composition and PD-L1 expression. Presentation: Thursday, June 15, 2023

Epigenomic response to albuterol treatment in asthma-relevant airway epithelial cells

BackgroundAlbuterol is the first-line asthma medication used in diverse populations. Although DNA methylation (DNAm) is an epigenetic mechanism involved in asthma and bronchodilator drug response (BDR), no study has assessed whether albuterol could induce changes in the airway epithelial methylome. We aimed to characterize albuterol-induced DNAm changes in airway epithelial cells, and assess potential functional consequences and the influence of genetic variation and asthma-related clinical variables.ResultsWe followed a discovery and validation study design to characterize albuterol-induced DNAm changes in paired airway epithelial cultures stimulated in vitro with albuterol. In the discovery phase, an epigenome-wide association study using paired nasal epithelial cultures from Puerto Rican children (n = 97) identified 22 CpGs genome-wide associated with repeated-use albuterol treatment (p < 9 × 10–8). Albuterol predominantly induced a hypomethylation effect on CpGs captured by the EPIC array across the genome (probability of hypomethylation: 76%, p value = 3.3 × 10–5). DNAm changes on the CpGs cg23032799 (CREB3L1), cg00483640 (MYLK4-LINC01600), and cg05673431 (KSR1) were validated in nasal epithelia from 10 independent donors (false discovery rate [FDR] < 0.05). The effect on the CpG cg23032799 (CREB3L1) was cross-tissue validated in bronchial epithelial cells at nominal level (p = 0.030). DNAm changes in these three CpGs were shown to be influenced by three independent genetic variants (FDR < 0.05). In silico analyses showed these polymorphisms regulated gene expression of nearby genes in lungs and/or fibroblasts including KSR1 and LINC01600 (6.30 × 10–14 ≤ p ≤ 6.60 × 10–5). Additionally, hypomethylation at the CpGs cg10290200 (FLNC) and cg05673431 (KSR1) was associated with increased gene expression of the genes where they are located (FDR < 0.05). Furthermore, while the epigenetic effect of albuterol was independent of the asthma status, severity, and use of medication, BDR was nominally associated with the effect on the CpG cg23032799 (CREB3L1) (p = 0.004). Gene-set enrichment analyses revealed that epigenomic modifications of albuterol could participate in asthma-relevant processes (e.g., IL-2, TNF-α, and NF-κB signaling pathways). Finally, nine differentially methylated regions were associated with albuterol treatment, including CREB3L1, MYLK4, and KSR1 (adjusted p value < 0.05).ConclusionsThis study revealed evidence of epigenetic modifications induced by albuterol in the mucociliary airway epithelium. The epigenomic response induced by albuterol might have potential clinical implications by affecting biological pathways relevant to asthma.

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.

Subtyping of Group 3/4 medulloblastoma as a potential prognostic biomarker among patients treated with reduced dose of craniospinal irradiation: a Japanese Pediatric Molecular Neuro-Oncology Group study

BackgroundOne of the most significant challenges in patients with medulloblastoma is reducing the dose of craniospinal irradiation (CSI) to minimize neurological sequelae in survivors. Molecular characterization of patients receiving lower than standard dose of CSI therapy is important to facilitate further reduction of treatment burden.MethodsWe conducted DNA methylation analysis using an Illumina Methylation EPIC array to investigate molecular prognostic markers in 38 patients with medulloblastoma who were registered in the Japan Pediatric Molecular Neuro-Oncology Group and treated with reduced-dose CSI.ResultsAmong the patients, 23 were classified as having a standard-risk and 15 as high-risk according to the classic classification based on tumor resection rate and presence of metastasis, respectively. The median follow-up period was 71.5 months (12.0–231.0). The median CSI dose was 18 Gy (15.0–24.0) in both groups, and 5 patients in the high-risk group received a CSI dose of 18.0 Gy. Molecular subgrouping revealed that the standard-risk cohort included 5 WNT, 2 SHH, and 16 Group 3/4 cases; all 15 patients in the high-risk cohort had Group 3/4 medulloblastoma. Among the patients with Group 3/4 medulloblastoma, 9 of the 31 Group 3/4 cases were subclassified as subclass II, III, and V, which were known to an association with poor prognosis according to the novel subtyping among the subgroups. Patients with poor prognostic subtype showed worse prognosis than that of others (5-year progression survival rate 90.4% vs. 22.2%; p < 0.0001). The result was replicated in the multivariate analysis (hazard ratio12.77, 95% confidence interval for hazard ratio 2.38–99.21, p value 0.0026 for progression-free survival, hazard ratio 5.02, 95% confidence interval for hazard ratio 1.03–29.11, p value 0.044 for overall survival).ConclusionAlthough these findings require validation in a larger cohort, the present findings suggest that novel subtyping of Group 3/4 medulloblastoma may be a promising prognostic biomarker even among patients treated with lower-dose CSI than standard treatment.

The effect of polyphenols on DNA methylation-assessed biological age attenuation: the DIRECT PLUS randomized controlled trial

BackgroundEpigenetic age is an estimator of biological age based on DNA methylation; its discrepancy from chronologic age warrants further investigation. We recently reported that greater polyphenol intake benefitted ectopic fats, brain function, and gut microbiota profile, corresponding with elevated urine polyphenols. The effect of polyphenol-rich dietary interventions on biological aging is yet to be determined.MethodsWe calculated different biological aging epigenetic clocks of different generations (Horvath2013, Hannum2013, Li2018, Horvath skin and blood2018, PhenoAge2018, PCGrimAge2022), their corresponding age and intrinsic age accelerations, and DunedinPACE, all based on DNA methylation (Illumina EPIC array; pre-specified secondary outcome) for 256 participants with abdominal obesity or dyslipidemia, before and after the 18-month DIRECT PLUS randomized controlled trial. Three interventions were assigned: healthy dietary guidelines, a Mediterranean (MED) diet, and a polyphenol-rich, low-red/processed meat Green-MED diet. Both MED groups consumed 28 g walnuts/day (+ 440 mg/day polyphenols). The Green-MED group consumed green tea (3–4 cups/day) and Mankai (Wolffia globosa strain) 500-ml green shake (+ 800 mg/day polyphenols). Adherence to the Green-MED diet was assessed by questionnaire and urine polyphenols metabolomics (high-performance liquid chromatography quadrupole time of flight).ResultsBaseline chronological age (51.3 ± 10.6 years) was significantly correlated with all methylation age (mAge) clocks with correlations ranging from 0.83 to 0.95; p < 2.2e − 16 for all. While all interventions did not differ in terms of changes between mAge clocks, greater Green-Med diet adherence was associated with a lower 18-month relative change (i.e., greater mAge attenuation) in Li and Hannum mAge (beta = − 0.41, p = 0.004 and beta = − 0.38, p = 0.03, respectively; multivariate models). Greater Li mAge attenuation (multivariate models adjusted for age, sex, baseline mAge, and weight loss) was mostly affected by higher intake of Mankai (beta = − 1.8; p = 0.061) and green tea (beta = − 1.57; p = 0.0016) and corresponded with elevated urine polyphenols: hydroxytyrosol, tyrosol, and urolithin C (p < 0.05 for all) and urolithin A (p = 0.08), highly common in green plants. Overall, participants undergoing either MED-style diet had ~ 8.9 months favorable difference between the observed and expected Li mAge at the end of the intervention (p = 0.02).ConclusionsThis study showed that MED and green-MED diets with increased polyphenols intake, such as green tea and Mankai, are inversely associated with biological aging. To the best of our knowledge, this is the first clinical trial to indicate a potential link between polyphenol intake, urine polyphenols, and biological aging.Trial registrationClinicalTrials.gov, NCT03020186.

PL02.2.A RAPID, COMPREHENSIVE REPORTING OF MOLECULAR DIAGNOSTICS WITH RAPID-CNS2: A PROSPECTIVE VALIDATION COHORT

Abstract BACKGROUND The WHO classification of CNS tumours 2021 recommends reporting a wide range of molecular alterations for WHO-compatible diagnoses. Conventional molecular diagnostic workflows warrant considerable investment and long turnaround times limited by batching. Nanopore sequencing enables long read sequencing, real-time targeting and simultaneous base modification detection with compact devices. Rapid-CNS2- a rapid, comprehensive adaptive sampling based sequencing pipeline with a turnaround time of 5 days for CNS tumours was previously described. This study presents an optimised Rapid-CNS2 pipeline and prospective validation cohort that successfully reports hard-to-detect variants. MATERIAL AND METHODS We performed targeted adaptive sampling-based sequencing on 156 samples comprising a mix of archival and current diagnostic cases from the Department of Neuropathology, University Hospital Heidelberg. We tested variable conditions including minimum DNA input, target size, flowcell reuse and sequencing times. An analysis pipeline for detection of single nucleotide variants (SNV), indels, copy number alterations (CNA), fusions, complex structural variants (SV), target gene methylation and methylation classification incorporating multi-GPU optimization was deployed. Results were compared to NGS panel sequencing and EPIC array analyses. RESULTS Integrated diagnoses for all cases were in accordance with those issued using molecular alterations reported by conventional analyses. Rapid-CNS2 showed improved resolution over NGS panel sequencing for CNAs. Complete concordance was achieved for MGMT promoter methylation status and methylation classification. High accuracy was obtained for detection of SNV/Indels. Long reads spanning the breakpoint identified clinically relevant fusions. Subclonal SVs including EGFR vIII missed by NGS panel sequencing were detected with high confidence. The optimized workflow shortened the turnaround time to 30 hours from sample receipt to report and minimum input DNA required was reduced to 500ng. Due to the versatility of adaptive sampling, the analyses can readily be limited to a smaller set of targets or methylation and CNAs only, further reducing the turnaround time. CONCLUSION We present a thorough validation and prospective clinical application of Rapid-CNS2 with cases spanning a range of brain tumours and metastases. The improved custom neurooncology adaptive sampling-based sequencing pipeline enables simultaneous copy-number, mutational, structural variant and methylation analysis with flexible target selection, no additional library preparation, competitive costs and next-day reporting of RESULTS

P12.08.A CELL-EXTRINSIC D-2-HYDROXYGLUTARATE PROMOTES HYPERMETHYLATION AT LINEAGE-SPECIFIC REGULATORY REGIONS TO PREVENT MICROGLIAL ACTIVATION IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in diffuse gliomas. Their composition and transcriptional programs differ according to the IDH mutation status of glioma cells. The underlying mechanisms remain until now little known. MATERIAL AND METHODS We compared bulk DNA methylome (Methylation EPIC array) and transcriptome of TAMs (CD11b+ purified by magnetic-activated cell sorting) from 25 IDH-mutant and 11 IDH-wildtype gliomas. The contamination of the CD11b+ fraction by tumor cells was determined by the detection of IDH and/or TERT mutations using droplet digital PCR. Only samples with a purity of at least 95% were used for analyses. To determine experimentally direct effects of D-2-hydroxyglutarate (D-2-HG), which is the oncometabolite produced and realeased into the microenvironment by IDH-mutated glioma cells, we used primary cultures of human microglial cells obtained from glioma surgery or epilepsy surgery. We exposed microglia or not to (D-2-HG) during 14 days, and analyzed their DNA methylome and transcriptome. RESULTS TAMs from IDH-mutant gliomas compared with TAMs from IDH-wildtype gliomas exhibit decreased expression of pro-inflammatory pathways (mainly IFN-γ, IFN-α, and TNF-α signaling) and genes related to G2-M checkpoint, glycolysis and Epithelial-Mesenchymal Transition (EMT). We uncovered that TAMs from IDH-mutant gliomas exhibit significant hypermethylation at promoters and enhancers linked to decreased expression of genes involved in inflammatory responses. Notably, these hypermethylated regions were enriched for binding motifs of transcription factors controlling an environment-dependent transcriptional network in microglial cells (such as PU.1, AP-1, C/EBP and RUNX). These findings were confirmed by comparing methylome data of TAMs from IDH-mutant gliomas and methylome of microglia from normal brains available in the literature. A 14 days exposition to D-2-HG of human microglial cells in vitro partially recapitulates the observed methylation changes. Transcriptome analyses of these cells show that this oncometabolite dampens the IFN-α/γ pathways and attenuates the response to LPS while inducing a metabolic shift toward oxidative phosphorylation. CONCLUSION Our findings establish a link between an extrinsic cell-effect of D-2-HG and the hypermethylation of DNA of microglial cells at regulatory regions in IDH-mutant gliomas. Our observations support the concept that oncometabolites may disrupt directly the function of immune cells of the tumor microenvironment.

Integrated epigenome, whole genome sequence and metabolome analyses identify novel multi-omics pathways in type 2 diabetes: a Middle Eastern study

BackgroundT2D is of high prevalence in the middle east and thus studying its mechanisms is of a significant importance. Using 1026 Qatar BioBank samples, epigenetics, whole genome sequencing and metabolomics were combined to further elucidate the biological mechanisms of T2D in a population with a high prevalence of T2D.MethodsAn epigenome-wide association study (EWAS) with T2D was performed using the Infinium 850K EPIC array, followed by whole genome-wide sequencing SNP-CpG association analysis (> 5.5 million SNPs) and a methylome-metabolome (CpG-metabolite) analysis of the identified T2D sites.ResultsA total of 66 T2D-CpG associations were identified, including 63 novel sites in pathways of fructose and mannose metabolism, insulin signaling, galactose, starch and sucrose metabolism, and carbohydrate absorption and digestion. Whole genome SNP associations with the 66 CpGs resulted in 688 significant CpG-SNP associations comprising 22 unique CpGs (33% of the 66 CPGs) and included 181 novel pairs or pairs in novel loci. Fourteen of the loci overlapped published GWAS loci for diabetes related traits and were used to identify causal associations of HK1 and PFKFB2 with HbA1c. Methylome-metabolome analysis identified 66 significant CpG-metabolite pairs among which 61 pairs were novel. Using the identified methylome-metabolome associations, methylation QTLs, and metabolic networks, a multi-omics network was constructed which suggested a number of metabolic mechanisms underlying T2D methylated genes. 1-palmitoyl-2-oleoyl-GPE (16:0/18:1) – a triglyceride-associated metabolite, shared a common network with 13 methylated CpGs, including TXNIP, PFKFB2, OCIAD1, and BLCAP. Mannonate – a food component/plant shared a common network with 6 methylated genes, including TXNIP, BLCAP, THBS4 and PEF1, pointing to a common possible cause of methylation in those genes. A subnetwork with alanine, glutamine, urea cycle (citrulline, arginine), and 1-carboxyethylvaline linked to PFKFB2 and TXNIP revealed associations with kidney function, hypertension and triglyceride metabolism. The pathway containing STYXL1-POR was associated with a sphingosine-ceramides subnetwork associated with HDL-C and LDL-C and point to steroid perturbations in T2D.ConclusionsThis study revealed several novel methylated genes in T2D, with their genomic variants and associated metabolic pathways with several implications for future clinical use of multi-omics associations in disease and for studying therapeutic targets.

Blood-based genome-wide DNA methylation correlations across body-fat- and adiposity-related biochemical traits

The recent increase in obesity levels across many countries is likely to be driven by nongenetic factors. The epigenetic modification DNA methylation (DNAm) may help to explore this, as it is sensitive to both genetic and environmental exposures. While the relationship between DNAm and body-fat traits has been extensively studied, there is limited literature on the shared associations of DNAm variation across such traits. Akin to genetic correlation estimates, here, we introduce an approach to evaluate the similarities in DNAm associations between traits: DNAm correlations. As DNAm can be both a cause and consequence of complex traits, DNAm correlations have the potential to provide insights into trait relationships above that currently obtained from genetic and phenotypic correlations. Utilizing 7,519 unrelated individuals from Generation Scotland with DNAm from the EPIC array, we calculated DNAm correlations between body-fat- and adiposity-related traits by using the bivariate OREML framework in the OSCA software. For each trait, we also estimated the shared contribution of DNAm between sexes. We identified strong, positive DNAm correlations between each of the body-fat traits (BMI, body-fat percentage, and waist-to-hip ratio, ranging from 0.96 to 1.00), finding larger associations than those identified by genetic and phenotypic correlations. We identified a significant deviation from 1 in the DNAm correlations for BMI between males and females, with sex-specific DNAm changes associated with BMI identified at eight DNAm probes. Employing genome-wide DNAm correlations to evaluate the similarities in the associations of DNAm with complex traits has provided insight into obesity-related traits beyond that provided by genetic correlations.