Methylation Status Research Articles

A novel family of beta mixture models for the differential analysis of DNA methylation data: An application to prostate cancer.

Identifying differentially methylated cytosine-guanine dinucleotide (CpG) sites between benign and tumour samples can assist in understanding disease. However, differential analysis of bounded DNA methylation data often requires data transformation, reducing biological interpretability. To address this, a family of beta mixture models (BMMs) is proposed that (i) objectively infers methylation state thresholds and (ii) identifies differentially methylated CpG sites (DMCs) given untransformed, beta-valued methylation data. The BMMs achieve this through model-based clustering of CpG sites and by employing parameter constraints, facilitating application to different study settings. Inference proceeds via an expectation-maximisation algorithm, with an approximate maximization step providing tractability and computational feasibility. Performance of the BMMs is assessed through thorough simulation studies, and the BMMs are used for differential analyses of DNA methylation data from a prostate cancer study. Intuitive and biologically interpretable methylation state thresholds are inferred and DMCs are identified, including those related to genes such as GSTP1, RASSF1 and RARB, known for their role in prostate cancer development. Gene ontology analysis of the DMCs revealed significant enrichment in cancer-related pathways, demonstrating the utility of BMMs to reveal biologically relevant insights. An R package betaclust facilitates widespread use of BMMs.

DNA methylation profiling of CpG islands in trigeminal ganglion of rats with orofacial pain induced by experimental tooth movement

BackgroundTooth movement induced orofacial pain is the most cited negative effect during orthodontic treatment, while treatment options without side effects are limited. The differential expression of pain-related genes due to DNA methylation and demethylation is instrumental in pain. The purpose of the study was to evaluate the DNA methylation profiling of CpG islands (CGI) and CGI shores in promoter regions in trigeminal ganglions (TG) of tooth movement induced orofacial pain rats, thus to further insight the DNA methylation regulation in orofacial pain.Materials and methodsAn orofacial pain rat model was constructed by ligating coil springs between the incisor and first maxillary molar with 40 g of force. The Rat Grimace Score (RGS) was used for pain evaluation. The genome methylation status was analyzed by the reduced representation bisulfite sequencing (RRBS) technique. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analyses were conducted in the differentially methylated regions (DMRs). Moreover, a protein-protein interaction (PPI) network was established to detect annotated genes associated with pain.ResultsRGS was significantly higher in orofacial pain rats than in sham rats. RRBS showed widespread methylation changes in CGI and CGI shores in TG promoter regions. Both 902 hypermethylated DMRs and 862 hypomethylated DMRs were found in the CGIs of promoter regions. KEGG analysis revealed that annotated genes are participated in endocrine, nervous, immune, and sensory systems. Moreover, the “Calcium signaling pathway”, “Wnt signaling pathway” and “Neuroactive ligand-receptor interaction” were significantly enriched pathways. Furthermore, PPI network showed several genes (Ctnnb1, Dlg4, Creb1, Camk2g, Bmp2, etc.) with different methylation statuses were reported to be associated with pain.ConclusionsThis study demonstrated methylation changes were existed in CGI and CGI shores in TG promoter regions when pain occurs, thus providing a basis for further study on the mechanism of DNA methylation in orofacial pain.

Epigenetic Suppression of miR-137 Induces RNF4 Expression, Facilitating Wnt Signaling in Colorectal Cancer.

Colorectal cancer (CRC) is a significant health issue worldwide. Recent studies highlight the critical role of miRNAs in CRC development, particularly miR-137, which acts as a key tumor suppressor. Despite its known role, further exploration of miR-137's downstream signaling is needed to understand its biology and therapeutic potential. We examined the methylation status of miR-137 using one TCGA data and three GEO data sets. A clinical validation cohort of 78 samples was analyzed using MSP for miR-137 promoter methylation. Various in vitro molecular/cellular and animal experiments were conducted to elucidate miR-137's role in CRC. Bioinformatic analysis indicated frequent methylation of miR-137 in CRC tissues, correlating with suppressed expression. EZH2-mediated H3K27 trimethylation silences miR-137 in CRC cells by increasing chromatin compaction, reversible by EZH2 siRNA or inhibitor GSK343. miR-137 inhibits CRC cell proliferation, migration, invasion, and xenograft tumor growth, confirming its tumor-suppressive role. Using the miRWalk repository showed that miR-137 regulates the Wnt signaling pathway by reducing typical protein expression in HCT116 and SW480 cells. miR-137 directly targets RNF4, leading to its downregulation at transcriptional and protein levels, with an observed inverse correlation in CRC tissues. miR-137 accelerates c-Myc and β-catenin degradation by inhibiting RNF4, impacting protein stability and Wnt pathway inhibition. miR-137 is epigenetically silenced through DNA methylation and EZH2-mediated H3K27 trimethylation. It regulates the Wnt signaling pathway by targeting RNF4, leading to c-Myc and β-catenin destabilization. Restoring miR-137 or inhibiting RNF4 suppresses CRC cell proliferation, migration, invasion, and tumor growth, highlighting its therapeutic potential in CRC.

Methylation and expression of imprinted genes in circulating extracellular vesicles from women experiencing early onset preeclampsia

Preeclampsia (PE) is a pregnancy complication marked by high blood pressure, posing risk to maternal and fetal health. "Genomic imprinting", an epigenetic phenomenon regulated by DNA methylation at Differently Methylated Regions (DMR's), influences placental development. Research on circulating extracellular vesicles (EVs) in PE suggests them as potential source for early biomarkers, but methylation status of EV-DNA in Preeclampsia is not reported yet. This study examines the methylation and expression profile of imprinted genes - PEG10, PEG3, MEST, and DLK1 in circulating EVs of 1st and 3rd trimester control and early onset preeclampsia (EOPE) pregnant women (n=15) using pyrosequencing and qRT-PCR respectively. In 1st trimester, PEG3 was significantly hypermethylated, whereas no significant methylation changes were noted in PEG10 and MEST in EOPE. In 3rd trimester, significant hypomethylation in PEG10, PEG3 and IGDMR was observed whereas significant hypermethyaltion noted in MEST. mRNA expression of PEG10, PEG3 and DLK1 was not affected in circulating EVs of 1st trimester EOPE. However, in 3rd trimester significant increased expression in PEG10, PEG3 and DLK1 noted. MEST expression was reduced in 3rd trimester EOPE. No correlation was observed between average DNA methylation and gene expression in PEG10 and PEG3 in 1st trimester. However, in 3rd trimester, significant negative correlation was noted in PEG10 (r=-0.426, p=0.04), PEG3 (r=-0.496, p=0.01), MEST (r=-0.398, p=0.03) and DLK1 (r=-0.403, p=0.03). The results of our study strengthen the potential of circulating EVs from maternal serum as non-invasive indicators of placental pathophysiology, including preeclampsia.

Exon 1 methylation status of CDH13 is associated with decreased overall survival and distant metastasis in patients with postoperative colorectal cancer

BackgroundCadherin 13 (CDH13) is a member of the cadherin superfamily that exerts tumor-suppressive effects on cancers derived from epithelial cells. Although hypermethylation of CDH13 promoter has been reported in various cancers, its prognostic value for colorectal cancer (CRC) is still controversial. The methylation alterations of CDH13 within exon 1 have not yet been investigated.MethodsA total of 49 CRC patients were recruited for the prospective study. The methylation status of CpG sites was quantified by Bisulfite Amplicon Sequencing (BSAS) in malignant tissues and adjacent normal tissues. The primary endpoint of the study was overall survival (OS) after surgery. The relationship between methylation level with pathological stage and OS was also evaluated.ResultsCompared with adjacent normal tissues, the overall average methylation level within exon 1 was significantly increased in tumor tissues (p < 0.001). The association study showed that the hypermethylation status of the CpG1 site was non-significantly associated with the presence of distant metastasis (p = 0.032). Moreover, the hypermethylation of two CpG sites, including CpG1 (p = 0.003) and CpG5 (p = 0.032), was associated with worse OS in CRC. Co-hypermethylation of CpG1 and CpG5 sites was significantly associated with a worse clinical outcome (HR: 4.43 [95% CI 1.27–15.46]; p = 0.019) in multivariate Cox regression analysis.ConclusionThe methylation level of CDH13 exon 1 in CRC tissue was significantly higher than in adjacent normal tissues. Hypermethylation at the CpG1 site suggests a risk of distant metastasis in CRC. The hypermethylation of the CpG1 site and CpG5 site, including the co-hypermethylation of these two sites, may serve as a valuable prognostic biomarker.

Dynamic visualization of DNA methylation in cell cycle genes during iPSC cardiac differentiation.

Existing analyses with conventional assays have generated significant insights into static states of DNA methylation but were unable to visualize the dynamics of epigenetic regulation. We utilized a genomic DNA methylation reporter (GMR) system carrying Snrpn minimal promoter and CpG regions of Cdk1 (Cyclin-dependent kinase 1) or Sox2 (SRY-Box Transcription Factor 2). Mouse Sox2 GMR iPSCs rapidly lost fluorescent reporter signal upon the induction of cardiac differentiation. Cdk1 GMR reporter signal was strong in undifferentiated iPSCs, and gradually decreased during cardiomyocyte differentiation. RT-qPCR and pyrosequencing demonstrated that the reduction of Sox2 and Cdk1 was regulated by hypermethylation of their promoters' CpG regions during cardiac differentiation. The GMR reporter system can be useful for monitoring real-time epigenetic DNA modification at single-cell resolution.

Transposable element 5mC methylation state of blood cells predicts age and disease.

Transposable elements (TEs) are DNA sequences that expand selfishly in the genome, possibly causing severe cellular damage. While normally silenced, TEs have been shown to activate during aging. DNA 5-methylcytosine (5mC) is one of the main epigenetic modifications by which TEs are silenced and has been used to train highly accurate age predictors. Yet, one common criticism of such predictors is that they lack interpretability. In this study, we investigate the changes in TE 5mC methylation that occur during aging in human blood using published methylation array data. We find that evolutionarily young long interspersed nuclear elements 1 (L1s), the only known TEs capable of autonomous transposition in humans, undergo the fastest loss of 5mC methylation, suggesting an active mechanism of de-repression. The same young L1s also showed preferential gain in chromatin accessibility but not expression. The long terminal repeat retrotransposons THE1A and THE1C also showed very rapid 5mC loss. We then show that accurate age predictors can be trained on both 5mC methylation of individual TE copies and average methylation of TE families genome wide. Lastly, we show that while old L1s gradually lose 5mC during the entire lifespan, demethylation of young L1s only happens late in life and is associated with cancer.

Detection of Mismatch Repair Deficiency in Endometrial Cancer: Assessment of IHC, Fragment Length Analysis, and Amplicon Sequencing Based MSI Testing.

Background/Objectives: Mismatch repair (MMR) deficiency can be indicative of Lynch syndrome (LS) and guide treatment with immune checkpoint inhibitors. Colorectal cancers (CRCs) and endometrial cancers (ECs) are routinely screened to identify LS, primarily using immunohistochemistry (IHC) or microsatellite instability (MSI) testing, but concordance between these methods is variable in ECs. Here, we investigate this variability in 361 ECs from the Ohio OCCPI/OPTEC (n = 196) and Manchester PETALS (n = 165) trials, where concordance between assays differed significantly. Methods: Samples were re-tested using the amplicon-sequencing-based Newcastle MSI assay (NCL_MSI), and analysed with respect to existing IHC, MSI and MLH1 promoter hypermethylation data. Results: NCL_MSI showed consistency with the Ohio results (94% and 97% concordance with IHC and original MSI assays, respectively) and increased concordance within the Manchester cohort from 78% to 86% (MSI) and 84% (IHC). Among discordant Manchester samples, NCL_MSI was significantly associated with MLH1 promoter methylation status (p = 0.0028) and had the highest concordance with methylation, (62/69 samples, 90%), indicating utility as a screening tool in this tumour type. However, tumours with germline MSH6 defects were only detected efficiently with IHC; seven out of eight LS tumours classified as MSS by either MSI assay had isolated MSH6 loss, compared to four out of twelve classified as MSI-H by both (p = 0.028). Furthermore, reduced MSI signal was observed in tumours with isolated MSH6 loss (p = 0.009 Ohio, p = 6.2 × 10-5 Manchester) and in both ECs and CRCs with germline defects, although this only reached significance in CRCs (p = 0.002). Conclusions: These results provide further evidence that ECs with MSH6 loss in particular and LS tumours in general have an attenuated MSI signal, providing support for current guidelines specifically recommending IHC for LS detection and immune checkpoint therapy assessment in EC.

Methylation assay in KMT2B-related dystonia: a novel diagnostic validation tool

Background/objectivesKMT2B-related dystonia (DYT28, OMIM #617284) is a progressive neurological condition characterized by early onset movement disorders with autosomal dominant inheritance. In this study, we describe the use of a genome methylation episignature methodology to functionally validate two variants of uncertain significance (VUS) in the KMT2B gene.MethodsGenome-wide methylation status was assessed using the EPIC methylation assay in peripheral blood samples from two subjects with early onset movement disorder and missense variants of uncertain significance in the KMT2B gene (p.Leu1720Phe and p.Tyr2515Cys). After QC and normalization steps, we compared the M values for all 144 probes, previously described as an EpiSign for KMT2B-related dystonia, between the two subjects and 14 controls individuals.ResultsThe individual harboring the p.Tyr2515Cys variant exhibited a hypermethylation profile compatible with pathogenic/likely pathogenic variants in KMT2B, allowing for variant reclassification, conclusive genetic counseling, and patient stratification for deep brain stimulation. In contrast, the individual harboring the p.Leu1720Phe variant had a methylation status similar to controls, practically ruling out KMT2B-related dystonia.ConclusionInvestigation of methylation status can be a powerful tool to determine pathogenicity when facing KMT2B variants of uncertain significance. Methylation results may optimize genetic counseling and positively impact patient care.

Effect of The Duration of The Diagnosis on DNA Methylation Changes in Three Different Diabetic Genes in Type 2 Diabetes Mellitus

The primary epigenetic alteration is DNA methylation. This study examined variations in DNA methylation in patients with type 2 diabetes at various time intervals, concentrating on the Calpain-10 (CAPN10), ATP binding cassette subfamily C member 8 (ABCC8), and Transcription factor 7-like 2 (TCF7L2) genes. Since persistent diabetes can lead to aberrant methylation patterns, it is interested in how long these changes may last. The blood samples from 140 individuals with Type 2 Diabetes Mellitus (T2DM) were collected, and the patients were grouped according to how long they had been diagnosed. Four groups of individuals were created based on their time on the disease: those with T2DM &lt;1 year, those with T2DM 1-3 years, and those with T2DM 3-5 years. The genders and ages of the participants were also noted. Using the Promega technique, bisulfite conversion and DNA extraction were completed. Methylation-specific PCR amplification was used to detect DNA methylation. In the study, ROC curve analysis, Chi-square and Spearman's correlation coefficients, as well as non-parametric tests, were employed to analyze the methylation percentage variation and methylation patterns among groups. A significant threshold of p &lt; 0.05 was established. The results of the study indicated that the DNA methylation rates of T2DM patients and the control group differed significantly. Patients with type 2 diabetes, particularly those who had just received a diagnosis, showed higher levels of methylation than the control group. The study also found that the length of diagnosis may have an impact on the discriminative strength of the DNA methylation status of the ABCC8 gene, resulting in varying degrees of T2DM prediction.

ZBTB24 is a conserved multifaceted transcription factor at genes and centromeres that governs the DNA methylation state and expression of satellite repeats.

Since its discovery as a causative gene of the Immunodeficiency with Centromeric instability and Facial anomalies syndrome, ZBTB24 has emerged as a key player in DNA methylation, immunity and development. By extensively analyzing ZBTB24 genomic functions in ICF-relevant mouse and human cellular models, we document here its multiple facets as a transcription factor, with key roles in immune response-related genes expression and also in early embryonic development. Using a constitutive Zbtb24 ICF-like mutant and an auxin-inducible degron system in mouse embryonic stem cells, we showed that ZBTB24 is recruited to centromeric satellite DNA where it is required to establish and maintain the correct DNA methylation patterns through the recruitment of DNMT3B. The ability of ZBTB24 to occupy centromeric satellite DNA is conserved in human cells. Together, our results unveiled an essential and underappreciated role for ZBTB24 at mouse and human centromeric satellite repeat arrays by controlling their DNA methylation and transcription status.

Histone H3 N-terminal recognition by the PHD finger of PHRF1 is required for proper DNA damage response.

Plant homeodomain (PHD) fingers are critical effectors of histone post-translational modifications (PTMs), acting as regulators of gene expression and genome integrity, and frequently presenting in human disease. While most PHD fingers recognize unmodified and methylated states of histone H3 lysine 4 (H3K4), the specific functions of many of the over 100 PHD finger-containing proteins in humans remain poorly understood, despite their significant implications in disease processes. In this study, we undertook a comprehensive analysis of one such poorly characterized PHD finger-containing protein, PHRF1. Using biochemical, molecular, and cellular approaches, we show that PHRF1 robustly binds to histone H3, specifically at its N-terminal region. Through RNA-seq and proteomic analyses, we also find that PHRF1 is intricately involved in transcriptional and RNA splicing regulation and plays a significant role in DNA damage response (DDR). Crucially, mutagenesis of proline 221 to leucine (P221L) in the PHD finger of PHRF1 abolishes histone interaction and fails to rescue defective DDR. These findings underscore the importance of PHRF1-H3 interaction in maintaining genome integrity and provide insight into how PHD fingers contribute to chromatin biology.

ScCancerExplorer: a comprehensive database for interactively exploring single-cell multi-omics data of human pan-cancer.

Genomic, epigenomicand transcriptomic alterations are hallmarks of cancer cells, and are closely connected. Especially, epigenetic regulation plays a critical role in tumorigenesis and progression. The growing single-cell epigenome data in cancer research provide new opportunities for data mining from a more comprehensive perspective. However, there is still a lack of databases designed for interactively exploring the single-cell multi-omics data of human pan-cancer, especially for the single-cell epigenome data. To fill in the gap, we developed scCancerExplorer, a comprehensive and user-friendly database to facilitate the exploration of the single-cell genome, epigenome (chromatin accessibility and DNA methylation), and transcriptome data of 50 cancer types. Five major modules were provided to explore those data interactively, including 'Integrated multi-omics analysis', 'Single-cell transcriptome', 'Single-cell epigenome', 'Single-cell genome' and 'TCGA analysis'. By simple clicking, users can easily investigate gene expression features, chromatin accessibility patterns, transcription factor activities, DNA methylation states, copy number variationsand TCGA survival analysis results. Taken together, scCancerExplorer is distinguished from previous databases with rich and interactive functions for exploring the single-cell multi-omics data of human pan-cancer. It bridges the gap between single-cell multi-omics data and the end-users, and will facilitate progress in the field of cancer research. scCancerExplorer is freely accessible via https://bianlab.cn/scCancerExplorer.

Meta-analyses of epigenetic age acceleration and GrimAge components of schizophrenia or first-episode psychosis

Schizophrenia is a common chronic psychiatric disorder that causes age-related dysfunction. The life expectancy in patients with schizophrenia is ≥10 years shorter than that in the general population because of the higher risk of other diseases, such as cardiovascular diseases. Aging studies based on DNA methylation status have received considerable attention. Several epigenetic age accelerations and predicted values of aging-related proteins (GrimAge and GrimAge2 components) have been analyzed in multiple diseases. However, no studies have investigated up to GrimAge and GrimAge2 components between patients with schizophrenia and controls. Therefore, we aimed to conduct multiple regression analyses to investigate the association between schizophrenia and epigenetic age accelerations and GrimAge and GrimAge2 components in seven cohorts. Furthermore, we included patients with first-episode psychosis whose illness duration was often shorter than schizophrenia in our analysis. We integrated these results with meta-analyses, noting the acceleration of GrimAge, GrimAge2, and DunedinPACE, and increase in adrenomedullin, beta-2 microglobulin, cystatin C, and plasminogen activation inhibitor-1 levels, in patients with schizophrenia or first-episode psychosis. These results corroborated the finding that patients with schizophrenia had an increased risk of diabetes, cardiovascular disease, and cognitive dysfunction from a biological perspective. Patients with schizophrenia and first-episode psychosis showed differences in the results when compared with controls. Such analyses may lead to the development of novel therapeutic targets to patients with schizophrenia or relevant diseases from the perspective of aging in the future.

NS1-mediated DNMT1 degradation regulates human bocavirus 1 replication and RNA processing.

Methylation of the DNA genome plays an important role in viral gene inactivation. However, the role of DNA methylation in human bocavirus (HBoV) remains unclear. In this study, the HBoV1 genomic DNA was found extensively methylated at the CHG and CHH sites. Inhibiting DNA methylation with 5-aza-2'-deoxycytidine (DAC) altered the methylation status and reduced viral DNA production, while enhanced the RNA splicing at D1 and D3 sites and the polyadenylation at the proximal polyadenylation site, (pA)p. Knockdown of DNA methyltransferase 1 (DNMT1) had the same effect on viral DNA synthesis and RNA processing as the DAC treatment, indicating that DNMT1 is the major host methyltransferase involved in viral DNA methylation. In addition, the nonstructural protein NS1 promoted DNMT1 degradation through the ubiquitin-proteasome pathway to regulate viral replication and RNA processing. Collectively, the results suggest that DNA methylation and DNMT1 facilitate HBoV replication and are essential for appropriate NS1 localization in the nucleus. DNMT1 degradation through NS1 promotes the virus RNA processing, leading to viral protein expression.

Comparison of cell type and disease subset chromatin modifications in SLE

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease with protean manifestations. There is little understanding of why some organs are specifically impacted in patients and the mechanisms of disease persistence remain unclear. While much work has been done characterizing the DNA methylation status in SLE, there is less information on histone modifications, a more dynamic epigenetic feature. This study identifies two histone marks of activation and the binding of p300 genome-wide in three cell types and three clinical subsets to better understand cell-specific effects and differences across clinical subsets.ResultsWe examined 20 patients with SLE and 8 controls and found that individual chromatin marks varied considerably across T cells, B cells, and monocytes. When pathways were examined, there was far more concordance with conservation of TNF, IL-2/STAT5, and KRAS pathways across multiple cell types and ChIP data sets. Patients with cutaneous lupus and lupus nephritis generally had less dramatically altered chromatin than the general SLE group. Signals also demonstrated significant overlap with GWAS signals in a manner that did not implicate one cell type more than the others.ConclusionsThe pathways identified by altered histone modifications and p300 binding are pathways known to be important from RNA expression studies and recognized pathogenic mechanisms of disease. NFκB and classical inflammatory pathways were strongly associated with increased peak heights across all cell types but were the highest-ranking pathway for all three antibodies in monocytes according to fgsea analysis. IL-6 Jak/STAT3 signaling was the most significant pathway association in T cells marked by H3K27ac change. Therefore, each cell type experiences the disease process distinctly although in all cases there was a strong theme of classical inflammatory pathways. Importantly, this NFκB pathway, so strongly implicated in the patients with generalized SLE, was much less impacted in monocytes when cutaneous lupus was compared to the general SLE cohort and also less impacted in lupus nephritis compared to general SLE. These studies define important cell type differences and emphasize the breadth of the inflammatory effects in SLE.

Serum lactate dehydrogenase as a prognostic marker for treatment response in IDH wild-type glioblastoma patients undergoing stupp protocol.

Elevated lactate dehydrogenase (LDH), a marker of tumor aggressiveness and metabolic alterations, may predict treatment response and overall survival across various tumors. This study investigates the correlation between serum LDH levels and clinical outcomes in glioblastoma patients treated with radiotherapy (RT) and temozolomide (TMZ). This retrospective study analysed patients with IDH wild-type glioblastoma (IDH-wt GB) treated at the Radiotherapy Department of Azienda Ospedaliero-Universitaria Senese from 2018 to 2023. Clinical data, including hematologic parameters (e.g., LDH), imaging (MRI), and MGMT promoter methylation status, were collected. All patients received RT and TMZ following the Stupp protocol. Serum LDH levels were measured one week before RT, and Radiological Response (RR) was assessed using RANO criteria. Overall survival (OS), progression-free survival (PFS), and RR were primary endpoints. Statistical analyses included Kaplan-Meier, Cox regression, and decision tree analysis for LDH cut-off determination. In a cohort of 147 IDH wild-type glioblastoma patients treated with the Stupp protocol, the median OS was 14 months and median PFS was 8 months. Elevated baseline LDH levels were associated with significantly poorer outcomes, showing a median OS of 9 months versus 20 months and a median PFS of 6 months versus 13 months for lower LDH levels (p < 0.001 and p = 0.0001, respectively). LDH levels also correlated with RR (p = 0,001), Multivariate analysis confirmed high LDH as an independent predictor of worse OS (HR = 2.31) and PFS (HR = 2.60), suggesting its utility as a prognostic biomarker. Elevated LDH levels before starting the Stupp protocol are clinically significant as they predict poorer overall survival and progression-free survival in glioblastoma patients and worse RR. Incorporating LDH measurements into treatment planning can help identify patients at higher risk of poor outcomes, allowing for more tailored and potentially aggressive treatment strategies to improve management and therapeutic responses in glioblastoma.

AI tool for predicting MGMT methylation in glioblastoma for clinical decision support in resource limited settings

Glioblastoma is an aggressive brain cancer with a poor prognosis. The O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status is crucial for treatment stratification, yet economic constraints often limit access. This study aims to develop an artificial intelligence (AI) framework for predicting MGMT methylation. Diagnostic magnetic resonance (MR) images in public repositories were used for training. The algorithm created was validated in data from a single institution. All images were segmented according to widely used guidelines for radiotherapy planning and combined with clinical evaluations from neuroradiology experts. Radiomic features and clinical impressions were extracted, tabulated, and used for modeling. Feature selection methods were used to identify relevant phenotypes. A total of 100 patients were used for training and 46 for validation. A total of 343 features were extracted. Eight feature selection methods produced seven independent predictive frameworks. The top-performing ML model was a model post-Least Absolute Shrinkage and Selection Operator (LASSO) feature selection reaching accuracy (ACC) of 0.82, an area under the curve (AUC) of 0.81, a recall of 0.75, and a precision of 0.75. This study demonstrates that integrating clinical and radiotherapy-derived AI-driven phenotypes can predict MGMT methylation. The framework addresses constraints that limit molecular diagnosis access.

Abstract A014: A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction

Abstract Liquid biopsy continues to be a focus of the cancer diagnostics industry, primarily due to the non-invasive nature of sample collection, however the low levels of circulating tumor DNA (ctDNA) remain a challenge. Methylation has emerged as a promising biomarker for detecting ctDNA, yet detecting the cancerous signal in an excess of non-cancerous, unmethylated cfDNA in the background has been proven difficult. Harbinger Health has developed a novel method that depletes unmethylated background at the CpG-level from specific genomic regions of interest, resulting in an enrichment of the ctDNA fraction. We developed a background cfDNA depletion method that utilizes the endonuclease activity and the specificity of CRISPR/Cas12a to remove unmethylated DNA fragments at ctDNA methylation biomarker sites. First, we identified cancer-informative regions that showed consistent contiguous elevated methylation states in cancer compared to normal samples. We then designed guide RNAs that specifically bind and cut unmethylated CpG sites within the target regions. Cas depletion of bisulfite-converted DNA enriches cancer informative signal for bioanalysis. To test validity of our Cas complex designs, we created model samples that mimic fully unmethylated and fully methylated species. We spiked these cfDNA models into a background of genomic DNA from 5% to 0.01%. In triplicate, we examined cutting efficiency, multiplexing and specificity of Cas to unmethylated species of model DNA. Results were assessed using qPCR by comparing differences in Ct with and without Cas treatment. Additionally, we applied this depletion step during our library preparation process to show utility in methylation analysis of low tumor content liquid biopsy samples. Using both the model sample dilution series and bisulfite converted cfDNA samples, which represent a range of cancerous and background methylation signal, we deplete unmethylated targets using Cas prior to indexing PCR. This rendered unmethylated targets non-amplifiable, removing them from analysis. Results were assessed using both qPCR and sequencing data, such as methylated read counts over the target region. We demonstrated our analytical approach efficiently depletes non-cancer cfDNA by on average 9-fold, which increases the fraction of ctDNA signal and can be multiplexed. This background signal depletion driven by CRISPR is sensitive and specific, maintaining methylated DNA signal detection down to 0.01% at inputs of 10ng DNA. Our Cas-mediated depletion of background signal method has proven useful for enriching rare methylated fragments over background by improving the signal to noise ratio. This technique is novel by harnessing the specificity of CRISPR to target DNA in both a sequence and methylation state specific manner. The depletion of precise methylation states at the CpG-level make it suitable for use in multiple bioassays. Citation Format: Caitlin Gilley, Miguel Williams, Emily Neaga, Sarah Falotico, Kade Pettie, Anthony Shuber. A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A014.

Abstract A035: A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA

Abstract Non-invasive liquid biopsy tests are proving to be the next frontier in cancer diagnostics with methylation emerging as a reliable biomarker for low levels of circulating tumor DNA (ctDNA). There is a rising interest in sensitive, low-cost liquid biopsy assays to detect low copies of hypermethylated ctDNA in an excess background of non-cancer-associated hypomethylated cell- free DNA (cfDNA). Real-time quantitative methylation-specific PCR (qMSP) allows for the detection of low abundance methylated fragments. qMSP approaches have previously been developed but are predominantly designed to target a specific CpG site. To date we are not aware of any qMSP approaches that target pan-cancer-associated methylation patterns across multiple CpG sites. Harbinger Health has developed a qMSP method with locked nucleic acid (LNA) blockers that target methylation haplotypes, allowing for multiplex enrichment and quantification of cancer-specific methylation patterns. Using targeted bisulfite sequencing, we identified ten pan-cancer-informative CpG-dense regions which showed consistent contiguous elevated methylation states in cancer cfDNA and low levels of methylation in non-cancer cfDNA. We designed qMSP assays for these regions and LNA blockers which bind to unmethylated target CpG sites, outcompeting the hydrolysis probe through a high Tm differential. This prevented non-specific probe hybridization. The qMSP-LNA assays were evaluated in simplex and multiplex using cell line DNA in a titration of hypermethylated “ctDNA” spiked into hypomethylated “cfDNA” to simulate varying ctDNA levels, and then validated with cancer and non-cancer cfDNA samples at 5 ng input, equivalent to 1450 haploid copies. The percentage of methylated DNA was estimated by the ratio of methylated copies (qMSP-LNA) to total copies (internal reference), which were quantified by standard curves. The qMSP-LNA assays detected low abundance methylated DNA fragments down to 23 target copies. LNA blockers completely depleted background signal originating from unmethylated DNA with high specificity, showing no inhibition of methylated DNA detection. When tested in cfDNA samples which varied in conversion efficiency, discordant methylation states, and methylation abundance, the assays reproducibly amplified and detected highly methylated fragments which correspond to cancer signal. Harbinger Health has developed qMSP-LNA assays for contiguous CpG biomarkers which differentiate cancer and non-cancer samples from only ten pan-cancer-informative regions. The qMSP-LNA method detected low abundance fragments with specific methylation patterns with high specificity and sensitivity. The assay reproducibly detected methylated DNA in cfDNA samples that represent a clinically relevant range of methylation signal and will be further validated in a larger cohort of cancer and non-cancer cfDNA samples. Due to the high specificity and sensitivity, this cost-effective PCR method may provide utility and improve access for early cancer detection and monitoring. Citation Format: Emily Neaga, Caitlin Gilley, Sarah Falotico, Mayur Gurnani, Zhenyu Zhang, Jocelyn Charlton, Miguel Williams, Anthony Shuber. A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A035.