Methylation Analysis Research Articles

EPCO-15. INTRA-TUMORAL HETEROGENEITY OF DNA METHYLATION PROFILING AND CELLULAR COMPOSITION IN GLIOBLASTOMA

Abstract BACKGROUND DNA methylation profiling has become an important diagnostic and exploratory tool in neuro-oncology. Intra-tumoral molecular heterogeneity is a crucial factor in the treatment resistance of IDH-wildtype glioblastoma (GBM). However, the intra-tumoral heterogeneity of methylation profiles in GBM remains poorly elucidated. This study aimed to investigate the intra-tumoral heterogeneity of DNA methylation subclasses and deconvolve methylation profiles to infer the cellular composition of the tumor. METHODS We conducted genome-wide DNA methylation analysis and applied the DKFZ/Heidelberg CNS tumor classifier to FFPE tissue samples from a multi-sampling cohort with multiple samples per patient and a single-sampling cohort with one sample per patient. Only samples classified as GBM were included. We conducted two different methylation-based deconvolution analyses. Deconvolution 1 inferred the fractions of tumor subtypes (RTK_I, RTK_II, MES_TYP, and MES_ATYP) and cell types in the microenvironment. Deconvolution 2 estimated the abundance of malignant cell states (stem-like and differentiated cell components) and cell types in the microenvironment. RESULTS The multi-sampling cohort included 32 patients with 113 samples, and the single-sampling cohort included 87 patients with 87 samples. In the multi-sampling cohort, 8 patients (25%) exhibited intra-patient heterogeneity in methylation subclasses across their samples. Deconvolution 1 indicated that each tumor subclass component was heterogeneously distributed within samples, and the abundance of the myeloid cell component correlated with the MES_TYP component across samples. Deconvolution 2 similarly revealed heterogeneity in the immune component abundance across samples within patients. Integrating the results of Deconvolution 1 and Deconvolution 2 using the entire cohort revealed significant correlations between RTK_I and stem-like, RTK_II and differentiated, and MES_TYP and immune components. These findings were also replicated in TCGA dataset. CONCLUSIONS Our findings underscore the importance of considering intra-tumoral heterogeneity in methylation profiles and the biological characteristics of each methylation subclass in developing novel GBM therapies.

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.

EPCO-54. MULTIOMIC ANALYSIS OF SPINAL EPENDYMOMAS REVEALS MECHANISMS OF TUMOR AGGRESSIVENESS WITH MYCN AMPLIFICATION

Abstract Grade-3 spinal ependymomas, particularly those harboring MYCN amplification, have dismal prognoses despite optimal treatment regimens. MYCN functions as a transcription factor (TF) to drive the expression of key tumor driver genes. Our aim is to identify mechanisms underlying tumor aggressiveness and treatment resistance in MYCN-amplified ependymomas. MYCN amplification in spinal ependymomas was identified with custom next generation sequencing panels and bulk methylation analysis. We then conducted paired single-nucleus RNA (snRNA-seq) and chromatin accessibility (snATAC-seq) sequencing of grade-2 (n=2, 1 patient) and grade-3 spinal ependymomas (n=6, 6 patients, 4 MYCN-amplified). Comparator snRNA-seq datasets (grade-2/grade-3; 8/2) were included from a publicly available dataset (GSE163686; all non-MYCN-amplified). Canonical cell classes were identified with a combination of cluster- and cell-based strategies. Downstream analysis was conducted using R 4.3 (Seurat, Signac, clusterProfiler, CellChat), and Python 3.11 (scanpy) packages. We identified ependymoma tumor cells distinct from canonical immune cell classes with gene expression analysis and confirmed a distinct copy-number-variation pattern in these cells with chromosome 1 and 2p gain, and chromosome 6p and 22q loss. MYCN-amplified tumor cells (compared to non-MYCN-amplified grade2/3 tumor cells) showed increased differential accessibility at genes including VEGFA. Increased MYCN TF activity was confirmed with gene overexpression of downstream targets including NOTCH3, CREB5, PLK1, and VEGFA. Other genes that were highly accessible and overexpressed in MYCN-amplified tumor cells were related to oxidative stress, differentiation, and anti-apoptotic pathways. MYCN-amplified tumor cells modified the tumor microenvironment with increased VISTA signaling between MYCN-amplified tumor cells and tumor-associated macrophages, creating an immunosuppressive environment. We confirmed these effects by observing a higher proportion of M2-like macrophages in MYCN-amplified tumors (29.6% vs 18.2%). In conclusion, MYCN amplification in spinal ependymomas upregulates tumor cell proliferation, angiogenesis, and M2-like macrophage recruitment/programming.

PATH-53. DNA MUTATION SEQUENCING AND METHYLATION ANALYSIS OF SOMATICNF1 MUTANT IDH-WILDTYPE GLIOBLASTOMA IDENTIFIES THREE EPIGENETIC GROUPS ANDCDKN2A/B LOSS AS A NEGATIVE PROGNOSTIC BIOMARKER

Abstract The tumor suppressor NF1 is frequently mutated in IDH-wildtype glioblastoma. However, the molecular subgroups and clinically relevant biomarkers within somatic NF1 mutant, IDH-wildtype glioblastomas remain incompletely understood. Here, we combine methylation arrays and targeted DNA sequencing to identify epigenetic subgroups and clinical biomarkers within non-syndromic NF1 mutant IDH-wildtype glioblastoma. We identified 186 patients treated at the University of California San Francisco between 2016-2024 with newly diagnosed IDH-wildtype glioblastoma containing a somatic pathogenic NF1 mutation on a CLIA certified targeted DNA sequencing assay. Illumina EPIC DNA methylation arrays (n=129 patients) were processed using the minfi package and Heidelberg random forest classifier (v12.8). Downstream analyses including hierarchical clustering and survival analyses were performed in R. DNA sequencing identified 12 recurrently altered genes in greater than 10% of cases across cell cycle regulators (CDKN2A/B, TP53, RB1), PI3K signaling (PTEN, PIK3R1, PIK3CA), epigenetic modifiers (ATRX, SETD2), and growth factor signaling (EGFR, PDGFRA, PTPN11). DNA methylation analysis revealed three epigenetic subgroups. Group 1 showed lower median patient age and significantly increased TP53 co-mutation rate. Group 2 contained a significantly higher proportion of male patients and was significantly enriched for RB1 and TERT promoter alterations, with a majority of tumors classified as GBM-MES using the Heidelberg classifier. Group 3 showed a significantly higher proportion of female patients and was significantly enriched for homozygous CDKN2A/B deletion. Homozygous CDKN2A/B loss was associated with significantly worse overall survival across the entire non-syndromic NF1 mutant glioblastoma cohort but not in a propensity score matched NF1 wildtype glioblastoma cohort. DNA methylation profiling revealed three epigenetic subgroups characterized by distinct clinical features, co-mutation patterns, and reference DKFZ methylation classifier identities. CDKN2A/B loss may be a negative prognostic biomarker specifically in NF1 mutant glioblastomas; future multi-institutional studies are required to validate these findings and their therapeutic implications in a larger, representative cohort.

Water-Soluble Intracellular Polysaccharides (IPSW-2 to 4) from Phellinus igniarius Mycelia: Fractionation, Structural Elucidation, and Antioxidant Activity

Phellinus igniarius is a medicinal fungus. Nonetheless, research on its water-soluble intracellular polysaccharides (IPSW-2 to 4) fractionation, structural elucidation, and antioxidant activity is limited. In this study, water-soluble intracellular polysaccharides (IPSW-2 to 4) were extracted and fractionated from P. igniarius mycelia, and their antioxidant and structural properties were assessed using GC-FID, GC-MS, FTIR, and NMR spectroscopy (1H and 13C). In the water-eluted P. igniarius polysaccharide fractions (IPS30W, IPS60W, and IPS80W) of anion-exchange chromatography, the polysaccharide content was 79.05%, 68.25%, and 62.06%, with higher yields of 25.07%, 21.38%, and 20.34%, respectively. In contrast, the salt (NaCl) elution fractions (IPS30S1, IPS60S1, IPS60S2, and IPS80S1) of anion-exchange chromatography had lower polysaccharide content and yield. Hence, water elution fractions (IPS30W, IPS60W, and IPS80W) were selected for further purification. After repeated purification using size-exclusion chromatography, IPSW-2 to 4 were obtained with a yield of 8% to 15.83%. The IPSW-2 to IPSW-4 structures were elucidated, and they showed no triple helical conformation. Based on periodate oxidation, Smith degradation, methylation analysis, and 1H and 13C NMR spectroscopy, the primary structures of IPSW-2, IPSW-3, and IPSW-4 were all glucan, with the main chain consisting of (1→6)-α-D-Glcp, (1→3,4)-α-D-Glcp, and (1→3, 6)-α-D-Glcp, with α-D-Glcp as a side chain. Finally, antioxidant analysis showed that IPS30W, IPS60W, and IPS80W were all more capable of scavenging superoxide anions than the polysaccharides of Phyllostachys (13.8%) and floribunda (15.1%) at the same concentration (0.40 mg/mL). This will serve as a guide for the development of functional foods.

Methylation Patterns of Diabetes and Obesity Susceptibility Genes in Gestational Diabetes Mellitus: A Cross-Sectional Analysis from Karachi, Pakistan.

Background: Women with gestational diabetes mellitus (GDM) and their offspring have an increased risk of adverse perinatal and long-term health outcomes, which may be attributable to epigenetic modification of diabetes and obesity susceptibility genes. We aimed to investigate the methylation patterns of eight genes in GDM and normoglycemic (NG) mothers, and their respective offspring. Methods: This cross-sectional study, conducted at Aga Khan University from August 2019 to December 2022, recruited pregnant women in the first trimester of gestation from the outpatient obstetrics clinic. Participants were classified as NG or GDM based on the Society of Obstetricians and Gynecologists Pakistan. Venous blood samples were collected from mothers and cord blood from neonates. Peripheral blood mononuclear cells were used for DNA extraction and methylation analysis using methylation-specific PCR. Maternal and neonatal clinical data were recorded. Statistical analysis was performed using R, including binary logistic regression to assess the association between various gene methylation levels and GDM. Results: The study found that GDM mothers had significantly higher fasting blood glucose, 2-hr OGTT, and serum carboxymethyl lysine (CML) levels compared to NG mothers, but no significant differences in neonatal birth weight or serum CML levels. Chemerin methylation was significantly lower in GDM mothers and their babies, while NAMPT, MTNR1B, FNDC5, FAT4, and FTO methylation levels were higher in GDM offspring compared to NG offspring. GDM mothers also had higher methylation levels of brain-derived neurotrophic factor gene (BDNF). Multivariable binary logistic regression identified methylation levels of maternal BDNF and neonatal MTNR1B to be independently associated with GDM. Conclusions: Our study shows a trend of epigenetic modifications in both GDM mothers and their offspring in various genes related to metabolism and inflammation, suggesting an intergenerational transmission of increased risk of developing metabolic disorders. These findings emphasize the need for high throughput studies, early screening, tight glucose control during pregnancy, and postnatal follow-up to mitigate long-term health risks.

DNA methylation analysis of multiple genes in thymic epithelial tumors.

Aim: To investigate DNA methylation levels of a panel of genes in thymic epithelial tumors (TETs).Materials & methods: We selected 15 genes among the most promising epigenetic biomarkers of TETs and evaluated their methylation levels in 71 TET samples.Results: thymic carcinomas (TCs) showed hypermethylation of GHSR and ELF3 genes and reduced IL1RN methylation levels compared with thymomas (TMs) and healthy thymic tissues. RAG1 was hypomethylated in TMs compared with healthy thymic tissues. No difference in the methylation levels of the investigated genes was seen among TM stages and subtypes. No changes in blood methylation levels of the investigated genes were seen among TET subtypes.Conclusion: The present study confirms GHSR, ELF3, IL1RN and RAG1 as TET epigenetic biomarkers.

Blood and Bone-Derived DNA Methylation Ages Predict Mortality After Geriatric Hip Fracture: A Pilot Study.

The purpose of this study was to (1) perform the first analysis of bone-derived DNA methylation, (2) compare DNA methylation clocks derived from bone with those derived from whole blood, and (3) establish a relationship between DNA methylation age and 1-year mortality within the geriatric hip fracture population. Patients ≥65 years old who presented to a Level-I trauma center with a hip fracture were prospectively enrolled from 2020 to 2021. Preoperative whole blood and intraoperative bone samples were collected. Following DNA extraction, RRBS (reduced representation bisulfite sequencing) libraries for methylation clock analysis were prepared. Sequencing data were analyzed using computational algorithms previously described by Horvath et al. to build a regression model of methylation (biological) age for each tissue type. Student t tests were used to analyze differences (Δ) in methylation age versus chronological age. Correlation between blood and bone methylation ages was expressed using the Pearson R coefficient. Blood and bone samples were collected from 47 patients. DNA extraction, sequencing, and methylation analysis were performed on 24 specimens from 12 subjects. Mean age at presentation was 85.4 ± 8.65 years. There was no difference in DNA extraction yield between the blood and bone samples (p = 0.935). The mean follow-up duration was 12.4 ± 4.3 months. The mortality cohort (4 patients, 33%) showed a mean ΔAgeBone of 18.33 ± 6.47 years and mean ΔAgeBlood of 16.93 ± 4.02 years. In comparison, the survival cohort showed a significantly lower mean ΔAgeBone and ΔAgeBlood (7.86 ± 6.7 and 7.31 ± 7.71 years; p = 0.026 and 0.039, respectively). Bone-derived methylation age was strongly correlated with blood-derived methylation age (R = 0.81; p = 0.0016). Bone-derived DNA methylation clocks were found to be both feasible and strongly correlated with those derived from whole blood within a geriatric hip fracture population. Mortality was independently associated with the DNA methylation age, and that age was approximately 17 years greater than chronological age in the mortality cohort. The results of the present study suggest that prevention of advanced DNA methylation may play a key role in decreasing mortality following hip fracture. Prognostic Level I. See Instructions for Authors for a complete description of levels of evidence.

An Investigation of RNA Methylations with Biophysical Approaches in a Cervical Cancer Cell Model

RNA methylation adds a second layer of genetic information that dictates the post-transcriptional fate of RNAs. Although various methods exist that enable the analysis of RNA methylation in a site-specific or transcriptome-wide manner, whether biophysical approaches can be employed to such analyses is unexplored. In this study, Fourier-transform infrared (FT-IR) and circular dichroism (CD) spectroscopy are employed to examine the methylation status of both synthetic and cellular RNAs. The results show that FT-IR spectroscopy is perfectly capable of quantitatively distinguishing synthetic m6A-methylated RNAs from un-methylated ones. Subsequently, FT-IR spectroscopy is successfully employed to assess the changes in the extent of total RNA methylation upon the knockdown of the m6A writer, METTL3, in HeLa cells. In addition, the same approach is shown to accurately detect reduction in total RNA methylation upon the treatment of HeLa cells with tumor necrosis factor alpha (TNF-α). It is also demonstrated that m1A and m6A methylation induce quite a distinct secondary structure on RNAs, as evident from CD spectra. These results strongly suggest that both FT-IR and CD spectroscopy methods can be exploited to uncover biophysical properties impinged on RNAs by methyl moieties, providing a fast, convenient and cheap alternative to the existing methods.

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.

Genome-wide DNA methylation and mRNA transcription analysis revealed aberrant gene regulation pathways in dermatomyositis and polymyositis patients.

Genome-wide DNA methylation and mRNA transcription analysis revealed aberrant gene regulation pathways in dermatomyositis and polymyositis patients.

Best practices for germline variant and DNA methylation analysis of second- and third-generation sequencing data.

This comprehensive review provides insights and suggested strategies for the analysis of germline variants using second- and third-generation sequencing technologies (SGS and TGS). It addresses the critical stages of data processing, starting from alignment and preprocessing to quality control, variant calling, and the removal of artifacts. The document emphasized the importance of meticulous data handling, highlighting advanced methodologies for annotating variants and identifying structural variations and methylated DNA sites. Special attention is given to the inspection of problematic variants, a step that is crucial for ensuring the accuracy of the analysis, particularly in clinical settings where genetic diagnostics can inform patient care. Additionally, the document covers the use of various bioinformatics tools and software that enhance the precision and reliability of these analyses. It outlines best practices for the annotation of variants, including considerations for problematic genetic alterations such as those in the human leukocyte antigen region, runs of homozygosity, and mitochondrial DNA alterations. The document also explores the complexities associated with identifying structural variants and copy number variations, underscoring the challenges posed by these large-scale genomic alterations. The objective is to offer a comprehensive framework for researchers and clinicians, ensuring that genetic analyses conducted with SGS and TGS are both accurate and reproducible. By following these best practices, the document aims to increase the diagnostic accuracy for hereditary diseases, facilitating early diagnosis, prevention, and personalized treatment strategies. This review serves as a valuable resource for both novices and experts in the field, providing insights into the latest advancements and methodologies in genetic analysis. It also aims to encourage the adoption of these practices in diverse research and clinical contexts, promoting consistency and reliability across studies.

LINE-1 cfDNA Methylation as an Emerging Biomarker in Solid Cancers

Epigenetic dysregulation is a hallmark of many human malignancies, with DNA methylation being a primary mechanism influencing gene expression and maintaining genomic stability. Genome-wide hypomethylation, characteristic of many cancers, is partly attributed to the demethylation of repetitive elements, including LINE-1, a prevalent non-LTR retrotransposon. The methylation status of LINE-1 is closely associated with overall genomic methylation levels in tumors. cfDNA comprises extracellular DNA fragments found in bodily fluids such as plasma, serum, and urine, offering a dynamic snapshot of the genetic and epigenetic landscape of tumors. This real-time sampling provides a minimally invasive avenue for cancer diagnostics, prognostics, and monitoring. The methylation status of LINE-1 in cfDNA has emerged as a promising biomarker, with several studies highlighting its potential in diagnosing and predicting outcomes in cancer patients. Recent research also suggests that cfDNA-based LINE-1 methylation analysis could serve as a valuable tool in evaluating the efficacy of cancer therapies, including immunotherapy. The growing clinical significance of cfDNA calls for a closer examination of its components, particularly repetitive elements like LINE-1. Despite their importance, the role of LINE-1 elements in cfDNA has not been thoroughly gauged. We aim to address this gap by reviewing the current literature on LINE-1 cfDNA assays, focusing on their potential applications in diagnostics and disease monitoring.

Comparative Analysis of Two Soybean Cultivars Revealed Tolerance Mechanisms Underlying Soybean Adaptation to Flooding

Flooding stress poses a significant challenge to soybean cultivation, impacting plant growth, development, and ultimately yield. In this study, we investigated the responses of two distinct soybean cultivars: flooding-tolerant Nanxiadou 38 (ND38) and flooding-sensitive Nanxiadou 45 (ND45). To achieve this, healthy seedlings were cultivated with the water surface consistently maintained at 5 cm above the soil surface. Our objective was to elucidate the physiological and molecular adaptations of the two cultivars. Under flooding stress, seedlings of both cultivars exhibited significant dwarfing and a notable decrease in root length. While there were no significant differences in the dry weight of aboveground shoots, the dry weight of underground shoots in ND38 was strikingly decreased following flooding. Additionally, total chlorophyll content decreased significantly following flooding stress, indicating impaired photosynthetic performance of the cultivars. Moreover, malondialdehyde (MDA) levels increased significantly after flooding, particularly in the ND45 cultivar, suggesting heightened oxidative stress. Expression analysis of methylation and demethylation genes indicated that MET1 and DME play crucial roles in response to flooding stress in soybeans. Meanwhile, analysis of the hemoglobin family (GLBs), aquaporin family (AQPs), glycolytic pathway-related genes, and NAC transcription factor-related genes identified GLB1-1 and GLB1-2, GLB2-2, PIP2-6, PIP2-7, TIP2-2, TIP4-1, TIP5-1, Gm02G222400 (fructose-bisphosphate aldolase), Gm19G017200 (glucose-6-phosphate isomerase), and Gm04G213900 (alcohol dehydrogenase 1) as key contributors to flooding tolerance in both soybean cultivars. These findings provide crucial insights into the physiological and molecular mechanisms underlying flooding tolerance in soybeans, which could guide future molecular breeding strategies for the development of flooding-tolerant soybean cultivars.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Epigenetic Landscape of DNA Methylation in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, characterized by its aggressive progression and dismal prognosis. Advances in epigenetic profiling, specifically DNA methylation analysis, have significantly deepened our understanding of PDAC pathogenesis. This review synthesizes findings from recent genome-wide DNA methylation studies, which have delineated a complex DNA methylation landscape differentiating between normal and cancerous pancreatic tissues, as well as across various stages and molecular subtypes of PDAC. These studies identified specific differentially methylated regions (DMRs) that not only enhance our grasp of the epigenetic drivers of PDAC but also offer potential biomarkers for early diagnosis and prognosis, enabling the customization of therapeutic approaches. The review further explores how DNA methylation profiling could facilitate the development of subtype-tailored therapies, potentially improving treatment outcomes based on precise molecular characterizations. Overall, leveraging DNA methylation alterations as functional biomarkers holds promise for advancing our understanding of disease progression and refining PDAC management strategies, which could lead to improved patient outcomes and a deeper comprehension of the disease’s underlying biological mechanisms.

Proximal and classic epithelioid sarcomas are distinct molecular entities defined by MYC/GATA3 and SOX17/endothelial markers, respectively

Epithelioid sarcoma (ES) is a rare tumor hallmarked by the loss of INI1/SMARCB1 expression. Apart from this alteration, little is known about the biology of ES. Despite recent advances in treatment, the prognosis of ES remains unsatisfactory. To elucidate the molecular underpinnings of ES, and to identify diagnostic biomarkers and potential therapeutic vulnerabilities, we performed an integrated omics profiling (RNA sequencing and methylation array) of 24 primary, untreated ESs. Transcriptome and methylome analysis identified two distinct molecular clusters that essentially corresponded to the morphologic variants of ES, classic ES (C-ES) and the more aggressive proximal ES (P-ES). The P-ES group was characterized by hyperactivation of GATA3 and MYC pathways, with extensive epigenetic rewiring associated with EZH2 overexpression. Both DNA methylation and gene expression analysis indicated a striking similarity with the “MYC subgroup” of ATRT, another SMARCB1-deficient tumor, implying a shared molecular background and potential therapeutic vulnerabilities. Conversely, the C-ES group exhibited an endothelial-like molecular profile, with expression of vascular genes and elevated pro-angiogenic SOX17 signaling. Immunohistochemistry validated the overexpression of the chromatin regulators GATA3 (9/12 vs. 0/16) and EZH2 (7/7 vs. 2/6) in P-ESs, and of the vascular factors SOX17 (8/8 vs. 1/10) and N-cadherin (5/9 vs 0/10) in C-ESs. Therefore, these molecules emerge as potential diagnostic tools to fill the gap represented by the lack of ES subtype-specific biomarkers. In summary, our study shows that P-ES and C-ES represent distinct molecular entities defined by MYC/GATA3 and SOX17/endothelial molecular traits, respectively. Besides providing insights into the biology of ES, our study pinpoints subtype-specific biomarkers and potential therapeutic vulnerabilities.

Separation, purification, structure characterization, and immune activity of a polysaccharide from Alocasia cucullata obtained by freeze-thaw treatment

In this study, ACP-1, a water-soluble polysaccharide was isolated from the roots of Alocasia cucullata (AC) using freeze-thaw treatment (FTT). Structural characterization revealed ACP-1 (2.10×105 Da) to be a homogeneous heteropolysaccharide primarily consisting of glucose, galactose, and arabinose, and a trace of fucose, rhamnose, and glucuronic acid. Methylation analysis and nuclear magnetic resonance spectroscopy revealed that the backbone of ACP-1 consisted of →[3)-β-D-Galp-(1]4→3,6)-β-D-Glcp-(1→3,6)-β-D-Glcp-(1→, with a branch at C-3. In vitro experiments demonstrated that ACP-1 significantly enhanced the proliferation and phagocytosis of RAW264.7 cells, upregulated the expression of co-stimulatory molecules (CD80 and CD86), and activated RAW264.7 cells via the nuclear factor kappa-B signal transduction pathway, resulting in nitric monoxide release and the secretion of the cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. Overall, ACP-1 shows potential as a novel immunostimulant.

Methylation aberrations in partner spermatozoa and impaired expression of imprinted genes in the placentae of early-onset preeclampsia

IntroductionDisturbed paternal epigenetic status of imprinted genes has been observed in infertility and recurrent spontaneous abortions. Shallow placentation has been associated with early-onset preeclampsia. Hence, the present study aimed to investigate the methylation patterns of imprinted genes involved in placental development, in the spermatozoa of partners of women experiencing preeclampsia. MethodsThe study involved recruitment of couples into preeclampsia (n = 14) and control (n = 25) groups. Methylation analysis of imprinted gene differentially methylated regions (DMRs) and LINE1 repetitive element was carried out by pyrosequencing in the spermatozoa and placental villi. Global 5mC levels in the spermatozoa were measured through ELISA. Expression of imprinted genes was quantified in the placental villi by real time qPCR. Association of birth weight with DNA methylation and gene expression was assessed. ResultsKvDMR, PEG3 DMR, PEG10 DMR and DLK1-GTL2 IG-DMR were differentially methylated in the spermatozoa and placental villi of preeclampsia group. Global 5mC content and LINE1 methylation levels did not differ between the spermatozoa of the two groups. Increased transcript levels of PEG3, IGF2, DLK1, PHLDA2 and CDKN1C were observed in the preeclamptic placental villi. Birth weight showed significant association with KvDMR, PEG10 DMR, DLK1-GTL2 IG-DMR and LINE1 methylation levels in the spermatozoa. DLK1 expression levels showed a negative association with birth weight. DiscussionThe study highlighted the paternal contribution to early-onset preeclampsia, in the form of disrupted sperm DNA methylation patterns at imprinted gene loci. These loci, after further evaluation in future studies, could serve as sperm-based preeclampsia predictive markers, for couples planning pregnancy.

Clinicopathological and epigenetic differences between primary neuroendocrine tumors and neuroendocrine metastases in the ovary.

Currently, the available literature provides insufficient support to differentiate between primary ovarian neuroendocrine tumors (PON) and neuroendocrine ovarian metastases (NOM) in patients. For this reason, patients with a well-differentiated ovarian neuroendocrine tumor (NET) were identified through electronic patient records and a nationwide search between 1991 and 2023. Clinical characteristics were collected from electronic patient files. This resulted in the inclusion of 71 patients with NOM and 17 patients with PON. Histologic material was stained for Ki67, SSTR2a, CDX2, PAX8, TTF1, SATB2, ISLET1, OTP, PDX1, and ARX. DNA methylation analysis was performed on a subset of cases. All PON were unilateral and nine were found within a teratoma (PON-T+). A total of 78% of NOM were bilateral, and none were associated with a teratoma. PON without teratomous components (PON-T-) displayed a similar insular growth pattern and immunohistochemistry as NOM (p > 0.05). When compared with PON-T+, PON-T- more frequently displayed ISLET1 positivity and were larger, and patients were older at diagnosis (p < 0.05). Unsupervised analysis of DNA methylation profiles from tumors of ovarian (n = 16), pancreatic (n = 22), ileal (n = 10), and rectal (n = 7) origin revealed that four of five PON-T- clustered together with NOM and ileal NET, whereas four of five PON-T+ grouped with rectum NET. In conclusion, unilateral ovarian NET within a teratoma should be treated as a PON. Ovarian NET localizations without teratomous components have a molecular profile analogous to midgut NET metastases. For these patients, a thorough review of imaging should be performed to identify a possible undetected midgut NET and a corresponding follow-up strategy may be recommended.