Distinct DNA Methylation Patterns Research Articles

Molecular subgroups of T-cell acute lymphoblastic leukemia in adults treated according to pediatric-based GMALL protocols.

In contrast to B-cell precursor acute lymphoblastic leukemia (ALL), molecular subgroups are less well defined in T-lineage ALL. Comprehensive studies on molecular T-ALL subgroups have been predominantly performed in pediatric ALL patients. Currently, molecular characteristics are rarely considered for risk stratification. Herein, we present a homogenously treated cohort of 230 adult T-ALL patients characterized on transcriptome, and partly on DNA methylation and gene mutation level in correlation with clinical outcome. We identified nine molecular subgroups based on aberrant oncogene expression correlating to four distinct DNA methylation patterns. The subgroup distribution differed from reported pediatric T-ALL cohorts with higher frequencies of prognostic unfavorable subgroups like HOXA or LYL1/LMO2. A small subset (3%) of HOXA adult T-ALL patients revealed restricted expression of posterior HOX genes with aberrant activation of lncRNA HOTTIP. With respect to outcome, TLX1 (n = 44) and NKX2-1 (n = 4) had an exceptionally favorable 3-year overall survival (3y-OS) of 94%. Within thymic T-ALL, the non TLX1 patients had an inferior but still good prognosis. To our knowledge this is the largest cohort of adult T-ALL patients characterized by transcriptome sequencing with meaningful clinical follow-up. Risk classification based on molecular subgroups might emerge and contribute to improvements in outcome.

Circulating-tumour DNA methylation of HAND1 gene: a promising biomarker in early detection of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the significant global health concerns with an increase in cases. Regular screening tests are crucial for early detection as it is often asymptomatic in the initial stages. Liquid biopsies, a non-invasive approach that examines biomarkers in biofluids, offer a promising future in diagnosing and screening cancer. Circulating-tumour DNA (ctDNA) is the genetic material in biofluids released into the circulatory system by cells. ctDNA is a promising marker for monitoring patients since cancer cells display distinct DNA methylation patterns compared to normal cells. The potential of our research to contribute to early detection and improved patient outcomes is significant.AimsThe primary objective of this research project was to explore the HAND1 methylation levels in plasma ctDNA as a potential biomarker for diagnosing CRC and evaluate the methylation level of the well-established gene SPET9 to compare it with the methylation level of HAND1.Materials and methodsPlasma samples were collected from 30 CRC patients and 15 healthy individuals, with CRC samples obtained pre-treatment. ctDNA was extracted and treated with bisulfite for methylation status assessment. Quantitative methylation-specific PCR (qMS-PCR) was performed for HAND1 and SEPT9, using β-actin (ACTB gene) as a reference. The study aims to evaluate the potential of these genes as diagnostic biomarkers for CRC, contributing to early detection and improved patient outcomes.ResultsOur study yielded significant results: 90% of CRC patients (27 out of 30) had hypermethylation in the SEPT9 gene, and 83% (25 out of 30) exhibited hypermethylation in the HAND1 gene. The methylation levels of both genes were significantly higher in CRC patients than in healthy donors. These findings underscore the potential of SEPT9 and HAND1 methylation as promising biomarkers for diagnosing CRC, potentially leading to early detection and improved patient outcomes.ConclusionThese findings highlight the potential of SEPT9 and HAND1 methylation as promising biomarkers for diagnosing CRC. However, further research and validation studies are needed to confirm these findings and to explore their clinical utility in CRC diagnosis and management.

Multi-omics cluster defines the subtypes of CRC with distinct prognosis and tumor microenvironment

BackgroundColorectal cancer (CRC) is a complex malignancy characterized by diverse molecular profiles, clinical outcomes, and limited precision in prognostic markers. Addressing these challenges, this study utilized multi-omics data to define consensus molecular subtypes in CRC and elucidate their association with clinical outcomes and underlying biological processes.MethodsConsensus molecular subtypes were obtained by applying ten integrated multi-omics clustering algorithms to analyze TCGA-CRC multi-omics data, including mRNA, lncRNA, miRNA, DNA methylation CpG sites, and somatic mutation data. The association of subtypes with prognoses, enrichment functions, immune status, and genomic alterations were further analyzed. Next, we conducted univariate Cox and Lasso regression analyses to investigate the potential prognostic application of biomarkers associated with multi-omics subtypes derived from weighted gene co-expression network analysis (WGCNA). The function of one of the biomarkers MID2 was validated in CRC cell lines.ResultsTwo CRC subtypes linked to distinct clinical outcomes were identified in TCGA-CRC cohort and validated with three external datasets. The CS1 subtype exhibited a poor prognosis and was characterized by higher tumor-related Hallmark pathway activity and lower metabolism pathway activity. In addition, the CS1 was predicted to have less immunotherapy responder and exhibited more genomic alteration compared to CS2. Then a prognostic model comprising five genes was established, with patients in the high-risk group showing substantial concordance with the CS1 subtype, and those in the low-risk group with the CS2 subtype. The gene MID2, included in the prognostic model, was found to be correlated with epithelial–mesenchymal transition (EMT) pathway and distinct DNA methylation patterns. Knockdown of MID2 in CRC cells resulted in reduced colony formation, migration, and invasion capacities.ConclusionThe integrative multi-omics subtypes proposed potential biomarkers for CRC and provided valuable knowledge for precision oncology.

Abstract 2437: Distinct DNA methylation patterns in male urogenital cancers

Abstract Background: Male urogenital cancers including prostate cancer (PCa), bladder cancer (BLCA) and kidney renal cancer (KIRC), account for 38% of the total number of new male cancers. Previous studies have shown that changes in DNA methylation patterns are associated with the occurrence and progression of these cancers. Here, we performed whole-genome bisulfite methylation sequencing (WGBS) on tissue samples from these cancers to comprehensively characterize the methylation patterns to provide insights into the potential biological functions of methylation and to identify possible early detection biomarkers. Methods: Tissue samples were collected from patients with PCa, BLCA and KIRC who had not received any treatment prior to surgery from Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine. Samples with the cancer cells content ≥30% in cancer tissues and no cancer cells in the adjacent tissues was deemed qualified. A total of 43 samples were sequenced by whole-genome bisulfite methylation sequencing (WGBS) with a depth of 30X, including PCa, BLCA, KIRC cancer tissues, and their adjacent cancer tissues. Results: Distinct methylation patterns were observed in the 3 cancers. By comparing cancer and adjacent tissues, the numbers of differentially methylated sites (DMSs) were 21,195 (31% hyperDMSs and 69% hypoDMSs), 587,010 (23% hyperDMSs and 77% hypoDMSs) and 7,855,162 (5% hyperDMSs and 95% hypoDMSs) in PCa, BLCA and KIRC, respectively. The distribution of hyperDMSs and hypoDMSs across various chromosomes was relatively consistent in KIRC and BLCA. However, in PCa, the hypoDMSs were mainly located on chromosome 8 (5265/14666, 36%). Among the three cancers, only 2-3% of hypoDMSs were located in gene promoter regions, while the proportions of hyperDMSs in gene promoter regions were higher with 4%, 8% and 17% for PCa, BLCA and KIRC, respectively. Among the above DMSs, 1901 DMSs-related genes were shared by PCa, BLCA and KIRC and 251, 4252, 2475 DMSs-related genes were unique in PCa, BLCA and KIRC, respectively. Gene Ontology analysis revealed that the shared DMSs-related genes were enriched in Wnt signaling pathway and positive regulation of MAPK cascade. Conclusions: Our present study demonstrated the differential methylation patterns in male urogenital cancers, which may provide insights into the potential biological functions of methylation and to identify possible early detection biomarkers. Citation Format: Guopeng Yu, Jiangyi Wang, Long Li, Wenchuan Xie, Yushan Liu, Qing Yang, Bin Xu. Distinct DNA methylation patterns in male urogenital cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2437.

Abstract 7005: Distinct DNA methylation patterns between early-onset and average-onset colorectal cancer patients

Abstract Objective: Early onset colorectal cancer (EO CRC) is rising at an alarming rate. While average onset colorectal cancer (AO CRC) affects patients mostly above 65 years of age, EO CRC affects patients under 50 years of age, disproportionately affecting males from low-income communities and racial/ethnic minorities. Chronic inflammation has been identified as a strong contributor to pathogenesis. We hypothesize that methylation of DNA may serve as a potential mechanism associated with chronic inflammation that contributes to the etiology differentially between EO and AO CRC. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue specimens from four AO and EO CRC patients each were retrieved from the Tissue Repository. Paraffin curls containing tumor and adjacent normal tissues were generated from appropriate EO and AO CRC patient tissues. The Qiagen GeneRead DNA FFPE genomic DNA kit was then used to extract the DNA from the paraffin curls. Illumina Methylation EPIC v2.0 kit was used to evaluate DNA methylation (DNAm) patterns after the restoration process from deamination in the fixed/embedded sample using the Uracil-N-Glycosylase enzyme. Unreliable and non-specific probes were removed before statistical analysis for differentially methylated CpG sites. Age acceleration for each tissue was calculated by comparing the estimated DNAm age with chronological age using the Horvath approach. Results: A clear clustering of DNAm patterns by age of onset were observed for both normal and tumor tissues. However, there was no difference when comparing tumor and normal tissues after multiple corrections. Four-way ANCOVA adjusting for race, gender, BMI, and tissue type suggested that age onset was the most significant factor. We also found that age acceleration in EO CRC tissue is greater than in AO CRC. After applying Bonferroni correction, significant differentially methylated regions (DMRs) were found in the promoter and the gene body of SNTB1, NFATC3, SLC25A24, RABGAP1L, WDSUB1, WWOX, SELB, SLC28A3, and PPP6R2 in EO CRC patients when compared to AO CRC. SNTB1 regulates colorectal cancer progression and stemness, while NFATC3 has been associated with inflammation and cancer. Conclusions: The preliminary results from this study showed a significant difference in the DNAm profile of FFPE samples between AO and EO CRC. The finding suggests that epigenetics may play a role in the etiology of CRC, potentially involved in the chronic inflammation pathway. The analysis of a larger patient number is underway to strengthen these findings. Citation Format: L. Joseph Su, Ping-Ching Hsu, Aniruddha Rathod, Yulun Liu, Jacopo Ferruzzi, Syed Kazmi, Emina Huang. Distinct DNA methylation patterns between early-onset and average-onset colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7005.

Revealing distinct DNA methylation patterns in hepatic carcinoma through high-throughput sequencing

Abstract Objectives To study the relationship between DNA methylation and tumour development and provide experimental evidence for the personalized diagnosis and treatment of hepatic carcinoma. Methods The DNA of hepatic carcinoma tissue (Ca group) and adjacent normal tissue (T group) were extracted using the phenol-chloroform method and then treated with bisulfite. Twenty-five genes including 45 subtypes were amplified by PCR. The PCR products were sequenced via the Illumina 450k methylation array assay. The changes of methylated DNA performance were analysed through principal component analysis (PCA). Cluster analysis was used to evaluate the classification of methylated DNA regions. Haplotype abundance variation was tested for methylation differences. Statistical analysis was performed using the chi-square (χ2) test or Fisher’s exact test. Results Sequencing discoveries indicated CG-type methylation pervading all amplicons. However, CHG-type and CHH-type methylations were confined to only four amplicons (or nine subtypes). The methylation ratios of three specific amplicons (DAB2IP, PRDM14-1, Rab31-1) out of 45 amplicon subtypes in the Ca group significantly increased (over 10 %) compared to the T group (p<0.05). Nineteen amplicons demonstrated minor distinction (methylation pattern variations between 1 and 10 %), with the remaining 23 amplicons showing only minimal disparities (under 1 %). PCA and cluster analysis unveiled a marked difference in methylation levels between cancerous and healthy tissues (p<0.05). Conclusions The changes in haplotypes and methylation sites could serve as a biomarker for the clinical diagnosis of hepatic carcinoma. Methylation patterns might play an important role in the occurrence and development of hepatic carcinoma.

(060) Epigenetic and Gene Expression Profiles in Non-Plaque Tissues from Men with Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) can significantly impact sexual function. A hereditary predisposition to PD suggests that genetic variants may exist, and gene expression may modulate the penetrance of the disease. Identification of changes in epigenetic and gene expression profiles will facilitate a deeper understanding of the disease and provide insight for more targeted therapies. Objective Our objective is to determine if PD is correlated with epigenetic variations and/or gene expression changes, as indicated by DNA methylation assessment and RNA sequencing. Methods Penile tunica albuginea samples (not from fibrotic plaques) were obtained from men with PD undergoing penile prosthesis placement. Control samples were collected from men with erectile dysfunction (ED) without PD undergoing penile prosthesis placement. DNA methylation analyses were performed on homogenized penile tissue samples via an Illumina Human MethylationEPIC BeadChip v2 array. Using the minfi package in R, beta values were produced for all 936,990 CpG sites for each sample and SWAN normalization was applied. Differentially methylated regions (DMR) were found via USEQ with a threshold Wilcoxon FDR score of 40. Gene expression levels were examined in PD and ED samples via RNA-sequencing. Gene expression data was compared against epigenetic data to determine if any epigenetics changes identified affected gene expression profiles. Results A total of 24 tissue samples were collected: 12 ED and 12 PD. USEQ yielded 36 total DMRs with an FDR score of 40 or greater (p-value<0.0001). Of these 36 DMRs, a Stanford GREAT analysis revealed 60 total gene-region associations and five implicated biological processes: anterior/posterior pattern specification, chordate embryonic development, embryo development (ending in birth or egg hatching), somitogenesis, and pattern specification process. Furthermore, differential gene expression analysis identified 76 genes with significant expression changes between PD and ED groups, of which 17 genes were upregulated while 59 were downregulated. Long noncoding RNAs and the keratocan gene showed notable expression differences between PD and ED groups. GO enrichment analysis revealed significant terms related to DNA binding and protein binding. None of the differentially regulated genes overlapped with identified DMRs. Conclusions This study identified distinct gene expression and DNA methylation patterns between PD and ED groups, despite PD samples not being taken from plaque regions, suggesting that more systemic changes may be present. Upregulated genes in PD included long noncoding RNAs and the keratocan gene, suggesting their involvement in disease pathogenesis. Long noncoding RNA overexpression may serve a regulatory function in gene expression or cell processes while overexpression of the keratocan gene may promote collagen accumulation and remodeling, leading to fibrous plaque formation. Likewise, DMRs and their region-gene associations suggest a role in early embryonic development for pathogenesis. Although RNA-seq did not indicate any significant differentially expressed genes that matched with gene-region associations correlated with DMRs, the significance and quantity of DMRs between ED and PD sample groups is indicative of affected biological processes that manifest in distinct pathophysiologies. Further research is needed to investigate if differentially regulated genes exist in DMRs that exhibited changes but did not meet the criteria threshold for inclusion here. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Contraline, Techfields Inc, Alto Neurosciences, Curebase.

Comparative hepatotoxicity of novel lithium bis(trifluoromethanesulfonyl)imide (LiTFSI, ie. HQ-115) and legacy Perfluorooctanoic acid (PFOA) in male mice: Insights into epigenetic mechanisms and pathway-specific responses

Lithium Bis(trifluoromethanesulfonyl)imide (LiTFSI ie. HQ-115), a polymer electrolyte used in energy applications, has been detected in the environment, yet its health risks and environmental epigenetic effects remain unknown. This study aims to unravel the potential health risks associated with LiTFSI, investigate the role of DNA methylation-induced toxic mechanisms in its effects, and compare its hepatotoxic impact with the well-studied Perfluorooctanoic Acid (PFOA). Using a murine model, six-week-old male CD1 mice were exposed to 10 and 20 mg/kg/day of each chemical for 14 days as 14-day exposure and 1 and 5 mg/kg/day for 30 days as 30-day exposure. Results indicate that PFOA exposure induced significant hepatotoxicity, characterized by liver enlargement, and elevated serum biomarkers. In contrast, LiTFSI exposure showed lower hepatotoxicity, accompanied by mild liver injuries. Despite higher bioaccumulation of PFOA in serum, LiTFSI exhibited a similar range of liver concentrations compared to PFOA. Reduced Representative Bisulfite Sequencing (RRBS) analysis revealed distinct DNA methylation patterns between 14-day and 30-day exposure for the two compounds. Both LiTFSI and PFOA implicated liver inflammatory pathways and lipid metabolism. Transcriptional results showed that differentially methylated regions in both exposures are enriched with cancer/disease-related motifs. Furthermore, Peroxisome proliferator-activated receptor alpha (PPARα), a regulator of lipid metabolism, was upregulated in both exposures, with downstream genes indicating potential oxidative damages. Overall, LiTFSI exhibits distinct hepatotoxicity profiles, emphasizing the need for comprehensive assessment of emerging PFAS compounds.

Tissue of origin prediction for cancer of unknown primary using a targeted methylation sequencing panel

RationaleCancer of unknown primary (CUP) is a group of rare malignancies with poor prognosis and unidentifiable tissue-of-origin. Distinct DNA methylation patterns in different tissues and cancer types enable the identification of the tissue of origin in CUP patients, which could help risk assessment and guide site-directed therapy.MethodsUsing genome-wide DNA methylation profile datasets from The Cancer Genome Atlas (TCGA) and machine learning methods, we developed a 200-CpG methylation feature classifier for CUP tissue of origin prediction (MFCUP). MFCUP was further validated with public-available methylation array data of 2977 specimens and targeted methylation sequencing of 78 Formalin‐fixed paraffin‐embedded (FFPE) samples from a single center.ResultsMFCUP achieved an accuracy of 97.2% in a validation cohort (n = 5923) representing 25 cancer types. When applied to an Infinium 450 K array dataset (n = 1052) and an Infinium EPIC (850 K) array dataset (n = 1925), MFCUP achieved an overall accuracy of 93.4% and 84.8%, respectively. Based on MFCUP, we established a targeted bisulfite sequencing panel and validated it with FFPE sections from 78 patients of 20 cancer types. This methylation sequencing panel correctly identified tissue of origin in 88.5% (69/78) of samples. We also found that the methylation levels of specific CpGs can distinguish one cancer type from others, indicating their potential as biomarkers for cancer diagnosis and screening.ConclusionOur methylation-based cancer classifier and targeted methylation sequencing panel can predict tissue of origin in diverse cancer types with high accuracy.

DNA-methylome-derived epigenetic fingerprint as an immunophenotype indicator of durable clinical immunotherapeutic benefits in head and neck squamous cell carcinoma.

Cancer immunotherapy provides durable response and improves survival in a subset of head and neck squamous cell carcinoma (HNSC) patients, which may due to discriminative tumor microenvironment (TME). Epigenetic regulations play critical roles in HNSC tumorigenesis, progression, and activation of functional immune cells. This study aims to identify an epigenetic signature as an immunophenotype indicator of durable clinical immunotherapeutic benefits in HNSC patients. Unsupervised consensus clustering approach was applied to distinguish immunophenotypes based on five immune signatures in The Cancer Genome Atlas (TCGA) HNSC cohort. Two immunophenotypes (immune 'Hot' and immune 'Cold') that had different TME features, diverse prognosis, and distinct DNA methylation patterns were recognized. Immunophenotype-related methylated signatures (IPMS) were identified by the least absolute shrinkage and selector operation algorithm. Additionally, the IPMS score by deconvolution algorithm was constructed as an immunophenotype classifier to predict clinical outcomes and immunotherapeutic response. The 'Hot' HNSC immunophenotype had higher immunoactivity and better overall survival (p = 0.00055) compared to the 'Cold' tumors. The immunophenotypes had distinct DNA methylation patterns, which was closely associated with HNSC tumorigenesis and functional immune cell infiltration. 311 immunophenotype-related methylated CpG sites (IRMCs) was identified from TCGA-HNSC dataset. IPMS score model achieved a strong clinical predictive performance for classifying immunophenotypes. The area under the curve value (AUC) of the IPMS score model reached 85.9% and 89.8% in TCGA train and test datasets, respectively, and robustness was verified in five HNSC validation datasets. It was also validated as an immunophenotype classifier for predicting durable clinical benefits (DCB) in lung cancer patients who received anti-PD-1/PD-L1 immunotherapy (p = 0.017) and TCGA-SKCM patients who received distinct immunotherapy (p = 0.033). This study systematically analyzed DNA methylation patterns in distinct immunophenotypes to identify IPMS with clinical prognostic potential for personalized epigenetic anticancer approaches in HNSC patients. The IPMS score model may serve as a reliable epigenome prognostic tool for clinical immunophenotyping to guide immunotherapeutic strategies in HNSC.

(059) Epigenetic Profiles of Men with Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) can significantly impact sexual function. A hereditary predisposition to PD suggests that genetic variants may exist, and gene expression may modulate the penetrance of the disease. Identification of changes in epigenetic or gene expression profiles would facilitate a deeper understanding of the disease and provide insight for more targeted therapies. Objective Our objective is to determine if predisposition to PD is correlated with epigenetic variations and/or gene expression changes, as indicated by DNA methylation assessment and RNA sequencing. Methods Penile tunica albuginea samples were obtained from men with PD undergoing penile prosthesis placement. Penile sample from these men were not from fibrotic plaques. Control samples were collected from men with erectile dysfunction (ED) without PD undergoing penile prosthesis placement. DNA methylation analyses were performed on homogenized penile tissue samples via an Illumina Human MethylationEPIC BeadChip v2 array. Using the minfi package in R, beta values were produced for all 936,990 CpG sites for each sample and SWAN normalization was applied. Differentially methylated regions (DMR) were found via USEQ with a threshold Wilcoxon FDR score of 40. Gene expression levels were examined in PD and ED samples via RNA-sequencing. Gene expression data was compared against epigenetic data to determine if any epigenetics changes identified affected gene expression profiles. Results A total of 24 tissue samples were collected: 12 ED and 12 PD. USEQ yielded 36 total DMRs with a FDR score of 40 or greater (p-value<0.0001). Of these 36 DMRs, a Stanford GREAT analysis revealed 60 total gene-region associations and five implicated biological processes: anterior/posterior pattern specification, chordate embryonic development, embryo development (ending in birth or egg hatching), somitogenesis, and pattern specification process. Furthermore, differential gene expression analysis identified 76 genes with significant expression changes between PD and ED groups, of which 17 genes were upregulated while 59 were downregulated. Long noncoding RNAs and the keratocan gene showed notable expression differences between PD and ED groups. GO enrichment analysis revealed significant terms related to DNA binding and protein binding. None of the differentially regulated genes overlapped with identified DMRs. Conclusions This study identified distinct gene expression and DNA methylation patterns between PD and ED groups, despite PD samples not being taken from plaque regions. Upregulated genes in PD included long noncoding RNAs and the keratocan gene, suggesting their involvement in disease pathogenesis. Long noncoding RNA overexpression may serve a regulatory function in gene expression or cell processes while overexpression of the keratocan gene may promote collagen accumulation and remodeling, leading to fibrous plaque formation in the affected area. Likewise, DMRs and their region-gene associations suggest a role in early embryonic development for pathogenesis. Although RNA-seq did not indicate any significant differentially expressed genes that matched with gene-region associations correlated with DMRs, the significance and quantity of DMRs between ED and PD sample groups is indicative of affected biological processes that manifest in distinct pathophysiologies. Further research is needed to investigate if differentially regulated genes exist in DMRs that exhibited changes but did not meet the criteria threshold for inclusion here. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Paterna bio, Firmtech, Turtle health, Maximus, Carrot, Contraline, endo pharmaceuticals, inherent biosciences, techfields inc, macro trials, alto neurosciences, curebase, vivify medical.

Unveiling DNA methylation in Alzheimer's disease: a review of array-based human brain studies.

The intricacies of Alzheimer's disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms, particularly DNA methylation. This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer's disease neuropathology. The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer's disease progression. The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus. Notably, ANK1 hypermethylation, a protein implicated in neurofibrillary tangle formation, was recurrently identified in the entorhinal cortex. Further, the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3, RHBDF2, and MCF2L, potentially influencing neuroinflammatory processes. The complex role of BIN1 in late-onset Alzheimer's disease is underscored by its association with altered methylation patterns. Despite the disparities across studies, these findings highlight the intricate interplay between epigenetic modifications and Alzheimer's disease pathology. Future research efforts should address methodological variations, incorporate diverse cohorts, and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer's disease progression.

Chromatinopathies: insight in clinical aspects and underlying epigenetic changes.

Chromatinopathies (CPs), a group of rare inborn defects characterized by chromatin state imbalance, have evolved from initially resembling Cornelia de Lange syndrome to encompass a wide array of genetic diseases with diverse clinical presentations. The CPs classification now includes human developmental disorders caused by germline mutations in epigenes, genes that regulate the epigenome. Recent advances in next-generation sequencing have enabled the association of 154 epigenes with CPs, revealing distinctive DNA methylation patterns known as episignatures.It has been shown that episignatures are unique for a particular CP or share similarities among specific CP subgroup. Consequently, these episignatures have emerged as promising biomarkers for diagnosing and treating CPs, differentiating subtypes, evaluating variants of unknown significance, and facilitating targeted therapies tailored to the underlying epigenetic dysregulation.The following review was conducted to collect, summarize, and analyze data regarding CPs in such aspects as clinical evaluation encompassing long-term patient care, underlying epigenetic changes, and innovative molecular and bioinformatic methodologies that have been devised for the assessment of CPs. We have also shed light on promising novel treatment options that have surfaced in recent research and presented a synthesis of ongoing clinical trials, contributing to the current understanding of the dynamic and evolving nature of CPs investigation.

Blood-based DNA methylation in advanced Nasopharyngeal Carcinoma exhibited distinct CpG methylation signature

The TNM staging system is currently used to detect cancer stages. Regardless, a small proportion of cancer patients recur even after therapy, suggesting more specific molecular tools are required to justify the stage-specific detection and prompt cancer diagnosis. Thus, we aimed to explore the blood-based DNA methylation signature of metastatic nasopharyngeal carcinoma (NPC) to establish a holistic methylation biomarker panel. For the identification of methylation signature, the EPIC BeadChip-based array was performed. Comparative analysis for identifying unique probes, validation, and functional studies was investigated by analyzing GEO and TCGA datasets. We observed 4093 differentially methylated probes (DMPs), 1232 hydroxymethylated probes, and 25 CpG islands. Gene expression study revealed both upregulated and downregulated genes. Correlation analysis suggested a positive (with a positive r, p ≤ 0.05) and negative (with a negative r, p ≤ 0.05) association with different cancers. TFBS analysis exhibited the binding site for many TFs. Furthermore, gene enrichment analysis indicated the involvement of those identified genes in biological pathways. However, blood-based DNA methylation data uncovered a distinct DNA methylation pattern, which might have an additive role in NPC progression by altering the TFs binding. Moreover, based on tissue-specificity, a variation of correlation between methylation and gene expression was noted in different cancers.

Abstract C055: Distinct epigenomic regulation of gene expression in Hispanic/Latino children with high-risk B-cell acute lymphoblastic leukemia

Abstract B-Cell Acute Lymphoblastic Leukemia (B-ALL) is the most common childhood malignancy. Hispanic/Latino (H/L) children have a 40% higher death rate than non-Hispanic white (NHW) children after correcting for socioeconomic factors. Recently, we identified a 3.5-fold increased incidence of deletion of one IKZF1 allele (IKZF1-del) and over 15-fold increased incidence of deletion of one IKZF1 allele concomitant with IGH-CRLF2 translocation (IKZF1-del/IGH-CRLF2) in B-ALL of H/L children. This provided a biological rationale for the worse prognosis of B-ALL in H/L children. The very high incidence (29% in all H/L children and 60% in ≥10yr old H/L children) makes IKZF1-del the most frequent genetic alteration that confers adverse prognosis in B-ALL in H/L children. The IKZF1 gene encodes a DNA-binding protein, IKAROS, which regulates transcription of its target genes via chromatin remodeling. The purpose of the study is to analyze the global epigenomic regulation of gene expression in H/L and NHW children with B-ALL and identify biological determinants of altered gene expression that result in worse prognosis for H/L children with B-ALL. /. B-ALL primary cells from 60 H/L and NHW children were analyzed with whole genome sequencing and gene expression profiling using RNA-seq. A subset of B-ALL from each group was analyzed for epigenomic changes by Reduced Representation Bisulfite Sequencing (RRBS), ChIP-seq, CUT&RUN, and CUT&Tag. Results showed that gene expression pattern in B-ALL of H/L children is quite different from B-ALL of NHW children. GSEA and Ingenuity pathway analysis identified oncogenic drivers of B-ALL in H/L children. B-ALL of H/L children have elevated expression of genes that promotestemness, inflammation, and with antiapoptotic activity. RBBS analysis showed a distinct DNA methylation pattern in B-ALL in H/L vs NHW children at promoters of a large set of genes that are involved in cellular signaling, RNA processing, and cellular proliferation. Presence of IKZF1-del results in the alteration of global epigenomic regulation of gene expression with altered enhancer and super-enhancer landscape. Epigenomic alterations due to IKZF1-del resulted in profound redistribution of enhancers and super-enhancers in B-ALL of both H/L vs. NHW children. This was associated with a distinct alteration in gene expression and uncovered that IKAROS regulates different sets of genes in B-ALL in H/L vs. NHW children. These data identified a unique oncogenic signaling that are responsible for aggressive course of B-ALL in H/L children. In conclusion, the multiomics analysis of primary B-ALL cells revealed a distinct global epigenomic regulation of gene expression between B-ALL from H/L and NHW children. This was demonstrated by alterations in DNA methylation and enhancer landscape. Presence of IKZF1-del has a different effect on gene expression in B-ALL of H//L vs NHW children. Results of the study provide a biological rationale for the worse prognosis of B-ALL in H/L children and identified novel therapeutic targets for this disease. Citation Format: Laura Budurlean, Bing He, Yali Ding, Diwakar Bastihalli Tukaramrao, Chingakham Singh, Joseph Schramm, Daniel Bogush, James Broach, Sinisa Dovat. Distinct epigenomic regulation of gene expression in Hispanic/Latino children with high-risk B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C055.

Mapping Chemo-Resistance Profiles of Pediatric Acute Leukemia through Integration of Ex-Vivo Drug Screens with Molecular Data

Introduction While novel approaches for treatment of acute lymphoblastic leukemia (ALL) in children have resulted in significant improvements in survival rates, survivors are faced with potentially life-long challenges as a result of the highly toxic treatment and the outcome for patients who relapse remains poor. The current risk-adapted treatment for ALL in children includes multiple drugs administered sequentially or in combination, with little variation in timing or intensity. Factors that affect varying treatment response between patients and cellular mechanisms that underly response to individual drugs are still under exploration. In this study, we investigate the relationship between ex-vivo drug resistance, transcriptomes, epigenomes and clinical outcome, with the aim of better understanding underlying determinants of treatment response. Methods Pediatric patients diagnosed with B-ALL between 1992 and 2008 in the Nordic countries were included in the study (n = 598). For these patients, ex-vivo drug response to ten clinically used drugs was determined using the fluorometric microculture cytotoxicity assay (FMCA). Clinical data, including follow-up information (overall survival and relapse free survival) was available for all patients. Additionally, DNA-methylation (450K array, n = 383) and gene expression profiling (RNA-seq, n = 119) was performed on a subset of diagnostic patient samples. The patients were stratified into low, medium and high resistance groups based on ex-vivo drug response. The low and high resistance groups were compared with regard to differentially methylated CpG sites or expressed genes associated with individual drug response profiles. Results Overall survival was significantly lower for patients with higher ex-vivo drug resistance to dexamethasone, doxorubicin and thioguanine compared to patients that displayed sensitivity to these drugs (p < 0.05). Similarly, relapse free survival was significantly lower for patients with ex-vivo resistance to cytarabine, dexamethasone, doxorubicin, prednisolone and thioguanine (p < 0.05). After adjusting for sex and risk group, according to the Nordic Society of Pediatric Hematology and Oncology (NOPHO) protocol, in a Cox proportional hazards model, higher hazard ratios for relapse were observed in the highly resistant group across all drugs. We identified distinct DNA methylation and gene expression patterns in patients with low and high resistance, indicating the presence of predetermined molecular resistance fates. These resistance patterns included 665 genes that were differentially expressed (absolute log2FC > 1, Benjamini-Hochberg adjusted p value < 0.05) and 1 422 CpG sites with differential methylation status (absolute mean β-value difference > 0.2, Benjamini-Hochberg adjusted p value < 0.05) in resistant compared to sensitive groups, for one or multiple drugs. With the aim of identifying the genes with the largest impact on drug resistance, we selected the most differentially expressed genes and the genes with the largest difference in methylation status between resistant and sensitive samples. Among these, we identified ~20 putative genes of interest for future studies into resistance mechanisms based on potential functional prioritization. Conclusion Our findings confirm that drug response profiles are associated with outcome in pediatric ALL, reflecting response in vivo. We show molecular evidence for differentiating low- and high-resistant types based on diagnostic gene expression and DNA methylation profiles, suggesting contribution of certain molecular signatures to drug response. Our data integrating molecular data with drug response profiles may help identifying such changes of importance in drug resistance mechanisms.

Identifying key DNA methylation sites and their cis-methylation quantitative loci for intramuscular fatty acid traits using genome and methylome data in Yorkshire pigs

Single-nucleotide polymorphisms (SNPs) that are located near CpG sites have the potential to influence DNA methylation levels, leading to distinct patterns of DNA methylation. These methylation quantitative trait loci (meQTLs) can influence methylation levels across the entire genome, thereby impacting phenotypic traits. In this study, we utilised reduced representation bisulphite sequencing (RRBS) to analyse DNA methylation in muscle tissue samples collected from 140 Yorkshire pigs. Additionally, we assessed SNPs using the Porcine 50K chip, examined 17 fatty acid traits, and conducted epigenome-wide association studies (EWAS) and genome-wide association studies (GWAS). Our investigation revealed numerous associations between DNA methylation and fatty acid traits, including C16:0, C18:0, C20:3n-3, and C22:6n-3. Notably, a substantial portion of these associations was specifically identified by EWAS and not GWAS. Subsequently, we performed GWAS to identify meQTLs using methylation levels of significant CpGs from the EWAS as traits. The results revealed the predominance of trans-meQTLs; however, we also discovered a cis-meQTL on SSC12. Finally, we reported a crucial CpG site (SSC12:3735523) and its cis-meQTL on SSC12, which are located in close proximity to the FASN gene. Our findings contribute to the identification of CpG sites and their cis-meQTLs associated with fatty acid traits, utilising comprehensive genome and methylome data. Moreover, our study provides novel insights into the molecular mechanisms underlying fatty acid traits in pigs.

Whole-genome DNA methylation profiling reveals epigenetic signatures in developing muscle in Tan and Hu sheep and their offspring.

The Tan sheep is a popular local breed in China because of its tenderness and flavor. The Hu sheep breed is also famous for its high litter size, and its muscle growth rate is faster than that of Tan sheep. However, the epigenetic mechanism behind these muscle-related phenotypes is unknown. In this study, the longissimus dorsi tissue from 18 6 month-old Tan sheep, Hu sheep, and Tan-Hu F2 generation (6 sheep per population) were collected. After genomic DNA extraction, whole-genome bisulfite sequencing (WGBS) and bioinformatics analysis were performed to construct genome-wide DNA methylome maps for the Tan sheep, Hu sheep and their Tan-Hu F2 generation. Distinct genome-wide DNA methylation patterns were observed between Tan sheep and Hu sheep. Moreover, DNA methylated regions were significantly increased in the skeletal muscle from Tan sheep vs. the F2 generation compared to the Hu sheep vs. F2 generation and the Tan sheep vs. Hu sheep. Compared with Hu sheep, the methylation levels of actin alpha 1 (ACTA1), myosin heavy chain 11 (MYH11), Wiskott-Aldrich syndrome protein (WAS), vav guanine nucleotide exchange factor 1 (VAV1), fibronectin 1 (FN1) and Rho-associated protein kinase 2 (ROCK2) genes were markedly distinct in the Tan sheep. Furthermore, Gene Ontology analysis indicated that these genes were involved in myotube differentiation, myotube cell development, smooth muscle cell differentiation and striated muscle cell differentiation. The findings from this study, in addition to data from previous research, demonstrated that the ACTA1, MYH11, WAS, VAV1, FN1, and ROCK2 genes may exert regulatory effects on muscle development.

DNA methylation alterations at RE1-silencing transcription factor binding sites and their flanking regions in cancer

BackgroundDNA methylation changes, frequent early events in cancer, can modulate the binding of transcription factors. RE1-silencing transcription factor (REST) plays a fundamental role in regulating the expression of neuronal genes, and in particular their silencing in non-neuronal tissues, by inducing chromatin modifications, including DNA methylation changes, not only in the proximity of its binding sites but also in the flanking regions. REST has been found aberrantly expressed in brain cancer and other cancer types. In this work, we investigated DNA methylation alterations at REST binding sites and their flanking regions in a brain cancer (pilocytic astrocytoma), two gastrointestinal tumours (colorectal cancer and biliary tract cancer) and a blood cancer (chronic lymphocytic leukemia).ResultsDifferential methylation analyses focused on REST binding sites and their flanking regions were conducted between tumour and normal samples from our experimental datasets analysed by Illumina microarrays and the identified alterations were validated using publicly available datasets. We discovered distinct DNA methylation patterns between pilocytic astrocytoma and the other cancer types in agreement with the opposite oncogenic and tumour suppressive role of REST in glioma and non-brain tumours.ConclusionsOur results suggest that these DNA methylation alterations in cancer may be associated with REST dysfunction opening the enthusiastic possibility to develop novel therapeutic interventions based on the modulation of this master regulator in order to restore the aberrant methylation of its target regions into a normal status.

Abstract 5516: Identifying DNA methylation biomarkers in Brazilian women of African descent

Abstract Breast cancer is the most common cancer, and the leading cause of cancer-related deaths in Brazilian women. Studies have shown that in Brazil while breast cancer mortality has increased in the last decade for all women, mortality in women of African descent has doubled. A recent study showed that Afro-Brazilian women suffer from higher excess breast cancer mortality even after adjusting for extent of disease, year of diagnosis, age and socioeconomic status, which suggests biological factors also contribute to the differences in the clinicopathological make-up of the disease in women in this group. DNA methylation biomarkers, representing a combination of genetic and non-genetic factors, have the potential to be especially valuable when considering the combined contribution of the environment and genetics in breast cancer etiology. Several groups have found differences in DNA methylation levels associated with race and ethnicity in breast cancer. In addition, research has shown that women of different races/ethnicities with similar breast cancer tumor subtypes have distinct DNA methylation patterns. To explore further the association between race, ancestry and DNA methylation, we conducted a study in paired tumor and non-tumor tissue samples of forty-eight Brazilian women who self-identified as Black or Brown and were treated at National Cancer Institute Breast Cancer Hospital in Rio de Janeiro, Brazil. We carried out a DNA methylation profiling analysis using Illumina EPIC (850k) arrays in bisulfite converted DNA originally extracted from frozen tissue samples. A total of 739,883 differentially methylated probes (DMP) were identified, of which a large proportion 6,241 (78.4%) were hypomethylated. Lower DNA methylation was more common on intragenic regions and gene bodies, while hypermethylation was more commonly found on the promoter regions. DNA methylation was often higher in non-tumor than tumor samples. Among our top hits we found genes previously associated with estrogen receptor negative tumor types, CDH4, CERK, PROX1 and ADHFE1, and with high risk of breast cancer mortality (ST6GAL1, LYN, TFF1 and BMP3). A DPM enrichment pathway analysis predicted effects in known signaling pathways and transcriptional misregulation. We are currently analyzing the associations between the top differentially methylated regions with ancestry informative markers, and the clinicopathological characteristics of disease, to gain a better understanding of the role of DNA methylation marks in this population. This study adds to our current knowledge on epigenetics marks that will ultimately help us address mortality due to this disease in this population. Citation Format: Lissette Delgado-Cruzata, Diego J. Gomes de Paula, Jennifer Vieira Gomes, Tatiana A. Simão, Leonor Gusmão, Luis Felipe Ribeiro Pinto, Sheila C. Soares-Lima. Identifying DNA methylation biomarkers in Brazilian women of African descent. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5516.